The degree to which cKit-expressing progenitors generate cardiomyocytes in the heart is controversial. Genetic fate-mapping studies suggest minimal contribution; however, whether or not minimal contribution reflects minimal cardiomyogenic capacity is unclear because the embryonic origin and role in cardiogenesis of these progenitors remain elusive. Using high-resolution genetic fate-mapping approaches with cKit CreERT2/+ -induced pluripotent stem cells, we show that, paradoxically, the cardiogenic fate of CNC kit is regulated by bone morphogenetic protein antagonism, a signaling pathway activated transiently during establishment of the cardiac crescent, and extinguished from the heart before CNC invasion. Together, these findings elucidate the origin of cKit + cardiac progenitors and suggest that a nonpermissive cardiac milieu, rather than minimal cardiomyogenic capacity, controls the degree of CNC kit contribution to myocardium.

Whether the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis exerts cardioprotective effects remains controversial; and the underlying mechanism(s) for such actions are unclear.Here we tested the hypothesis that growth hormone-releasing hormone (GHRH) directly activates cellular reparative mechanisms within the injured heart, in a GH/IGF-1 independent fashion. After experimental myocardial infarction (MI), rats were randomly assigned to receive, during a 4-week period, either placebo (n = 14), rat recombinant GH (n = 8) or JI-38 (n = 8; 50 µg/kg per day), a potent GHRH agonist. JI-38 did not elevate serum levels of GH or IGF-1, but it markedly attenuated the degree of cardiac functional decline and remodeling after injury. In contrast, GH administration markedly elevated body weight, heart weight, and circulating GH and IGF-1, but it did not offset the decline in cardiac structure and function. Whereas both JI-38 and GH augmented levels of cardiac precursor cell proliferation, only JI-38 increased antiapoptotic gene expression. The receptor for GHRH was detectable on myocytes, supporting direct activation of cardiac signal transduction. Collectively, these findings demonstrate that within the heart, GHRH agonists can activate cardiac repair after MI, suggesting the existence of a potential signaling pathway based on GHRH in the heart. The phenotypic profile of the response to a potent GHRH agonist has therapeutic implications.cardiac stem cells | apoptosis | remodeling | heart failure C ongestive heart failure remains a leading cause of morbidity and mortality in developed countries. Despite major therapeutic advances, current therapies fail to fully reverse heart failure and/or left ventricular (LV) dysfunction. One major therapeutic avenue is that of cytokine and/or hormonal signaling pathways, and in this regard, various experimental and clinical studies have suggested an important role for the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis in the regulation of cardiac growth and function (1, 2). Moreover, several clinical studies have tested the impact of GH replacement on the failing human heart, with controversial results (3, 4).In addition to GH itself and IGF-1, GH-releasing peptides such as ghrelin and synthetic GH secretagogues (GHS) are also suggested to have cardiac effects (5-8), and growth hormone-releasing hormone (GHRH) mRNA is detected in peripheral tissues, including the heart (9, 10), consistent with widespread biologic signaling potential beyond the hypothalamic-pituitary axis.Recently, Granata et al. (10) reported that rat GHRH (1-44) promoted survival of cardiomyocytes in vitro and protected rat hearts from ischemia-reperfusion injury. The detection of the GHRH receptor (GHRHR) on the cardiomyocyte sarcolemmal membrane supports the view that GHRH may elicit direct signal transduction within the heart, independent of the GH/IGF-1 axis per se (10). Ghrelin and other GHS may have pharmacologic potential (10) but also have pleiotropic actions with a high possibility...

