Ethical concerns about stem cell-based research have delayed important advances in many areas of medicine, including cardiology. The introduction of induced pluripotent stem cells (iPSCs) has supplanted the need to use human stem cells for most purposes, thus eliminating all ethical controversies. Since then, many new avenues have been opened in cardiology research, not only in approaches to tissue replacement but also in the design and testing of antiarrhythmic drugs. This methodology has advanced to the point where induced human cardiomyocyte cell lines can now also be obtained from commercial sources or tissue banks. Initial studies with readily available iPSCs have generally confirmed that their behavioral characteristics accurately predict the behavior of beating cardiomyocytes in vivo. As a result, iPSCs can provide new ways to study arrhythmias and heart disease in general, accelerating the development of new, more effective antiarrhythmic drugs, clinical diagnoses, and personalized medical care. The focus on producing cardiomyocytes that can be used to replace damaged heart tissue has somewhat diverted interest in a host of other applications. This manuscript is intended to provide non-specialists with a brief introduction and overview of the research carried out in the field of heart rhythm disorders.

Human induced pluripotent stem cells (iPSCs) are produced by reprogramming adult mesenchymal cells, most often fibroblasts. The process is complicated, requiring the introduction and activation of four gene regulatory networks, each comprised of molecular regulators that interact with each other and with other substances in the cell to control gene expression of mRNA and protein-specific genes. Acting together these four transcription factors can produce mature cells that behave in a completely different manner to the original fibroblasts used to form them, leading to the formation of multiple cell types[1-3].

Myocytes created from fibroblasts are basically identical to native cardiomyocytes. There are three different types of native cardiomyocytes, and iPSC production yields all three types in variable and unpredictable proportions, presenting difficulties for researchers. The most obvious and most publicized cardiological application of iPSCs is the production of new cardiac tissue to replace tissues destroyed by infarction or other diseases[4-9], but this goal has yet to be successfully realized in humans. Initially, this was partly because the subtype of iPSCs could not be assured. Obviously, atrial cardiomyocytes would not be a suitable substitute for damaged ventricular cardiomyocytes and, regardless, there is always the danger that introducing a mixture of cells might lead to teratoma formation[10]. Nonetheless, substantial advances have already been made, and success seems to be mainly a matter of time. Once these problems have been fully resolved, iPSCs, in various configurations, could be used to repair damaged hearts. They could also be used to predict interactions between drugs and the cardiac conduction system.

The occurrence of any specific conduction abnormality - including QT prolongation, altered action potential duration, triggered activity, the blockade of human-ether-a-go-go-related channel (hERG) and other ion conduction channels-and the occurrence of lethal arrhythmias-such as Torsades de Pointes (TdP)-cannot reliably be predicted with currently available screening methods (Langendorf preparations, patch-clamp, or even arterially perfused isolated rabbit left ventricular wedge)[11]. Animal models are problematic predictors of arrhythmia occurrence because of anatomic variations[12-16]. Such an issue poses a huge difficulty for drug makers trying to produce effective antiarrhythmic drugs; animal-to-human extrapolation is an uncertain process, which can pose a danger to patients if unrecognized differences emerge between animal and human models[17].

The problems associated with the use of amiodarone and sotalol illustrate the difficulties of drug development[18,19]. Both drugs are used to treat atrial and ventricular tachyarrhythmias and can be life-saving, but can also produce lethal side effects. Unfortunately, predicting side effects is, at this moment, impossible. Amiodarone’s most feared side effect is fatal pulmonary interstitial fibrosis, but hepatitis, hypothyroidism (probably irreversible) and mixed sensorimotor polyneuropathy have all been reported with some regularity. Amiodarone’s most important complications are QT prolongation and TdP[20]. If new and safer replacement drugs are ever to be developed and approved by the United States Food and Drug Administration and the European Medicines Agency, developers will first have to establish that new drugs do not produce predictable untoward side effects or exacerbate the conditions they were designed to treat.

The availability of iPSCs allows researchers to make reasonably accurate predictions about what effect any new drug will have on the heart and its electrical system. Cultured iPSCs, can be used to construct in vitro models of the human cardiac conduction system. The effects observed in vitro can then be used to predict how, and/or whether, a drug will alter electrical conduction, or produce structural alterations in humans. The process is not as simple as it sounds and some knowledge of the subject is crucial to clinicians for the safe use of new drugs.

