Cardiomyocyte GSK-3β deficiency induces cardiac progenitor cell proliferation in the ischemic heart through paracrine mechanisms

Cardiomyopathy is an irreparable loss and novel strategies are needed to induce resident cardiac progenitor cell (CPC) proliferation in situ to enhance the possibility of cardiac regeneration. Here we identify a potential role for glycogen synthase kinase-3β (GSK-3β), a critical regulator of cell proliferation and differentiation, in CPC proliferation that occurs after myocardial infarction (MI). Cardiomyocyte-specific conditional GSK-3β knockout (cKO) and littermate control mice were employed and challenged with MI. Though cardiac left ventricular chamber dimension (LVID) and contractile functions were comparable at two week post-MI, cKO mice displayed significantly preserved LV chamber and contractile function vs. control mice at four-weeks post-MI. Consistent with protective phenotypes, an increased percentage of c-kit positive cells (KPCs) were observed in the cKO hearts at four and six weeks post-MI which was accompanied by increased levels of cardiomyocyte proliferation. Further analysis revealed that the observed increased number of KPCs in the ischemic cKO hearts was mainly from a cardiac lineage as the majority of identified KPCs were negative for the hematopoietic marker, CD45. Mechanistically, cardiomyocyte-GSK-3β profoundly suppresses the expression of growth factors (GFs), including basic-FGF angiopoietin-2, erythropoietin, stem cell factor (SCF), PDGF-BB, G-CSF, and VEGF, post-hypoxia. In conclusion, our findings strongly suggest that loss of cardiomyocyte-GSK-3β promotes cardiomyocyte and resident CPC proliferation post-MI. The induction of cardiomyocytes and CPC proliferation in the ischemic cKO hearts is potentially regulated by autocrine and paracrine signaling governed by dysregulated growth factors post-MI. A strategy to inhibit cardiomyocyte GSK-3β could be helpful for promotion of in-situ cardiac regeneration post-MI injury.

The Spatiotemporal Expression of Notch1 and Numb and Their Functional Interaction during Cardiac Morphogenesis

Numb family proteins (NFPs), including Numb and Numblike (Numbl), are commonly known for their role as cell fate determinants for multiple types of progenitor cells, mainly due to their function as Notch inhibitors. Previous studies have shown that myocardial NFP double knockout (MDKO) hearts display an up-regulated Notch activation and various defects in cardiac progenitor cell differentiation and cardiac morphogenesis. Whether enhanced Notch activation causes these defects in MDKO is not fully clear. To answer the question, we examined the spatiotemporal patterns of Notch1 expression, Notch activation, and Numb expression in the murine embryonic hearts using multiple approaches including RNAScope, and Numb and Notch reporter mouse lines. To further interrogate the interaction between NFPs and Notch signaling activation, we deleted both Notch1 or RBPJk alleles in the MDKO. We examined and compared the phenotypes of Notch1 knockout, NFPs double knockout, Notch1; Numb; Numbl and RBPJk; Numb; Numbl triple knockouts. Our study showed that Notch1 is expressed and activated in the myocardium at several stages, and Numb is enriched in the epicardium and did not show the asymmetric distribution in the myocardium. Cardiac-specific Notch1 deletion causes multiple structural defects and embryonic lethality. Notch1 or RBPJk deletion in MDKO did not rescue the structural defects in the MDKO but partially rescued the defects of cardiac progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis. Our study concludes that NFPs regulate progenitor cell differentiation, cardiomyocyte proliferation, and trabecular morphogenesis partially through Notch1 and play more roles than inhibiting Notch1 signaling during cardiac morphogenesis.

The Drosophila Forkhead/Fox transcription factor Jumeau mediates specific cardiac progenitor cell divisions by regulating expression of the kinesin Nebbish

Forkhead (Fkh/Fox) domain transcription factors (TFs) mediate multiple cardiogenic processes in both mammals and Drosophila. We showed previously that the Drosophila Fox gene jumeau (jumu) controls three categories of cardiac progenitor cell division—asymmetric, symmetric, and cell division at an earlier stage—by regulating Polo kinase activity, and mediates the latter two categories in concert with the TF Myb. Those observations raised the question of whether other jumu-regulated genes also mediate all three categories of cardiac progenitor cell division or a subset thereof. By comparing microarray-based expression profiles of wild-type and jumu loss-of-function mesodermal cells, we identified nebbish (neb), a kinesin-encoding gene activated by jumu. Phenotypic analysis shows that neb is required for only two categories of jumu-regulated cardiac progenitor cell division: symmetric and cell division at an earlier stage. Synergistic genetic interactions between neb, jumu, Myb, and polo and the rescue of jumu mutations by ectopic cardiac mesoderm-specific expression of neb demonstrate that neb is an integral component of a jumu-regulated subnetwork mediating cardiac progenitor cell divisions. Our results emphasize the central role of Fox TFs in cardiogenesis and illustrate how a single TF can utilize different combinations of other regulators and downstream effectors to control distinct developmental processes.