Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1–2% of total c-kitpos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC’s myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

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An overdose of Isoproterenol (ISO) causes acute cardiomyocyte (CM) dropout and activates the resident cardiac c-kit pos stem/progenitor cells (CSCs) generating a burst of new CM formation that replaces those lost to ISO. Recently, unsuccessful attempts to reproduce these findings using c-kit Cre knock-in (KI) mouse models were reported. We tested whether c-kit haploinsufficiency in c-kit Cre KI mice was the cause of the discrepant results in response to ISO. Male C57BL/6J wild-type (wt) mice and c-kit Cre KI mice were given a single dose of ISO (200 and/or 400 mg/Kg s.c.). CM formation was measured with different doses and duration of BrdU or EdU. We compared the myogenic and regenerative potential of the c-kit Cre CSCs with wtCSCs. Acute ISO overdose causes LV dysfunction with dose-dependent CM death by necrosis and apoptosis, whose intensity follows a basal-apical and epicardium to sub-endocardium gradient, with the most severe damage confined to the apical sub-endocardium. The damage triggers significant new CM formation mainly in the apical sub-endocardial layer. c-kit haploinsufficiency caused by c-kit Cre KIs severely affects CSCs myogenic potential. c-kit Cre KI mice post-ISO fail to respond with CSC activation and show reduced CM formation and suffer chronic cardiac dysfunction. Transplantation of wtCSCs rescued the defective regenerative cardiac phenotype of c-kit Cre KI mice. Furthermore, BAC-mediated transgenesis of a single c-kit gene copy normalized the functional diploid c-kit content of c-kit Cre KI CSCs and fully restored their regenerative competence. Overall, these data show that c-kit haploinsufficiency impairs the endogenous cardioregenerative response after injury affecting CSC activation and CM replacement. Repopulation of c-kit haploinsufficient myocardial tissue with wtCSCs as well c-kit gene deficit correction of haploinsufficient CSCs restores CM replacement and functional cardiac repair. Thus, adult neo-cardiomyogenesis depends on and requires a diploid level of c-kit.

BackgroundDiabetes 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 ResultsWe 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/mm2). 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 μm2), and apoptotic death (830±54 versus 470±34 per 106 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.ConclusionsMyocardial CA activation is significantly elevated in human diabetic ischemic cardiomyopathy. These data may open new avenues for targeted treatment of diabetic heart failure.