Both cardiac myocytes and cardiac stem cells (CSCs) express the receptor of growth hormone releasing hormone (GHRH), activation of which improves injury responses after myocardial infarction (MI). Here we show that a GHRH-agonist (GHRH-A; JI-38) reverses ventricular remodeling and enhances functional recovery in the setting of chronic MI. This response is mediated entirely by activation of GHRH receptor (GHRHR), as demonstrated by the use of a highly selective GHRH antagonist (MIA-602). One month after MI, animals were randomly assigned to receive: placebo, GHRH-A (JI-38), rat recombinant GH, MIA-602, or a combination of GHRH-A and MIA-602, for a 4-wk period. We assessed cardiac performance and hemodynamics by using echocardiography and micromanometry derived pressure-volume loops. Morphometric measurements were carried out to determine MI size and capillary density, and the expression of GHRHR was assessed by immunofluorescence and quantitative RT-PCR. GHRH-A markedly improved cardiac function as shown by echocardiographic and hemodynamic parameters. MI size was substantially reduced, whereas myocyte and nonmyocyte mitosis was markedly increased by GHRH-A. These effects occurred without increases in circulating levels of growth hormone and insulin-like growth factor I and were, at least partially, nullified by GHRH antagonism, confirming a receptor-mediated mechanism. GHRH-A stimulated CSCs proliferation ex vivo, in a manner offset by MIA-602. Collectively, our findings reveal the importance of the GHRH signaling pathway within the heart. Therapy with GHRH-A although initiated 1 mo after MI substantially improved cardiac performance and reduced infarct size, suggesting a regenerative process. Therefore, activation of GHRHR provides a unique therapeutic approach to reverse remodeling after MI.heart failure | cardiac stem cells | cardiac regeneration

Rationale Culture expanded cells originating from cardiac tissue that express the cell surface receptor cKit are undergoing clinical testing as a cell source for heart failure and congenital heart disease. While accumulating data support that mesenchymal stem cells (MSCs) enhance the efficacy of cardiac cKit+ cells (CSCs), the underlying mechanism for this synergistic effect remain incompletely understood. Objective To test the hypothesis that MSCs stimulate endogenous CSCs to proliferate, migrate, and differentiate via the SDF1/CXCR4 and SCF/cKit pathways. Methods and Results Using genetic lineage-tracing approaches we show that in the postnatal murine heart, cKit+ cells proliferate, migrate, and form cardiomyocytes, but not endothelial cells. CSCs exhibit marked chemotactic and proliferative responses when co-cultured with MSCs but not cardiac stromal cells. Antagonism of the CXCR4 pathway with AMD3100 inhibited MSC-induced CSC chemotaxis but stimulated CSC cardiomyogenesis (p<0.0001). Furthermore, MSCs enhanced CSC proliferation via the SCF/cKit and SDF1/CXCR4 pathways (p<0.0001). Conclusions Together these findings show that MSCs exhibit profound, yet differential, effects upon CSC migration, proliferation and differentiation, and suggest a mechanism underlying the improved cardiac regeneration associated with combination therapy using CSCs and MSCs. These findings have important therapeutic implications for cell-based therapy strategies that employ mixtures of CSCs and MSCs.

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BackgroundMammalian heart regenerative activity is lost before adulthood but increases after cardiac injury. Cardiac repair mechanisms, which involve both endogenous cardiac stem cells (CSCs) and cardiomyocyte cell-cycle reentry, are inadequate to achieve full recovery after myocardial infarction (MI). Mice deficient in S-nitrosoglutathione reductase (GSNOR−⁄−), an enzyme regulating S-nitrosothiol turnover, have preserved cardiac function after MI. Here, we tested the hypothesis that GSNOR activity modulates cardiac cell proliferation in the post-MI adult heart.Methods and ResultsGSNOR−⁄− and C57Bl6/J (wild-type [WT]) mice were subjected to sham operation (n=3 GSNOR−⁄−; n=3 WT) or MI (n=41 GSNOR−⁄−; n=65 WT). Compared with WT,GSNOR−⁄− mice exhibited improved survival, cardiac performance, and architecture after MI, as demonstrated by higher ejection fraction (P<0.05), lower endocardial volumes (P<0.001), and diminished scar size (P<0.05). In addition, cardiomyocytes from post-MI GSNOR−⁄− hearts exhibited faster calcium decay and sarcomeric relaxation times (P<0.001). Immunophenotypic analysis illustrated that post-MI GSNOR−⁄− hearts demonstrated enhanced neovascularization (P<0.001), c-kit+ CSC abundance (P=0.013), and a ≈3-fold increase in proliferation of adult cardiomyocytes and c-kit+/CD45− CSCs (P<0.0001 and P=0.023, respectively) as measured by using 5-bromodeoxyuridine.ConclusionsLoss of GSNOR confers enhanced post-MI cardiac regenerative activity, characterized by enhanced turnover of cardiomyocytes and CSCs. Endogenous denitrosylases exert an inhibitory effect over cardiac repair mechanisms and therefore represents a potential novel therapeutic target.