Cures for cardiac conduction diseases will only be found when their root causes are fully elucidated. Even physicians who have nothing to do with arrhythmia research should retain some knowledge of the molecular biology that underlies cardiac conduction.

The cardiomyocyte repolarization/depolarization cycle begins with a current generated by the outward flow of potassium ions through specific pores or channels. Potassium pores exist in all life forms and many different types have been identified (more than 20). Two types of potassium channels are absolutely critical to the process of cardiac repolarization: The rapid delayed rectifier current (identified as IKr) and the slow delayed rectifier current (identified as IKs). If a drug or a mutation disrupts either of these two currents, the action potential of the cell is prolonged with an increase in the time required for electrical depolarization and repolarization of the ventricles[21]. Prolonged repolarization leads to the occurrence of early after depolarization (EAD) currents. EADs are dangerous because they favor the occurrence of triggered activity (defined as the occurrence of spontaneous action potentials occurring during phase 2 or phase 3 of repolarization, leading to the production of inappropriate action potentials and arrhythmia)[12]. Blockade of the IKr also causes the QT interval to be prolonged, leading to the triggered activity via a slightly different mechanism[22]. Such a situation is likely to occur when a drug molecule interferes with potassium channels as in the case of type III antiarrhythmic drugs. Slowing of the potassium current is associated with a repolarization dispersion, where one area of the myocardium recovers from depolarization faster than an adjoining region, which also makes TdP more likely to occur[23]. Repolarization dispersion is thought to be the reason that myocardial hypertrophy is associated with arrhythmias[24]. The farther the depolarization front has to travel, the greater the interval between depolarization and repolarization. Dispersion is especially likely to occur if the area of abnormal delay and dispersion is located within the Purkinje system or, alternatively, if the area is located in the mid-wall of the left ventricle where the “M cells” are located. These cells have prolonged action potentials that act to further increase the dispersion of repolarization, making the occurrence of TdP ever more likely[25]. For a new antiarrhythmic drug to be introduced, it must first be proven that it exerts none of the effects enumerated above.

Sudden cardiac death (SCD) due to ventricular tachycardia (VT) or ventricular fibrillation (VF) accounts for approximately half of all deaths in patients with heart failure (HF) and may be considered a heritable trait[26-32]. Current guidelines[33] recommend an implantable cardioverter-defibrillator (ICD) in patients with symptomatic and severe left ventricular dysfunction of any origin. However, SCD may occur in asymptomatic patients with only mild HF. On the contrary, as many as two-thirds of patients with severe HF implanted with an ICD do not experience device interventions over 3 to 5 years follow-up[34]. A similar clinical scenario leaves unanswered the question of whether selected gene variants may affect the risk of SCD in HF patients. Genomic science provides us with new approaches to identify gene variants or mutations that predispose patients with inherited electrical diseases to SCD. However, a growing body of evidence suggests that DNA changes in the same genes that convey risk in primary electrical diseases may enhance susceptibility to VT/VF even in a polygenic condition such as HF. Sustained VT and VF often occur as a consequence of delayed after-depolarizations triggered by diastolic sarcoplasmic reticulum (SR) calcium leak[35]. Genes encoding calcium handling proteins involved in electrical homeostasis of the failing heart may represent suitable candidates for defining individual susceptibility to life-threatening arrhythmia[26,27]. However, only very few genes belonging to the major candidate systems have been characterized and screened for possible association with SCD in HF. The cardiac ryanodine receptor 2 (RyR2), a calcium-releasing channel located in the SR membrane, plays a key role in the electrical homeostasis of cardiomyocytes. RyR2 dysfunction has been described in both HF patients and animal models and is critical to many of the aspects of the disease, including life-threatening arrhythmia[36]. In a large cohort of HF patients, Ran et al[37] found that the A allele of RYR2 c.5656G>A was associated with an increased risk of SCD. Arvanitis et al[38] reported that the Ser96Ala variant in histidine-rich calcium-binding protein was associated with ventricular arrhythmia in idiopathic dilated cardiomyopathy. It is known that a serine residue replacing glycine at position 1886 (G1886S or rs3766871) in the RyR2 gene prompts a significant increase in intracellular calcium oscillation and creates a site of phosphorylation for protein kinase C (PKC) entailing PKC-mediated calcium diastolic leak from the SR[39,40]. While the RYR2 rs3766871 variant has been previously described only in the setting of arrhythmogenic right ventricular cardiomyopathy, a role of RyR2 rs3766871 minor allele for increased susceptibility to VT/VF has been recently reported also in patients with HF[41]. The SERCA calcium ATPase (ATP2A2) belongs to a large family of P-type cation pumps that couple adenosine triphosphate (ATP) hydrolysis with cation transport across membranes[42]. Alternative splicing of the ATP2A2 gene produces two isoforms, SERCA2a (primarily located in the heart and slow-twitch skeletal muscle) and SERCA2b (present in smooth muscle and non-muscle tissues). Mutations in the ATP2A2 gene affect the expression level, ATP affinity, calcium affinity, and phosphorylation of ATP. In an attempt to investigate whether variants of the genes encoding major calcium handling proteins affect the occurrence of VT/VF in HF patients, it was found that the ATP2A2 c.2741+54G>A gene variant was associated with decreased susceptibility to life-threatening arrhythmia. Indeed, patients carrying the ATP2A2 c.2741+54A allele variant had an approximately 70% reduction in the relative risk of VT/VF during follow-up[43]. Defective calcium handling in failing cardiomyocytes has long been recognized as a cause of ventricular arrhythmia, and recent evidence suggests that selected calcium gene variants may modify the risk of SCD even in a complex and polygenic disease such as HF. While statistically associated with a modified risk of SCD, the biological role of many of these gene variants is presently unknown. The recent breakthrough discovery of iPSCs could enable the investigation of mutated cardiomyocytes generated from patient’s somatic cells, allowing functional characterization of iPSC-derived mutated cardiomyocytes. A similar approach represents an interesting and promising solution for the biological relevance of genetic substrates in secondary arrhythmogenic conditions.

