Biological Modification of Arrhythmogenic Substrates by Cell-Free Therapeutics

Abstract

Ventricular arrhythmias (VAs) represent a major cause of sudden cardiac death and afflict patients with heart failure from both ischaemic and non-ischaemic origins, and inherited cardiomyopathies. Current VA management, including anti-arrhythmic medications, autonomic modulation, implantable cardioverter–defibrillator implantation, and catheter ablation, remains suboptimal. Catheter ablation may even cause significant cardiomyocyte loss. Cell-based therapies and exosome treatment have been proposed as promising strategies to lessen cardiomyocyte death, modulate immune reaction, and reduce myocardial scarring, and, therefore, are potentially beneficial in treating VAs. In this review, we summarise the current cornerstones of VA management. We also discuss recent advances and ongoing evidence regarding cell-based and exosome therapy, with special attention to VA treatment.

Introduction

Ventricular arrhythmias (VAs) continue to be a major contributor to cardiac morbidity and mortality worldwide, despite early coronary interventions and continuous progress in pharmacological therapies for heart failure [1]. Myocardial ischaemic insults [2,3], genetic defects [4,5], and acute/chronic inflammation [6,7] can individually or jointly contribute to arrhythmogenic substrates, facilitating VAs. Current management strategies for VAs include anti-arrhythmic medications, autonomic modulation, implantable cardioverter–defibrillator (ICD) implantation, cardiac stereotactic body radiotherapy (SBRT), and catheter ablation [1,8,9]. Although ICD placement is dramatically effective for terminating VAs and improving survival, ICD shocks can damage myocardial cells and elicit sympathetic responses [10].

Multiple shocks ensue in patients with frequent or incessant VAs, leading to electrical storms and myocardial stunning [10,11]. Superior to the escalation of anti-arrhythmic agents, substrate-based catheter ablation is associated with a higher rate of VA suppression [12,13]; however, it causes some collateral viable myocardium loss [14,15]. The resulting myocardial damage may be pro-arrhythmic, which could contribute to subsequent VAs and worsening cardiac function [15]. Notably, the arrhythmogenic substrates in certain cardiomyopathies remain active and evolve over time. Even though a single ablation procedure is initially effective, patients still encounter recurrent VA events [16,17]. Repeated ablation or “destruction” of heart tissue diminishes more “contractile units” and can exacerbate heart failure [15,16,18]. Alternative approaches to modify VA substrates without further affecting myocardial viability are clearly desired.

Cell-based and cell-derived therapies have been proposed as promising strategies to lessen cardiomyocyte death, modulate immune reaction, and reduce myocardial scarring, leading to improved cardiac function [19–21]. These salutary effects of cells and exosomes merit consideration as potential therapeutic strategies for VAs. Therefore, in this review, we discuss the conventional VA management and current approaches to treating VAs with cell-based and cell-derived therapies. We further discuss the potential mechanisms, including immune modulation and anti-fibrotic effects.

Current Cornerstones of VA Management

The aetiology and pathophysiological mechanisms of VA are complex, thus making the current VA therapeutic effects inconsistent for some patients. In addition to patient- and disease-related factors, beta-blockers and other anti-arrhythmic drugs have limited effectiveness and can sometimes cause undesired adverse drug effects [8]. Many of these drugs may even lead to unintended pro-arrhythmic consequences [8,22]. In patients with refractory VAs, autonomic modulation, such as stellate ganglion block or surgical sympathetic denervation, could reduce VA burden, but temporarily, and thus are usually used for bridging to advanced therapeutic interventions [23]. Ironically, ICDs are dramatically effective for terminating VAs and prolonging survival, yet they do not prevent VA recurrence [10]. VAs recurrence is associated with increased mortality and heart failure hospitalisations in ICD patients, despite effective termination of the arrhythmias [24]. Life-saving shocks themselves have been shown to be independently associated with myocardial tissue damage and an increased risk of heart failure exacerbation, recurrent VAs, and mortality [25,26]. This highlights the importance of blocking VA development rather than terminating VAs. Thus, targeting arrhythmogenic substrates with catheter ablation has emerged as an effective mechanistic-based approach.

