Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

Cardiac adaptations from 4 weeks of intensity-controlled vigorous exercise are lost after a similar period of detraining.

Intensity-controlled (relative to VO2max) treadmill exercise training in adult rats results in the activation and ensuing differentiation of endogenous c-kit(pos) cardiac stem/progenitor cells (eCSCs) into newly formed cardiomyocytes and capillaries. Whether these training-induced adaptations persist following detraining is undetermined. Twelve male Wistar rats (~230 g) were exercised at 80-85% of their VO2max for 30 min day(-1), 4 days week(-1) for 4 weeks (TR; n = 6), followed by 4 weeks of detraining (DTR; n = 6). Twelve untrained rats acted as controls (CTRL). Exercise training significantly enhanced VO2max (11.34 mL kg(-1) min(-1)) and wet heart weight (29%) above CTRL (P < 0.05). Echocardiography revealed that exercise training increased LV mass (~32%), posterior and septal wall thickness (~15%), ejection fraction and fractional shortening (~10%) compared to CTRL (P < 0.05). Cardiomyocyte diameter (17.9 ± 0.1 µm vs. 14.9 ± 0.6 µm), newly formed (BrdU(pos)/Ki67(pos)) cardiomyocytes (7.2 ± 1.3%/1.9 ± 0.7% vs. 0.2 ± 0.1%/0.1 ± 0.1%), total cardiomyocyte number (45.6 ± 0.6 × 10(6) vs. 42.5 ± 0.4 × 10(6)), c-kit(pos) eCSC number (884 ± 112 per 10(6) cardiomyocytes vs. 482 ± 132 per 10(6) cardiomyocytes), and capillary density (4123 ± 227 per mm(2) vs. 2117 ± 118 per mm(2)) were significantly greater in the LV of trained animals (P < 0.05) than CTRL. Detraining removed the stimulus for c-kit(pos) eCSC activation (640 ± 98 per 10(6) cardiomyocytes) and resultant cardiomyocyte hyperplasia (0.4 ± 0.3% BrdU(pos)/0.2 ± 0.2% Ki67(pos) cardiomyocytes). Capillary density (3673 ± 374 per mm(2)) and total myocyte number (44.7 ± 0.5 × 10(6)) remained elevated following detraining, but cardiomyocyte hypertrophy (15.0 ± 0.4 µm) was lost, resulting in a reduction of anatomical (wall thickness ~4%; LV mass ~10% and cardiac mass ~8%, above CTRL) and functional (EF & FS ~2% above CTRL) parameters gained through exercise training. These findings demonstrate that cardiac adaptations, produced by 4 weeks of intensity-controlled exercise training are lost after a similar period of detraining.

Porcine skeletal muscle-derived multipotent PW1pos/Pax7neg interstitial cells: isolation, characterization, and long-term culture.

Developing effective strategies for the regeneration of solid tissue requires an understanding of the biology underlying the tissue's endogenous repair mechanisms. PW1/Peg3(pos)/Pax7(neg) skeletal muscle-derived interstitial progenitor cells (PICs) were first identified recently in the interstitium of murine skeletal muscle and shown to contribute to muscle fiber regeneration in vivo. PICs, therefore, represent a novel candidate resident progenitor cell for muscle regeneration. To explore the potential of these cells for clinical translation, we must ascertain the presence of PICs in larger mammalian species and identify criteria to successfully isolate and expand this population. In this study, we report the isolation, characterization, and maintenance of multipotent PICs from juvenile porcine skeletal muscle. We show that porcine PICs can be reproducibly isolated from skeletal muscle, express stem/progenitor cell markers, and have a stable phenotype and karyotype through multiple passages. Furthermore, porcine PICs are clonogenic and multipotent, giving rise to skeletal myoblast/myotubes, smooth muscle, and endothelial cells. In addition, PICs can be induced to differentiate into cardiomyocyte-like cells. These results demonstrate, in an animal model with size and physiology extrapolatable to the human, that porcine skeletal muscle-derived PW1(pos)/Pax7(neg) PICs are a source of stem/progenitor cells. These findings open new avenues for a variety of solid tissue engineering and regeneration using a single multipotent stem cell type isolated from an easily accessible source, such as skeletal muscle.

Carbonic anhydrase activation is associated with worsened pathological remodeling in human ischemic diabetic cardiomyopathy.

