Academic Editor: Calogera Pisano
Ischemic heart disease resulting from a myocardial infarction (MI), is a major health issue. Stem cell therapies may play an important role in this field. However, cardiomyocytes induced from stem cells are characterized by low rates of differentiation and immaturity. After transplantation into the damaged heart, they may even increase the risk of arrhythmias. Studies have demonstrated that electrical stimulation (ES) can promote the cardiac differentiation of stem cells. This review summarizes the latest research on the effects of applying different electrical stimulation (ES) parameters to different types of stem cells and the related mechanisms that may be involved.
Cardiovascular disease is a major world-wide health concern [1]. When an acute myocardial infarction (MI) occurs, the number of viable cardiomyocytes will decrease. The formation of fibrous scar tissue and the remodeling of ventricular tissue after a myocardial infarction, results in myocardial dilatation, aneurysm formation and will eventually lead to heart failure. Currently, there is no treatment that can reverse all the damage in the infarcted myocardium to improve heart function. Therefore, there is a need for new treatment methods to improve cardiomyocyte viability to preserve heart function. Transplanting cardiomyocytes derived from stem cells into an area of myocardial infarction to repair the damaged myocardium and restore heart function is a promising treatment for myocardial infarction [2, 3].
Cardiomyocytes (CM) derived from stem cells are characterized by low rates of differentiation and immaturity. Following transplantation into a damaged heart, they may further increase the risk of arrhythmias [4, 5]. During myocardial contraction, pacemaker cells send out rhythmic electrical signals from the sinoatrial node, which are transmitted to the cardiomyocytes through the cell gap junctions, and then transmitted to other areas of the heart through the highly conductive Purkinje fibers. During this process, electrical signals and gap connections are vital to the heart’s synchronized contractions [6]. In recent years, electrical stimulation (ES) has received widespread attention due safety, convenience, and the avoidance of drugs. Therefore, it has become a promising method to induce stem cell cardiac differentiation [7, 8], which will further promote the clinical application of cardiac stem cells in the treatment of myocardial infarction.
Cardiomyocytes form gap junctions through the intercalated disk, which is conducive to the conduction of electrical stimuli between the cells and the rhythmic contraction in accordance with electrical signals [9]. In order to promote the cardiac differentiation of stem cells, it is important to optimize the parameters of the applied electric stimulation when applying ES to simulate cardiac electrophysiological signals [10].
Pulse direction can be divided into monophase pulse and biphase pulse. Although monophase pulse can effectively stimulate cardiomyocytes to produce action potentials, it can produce reactive oxygen species which can result in tissue damage. Biphasic pulses can effectively prevent tissue damage, but the hyperpolarization of the biphasic pulses generated may suppress action potential initiation [11].
Sven et al. [12] applied monophasic pulses (amplitude: 1 V, pulse
duration: 5 ms) at a stimulation frequency of 10 Hz to fetal rat cardiomyocytes
for 6 consecutive days. ES promoted cell morphological elongation and parallel
arrangement as well as the expression of connexin 43 (Cx43). Pietronave
et al. [13] applied monophasic (2 ms, 1 Hz, 5 V amplitude) or biphasic
(2 ms, 1 Hz,
For the same electric field strength, the cell apoptosis rate is higher when the pulse duration is longer [14]. Therefore, the optimal combination of intensity and duration is crucial.
In order to explore the effect of the duration of ES on the myocardial
differentiation of human cardiosphere-derived cells (hCDCs), Nazari et
al. [15] applied a charge balance biphasic pulse (I = 2 mA, V = 150 mV,
The impact of ES depends on the degree of differentiation of the cells during the first ES. If applied too early, ES will inhibit the accumulation of myocardial protein and produce undesirable contraction behavior. If it is applied too late, ES no longer contributes to the functional development of cardiomyocytes.
Radisic et al. [17] applied ES to neonatal rat ventricular myocytes of
1, 3, and 5 days. After the 1 d-old ventricular myocytes are electrically
stimulated, the contents of Cx43 and
The frequency of ES can affect the contraction and contractile behavior of
cardiomyocytes. ES with a frequency of 1–2 Hz can induce a transient surge of
intracellular Ca
Tandon et al. [20] applied monophasic pulses of 3 V/cm, pulse width 2 ms, and frequencies of 1, 3, and 5 Hz to the cardiac tissue of neonatal rat ventricular myocytes. Studies have shown that cardiac tissues stimulated at 3 Hz frequency had the highest tissue density, the highest concentrations of cardiac troponin-I and Cx43, and the best contractility. Zhang et al. [21] applied ES to hiPSC-CMs after 2 days of cell preculture. Cardiomyocytes were first stimulated at 1 Hz for 5 continuous days, and followed by 1 Hz daily step-up stimulation until the frequency reached 6 Hz. And after maintaining stimulation at 6 Hz for 2 days, the researchers continued to apply 1 Hz stimulation for 14 days. The results indicated that, after ES, hiPSC-CMs highly expressed the Cx43 and achieved a more mature phenotype, as confirmed by the more organized sarcomeres. Marc et al. [22] tested two pacing protocols: biphasic pulses with a frequency at 0.5 Hz for the entire period and pacing at 2 Hz during the first week and 1.5 Hz thereafter, a field strength of 2 V/cm and a pulse width of 4 ms, act on hiPSC-CMs respectively. The high frequency paced human engineered heart tissues (hEHTs) had markedly higher forces. Ronaldson-Bouchard et al. [18] also applied two schemes of ES. Under the condition that the other ES parameters are the same, 3 weeks at 2 Hz and 2 weeks at a frequency increasing from 2 Hz to 6 Hz by 0.33 Hz per day, followed by one week at 2 Hz. The intensity-trained tissues had higher maturation of contractile function. Nunes et al. [23] utilized two different protocols of ES (5 V/cm, 1 ms, monophasic square waveform) to hiPSC-CMs: the stimulation frequency was progressively and daily increased from 1 to 3 Hz (Low frequency ramp-up regimen) and from 1 to 6 Hz (High frequency ramp-up regimen). They found that compared with the low frequency ramp-up regimen, the high frequency ramp-up regimen within one week could further enhance the structure and electrophysiological function of the engineered myocardial tissue.
