IMR Press / RCM / Volume 23 / Issue 8 / DOI: 10.31083/j.rcm2308284
Open Access Review
The Remaining Conundrum of the Role of the Na+/H+ Exchanger Isoform 1 (NHE1) in Cardiac Physiology and Pathology: Can It Be Rectified?
Show Less
1 Department of Physiology and Pharmacology, University of Western Ontario, London, ON N6A 5C1, Canada
2 Institute of Cardiovascular Sciences, Albrechtsen Research Centre, St. Boniface Hospital, and Department of Physiology and Pathophysiology, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB R2H 2A6, Canada
3 Department of Biochemistry, University Alberta, Edmonton, AB T6G 2H7, Canada
*Correspondence: (Larry Fliegel)
Academic Editor: Fabian Sanchis-Gomar
Rev. Cardiovasc. Med. 2022, 23(8), 284;
Submitted: 8 June 2022 | Revised: 29 June 2022 | Accepted: 8 July 2022 | Published: 15 August 2022
(This article belongs to the Special Issue Highlighting Excellence in Cardiovascular Research in Canada)
Copyright: © 2022 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.

The mammalian Na+/H+ exchanger (NHE) is a family of ubiquitous membrane proteins present in humans. Isoform one (NHE1) is present on the plasma membrane and regulates intracellular pH by removal of one intracellular proton in exchange for one extracellular sodium thus functioning as an electroneutral process. Human NHE1 has a 500 amino acid membrane domain plus a C-terminal 315 amino acid, regulatory cytosolic tail. It is regulated through a cytosolic regulatory C-terminal tail which is subject to phosphorylation and is modulated by proteins and lipids. Substantial evidence has implicated NHE1 activity in both myocardial ischemia and reperfusion damage and myocardial remodeling resulting in heart failure. Experimental data show excellent cardioprotection with NHE1 inhibitors although results from clinical results have been mixed. In cardiac surgery patients receiving the NHE1 inhibitor cariporide, subgroups showed beneficial effects of treatment. However, in one trial this was associated with a significantly increased incidence of ischemic strokes. This likely reflected both inappropriate dosing regimens as well as overly high drug doses. We suggest that further progress towards NHE1 inhibition as a treatment for cardiovascular disease is warranted through the development of novel compounds to inhibit NHE1 that are structurally different than those previously used in compromised clinical trials. Some novel pyrazinoyl guanidine inhibitors of NHE1 are already in development and the recent elucidation of the three-dimensional structure of the NHE1 protein and identity of the inhibitor binding site may facilitate development. An alternative approach may also be to control the endogenous regulation of activity of NHE1, which is activated in disease.

NHE1 regulation
NHE1 inhibitors
cardiac hypertrophy and remodelling
ischemia/reperfusion injury
pyrazinoyl guanidine
1. Introduction

The ubiquitously expressed mammalian Na+/H+ exchanger (NHE) is a family of membrane proteins of human cells of which there are currently 10 known isoforms. Isoform one (NHE1) removes a single intracellular proton in exchange for a single extracellular sodium ion (Fig. 1A) and is ubiquitously present throughout the tissues and cell types of the body [1, 2]. NHE maintains intracellular pH (pH𝑖), thus protecting cells from acidification which results from metabolism. It also responds to osmotic challenge regulating cell volume [3, 4]. There are nine SLC9A type isoforms of NHE, two SLC9B types and also two SLC9C types. Most isoforms of NHEs have restricted cellular locations or intracellular locations but NHE1 (SLC9A1) is the primary plasma membrane isoform found in virtually all mammalian cells [4, 5, 6, 7, 8, 9, 10]. NHE1 consists of two general domains. One is the membrane transport domain which moves ions, and the second is a regulatory cytosolic domain (Fig. 1B). The human N-terminal membrane transport domain is approximately 500 amino acids and its atomic structure has recently been determined [11]. The human cytosolic regulatory domain is an additional 315 amino acids that functions to regulate the membrane domain [12].

Fig. 1.

Schematic diagrams of the Na+/H+ exchanger (NHE1) within the plasma membrane. (A) Schematic diagram illustrating dimeric structure of NHE1 within a lipid bilayer. Arrows indicate direction of transport. (B) Schematic diagram of NHE1 withing the lipid bilayer illustrating the two-domain structure, and approximate locations within the membrane.

The physiological and pathological roles of NHE1 are many. Outside of the myocardium NHE1 plays a role in cell growth, proliferation and differentiation [2, 13, 14, 15, 16]. NHE1 is also an important trigger of growth and metastasis in cancer, notably as a trigger of metastasis in breast cancer [17, 18, 19, 20, 21]. Genetic mutations in NHE1 and its absence, have been shown to be responsible for the disease Lichtenstein-Knorr syndrome which manifests itself through many developmental defects and ataxia and hearing loss [22, 23].

In the myocardium, NHE1 is the only plasma membrane isoform of Na+/H+ exchanger present. Its activity was demonstrated as early as 1984–1985 [24, 25, 26, 27, 28] and a human clone was initially isolated from the myocardium in 1993 [29]. NHE1 is associated with both ischemic reperfusion damage to the myocardium and heart hypertrophy and its inhibition shows beneficial effects in animal models of this disease [30, 31, 32, 33]. It is inhibited by pyrazinoyl guanidines including amiloride, a potassium-sparing diuretic used for decades to treat hypertension and heart failure (in combination with other drugs). NHE1 is also inhibited by benzoyl guanidines such as cariporide which were later developed for clinical experimentation (Fig. 2A). Despite its key role in cardiac physiology and pathology, there is as yet no known NHE1-based therapy developed for clinical protection of the myocardium. Why is that?

Fig. 2.

Structure of amiloride, cariporide and hexamethylene amiloride (HMA) (A-C, respectively). Amiloride and HMA are pyrazinoyl guanidines that differ in the aromatic core from cariporide a benzoyl guanidine.

This review presents a discussion of the structure, chemistry and regulation of NHE1 in the myocardium as well as location of inhibitor binding sites and the potential development of novel NHE1 inhibitors. This is followed by assessment of the role of NHE1 in cardiac pathologies including ischemic and reperfusion injury, myocardial hypertrophy and remodeling resulting in heart failure as well as its role in diabetes related cardiac pathology. Finally, we discuss the clinical potential of NHE1 inhibitors to treat heart disease reflecting on completed, ongoing and future clinical trials. Our goal is to both update the field and to stimulate potential development of new inhibitors that can be useful in treating heart disease and indeed other common human afflictions.

2. Expression and Localization of Myocardial NHE1

NHE1 plays an important physiological role in regulating intracellular pH (pH𝑖) in the myocardium. Through the generation of protons by intermediary metabolism, and also because of the negative membrane potential, protons accumulate within the cytosol and inhibit contractility. NHE1 removes these protons. Cardiac NHE1 has distinct activity characteristics, having a very steep relationship between pH𝑖 and activity [34]. While HCO3- based transporters can also contribute partially to recovery from intracellular proton accumulation [35, 36, 37, 38, 39, 40, 41], as can lactate proton symport [42], so when NHE1 is inhibited or absent, these other mechanisms can aid in pH recovery from acidosis. Additionally, evaluation of the relative contribution of bicarbonate dependent and the NHE transporter to acid extrusion showed that NHE1 is the dominant transporter for proton efflux following intracellular acid load [43]. NHE1 appears to be the principal pH regulatory mechanism in cardiomyocytes. It is the only plasma membrane isoform present in the myocardium that localizes to the intercalated disks and transverse tubules [44, 45]. Cardiac cells do not possess NHE2–5 [46, 47, 48, 49] and NHE6–9 are localized to intracellular organelle membranes such as mitochondria, endosomes and the Golgi network so they do not directly contribute to proton extrusion from cardiomyocytes [50, 51]. cDNA for NHE1 codes for the identical NHE1 message as in other tissues [29] and though a different size mRNA for NHE1 has been shown to occur in ischemic conditions [52], this does not code for a functional protein. Na+/H+ exchange has been clearly demonstrated in cardiac sarcolemma vesicles where it was inhibited by amiloride [28, 53]. The level of NHE1 protein is low, similar to other tissues, but it is clearly very active. It has been possible to immunoprecipitate NHE1 in vivo from isolated cardiomyocytes and tissue, and in vivo phosphorylation was demonstrated [54, 55]. Expression levels of NHE1 in the heart can vary. Ischemia, with or without reperfusion, increases NHE1 mRNA up to seven-fold [56, 57]. Expression in the myocardium also varies developmentally. In rabbit fetal and neonatal hearts mRNA levels are elevated [58]. These results correlate well with gene expression from the NHE1 promoter which was examined in transgenic mice and showed that NHE1 transcription was maximum in the heart and liver in 12-day-old embryonic mice [59].

3. Regulation of NHE1 in the Myocardium
3.1 Hormonal Regulation

NHE1 is normally quiescent in the myocardium at neutral pH, however the protein is activated when intracellular pH decreases and is also activated by stimuli such as growth factors, hormones and osmotic stress. These tend to shift the activity curve such that the protein is active at more alkaline pH𝑖. Regulation of NHE1 occurs in all tissues. This review is restricted in large part to regulation of NHE1 in the myocardium (see also reviews in [12, 60]. Regulation of NHE1 in the myocardium is extremely important. Evidence has shown that activating NHE1 activity through changes in regulation of the protein, accentuate NHE1-induced damage to the myocardium [61, 62, 63, 64, 65]. As noted above, NHE1 activity and mRNA levels are elevated by myocardial ischemia, with or without reperfusion [56, 57] and this may exacerbate NHE1’s detrimental effects in disease. Additionally, targeting regulation of NHE1 has been suggested to be an important approach to treat myocardial disease [66]. Hormones and growth factors modulate cardiac NHE1activity and contribute to its role in cardiac pathology. Endothelin-I, angiotensin II, α-adrenergic agonists, thrombin, and epidermal growth factor are known to stimulate NHE1 in the myocardium. Hormonal regulation often works through activation of protein kinases that phosphorylate the regulatory cytosolic domain of NHE1. Angiotensin II and endothelin stimulate NHE1 activity and their release can occur locally after stretch [67, 68]. The stimulatory action of angiotensin II occurs via the AT1 receptor and occurs through protein kinase C and an epidermal growth factor mediated mechanism. The AT2 receptor mediates an opposing, counteracting inhibition of NHE1 [69]. Endothelin-1 stimulates NHE1 activity [70] as noted above, and inotropic effects of endothelin on the heart may be at least partially attributed to its stimulation of NHE1 [71]. α1-adrenergic agonists like phenylephrine stimulate NHE1 activity by the α1A-adrenoceptor/extracellular signal-regulated kinase (ERK) pathway [55, 72, 73] and this is blocked by Ras-mitogen-activated protein kinase (RSK) [74] and mitogen activated protein kinase (MAPK) [72] inhibition. NHE1 stimulation by α1-adrenergic agonists may also play a role in exacerbation of reperfusion-induced arrhythmias [75]. Thrombin also activates NHE1 in cardiomyocytes through a protein kinase C-mediated mechanism [76] though protein kinase C does not directly phosphorylate the NHE1 cytosolic domain [77]. Fig. 3 illustrates some of the hormones acting to stimulate NHE1.

Fig. 3.

Schematic illustration of regulators of NHE1 in the myocardium. Hormones regulating NHE1 are indicated. The approximate location of lipid, protein and phosphorylation sites on the cytosolic regulatory tail is indicated. P, phosphorylation sites. The kinases phosphorylation sites of AKT, ERK and p90RSK sites are shown. CaM, Calmodulin; CHP, calcineurin homologous protein; 14-3-3,14-3-3 protein; HSP, heat shock protein. Some sites overlap.

3.1.1 Hormonal Regulation of NHE1 through Kinase-Dependent Phosphorylation

Hormonal activation of NHE1 occurs at least partially through protein kinase-mediated phosphorylation or through interaction with other regulatory proteins (or lipids). While the exact percentage of activation in the myocardium that occurs through phosphorylation is not known, it has generally been estimated to be 50% of hormonal regulation though this surely varies with cell type (see reviews [12, 44]). A number of different protein kinases phosphorylate the regulatory tail which is thought to occur mainly in the C-terminal 180 amino acids [12, 60]. In brief, phosphorylation-mediated regulation of NHE1 in several tissues was described earlier [4, 10, 12, 44, 78]. Amino acids phosphorylated include Ser648 by Protein kinase B (PKB or Akt) and [79, 80] amino acids Thr718, Ser723, Ser726, Ser729 by p38 MAPK (equivalent human numbering) [81], and see also [4] for review).

The MAPK phosphorylation pathway was identified as being important in NHE1 regulation in the myocardium. This pathway is regulated by many hormones including endothelin, angiotensin II, catecholamines and some cytokines [82]. This pathway has also been shown to play a role in ischemia reperfusion activation of NHE1 leading to cardiac injury [83] and this p90RSK containing pathway is activated by several other stimuli. One way to activate NHE1 in the heart is by sustained intracellular acidosis which leads to activation of Ras signaling and the kinases ERK1/2 and p90RSK that directly phosphorylate the NHE1 C-terminus [55, 84, 85, 86]. The ERK1/2 and p90RSK pathway has also been shown to be activated and to phosphorylate NHE1 during cardiac ischemia reperfusion injury [87]. Several studies have tried to localize the precise sites phosphorylated by the activated kinases. Early in vitro studies [44, 88] identified four general regions of phosphorylation of NHE1 in the cytosolic tail, (1) S693; (2) T718,S723/726/729; (3) S766/770/771; and (4) T779,S785 and of these, Ser770 and Ser771 of region three were found to mediate ERK1/2 activation of NHE1 by sustained intracellular acidosis in heart cells [55]. Additionally, Ser703 was also earlier identified as being phosphorylated by the kinase p90RSK in several studies [89, 90] including a study on vascular smooth muscle cells, and this amino acid has also been suggested to be important in ischemic and reperfusion injury in the myocardium [90], though sustained intracellular acidosis can activated NHE1 independent of Ser703 and p90RSK [91] (Fig. 3). A way to activate NHE1 through ERK1/2 during cardiac ischemia reperfusion, is by the elevated bursts of reactive oxygen species. Hydrogen peroxide has been shown to activate ERK1/2 and this increases phosphorylation and activation of the Na+/H+ exchanger [92, 93, 94]. How phosphorylation activates NHE1 is still somewhat of a mystery, but it is clear that it results in structural changes in the cytosolic regulatory domain which somehow affect activity of the membrane domain [1, 95, 96].

Some other protein kinases also phosphorylate NHE1 though these are less well studied. Heart β-Raf protein can associate with NHE1 C-terminal domain and can phosphorylate the cytosolic tail at Thr653 [97]. Another regulatory kinase of cardiac NHE1 is PKB [79]. It phosphorylates amino acid Ser648 and this phosphorylation produces an inhibitory effect. Ser648, is within the calmodulin (CaM) high-affinity binding region (see below).

3.1.2 Role of Phosphatases

The requisite dephosphorylation of NHE1 protein must occur sometime after activation of the protein. It is not as well studied in the myocardium. Both protein phosphatases 1 and protein phosphatase 2A (PP1 and PP2A) directly associate with NHE1 [98, 99]. Colocalization of NHE1 and PP2A was shown in ventricular cardiomyocytes [98]. The calcineurin A subunit also binds to NHE1 [100], but its role in dephosphorylation of NHE1 is not yet known. Its binding may facilitate NHE1-induced translocation of NFAT and myocardial hypertrophy progression [100]. An interaction between NHE1 and the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-2) has also been confirmed. Functionally, SHP-2 overexpression caused a higher steady state pH𝑖, and increased recovery from an acid load [101].

3.2 Other Regulatory Processes
3.2.1 Osmotic Regulation

It is known that Na+/H+ exchanger is activated by osmotic regulation. Upon exposure to hyperosmotic solutions NHE1 rapidly increases activity which results in cellular alkalinization. This is part of the regulatory volume increase in cells whereby they compensate for shrinkage that is induced by hyperosmolar external media [102]. Cardiomyocytes exposed to hyperosmolar solutions also show this osmotic activation of the Na+/H+ exchanger. The effect is blocked by calmodulin antagonists and the myosin light chain kinase inhibitor ML-7 [103]. Experiments in intact hearts also show hyperosmotic activation of NHE1 which produces intracellular alkalinization [104]. During myocardial ischemia, accumulating metabolites can cause a hyperosmotic extracellular milieu [105] which could be a mechanism of activation of NHE1 in vivo.

3.2.2 Regulation by Nitric Oxide

Nitric oxide (NO) has been shown to regulate NHE1 activity in adult ventricular cardiomyocytes in a study where which NO levels were manipulated through various approaches [106]. The response was biphasic such that NHE1 flux was activated by low NO levels but inhibited by high NO amounts. These responses were dependent on two pathways, namely a cGMP-dependent NHE1 activation and a cAMP-dependent inhibition. The protein kinases PKG and PKA were tested for their ability to phosphorylate the NHE1 C-terminus and multiple residues were phosphorylated including Ser648 and Ser703. PKA was more selective for Ser648, possibly accounting for the inhibitory effect of cAMP. The biphasic effect of nitic oxide was specific to adult cardiomyocytes and was not observed in neonatal myocytes or in MDA-MB-468 breast cancer cells [106].

3.2.3 Protein-Mediated Regulation

Protein mediated regulation of NHE1 also occurs through the cytosolic regulatory tail. Binding occurs by regulatory proteins, and also by other proteins that may be using NHE1 as a scaffold for other cellular functions aside from regulation of NHE1 activity. This regulatory mechanism has been suggested to account for 50% of the regulation of NHE1 (see reviews [12, 44]) though this is clearly difficult to quantitate and will surely vary with cell type. Most of this type of regulation has been studied in non-myocardial tissue and is briefly reviewed. The “scaffolding” of proteins may vary in response to cellular stimuli [107]. As noted above, protein phosphatases bind to the NHE1 tail and facilitate de-phosphorylation of the tail and have other physiological consequences [100]. In “opposition” to the phosphatases are kinases which also bind to the NHE1 tail. NHE1 acts as a scaffold for ERK and Raf [108]. ERK binds to the cytosolic domain at specific D-domain and F-sites binding sites. These binding sites, and the interaction of NHE1 with ERK affect not only the phosphorylation and activation of NHE1 but also the regulation and activation of ERK itself [96]. Direct binding of ERK to NHE in the myocardium has not, to our knowledge, been demonstrated.

Other interaction partners of NHE1 were reviewed [4] for all general tissues and will be briefly summarized. Several studies have also examined the “interactome” of the regulatory NHE1 cytosolic tail, describing protein that bind to the NHE1 C-terminus from the kidney [109] and from breast cancer cells [110]. Results from some of these are outlined below.

3.2.4 Regulation by Calmodulin

The calcium-binding second messenger protein known as calmodulin mediates Ca2+-induced activation of NHE1. It binds in the presence of Ca2+on two locations on the tail of NHE1. One is the high affinity binding region (amino acids 637-656) and a second is a lower, intermediate affinity region with binding at amino acids 657-700 [4, 12, 44]). Calmodulin regulates NHE1 activity through its high affinity binding site on the NHE1 tail. It binds there preventing this autoinhibitory domain from inhibiting the membrane domain. As noted above, protein kinase B phosphorylates NHE1 within the calmodulin high-affinity binding region at amino acid Ser648 (Fig. 3). This results in a reduction in NHE1 activity by preventing calmodulin binding to NHE1, and thereby preventing blocking of the autoinhibitory site on the cytosolic NHE1 tail. Snabaitis et al. [79] suggest that during ischemic and reperfusion injury this may be a cardioprotective mechanism. There are not many studies on the regulation of NHE1 by calmodulin in the myocardium. It has been shown that the calmodulin blocker W7, inhibits NHE1 activity in isolated cardiomyocytes [103, 111].

3.2.5 Regulation by Calcineurin B Homologous Proteins

There are several isoforms of Calcineurin B homologous proteins (CHPs, CHP1, CHP2 and CHP3) [112, 113, 114, 115]. These are Ca2+-binding proteins with EF-hand motifs that bind Ca2+ ions similar to calmodulin. CHP1 is expressed in the heart and many other tissues. CHP2 expression is mostly restricted to intestinal epithelial cells and malignant tumor cells. CHP3, was initially detected in mouse testis. It is also expressed in the heart, stomach and brain, and some specialized cells such as hematopoietic cells. CHP1 binds to the NHE1 tail at amino acids 518-537 and this binding enhances NHE1 activity [116, 117] (Fig. 3). Mutation of the CHP1 binding site causes NHE1 to have a shorter cellular half-life and causes reduced cell surface expression [118]. CHP3 also has Ca2+-dependent binding to NHE1. CHP3 can also enhance NHE1 stability and activity at the plasma membrane [119]. However, while CHP1 and CHP3 are both expressed in the heart, their role in NHE1 regulation in this specific tissue has not been studied.

3.2.6 Regulation by ERM Protein Family

Ezrin, radixin and moesin, the ERM family form links between NHE1 and actin filaments of the cytoskeleton [120, 121]. This linkage helps facilitate cell migration [122]. NHE1 has ERM binding motifs in amino acids 552-560 of its cytoplasmic tail [121]. While not a great deal has been studied with regards to ERM proteins and the myocardium, one study [123] examined the intracellular location of ERM proteins in left ventricular cardiomyocytes and found that they were localized predominantly at the intercalated disc regions. With intracellular acidification this localization changed, with more localization of activated phospho-ERM in the transverse tubules, which is where NHE1 was localized. This effect was blocked with the NHE1 inhibitor cariporide. These results suggested that ERM proteins may mediate at least some of NHE1 activation in the myocardium.

3.2.7 Regulation by Heat Shock Proteins

Heat shock proteins, both Hsp70 and Hsp90, have been shown to be associated with NHE1 in several studies [101, 109, 110, 124] and inhibition of heat shock proteins can affect NHE1 activity [109, 125]. Hsp90 may affect NHE1 function through alteration of phosphorylation of the protein via AKT kinase [109]. A role of the association in inflammatory response has been suggested [126] and though the association has been shown in different tissues such as kidney and breast cancer cells [109, 110], it has not been extensively studied in the myocardium. One study has examined cardiofibroblasts [127] and showed the NHE1 interacted with Hsp70 by immunoprecipitation.

3.2.8 Regulation by Carbonic Anhydrase II (CAII)

Having an association with a membrane protein which moves ions such as those which CAII or other proteins produce, is thought to facilitate transport of the ions and is called a membrane transport metabolon [128]. CAII is a protein that catalyzes the hydration of carbon dioxide which leads to production of bicarbonate ions and protons. It occurs in a metabolon with NHE1 and other membrane ion transport proteins [128, 129]. A CAII-NHE1 interaction of this type has been studied in some detail in the myocardium. Initial studies characterized the fundamentals of the interaction and demonstrated that CAII binds to NHE1 in vivo, at the penultimate 13 amino acids of the regulatory cytosolic tail (Fig. 3). Ser796 and Asp797 form part of the CAII binding site on NHE1. The association of NHE1 and CAII was dependent on NHE1 being phosphorylated upstream of the CAII binding site [130, 131]. The association of these two proteins was then studied in the myocardium when varying myocardial stretch. Stretch is known to activate NHE1 (see Section 3.2.11) so the association of NHE1 with CAII was examined following stretch of rat papillary muscle by co-immunoprecipitation of the two proteins. Stretch increased association of NHE1 and CAII and inhibition of p90RSK reduced the interaction, suggesting that phosphorylation was involved [132]. The same group [133] examined the association of NHE1 and CAII in obese type 2 diabetic mice. Both control (heterozygote) and obese mice showed co-immunoprecipitation of NHE1 and CAII, and they observed an increase in the amount of CAII attached to NHE1 in homozygote obese diabetic mice. It was suggested that there is an increase in the amount of this “membrane transport metabolon” in the failing mouse heart [133].

3.2.9 Lipid Regulators of NHE1

Phosphatidylinositol 4, 5-bisphosphate is a different binding cofactor of NHE1, being a lipid and not a protein. It binds in two cationic juxtamembrane binding regions of NHE1, at amino acids 513-520 and 556–564 of the rat protein which are equal to amino acids 509-516 and 552-560 of human NHE1 [134]. Mutation of these binding sites decreases NHE1 transport efficiency [134]. The second region overlaps with a region between amino acids 542-598 which is called a lipid interacting domain, with a hydrophobic sequence 573LIAFY577 within it that binds the lipids diacyl glycerol and phorbol esters. These directly activate NHE1 [135]. Lipid regulators of NHE1 in the myocardium were examined some time ago, though not in the context of these lipid binding domains. Green et al. [136] showed that in cultured cardiac cells phorbol esters activate NHE and produce cellular alkalinization. Phorbol esters have also been shown to activate NHE1 in rat vascular myocytes [137] and Vigne et al. [138] showed that phorbol esters activate NHE in skeletal muscle myoblasts. Phorbol esters almost certainly act through these lipid binding sites since it has been shown that protein kinase C cannot directly phosphorylate the C-terminus of NHE1 directly [77] and NHE1 phosphorylation does not to correlate directly with protein kinase C activity [137].