Advances in three-dimensional electroanatomical mapping systems (3D EAM) have greatly facilitated catheter ablation of complex VAs. The multicentre randomised PARTITA trial recently demonstrated that early catheter ablation after the first ICD shock diminished the composite endpoint of worsening heart failure hospitalisations or death compared with standard therapy [27]. Common catheter ablation strategies include scar de-channelling, core isolation, endocardial/epicardial scar homogenisation, and eradication of late potentials or local abnormal activity [28]. In some instances, simultaneous endocardial and epicardial delineation technique addresses arrhythmogenic tissue harbored in the mid-myocardium and subepicardium and enhances the precision of ablation therapy [29]. Although catheter ablation effectively destroys the myocardial tissue underlying critical arrhythmia circuits, patients may lose more heart tissue and have extensive dense scarring at the same time (Figure 1). Electrical isolation of arrhythmogenic substrates results in regional non-contractile myocardium [30], which dampens ventricular systolic and diastolic functions. Another therapeutic strategy that has gained attention is the SBRT. Although it was first developed to treat solid tumours, SBRT has shown promising results by ablating arrhythmogenic sites with highly precise action [9]. Similar to radiofrequency ablation, radioablation induces double-strand deoxyribonucleic acid (DNA) breaks and programmed cell death, which leads to late myocardial fibrosis and electrical inert tissue [31]. In many patients with VA, the arrhythmogenic substrates remain active and evolve over time [16].

Figure 1.

Summary of the effects of catheter ablation and exosome-based therapies on the ventricular arrhythmogenic substrate.

In an arrhythmogenic cardiomyopathy (ACM) cohort, 3D EAM showed progressive scarring and right ventricular dilation during each VA recurrence [16]. Despite aggressive catheter ablation with simultaneous endocardial and epicardial approach, recurrent VAs were still observed in more than a quarter of patients [17]. Sixty (60%) to 100% of patients who underwent cardiac SBRT also suffered from recurrent VAs. Inhomogeneity of radioablation effects on targeted cardiac tissue created new diseased paths, thus altering arrhythmic circuits and limiting therapeutic efficacy [32,33]. An alternative therapeutic concept for healing/repairing arrhythmogenic myocardial tissue has been proposed to alleviate the VA burden to compensate for the loss of more cardiac fibres with each catheter ablation.

Cell-Based Therapy in VA Management: Anti-Arrhythmic or Pro-Arrhythmic?

The delivery of different cell types, which has been shown to halt abnormal cardiac remodelling and restore ventricular contractility, is considered beneficial in the management of VAs. By direct remuscularisation [34,35], indirect remuscularisation [36,37], or non-myogenic paracrine effects [38,39], depending on the type of cells delivered, cell-based cardiac repair seeks to modify arrhythmogenic substrates and thus reduce arrhythmogenicity.

A pioneering phase I trial using autologous skeletal muscle myoblast transplantation in severe ischaemic cardiomyopathy patients reduced the New York Heart Association (NYHA) score and improved systolic function [40]. However, 44% of the patients presented episodes of sustained ventricular tachycardia. Although it elicited arrhythmias, this study showed the feasibility of using cell therapy in patients and opened the possibilities to improve it.

Pluripotent stem cells (PSC), including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), are believed to differentiate into de novo cardiomyocytes (CMs) [41], and are therefore able to repair cardiac conduction and eliminate re-entry circuits. Several studies have shown that human (h)ESC-CM grafts transplanted into injured hearts contracted synchronously with host muscle and demonstrated 1:1 host-graft electromechanical coupling [42]. Interestingly, although injured hearts treated with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced VAs, the most concerning obstacle to achieving the clinical translation of the PSC-CM is still arrhythmogenicity [43].

VAs have also been identified post-PSC transplantation in many large animal studies, which unequivocally refute the anti-arrhythmic effects of PSC-CM [43]. Electrophysiological studies performed in large animals (swine and non-human primates) with sustained engraftment have shown increased ventricular arrhythmias at the site of engraftment [34,44,45]. A recent computer simulation study of PSC-CM patches in the human post-myocardial infarction model showed arrhythmogenicity of remuscularisation, which was closely linked to re-entry mechanism [46]. Nonetheless, preclinical studies have suggested that automaticity is the main cause of these VAs [45,47]. Addressing the cellular heterogeneity of current differentiation protocols, improving maturity and gap junctions of engrafted cardiomyocytes, conducting dose escalation experiments, and optimising pharmacological therapy are all paramount in balancing pro-arrhythmic and anti-arrhythmic effects after cell transplantation [43]. To date, barriers remain to cell-based therapies targeting remuscularisation for treating VAs, and an effective solution is yet to be defined.

Transplantation of non-PSC cells, including bone-marrow-derived stem cells (BMSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and cardiosphere-derived cells (CDCs), can exert its salutary effects through non-myogenic paracrine and indirect remuscularisation mechanisms, without raising the concern of developing VAs [41]. Specifically, transplanted cells secrete exosomes that contain bioactive factors promoting angiogenesis, preventing fibrosis, and apoptosis, which results in reduced CM loss and increased native CM proliferation [48]. Furthermore, in a rodent model of heart failure with preserved ejection fraction, CDC therapy decreased VAs by shortening action potential duration (APD), improving APD homogeneity, and decreasing fibrosis [49], suggesting that other non-myogenic mechanisms are beneficial in VA treatment, such as anti-fibrosis and anti-inflammation (Figure 1). The effects of CDC-secreted exosomes on VAs will be further discussed in the following section.