Therapies for heart failure with preserved ejection fraction (HFpEF) are lacking. Growth hormone-releasing hormone agonists (GHRH-As) have salutary effects in ischemic and nonischemic heart failure animal models. Accordingly, we hypothesized that GHRH-A treatment ameliorates chronic kidney disease (CKD)-induced HFpEF in a large-animal model. Female Yorkshire pigs (n = 16) underwent 5/6 nephrectomy via renal artery embolization and 12 wk later were randomized to receive daily subcutaneous injections of GHRH-A (MR-409; n = 8; 30 µg/kg) or placebo (n = 8) for 4 to 6 wk. Renal and cardiac structure and function were serially assessed postembolization. Animals with 5/6 nephrectomy exhibited CKD (elevated blood urea nitrogen [BUN] and creatinine) and faithfully recapitulated the hemodynamic features of HFpEF. HFpEF was demonstrated at 12 wk by maintenance of ejection fraction associated with increased left ventricular mass, relative wall thickness, end-diastolic pressure (EDP), end-diastolic pressure/end-diastolic volume (EDP/EDV) ratio, and tau, the time constant of isovolumic diastolic relaxation. After 4 to 6 wk of treatment, the GHRH-A group exhibited normalization of EDP (P = 0.03), reduced EDP/EDV ratio (P = 0.018), and a reduction in myocardial pro-brain natriuretic peptide protein abundance. GHRH-A increased cardiomyocyte [Ca2+] transient amplitude (P = 0.009). Improvement of the diastolic function was also evidenced by increased abundance of titin isoforms and their ratio (P = 0.0022). GHRH-A exerted a beneficial effect on diastolic function in a CKD large-animal model as demonstrated by improving hemodynamic, structural, and molecular characteristics of HFpEF. These findings have important therapeutic implications for the HFpEF syndrome.

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder characterized by ventricular hypertrophy, myofibrillar disarray and fibrosis, and is primarily caused by mutations in sarcomeric genes. With no definitive cure for HCM, there is an urgent need for the development of novel preventive and reparative therapies. This study is focused on Aspartic acid-to-Valine (D166V) mutation in the myosin regulatory light chain, RLC (MYL2 gene), associated with a malignant form of HCM. Since myosin RLC phosphorylation is critical for normal cardiac function, we aimed to exploit this post-translational modification via phosphomimetic-RLC gene therapy. We hypothesized that mimicking/modulating cardiac RLC phosphorylation in nonphosphorylatable D166V myocardium would improve heart function of HCM-D166V mice. Adeno-Associated Virus, serotype-9 (AAV9) was used to deliver phosphomimetic human RLC variant with Serine-to-Aspartic acid substitution at Ser15-RLC phosphorylation site (S15D-RLC) into the hearts of humanized HCM-D166V mice. Improvement of heart function was monitored by echocardiography, invasive hemodynamics (PV-loops) and muscle contractile mechanics. A significant increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM mice, were observed in AAV-vs. PBS-injected HCM mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated vs. control mice. In addition, increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM hearts. Our data suggest that myosin RLC phosphorylation may have important translational implications for the treatment of RLC-induced HCM and possibly play a role in other disease settings accompanied by depressed Ser15-RLC phosphorylation.