3.2.10 Role of ATP Binding

NHE1 is an ATP binding protein. Early studies showed that depletion of intracellular ATP levels inhibits NHE1 activity [139, 140, 141, 142, 143]. More recently, direct binding of ATP to the NHE1 cytosolic domain was demonstrated by photoaffinity labeling and equilibrium dialysis. The location of ATP binding was localized to amino acids Gly542-Pro598 of human NHE1 (Fig. 3). ATP binding affected the pH dependence of NHE1 activity, ATP depletion caused an acidic shift in the pH𝑖 required for activation of NHE1 [144] which can shift the threshold for activation by about a half a pH unit [139, 145]. In cultured rat aortic smooth muscle cells, the activity of NHE1 has been shown to be reduced in response to hypoxic conditions with an increase in the threshold for activation of NHE1 [146]. ATP would be reduced under hypoxia which may account for this change in activity though direct ATP binding effects were not shown in this study [147]. A similar result was also demonstrated in cultured rat ventricular cells. Treatment of cells with 2-deoxyglucose demonstrated NHE-dependence on ATP levels [111]. Given that ischemia causes depletion of intracellular ATP levels, this study also suggests that NHE1 activity would be reduced during cardiac ischemia in the intact heart.

3.2.11 Regulation of NHE1 by Stretch

Stretch enhances myocardial contractility by two mechanisms. One rapid mechanism is the classic Frank-Starling mechanism that is attributed to enhanced myofilament calcium responsiveness. The second mechanism is the “slow force response” which occurs more slowly, as its name suggests. It is due to an increase in calcium transient size as a consequence of stretch activating autocrine and paracrine mechanisms [148, 149]. It is in the slow force response that NHE makes a significant contribution. Knockdown or inhibition of NHE1 blunts the slow force response [150, 151]. The mechanism by which this response works has been studied in several authors and was reviewed earlier [67, 148]. Briefly, stretch caused release of Angiotensin II, and activation of the AT1 receptor. This results in formation and release of endothelin which causes NHE1 hyperactivity. The increased NHE1 activity causes an increase in intracellular sodium and results in an increase in intracellular calcium though the reversal activity of the Na+/Ca2+ exchanger. Concurrently elevated reactive oxygen species can trigger NHE1 phosphorylation. Some of this activation may be through the local hormones [148, 152]. Stretch activates angiotensin/endothelin dependent chain of events leading to increasing phosphorylation of Ser703 of NHE1 and elevated ERK phosphorylation [153]. Inhibition or knockdown of the mineralocorticoid receptor also reduces the activation by stretch, blocks reactive oxygen species elevation and blocks ERK and p90RSK phosphorylation [152]. Additionally, knock down or blockade of the epidermal growth factor receptor also blocks this slow force response [153, 154]. Conversely, p38-MAPK activation after myocardial stretch limits ERK and p90RSK phosphorylation, and NHE1 phosphorylation through activation of a dual specificity phosphatase which inhibits the slow force response [155]. This NHE1-dependent stretch induced slow force response is important since the increase in the calcium transient is thought to be involved in development of cardiac hypertrophy and therefore a contributor to heart failure. This mechanism possibly acts as an early step towards cardiac pathology if the stimulus remains over time. The activation of NHE1 after stretch may thus have important clinical implications (reviewed in [156]).

3.2.12 Alteration in NHE1 Activity by Diet

Relatively little research has been carried out to identify the impact diet has on NHE1 function and expression. Generally, high fat diets have been shown to increase oxidative stress and heart dysfunction. This may be associated with an activation of NHE1 because long term ablation of NHE1 activity via the use of NHE1-null mice mitigated the deleterious cardiac effects of the high fat diet [157]. However, perhaps the most powerful dietary intervention that demonstrated NHE-1 inhibition was provided by ginseng, a widely used medicinal herb particularly in Asian societies. Ginseng provided a direct anti-hypertrophic action in cultured cardiomyocytes, and importantly, inhibited heart failure through an attenuation of the upregulation of NHE1 activity typically exhibited in hypertrophic responses [158].

The mechanism whereby diet alters NHE1 activity is unclear. However, one may speculate that the membrane lipid composition which influences related membrane transporters like the Na+-Ca2+ exchanger (NCX) [159, 160, 161, 162] may induce similar effects on NHE1 activity. In support of this hypothesis, acute administration to isolated cardiomyocytes of the omega-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibited NHE1 activity [163]. Consistent with this effect, supplementing the diet with EPA and DHA in a rabbit model of volume and pressure overloaded cardiac hypertrophy and failure attenuated the upregulation in NHE1 activity [164].

3.3 SLC9A1 Gene Regulation

The NHE1 gene has been cloned from several species including the human, porcine and rabbit forms of the promoter [165, 166, 167]. The human gene has 12 exons and 11 introns and the mouse gene has a similar design [168]. There is a very large (41.5 kb) intron between the first and second exon while the other introns are much smaller [169]. Studies in vitro and in transgenic mice with the 5’ flanking region of the mouse promoter showed that NHE1 expression in the myocardium is high during early embryonic development and showed that the NHE1 protein is at relatively high levels in the neonate and declines with age [58, 59, 170, 171]. This effect may be comparable to the myosin heavy chain where a switch to fetal type of gene expression during hypertrophy increases expression.

A number of studies characterized transcription factors and regions important in expression in tissues outside the myocardium AP-1. The C/EBP family have been shown to be important in some cell types [172, 173]. The porcine, rabbit and mouse and human NHE1 promoter, are homologous particularly in the proximal 500 bp of the 5’ flanking region [165, 166, 167, 169]. Consensus sequences for the transcription factors AP-1, C/EBP and Sp1 are conserved between pig and human [167]. A proximal AP-2 binding site in the mouse NHE1 promoter is important in expression in fibroblasts and in P19 embryonal carcinoma cells [57, 165]. The NHE1 promoter is activated in some models of cell differentiation including in P19 cells or in L6 muscle cells [174, 175]. Some other regions of the promoter important in expression are a poly (dA:dT) region of the NHE1 promoter is located at bp -155 to -169 of the mouse gene which was tested in L6 and NIH 3T3 cells [176]. Several other regions of the NHE1 promoter were studied in a variety of cells. COUP-TF (Chicken ovalbumin upstream promoter transcription factor) type I and II is more distal (-841 to -800 bp) and is also important in expression [177]. Thyroid hormone receptor TRα1 is also implicated in regulation of the promoter [178].

Studies directly examining NHE1 promoter regulation in the myocardium are rare. A 1.1 kb region of the mouse promoter drove expression in cardiomyocytes that was stimulated by serum. Deletion of a proximal AP-2 site decreased promoter activity 4-fold [179]. Mutation of that site, combined with deletion of distal regions of the promoter, virtually eliminated promoter activity in cardiomyocytes. DNase I footprinting analysis showed that the poly(dA:dT) rich region (-155 to -169), is protected by heart nuclear extracts as is the COUP-TF element [176, 180]. Thyroid hormone may regulate the NHE1 gene in the heart. Treatment of heart cells with thyroid hormone increased protein binding of nuclear extracts in the COUP-TF region and treatment of cardiomyocytes with thyroid hormone increases NHE1 protein expression [178].

NHE1 levels have been shown to be elevated in the myocardium following ischemic heart disease and some of this could be mediated through reactive oxygen species (ROS). Increasing serum from 0.5 to 10% induces NHE1 promoter activity in NIH3T3 fibroblasts [179]. The increase correlates with increased O2 superoxide production and NHE1 promoter activity and O2 superoxide production could be blocked by the oxidase inhibitor diphenyleniodonium [181]. Tiron, a specific O2 superoxide scavenger, could also block increases in NHE1 promoter activity and protein levels [181].

Aside from these earlier studies, there has been a surprising dearth of recent work on the promoter in the myocardium. It would be interesting to examine NHE1 transcription in detail during cardiac hypertrophy and in response to ischemia and reperfusion.

4. Structure of NHE1 and Location of Inhibitor Binding Site

The structure of NHE1 and the inhibitor binding site is of paramount interest as clearly the binding site will affect the efficacy and potency of inhibitors and the site itself will affect the design of novel inhibitors. Traditionally, human NHE1 has been thought of as a 12 transmembrane protein with a large cytoplasmic tail (Fig. 4A, Ref. [182, 183, 184]). This type of low-resolution model was initially based on hydrophobicity plots and later more experimental evidence was added, mostly using cysteine accessibility studies along with hydrophobicity plot [182, 183]. These studies initially suggested 12 transmembrane segments were present with two intracellular re-entrant loops, one between TM4 and TM5 and one between TM8 and TM9. There was a larger extracellular loop predicted between TM9 and T10.

Fig. 4.

Molecular models of human NHE1. (A) Two-dimensional topology model of human NHE1 based on hydrophobicity analysis and cysteine accessibility studies [182, 183]. (B) Novel two-dimensional topology model of human NHE1. TM’s 2-14 are based on the Cryo-EM structure [11]. TM1 is added as per [182, 183, 184]. The core domain TMs are colored light green and the dimerization domains TMs are light blue. Amino acid numbering indicates the end of the transmembrane segments. EL, extracellular loop; IL, intracellular loop. The transmembrane segment number is indicated beginning from the N-terminus and including the first TM. The unwound region of TMs 6 and 13 is indicated by a discontinuity. Amino acids important in cation coordination, proton sensing or inhibitor binding are indicated in red. (C) Structure of human NHE1 fold from [11]. TM 6 (green) and 13 (red) of human NHE1 are shown (amino acids 223-249 and 445-469 respectively). (D) Lateral view of cariporide binding site in the Cryo-EM structure [11] of human NHE1. Most of the protein is not shown. Cariporide cyan, E346 red, D267 magenta, F162 yellow, L163 grey. (E) Lateral/extracellular view, color scheme retained as in D, plus D159 orange.

The structure of the E. coli NhaA (Na+/H+ antiporter type A) protein was deduced in 2005 [185] and this led to attempts to model human NHE1 based on the structure of the E. coli protein [186]. However, this led to some conflicts in interpretation [183, 187, 188]. The E. coli protein, NhaA (for Na+/H+ antiporter type A) has significant differences from the mammalian protein. For example, the stoichiometry of exchange in bacteria is 1Na+/2H+ [189] compared to the electroneutral 1:1 exchange of mammalian NHE1. However, it is worth briefly reviewing a few fundamental aspects of this structure which are similar in other forms of this type of transport (see also reviews [190, 191, 192]). The crystal structure of acid-inactivated NhaA has 12 TM segments. A critical feature is a TMIV-XI assembly. Both these helices are discontinuous, interrupted by an extended segment. The discontinuous helices form mid-membrane dipoles. It was suggested that the charge on D133 compensates for the opposing N-terminal end and K300 for the opposing C-terminal end of the helix [185, 191]. The pore contains cytoplasmic and periplasmic funnels, both of which narrow so that hydrated cations cannot pass. The ion binding site of NhaA is formed around the extended segments of TMs IV and XI and includes D164, D163, and T132 [193, 194]. It is hypothesized that Na+ binding to the active site from the cytoplasm causes a charge imbalance. This triggers movements of the TMIV-XI assembly, exposure of Na+ to the periplasm, its release, and proton. Although this model for the function of NhaA is well developed and elegant, there is some controversy. Arkin et al. [195] proposed a variation of the model, in which 3 conserved Asp residues are key to Na+/H+ antiport. D164 is the Na+ binding site, D163 controls accessibility, and D133 mediates pH regulation. More recently, K300 was proposed to be essential for stability but not for electrogenic transport [196].

Structures of other Na+/H+ exchangers have been more recently elucidated. This includes that of the archaeal Na+/H+ antiporter NhaP1 from Methanococcus jannaschii [197, 198], NapA from Thermus thermophilus [199, 200], NhaP [201] (a Na+/H+ exchanger of Pyrococcus abyssi, an archaeon from deep-sea hydrothermal vents), NHE9 [202] (another mammalian isoform related to NHE1), NHA2 [203] (a more distant mammalian Na+/H+ exchanger more closely related to NhaA). Generally, a summary would be to that these Na+/H+ exchanger types have significant similarities to NhaA of E. coli despite their low homology to the E. coli protein. Functionally this includes acid residues for cation coordination, and the presence of a Na+/H+ exchanger fold consisting of discontinuous transmembrane segments with a mid-membrane unwound stretch of amino acids. Some publications have also suggested alternating access to key acidic (Asp) residues using elevator like structural movements of transporter domains [193, 200, 202].

Very recently, the structure of human NHE1 in complex with the regulatory protein CHP1, was determined by cryogenic electron microscopy (Cryo EM) [11]. The structure was of amino acids 87-590, which formed 13 transmembrane helices and 3 cytosolic helices. The topology was generally consistent with previous models except a previous re-entrant loop between a previously labeled TM9 and TM10, is actually two TM helices. A simplified model of the topology is shown in Fig. 4B. The model is based on the Cryo EM structure of human NHE1. TM 1 was added based on an earlier molecular model of NHE1 and experiments involving cysteine labeling of TM 1 which showed that TM 1 was present in the intact protein [182, 183, 184] (Numbering of NHE1 TMs will from here on include TM1 as shown in Fig. 4B.)

NHE1 was a homodimer in the Cryo EM structure. Each monomer was made of a dimerization domain of TM segments (TMs 2-4, and 8-11, amino acids 99-176, plus 288-405) plus a core domain (TMs 5-7 and 12-14, amino acids 187-283 and 411-505) [11] (see Fig. 4B). TMs 6 and 13 (amino acids 223-253 and 445-469) have the NHE-fold with an unwound region in the middle and they cross each other (Fig. 4C). The helix breaks are thought to participate in forming the ion permeation pathway. In their structure there was a funnel between the dimerization domain and the core domain of each protomer. The funnel is formed by TMs 2,3,4,6,7 and 11. Proton-titratable residues in the funnel are E131, D172, D238, D267 and E391 (indicated in Fig. 4B). This results in a negatively charged cavity which was thought to participate in cation binding and proton sensing [11]. D267 was thought to participate in cation binding and is critical to activity [204, 205]. The S263 sidechain was also thought to participate in ion transport and D238 was thought to indirectly participate, coordinating a water molecule [11]. E391 is essential [204] but because of its location away from the cation binding site it may affect folding or stability of the protein and was not thought to directly binding cations. E131 was thought to be a pH sensor functioning when protonated to accelerate cation release [11]. Mutation of F162 has been shown to have a large effect, reducing efficacy of cariporide and mutation of I169 and I170 accentuate this effect [206]. Mutation of E346 has also been shown to have large effects on inhibitor efficacy [207, 208].

Considering the potential benefits of NHE1 inhibition in cardiac disease, there has always been great interest in the determination of the exact site of inhibitor binding. Classically, a number of studies carried out site specific mutagenesis on amino acids believed to be involved in inhibitor binding. Then any changes in inhibitor efficacy were examined. Some of these studies have suggested a number of amino acids with large effects on inhibitor potency as being important in human NHE1 inhibition that includes human F167 [209], L167 (rat) [208], Gly174, Leu163 [210] while other studies have showed smaller changes in drug potency with mutation of human L265 and L255 [211]. Other studies [208, 212, 213] used large scale replacement of pieces of NHE1 to try to determine which regions are critical in drug interaction, but these have the disadvantage of increased likelihood of disruption of the structure of NHE1. Molecular modeling of NHE1 and inhibitor docking was also attempted [184] and a number of amino acids were suggested to interact with inhibitors at different potential binding sites.

While these studies provided interesting information, the recent Cryo-EM structure directly showed the inhibitor binding site of the benzoylguanidine cariporide [11]. Cariporide bound from the extracellular side of NHE1 in a pocket located between the dimerization and core domain. It was surrounded by TMs, 4, 7, 9, 10 and 13. The extracellular loop #1 from the opposing subunit, and resides of TM 9 and 10 may be essential for the binding pocket. Amino acid D267 of TM7 points up into the inhibitor binding pocket and interacts with the positively charged group of the guanidine (Fig. 4D,E) [11]. The side chain of F162 (TM4) also interacts with the guanidine group and the phenyl ring of cariporide which agrees with mutational studies [206]. The guanidine group is also coordinated by the side chain of E346 (TM9) which agrees with another mutational studies [207]. Cariporide also has a methylsulfonyl group at the meta- and para- position of the phenyl ring (Fig. 2B). These are buried in a sub pocket formed by D159, L163, D95, H98 and V99. Mutational studies, including of L163, support the importance of these amino acids in inhibitor binding [209].

It is also important to note that it has long been suggested that inhibitors such as amiloride, bind to the same or are overlapping with, the cation binding site (reviewed in [214]). The Cryo-EM structure of NHE1 could not visualize the precise location of the Na+ ion, which is thought to be partially hydrated, and is about the same size of the guanidine group [11, 214]. Given the coordinating amino acids, their characteristic charges, location, and the size of the pocket, the authors hypothesized that F162, D267 and E346 coordinate the Na+ ion in the outward facing site [11]. Mutation of these residues affects both inhibitor efficacy and activity, supporting this hypothesis [215].

5. Development of Inhibitors of NHE1: Past and Present

Because of the extensive experimental evidence that NHE1 participates in cardiac pathologies including ischemia and reperfusion injury as well as myocardial remodeling and heart failure (discussed in Sections 6 and 7) there has been substantial interest in inhibition of NHE1 and in the development of clinically useful compounds for treatment of the diseased myocardium. Early pharmacological studies probing the function of NHE1 in biological systems including heart disease employed the use of amiloride (Fig. 2A), a potassium-sparing diuretic or its derivatives such as ethylisopropylamiloride (EIPA), methylisobutyl amiloride (MIA), hexamethyl amiloride (HMA), dimethylamiloride (DMA) (Fig. 2C) and others. While these agents are effective in inhibiting NHE1, they lack specificity against the NHE1 isoform, have non-specific effects on many aspects of cardiac performance [216] and are also effective against other ion regulatory systems. This concern regarding non-specificity was rectified to a large degree by the development of novel second generation NHE-1 specific inhibitors by the pharmaceutical industry. The first among these was the benzoylguanidine derivative HOE694 ((3-methylsulphonyl-4-piperidino-benzoyl) guanidine methanesulphonate developed by Hoechst AG (now part of Sanofi) and which was demonstrated to exert cardioprotective and antiarrhythmic effects in ischemic and reperfused hearts [32]. This was followed by the development of a new NHE1-specific inhibitor initially designated as HOE642 (N-(Aminoiminomethyl)-4-(1-methylethyl)-3-(methylsulfonyl)-benzamide) [31] and subsequently renamed as cariporide (Fig. 2B) in preparation for clinical development. As will be evident from the discussion below, cariporide is the most extensively studied of the NHE1-specific inhibitors both experimentally as well as in clinical studies particularly with respect to mitigating ischemic and reperfusion injury.

The development of these benzoyl guanidine derivatives has led to the rapid formulation of numerous newer and more potent NHE1-specific inhibitors. For an in-depth description of the development and chemistry of NHE1-specific inhibitors please refer to [217]. A partial list of these agents is provided in Table 1 (Ref. [32, 218, 219, 220, 221, 222, 223, 224, 225]). Virtually all of the agents listed in this table possess the monocyclic acylguanidine structure found in the amiloride-based inhibitors (with the exception of SL 59.1227) but demonstrate markedly enhanced specificity towards NHE1 as well as greater potency. Preclinical and clinical studies with a number of these agents are discussed in greater detail below.

Table 1.Some examples of NHE1-specific inhibitors developed by the pharmaceutical industry.
Drug Chemical name Developer Reference
HOE694 (3-methylsulphonyl-4- piperidino -benzoyl) guanidine methanesulphonate Hoechst AG4 [32]
HOE6421 (N-(Aminoiminomethyl)-4-(1-methylethyl)-3-(methylsulfonyl) -benzamide) Hoechst AG [218]
MS31-038 2-phenyl-8-(2-methoxyethoxy)-4-quinolyl carbonylguanidine bismethanesulfonate Mitsui [219]
EMD875802 N-carbamimidoyl-2-methyl-4,5-bis(methylsulfonyl) benzamide Merck KGaA5 [220]
EMD851313 (2-Methyl-5-(methylsulfonyl)benzoyl)guanidine Merck KGaA [221]
T162559 ((5E,7S)-[7-(5-fluoro-2-methylphenyl)-4-methyl-7,8-dihydro-5(6H)-quinolinylideneamino] guanidine dimethanesulfonate) Takeda [222]
BIX N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine BI6 [223]
SL59.1227 3-[(cyclopropylcarbonyl)amino]-N-[2-(dimethylamino)ethyl]-4-[4-(5-methyl-1H-imidazol-4-yl)piperidin-1-yl]benzamide Sanofi [224]
Zoniporide [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine Pfizer [225]
1cariporide; 2rimeporide; 3eniporide; 4currently a wholly-owned subsidiary of Sanofi; 5now Merck Serono (EMD Serono in the United States and Canada); 6Boehringer Ingelheim.

Recently one group has developed a novel series of compounds of 6-substituted amiloride and hexamethylene amiloride (Fig. 2C) derivatives as inhibitors of NHE1. Depending on the precise compound made, they may also inhibit human urokinase plasminogen activator [226, 227, 228]. The reasoning behind modification of amiloride (and its derivative) for use as an inhibitor is that amiloride is a pyrazinoyl guanidine, a clinically safe compound used as a potassium-sparing diuretic. Given the problems the benzoyl guanidine derivative cariporide for treatment of heart failure (see below) it was reasoned that using a pyrazinoyl guanidine core that differs in the aromatic core from cariporide (a benzoyl guanidine, Fig. 2) and related NHE1 inhibitors might be a prudent approach to avoid side effects and regulatory issues for drug development. 6-substituted amiloride/HMA compounds have increased potency towards NHE1. Amiloride is an ideal candidate for a scaffold to build a dual-targeting compound with cardiovascular beneficial properties. HMA is both significantly more potent and more specific towards NHE1; amiloride inhibits sodium channels but HMA much less so [229]. Inhibitors have been developed with nM potency towards NHE1 and show no effects on diuresis or urinary Na+/K+ level in rats [226, 227, 230]. While in theory these compounds sound extremely promising, they have not yet been tested for efficacy in prevention of ischemic and reperfusion injury or in the prevention of deleterious cardiac remodeling.

It is of note that some of these compounds have a dual action, inhibiting urokinase plasminogen activator (uPA) [226, 227, 230]. In the myocardium, uPA induces cardiac fibrosis and human hearts with end stage failure and fibrosis have elevated plasminogen activator activity [231]. Excess uPA promotes cardiac fibrosis in association with M2 macrophages [232]. Blocking the uPA pathway reduces cardiac macrophage accumulation, excess collagen formation and heart fibrosis [233]. Thus, it may also be of interest to test the effect of dual inhibition of NHE1 and uPA, in heart failure models. To our knowledge this has not been done. Caution is advised though, as urokinase inhibition could increase thrombolysis and thrombosis. Fortunately, a large number of NHE1 inhibitory compounds have been made with varying degrees of uPA inhibition from none, to little, to potent inhibitors of both uPA and NHE1 [226, 227, 230].

6. Role of NHE1 in Cardiac Ischemic and Reperfusion-Induced Injury
6.1 Theoretical Concepts

Although the entry of excessive extracellular Ca2+ into the cardiomyocyte was recognized early on as a key event in the toxic effects of ischemic injury to the heart (Ca2+ overload) [234], the mechanism whereby this excess Ca2+ entered the cell remained unknown until decades later. Blocking the L-type Ca2+ channel had limited clinical utility in protecting the heart from ischemic and reperfusion injury so that was clearly not the mechanism for Ca2+ entry [235]. Lazdunski proposed in 1985 [24] that a mechanism involving NHE and NCX activation was responsible for the toxic entry of Ca2+ into the myocardium as is summarized in Fig. 5. This scheme reinforces the concept of a close link between NHE1 and NCX not only with regards to ischemia and reperfusion but also pertaining to NHE1-dependent cardiac hypertrophy, as discussed in section 7 of this review. However, NHE1 inhibition has been shown to attenuate the deleterious effects of many factors including to varying degrees, interactions with G-protein-coupled receptors [236], the α1A adrenoceptor subtype [75], protein kinases [84, 87, 237], angiotensin I and II receptors [69], membrane lipids [238] and endothelin-1 [239]. The primary cardioprotective action of the inhibitors, however, remains in their ability to block Na+, H+ and Ca2+ movements via the NHE1 and NCX transport pathways in the myocardial cell (Fig. 5).

Fig. 5.

Schematic of the mechanism by which the Na+/H+ exchanger (NHE1) and the Na+/Ca2+ exchanger (NCX) interact to generate cardiac injury and dysfunction during ischemia/reperfusion challenge. (1) In the presence of reduced coronary blood flow and absence or reduced oxygen, ischemia causes a reduction in intracellular ATP levels and an accumulation of intracellular H+. The decreased levels of ATP stores impair Na+/K+ ATPase activity reducing Na+ export. (2) The elevated intracellular H+ activates NHE1 to remove the H+ in exchange for extracellular Na+. (3) The increasing intracellular Na+ via NHE1, slows the removal of intracellular calcium by NCX or drives NCX in the reverse direction thereby increasing intracellular calcium concentrations in the cell. (4) The excess entry of Ca2+ in the cell results in contractile dysfunction and structural damage to the myocardial cell.

6.2 Studies with Amiloride Analogues

Karmazyn’s laboratory was the first to demonstrate the likely validity of this concept by demonstrating a cardioprotective effect of amiloride, in isolated perfused rat hearts subjected to ischemia and reperfusion [240]. This seminal observation using a well-known inhibitor of Na+ transport (amiloride) was independently confirmed and extended by two other labs shortly thereafter. Tani and Neely demonstrated both metabolic and ionic data consistent with an involvement of the NHE and NCX pathways in ischemic/reperfusion injury [241]. By this time, Edwin Cragoe, Jr. had established a large library of amiloride analogues at the Merck laboratories [242] that were more specific inhibitors of the NHE pathway than amiloride and this significantly accelerated the work in this area. Several laboratories took advantage of this advancement and employed several of these amiloride analogues in a series of studies to again elicit significant cardioprotection during ischemia and reperfusion challenge to the heart [243, 244, 245, 246, 247].

