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 pH${}_{\text{𝑖}}$. 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, $\alpha$-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 AT${}_1$ receptor and occurs through protein kinase C and an epidermal growth factor mediated mechanism. The AT${}_2$ 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]. $\alpha$${}_1$-adrenergic agonists like phenylephrine stimulate NHE1 activity by the $\alpha$${}_{1A}$-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 $\alpha$${}_1$-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 p90${}^{\text{RSK}}$ sites are shown. CaM, Calmodulin; CHP, calcineurin homologous protein; 14-3-3,14-3-3 protein; HSP, heat shock protein. Some sites overlap.

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 TR$\alpha$${}_1$ 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 O${}_2$ superoxide production and NHE1 promoter activity and O${}_2$ superoxide production could be blocked by the oxidase inhibitor diphenyleniodonium [181]. Tiron, a specific O${}_2$ 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.

Although the entry of excessive extracellular Ca${}^{2+}$ into the cardiomyocyte was recognized early on as a key event in the toxic effects of ischemic injury to the heart (Ca${}^{2+}$ overload) [234], the mechanism whereby this excess Ca${}^{2+}$ entered the cell remained unknown until decades later. Blocking the L-type Ca${}^{2+}$ channel had limited clinical utility in protecting the heart from ischemic and reperfusion injury so that was clearly not the mechanism for Ca${}^{2+}$ entry [235]. Lazdunski proposed in 1985 [24] that a mechanism involving NHE and NCX activation was responsible for the toxic entry of Ca${}^{2+}$ 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 $\alpha$${}_{1A}$ 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 Ca${}^{2+}$ movements via the NHE1 and NCX transport pathways in the myocardial cell (Fig. 5).

Fig. 5.

Schematic of the mechanism by which the Na${}^{\mathrm{+}}$/H${}^{\mathrm{+}}$ exchanger (NHE1) and the Na${}^{\mathrm{+}}$/Ca${}^{\mathrm{2}\mathrm{+}}$ 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 Ca${}^{2+}$ in the cell results in contractile dysfunction and structural damage to the myocardial cell.

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 Ca${}^{2+}$ paradox protocol of myocardial Ca${}^{2+}$ overload [257].

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].

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].

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].

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 $\alpha$1 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].

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 $\alpha$1 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].

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 $\beta$${}_1$ 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).

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 AT${}_1$ 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 $\beta$${}_1$ adrenoceptor activation [reviewed in [343]], indeed $\alpha$${}_1$ blockers are generally contraindicated due to vasodilator effects of these agents resulting in reflex sympathetic activity [344]. Nonetheless, $\alpha$${}_1$ 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 AT${}_1$ 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].

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 3$\beta$ (GSK-3$\beta$) 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.

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].

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.

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]${}_{\text{𝑖}}$ 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 Ca${}^{2+}$${}_{\text{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].