Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host–graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair. Although hESC-CMs form gap junctions and beat synchronously in vitro, there is only indirect evidence for their electromechanical integration after transplantation. We do not know whether hESCCMgrafts contract synchronously at physiological human rates, integrate in injured hearts despite scar tissue, or affect electrical stability. Indeed, both pro-arrhythmic 9 and anti-arrhythmic effects have been reported for mouse cardiomyocyte grafts in injured mouse hearts. Human cardiac grafts could plausibly contribute to arrhythmogenesis through automaticity and triggered activity, and their irregular graft geometry could promote reentry. To address these uncertainties, we employed a new guinea-pig model of cardiac injury. Priorworkwith hESC-CMs in infarcted hearts was carried out in mice and rats, but these species’ rapid heart rates (approximately 600 and 400 beats per minute (b.p.m.), respectively) may prevent host–graft coupling or arrhythmias that could occur in humans. In vitro hESC-CMs show a spontaneous rate of approximately 50 to 150 b.p.m. and can be paced up to 240 b.p.m., suggesting that they can keep up with the guinea-pig heart (approximately 200 to 250 b.p.m.). We first examined the structural, mechanical and electrocardiographic consequences of hESC-CM transplantation in injured hearts of immunosuppressed guinea-pigs (Supplementary Fig. 1a). hESCCMs were derived from H7 hESCs, as previously described. Adult guinea-pigs were subjected to cardiac cryoinjury and implanted with telemetric electrocardiographic (ECG) transmitters. Ten days later, they underwent a repeat thoracotomy and intra-cardiac injection of either 1 3 10 hESC-CMs in a pro-survival cocktail (PSC) of factors previously shown to enhance hESC-CM engraftment (n 5 15), 1 3 10 non-cardiac hESC-derivatives in PSC (non-CMs; n 5 13), or PSC vehicle alone (n 5 14). hESC-CMs were 63% pure by anti-aactinin flow cytometry, whereas non-CMs included no detectable cardiomyocytes (Supplementary Fig. 2). Twenty-eight days after transplantation, all animals showed transmural scar and thinning of the left ventricle. Scar area was not different among the groups (13.2 6 0.9% of the left ventricle in hESC-CM, 14.8 6 1.4% in non-CM, and 15.3 6 1.9% in PSC-only recipients). However, hESC-CM recipients showed partial remuscularization with islands of human myocardium occupying 8.4 6 1.5% of the scar area (Fig. 1a). The human origin of these grafts was confirmed by in situ hybridization with a human pan-centromeric probe, and more than 99% of the human cells immunostained positively with the cardiac marker b-myosin heavy chain (bMHC; also known as MYH7) (Fig. 1b and Supplementary Fig. 3a–f). No teratomas developed, and hESC-CM grafts were negative for multiple non-cardiac markers (Supplementary Fig. 3g–k). Most of the graft myocardium was located in the central scar, but there were occasional points of host–graft contact in the border zone with shared intercalated discs identified by anti-connexin 43 and cadherin immunostaining (Fig. 1c–h and Supplementary Fig. 3d, e). Minimal immune reaction was observed in sections stained with a guinea-pig-specific pan-leukocyte marker (Supplementary Fig. 3l). Grafts were supplied by host-derived neovessels that contained erythrocytes, indicating perfusion by the host coronary circulation (Supplementary Fig. 3h, m–o). Surviving human cells were found in only 7 of 13non-CMrecipients at 28 days after transplantation, and these grafts were smaller than those in hESC-CM recipients (less than 1% of the scar area). No bMHC-positive graft was detected in non-CM recipients; instead, the surviving human cells consisted of small epithelial nests and scattered fibroblastic cells (Supplementary Fig. 4). Hearts receiving hESC-CMs, non-CMs and PSC only, were assessed by echocardiography on days 22 and 128 relative to cell transplantation. All groups showed increased left-ventricle dimensions and reduced fractional shortening on day22 relative to uninjured controls