A biochemical approach further indirectly implicated the NHE pathway in the cardiac damage by modifying the extracellular acidity and Na+ levels during the ischemia and reperfusion insult [243, 245, 248, 249]. Ionic changes consistent with a role for NHE during ischemia/reperfusion and its blockage by NHE inhibitors were demonstrated by radioisotopic [241], atomic absorption spectrometric [245], electrophysiological [248] and nuclear magnetic resonance methods [250]. The cardioprotection was observed whether the NHE inhibitors were delivered prior to ischemic insult or solely during reperfusion although protection was generally greater when the drug was present during the ischemic period [251, 252]. The cardioprotective effects of NHE blockers were not species specific. Protection was observed in hearts from rats and guinea pigs [247].

All of these studies were consistent with a critical involvement of NHE in the cardiac damage [240, 241, 253, 254], cardiac contractile dysfunction [240, 241, 243], and arrhythmias [255, 256] that are evident during the ischemia and reperfusion insult. NHE inhibitors were cardioprotective not only during ischemia and reperfusion challenge but protected the myocardium during hypoxia/reoxygenation insult [257], hypothermic conditions [258, 259], oxidative stress [260, 261], and pacing induced-heart failure [262]. Conversely, NHE inhibitors did not provide cardioprotection during ischemic pre-conditioning [259] or the Ca2+ paradox protocol of myocardial Ca2+ overload [257].

6.3 Studies with New Generation NHE1 Specific Inhibitors

Earlier studies using amiloride or its analogues as cardioprotective agents presented some difficulty in interpretation as they lacked specificity in terms of targeting NHE1. Moreover, virtually all studies with these agents were carried out using in vitro cardiac preparations with no studies on more clinically-relevant in vivo animal models. This situation was rectified to large degree by the development of highly specific NHE1 inhibitors as discussed in section 5. The first of these agents to be developed was the benzoyl guanidine derivative HOE 694 which was shown to produce extensive protection in both isolated rat hearts as well as in rats subjected to coronary artery ligation [32]. Although HOE 694 was not destined for clinical development this was not the case for HOE 642 (cariporide) (Fig. 2B), a more potent NHE1 inhibitor, developed shortly thereafter [218] and which, in an initial study was shown to exert potent cardioprotective effects in both isolated hearts as well as in vivo coronary artery ligation followed by reperfusion [31]. This protection was manifested in terms of reduction in the incidence of arrhythmias, reduction in tissue injury as well as preservation of energy metabolites [31]. Cariporide subsequently emerged as the most widely studied of the newer generation of NHE1 inhibitors as a cardioprotective strategy. Indeed, these studies using cariporide have shown excellent cardioprotection across different animal species and experimental models [253, 254, 263, 264, 265, 266, 267].

The consistent protection seen with cariporide was clearly evident in subsequent studies utilizing newer NHE1 specific inhibitors including AVE 4890 [268], MS-31-038 [219], BIX [223], BIIB 513 [269], EMD 85131 [221] and others. Indeed, it can be safely stated that virtually all animal studies using these agents have consistently demonstrated excellent cardioprotection irrespective of experimental model or animal species. This consistency in demonstrating cardioprotective efficacy of NHE1 inhibitors is likely unmatched by any other strategy. Indeed, Garrett Gross’ laboratory at the Medical College of Wisconsin has reported that NHE1 inhibition with BIIB 513 was superior to ischemic preconditioning particularly when ischemic preconditioning was no longer effective as a result of prolonged ischemic duration [221].

6.4 Studies with NHE1 Transgenic Mice

A number of studies were undertaken to determine whether genetic modulation of NHE1 alters the cardiac response to ischemia and reperfusion. Surprisingly, overexpression of NHE1 in transgenic mouse hearts resulted in enhanced recovery after reperfusion of ischemic isolated perfused hearts which the authors attributed to improved metabolic parameters [270, 271]. Ironically, in one study the protection seen with NHE1 overexpression was similarly observed with cariporide [271]. In another study, cardiac overexpression of NHE1 resulted in modest protection against ischemia and reperfusion in vivo although this was not affected by zoniporide, a highly specific NHE1 inhibitor suggesting that the protection was not NHE1 dependent [272]. With aging, NHE1 overexpressing mice exhibited increased apoptosis, left ventricular contractile dysfunction, myocardial remodeling and premature death which the authors attributed to sustained endoplasmic reticulum stress [272]. While further work is required in this area, when taken together the results suggest that NHE1 overexpression per se may not be necessarily deleterious to the ischemic myocardium although inhibiting the exchanger to a critical level confers protection. This concept is reinforced by the finding that genetic ablation of NHE1 in mice confers protection against ischemic and reperfusion injury in isolated perfused hearts [273].

6.5 NHE1 Inhibition for Post-Cardiac Arrest Resuscitation: An Ischemia and Reperfusion Scenario

Successful cardiac resuscitation, particularly after out-of-hospital sudden cardiac arrest, represents a major medical challenge. This reflects the very poor outcome in terms of successful resuscitation which is seen in only about 10% or less of cardiac arrest victims, although these rates vary depending on various factors [274]. Cardiac resuscitation is in essence an ischemia (cardiac arrest) and reperfusion (resuscitation) scenario thus suggesting a potential benefit of cardioprotective agents in terms of improving resuscitation efforts. Indeed, NHE1 inhibitors have been proven to be of immense benefit for improving post-cardiac arrest resuscitation in experimental animal models. Much of the original and pioneering work in this area comes from Gazmuri’s laboratory in Chicago who first proposed this concept (see below) and who recently published a comprehensive review of the role of NHE1 in cardiac resuscitation [275]. As such only a brief discussion is presented here.

As just noted, the first report demonstrating a beneficial effect of NHE1 inhibition was presented by Gazmuri and colleagues who showed that cariporide improved post ventricular fibrillation recovery in isolated rat hearts as well as rats in vivo subjected to cardiac arrest [276]. It is interesting that with respect to the latter, cariporide reduced the degree of chest compression required to attain sinus rhythm and precluded the necessity for electrical defibrillation in six of eight animals studied whereas electrical defibrillation was required in all 8 control rats [276]. Moreover, and importantly, over 90% of cariporide treated animals survived the cardiac arrest-resuscitation protocol compared to just over 60% seen in control animals [276]. A similar beneficial effect of cariporide was also convincingly demonstrated by these and other investigators using a pig model of cardiac arrest followed by attempted resuscitation [277, 278]. Thus, cariporide improved hemodynamics following resuscitation compared to control animals, completely prevented mortality while reducing neurological deficit [278].

From a mechanistic perspective, the beneficial effects of NHE1 inhibition in improving cardiac resuscitation appear to be similar to mechanisms seen in classic cardioprotection seen in ischemic and reperfused hearts with NHE1 inhibitors. Thus, it has been reported that the NHE1 specific inhibitor AVE4454B, although less effective than cariporide in terms of improving cardiac resuscitation [279], improved cardiac resuscitation in the rat which was associated with diminished cardiac mitochondrial calcium overload [280]. The beneficial effects of cariporide were also associated with diminished arrhythmogenesis including ectopic activity and reduced the shortening of the action potential duration associated with resuscitation [277, 281]. Further mechanistic insights into the improved resuscitation produced by NHE1 inhibitors were provided in a study using the NHE1 specific inhibitor zoniporide. In this regard zoniporide, as expected, improved ventricular recovery following ventricular fibrillation-induced cardiac arrest in pigs, effects associated with improved myocardial metabolic status including preserved myocardial creatine phosphate to creatine ratios thus indicating improved oxidative phosphorylation [282]. Mitochondrial as well as neuronal protection with cariporide was also demonstrated in an asphyxia model of cardiac arrest in the rat although the protection was evidently dependent on cariporide dose [283]. Thus, while a protective response was seen using either 1 or 3 mg/kg cariporide no protection was evident when the cariporide dose was increased to 5 mg/kg [283]. The protective effect of the two lower cariporide doses was enhanced under hypothermic resuscitation conditions whereas no additional benefit was observed with 5 mg/kg cariporide [283]. Taken together, the latter results suggest that the optimal beneficial effect of NHE1 inhibitors in cardiac resuscitation may be dependent on various factors including drug dose and resuscitation conditions.

Finally, it should be added that a contribution to the beneficial effect of cariporide in cardiac resuscitation may reflect improved hemodynamics, particularly in conjunction with the initial chest compression. Thus, Gazmuri’s group has shown, using a rat closed chest ventricular fibrillation model, that cariporide enhances hemodynamic efficacy of resuscitation by producing comparable or higher systemic as well as regional blood flows while at the same time reducing depth of compression, a finding of substantial clinical relevance in terms of improving efficacy of closed chest cardiopulmonary resuscitation [284].

7. Role of NHE1 in Cardiac Hypertrophy, Remodeling and Development of Heart Failure
7.1 Sodium as a Key Factor in the Hypertrophic Program

The role of sodium in the development of cardiac hypertrophy is well established. High dietary sodium is associated with an increased incidence of cardiac hypertrophy and heart failure. Indeed, heart failure patients exhibit defective sodium cellular regulation which could compound the deleterious effects of sodium on cardiac pathology [285]. Although one of the mechanisms associated with sodium-induced cardiac hypertrophy involves the secondary response to chronic hypertension, substantial evidence supports a direct effect of sodium on myocardial hypertrophy thus directly contributing to myocardial remodeling and the development of heart failure. Thus, elevations in sodium concentrations directly produce hypertrophy in cultured myocardial myoblasts [286] and administering a high sodium diet to rats produces cardiac hypertrophy independently of blood pressure elevation [287]. Moreover, the sodium channel blocker tetrodotoxin prevents isoproterenol-induced hypertrophy in cultured H9c2 cardiomyoblasts [288]. One of the multiple sodium-dependent direct mechanisms proposed to induce cardiac hypertrophy involves the elevation of intracellular calcium concentrations due to reduced calcium efflux via the NCX or reverse mode NCX activity [289]. This mechanism is supported by studies showing that selective chronic inhibition of NCX inhibits cardiac hypertrophy in nephrectomized hypertensive rats, a model of heart failure with preserved ejection fraction, independently of blood pressure reduction [290]. However, elevations in intracellular sodium concentrations can directly produce hypertrophy by stimulation of intracellular signaling molecules linked to the hypertrophic program, including protein kinase C [291] as well as reactive oxygen species [292]. Moreover, high intracellular sodium concentrations may also contribute to cardiac hypertrophy by altering mitochondrial dynamics resulting in metabolic remodeling although the precise mechanisms underlying these events have not been fully elucidated [293]. As discussed below, studies using NHE1-specific inhibitors have demonstrated a multitude of intracellular mechanisms potentially contributing to a sodium dependent hypertrophic program. It is interesting to point out that NHE-dependent cardiac hypertrophy manifests primarily as pathological hypertrophy and does not seem to be involved in physiological adaptive hypertrophic responses. In this regard, it has been proposed that AKT (protein kinase B)-dependent NHE1 phosphorylation prevents NHE1 overactivation in physiological hypertrophy [294] and thus it is likely that NHE1 activation does not partake in the physiological hypertrophic program. High intake of dietary sodium in experimental animals has also been linked to activation of the adrenergic system, particularly that involving α1 receptor activation resulting in a myocardial hypertrophic response [295]. The critical role of sodium in the development of cardiac hypertrophy has been shown not only in experimental animals but also clinically in studies demonstrating that high urinary sodium excretion is independently associated with the development of left ventricular hypertrophy in both non-diabetic hypertensive subjects [296] as well as in patients with Type 2 diabetes [297].

7.2 Studies with NHE1 Inhibitors

A number of studies have documented an important role of NHE1 in mediating the enhanced intracellular sodium elevations, particularly under ischemic insult. For example, Bak and Ingwall showed that amiloride, a nonspecific NHE inhibitor, blunted the rise in intracellular sodium in ischemic isolated rat hearts [298]. However, as mentioned, amiloride is a nonspecific agent which affects other cellular processes in addition to NHE inhibition and thus, studies using this drug alone should be interpreted cautiously. However, Baartscheer and colleagues showed that cariporide markedly inhibited intracellular sodium overload and improved calcium regulation in rabbit hearts ex vivo in which the animals were subjected to 12 weeks of chronic combined pressure and volume overload [299].

While NHE1 activation likely represents a major mechanism for intracellular sodium elevation, other cellular processes may also contribute and should be mentioned. For example, inhibition of the late sodium current with the selective inhibitor ranolazine inhibited both hypertrophy and fibrosis as well as improving cardiac function in mice exposed to chronic pressure overload produced by aortic banding [238]. Importantly, this effect was associated with a suppression of sodium overload and improved intracellular calcium homeostasis [238].

A number of lines of evidence suggest that NHE1 is a major contributor to the development of cardiac hypertrophy and heart failure. First, cardiomyocyte hypertrophy is associated with upregulation of NHE1 expression and activity in cardiomyocytes in a number of diverse experimental models [262, 299, 300, 301, 302, 303, 304, 305] as well as in cardiomyocytes harvested from humans with end stage heart failure [306]. Secondly, many paracrine, autocrine and hormonal factors which are important initiators of the hypertrophic program are also potent activators of NHE1 activity in the heart, among these being angiotensin II, endothelin 1 and α1 adrenergic agonists [reviewed in [307]]. Indeed, paracrine and autocrine factors including angiotensin II and endothelin-1 have been shown to be key initiators of NHE1 activation (see Section 3) and the hypertrophic program following the production of myocardial stretch [308]. Thirdly, the importance of NHE1 to the development of cardiac hypertrophy and indeed myocardial remodeling and heart failure in general has been borne out by a large number of studies employing a variety of experimental models as summarized in Table 2 (Ref. [33, 220, 262, 299, 301, 302, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333]). One of the first observations documenting an antihypertrophic effect of NHE1 inhibition originated from the Karmazyn laboratory which showed that cariporide effectively attenuated early (one week) and late (twelve week) hypertrophic responses and left ventricular dysfunction following sustained coronary artery ligation in the rat [309, 310]. This laboratory further demonstrated that NHE1 inhibition also can reverse myocardial remodeling and heart failure when treatment is delayed for up to four weeks following coronary artery ligation [220]. The ability of NHE1 inhibition to reverse remodeling and heart failure is an important observation from a clinical standpoint. This beneficial effect has also been demonstrated in a pressure/volume overload model of heart failure in the rabbit when cariporide was started one month after the initiation of heart failure [311]. It should be noted that the salutary effects of NHE1 inhibition occurred in the absence of infarct size reduction thus demonstrating a direct antihypertrophic effect of NHE1 inhibition. It is interesting that early and transient treatment of rats with a potent NHE1-specific inhibitor for one week, followed by a five-week period in the absence of any pharmacological intervention, resulted in substantial reduction in hypertrophy and heart failure, suggesting that early activation of NHE1 following insult may be critical for the subsequent development of heart failure [312]. While NHE1 inhibition exerts antihypertrophic effects on its own, it is interesting that the benefit was enhanced with coadministration of cariporide with an angiotensin converting enzyme inhibitor (ramipril) in rats subjected to 18 weeks of sustained coronary artery occlusion [313].

Table 2.Summary of studies demonstrating antihypertrophic and anti-remodeling effects of NHE1 inhibitors.
Experimental model NHE1 inhibitor Reference Main Result of inhibitor treatment
Rat 1 wk CAL cariporide [309] Attenuate HY and HF
Rat 13–15 wk CAL cariporide [310] Attenuate HY and HF
Rat PH/RVH cariporide [302] Attenuate RV HY & fibrosis
SHR cariporide [33, 314, 315, 316] Attenuate HY, apoptosis & antiarrhythmic
β1AR TG mouse cariporide [317] Prevent HY, HF & fibrosis
Rabbit P/V overload cariporide [299, 301, 311] Nai & Cai increases
Attenuate HY, regress remodeling
Isoproterenol treated rats BIIB723 [33] Prevent HY & fibrosis
Aldosterone treated NRVM EMD87580 [318] Prevent HY & Nai increases
Rat 12 wk CAL EMD87580 [220] & reverse remodeling & HF
Mouse 5 wk TAB cariporide [319] remodeling, preserve systolic function
LV paced rabbits BIIB722 [262] HF & ventricle dysfunction
Rat 12 or 18 wk CAL EMD87580 [320, 321] Protect mito function
Hamster HHC EMD87580 [322] Prevent Nai & Cai overload
[323] Prevent early death
Rat 18 wk CAL cariporide [313] LV remodeling & HY
GC-A deficient mice cariporide [324] Normalize pHi, Cai, HY & fibrosis
PE-treated NRVM EMD87580 [325] mito integrity & ROS
ET1-treated NRVM cariporide [33, 326] Prevent ET-1 HY & Nai & Cai
NHE1 overexpressing TG mice cariporide [327] block NHE1-induced HY & Cai
Estrogen treated ARVM AVE4890 [328] Block NHE1, HY & pHi
ET1-treated NRVM AVE4890 [329] ET-1-induced mito dysfunction
Glycoside-treated NRVM AVE4890 or EMD87580 [330] induced HY
ISO-infused rats BIIB723 [331] HY & improve Ca2+ handling
Ang II-treated ACVM cariporide [332] HY & ROS
Rat 6 wk CAL BIX [312] HY & HF& calcineurin
PE-treated NRVM BIX [312] HY HF
Ang II-treated H9c2 cells EMD87580 [333] HY & cathepsin B
, increase; , decrease; ACVM, adult cultured ventricular myocytes; Ang II, angiotensin II; β1AR TG mouse, beta 1 adrenergic receptor transgenic mouse; Cai, intracellular calcium; CAL, coronary artery ligation; ET1, endothelin-1; GC-A, guanylyl cyclase-A; HF, heart failure; HY, hypertrophy; HHC, hereditary hypertrophic cardiomyopathy; ISO, isoproterenol; LV, left ventricle; MI, myocardial infarction; mito, mitochondria; Nai, intracellular sodium; NRVM, neonatal rat ventricular myocytes; PE, phenylephrine; PH/RVH, pulmonary hypertension with right ventricular hypertrophy; P/V, pressure/volume; ROS, reactive oxygen species; RV, right ventricle; SHR, spontaneously hypertensive rat; TAB, thoracic aorta banding.
7.3 NHE1 Inhibition in Different Heart Failure Models Not Involving Myocardial Ischemia

The ability of NHE1 inhibition to attenuate cardiac hypertrophy is not restricted to experimental models involving myocardial ischemia. For example, Cingolani’s group showed that cariporide reduced the hypertrophic response as well as myocardial fibrosis in the spontaneously hypertensive rat (SHR) which was dissociated from blood pressure reduction [314, 334]. Moreover, cariporide effectively prevented cardiac hypertrophy, fibrosis and left ventricular dysfunction in a transgenic mouse model overexpressing the β1 adrenergic receptor [317]. This study strongly suggests that NHE1 inhibition could be an effective treatment for the prevention of hypertrophy and heart failure due to increased sympathetic drive. Indeed, this is borne out by studies showing that cardiac hypertrophy and fibrosis caused by 30-day infusion of isoproterenol to rats, can be prevented by the NHE1 specific inhibitor BIIB723 [33]. Genetically induced ablation of the cardiac atrial natriuretic peptide (ANP) receptor similarly produces a cardiac hypertrophy phenotype with accompanying heart failure that can be significantly inhibited by cariporide in the absence of blood pressure reduction [324]. Hearts from these animals exhibited enhanced NHE1 activity thus suggesting that the ANP-guanylate cyclase system is an inhibitory regulator of cardiac NHE1 activity thereby mitigating the cardiac hypertrophic response to hypertension-related pressure overload [324]. In addition, cariporide inhibited the myocardial remodelling and heart failure in mice subjected to pressure overload produced by five-week thoracic aortic banding [319].

NHE1 likely plays an important role in the development of heredity hypertrophic cardiomyopathy. Bkaily’s group has shown that dietary administration of the NHE1-specific inhibitor EMD 87580 (rimeporide) prevented the development of hypertrophy, necrosis, intracellular sodium and calcium overload, as well as preventing early mortality in a dystrophic hamster model [322, 323]. Moreover, rimeporide administration to dogs with muscular dystrophy resulted in a reduction in left ventricular function deterioration in these animals [335]. Such promising results in animal models has led to clinical testing of rimeporide in young boys with Duchenne muscular dystrophy (DMD, N = 20). A phase 1B clinical trial revealed that four-week treatment with rimeporide is well tolerated and produces no safety concerns [336]. Coupled with encouraging, although preliminary biomarker data suggesting some clinical efficacy, further larger scale placebo-controlled studies are planned to demonstrate the effectiveness of rimeporide in reducing cardiomyopathy associated with DMD. The mechanisms underlying the beneficial effects of NHE1 inhibition in dystrophic cardiomyopathy are not known with certainty but, as already alluded to, likely involve attenuation of calcium and sodium overload. Moreover, as DMD is associated with mitochondrial dysfunction [337, 338], protection by NHE1 inhibitors may involve mitochondrial protection as observed in other models of heart failure (see Section 7.5).

7.4 Induction of Hypertrophy by NHE1 Activation

Direct effects of various hormonal as well as paracrine and autocrine factors, some of which have already been referred to, can produce hypertrophy either directly on cultured cardiomyocytes or via chronic in vivo infusion through NHE1-dependent mechanisms. Among these are aldosterone [318, 339], estrogen [328], cardiac glycosides [330], isoproterenol [331] and angiotensin II acting via the angiotensin AT1 receptor [340, 341], the effect of the latter possibly mediated by endogenous endothelin-1 and NHE1 activation [332]. These NHE1-dependent effects of pro-hypertrophic factors may be important in understanding their roles in cardiovascular diseases such as heart failure. For example, aldosterone has been shown in a landmark clinical study (the RALES study) to play an important role in development of heart failure, as demonstrated by a significant reduction in mortality and morbidity in heart failure patients treated with the mineralocorticoid receptor blocker spironolactone [reviewed in [342]]. The role of increased catecholamine drive and angiotensin II in cardiac pathology are well established and represent targets for established therapies for treating cardiovascular disorders. The deleterious effects of catecholamines on the development of heart failure are mostly mediated by β1 adrenoceptor activation [reviewed in [343]], indeed α1 blockers are generally contraindicated due to vasodilator effects of these agents resulting in reflex sympathetic activity [344]. Nonetheless, α1 adrenoceptor activation in the heart can produce deleterious effects such as increased cardiac fibrosis via calcineurin activation (of relevance see section 7.5) which would contribute to the severity of myocardial remodelling and heart failure [345] With respect to angiotensin II, targeting this hormone for the treatment of heart failure has been a mainstay for therapy for decades. This is generally achieved either by inhibition of angiotensin converting enzyme (ACE) or by the use of AT1 receptor antagonists (ARBs) although there is some evidence based on meta-analysis of clinical trials that ACE inhibition is more effective than ARBs in reducing mortality in heart failure patients [346]. We believe that benefit would also be achieved by NHE1-specific inhibitors. In fact, such protection could in theory be superior to that seen with targeting individual agonists as pro-remodelling effects of numerous autocrine, paracrine and hormonal factors would be inhibited by targeting NHE1. It must be added however that beneficial endogenous factors have also been identified such as insulin-like growth factor 1, which has been shown to improve cardiac function in hypertrophied hearts of spontaneously hypertensive rats while suppressing NHE1 activity [54].

A non-pharmacological mode of NHE1 upregulation involves genetic modification of the antiporter resulting in enhanced NHE1 activity. In this regard, Fliegel’s group showed that transgenic mice expressing an overactive form of NHE1 exhibit cardiac hypertrophy in the absence of any pro-hypertrophic insult, when compared to mice expressing the wild type NHE1 [64, 65]. Additionally, infection of neonatal rat ventricular myocytes with an adenoviral vector expressing a constitutively active NHE1 resulted in a hypertrophic response in the absence of any other pro-hypertrophic intervention [63].

7.5 Potential Mechanisms Underlying NHE1-Dependent Cardiac Hypertrophy

The mechanisms by which NHE1 activation induces cardiomyocyte hypertrophy are likely complex and associated with the stimulation of a number of intracellular pathways. As discussed above, NHE1 interacts with various intracellular cofactors and binding partners which in general, enhance the antiporter’s activity. With respect to the development of hypertrophy specifically, it has been reported that the ubiquitous multifunctional protein osteopontin originally identified in bone may be an important cofactor in mediating the hypertrophic influence of NHE1 activation. Thus, a close relationship between NHE1 and osteopontin expression was identified in cultured cardiomyocytes and silencing osteopontin in these cells supressed the hypertrophic effect of NHE1 overexpression [62]. Furthermore, osteopontin expression upregulation and the hypertrophic response in H9c2 cardiomyoblasts treated with angiotensin II was prevented by rimeporide [347].

Among the strongest candidates as a key factor in mediating the pro-hypertrophic effect of NHE1 activation is stimulation of calcineurin, a serine/threonine protein phosphatase which is an activator of transcriptional factors well known to be important in the pathological hypertrophic program as well as evolution to heart failure, among these being nuclear factor of activated T cells (NFAT) and myocyte enhancer factor 2 (Mef2) [348, 349]. As calcineurin can be activated by increased intracellular calcium concentrations or more specifically by formation of a calcium/calmodulin complex, it is not surprising that stimulation of NHE1 is a likely contributor to increased calcineurin activity leading to cardiomyocyte hypertrophy based on the concepts discussed in section 7.1 related to increased intracellular calcium concentrations. Indeed, overexpression of cardiac NHE1 per se has been shown to be sufficient to increase intracellular calcium levels, upregulate the calcineurin pathway and induce cardiomyocyte hypertrophy, thus demonstrating a strong link between NHE1 and the calcineurin pathway in promoting the hypertrophic program [327]. Inhibition of cardiac hypertrophy both in vivo as well as in cultured cardiomyocytes is associated with concomitant regression of calcineurin/NFAT expression [304]. Kilić et al. [312] showed that early and transient NHE1 inhibition was sufficient in preventing the hypertrophic response and calcineurin activation both in rats subjected to sustained coronary artery ligation as well as myocytes exposed to phenylephrine treatment. The relationship between NHE1 and calcineurin activity has also been reported with other antihypertrophic strategies including treatment with ginseng [158] or a chimeric natriuretic peptide [350]. Thus, NHE1-dependent calcineurin activation and subsequent cardiomyocyte hypertrophy likely follows the series of events summarized in Fig. 6.