BACKGROUND: Diabetes mellitus (DM) has multifactorial detrimental effects on myocardial tissue. Recently, carbonic anhydrases (CAs) have been shown to play a major role in diabetic microangiopathy but their role in the diabetic cardiomyopathy is still unknown. METHODS AND RESULTS: We obtained left ventricular samples from patients with DM type 2 (DM-T2) and nondiabetic (NDM) patients with postinfarct heart failure who were undergoing surgical coronary revascularization. Myocardial levels of CA-I and CA-II were 6- and 11-fold higher, respectively, in DM-T2 versus NDM patients. Elevated CA-I expression was mainly localized in the cardiac interstitium and endothelial cells. CA-I induced by high glucose levels hampers endothelial cell permeability and determines endothelial cell apoptosis in vitro. Accordingly, capillary density was significantly lower in the DM-T2 myocardial samples (mean±SE=2152±146 versus 4545±211/mm(2)). On the other hand, CA-II was mainly upregulated in cardiomyocytes. The latter was associated with sodium-hydrogen exchanger-1 hyperphosphorylation, exaggerated myocyte hypertrophy (cross-sectional area 565±34 versus 412±27 µm(2)), and apoptotic death (830±54 versus 470±34 per 10(6) myocytes) in DM-T2 versus NDM patients. CA-II is activated by high glucose levels and directly induces cardiomyocyte hypertrophy and death in vitro, which are prevented by sodium-hydrogen exchanger-1 inhibition. CA-II was shown to be a direct target for repression by microRNA-23b, which was downregulated in myocardial samples from DM-T2 patients. MicroRNA-23b is regulated by p38 mitogen-activated protein kinase, and it modulates high-glucose CA-II-dependent effects on cardiomyocyte survival in vitro. CONCLUSIONS: Myocardial CA activation is significantly elevated in human diabetic ischemic cardiomyopathy. These data may open new avenues for targeted treatment of diabetic heart failure.

MicroRNA-1 downregulation increases connexin 43 displacement and induces ventricular tachyarrhythmias in rodent hypertrophic hearts.

Downregulation of the muscle-specific microRNA-1 (miR-1) mediates the induction of pathologic cardiac hypertrophy. Dysfunction of the gap junction protein connexin 43 (Cx43), an established miR-1 target, during cardiac hypertrophy leads to ventricular tachyarrhythmias (VT). However, it is still unknown whether miR-1 and Cx43 are interconnected in the pro-arrhythmic context of hypertrophy. Thus, in this study we investigated whether a reduction in the extent of cardiac hypertrophy could limit the pathological electrical remodeling of Cx43 and the onset of VT by modulating miR-1 levels. Wistar male rats underwent mechanical constriction of the ascending aorta to induce pathologic left ventricular hypertrophy (LVH) and afterwards were randomly assigned to receive 10mg/kg valsartan, VAL (LVH+VAL) delivered in the drinking water or placebo (LVH) for 12 weeks. Sham surgery was performed for control groups. Programmed ventricular stimulation reproducibly induced VT in LVH compared to LVH+VAL group. When compared to sham controls, rats from LVH group showed a significant decrease of miR-1 and an increase of Cx43 expression and its ERK1/2-dependent phosphorylation, which displaces Cx43 from the gap junction. Interestingly, VAL administration to rats with aortic banding significantly reduced cardiac hypertrophy and prevented miR-1 down-regulation and Cx43 up-regulation and phosphorylation. Gain- and loss-of-function experiments in neonatal cardiomyocytes (NCMs) in vitro confirmed that Cx43 is a direct target of miR-1. Accordingly, in vitro angiotensin II stimulation reduced miR-1 levels and increased Cx43 expression and phosphorylation compared to un-stimulated NCMs. Finally, in vivo miR-1 cardiac overexpression by an adenoviral vector intra-myocardial injection reduced Cx43 expression and phosphorylation in mice with isoproterenol-induced LVH. In conclusion, miR-1 regulates Cx43 expression and activity in hypertrophic cardiomyocytes in vitro and in vivo. Treatment of pressure overload-induced myocyte hypertrophy reduces the risk of life-threatening VT by normalizing miR-1 expression levels with the consequent stabilization of Cx43 expression and activity within the gap junction.