Embryonic stem cell therapy of heart failure in genetic cardiomyopathy

Abstract

Pathogenic causes underlying nonischemic cardiomyopathies are increasingly being resolved, yet repair therapies for these commonly heritable forms of heart failure are lacking. A case in point is human dilated cardiomyopathy 10 (CMD10; Online Mendelian Inheritance in Man #608569), a progressive organ dysfunction syndrome refractory to conventional therapies and linked to mutations in cardiac ATP-sensitive K(+) (K(ATP)) channel subunits. Embryonic stem cell therapy demonstrates benefit in ischemic heart disease, but the reparative capacity of this allogeneic regenerative cell source has not been tested in inherited cardiomyopathy. Here, in a Kir6.2-knockout model lacking functional K(ATP) channels, we recapitulated under the imposed stress of pressure overload the gene-environment substrate of CMD10. Salient features of the human malignant heart failure phenotype were reproduced, including compromised contractility, ventricular dilatation, and poor survival. Embryonic stem cells were delivered through the epicardial route into the left ventricular wall of cardiomyopathic stressed Kir6.2-null mutants. At 1 month of therapy, transplantation of 200,000 cells per heart achieved teratoma-free reversal of systolic dysfunction and electrical synchronization and halted maladaptive remodeling, thereby preventing end-stage organ failure. Tracked using the lacZ reporter transgene, stem cells engrafted into host heart. Beyond formation of cardiac tissue positive for Kir6.2, transplantation induced cell cycle activation and halved fibrotic zones, normalizing sarcomeric and gap junction organization within remuscularized hearts. Improved systemic function induced by stem cell therapy translated into increased stamina, absence of anasarca, and benefit to overall survivorship. Embryonic stem cells thus achieve functional repair in nonischemic genetic cardiomyopathy, expanding indications to the therapy of heritable heart failure. Disclosure of potential conflicts of interest is found at the end of this article.

Figure 1

K_ATP channel knockout model recapitulates human cardiomyopathy. K_ATP channel Kir6.2-null mutants prior to (prestress) (A) and following transverse aortic constriction (stressed) (B). Under hemodynamic overload, stressed Kir6.2-null mutants displayed signs of biventricular failure and anasarca ([B], acute) with reduced ambulation and cachexia ([B], chronic). Predisposition to ventricular tachyar-rhythmia was demonstrated by telemetry either spontaneously (acute) or under isoproterenol challenge (chronic) (C), and left ventricular dilatation with compromised contractility was documented by echocardiography (D). In (D), dotted line, LVDd; solid line, LVDs; velocity, peak velocity at the left ventricular outflow tract. Continuous stress induced a significant increase in left ventricular mass ([E], inset), with survivorship curves demonstrating a poor survival of the stressed cohort (E). On electron microscopy (F, G), CB were present in cardiomyopathic stressed myocardium displaying sarcomeric disarray (G). From the initial cohort of 98 Kir6.2-null mutants, following 2 weeks of stress, 24 surviving cardiomyopathic mutants were randomized 2:1, in ES(−) or ES(+) treatment groups (H). Numbers of animals per group (n) are indicated in (E, H). Abbreviations: CB, contraction band; ES, embryonic stem cell; LVDd, left ventricular end-diastolic dimension; LVDs, left ventricular end-systolic dimension; ms, millisecond.

Figure 2

Safety profile of ES transplantation. (A): Wild-type ES were engineered to express the lacZ reporter transgene visualized by optical analysis. (B): Cardiomyopathic Kir6.2-knockout mice treated with 200,000 ES per heart (ES(+); unless otherwise indicated) had no arrhythmic events on four-limb electrocardiography throughout the follow-up period. (C): Uncontrolled growth was not detectable at this dose on echocardiography (top left) or macroscopic (middle left and bottom left) evaluation. (D): Microscopy confirmed the absence of multiorgan seeding in heart, lung, liver, and spleen following treatment with 200,000 ES per heart. Treatment with 3,000,000 ES, however, regularly demonstrated teratoma formation ([C], right panels) containing multiple lineages (E). Scale bars = 5 mm. Abbreviations: ES, embryonic stem cell; LV, left ventricle; ms, millisecond; wks, weeks.