Fig. 6.

Proposed pathway for calcineurin-mediated hypertrophy following NHE1 activation.

The evidence for calcineurin notwithstanding, other intracellular mechanisms may also contribute to protection of the hypertrophied myocardium shown by NHE1 inhibitors particularly concerning mitochondrial preservation during the remodeling process. Recent evidence, for example, suggests that the antihypertrophic effect of NHE1 inhibition with rimeporide in cultured H9c2 cells treated with angiotensin II is associated with suppression of the activation of Cathepsin B, a cysteine protease involved in cardiovascular and other pathologies [333]. Mitochondrial protection as demonstrated by reduced mitochondrial dependent generation of reactive oxygen species may also be of importance in understanding the anti-remodeling effects of NHE1 inhibition [351]. Karmazyn’s laboratory has previously shown that rimeporide reduced MAPK activity, preserved mitochondrial membrane potential, attenuated permeability transition pore opening and reduced superoxide generation in phenylephrine-treated neonatal rat cardiomyocytes while supressing the hypertrophic response [320, 325]. Further evidence suggests that NHE1 inhibition reduces mitochondrial dysfunction in the hypertrophied heart by attenuating phosphorylation of AMP-activated protein kinase (AMPK)/glycogen synthase kinase 3β (GSK-3β) during hypertrophy both in vivo following sustained coronary artery ligation or in cultured myocytes in which hypertrophy was induced by endothelin-1 [329]. A potential mitochondria-related locus of protection by NHE1 inhibitors may be related to attenuation of mitochondrial fission during the post-infarction remodeling process thus preserving mitochondrial fission/fusion balance. Indeed, excessive mitochondrial fission is known to contribute to cardiac pathology associated with the development of cardiac remodeling and failure [352]. In this regard, upregulation of the primary fission protein Fis1 was significantly blunted by EMD87580 in hearts of rats subjected to 12 or 18 weeks of sustained coronary artery ligation [321]. Thus, when taken together, it appears that mitochondrial protection represents an important component underlying the salutary effects of NHE1 inhibition in cardiac hypertrophy, acting via multifaceted mechanisms to preserve mitochondrial integrity.

7.6 Anti-Remodeling Effects of Drugs Developed for the Treatment of Type 2 Diabetes: Evidence for NHE1 Inhibition

It is interesting to note that NHE1 inhibition may also contribute to the antihypertrophic and remodelling effects of drugs not initially developed for this purpose. An example of this are the sodium-glucose cotransport 2 inhibitors, the so-called gliflozins, developed for the treatment of type 2 diabetes mellitus but which exert a number of beneficial effects on the heart not related to glucose regulation but which likely involve NHE1 inhibition [353, 354, 355, 356, 357]. For example, dapagliflozin reduced fibrosis, inflammation and left ventricular dysfunction in db/db diabetic mice chronically (30 days) infused with angiotensin II [358]. These beneficial effects were associated with a number of cellular effects including decreased intracellular calcium transients, decreased inflammation, decreased ROS production as well as decreased expression of the voltage-dependent L-type calcium channel and decreased NCX levels while inhibiting NHE1 [328]. The relative contribution of each of these mechanisms awaits concrete elucidation. Gliflozins, are drugs that work primarily on the kidney to aid in glucose homeostasis in diabetic patients and may have effects through NHE1 (see section 8.1). In addition to the gliflozins, teneligliptin, a dipeptidyl peptidase-4 inhibitor used for the treatment of type 2 diabetes in some countries has been shown to reduce cardiac hypertrophy and the concomitant increase in NHE1 expression in spontaneously hypertensive rats that was attributed to normalization of the elevated plasma angiotensin II levels observed in these animals [359].

7.7 Potential Role for Genetic Polymorphisms in Human Disease

Genetic polymorphisms of NHE1 have been reported and this includes at least one that results in human disease (recently reviewed in [22]). Briefly, the first genetic defect in humans that was found to be attributable to NHE1 was reported by Guissart et al. [23]. Lichtenstein-Knorr syndrome is one of several autosomal recessive cerebellar ataxias with a variety of neurological symptoms, cardiomyopathies and ataxias [360]. It is of juvenile or adolescent onset with ataxia and sensorineural hearing loss [361]. Guissart et al. [23] showed that the SLC9A1 (NHE1) gene, is responsible for the defect in this disease. A mutation in the SLC9A1 gene changed Gly305 to Arg. The Gly305Arg mutation causes reduced expression and decreased protein glycosylation. It also caused almost a complete absence of targeting of the protein to the cell surface and virtually no protein activity at the cell surface [23]. After this report another study [362] reported a different human mutation resulting in similar symptoms including cerebellar ataxia. In this case the mutation at amino acid Ile288 caused a premature truncation of most of the protein.

NHE1 knockout mice have also been characterized. One spontaneous mutation of NHE1 in mice was a change causing Lys442 to become a stop codon and terminate NHE1 within the transmembrane domain. Homozygous defective mice had a slow-wave epilepsy (swe) mutation. They also had an ataxic gait including locomotor ataxia that was prominent in their hind limbs Mutant homozygous mice were of small size and less than half survived to weaning [363]. A second study in mice confirmed the above physiological effects with a targeted disruption of NHE1 [364]. The genetic knockout of NHE1 allowed an interesting insight into NHE1 physiology in the myocardium. When mice with the genetic knockout of NHE1 were subjected to cardiac ischemia reperfusion injury, they were resistant in comparison to the controls [273]. This confirmed the role of NHE1 in ischemia reperfusion damage.

Various specific genetic polymorphisms have been identified in the NHE1 gene though a thorough study of their total incidence and effect on the myocardium is lacking. One polymorphism was a change of Asn266 to His [365]. The mutation was not fully characterized clinically. Mutant N226H protein was expressed and targeted properly however, the N266H protein had no detectable activity. The NHE1 cytoplasmic tail is responsible for regulation of NHE1. Another study characterized the effect of stop codon polymorphisms in the regulatory tail [366]. Stop codons at amino acid 321, 449 and 735 were examined (mutations at 321 and 449 were actually within the membrane domain). Mutants stopping at amino acids 321 and 449 lost NHE1 activity and did not target properly to the plasma membrane. They were also more rapidly degraded than wild type protein. The mutant protein ending at amino acid 735 had reduced expression and activity.

Another study [367] examined the effect of change of two polymorphisms in the phosphorylatable amino acids in the regulatory tail. It examined the Ser703 and Ser771, to proline polymorphisms [367]. Ser703 is critical to 14-3-3 binding to NHE1 and to NHE1 activation by growth factors [368, 369] (Fig. 3). Ser771 is also important in Erk 1/2 dependent activation of NHE1 [370, 371] (see section 3.1.1). The Ser703Pro mutant had virtually the same activity, targeting and expression levels as the wild type NHE1 protein. However, the Ser771Pro mutant protein had reduced activity and expression levels, but normal cell surface targeting. The Ser771Pro mutant showed abnormal regulation. It was not strongly activated by sustained intracellular acidosis, but was activated partially even by very short periods of acidosis. It was hypothesized that insertion of a Pro in this location leads to an abnormal conformation that alters synthesis or degradation of the protein and causes an abnormal regulation of the protein by conformational changes in the tail, and by the inability to be phosphorylated [367]. It is not known how mutation of these regulatory amino acids could affect NHE1 function in humans. However, Ser703 is phosphorylated by p90 ribosomal S6 kinase. A mouse heart dominant negative p90 ribosomal S6 kinase mutant was resistant to myocardial injury induced by left coronary artery occlusion [90]. One might postulate that humans carrying the Ser703Pro polymorphism might also be resistant to coronary artery occlusion, but this remains to be demonstrated.

8. NHE1 in Diabetes and Related Heart Diseases

Because of the ubiquitous expression of the NHE throughout the tissues and cell types of the body [1, 2] and its important function in each of these tissues, it is perhaps not surprising that changes in NHE expression and function have been implicated in a variety of diseases in a wide range of tissues and cell types. This includes brain development and function [372], dental pulp [373], immune function and inflammation [374], kidney function [375], epilepsy [363, 376], gallstone formation [377], cataracts [378] and muscular dystrophies [379]. These areas of NHE lesions have, in some cases, led to heart related problems as well. For example, it has been suggested that the heart failure associated with Becker and Duchenne muscular dystrophies may be in part due to NHE1 over-activation and the subsequent Na+ overload [379]. In support of this hypothesis, the chronic administration of a NHE-1 inhibitor to a dystrophic animal model prevented the intracellular Na+ overload and early death due to heart failure [379]. Additionally, with the predominant place inflammation and infection has in cardiovascular disease [380], it may be a particularly fruitful avenue for future research to determine if the beneficial cardiovascular effects of NHE inhibitors may be due in part to an action on the immune system and the pathways that ultimately produce inflammation. However, the two disease conditions that have been associated with NHE malfunction and may also have the most significant impact upon the cardiovascular system are diabetes mellitus and renal disease.

Diabetes, Heart Disease and Na+/H+ Exchange

A significant change in how diabetic heart disease was viewed was first proposed with experimental evidence in the 1970’s and early 1980’s [381, 382, 383, 384, 385, 386, 387, 388, 389]. Instead of diabetic heart disease and failure being viewed as a primarily vascular lesion [390], the aforementioned works clearly identified a subcellular basis for a cardiomyopathy independent of vascular complications. The cardiac dysfunction was demonstrated in both insulin dependent and non-insulin dependent models of diabetes [382, 383, 391, 392].

Insulin dependent models of diabetes exhibited hearts resistant to ischemic/reperfusion insult [393]. A reduced [pH]𝑖 recovery was found in response to an acid load in cardiac muscle preparations from insulin deficient diabetic animals [394]. This suggested that a decrease in NHE activity or expression levels was present in the diabetic heart. A direct demonstration of a reduced Na+/H+ exchange activity was found in cardiac sarcolemmal vesicles isolated from diabetic animals in comparison to control preparations [395]. However, expression levels of NHE1 mRNA were unchanged in hearts from streptozotocin-induced diabetic rats [396]. The decrease in Na+-H+ exchange in the heart would be expected to lessen the influx of Ca2+i, much as a drug that inhibits NHE, as discussed earlier in this manuscript. This, in turn, would result in a protection of the diabetic heart from ischemic/reperfusion injury.

However, whereas type 1 diabetic animals are more resistant to ischemia, insulin resistant Type 2 diabetic animals were conversely more sensitive to ischemic challenge. The decreased post-ischemic cardiac performance exhibited by hearts from late-stage insulin-resistant models of diabetes, may be due to greater endogenous stores of glycolytic substrates and the resultant excessive production of lactate and H+ [397]. This would tend to enhance the exchange of ions through the NHE pathway and generate augmented cardiac damage [398]. This enhanced NHE activation in Type 2 diabetes agrees well with its activation during hyperinsulinemia [399]. Packer [400, 401] has proposed a key role of cardiac and vascular NHE1 as well as renal NHE3 as principal factors linking diabetes with the development of heart failure. Thus, it was proposed that neurohormonal-dependent upregulation of NHE1 and NHE3 would result in NHE1-dependent cardiac remodelling coupled with NHE3-dependent renal sodium retention, the combination accelerating the progression to heart failure [401].

As noted above in section 7.6, sodium/glucose cotransporter 2 (SGLT2) inhibitors, gliflozins, are drugs that work primarily on the kidney to aid in glucose homeostasis in diabetic patients. Empagliflozin has been reported to attenuate both sodium and calcium dysregulation in mouse ventricular myocytes treated with ouabain potentially via NHE1 inhibition [402]. It is also important to note that empagliflozin has recently been shown to reduce oxidative stress in cultured human umbilical vein endothelial cells and coronary artery endothelial cells through a mechanism involving NHE1 inhibition [403] thus providing further supporting evidence for the beneficial effects of gliflozins on the heart through NHE1 inhibition. However, several recent studies have suggested that these compounds also inhibit cardiac NHE1 activity [353, 354, 358, 404] and expression [358]. This inhibition appears to be the mechanism for a lowering of cytosolic Na+, vasodilation [354], a decrease in lactate generation [404], an attenuation of the diabetic cardiomyopathy [358] and a reduction in infarct size in the post-ischemic reperfused heart [405]. This inhibition of NHE1 by SGLT2 inhibitors appears to be a class action effect as empagliflozin, dapagliflozin and canagliflozin have been reported to inhibit NHE [358]. It should be noted however that there is controversy in this area and there are reports that SGLT2 blockers do not directly inhibit NHE1 [406, 407]. The different results reported may be due to species differences or due to the method of drug application. Indeed, in a recent report [408] the long-term treatment of H9c2 cells with empagliflozin was shown to inhibit expression of the NHE1 protein while short term treatment did not inhibit NHE1 expression. This also raises the possibility that some of the effects observed are due to inhibition of protein expression, rather than a direct inhibitor effect on the protein.

The glifozins are not the only non-specific drug interactions that may have their biological action via a primary effect on the NHE. NHE3 in the kidney is affected by incretin-based agents, antagonists of the renin-angiotensin system, insulin and insulin sensitizers, statins and spironolactone [409].

9. The Effects of NHE1 Inhibitors in Clinical Settings

The robust experimental data demonstrating substantive cardioprotective properties of NHE1 inhibitors, unmatched by other cardioprotective strategies, rapidly progressed to clinical evaluation of NHE1 inhibitors in patients with coronary artery disease, mostly employing cariporide as the drug of choice. The first such study recruited a total of 100 patients who had experienced an acute myocardial infarction (AMI) and who were subjected either to percutaneous transluminal coronary angioplasty (PTCA) with cariporide administered at the time of reperfusion or with a placebo [410]. Patients receiving cariporide exhibited improved left ventricular function three weeks post-PTCA and reduced plasma enzyme levels within 72 hours after reperfusion, the latter indicating an inhibition of reperfusion injury by cariporide [410].

The results of the above study were somewhat surprising in view of the small number of patients recruited but also because experimental studies have demonstrated that optimal protection by NHE1 inhibitors occurs when the drug is also present during the ischemic period before the onset of reperfusion. Clinically, this can be achieved under controlled I/R conditions such as in coronary artery bypass grafting (CABG, discussed below). Indeed, a Phase II clinical study named “Evaluation of the Safety and Cardioprotective Effects of Eniporide in AMI” (ESCAMI) study was conducted to investigate the hypothesis that eniporide would reduce injury given as an adjunct to reperfusion performed either by thrombolysis or PTCA [411]. This was a phase 2 international multicenter randomized, double-blinded, placebo-controlled, dose-finding trial which was carried out in two stages: in Stage 1 (433 patients), eniporide was administered at 50 mg, 100 mg, 150 mg or 200 mg whereas in Stage 2 (978 patients), based on the results of Stage 1 eniporide was further studied at doses of 100 mg or 150 mg. For both stages, eniporide or placebo was administered over a 10-minute infusion period. Specifically, in patients subjected to thrombolytic therapy, infusion was completed at least 15 minutes after starting thrombolytic treatment, whereas in angioplasty, the patient’s infusion was completed at least 10 minutes before the start of PTCA. The results of the Stage 1 study were encouraging in that there were significant reductions in infarct size, the primary efficacy end point of the study, of 25.7% and 41.7%, were found with 100 mg and 150 mg eniporide, respectively. This effect was more evident in the PTCA treated patients. However, no protection was observed in Stage 2 of this trial using these two eniporide doses. It is interesting to add that in a subgroup of over 300 patients who were subjected to delayed reperfusion (>4 hours after onset symptoms) a significant reduction in heart failure symptoms was observed in the 150 mg eniporide group when compared with placebo (placebo 21.9%, eniporide 11.1%).

Further evaluation of the protective effects of NHE1 inhibitors was then carried out in two important clinical trials. The first of these was the Guard During Ischemia Against Necrosis (GUARDIAN) trial which was designed to determine whether cariporide could reduce mortality and MI in patients at risk of myocardial necrosis as well as to determine the drug’s safety [412]. GUARDIAN was a combined phase 2/phase 3 international multicenter, double-blind, randomized and dose-finding study in which the primary objective was to evaluate the efficacy of cariporide in reducing all-cause mortality and/or MI across the various entry populations 36 days after randomization. This study recruited 11,590 patients who were either hospitalized for an acute coronary syndrome (unstable angina or non–Q-wave myocardial infarction) or who were subjected to either PTCA or CABG. Patients were randomized to receive either one of three cariporide doses of 20 mg, 80 mg or 120 mg or placebo which were administered every eight hours for two to seven days as a 60-minute infusion. Starting time for cariporide administration differed based on the underlying condition and as decided by individual investigators. Generally, cariporide was initiated as soon as possible after admission in patients with acute coronary syndrome and between 15 minutes and 2 hours before PTCA or CABG. Doses of 20 and 80 mg were ineffective across all clinical settings. However, at day 36 CABG patients who were treated with 120 mg cariporide exhibited a significant 25% risk reduction in either death or myocardial infarction which primarily reflected a 32% risk reduction in nonfatal infarctions.

The GUARDIAN study, while showing no overall benefit of cariporide when assessed across all clinical settings, did demonstrate substantial benefit, as noted above, with fewer end-point events when administered at the 120 mg dose to high-risk CABG patients. This encouraging result was subsequently used as the major basis for the phase 3 Na+/H+ Exchange inhibition to Prevent coronary Events in acute cardiac condition (EXPEDITION) trial to study the potential benefit of cariporide on death and non-fatal myocardial infarction in CABG patients [413]. Thus, in this trial a total of 5761 patients were randomized to receive intravenous cariporide as a 180 mg 1-hour preoperative loading dose followed by 40 mg per hour over a 24 hour and then by 20 mg per hour over the subsequent 24 hours, or placebo. The primary composite endpoint of death or MI in the EXPEDITION study was assessed at 5 days with patients followed for up to 6 months.

The results from the EXPEDITION trial were promising vis a vis the cardioprotective effects of cariporide in that the incidence of death or MI was reduced from 20.3% in the placebo group to 16.6% in patients treated with cariporide as was the incidence of MI alone (18.9% in the placebo group vs 14.4% in cariporide-treated patients), both highly significant reductions. These beneficial effects were maintained at 6 months follow up. Unfortunately, the beneficial cardiac effects of cariporide were associated with increased mortality from 1.5% in the placebo group to 2.2% with cariporide. These increases were statistically significant at 5 days and 3 months follow up but not at 6 months and were caused almost exclusively by a significantly higher incidence of thromboembolic strokes in patients receiving cariporide [413].

10. Perspectives and Future Directions

This paper has presented an overview of NHE1 in terms of its chemistry, regulation and its role in cardiac pathologies, the latter pertaining primarily to myocardial ischemic and reperfusion injury as well as myocardial remodeling resulting in heart failure. Much has been learned about the role of the antiporter as a critical regulator of intracellular pH but of greater relevance to the present discussion, its potential as a target for pharmacological intervention for cardiac therapeutics. The robust experimental evidence demonstrating salutary effects of NHE1 specific inhibitors has led to a rapid evaluation of these agents in the clinical setting particularly with respect to the assessment of cariporide as a cardioprotective agent in patients subjected to reperfusion protocols. Needless to say, the overall results seen in clinical trials with cariporide, as well as with eniporide, have been disappointing as evidenced by lack of efficacy and unexpected side effects as seen in the EXPEDITION study (even though a cardioprotective influence was demonstrated). A thorough evaluation of these results has previously been presented [414] and will, therefore, be discussed briefly here.

The results of the initial small study [410] notwithstanding, the failure to demonstrate efficacy in either the ESCAMI study or in either the thrombolysis or PTCA arms in the GUARDIAN trial, is surprising as animal data clearly demonstrated optimal protective efficacy of NHE1 inhibitors when the drug is present during the ischemic period, as noted in Sections 6.2 and 6.3. Indeed, when CABG patients were treated prior to surgery with the highest cariporide dose (120 mg group) in the GUARDIAN study, significant cardioprotection was observed [412]. Thus, expectations were high for favorable results in EXPEDITION as only CABG patients were recruited to this study with one standard cariporide dosing regimen. In fact, a significant cardioprotection was seen in EXPEDITION but the results were associated with a significantly increased incidence of ischemic strokes, thus resulting in an early cessation of the trial. The reasons for the increased incidence of strokes in cariporide-treated patients are not known with certainty. Importantly, we do not know for example whether this reflects a property of NHE1 inhibitors in general or cariporide specifically. The former is unlikely as NHE1 inhibitors have been extensively shown to exert cerebral protective effects and have been proposed as a potential treatment for strokes [415]. In addition, inhibitors of platelet NHE1 inhibit platelet aggregation [416].

There is a strong possibility that the increased incidence of strokes seen in EXPEDITION reflected the substantially different dosing regimen as well as total dose of cariporide administered when compared to that administered in the 120 mg CABG group in the GUARDIAN study. Thus, the total cariporide administered to patients in EXPEDITION was 1620 mg over a 48-hour period compared to 720 mg during the same time period in the GUARDIAN study. It is therefore possible, or even likely, that the increased incidence of strokes seen in cariporide-treated patients in EXPEDITION reflected an unnecessary overdosing with cariporide, particularly when compared with the highest dose CABG group in the GUARDIAN study which resulted in cardioprotection but no increase in the incidence of strokes [412]. As the treatment regimen in the highest cariporide dose CABG group in GUARDIAN resulted in cardioprotection, it is difficult to rationalize the more than two-fold increase in cariporide dosing in EXPEDITION during the pre-surgery 48-hour period. As outlined by the late Dr Gerald Buckberg, a renowned cardiac surgeon who was a participating investigator and a member of the EXPEDITION Steering Committee, the sponsoring company of the trial altered the steering committee’s recommendations regarding dosing by extending drug delivery duration and adding a high dose delivery. “They did this despite our emphasizing that these patients did not need elevated doses” [417].

The increased incidence of cerebrovascular events seen in EXPEDITION had a profoundly negative effect on the clinical development of NHE1 inhibitors as evidenced by a total cessation of all NHE1 inhibitor-related research and development by the pharmaceutical industry. Whether this action was justified is difficult to address as there is a complete paucity of data in the literature addressing the question of a possible pro-thrombotic effect of cariporide specifically or NHE1-specific inhibitors in general. For example, was the thrombotic effect of cariporide due to high dosing and can this be confirmed in the laboratory setting? Are there any insights into potential mechanisms for the pro-thrombotic effect of cariporide and does this involve NHE1 inhibition or non-specific effects of cariporide? This question would be particularly important to address since NHE1 activity likely contributes to platelet aggregation with an attenuation of the latter by NHE1 inhibitors, as already noted above. Do other NHE1-specific inhibitors share this pro-thrombotic effect particularly at high doses? Does the method of administration influence the deleterious effect of cariporide? In this regard, cariporide and other NHE1 inhibitors would obviously be most effective as cardioprotective agents in CABG patients, based on existing clinical data with cariporide. Would an oral preparation be effective in producing cardioprotection and would this minimize any possible prothrombotic risk compared to drug infusion? In view of the immense potential of NHE1 inhibitors for the treatment of heart disease as outlined in this review, the results of the EXPEDITION study should likely not have precluded the development of newer NHE1-specific inhibitors without addressing the issues just raised. The potential for NHE1 inhibitors to benefit patients with cardiovascular disorders warrants further research and possible clinical development of these agents. As stated by the EXPEDITION study authors “the use of NHE inhibitors could lead to significant improvement in medium- and long-term survival among patients undergoing heart surgery as well as those at risk of MI at any time” [413]. This potential benefit should not go unexplored.

With the recent elucidation of the 3D structure of NHE1 [11] and the development of novel inhibitors towards NHE1, there are also new opportunities. Understanding the precise location of the inhibitor binding pocket and its coordination may allow medicinal chemists to design novel inhibitors with even higher specificity and potency towards NHE1. Additionally, some such novel NHE1 inhibitors have recently been developed [226, 227, 228, 230] though they have not been tested in a cardiovascular disease setting. The advantage of novel inhibitors that are structurally different from cariporide is that they may avoid the stigma of the previous problems and it may be easier to obtain regulatory approval for them. There has also been little attention to altering NHE1 regulation in the disease state, which is another approach that has shown some promise [66, 83, 90], but has not been further developed in the clinic.

11. Conclusions

In summary, we propose that NHE-specific inhibition remains a worthwhile endeavour in the development of effective therapeutics for the treatment of heart disease. The scientific evidence for an effective approach to mitigating damage to the myocardium remains very strong as is the concept of NHE1 inhibition for the treatment of heart failure. Despite concerns with earlier clinical trials, the rationale and approach remain sound and new compounds for proposed treatment along with a more careful application based on experimental data could lead to useful clinical treatments for this major health problem.

Author Contributions

LF, GP and MK designed the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Ethics Approval and Consent to Participate

Not applicable.


We thank B. Buckley, University of Wollongong, for his assistance drawing the structures of amiloride, cariporide and HMA. We thank the peer reviewers for their opinions and suggestions.


Work cited from the authors’ laboratory was funded by past Canadian Institutes of Health Research Operating Grants (MK, LF #MOP 97816) or a Foundation Grant (GNP) from the Canadian Institutes of Health Research.