The biomimetic conductive microenvironment may affect the development of the heart by promoting electrical conduction between cardiomyocytes in vitro.
You et al. [24] found that when seeding neonatal rat cardiomyocytes on Au nanoparticles homogeneously synthesized throughout a polymer templated gel without ES, the expression level of Cx43 was significantly increased. Similarly, Wang et al. [25] found that flexible and conductive graphene sheets can promote the intrinsic electrical propagation by mimicking natural biomimetic conductive microenvironment without applying external ES, not only regenerating CMs in vitro, but also accelerating the maturation of functional CMs. These results highlight the possibility of conductive substrates affecting the development of cardiomyocytes.
Although studies have shown that ES can play a key role in the process of heart development and maturation, the mechanisms involved are not yet fully understood.
(1) Wu Chang-xue et al. [26] found that as the time of ES progressed, the expression of myocyte enhancer factor 2C gene was up-regulated, which in turn promoted the expression of troponin I and the formation of cardiomyocytes.
(2) Genovese et al. [27] found that the expression of follistatin (FST) was up-regulated in hMSCs after ES, and demonstrated that short-term ES promotes the differentiation of cardiomyocytes of hMSCs. In recent years, FST has been proven to be the key to muscle development, differentiation, and regeneration. It can promote cell proliferation and limit fibrogenesis by participating in the repair of mesodermal- and endodermal- tissues [28].
(1) Liang Li et al. [29] found that during ES, one of the mechanisms
for the cardiac differentiation of bone marrow mesenchymal stem cells (BMSCs) is
the upregulation of transforming growth factor-
(2) He et al. [15] applied ES to human cardiosphere-derived cells (hCDCs) and found that the differentiation of hCDCs is highly dependent on the synchronous transmission of electric current through the optimally aligned and elongated cardiomyocytes, which is called a “Syncytium”. When a pulsatile electric current with constant amplitude is applied, the aligned hCDCs is perpendicular to the direction of the current. The aligned cardiomyocytes start to act like a capacitor that is charged and discharged in response to the applied electric stimulation. This pulsatile property of cardiomyocytes facilitates the formation of synchronous myofiber contractions.
(3) Crestani et al. [30] found that the expression of cardiac
intercalated disk proteins (Nebulin-related-anchoring protein (Nrap) and
(4) Ma et al. [16] found that the possible mechanism of ES to promote
cardiac differentiation of hiPSCs is to activate the Ca
(5) Kanwal Haneef et al. [34] found that applying ES to stem cells on 3D collagen scaffolds can promote the expression of cardiac markers, and found that 3D collagen scaffolds have natural extra cellular matrix (ECM) capabilities, providing structural strength and resistance to deformation. ECM is mainly composed of collagen that gives structural strength to the left ventricle. The latest research [28] suggests, this may be due to the following reasons: Glycosaminoglycans (GAGs) are widely distributed in ECM. GAGs are rich in ionizable carboxyl and sulfate groups, which have the characteristics of charge transfer. GAGs act as an electrical conduit from the ECM to the cell during electro-stimulatory applications. At the same time, ES will increase the synthesis of proteoglycans, each of which contains a specific GAG side chain. In contrast, collagen tissue has piezoelectric properties; so that when the tissue is compressed, it generates an electric charge. It is caused by highly aligned fibrillar type I and II collagen packing and the cross-linked structures, mainly exists in bone and cartilaginous tissues [35], and the myocardium [36].
Under different conditions, different ES parameters may correspond to different differentiation of myocardial stem cells, such as atria, ventricles, conduction cells, etc. Chan et al. [37] found that biphasic pulse stimulation with electric field strength of 6.6 V/cm, pulse width of 2 ms, and frequency of 1 Hz is the best ES parameter for hiPSC-CMs. Hernández et al. [38] found that cardiac differentiation induced by ES is affected by the cell line used. Crestani et al. [30] found that hiPSCs were more likely to differentiate into cardiac conduction cell phenotype under ES, with the increase of connexin 40 (Cx40) expression and the reduction of Cx43 expression. The Irx3 gene plays a key role in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart. It can directly suppress Cx43 transcription and indirectly activate Cx40 transcription [39].
Areas of future research include determining the ES conditions corresponding to the model standards of different stem cells, in order to further improve our understanding of the mechanism of ES induced differentiation, and to employ tissue engineering for cardiac differentiation of stem cells by ES. This has the potential for the clinical application of ES to minimize damage after an MI, and reduce the incidence of chronic heart failure.
In conclusion, ES is one of the effective methods to solve the low differentiation rate and immaturity of cardiomyocytes derived from stem cells. In clinical practice, electrical stimulation programs should be individually tailored according to different stem cells to produce the best promotion effect. In addition, if other stimuli are applied at the same time as electrical stimulation, it may have a synergistic or antagonistic effect, such as mechanical stimulation, matrix, etc.
YD drafted the manuscript and participated in its design and so on. JM and FZ conceived of the study, and participated in its coordination and helped to draft the manuscript and so on. All authors read and approved the final manuscript.
Not applicable.
We would like to express our gratitude to all those who helped us during the writing of this manuscript. Thanks to all the peer reviewers for their opinions and suggestions.
This research was funded by 2018 National Natural Science Foundation, grant number 81870181.
The authors declare no conflict of interest.