Role of Inflammation and Fibrosis in VA Substrates

Cardiomyocyte death, hyperinflammation, and fibrosis embrace a vicious triangle that underlies most cardiomyopathies, directly or indirectly, responsible for forming arrhythmogenic substrates [50]. Inflammation can exacerbate cell loss and increase cardiac fibrosis, which is associated with altered cardiac electrical properties, namely conduction slowing or block, favouring re-entrant tachyarrhythmias [51]. Specifically, inflammatory cytokines (interleukin [IL]-17, tumour necrosis factor-α, and IL-6) have been suggested to cause gap junction dislocation and downregulation [52,53]. IL-1β decreases inward potassium current, prolongs repolarisation, and increases diastolic SR Ca²⁺ leak, making cardiomyocytes susceptible to early and late afterdepolarisation [54]. Moreover, infiltrating macrophages can couple to cardiomyocytes via gap junctions and change APD [55]. The increased APD heterogeneity thus serves as a non-fibrotic, “functional” arrhythmogenic substrate. Consistent with this notion, a recent study compared the secretome of human MSCs from failing and nonfailing hearts. MSCs from heart failure patients prolonged APD, increased Ca²⁺ alternans, and promoted spontaneous calcium release activity [56]. Failing MSCs exhibited increased secretion of IL-1β and IL-6, and anti-cytokine therapies rescued the arrhythmia substrates [56]. Together, inflammation and fibrosis are also critical in arrhythmogenesis. This evidence highlights the potential of immune modulation and antifibrosis therapies to pave new avenues for VA management.

Towards an Exosome Therapy for VAs

Current literature has shown that cell-based therapy stimulates cardiac regeneration largely via indirect non-myogenic paracrine effects, but not through direct remuscularisation [21,48]. Among the biological products secreted by transplanted cells, the role of exosomes is still considered the most important. Exosomes are small extracellular vesicles that contain a variety of bioactive molecules, including nucleic acid, proteins, lipids, and some metabolites [21,57]. Exosomes secreted by progenitor and stromal cells deliver a “regenerative cargo” to the damaged myocardium and exert anti-apoptotic, anti-inflammatory, and anti-fibrotic effects [57,58]. The administration of CDC-derived exosomes has been shown to recapitulate the regenerative and functional effects produced by CDC transplantation; however, inhibition of exosome production by CDCs blocks these benefits [48]. In an acute myocardial infarction model, exosome injection into the border zone improves cardiac function and decreases the level of proinflammatory cytokines. Reduction in scar mass and increased viable myocardium was seen after exosome therapy [48]. The favourable biological effects suggest that exosome therapy can potentially modify the microenvironment and remodel arrhythmogenic substrates with a consequent reduction in arrhythmias. Pro-arrhythmic effects after exosome therapies remain an important concern but have not been shown in animal studies [48,59,60].

Exosomes Attenuate ACM

ACM is a hereditary disease characterised by progressive myocyte loss, hyperinflammation, and fibrofatty replacement, which disposes to VAs and sudden cardiac death [50,61]. In a mouse ACM model, weekly injection of exosomes secreted by immortalised CDCs with high β-catenin expression prevented abnormal biventricular remodelling. It is noteworthy that ambulatory telemetry and programmed electrical stimulation showed that spontaneous and inducible VA were reduced after exosomes treatment (Figure 2) [59]. Ex vivo electrophysiology studies using high-resolution optical mapping showed that exosomes restore cardiac conduction, shorten APD and reduce APD dispersion. Immunohistochemistry and transcriptome analyses of the exosome-treated hearts exhibited decreased cell death, tempered inflammation, and reduced fibrosis. Exosomes from engineered CDCs contain a distinct content of microRNAs, with enrichment of miR-4488. Antagonising miR-4488 caused increased inflammation and partially reversed the salutary properties of exosomes, including the anti-arrhythmic effects [59]. Together, this study demonstrated that inflammation plays a vital role in ACM arrhythmogenic substrate, while exosomes can modulate the inflammation, thus alleviating the VA burden. This knowledge may be valuable to other inherited diseases with inflammation as the main driver for arrhythmogenesis. As far as we know, there is no report of exosome-based therapy use for other genetic conditions, which brings the need for more studies in the field.

Figure 2.

Effects of exosome-based therapy on arrhythmogenic cardiomyopathy. (A) Quantification of ventricular arrhythmias over twenty-four hours. (B) Incidence of ventricular tachycardia and fibrillation induced by programmed electrical stimulation. (C) Representative gross pathology (top) and Masson’s trichrome-stained micrographs. (D) Representative images from immunohistochemical staining for nuclear factor-kB and α-sarcomeric actinin (ASA) and quantification of nuclei positive for nuclear factor-kB.