Conflict of Interest

The authors declare no conflict of interest. Morris Karmazyn and Grant N. Pierce are serving as the Guest editors of this journal. Grant N. Pierce is serving as one of the Editorial Board members of this journal. We declare that Morris Karmazyn and Grant N. Pierce had no involvement in the peer review of this article and have no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Fabian Sanchis-Gomar.

Publisher’s Note: IMR Press stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Fliegel L. Structural and functional changes in the Na(+)/H(+) exchanger isoform 1, induced by Erk1/2 phosphorylation. International Journal of Molecular Sciences. 2019; 20: 2378.
Pedersen SF, Counillon L. The SLC9A-C mammalian Na(+)/H(+) exchanger family: molecules, mechanisms, and Physiology. Physiological Reviews. 2019; 99: 2015–2113.
Fliegel L. The Na+/H+ exchanger isoform 1. International Journal Biochemistry and Cell Biology. 2005; 37: 33–37.
Hendus-Altenburger R, Kragelund BB, Pedersen SF. Structural dynamics and regulation of the mammalian SLC9A family of Na(+)/H(+) exchangers. Current Topics in Membranes. 2014; 73: 69–148.
Orlowski J, Kandasamy RA, Shull GE. Molecular cloning of putative members of the Na+/H+ exchanger gene family. Journal of Biological Chemistry. 1992; 267: 9331–9339.
Takaichi K, Wang D, Balkovetz DF, Warnock DG. Cloning, sequencing, and expression of Na+/H+ antiporter cDNAs from human tissues. American Journal of Physiology. 1992; 262: C1069–C1076.
Orlowski J, Grinstein S. Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflügers Archiv - European Journal of Physiology. 2004; 447: 549–565.
Holmes RS, Spradling-Reeves KD, Cox LA. Evolution of vertebrate zolute carrier family 9B genes and proteins (SLC9B): Evidence for a marsupial origin for testis specific SLC9B1 from an ancestral vertebrate SLC9B2 gene. Journal of Phylogenetics and Evolutionary Biology. 2016; 4: 167.
Fuster DG, Alexander RT. Traditional and emerging roles for the SLC9 Na+/H+ exchangers. Pflügers Archiv - European Journal of Physiology. 2014; 466: 61–76.
Odunewu-Aderibigbe A, Fliegel L. The Na(+) /H(+) exchanger and pH regulation in the heart. IUBMB Life. 2014; 66: 679–685.
Dong Y, Gao Y, Ilie A, Kim D, Boucher A, Li B, et al. Structure and mechanism of the human NHE1-CHP1 complex. Nature Communications. 2021; 12: 3474.
Malo ME, Fliegel L. Physiological role and regulation of the Na+/H+ exchanger. Canadian Journal of Physiology and Pharmacology. 2006; 84: 1081–1095.
Li X, Karki P, Lei L, Wang H, Fliegel L. Na+/H+ exchanger isoform 1 facilitates cardiomyocyte embryonic stem cell differentiation. American Journal of Physiology-Heart and Circulatory Physiology. 2009; 296: H159–H170.
Flinck M, Kramer SH, Schnipper J, Andersen AP, Pedersen SF. The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells. Cell Cycle. 2018; 17: 1056–1067.
Flinck M, Kramer SH, Pedersen SF. Roles of pH in control of cell proliferation. Acta Physiologica (Oxford). 2018; 223: e13068.
Carraro-Lacroix LR, Ramirez MA, Zorn TM, Reboucas NA, Malnic G. Increased NHE1 expression is associated with serum deprivation-induced differentiation in immortalized rat proximal tubule cells. American Journal of Physiology - Renal Physiology. 2006; 291: F129–F139.
Amith SR, Fong S, Baksh S, Fliegel L. Na(+)/H(+) exchange in the tumour microenvironment: does NHE1 drive breast cancer carcinogenesis? International Journal of Developmental Biology. 2015; 59: 367–377.
Amith SR, Fliegel L. The Na+/H+ exchanger in metastasis. Aging (Albany NY). 2016; 8: 1291.
Takatani-Nakase T, Matsui C, Hosotani M, Omura M, Takahashi K, Nakase I. Hypoxia enhances motility and EMT through the Na(+)/H(+) exchanger NHE-1 in MDA-MB-231 breast cancer cells. Experimental Cell Research. 2022; 412: 113006.
Amith SR, Fliegel L. Na(+)/H(+) exchanger-mediated hydrogen ion extrusion as a carcinogenic signal in triple-negative breast cancer etiopathogenesis and prospects for its inhibition in therapeutics. Seminars in Cancer Biology. 2017; 43: 35–41.
Andersen AP, Samsoe-Petersen J, Oernbo EK, Boedtkjer E, Moreira JMA, Kveiborg M, et al. The net acid extruders NHE1, NBCn1 and MCT4 promote mammary tumor growth through distinct but overlapping mechanisms. International Journal of Cancer. 2018; 142: 2529–2542.
Fliegel L. Role of Genetic Mutations of the Na(+)/H(+) Exchanger Isoform 1, in Human Disease and Protein Targeting and Activity. Molecular and Cellular Biochemistry. 2021; 476: 1221–1232.
Guissart C, Li X, Leheup B, Drouot N, Montaut-Verient B, Raffo E, et al. Mutation of SLC9A1, encoding the major Na+/H+ exchanger, causes ataxia-deafness Lichtenstein-Knorr syndrome. Human Molecular Genetics. 2015; 24: 463–470.
Lazdunski M, Frelin C, Vigne P. The sodium/hydrogen exchange system in cardiac cells. Its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH. Journal of Molecular and Cellular Cardiology. 1985; 17: 1029–1042.
Frelin C, Vigne P, Lazdunski M. The role of Na+/H+ exchange system in the regulation of the internal pH in cultured heart cells. European Journal of Biochemistry. 1985; 149: 1–4.
Seiler SM, Cragoe EJ Jr., Jones LR. Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles. Journal of Biological Chemistry. 1985; 260: 4869–4876.
Frelin C, Vigne P, Lazdunski M. The role of the Na+/H+ exchange system in cardiac cells in relation to the control of the internal Na+ concentration. A molecular basis for the antagonistic effect of ouabain and amiloride on the heart. Journal of Biological Chemistry. 1984; 259: 8880–8885.
Pierce GN, Philipson KN. Na+/H+ exchange in cardiac sarcolemmal vesicles. Biochimica et Biophysica Acta. 1985; 818: 109–116.
Fliegel L, Dyck JRB, Wang H, Fong C, Haworth RS. Cloning and analysis of the human myocardial Na+/H+exchanger. Molecular and Cellular BIochemistry. 1993; 125: 137–143.
Karmazyn M. Role of NHE-1 in cardiac hypertrophy and heart failure. In M. Karmazyn M (ed.) The Na+/H+ Exchanger, From Molecular to Its Role in Disease Avkiran and L (pp. 211–219). Fliegel, Kluwer academic Publishers: Boston/Dordrecht/London. 2003.
Scholz W, Albus U, Counillon L, Gogelein H, Lang HJ, Linz W, et al. Protective effects of HOE642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischaemia and reperfusion. Cardiovascular Research. 1995; 29: 260–268.
Scholz W, Albus U, Lang HJ, Linz W, Martorana PA, Englert HC, et al. Hoe 694, a new Na+/H+ exchange inhibitor and its effects in cardiac ischaemia. Bristish Journal of Pharmacology. 1993; 109: 562–568.
Ennis IL, Escudero EM, Console GM, Camihort G, Dumm CG, Seidler RW, et al. Regression of isoproterenol-induced cardiac hypertrophy by Na+/H+ exchanger inhibition. Hypertension. 2003; 41: 1324–1329.
Wallert MA, Frohlich O. Na+-H+ exchange in isolated myocytes from adult rat heart. American Journal of Physiology - Cell Physiology. 1989; 257: C207–C213.
Liu S, Piwnica-Worms D, Lieberman M. Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO_3- exchange. Journal of General Physiology. 1990; 96: 1247–1269.
Lagadic-Gossmann D, Buckler KJ, Vaughan-Jones RD. Role of bicarbonate in pH recovery from intracellular acidosis in the guinea-pig ventricular myocyte. Journal of Physiology. 1992; 458: 361–384.
Lagadic-Gossmann D, Vaughan-Jones RD, Buckler KJ. Adrenaline and extracellular ATP switch between two modes of acid extrusion in the guinea-pig ventricular myocyte. Journal of Physiology. 1992; 458: 385–407.
Dart C, Vaughan-Jones RD. Na+-HCO3- symport in the sheep cardiac purkinje fibre. Journal of Physiology. 1992; 451: 365–385.
Grace AA, Kirschenlohr HL, Metcalfe JC, Smith GA, Weissberg PL, Cragoe EJ Jr., et al. Regulation of intracellular pH in the perfused heart by external HCO3- and Na(+)-H+ exchange. American Journal of Physiology. 1993; 265: H289-H298.
Vandenberg JI, Metcalfe JC, Grace AA. Mechanisms of pHi recovery after global ischemia in the perfused heart. Circulation Research. 1993; 72: 993–1003.
Vandenberg JI, Metcalfe JC, Grace AA. Intracellular pH recovery during respiratory acidosis in perfused hearts. American Journal of Physiology. 1994; 266: C489–C497.
Halestrap AP, Wang X, Poole RC, Jackson VN, Price NT. Lactate transport in heart in relation to myocardial ischemia. American Journal of Cardiology. 1997; 80: 17A–25A.
Vaughan-Jones RD, Villafuerte FC, Swietach P, Yamamoto T, Rossini A, Spitzer KW. pH-Regulated Na(+) influx into the mammalian ventricular myocyte: the relative role of Na(+)-H(+) exchange and Na(+)-HCO Co-transport. Journal of Cardiovascular Electrophysiology. 2006; 17 Suppl 1: S134–S140.
Fliegel L. Regulation of the Na+/H+ exchanger in the healthy and diseased myocardium. Expert Opinion on Therapeutic Targets. 2009; 13: 55–68.
Petrecca K, Atanasiu R, Grinstein S, Orlowski J, Shrier A. Subcellular localization of the Na+/H+ exchanger NHE1 in rat myocardium. American Journal of Physiology-Heart and Circulatory Physiology. 1999; 276: H709–H717.
Malakooti J, Dahdal RY, Schmidt L, Layden TJ, Dudeja PK, Ramaswamy K. Molecular cloning, tissue distribution, and functinal expression of the human Na+/H+ exchanger NHE2. American Journal of Physiology-Gastrointestinal and Liver Physiology. 1999; 277: G383–G390.
Amemiya M, Loffing J, Lotscher M, Kaissling B, Alpern RJ, Moe OW. Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb. Kidney International. 1995; 48: 1206–1215.
Biemesderfer D, Pizzonia J, Abu-Alfa A, Exner M, Reilly R, Igarashi P, et al. NHE3: a exchanger isoform of renal brush border. American Journal of Physiology - Renal Fluid and Electrolyte Physiology. 1993; 265: F736–F742.
Baird NR, Orlowski J, Szabo EZ, Zaun HC, Schultheis PJ, Menon AG, et al. Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain. Journal of Biological Chemistry. 1999; 274: 4377–4382.
Nakamura N, Tanaka S, Teko Y, Mitsui K, Kanazawa H. Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation. Journal of Biolgocial Chemistry. 2005; 280: 1561–1572.
Numata M, Petrecca K, Lake N, Orlowski J. Identification of a mitochondrial Na+/H+ exchanger. Journal of Biological Chemistry. 1998; 273: 6951–6959.
Dyck JRB, Lopaschuk GD, Fliegel L. Identification of a small Na+/H+ exchanger-like message in the rabbit myocardium. FEBS Letters. 1992; 310: 255–259.
Seiler SM, Cragoe EJJ, Jones LR. Demonstration of a Na+-H+ exchange activity in purified canine cardiac sarcolemmal vesicles. Journal of Biological Chemistry. 1985; 260: 4869–4876.
Yeves AM, Burgos JI, Medina AJ, Villa-Abrille MC, Ennis IL. Cardioprotective role of IGF-1 in the hypertrophied myocardium of the spontaneously hypertensive rats: A key effect on NHE-1 activity. Acta Physiological (Oxford). 2018; 224: e13092.
Coccaro E, Karki P, Cojocaru C, Fliegel L. Phenylephrine and sustained acidosis activate the neonatal rat cardiomyocyte Na+/H+ exchanger through phosphorylation of amino acids Ser770 and Ser771. American Journal of Physiology-Heart and Circulatory Physiology. 2009; 297: H846–H858.
Gan XT, Chakrabarti S, Karmazyn M. Modulation of Na+/H+ exchange isoform 1 mRNA expression in isolated rat hearts. American Journal of Physiology-Heart and Circulatory Physiology. 1999; 277: H993–H998.
Dyck JRB, Maddaford T, Pierce GN, Fliegel L. Induction of expression of the sodium-hydrogen exchanger in rat myocardium. Cardiovascular Research. 1995; 29: 203–208.
Chen F, Jarmakani JM, Van Dop C. Developmental changes in mRNA encoding cardiac Na+/H+ exchanger (NHE-1) in rabbit. Biochemical and Biophysical Research Communications. 1995; 212: 960–967.
Rieder CV, Fliegel L. Transcriptional regulation of Na+/H+ exchanger expression in the intact mouse. Molecular and Cellular Biochemistry. 2003; 243: 87–95.
Fliegel L. Molecular biology of the myocardial Na+/H+ exchanger. Journal of Molecular and Cellular Cardiology. 2008; 44: 228–237.
Jaballah M, Mohamed IA, Alemrayat B, Al-Sulaiti F, Mlih M, Mraiche F. Na+/H+ exchanger isoform 1 induced cardiomyocyte hypertrophy involves activation of p90 ribosomal s6 kinase. PLoS ONE. 2015; 10: e0122230.
Mohamed IA, Gadeau AP, Fliegel L, Lopaschuk G, Mlih M, Abdulrahman N, et al. Na+/H+ exchanger isoform 1-induced osteopontin expression facilitates cardiomyocyte hypertrophy. PLOS One. 2015; 10: e0123318.
Mraiche F, Fliegel L. Elevated expression of activated Na(+)/H(+) exchanger protein induces hypertrophy in isolated rat neonatal ventricular cardiomyocytes. Molecular and Cellular Biochemistry. 2011; 358: 179–187.
Mraiche F, Oka T, Gan XT, Karmazyn M, Fliegel L. Activated NHE1 is required to induce early cardiac hypertrophy in mice. Basic Research in Cardiology. 2011; 106: 603–616.
Xue J, Mraiche F, Zhou D, Karmazyn M, Oka T, Fliegel L, et al. Elevated myocardial Na+/H+ exchanger isoform 1 activity elicits gene expression that leads to cardiac hypertrophy. Physiological Genomics. 2010; 42: 374–383.
Avkiran M, Cook AR, Cuello F. Targeting Na+/H+ exchanger regulation for cardiac protection: a RSKy approach? Current Opinion in Pharmacology. 2008; 8: 133–140.
Cingolani HE, Perez NG, Pieske B, von Lewinski D, Camilion de Hurtado MC. Stretch-elicited Na+/H+ exchanger activation: the autocrine/paracrine loop and its mechanical counterpart. Cardiovascular Research. 2003; 57: 953–960.
Cingolani HE, Alvarez BV, Ennis IL, Camilion de Hurtado MC. Stretch-induced alkalinization of feline papillary muscle: an autocrine-paracrine system. Circulation Research. 1998; 83: 775–780.
Gunasegaram S, Haworth RS, Hearse DJ, Avkiran M. Regulation of sarcolemmal Na(+)/H(+) exchanger activity by angiotensin II in adult rat ventricular myocytes: opposing actions via AT(1) versus AT(2) receptors. Circulation Research. 1999; 85: 919–930.
Woo SH, Lee CO. Effects of endothelin-1 on Ca2+ signaling in guinea-pig ventricular myocytes: role of protein kinase C. Journal of Molecular and Cellular Cardiology. 1999; 31: 631–643.
Kramer BK, Smith TW, Kelly RA. Endothelin and increased contractility in adult rat ventricular myocytes. Role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na+-H+ exchanger. Circulation Research. 1991; 68: 269–279.
Snabaitis AK, Yokoyama H, Avkiran M. Roles of mitogen-activated protein kinases and protein kinase C in a_1A-adrenoreceptor-mediated stimulation of the sarcolemmal Na+-H+ exchanger. Circulation Research. 2000; 86: 214–220.
Yokoyama H, Yasutake M, Avkiran M. Alpha1-adrenergic stimulation of sarcolemmal Na+-H+ exchanger activity in rat ventricular myocytes: evidence for selective mediation by the alpha1A-adrenoceptor subtype. Circulation Research. 1998; 82: 1078–1085.
Cuello F, Snabaitis AK, Cohen MS, Taunton J, Avkiran M. Evidence for direct regulation of myocardial Na+/H+ exchanger isoform 1 phosphorylation and activity by 90-kDa ribosomal S6 kinase (RSK): effects of the novel and specific RSK inhibitor fmk on responses to alpha1-adrenergic stimulation. Molecular Pharmacology. 2007; 71: 799–806.
Yasutake M, Avkiran M. Exacerbation of reperfusion arrhythmias by alpha 1 adrenergic stimulation: a potential role for receptor mediated activation of sarcolemmal sodium-hydrogen exchange. Cardiovascular Research. 1995; 29: 222–230.
Yasutake M, Haworth RS, King A, Avkiran M. Thrombin activates the sarcolemmal Na+-H+ exchanger. Circulation Research. 1996; 79: 705–715.
Fliegel L, Walsh MP, Singh D, Wong C, Barr A. Phosphorylation of the carboxyl-terminal domain of the Na+/H+ exchanger by Ca2+/calmodulin-dependent protein kinase II. Biochemical Journal. 1992; 282: 139–145.
Amith SR, Fliegel L. Regulation of the Na+/H+ exchanger (NHE1) in breast cancer metastasis. Cancer Research. 2013; 73: 1259–1264.
Snabaitis AK, Cuello F, Avkiran M. Protein kinase B/Akt phosphorylates and inhibits the cardiac Na+/H+ exchanger NHE1. Circulation Research. 2008; 103: 881–890.
Meima ME, Webb BA, Witkowska HE, Barber DL. The sodium-hydrogen exchanger NHE1 is an Akt substrate necessary for actin filament reorganization by growth factors. Journal of Biological Chemistry. 2009; 284: 26666–26675.
Khaled AR, Moor AN, Li A, Kim K, Ferris DK, Muegge K, et al. Trophic factor withdrawal: p38 mitogen-activated protein kinase activates NHE1, which induces intracellular alkalinization. Molecular and Cellular Biology. 2001; 21: 7545–7557.
Clerico A, Giannoni A, Vittorini S, Passino C. Thirty years of the heart as an endocrine organ: physiological role and clinical utility of cardiac natriuretic hormones. American Journal of Physiology - Heart and Circulatory Physiology. 2011; 301: H12–H20.
Shi X, O’Neill MM, MacDonnell S, Brookes PS, Yan C, Berk BC. The RSK Inhibitor BIX02565 Limits Cardiac Ischemia/Reperfusion Injury. Journal of Cardiovascular Pharmacology and Therapeutics. 2016; 21: 177–186.
Moor AN, Fliegel L. Protein kinase mediated regulation of the Na+/H+ exchanger in the rat myocardium by MAP-kinase-dependent pathways. Journal of Biological Chemistry. 1999; 274: 22985–22992.
Haworth RS, Dashnyam S, Avkiran M. Ras triggers acidosis-induced activation of the extracellular-signal-regulated kinase pathway in cardiac myocytes. Biochemical Journal. 2006; 399: 493–501.
Haworth RS, McCann C, Snabaitis AK, Roberts NA, Avkiran M. Stimulation of the plasma membrane Na+/H+ exchanger NHE1 by sustained intracellular acidosis. Evidence for a novel mechanism mediated by the ERK pathway. Journal of Biological Chemistry. 2003; 278: 31676–31684.
Moor AN, Gan XT, Karmazyn M, Fliegel L. Activation of Na+/H+ exchanger-directed protein kinases in the ischemic and ischemic-reperfused rat myocardium. Journal of Biological Chemistry. 2001; 276: 16113–16122.
Liu H, Stupak J, Zheng J, Keller BO, Brix BJ, Fliegel L, et al. Open tubular immobilized metal–ion affinity chromatography combined with MALDI MS and MS/MS for identification of protein phosphorylation sites. Analytical Chemistry. 2004; 76: 4223–4232.
Diaz RG, Nolly MB, Massarutti C, Casarini MJ, Garciarena CD, Ennis IL, et al. Phosphodiesterase 5A inhibition decreases NHE-1 activity without altering steady state pH(i): role of phosphatases. Cellular Physiology and Biochemistry. 2010; 26: 531–540.
Maekawa N, Abe J, Shishido T, Itoh S, Ding B, Sharma VK, et al. Inhibiting p90 ribosomal S6 kinase prevents (Na+)-H+ exchanger-mediated cardiac ischemia-reperfusion injury. Circulation. 2006; 113: 2516–2523.
Karki P, Coccaro E, Fliegel L. Sustained intracellular acidosis activates the myocardial Na(+)/H(+) exchanger independent of amino acid Ser(703) and p90(rsk). Biochimica et Biophysica Acta. 2010; 1798: 1565–1576.
Sabri A, Byron KL, Samarel AM, Bell J, Lucchesi PA. Hydrogen peroxide activates mitogen-activated protein kinases and Na+-H+ exchange in neonatal rat cardiac myocytes. Circulation Research. 1998; 82: 1053–1062.
Wei S, Rothstein EC, Fliegel L, Dell’Italia LJ, Lucchesi PA. Differential MAP kinase activation and Na(+)/H(+) exchanger phosphorylation by H(2)O(2) in rat cardiac myocytes. Americal Journal of Physiology-Cell Physiology. 2001; 281: C1542–C1550.
Rothstein EC, Byron KL, Reed RE, Fliegel L, Lucchesi PA. H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway. American Journal of Physiology-Heart and Circulatory Physiology. 2002; 283: H598–H605.
Li X, Khan MF, Schriemer DC, Fliegel L. Structural changes in the C-terminal regulatory region of the Na(+)/H(+) exchanger mediate phosphorylation induced regulation. Journal of Molecular and Cellular Cardiology. 2013; 61: 153–163.
Hendus-Altenburger R, Pedraz-Cuesta E, Olesen CW, Papaleo E, Schnell JA, Hopper JT, et al. The human Na(+)/H(+) exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2. BMC Biology. 2016; 14: 31.
Li X, Augustine A, Sun D, Li L, Fliegel L. Activation of the Na+/H+ exchanger in isolated cardiomyocytes through beta-Raf dependent pathways. Role of Thr653 of the cytosolic tail. Journal of Molecular and Cellular Cardiology. 2016; 99: 65–75.
Snabaitis AK, D’Mello R, Dashnyam S, Avkiran M. A novel role for protein phosphatase 2A in receptor-mediated regulation of the cardiac sarcolemmal Na+/H+ exchanger NHE1. Journal of Biological Chemistry. 2006; 281: 20252–20262.
Misik AJ, Perreault K, Holmes CF, Fliegel L. Protein phosphatase regulation of Na+/H+ exchanger isoform I. Biochemistry. 2005; 44: 5842–5852.
Hisamitsu T, Nakamura TY, Wakabayashi S. Na(+)/H(+) exchanger 1 directly binds to calcineurin A and activates downstream NFAT signaling, leading to cardiomyocyte hypertrophy. Molecular and Cellular Biology. 2012; 32: 3265–3280.
Xue J, Zhou D, Yao H, Gavrialov O, McConnell MJ, Gelb BD, et al. Novel functional interaction between Na+/H+ exchanger 1 and tyrosine phosphatase SHP-2. American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. 2007; 292: R2406–2416.
Bianchini L, Kapus A, Lukacs G, Wasan S, Wakabayashi S, Pouyssegur J, et al. Responsiveness of mutants of NHE1 isoform of Na+/H+ antiport to osmotic stress. American Journal of Physiology - Cell Physiology. 1995; 269: C998–C1007.
Moor AN, Murtazina R, Fliegel L. Calcium and osmotic regulation of the Na+/H+ exchanger in neonatal ventricular myocytes. Journal of Molecular and Cellular Cardiology. 2000; 32: 925–936.
Falck G, Schjott J, Bruvold M, Krane J, Skarra S, Jynge P. Hyperosmotic perfusion of the beating rat heart and the role of the Na+/K+/2Cl- co-transporter and the Na+/H+ exchanger. Basic Research in Cardiology. 2000; 95: 19–27.
Wright AR, Rees SA. Cardiac cell volume: crystal clear or murky waters? A comparison with other cell types. Pharmacology & Therapeutics. 1998; 80: 89–121.
Richards MA, Simon JN, Ma R, Loonat AA, Crabtree MJ, Paterson DJ, et al. Nitric oxide modulates cardiomyocyte pH control through a biphasic effect on sodium/hydrogen exchanger-1. Cardiovascular Research. 2020; 116: 1958–1971.
Meima ME, Mackley JR, Barber DL. Beyond ion translocation: structural functions of the sodium-hydrogen exchanger isoform-1. Current Opinion in Nephrology and Hypertension. 2007; 16: 365–372.
Bandyopadhyay S, Chiang CY, Srivastava J, Gersten M, White S, Bell R, et al. A human MAP kinase interactome. Nature Methods. 2010; 7: 801–805.
Odunewu-Aderibigbe A, Fliegel L. Protein mediated regulation of the NHE1 isoform of the Na(+)/H(+) exchanger in renal cells. A regulatory role of Hsp90 and AKT kinase. Cellular Signalling. 2017; 36: 145–153.