Overcoming the ethical problems related to the use of stem cells through the introduction of iPSCs opens up an interesting scenario on the study of the cellular basis of diseases[61,62]. The use of pluripotent cells makes it possible to reproduce models for the study of cardiological pathologies which frequently cause SCD and are often diagnosed post-mortem such as structural cardiomyopathies and channelopathies[63-66].

Furthermore, iPSCs can be exploited in the personalization of therapies in relation to the possibility of carrying out pharmacological tests on cells derived from the patient[67-70].

Although the principles are easy to understand, at present there are some important caveats. One is that fibroblast generated iPSCs demonstrate an immature phenotype so that they more closely resemble mid-gestation human fetal hearts[71-73]. These differences may well alter final experimental and clinical results, depending on the stage of development of the iPSCs being used. When used in other fields, the same caveat applies. Now that this difference has been recognized, finding ways to make sure the cells are organized and function as adult cells is the object of intense research, which has already begun to generate results. Recent reports indicate that iPSCs can be stimulated and made to mature by a combination of pacing and increasing mechanical stress[74-77].

Another issue that had been delaying progress is that protocols used to produce iPSCs do not produce just one kind of cell, but rather yield a mixed population of cardiomyocyte subtypes including ventricular-, atrial- and pacemaker-like cells[78-81]. Birket and colleagues[82] made the early observation that even though the iPSCs can behave like normal human cardiomyocytes, the production process leads to unequal numbers of each of the subtypes. Obviously, different results will be generated depending on which type of cell predominates. Many laboratories are working on effective cell separation methods and standardized methods should soon be available.

In summary, the main applications of stem cells include disease modeling, cell diagnostics, and therapy personalization (Figure 1). Such tasks involve molecular profiling, the identification of biomarkers of the expression of the pathological phenotype, as well as the identification and testing of targeted therapies. The availability of pluripotent cardiac stem cells, especially networked beating cardiomyocytes, is likely to revolutionize our understanding of many cardiac rhythm disorders and diseases, provide a rational testing method for the development of drugs, permit clinicians to assess effectiveness before drug administration and, most importantly, save lives.

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Figure 1 Induced pluripotent stem cells can provide new ways to study arrhythmias and heart disease in general, accelerating the development of new, more effective antiarrhythmic drugs, clinical diagnoses, and personalized medical care. iPSCs: Induced pluripotent stem cells. Figure created with BioRender (https://biorender.com).