Reproduced with permission from (Lin et al., 2021) [59]. Copyright © 2023, Oxford University Press. Abbreviations: VAs, ventricular arrhythmias; VT, ; VF, ; Dsg2, ; WT, ; Veh, ; EV, ; DSG2; MT, ; RV, LV,

Exosomes Suppress VAs in Ischaemic Cardiomyopathy

Ischaemic heart disease is still the main cardiovascular disease and the primary cause of death in the USA [62]. Most of these deaths are due to sudden cardiac death [63], which is attributed to lethal ventricular arrhythmias [64]. The potential therapeutic effects of exosomes have been tested in a rodent model of acute myocardial infarction [65]. For this purpose, iPSC-derived exosomes were packaged into hydrogel patches and implanted after infarct induction. The treatment with exosomes led to reduced cardiac dilation and improved function two weeks after infarction. Also, hearts treated with exosome-based therapy showed reduced infarct size, apoptosis, hypertrophy, and VA. Interestingly, in contrast to what is observed after iPSC-CM implantation, this cell-free approach was not arrhythmogenic [65].

Exosome-based therapy has also been tested in a porcine model of chronic ischaemic cardiomyopathy and has shown anti-arrhythmic benefits (Figure 3) [60]. In this study, 3D EAM was performed at baseline and after exosome injection, and arrhythmogenic substrates were characterised, identifying areas with abundant isolated late potentials (a surrogate of delayed conduction). Exosome treatment reduced the amount of fibrosis, thus altering wavefront propagation, resulting in lack of sustained reentry [60]. Moreover, animals that received exosomes and were initially susceptible to inducible arrhythmias became non-inducible, whereby the arrhythmogenic state of the control animals remained the same at the endpoint [60]. Proteomic analysis of exosome-treated myocardial tissue showed major pattern differences in pathways related to cell proliferation, inflammation, and fibrosis. Sequencing the RNA content of exosomes identified several miRs involved in fibrosis regulation and immunomodulation [60]. In summary, exosomes derived from CDCs not only promote cardiomyocyte proliferation, but also antagonise inflammation and fibrosis. Such changes could synergistically decrease the arrhythmogenic substrate.

Figure 3.

Effects of exosome-based therapy on ischemia cardiomyopathy. (A) Representative traces of programmed electrical stimulation in a porcine model of myocardial infarction. (B) Representative isochronal maps of areas of late activation in myocardial infarction. (C) Picrosirius red-stained sections of the infarct zone of infarcted pigs.

Reproduced with permission from (Dawkins et al., 2022) [60]. Copyright © 2023, Oxford University Press.

Abbreviations: CDC_EXO,.

Conclusion

Despite decades of effort, the management of VA remains suboptimal and challenging. The current scenario shows that substrate modification by catheter ablation achieves superior outcomes compared to medical management alone, but VA recurrence rates are still high, and the ablation lesions may beget new arrhythmogenic substrates. In fact, most VA critical circuits are not mappable in haemodynamically unstable patients after induction of VAs. Successful ablation is usually achieved by extensive ablation at the expense of massive cardiomyocyte loss. Rather than destroying viable myocardium, a regenerative and anti-fibrotic approach provides an alternative for VA management. Moving from cell-based therapy to cell-derived therapies, growing evidence has shown the potential of exosomes for modifying arrhythmogenic substrates and exerting anti-arrhythmic effects in animal models of disease. From a future perspective, we believe substrate modifications with catheter ablation, SBRT, and cell-derived therapies are complementary. Cell-derived therapies could benefit patients with substrates and the risk of VAs, while those with incessant or sustained VAs could receive ablation or radiotherapy to eliminate culprit circuits (Table 1). Although additional studies are needed to assess the long-term efficacy and safety of exosomes, including direct comparison with ablation and radiotherapy prior to translating this approach to humans, “cell-free” exosomes-based therapies may open new avenues for the management of arrhythmias.

Table 1.

Comparisons of current and novel methods of arrhythmia substrate modification.

	Catheter Ablation	SBRT	Cell-Derived Therapy
Mechanism of action	Thermal injury of the arrhythmia substrates with resisted heat and conduction heat	Photon damaging the DNA of myocardial cells and cause subsequent mitotic catastrophe	Reduce inflammation and fibrosis and induce indirect remuscularisation within the arrhythmia substrates
Response to therapy	Immediate	In days to weeks	In weeks
Advantages	Modify the arrhythmia substrates directly and effectively	Target the arrhythmia substrates regardless of the vascular access, cardiac anatomy	Repair the arrhythmia substrates in a regeneration way without causing cardiomyocyte loss
Role of clinical application	Patients with incessant or sustained VAs	Patients with incessant or sustained VAs	Patients with substrate and risks of VAs

Abbreviations: SBRT, cardiac stereotactic body radiotherapy; DNA, deoxyribonucleic acid; VAs, ventricular arrhythmias.