Amith SR, Vincent KM, Wilkinson JM, Postovit LM, Fliegel L. Defining the Na(+)/H(+) exchanger NHE1 interactome in triple-negative breast cancer cells. Cell Signalling. 2017; 29: 69–77.
Weissberg PL, Little PJ, Cragoe EJJ, Bobik A. The pH of spontaneously beating cultured rat heart cells is regulated by an ATP-calmodulin-dependent Na+/H+ antiport. Circulation Research. 1989; 64: 676–685.
Lin X, Barber DL. A calcineurin homologous protein inhibits GTPase-stimulated Na-H exchange. Journal of BIological Chemistry. 1996; 93: 12631–12636.
Pang T, Wakabayashi S, Shigekawa M. Expression of calcineurin B homologous protein 2 protects serum deprivation-induced cell death by serum-independent activation of Na+/H+ exchanger. Journal of Biological Chemistry. 2002; 277: 43771–43777.
Li X, Liu Y, Kay CM, Muller-Esterl W, Fliegel L. The Na(+)/H(+) exchanger cytoplasmic tail: Structure, function, and interactions with tescalcin. Biochemistry. 2003; 42: 7448–7456.
Mailander J, Muller-Esterl W, Dedio J. Human homolog of mouse tescalcin associates with Na(+)/H(+) exchanger type-1. FEBS Letters. 2001; 507: 331–335.
Pang T, Su X, Wakabayashi S, Shigekawa M. Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers. Journal of Biological Chemistry. 2001; 276: 17367–17372.
Wakabayashi S, Hisamitsu T, Nakamura TY. Regulation of the cardiac Na(+)/H(+) exchanger in health and disease. Journal of Molecular and Cellular Cardiology. 2013; 61: 68–76.
Matsushita M, Tanaka H, Mitsui K, Kanazawa H. Dual functional significance of calcineurin homologous protein 1 binding to Na(+)/H(+) exchanger isoform 1. American Journal of Physiology - Cell Physiology. 2011; 301: C280–288.
Zaun HC, Shrier A, Orlowski J. N-myristoylation and Ca2+ binding of calcineurin B homologous protein CHP3 are required to enhance Na+/H+ exchanger NHE1 half-life and activity at the plasma membrane. Journal of Biological Chemistry. 2012; 287: 36883–36895.
Vaheri A, Carpen O, Heiska L, Helander TS, Jaaskelainen J, Majander-Nordenswan P, et al. The ezrin protein family: membrane-cytoskeleton interactions and disease associations. Current Opinions in Cell Biology. 1997; 9: 659–666.
Denker SP, Huang DC, Orlowski J, Furthmayr H, Barber DL. Direct binding of the Na-H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation. Molecular Cell. 2000; 6: 1425–1436.
Denker SP, Barber DL. Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1. Journal of Cell BIology. 2002; 159: 1087–1096.
Darmellah A, Rucker-Martin C, Feuvray D. ERM proteins mediate the effects of Na+/H+ exchanger (NHE1) activation in cardiac myocytes. Cardiovascular Research. 2009; 81: 294–300.
Silva NLCL, Haworth RS, Singh D, Fliegel L. The carboxyl-terminal region of the Na+/H+ exchanger interacts with mammalian heat shock protein. Biochemistry. 1995; 34: 10412–10420.
Huang C, Lu X, Wang J, Tong L, Jiang B, Zhang W. Inhibition of endogenous heat shock protein 70 attenuates inducible nitric oxide synthase induction via disruption of heat shock protein 70/Na(+) /H(+) exchanger 1-Ca(2+) -calcium-calmodulin-dependent protein kinase II/transforming growth factor beta-activated kinase 1-nuclear factor-kappaB signals in BV-2 microglia. Journal of Neuroscience Research. 2015; 93: 1192–1202.
Huang C, Wang J, Chen Z, Wang Y, Zhang W. 2-phenylethynesulfonamide Prevents Induction of Pro-inflammatory Factors and Attenuates LPS-induced Liver Injury by Targeting NHE1-Hsp70 Complex in Mice. PLoS ONE. 2013; 8: e67582.
Ye Y, Jia X, Bajaj M, Birnbaum Y. Dapagliflozin sttenuates Na(+)/H(+) exchanger-1 in cardiofibroblasts via AMPK activation. Cardiovascular Drugs and Therapy. 2018; 32: 553–558.
Sterling D, Reithmeier RAF, Casey JR. A transport metabolon: Functional interaction of carbonic anhydrase II and chloride/bicarbonate exchangers. Journal of Biological Chemistry. 2001; 276: 47886–47894.
McMurtrie HL, Cleary HJ, Alvarez BV, Loiselle FB, Sterling D, Morgan PE, et al. The bicarbonate transport metabolon. Journal of Enzyme Inhibition and Medicinal Chemistry. 2004; 19: 231–236.
Li X, Alvarez B, Casey JR, Reithmeier RA, Fliegel L. Carbonic anhydrase II binds to and enhances activity of the Na+/H+ exchanger. Journal of Biological Chemistry. 2002; 277: 36085–36091.
Li X, Liu Y, Alvarez BV, Casey JR, Fliegel L. A novel carbonic anhydrase II binding site regulates NHE1 activity. Biochemistry. 2006; 45: 2414–2424.
Vargas LA, Diaz RG, Swenson ER, Perez NG, Alvarez BV. Inhibition of carbonic anhydrase prevents the Na(+)/H(+) exchanger 1-dependent slow force response to rat myocardial stretch. American Journal of Physiology - Heart and Circulatory Physiology. 2013; 305: H228–H237.
Jaquenod De Giusti C, Blanco PG, Lamas PA, Carrizo Velasquez F, Lofeudo JM, Portiansky EL, et al. Carbonic anhydrase II/sodium-proton exchanger 1 metabolon complex in cardiomyopathy of ob(-/-) type 2 diabetic mice. Journal of Molecular and Cellular Cardiology. 2019; 136: 53–63.
Aharonovitz O, Zaun HC, Balla T, York JD, Orlowski J, Grinstein S. Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate. Journal of Cell Biology. 2000; 150: 213–224.
Wakabayashi S, Nakamura TY, Kobayashi S, Hisamitsu T. Novel phorbol ester-binding motif mediates hormonal activation of Na+/H+ exchanger. Journal of BIological Chemistry. 2010; 285: 26652–26661.
Green RD, Frelin C, Vigne P, Lazdunski M. The activity of the Na/H antiporter in cultured cardiac cells is dependent on the culture conditions used. FEBS Letters. 1986; 196: 163–166.
Siczkowski M, Ng LL. Phorbol ester activation of the rat vascular myocyte Na(+)-H(+) exchanger isoform 1. Hypertension. 1996; 27: 859–866.
Vigne P, Freline C, Lazdunski M. The Na+/H+ antiport is activated by serum and phorbol esters in proliferating myoblast but not in differentiated myoblast. Journal of BIological Chemistry. 1985; 260: 8008–8013.
Cassel D, Katz M, Rotman M. Depletion of cellular ATP inhibits Na+/H+ antiport in cultured human cells. Modulation of the regulatory effect of intracellular protons on the antiporter activity. Journal of Biological Chemistry. 1986; 261: 5460–5466.
Aharonovitz O, Demaurex N, Woodside M, Grinstein S. ATP dependence is not an intrinsic property of Na+/H+ exchanger NHE1: requirement for an ancillary factor. American Journal of Physiology - Cell Physiology. 1999; 276: C1303–C1311.
Demaurex N, Grinstein S. Na+/H+ antiport: modulation by ATP and role in cell volume regulation. Journal of Experimental Biology. 1994; 196: 389–404.
Goss GG, Woodside M, Wakabayashi S, Pouyssegur J, Waddell T, Downey GP, et al. ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter. Analysis of phosphorylation and subcellular localization. Journal of Biological Chemistry. 1994; 269: 8741–8748.
Wakabayashi S, Fafournoux P, Sardet C, Pouyssegur J. The Na+/H+ antiporter cytoplasmic domain mediates growth factor signals and controls ”H(+)-sensing”. Proceedings of the National Academy of Sciences USA. 1992; 89: 2424–2428.
Shimada-Shimizu N, Hisamitsu T, Nakamura TY, Wakabayashi S. Evidence that Na+/H+ exchanger 1 is an ATP-binding protein. FEBS Journal. 2013; 280: 1430–1442.
Birkeland ES, Koch LM, Dechant R. Another consequence of the warburg effect? Metabolic regulation of Na(+)/H(+) exchangers may link aerobic glycolysis to cell growth. Frontiers in Oncology. 2020; 10: 1561.
Little PJ, Weissberg PL, Cragoe EJJ, Bobik A. Dependence of Na+/H+ antiport activation in cultured rat aortic smooth muscle on calmodulin, calcium, and ATP. Evidence for the involvement of calmodulin-dependent kinases. Journal of BIological Chemistry. 1988; 263: 16780–16786.
Kandilci HB, Richards MA, Fournier M, Simsek G, Chung YJ, Lakhal-Littleton S, et al. Cardiomyocyte Na(+)/H(+) Exchanger-1 Activity Is Reduced in Hypoxia. Frontiers in Cardiovascular Medicine. 2020; 7: 617038.
Ennis IL, Aiello EA, Cingolani HE, Perez NG. The autocrine/paracrine loop after myocardial stretch: mineralocorticoid receptor activation. Current Cardiology Reviews. 2013; 9: 230–240.
Cingolani HE, Perez NG, Cingolani OH, Ennis IL. The Anrep effect: 100 years later. American Journal of Physiololgy- Heart and Circulatory Physiology. 2013; 304: H175–H182.
Perez NG, Nolly MB, Roldan MC, Villa-Abrille MC, Cingolani E, Portiansky EL, et al. Silencing of NHE-1 blunts the slow force response to myocardial stretch. Journal of Applied Physiology. 2011; 111: 874–880.
von Lewinski D, Kockskamper J, Zhu D, Post H, Elgner A, Pieske B. Reduced stretch-induced force response in failing human myocardium caused by impaired Na(+)-contraction coupling. Circulation: Heart Failure. 2009; 2: 47–55.
Diaz RG, Perez NG, Morgan PE, Villa-Abrille MC, Caldiz CI, Nolly MB, et al. Myocardial mineralocorticoid receptor activation by stretching and its functional consequences. Hypertension. 2014; 63: 112–118.
Villa-Abrille MC, Caldiz CI, Ennis IL, Nolly MB, Casarini MJ, Chiappe de Cingolani GE, et al. The Anrep effect requires transactivation of the epidermal growth factor receptor. Journal of Physiology. 2010; 588: 1579–1590.
Brea MS, Diaz RG, Escudero DS, Caldiz CI, Portiansky EL, Morgan PE, et al. Epidermal Growth Factor Receptor Silencing Blunts the Slow Force Response to Myocardial Stretch. Journal of the American Heart Association. 2016; 5: e004017.
Zavala MR, Diaz RG, Medina AJ, Acosta MP, Escudero DS, Ennis IL, et al. p38-MAP kinase negatively regulates the slow force response to stretch in rat myocardium through the up-regulation of dual specificity phosphatase 6 (DUSP6). Cellular Physiology and Biochemistry. 2019; 52: 172–185.
Ennis IL, Perez NG. Cardiac Mineralocorticoid Receptor and the Na(+)/H(+) Exchanger: Spilling the Beans. Frontiers in Cardiovascular Medicine. 2020; 7: 614279.
Prasad V, Lorenz JN, Lasko VM, Nieman ML, Al Moamen NJ, Shull GE. Loss of the AE3 Cl(-)/HCO(-) 3 exchanger in mice affects rate-dependent inotropy and stress-related AKT signaling in heart. Frontiers in Physiology. 2013; 4: 399.
Guo J, Gan XT, Haist JV, Rajapurohitam V, Zeidan A, Faruq NS, et al. Ginseng inhibits cardiomyocyte hypertrophy and heart failure via NHE-1 inhibition and attenuation of calcineurin activation. Circulation: Heart Failure. 2011; 4: 79–88.
Philipson KD, Ward R. Effects of fatty acids on Na+-Ca2+ exchange and Ca2+ permeability of cardiac sarcolemmal vesicles. Journal of Biological Chemistry. 1985; 260: 9666–9671.
Kutryk MJ, Pierce GN. Stimulation of sodium-calcium exchange by cholesterol incorporation into isolated cardiac sarcolemmal vesicles. Journal of Biological Chemistry. 1988; 263: 13167–13172.
Vemuri R, Philipson KD. Influence of sterols and phospholipids on sarcolemmal and sarcoplasmic reticular cation transporters. Journal of Biological Chemistry. 1989; 264: 8680–8685.
Kutryk MJ, Maddaford TG, Ramjiawan B, Pierce GN. Oxidation of membrane cholesterol alters active and passive transsarcolemmal calcium movement. Circultation Research. 1991; 68: 18–26.
Goel DP, Maddaford TG, Pierce GN. Effects of omega-3 polyunsaturated fatty acids on cardiac sarcolemmal Na(+)/H(+) exchange. American Journal of Physiology - Heart and Circulatory Physiology. 2002; 283: H1688–H1694.
van Borren MM, den Ruijter HM, Baartscheer A, Ravesloot JH, Coronel R, Verkerk AO. Dietary omega-3 polyunsaturated fatty acids suppress NHE-1 upregulation in a rabbit model of volume- and pressure-overload. Frontiers in Physiology. 2012; 3: 76.
Dyck JRB, Silva NLCL, Fliegel L. Activation of the Na+/H+ exchanger gene by the transcription factor AP-2. Journal of Biolgocial Chemistry. 1995; 270: 1375–1381.
Besson P, Fernandez-Rachubinski F, Yang W, Fliegel L. Regulation of Na+/H+ exchanger gene expression: Mitogenic stimulation increases NHE1 promoter activity. American Journal of Physiology-Cell Physiology. 1998; 274: C831–C839.
Facanha AL, dos Reis MC, Montero-Lomeli M. Structural study of the porcine Na+/H+ exchanger NHE1 gene and its 5’- flanking region. Molecular and Cellular Biochemistry. 2000; 210: 91–99.
Wang H, Singh D, Yang W, Dyck JR, Fliegel L. Structure and analysis of the mouse Na+/H+ exchanger (NHE1) gene: homology and conservation of splice sites. Molecular and Cellular Biochemistry. 1996; 165: 155–159.
Miller RT, Counillon L, Pages G, Lifton RP, Sardet C, Pouyssegur J. Structure of the 5’-flanking regulatory region and gene for the human growth factor-activatable Na+/H+ exchanger NHE-1. Journal of Biological Chemistry. 1991; 266: 10813–10819.
Haworth RS, Yasutake M, Brooks G, Avkiran M. Cardiac Na+/H+ exchanger during post-natal development in the rat: Changes in mRNA expression and sarcolemmal activity. Journal of Molecular and Cellular Cardiology. 1997; 29: 321–332.
Rieder CV, Fliegel L. Developmental regulation of Na(+)/H(+) exchanger expression in fetal and neonatal mice. American Journal of Physiology - Heart and Circulatory Physiology. 2002; 283: H273–H283.
Kolyada AY, Johns CA, Madias NE. Role of C/EBP proteins in hepatic and vascular smooth muscle transcription of human NHE1 gene. American Journal of Physiology-Cell Physiology. 1995; 269: C1408–C1416.
Kolyada AY, Lebedeva TV, Johns CA, Madias NE. Proximal regulatory elements and nuclear activities required for transcription of the human Na+/H+ exchanger (NHE-1) gene. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1994; 1217: 54–64.
Yang W, Dyck JRB, Fliegel L. Regulation of NHE1 expression in L6 muscle cells. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1996; 1306: 107–113.
Dyck JRB, Fliegel L. Specific activation of the Na+/H+ exchanger during neuronal differentiation of embryonal carcinoma cells. Journal of Biological Chemistry. 1995; 270: 10420–10427.
Yang W, Wang H, Fliegel L. Regulation of Na+/H+ exchanger gene expression. Role of a novel poly(dA:dT) element in regulation of the NHE1 promoter. Journal of BIological Chemistry. 1996; 271: 20444–20449.
Fernandez-Rachubinski F, Fliegel L. COUP-TF I and COUP-TFII regulate expression of the NHE through a nuclear hormone responsive element with enhancer activity. European Journal of Biochemistry. 2001; 268: 620–634.
Li X, Misik AJ, Rieder CV, Solaro RJ, Lowen A, Fliegel L. Thyroid hormone receptor alpha 1 regulates expression of the Na+/H+ exchanger (NHE1). Journal of Biological Chemistry. 2002; 277: 28656–28662.
Yang W, Dyck JRB, Wang H, Fliegel L. Regulation of the NHE-1 promoter in the mammalian myocardium. American Journal of Physiology - Heart and CIrculatory Physiology. 1996; 270: H259–H266.
Wang H, Yang W, Fliegel L. Identification of an HMG-like protein involved in regulation of Na+/H+ exchanger expression. Mocleular and Cellular Biochemistry. 1997; 176: 99–106.
Akram S, Teong HF, Fliegel L, Pervaiz S, Clement MV. Reactive oxygen species-mediated regulation of the Na+-H+ exchanger 1 gene expression connects intracellular redox status with cells’ sensitivity to death triggers. Cell Death and Differention. 2006; 13: 628–641.
Wakabayashi S, Pang T, Su X, Shigekawa M. A novel topology model of the human Na(+)/H(+) exchanger isoform 1. Journal of BIological Chemistry. 2000; 275: 7942–7949.
Liu Y, Basu A, Li X, Fliegel L. Topological analysis of the Na+/H+ exchanger. Biochimica et Biophysica Acta - Biomembranes. 2015; 1848: 2385–2393.
Dutta D, Fliegel L. Molecular modeling and inhibitor docking analysis of the Na(+)/H(+) exchanger isoform one (1). Biochemistry and Cell Biology. 2019; 97: 333–343.
Hunte C, Screpanti E, Venturi M, Rimon A, Padan E, Michel H. Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH. Nature. 2005; 435: 1197–1202.
Nygaard EB, Lagerstedt JO, Bjerre G, Shi B, Budamagunta M, Poulsen KA, et al. Structural Modeling and Electron Paramagnetic Resonance Spectroscopy of the Human Na+/H+ Exchanger Isoform 1, NHE1. Journal of Biological Chemistry. 2011; 286: 634–648.
Schushan M, Landau M, Padan E, Ben-Tal N. Two conflicting NHE1 model structures: compatibility with experimental data and implications for the transport mechanism. Journal of Biological Chemistry. 2011; 286: le9; author reply Ie10.
Cala PM, Pedersen SF. Response to Schushan et al.: Two conflicting NHE1 model structures: Compatibility with experimental data and implications for the transport mechanism. Journal of Biological Chemistry. 2011; 286: le10.
Goldberg B, Arbel T, Chen J, Karpel R, Mackie GA, Schuldiner S, et al. Characterization of Na+/H+ antiporter gene of E.coli. Proceedings of the National Academy of Sciences USA. 1987; 84: 2615–2619.
Slepkov ER, Rainey JK, Sykes BD, Fliegel L. Structural and functional analysis of the Na(+)/H(+) exchanger. Biochemical Journal. 2007; 401: 623–633.
Padan E. The enlightening encounter between structure and function in the NhaA Na+-H+ antiporter. Trends in Biochemical Sciences. 2008; 33: 435–443.
Dutta D, Fliegel L. Structure and function of yeast and fungal Na(+) /H(+) antiporters. IUBMB Life. 2018; 70: 23–31.
Lee C, Yashiro S, Dotson DL, Uzdavinys P, Iwata S, Sansom MS, et al. Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights. Journal of General Physiology. 2014; 144: 529–544.
Huang Y, Chen W, Dotson DL, Beckstein O, Shen J. Mechanism of pH-dependent activation of the sodium-proton antiporter NhaA. Nature Communications. 2016; 7: 12940.
Arkin IT, Xu H, Jensen MO, Arbely E, Bennett ER, Bowers KJ, et al. Mechanism of Na+/H+ antiporting. Science. 2007; 317: 799–803.
Calinescu O, Dwivedi M, Patino-Ruiz M, Padan E, Fendler K. Lysine 300 is essential for stability but not for electrogenic transport of the Escherichia coli NhaA Na(+)/H(+) antiporter. Journal of Biological Chemistry. 2017; 292: 7932–7941.
Goswami P, Paulino C, Hizlan D, Vonck J, Yildiz O, Kuhlbrandt W. Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1. Embo Journal. 2011; 30: 439–449.
Paulino C, Kuhlbrandt W. pH- and sodium-induced changes in a sodium/proton antiporter. eLife. 2014; 3: e01412.
Lee C, Kang HJ, von Ballmoos C, Newstead S, Uzdavinys P, Dotson DL, et al. A two-domain elevator mechanism for sodium/proton antiport. Nature. 2013; 501: 573–577.
Coincon M, Uzdavinys P, Nji E, Dotson DL, Winkelmann I, Abdul-Hussein S, et al. Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters. Nature Structural & Molecular Biology. 2016; 23: 248–255.
Wohlert D, Kuhlbrandt W, Yildiz O. Structure and substrate ion binding in the sodium/proton antiporter PaNhaP. eLife. 2014; 3: e03579.
Winkelmann I, Matsuoka R, Meier PF, Shutin D, Zhang C, Orellana L, et al. Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9. EMBO Journal. 2020; 39: e105908.
Matsuoka R, Fudim R, Jung S, Zhang C, Bazzone A, Chatzikyriakidou Y, et al. Structure, mechanism and lipid-mediated remodeling of the mammalian Na(+)/H(+) exchanger NHA2. Nature Structural and Molecular Biology. 2022; 29: 108–120.
Murtazina R, Booth BJ, Bullis BL, Singh DN, Fliegel L. Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger. European Journal of Biochemistry. 2001; 268: 4674–4685.
Wiebe CA, Rieder C, Young PG, Dibrov P, Fliegel L. Functional analysis of amino acids of the Na+/H+ exchanger that are important for proton translocation. Molecular and Cellular Biochemistry. 2003; 254: 117–124.
Touret N, Poujeol P, Counillon L. Second-site revertants of a low-sodium-affinity mutant of the Na+/H+ exchanger reveal the participation of TM4 into a highly constrained sodium-binding site. Biochemistry. 2001; 40: 5095–5101.
Noel J, Germain D, Vadnais J. Glutamate 346 of human Na+-H+ exchanger NHE1 is crucial for modulating both the affinity for Na+ and the interaction with amiloride derivatives. Biochemistry. 2003; 42: 15361–15368.
Khadilkar A, Iannuzzi P, Orlowski J. Identification of sites in the second exomembrane loop and ninth transmembrane helix of the mammalian Na+/H+ exchanger important for drug recognition and cation translocation. Journal of BIological Chemistry. 2001; 276: 43792–43800.
Counillon L, Franchi A, Pouyssegur J. A point mutation of the Na+/H+ exchanger gene (NHE1) and amplification of the mutated allele confer amiloride resistance upon chronic acidosis. Proceedings of the National Academy of Sciences USA. 1993; 90: 4508–4512.
Counillon L, Noel J, Reithmeier RAF, Pouyssegur J. Random mutagenesis reveals a novel site involved in inhibitor interaction within the fourth transmembrane segment of the Na+/H+ exchanger-1. Biochemistry. 1997; 36: 2951–2959.
Ding J, Rainey JK, Xu C, Sykes BD, Fliegel L. Structural and functional characterization of transmembrane segment VII of the Na+/H+ exchanger isoform 1. Journal of Biological Chemistry. 2006; 281: 29817–29829.
Orlowski J, Kandasamy RA. Delineation of transmembrane domains of the Na+/H+ exchanger that confer sensitivity to pharmacological antagonists. Journal of BIological Chemistry. 1996; 271: 19922–19927.
Pedersen SF, King SA, Nygaard EB, Rigor RR, Cala PM. NHE1 inhibition by amiloride- and benzoylguanidine-type compounds. Inhibitor binding loci deduced from chimeras of NHE1 homologues with endogenous differences in inhibitor sensitivity. Journal of Biological Chemistry. 2007; 282: 19716–19727.
Harris C, Fliegel L. Amiloride and the Na+/H+ exchanger protein. Mechanism and significance of inhibition of the Na+/H+ exchanger. International Journal of Molecular Medicine. 1999; 3: 315–321.
Slepkov E, Ding J, Han J, Fliegel L. Mutational analysis of potential pore-lining amino acids in TM IV of the Na(+)/H(+) exchanger. Biochimica et Biophysica Acta - Biomembranes. 2007; 1768: 2882–2889.
Pierce GN, Cole WC, Liu K, Massaeli H, Maddaford TG, Chen YJ, et al. Modulation of cardiac performance by amiloride and several selected derivatives of amiloride. Journal of Pharmacology and Experimental Therapeutics. 1993; 265: 1280–1291.
Masereel B, Pochet L, Laeckmann D. An overview of inhibitors of Na(+)/H(+) exchanger. European Journal of Medicinal Chemistry. 2003; 38: 547–554.
Weichert A, Faber S, Jansen HW, Scholz W, Lang HJ. Synthesis of the highly selective Na+/H+ exchange inhibitors cariporide mesilate and (3-methanesulfonyl-4-piperidino-benzoyl) guanidine methanesulfonate. Arzneimittel Forschung - Drug Research. 1997; 47: 1204–1207.
Banno H, Fujiwara J, Hosoya J, Kitamori T, Mori H, Yamashita H, et al. Effects of MS-31-038, a novel Na(+)-H+ exchange inhibitor, on the myocardial infarct size in rats after postischemic administration. Arzneimittel Forschung - Drug Research. 1999; 49: 304–310.