Figure 3

Benefit of ES therapy on cardiac function. Serial echocardiography (A–E) demonstrated significant improvement in systolic contractile function in ES-treated (ES(+)) compared with untreated (ES(−)) cardiomyopathic Kir6.2-null mutants. Fractional shortening in M-mode (A, B), velocity of circumferential shortening (C), and LV ejection fraction (D, E) all demonstrated significant improvement following cell therapy. In (A), dotted line, LVDd; solid line, LVDs. (F–H): Ventricular conduction intervals in ES-treated cardiomyopathic hearts were shorter than those in untreated ones. Data are presented as means ± SEM (B, G, H) or medians with 95% confidence intervals (C, E). †, p < .05 versus Pre (0 weeks); *, p < .05 versus ES(−). Abbreviations: ES, embryonic stem cell; LV, left ventricular; LVDd, left ventricular end-diastolic dimension; LVDs, left ventricular end-systolic dimension; ms, millisecond; Pre, prestress; s, second; wks, weeks.

Figure 4

Stem cell intervention prevents maladaptive remodeling and cardiomegaly in genetic cardiomyopathy. Untreated (ES(−)) Kir6.2-knockout cardiomyopathic hearts were larger (A, B) and heavier on echocardiographic (C) and post-mortem (D, E) examination compared with ES-treated (ES(+)) counterparts. LV (C) and heart (D) weights were adjusted to BW (at day 0 of transverse aortic constriction). In (F), LV weight was plotted as a function of relative wall thickness, calculated as the sum of interventricular septum thickness and posterior wall thickness divided by LVDd. The evolution of heart geometry was monitored up to 6 wks following initiation of stress load imposed following randomization at 2 wks (closed circle) into treated (ES(+); red) and untreated (ES(−); black) groups. Data points represent an average of 5–24 Kir6.2-knockout mice. (G, H): Pulmonary congestion was reduced following cell therapy. †, p < .05 versus Pre; *, p < .05 versus ES(−). Scale bars = 5 mm. Abbreviations: BW, body weight; ES, embryonic stem cell; LA, left atrium; LL, left lobe; LV, left ventricular; LVDd, left ventricular end-diastolic dimension; LVVd, left ventricular end-diastolic volume; Pre, prestress; RL, right lobe; RV, right ventricle; wks, weeks.

Figure 5

Engraftment of transplanted stem cells. K_ATP channel-deficient cardiomyopathic hearts treated with ES, engineered to express the lacZ reporter transgene (ES(+)) demonstrated β-gal ([A], pink) nuclear expression with cellular coexpression of actinin ([A], green), along with Kir6.2 expression ([B], red) within otherwise K_ATP channel-deficient cardiomyocytes. Treated hearts had a significant increase in cells positive for Ki67 ([C, D], pink), a cellular marker for proliferation, and a decrease in interstitial fibrosis ([E]; [F], blue), compared with untreated hearts (ES(−)). In (A–C), blue indicates DAPI. *, p < .05 versus ES(−). Abbreviations: actinin, sarcomeric α-actinin; DAPI, 4′,6′-diamidino-2-phenylindole; ES, embryonic stem cell; β-gal, β-galactosidase.

Figure 6

Ultrastructure in untreated versus treated cardiomyopathic hearts. Untreated Kir6.2-knockout cardiomyopathic hearts (ES(−)) displayed significant fibrosis and CBs (A), disorganized Mito (C), and a paucity of tight junctions (E). (B, D, F): In contrast, stem cell-treated hearts (ES(+)) demonstrated proper sarcomeric ultrastructure (B), dense mitochondrial clusters (D), and desmosome with tight junction formation ([F], arrowheads and arrows). Abbreviations: CB, contraction band; ES, embryonic stem cell; Mito, mitochondria.

Figure 7

ES therapy produces favorable systemic outcome on morbidity and mortality. Exercise running capacity and workload evaluated by treadmill (A, B), overall fitness (C), and mortality (D) were all favorably affected by a 1-month-long treatment with ES (ES(+)) in K_ATP channel-null mutants mice despite the stress of continuous transverse aortic constriction. †, p < .05 versus Pre; *, p < .05 versus ES(−). Abbreviations: ES, embryonic stem cell; min, minutes; Pre, prestress.