Chen L, Chen CX, Gan XT, Beier N, Scholz W, Karmazyn M. Inhibition and reversal of myocardial infarction-induced hypertrophy and heart failure by NHE-1 inhibition. American Journal of Physiology- Heart and Circulatory Physiology. 2004; 286: H381–H387.
Gumina RJ, Mizumura T, Beier N, Schelling P, Schultz JJ, Gross GJ. A new sodium/hydrogen exchange inhibitor, EMD 85131, limits infarct size in dogs when administered before or after coronary artery occlusion. Journal of Pharmacology and Experimental Therapeutics. 1998; 286: 175–183.
Kawamoto T, Kimura H, Kusumoto K, Fukumoto S, Shiraishi M, Watanabe T, et al. Potent and selective inhibition of the human Na+/H+ exchanger isoform NHE1 by a novel aminoguanidine derivative T-162559. European Journal of Pharmacology. 2001; 420: 1–8.
Huber JD, Bentzien J, Boyer SJ, Burke J, De Lombaert S, Eickmeier C, et al. Identification of a potent sodium hydrogen exchanger isoform 1 (NHE1) inhibitor with a suitable profile for chronic dosing and demonstrated cardioprotective effects in a preclinical model of myocardial infarction in the rat. Journal of Medicinal Chemistry. 2012; 55: 7114–7140.
Lorrain J, Briand V, Favennec E, Duval N, Grosset A, Janiak P, et al. Pharmacological profile of SL 59.1227, a novel inhibitor of the sodium/hydrogen exchanger. British Journal of Pharmacology. 2000; 131: 1188–1194.
Guzman-Perez A, Wester RT, Allen MC, Brown JA, Buchholz AR, Cook ER, et al. Discovery of zoniporide: a potent and selective sodium-hydrogen exchanger type 1 (NHE-1) inhibitor with high aqueous solubility. Bioorganic and Medicinal Chemistry Letters. 2001; 11: 803–807.
Buckley BJ, Majed H, Aboelela A, Minaei E, Jiang L, Fildes K, et al. 6-Substituted amiloride derivatives as inhibitors of the urokinase-type plasminogen activator for use in metastatic disease. Bioorganic & Medicinal Chemistry Letters. 2019; 29: 126753.
Buckley BJ, Kumar A, Aboelela A, Bujaroski RS, Li X, Majed H, et al. Screening of 5- and 6-Substituted Amiloride Libraries Identifies Dual-uPA/NHE1 Active and Single Target-Selective Inhibitors. International Journal of Molecular Sciences. 2021; 22: 2999.
Li X, Buckley B, Stoletov K, Jing Y, Ranson M, Lewis JD, et al. Roles of the Na+/H+ exchanger isoform 1 and urokinase in prostate cancer cell migration and invasion. International Journal of Molecular Medicine. 2021; 2021 22: 13263.
Matthews H, Ranson M, Tyndall JD, Kelso MJ. Synthesis and preliminary evaluation of amiloride analogs as inhibitors of the urokinase-type plasminogen activator (uPA). Bioorganic & Medicinal Chemistry Letters. 2011; 21: 6760–6766.
Buckley BJ, Aboelela A, Minaei E, Jiang LX, Xu Z, Ali U, et al. 6-Substituted Hexamethylene Amiloride (HMA) Derivatives as Potent and Selective Inhibitors of the Human Urokinase Plasminogen Activator for Use in Cancer. Journal of Medicinal Chemistry. 2018; 61: 8299–8320.
Stempien-Otero A, Plawman A, Meznarich J, Dyamenahalli T, Otsuka G, Dichek DA. Mechanisms of cardiac fibrosis induced by urokinase plasminogen activator. Journal of Biological Chemistry. 2006; 281: 15345–15351.
Moriwaki H, Stempien-Otero A, Kremen M, Cozen AE, Dichek DA. Overexpression of urokinase by macrophages or deficiency of plasminogen activator inhibitor type 1 causes cardiac fibrosis in mice. Circulation Research. 2004; 95: 637–644.
Heymans S, Lupu F, Terclavers S, Vanwetswinkel B, Herbert JM, Baker A, et al. Loss or inhibition of uPA or MMP-9 attenuates LV remodeling and dysfunction after acute pressure overload in mice. American Journal of Pathology. 2005; 166: 15–25.
Jennings RB, Sommers HM, Kaltenbach JP, West JJ. Electrolyte Alterations in Acute Myocardial Ischemic Injury. Circulation Research. 1964; 14: 260–269.
Bersohn MM, Shine KI. Verapamil protection of ischemic isolated rabbit heart: dependence on pretreatment. Journal of Molecular and Cellular Cardiology. 1983; 15: 659–671.
Avkiran M, Haworth RS. Regulatory effects of G protein-coupled receptors on cardiac sarcolemmal Na+/H+ exchanger activity: signaling and significance. Cardiovascular Research. 2003; 57: 942–952.
Haworth RS, Sinnett-Smith J, Rozengurt E, Avkiran M. Protein kinase D inhibits plasma membrane Na(+)/H(+) exchanger activity. American Journal of Physiology - Cell Physiology. 1999; 277: C1202–C1209.
Nie J, Duan Q, He M, Li X, Wang B, Zhou C, et al. Ranolazine prevents pressure overload-induced cardiac hypertrophy and heart failure by restoring aberrant Na(+) and Ca(2+) handling. Journal of Cellular Physiology. 2019; 234: 11587–11601.
Khandoudi N, Ho J, Karmazyn M. Role of Na+-H+ exchange in mediating effects of endothlin-1 on normal and ischemic/reperfused hearts. Circulation Research. 1994; 75: 369–378.
Karmazyn M. Amiloride enhances post ischemic recovery: possible role of Na+/H+ exchange. American Journal of Physiology- Heart and Circulatory Physiology. 1988; 255: H608–H615.
Tani M, Neely JR. Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts: Possible involvement of H+-Na+ and Na+ - Ca2+ exchange. Circulation Research. 1989; 65: 1045–1056.
Kleyman TR, Cragoe EJJ. Amiloride and its analogs as tools in the study of ion transport. Journal of Membrane Biology. 1988; 105: 1–21.
Meng H-P, Pierce GN. Protective effects of 5-(N,N-dimethyl) amiloride on ischemia-reperfusion injury in hearts. American Journal of Physiology- Heart and Circulatory Physiology. 1990; 258: H1615–H1619.
Meng H-P, Pierce GN. Involvement of sodium in the protective effect of 5-(N,N-dimethyl)-amiloride on ischemia-reperfusion injury in isolated rat ventricular wall. Journal of Pharmacology and Experimental Therapeutics. 1991; 256: 1094–1100.
Meng HP, Lonsberry B, Pierce GN. Influence of perfusate pH on the postischemic recovery of cardiac contractile function: involvement of sodium-hydrogen exchange. Journal of Pharmacology and Experimental Therapeutics. 1991; 258: 772–777.
Meng H-P, Maddaford TG, Pierce G. Effect of amiloride and selected analogues on postischemic recovery of cardiac contractile function. American Journal of Physiology - Heart and Circulatory Physiology. 1993; 264: H1831–H1835.
Moffat MP, Karmazyn M. Protective effects of the potent Na+/H+ exchange inhibitor methylisobutyl amiloride against post-ischemic contractile dysfunction in rat and guinea-pig hearts. Journal of Molecular and Cellular Cardiology. 1993; 25: 959–971.
Ward CA, Moffat MP. Modulation of sodium-hydrogen exchange activity in cardiac myocytes during acidosis and realkalinisation: effects on calcium, pHi, and cell shortening. Cardiovascular Research. 1995; 29: 247–253.
Shimada Y, Hearse DJ, Avkiran M. Impact of extracellular buffer composition on cardioprotective efficacy of Na+/H+ exchanger inhibitors. American Journal of Physiology - Heart and Circulatory Physiology. 1996; 270: H692–H700.
Docherty JC, Yang L, Pierce GN, Deslauriers R. Na(+)-H+ exchange inhibition at reperfusion is cardioprotective during myocardial ischemia-reperfusion; 31P NMR studies. Molecular and Cellular Biochemistry. 1997; 176: 257–264.
Maddaford TG, Pierce GN. Myocardial dysfunction is associated with activation of Na+/H+ exchange immediately during reperfusion. American Journal of Physiology - Heart and Circulatory Physiology. 1997; 273: H2232–H2239.
Hurtado C, Pierce GN. Inhibition of Na(+)/H(+) exchange at the beginning of reperfusion is cardioprotective in isolated, beating adult cardiomyocytes. Journal of Molecular and Cellular Cardiology. 2000; 32: 1897–1907.
Chakrabarti S, Hoque AN, Karmazyn M. A rapid ischemia-induced apoptosis in isolated rat hearts and its attenuation by the sodium-hydrogen exchange inhibitor HOE 642 (cariporide). Journal of Molecular and Cellular Cardiology. 1997; 29: 3169–3174.
Haist JV, Hirst CN, Karmazyn M. Effective protection by NHE-1 inhibition in ischemic and reperfused heart under preconditioning blockade. American Journal of Physiology - Heart and Circulatory Physiology. 2003; 284: H798–H803.
Ruiz Petrich E, Ponce Zumino A, Moffat MP, Rioux Y, Schanne OF. Modulation of the electrophysiological effects of ischemia reperfusion by methylisobutyl amiloride. Journal of Molecular and Cellular Cardiology. 1996; 28: 1129–1141.
Gazmuri RJ, Ayoub IM, Kolarova JD, Karmazyn M. Myocardial protection during ventricular fibrillation by inhibition of the sodium-hydrogen exchanger isoform-1. Critical Care Medicine. 2002; 30: S166–S171.
Karmazyn M, Ray M, Haist JV. Comparative effects of Na+/H+ exchange inhibitors against cardiac injury produced by ischemia/reperfusion, hypoxia/reoxygenation, and the calcium paradox. Journal of Cardiovascular Pharmacology. 1993; 21: 172–178.
Myers ML, Karmazyn M. Improved cardiac function after prolonged hypothermic ischemia with the Na+/H+ exchange inhibitor HOE 694. Annals of Thoracic Surgery. 1996; 61: 1400–1406.
Shipolini AR, Yokoyama H, Galinanes M, Edmondson SJ, Hearse DJ, Avkiran M. Na+/H+ exchanger activity does not contribute to protection by ischemic preconditioning in the isolated rat heart. Circulation. 1997; 96: 3617–3625.
Hoque AN, Haist JV, Karmazyn M. Na+-H+ exchange inhibition protects against mechanical, ultrastructural, and biochemical impairment induced by low concentrations of lysophosphatidylcholine in isolated rat hearts. Circulation Research. 1997; 80: 95–102.
Avkiran M, Snabaitis AK. Regulation of cardiac sarcolemmal Na+/H+ exchanger activity: potential pathophysiological significance of endogenous mediators and oxidant stress. Journal of Thrombosis and Thrombolysis. 1999; 8: 25–32.
Aker S, Snabaitis AK, Konietzka I, Van De Sand A, Bongler K, Avkiran M, et al. Inhibition of the Na+/H+ exchanger attenuates the deterioration of ventricular function during pacing-induced heart failure in rabbits. Cardiovascular Research. 2004; 63: 273–282.
Linz W, Albus U, Crause P, Jung W, Weichert A, Scholkens BA, et al. Dose-dependent reduction of myocardial infarct mass in rabbits by the NHE-1 inhibitor cariporide (HOE 642). Clinical and Experimental Hypertension. 1998; 20: 733–749.
Hartmann M, Decking UK. Blocking Na+-H+ exchange by cariporide reduces Na(+)-overload in ischemia and is cardioprotective. Journal of Molecular and Cellular Cardiology. 1999; 31: 1985–1995.
Klein HH, Pich S, Bohle RM, Lindert-Heimberg S, Nebendahl K. Na(+)/H(+) exchange inhibitor cariporide attenuates cell injury predominantly during ischemia and not at onset of reperfusion in porcine hearts with low residual blood flow. Circulation. 2000; 102: 1977–1982.
Anzawa R, Seki S, Nagoshi T, Taniguchi I, Feuvray D, Yoshimura M. The role of Na+/H+ exchanger in Ca2+ overload and ischemic myocardial damage in hearts from type 2 diabetic db/db mice. Cardiovascular Diabetology. 2012; 11: 33.
Sakurai S, Kuroko Y, Shimizu S, Kawada T, Akiyama T, Yamazaki T, et al. Effects of intravenous cariporide on release of norepinephrine and myoglobin during myocardial ischemia/reperfusion in rabbits. Life Sciences. 2014; 114: 102–106.
Javadov S, Choi A, Rajapurohitam V, Zeidan A, Basnakian AG, Karmazyn M. NHE-1 inhibition-induced cardioprotection against ischaemia/reperfusion is associated with attenuation of the mitochondrial permeability transition. Cardiovascular Research. 2008; 77: 416–424.
An J, Varadarajan SG, Camara A, Chen Q, Novalija E, Gross GJ, et al. Blocking Na(+)/H(+) exchange reduces [Na(+)](i) and [Ca(2+)](i) load after ischemia and improves function in intact hearts. American Journal of Physiology- Heart and Circulatory Physiology. 2001; 281: H2398–H2409.
Mraiche F, Wagg CS, Lopaschuk GD, Fliegel L. Elevated levels of activated NHE1 protect the myocardium and improve metabolism following ischemia/reperfusion injury. Journal of Molecular and Cellular Cardiology. 2011; 50: 157–164.
Imahashi K, Mraiche F, Steenbergen C, Murphy E, Fliegel L. Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium. American Journal of Physiology- Heart and Circulatory Physiology. 2007; 292: H2237–H2247.
Cook AR, Bardswell SC, Pretheshan S, Dighe K, Kanaganayagam GS, Jabr RI, et al. Paradoxical resistance to myocardial ischemia and age-related cardiomyopathy in NHE1 transgenic mice: a role for ER stress? Journal of Molecular and Cellular Cardiology. 2009; 46: 225–233.
Wang Y, Meyer JW, Ashraf M, Shull GE. Mice with a null mutation in the NHE1 Na+-H+ exchanger are resistant to cardiac ischemia-reperfusion injury. Circulation Research. 2003; 93: 776–782.
Yan S, Gan Y, Jiang N, Wang R, Chen Y, Luo Z, et al. The global survival rate among adult out-of-hospital cardiac arrest patients who received cardiopulmonary resuscitation: a systematic review and meta-analysis. Critical Care. 2020; 24: 61.
Gazmuri RJ, Radhakrishnan J, Ayoub IM. Sodium-Hydrogen Exchanger Isoform-1 Inhibition: A Promising Pharmacological Intervention for Resuscitation from Cardiac Arrest. Molecules. 2019; 24: 1765.
Gazmuri RJ, Ayoub IM, Hoffner E, Kolarova JD. Successful ventricular defibrillation by the selective sodium-hydrogen exchanger isoform-1 inhibitor cariporide. Circulation. 2001; 104: 234–239.
Ayoub IM, Kolarova J, Yi Z, Trevedi A, Deshmukh H, Lubell DL, et al. Sodium-hydrogen exchange inhibition during ventricular fibrillation: Beneficial effects on ischemic contracture, action potential duration, reperfusion arrhythmias, myocardial function, and resuscitability. Circulation. 2003; 107: 1804–1809.
Liakopoulos OJ, Hristov N, Buckberg GD, Triana J, Trummer G, Allen BS. Resuscitation after prolonged cardiac arrest: effects of cardiopulmonary bypass and sodium-hydrogen exchange inhibition on myocardial and neurological recovery. European Journal of Cardio-Thoracic Surgery. 2011; 40: 978–984.
Radhakrishnan J, Kolarova JD, Ayoub IM, Gazmuri RJ. AVE4454B–a novel sodium-hydrogen exchanger isoform-1 inhibitor–compared less effective than cariporide for resuscitation from cardiac arrest. Translational Research. 2011; 157: 71–80.
Wang S, Radhakrishnan J, Ayoub IM, Kolarova JD, Taglieri DM, Gazmuri RJ. Limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation prevents excess mitochondrial Ca2+ accumulation and attenuates myocardial injury. Journal of Applied Physiology. 2007; 103: 55–65.
Ayoub IM, Kolarova J, Gazmuri RJ. Cariporide given during resuscitation promotes return of electrically stable and mechanically competent cardiac activity. Resuscitation. 2010; 81: 106–110.
Ayoub IM, Kolarova JD, Kantola RL, Radhakrishnan J, Wang S, Gazmuri RJ. Zoniporide preserves left ventricular compliance during ventricular fibrillation and minimizes postresuscitation myocardial dysfunction through benefits on energy metabolism. Critical Care Medicine. 2007; 35: 2329–2336.
Wei L, Zhao W, Hu Y, Wang X, Liu X, Zhang P, et al. Exploration of the optimal dose of HOE-642 for the protection of neuronal mitochondrial function after cardiac arrest in rats. Biomedicine and Pharmacotherapy. 2019; 110: 818–824.
Kolarova JD, Ayoub IM, Gazmuri RJ. Cariporide enables hemodynamically more effective chest compression by leftward shift of its flow-depth relationship. American Journal of Physiology- Heart and Circulatory Physiology. 2005; 288: H2904–H2911.
Pieske B, Houser SR, Hasenfuss G, Bers DM. Sodium and the heart: a hidden key factor in cardiac regulation. Cardiovascular Research. 2003; 57: 871–872.
Gu JW, Anand V, Shek EW, Moore MC, Brady AL, Kelly WC, et al. Sodium induces hypertrophy of cultured myocardial myoblasts and vascular smooth muscle cells. Hypertension. 1998; 31: 1083–1087.
Takeda Y, Yoneda T, Demura M, Miyamori I, Mabuchi H. Sodium-induced cardiac aldosterone synthesis causes cardiac hypertrophy. Endocrinology. 2000; 141: 1901–1904.
Chen MZ, Bu QT, Pang SC, Li FL, Sun MN, Chu EF, et al. Tetrodotoxin attenuates isoproterenol-induced hypertrophy in H9c2 rat cardiac myocytes. Molecular and Cellular Biochemistry. 2012; 371: 77–88.
Cingolani HE, Ennis IL. Sodium-hydrogen exchanger, cardiac overload, and myocardial hypertrophy. Circulation. 2007; 115: 1090–1100.
Primessnig U, Bracic T, Levijoki J, Otsomaa L, Pollesello P, Falcke M, et al. Long-term effects of Na(+) /Ca(2+) exchanger inhibition with ORM-11035 improves cardiac function and remodelling without lowering blood pressure in a model of heart failure with preserved ejection fraction. European Journal of Heart Failure. 2019; 21: 1543–1552.
Hayasaki-Kajiwara Y, Kitano Y, Iwasaki T, Shimamura T, Naya N, Iwaki K, et al. Na(+)influx via Na(+)/H(+)exchange activates protein kinase C isozymes delta and epsilon in cultured neonatal rat cardiac myocytes. Journal of Molecular and Cellular Cardiology. 1999; 31: 1559–1572.
Rugale C, Delbosc S, Cristol JP, Mimran A, Jover B. Sodium restriction prevents cardiac hypertrophy and oxidative stress in angiotensin II hypertension. American Journal of Physiology - Heart and Circulatory Physiology. 2003; 284: H1744–H1750.
Aksentijevic D, O’Brien BA, Eykyn TR, Shattock MJ. Is there a causal link between intracellular Na elevation and metabolic remodelling in cardiac hypertrophy? Biochemical Society Transactions. 2018; 46: 817–827.
Yeves AM, Villa-Abrille MC, Perez NG, Medina AJ, Escudero EM, Ennis IL. Physiological cardiac hypertrophy: critical role of AKT in the prevention of NHE-1 hyperactivity. Journal of Molecular and Cellular Cardiology. 2014; 76: 186–195.
Harmsen E, Leenen FH. Dietary sodium induced cardiac hypertrophy. Canadian Journal of Physiology and Pharmacology. 1992; 70: 580–586.
du Cailar G, Ribstein J, Mimran A. Dietary sodium and target organ damage in essential hypertension. American Journal of Hypertension. 2002; 15: 222–229.
Liu J, Yang X, Zhang P, Guo D, Xu B, Huang C, et al. Association of Urinary Sodium Excretion and Left Ventricular Hypertrophy in People With Type 2 Diabetes Mellitus: A Cross-Sectional Study. Frontiers in Endocrinology. 2021; 12: 728493.
Bak MI, Ingwall JS. Contribution of Na+/H+ exchange to Na+ overload in the ischemic hypertrophied hyperthyroid rat heart. Cardiovascular Research. 2003; 57: 1004–1014.
Baartscheer A, Schumacher CA, van Borren MM, Belterman CN, Coronel R, Fiolet JW. Increased Na+/H+-exchange activity is the cause of increased [Na+]i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model. Cardiovascular Research. 2003; 57: 1015–1024.
van Borren MM, Vos MA, Houtman MJ, Antoons G, Ravesloot JH. Increased sarcolemmal Na(+)/H(+) exchange activity in hypertrophied myocytes from dogs with chronic atrioventricular block. Frontiers in Physiology. 2013; 4: 322.
Baartscheer A, Schumacher CA, van Borren MM, Belterman CN, Coronel R, Opthof T, et al. Chronic inhibition of Na+/H+-exchanger attenuates cardiac hypertrophy and prevents cellular remodeling in heart failure. Cardiovascular Research. 2005; 65: 83–92.
Chen L, Gan XT, Haist JV, Feng Q, Lu X, Chakrabarti S, et al. Attenuation of compensatory right ventricular hypertrophy and heart failure following monocrotaline-induced pulmonary vascular injury by the Na+-H+ exchange inhibitor cariporide. Journal of Pharmacology and Experimental Therapeutics. 2001; 298: 469–476.
Darmellah A, Baetz D, Prunier F, Tamareille S, Rucker-Martin C, Feuvray D. Enhanced activity of the myocardial Na+/H+ exchanger contributes to left ventricular hypertrophy in the Goto-Kakizaki rat model of type 2 diabetes: critical role of Akt. Diabetologia. 2007; 50: 1335–1344.
Ennis IL, Garciarena CD, Escudero EM, Perez NG, Dulce RA, Camilion de Hurtado MC, et al. Normalization of the calcineurin pathway underlies the regression of hypertensive hypertrophy induced by Na+/H+ exchanger-1 (NHE-1) inhibition. Canadian Journal of Physiology and Pharmacology. 2007; 85: 301–310.
van Borren MM, Zegers JG, Baartscheer A, Ravesloot JH. Contribution of NHE-1 to cell length shortening of normal and failing rabbit cardiac myocytes. Journal of Molecular and Cellular Cardiology. 2006; 41: 706–715.
Yokoyama H, Gunasegaram S, Harding SE, Avkiran M. Sarcolemmal Na+/H+ exchanger activity and expression in human ventricular myocardium. Journal of the American College of Cardiology. 2000; 36: 534–540.
Fliegel L, Karmazyn M. The cardiac Na-H exchanger: a key downstream mediator for the cellular hypertrophic effects of paracrine, autocrine and hormonal factors. Biochemistry and Cell Biology. 2004; 82: 626–635.
Cingolani HE, Perez NG, Aiello EA, Ennis IL, Garciarena CD, Villa-Abrille MC, et al. Early signals after stretch leading to cardiac hypertrophy. Key role of NHE-1. Frontiers in Bioscience. 2008; 13: 7096–7114.
Yoshida H, Karmazyn M. Na(+)/H(+) exchange inhibition attenuates hypertrophy and heart failure in 1-wk postinfarction rat myocardium. American Journal of Physiology- Heart and Circulatory Physiology. 2000; 278: H300–H304.
Kusumoto K, Haist JV, Karmazyn M. Na(+)/H(+) exchange inhibition reduces hypertrophy and heart failure after myocardial infarction in rats. American Journal of Physiology- Heart and Circulatory Physiology. 2001; 280: H738–H745.
Baartscheer A, Hardziyenka M, Schumacher CA, Belterman CN, van Borren MM, Verkerk AO, et al. Chronic inhibition of the Na+/H+ - exchanger causes regression of hypertrophy, heart failure, and ionic and electrophysiological remodelling. British Journal of Pharmacology. 2008; 154: 1266–1275.
Kilic A, Huang CX, Rajapurohitam V, Madwed JB, Karmazyn M. Early and transient sodium-hydrogen exchanger isoform 1 inhibition attenuates subsequent cardiac hypertrophy and heart failure following coronary artery ligation. Journal of Pharmacology and Experimental Therapeutics. 2014; 351: 492–499.
Ruetten H, Gehring D, Hiss K, Schindler U, Gerl M, Busch AE, et al. Effects of combined inhibition of the Na+-H+ exchanger and angiotensin-converting enzyme in rats with congestive heart failure after myocardial infarction. British Journal of Pharmacology. 2005; 146: 723–731.
Camilion de Hurtado MC, Portiansky EL, Perez NG, Rebolledo OR, Cingolani HE. Regression of cardiomyocyte hypertrophy in SHR following chronic inhibition of the Na(+)/H(+) exchanger. Cardiovascular Research. 2002; 53: 862–868.
Cingolani HE, Camilion de Hurtado MC. Na(+)-H(+) exchanger inhibition: a new antihypertrophic tool. Circulation Research. 2002; 90: 751-753.
Garciarena CD, Caldiz CI, Portiansky EL, Chiappe de Cingolani GE, Ennis IL. Chronic NHE-1 blockade induces an antiapoptotic effect in the hypertrophied heart. Journal of Applied Physiology. 2009; 106: 1325–1331.
Engelhardt S, Hein L, Keller U, Klambt K, Lohse MJ. Inhibition of Na(+)-H(+) exchange prevents hypertrophy, fibrosis, and heart failure in beta(1)-adrenergic receptor transgenic mice. Circulation Research. 2002; 90: 814–819.
Karmazyn M, Liu Q, Gan XT, Brix BJ, Fliegel L. Aldosterone increases NHE-1 expression and induces NHE-1-dependent hypertrophy in neonatal rat ventricular myocytes. Hypertension. 2003; 42: 1171–1176.
Marano G, Vergari A, Catalano L, Gaudi S, Palazzesi S, Musumeci M, et al. Na+/H+ exchange inhibition attenuates left ventricular remodeling and preserves systolic function in pressure-overloaded hearts. British Journal of Pharmacology. 2004; 141: 526–532.
Javadov S, Huang C, Kirshenbaum L, Karmazyn M. NHE-1 inhibition improves impaired mitochondrial permeability transition and respiratory function during postinfarction remodelling in the rat. Journal of Molecular and Cellular Cardiology. 2005; 38: 135–143.
Javadov S, Rajapurohitam V, Kilic A, Hunter JC, Zeidan A, Said Faruq N, et al. Expression of mitochondrial fusion-fission proteins during post-infarction remodeling: the effect of NHE-1 inhibition. Basic Research in Cardiology. 2011; 106: 99–109.
Chahine M, Bkaily G, Nader M, Al-Khoury J, Jacques D, Beier N, et al. NHE-1-dependent intracellular sodium overload in hypertrophic hereditary cardiomyopathy: prevention by NHE-1 inhibitor. Journal of Molecular and Cellular Cardiology. 2005; 38: 571–582.
Bkaily G, Chahine M, Al-Khoury J, Avedanian L, Beier N, Scholz W, et al. Na(+)-H(+) exchanger inhibitor prevents early death in hereditary cardiomyopathy. Canadian Journal of Physiology and Pharmacology. 2015; 93: 923–934.
Kilic A, Velic A, De Windt LJ, Fabritz L, Voss M, Mitko D, et al. Enhanced activity of the myocardial Na+/H+ exchanger NHE-1 contributes to cardiac remodeling in atrial natriuretic peptide receptor-deficient mice. Circulation. 2005; 112: 2307–2317.
Javadov S, Baetz D, Rajapurohitam V, Zeidan A, Kirshenbaum LA, Karmazyn M. Antihypertrophic effect of Na+/H+ exchanger isoform 1 inhibition is mediated by reduced mitogen-activated protein kinase activation secondary to improved mitochondrial integrity and decreased generation of mitochondrial-derived reactive oxygen species. Journal of Pharmacology and Experimental Therapeutics. 2006; 317: 1036–1043.
Dulce RA, Hurtado C, Ennis IL, Garciarena CD, Alvarez MC, Caldiz C, et al. Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: involvement of Na+/H+ and Na+/Ca2+ exchangers. Journal of Molecular and Cellular Cardiology. 2006; 41: 807–815.
Nakamura TY, Iwata Y, Arai Y, Komamura K, Wakabayashi S. Activation of Na+/H+ exchanger 1 is sufficient to generate Ca2+ signals that induce cardiac hypertrophy and heart failure. Circulation Research. 2008; 103: 891–899.
Kilic A, Javadov S, Karmazyn M. Estrogen exerts concentration-dependent pro-and anti-hypertrophic effects on adult cultured ventricular myocytes. Role of NHE-1 in estrogen-induced hypertrophy. Journal of Molecular and Cellular Cardiology. 2009; 46: 360–369.
Javadov S, Rajapurohitam V, Kilic A, Zeidan A, Choi A, Karmazyn M. Anti-hypertrophic effect of NHE-1 inhibition involves GSK-3beta-dependent attenuation of mitochondrial dysfunction. Journal of Molecular and Cellular Cardiology. 2009; 46: 998–1007.
Gan XT, Gong XQ, Xue J, Haist JV, Bai D, Karmazyn M. Sodium-hydrogen exchange inhibition attenuates glycoside-induced hypertrophy in rat ventricular myocytes. Cardiovascular Research. 2010; 85: 79–89.
Shibata M, Takeshita D, Obata K, Mitsuyama S, Ito H, Zhang GX, et al. NHE-1 participates in isoproterenol-induced downregulation of SERCA2a and development of cardiac remodeling in rat hearts. American Journal of Physiology- Heart and Circulatory Physiology. 2011; 301: H2154–H2160.
Correa MV, Nolly MB, Caldiz CI, de Cingolani GE, Cingolani HE, Ennis IL. Endogenous endothelin 1 mediates angiotensin II-induced hypertrophy in electrically paced cardiac myocytes through EGFR transactivation, reactive oxygen species and NHE-1. Pflügers Archiv - European Journal of Physiology. 2014; 466: 1819–1830.
Riaz S, Abdulrahman N, Uddin S, Jabeen A, Gadeau AP, Fliegel L, et al. Anti-hypertrophic effect of Na(+)/H(+) exchanger-1 inhibition is mediated by reduced cathepsin B. European Journal of Pharmacology. 2020; 888: 173420.
Cingolani HE, Rebolledo OR, Portiansky EL, Perez NG, Camilion de Hurtado MC. Regression of hypertensive myocardial fibrosis by Na(+)/H(+) exchange inhibition. Hypertension. 2003; 41: 373–377.
Ghaleh B, Barthelemy I, Wojcik J, Sambin L, Bize A, Hittinger L, et al. Protective effects of rimeporide on left ventricular function in golden retriever muscular dystrophy dogs. International Journal of Cardiology. 2020; 312: 89–95.
Previtali SC, Gidaro T, Diaz-Manera J, Zambon A, Carnesecchi S, Roux-Lombard P, et al. Rimeporide as a first- in-class NHE-1 inhibitor: Results of a phase Ib trial in young patients with Duchenne Muscular Dystrophy. Pharmacological Research. 2020; 159: 104999.
Kyrychenko V, Polakova E, Janicek R, Shirokova N. Mitochondrial dysfunctions during progression of dystrophic cardiomyopathy. Cell Calcium. 2015; 58: 186–195.
Dubinin MV, Talanov EY, Tenkov KS, Starinets VS, Mikheeva IB, Belosludtsev KN. Transport of Ca(2+) and Ca(2+)-dependent permeability transition in heart mitochondria in the early stages of Duchenne muscular dystrophy. Biochimica et Biophysica Acta - Bioenergetics. 2020; 1861: 148250.
De Giusti VC, Nolly MB, Yeves AM, Caldiz CI, Villa-Abrille MC, Chiappe de Cingolani GE, et al. Aldosterone stimulates the cardiac Na(+)/H(+) exchanger via transactivation of the epidermal growth factor receptor. Hypertension. 2011; 58: 912–919.
Medina AJ, Pinilla OA, Portiansky EL, Caldiz CI, Ennis IL. Silencing of the Na(+)/H(+) exchanger 1(NHE-1) prevents cardiac structural and functional remodeling induced by angiotensin II. Experimental and Molecular Pathology. 2019; 107: 1–9.
Sandmann S, Yu M, Kaschina E, Blume A, Bouzinova E, Aalkjaer C, et al. Differential effects of angiotensin AT1 and AT2 receptors on the expression, translation and function of the Na+-H+ exchanger and Na+- HCO_3- symporter in the rat heart after myocardial infarction. Journal of the American College of Cardiology. 2001; 37: 2154–2165.
Young MJ, Funder JW. Mineralocorticoid receptors and pathophysiological roles for aldosterone in the cardiovascular system. Journal of Hypertension. 2002; 20: 1465–1468.
Lefkowitz RJ, Rockman HA, Koch WJ. Catecholamines, cardiac beta-adrenergic receptors, and heart failure. Circulation. 2000; 101: 1634–1637.
Colucci WS, Williams GH, Braunwald E. Increased plasma norepinephrine levels during prazosin therapy for severe congestive heart failure. Annals of Internal Medicine. 1980; 93: 452–453.
Wang J, Wang Y, Zhang W, Zhao X, Chen X, Xiao W, et al. Phenylephrine promotes cardiac fibroblast proliferation through calcineurin-NFAT pathway. Frontiers in Bioscience-Landmark. 2016; 21: 502–513.
Tai C, Gan T, Zou L, Sun Y, Zhang Y, Chen W, et al. Effect of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers on cardiovascular events in patients with heart failure: a meta-analysis of randomized controlled trials. BMC Cardiovascular Disorders. 2017; 17: 257.
Mlih M, Abdulrahman N, Gadeau AP, Mohamed IA, Jaballah M, Mraiche F. Na(+)/H (+) exchanger isoform 1 induced osteopontin expression in cardiomyocytes involves NFAT3/Gata4. Molecular and Cellular Biochemistry. 2015; 404: 211–220.
Wilkins BJ, Dai YS, Bueno OF, Parsons SA, Xu J, Plank DM, et al. Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy. Circulation Research. 2004; 94: 110–118.
Putt ME, Hannenhalli S, Lu Y, Haines P, Chandrupatla HR, Morrisey EE, et al. Evidence for coregulation of myocardial gene expression by MEF2 and NFAT in human heart failure. Circulation: Cardiovascular Genetics. 2009; 2: 212–219.
Kilic A, Rajapurohitam V, Sandberg SM, Zeidan A, Hunter JC, Said Faruq N, et al. A novel chimeric natriuretic peptide reduces cardiomyocyte hypertrophy through the NHE-1-calcineurin pathway. Cardiovascular Research. 2010; 88: 434–442.
Garciarena CD, Caldiz CI, Correa MV, Schinella GR, Mosca SM, Chiappe de Cingolani GE, et al. Na+/H+ exchanger-1 inhibitors decrease myocardial superoxide production via direct mitochondrial action. Journal of Applied Physiology. 2008; 105: 1706–1713.
Knowlton AA, Chen L, Malik ZA. Heart failure and mitochondrial dysfunction: the role of mitochondrial fission/fusion abnormalities and new therapeutic strategies. Journal of Cardiovascular Pharmacology. 2014; 63: 196–206.
Baartscheer A, Schumacher CA, Wust RC, Fiolet JW, Stienen GJ, Coronel R, et al. Empagliflozin decreases myocardial cytoplasmic Na(+) through inhibition of the cardiac Na(+)/H(+) exchanger in rats and rabbits. Diabetologia. 2017; 60: 568–573.
Uthman L, Baartscheer A, Bleijlevens B, Schumacher CA, Fiolet JWT, Koeman A, et al. Class effects of SGLT2 inhibitors in mouse cardiomyocytes and hearts: inhibition of Na(+)/H(+) exchanger, lowering of cytosolic Na(+) and vasodilation. Diabetologia. 2018; 61: 722–726.
Iborra-Egea O, Santiago-Vacas E, Yurista SR, Lupon J, Packer M, Heymans S, et al. Unraveling the Molecular Mechanism of Action of Empagliflozin in Heart Failure With Reduced Ejection Fraction With or Without Diabetes. JACC: Basic to Translational Science. 2019; 4: 831–840.
Trum M, Riechel J, Lebek S, Pabel S, Sossalla ST, Hirt S, et al. Empagliflozin inhibits Na(+) /H(+) exchanger activity in human atrial cardiomyocytes. ESC Heart Failure. 2020; 7.6: 4429–4437.
Bayes-Genis A, Iborra-Egea O, Spitaleri G, Domingo M, Revuelta-Lopez E, Codina P, et al. Decoding empagliflozin’s molecular mechanism of action in heart failure with preserved ejection fraction using artificial intelligence. Scientific Reports. 2021; 11: 12025.
Arow M, Waldman M, Yadin D, Nudelman V, Shainberg A, Abraham NG, et al. Sodium-glucose cotransporter 2 inhibitor Dapagliflozin attenuates diabetic cardiomyopathy. Cardiovascular Diabetology. 2020; 19: 7.
Kawase H, Bando YK, Nishimura K, Aoyama M, Monji A, Murohara T. A dipeptidyl peptidase-4 inhibitor ameliorates hypertensive cardiac remodeling via angiotensin-II/sodium-proton pump exchanger-1 axis. Journal of Molecular and Cellular Cardiology. 2016; 98: 37–47.
Anheim M, Tranchant C, Koenig M. The autosomal recessive cerebellar ataxias. New England Journal of Medicine. 2012; 366: 636–646.
Lichtenstein H, Knorr A. Uber einige Fa¨lle von fortschreitender Schwerho¨rigkeit bei heredita¨rer Ataxie. Dtsch. Zeitung fu¨r Nervenheilkd. 1930; 114: 1–28.
Iwama K, Osaka H, Ikeda T, Mitsuhashi S, Miyatake S, Takata A, et al. A novel SLC9A1 mutation causes cerebellar ataxia. Journal of Human Genetics. 2018; 63: 1049–1054.
Cox GA, Lutz CM, Yang C-L, Biemesderfer D, Bronson RT, Fu A, et al. Sodium/Hydrogen exchanger gene defect in slow-wave epilepsy mice. Cell. 1997; 91: 139–148.
Bell SM, Schreiner CM, Schultheis PJ, Miller ML, Evans RL, Vorhees CV, et al. Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures. Journal of Biological Chemistry. 1999; 276: C788–C795.
Li X, Fliegel L. A novel human mutation in the SLC9A1 gene results in abolition of Na+/H+ exchanger activity. PLoS ONE. 2015; 10: e0119453.
Li X, Augustine A, Chen S, Fliegel L. Stop Codon Polymorphisms in the Human SLC9A1 Gene Disrupt or Compromise Na+/H+ Exchanger Function. PLoS ONE. 2016; 11: e0162902.
Alves C, Ma Y, Li X, Fliegel L. Characterization of human mutations in phosphorylatable amino acids of the cytosolic regulatory tail of SLC9A1. Biochemistry and Cell Biology. 2014; 92: 524–529.
Takahashi E, Abe J, Gallis B, Aebersold R, Spring DJ, Krebs EG, et al. p90(RSK) is a serum-stimulated Na+/H+ exchanger isoform-1 kinase. Regulatory phosphorylation of serine 703 of Na+/H+ exchanger isoform-1. Journal of Biological Chemistry. 1999; 274: 20206–20214.
Lehoux S, Abe J, Florian JA, Berk BC. 14-3-3 Binding to Na+/H+ exchanger isoform-1 is associated with serum-dependent activation of Na+/H+ exchange. Journal of Biological Chemistry. 2001; 276: 15794–15800.
Malo ME, Li L, Fliegel L. Mitogen-activated protein kinase-dependent activation of the Na+/H+ exchanger is mediated through phosphorylation of amino acids Ser770 and Ser771. The Journal of Biological Chemistry. 2007; 282: 6292–6299.
Odunewu A, Fliegel L. Acidosis-mediated regulation of the NHE1 isoform of the Na+/H+ exchanger in renal cells. The American Journal of Physiology - Renal Physiology. 2013; 305: F370–381.
Douglas RM, Schmitt BM, Xia Y, Bevensee MO, Biemesderfer D, Boron WF, et al. Sodium-hydrogen exchangers and sodium-bicarbonate co-transporters: Ontogeny of protein expression in the rat brain. Neuroscience. 2001; 102: 271–228.
Chen GS, Lee SP, Huang SF, Chao SC, Chang CY, Wu GJ, et al. Functional and molecular characterization of transmembrane intracellular pH regulators in human dental pulp stem cells. Archives of Oral Biology. 2018; 90: 19–26.
De Vito P. The sodium/hydrogen exchanger: a possible mediator of immunity. Cellular Immunology. 2006; 240: 69–85.
Zhuo JL, Soleimani M, Li XC. New insights into the critical importance of intratubular Na(+)/H(+) Exchanger 3 and Its potential therapeutic implications in hypertension. Current Hypertension Reports. 2021; 23: 34.
Ali A, Ahmad FJ, Dua Y, Pillai KK, Vohora D. Seizures and sodium hydrogen exchangers: potential of sodium hydrogen exchanger inhibitors as novel anticonvulsants. CNS and Neurological Disorders - Drug Targets. 2008; 7: 343–347.
Chen Y, Wu S, Tian Y, Kong J. Phosphorylation and subcellular localization of Na(+)/H(+) exchanger isoform 3 (NHE3) are associated with altered gallbladder absorptive function after formation of cholesterol gallstones. Journal of Physiology and Biochemistry. 2017; 73: 133–139.
Lupachyk S, Stavniichuk R, Komissarenko JI, Drel VR, Obrosov AA, El-Remessy AB, et al. Na+/H+-exchanger-1 inhibition counteracts diabetic cataract formation and retinal oxidative-nitrative stress and apoptosis. International Journal of Molecular Medicine. 2012; 29: 989–998.
Bkaily G, Jacques D. Na(+)-H(+) exchanger and proton channel in heart failure associated with Becker and Duchenne muscular dystrophies. Canadian Journal of Physiology and Pharmacology. 2017; 95: 1213–1223.
Pierce G, Resch C, Mourin M, Dibrov P, Dibrov E. Bacteria and the growing threat of multidrug resistance for invasive cardiac interventions. Reviews in Cardiovascular Medicine. 2022; 23: 15.
Regan TJ, Ettinger PO, Khan MI, Jesrani OU, Lyons MM, Oldewurtel HA, et al. Altered myocardial function and metabolism in chronic diabetes mellitus without ischemia in dogs. Circulation Research. 1974; 35: 222–237.
Penpargkul S, Schaible T, Yipintsoi T, Scheuer J. The effect of diabetes on performance and metabolism of rat hearts. Circulation Research. 1980; 47: 911–921.
Fein FS, Kornstein LB, Strobeck JE, Capasso JM, Sonnenblick EH. Altered myocardial mechanics in diabetic rats. Circulation Research. 1980; 47: 922–933.
Dillmann WH. Diabetes mellitus induces changes in cardiac myosin of the rat. Diabetes. 1980; 29: 579–582.
Pierce GN, Dhalla NS. Cardiac myofibrillar ATPase activity in diabetic rats. Journal of Molecular and Cellular Cardiology. 1981; 13: 1063–1069.
Pierce GN, Dhalla NS. Sarcolemmal Na+-K+-ATPase activity in diabetic rat heart. American Journal of Physiology- Cell Physiology. 1983; 245: C241–C247.
Pierce GN, Kutryk MJ, Dhalla NS. Alterations in Ca2+binding by and composition of the cardiac sarcolemmal membrane in chronic diabetes. Proceedings of the National Academy of Sciences USA. 1983; 80: 5412–5416.
Ganguly PK, Pierce GN, Dhalla KS, Dhalla NS. Defective sarcoplasmic reticular calcium transport in diabetic cardiomyopathy. American Journal of Physiology-Endocrinology and Metabolism. 1983; 244: E528–E535.
Lopaschuk GD, Tahiliani AG, Vadlamudi RV, Katz S, McNeill JH. Cardiac sarcoplasmic reticulum function in insulin- or carnitine-treated diabetic rats. American Journal of Physiology- Heart and Circulatory Physiology. 1983; 245: H969–H976.
Scott RC. Diabetes and the heart. American Heart Journal. 1975; 90: 283–289.
Schaffer SW, Tan BH, Wilson GL. Development of a cardiomyopathy in a model of noninsulin-dependent diabetes. American Journal of Physiology- Heart and Circulatory Physiology. 1985; 248: H179–H185.
Lopaschuk GD, Russell JC. Myocardial function and energy substrate metabolism in the insulin-resistant JCR:LA corpulent rat. Journal of Applied Physiology. 1991; 71: 1302–1308.
Tani M, Neely JR. Hearts from diabetic rats are more resistant to in vitro ischemia: possible role of altered Ca2+ metabolism. Circulation Research. 1988; 62: 931–940.
Khandoudi N, Bernard M, Cozzone P, Feuvray D. Intracelluar pH and role of Na+/H+ exchange during ischaemia and reperfusion of normal and diabetic rat hearts. Cardiovascular Research. 1990; 11: 873–878.
Pierce GN, Ramjiawan B, Dhalla NS, Ferrari R. Na+-H+ exchange in cardiac sarcolemmal vesicles isolated from diabetic rats. American Journal of Physiology- Heart and Circulatory Physiology. 1990; 258: H255–H261.
Pierce G, Slotin T, Fliegel L, Gilchrist J, Maddaford T. Expression and activity of the sodium-hydrogen exchanger in cardiac sarcolemma in health and disease. in The Na+/H+ Exchanger. RG Landes Publishing Co. Austin. 1996; : 217–228.
Maddaford TG, Russell JC, Pierce GN. Postischemic cardiac performance in the insulin-resistant JCR:LA-cp rat. American Journal of Physiology- Heart and Circulatory Physiology. 1997; 273: H1187–H1192.
Goel DP, Pierce GN. Role of the sodium-hydrogen exchanger in ischemia-reperfusion injury in diabetes. Journal of Thrombosis and Thrombolysis. 1999; 8: 45–52.
Masai M, Fujioka Y, Fujiwara M, Morimoto S, Miyoshi A, Suzuki H, et al. Activation of Na+/H+ exchanger is associated with hyperinsulinemia in borderline hypertensive rats. European Journal of Clinical Investigation. 2001; 31: 193–200.
Packer M, Anker SD, Butler J, Filippatos G, Zannad F. Effects of Sodium-Glucose Cotransporter 2 Inhibitors for the Treatment of Patients With Heart Failure: Proposal of a Novel Mechanism of Action. JAMA Cardiology. 2017; 2: 1025–1029.
Packer M. Activation and Inhibition of Sodium-Hydrogen Exchanger Is a Mechanism That Links the Pathophysiology and Treatment of Diabetes Mellitus With That of Heart Failure. Circulation. 2017; 136: 1548–1559.
Peng X, Li L, Lin R, Wang X, Liu X, Li Y, et al. Empagliflozin Ameliorates Ouabain-Induced Na(+) and Ca(2+) Dysregulations in Ventricular Myocytes in an Na(+)-Dependent Manner. Cardiovascular Drugs and Therapy. 2022. (in press)
Uthman L, Li X, Baartscheer A, Schumacher CA, Baumgart P, Hermanides J, et al. Empagliflozin reduces oxidative stress through inhibition of the novel inflammation/NHE/[Na(+)]c/ROS-pathway in human endothelial cells. Biomedicine & Pharmacotherapy. 2022; 146: 112515.
Zhang H, Uthman L, Bakker D, Sari S, Chen S, Hollmann MW, et al. Empagliflozin Decreases Lactate Generation in an NHE-1 Dependent Fashion and Increases alpha-Ketoglutarate Synthesis From Palmitate in Type II Diabetic Mouse Hearts. Frontiers in Cardiovascular Medicine. 2020; 7: 592233.
Andreadou I, Bell RM, Botker HE, Zuurbier CJ. SGLT2 inhibitors reduce infarct size in reperfused ischemic heart and improve cardiac function during ischemic episodes in preclinical models. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2020; 1866: 165770.
Chung YJ, Park KC, Tokar S, Eykyn TR, Fuller W, Pavlovic D, et al. Off-target effects of SGLT2 blockers: empagliflozin does not inhibit Na+/H+ exchanger-1 or lower [Na+]i in the heart. Cardiovascular Research. 2020.
Chung YJ, Park KC, Tokar S, Eykyn TR, Fuller W, Pavlovic D, et al. SGLT2 inhibitors and the cardiac Na+/H+ exchanger-1: the plot thickens. Cardiovascular Research. 2021; 117: 2702–2704.
Abdulrahman N, Ibrahim M, Joseph JM, Elkoubatry HM, Al-Shamasi AA, Rayan M, et al. Empagliflozin inhibits angiotensin II-induced hypertrophy in H9c2 cardiomyoblasts through inhibition of NHE1 expression. Molecular and Cellular Biochemistry. 2022; 477: 1865–1872.
Packer M. Role of the sodium-hydrogen exchanger in mediating the renal effects of drugs commonly used in the treatment of type 2 diabetes. Diabetes Obesity and Metabolism. 2018; 20: 800–811.
Rupprecht HJ, vom Dahl J, Terres W, Seyfarth KM, Richardt G, Schultheibeta HP, et al. Cardioprotective effects of the Na(+)/H(+) exchange inhibitor cariporide in patients with acute anterior myocardial infarction undergoing direct PTCA. Circulation. 2000; 101: 2902–2908.
Zeymer U, Suryapranata H, Monassier JP, Opolski G, Davies J, Rasmanis G, et al. The Na(+)/H(+) exchange inhibitor eniporide as an adjunct to early reperfusion therapy for acute myocardial infarction. Results of the evaluation of the safety and cardioprotective effects of eniporide in acute myocardial infarction (ESCAMI) trial. Journal of the American College of Cardiology. 2001; 38: 1644–1650.
Theroux P, Chaitman BR, Danchin N, Erhardt L, Meinertz T, Schroeder JS, et al. Inhibition of the sodium-hydrogen exchanger with cariporide to prevent myocardial infarction in high-risk ischemic situations. Main results of the GUARDIAN trial. Guard during ischemia against necrosis (GUARDIAN) Investigators. Circulation. 2000; 102: 3032–3038.
Mentzer RM Jr., Bartels C, Bolli R, Boyce S, Buckberg GD, Chaitman B, et al. Sodium-hydrogen exchange inhibition by cariporide to reduce the risk of ischemic cardiac events in patients undergoing coronary artery bypass grafting: results of the EXPEDITION study. Annals of Thoracic Surgery. 2008; 85: 1261–1270.
Karmazyn M. NHE-1: still a viable therapeutic target. Journal of Molecular and Cellular Cardiology. 2013; 61: 77–82.
Leng T, Shi Y, Xiong ZG, Sun D. Proton-sensitive cation channels and ion exchangers in ischemic brain injury: new therapeutic targets for stroke? Progress in Neurobiology. 2014; 115: 189–209.
Rosskopf D. Sodium-hydrogen exchange and platelet function. Journal of Thrombosis and Thrombolysis. 1999; 8: 15–23.
Buckberg GD. Solving the mysteries of heart disease: life-saving answers ignored by the medical establishment. Health House Press: USA. 2018.
Back to top