Background: Previously we reported the use of a small molecule in upregulating cardiac genes by GPCR signaling. We further investigated the small molecule mediated effect involve caveolin and critical miRs that modulate the signaling pathway for increased survival reduced apoptosis and cardiac regeneration in ischemic myocardium.
Methods and Results: IPs were treated with a small molecule (ZP-G;20uM) for three to five days. Increased cell survival and proliferative activity (p<0.01 vs non treated IPS) with reduced cytochrome c release in cytosol was observed in small molecule treated IPS. Given the upregulation of cardiac genes, ultrastructure feature of small molecule treated IPS cells showed caveolin formation on transmission electron microscopy. miR Microarray analysis of treated IPS cells showed upregulation of miR 705,149, cardiac specific miR −133,762 and down regulation of miR-16, miR 34a, miR-290/292-5p modulating the downstream targets Chemokines, VEGFA, Vesicles associated membrane protein (Vamp), Cdk, E2f, Wnt3a and GPCR receptors. mRNA Microarray further validate 2 fold downregulation of Dnmt1,3b, Max gene associated protein, CDK2, NFkB repressing factor with concomitant 2-3 fold upregulation of caveolin, chemokines and integrins. Western blot analysis displayed significant upregulation of Gα protein, phospho Akt, and Bcl-2 levels as compared to nontreated IPS cells. Small molecule mediated effect was abolished by treatment of IPS cells with GPCR blocker. No tumor formation when treated IPS cells were injected in nude mouse. For Invivo study, female mice with myocardial infarction after permanent LAD ligation were transplanted with either DMEM (Gp1),or 2х105 non treated IPS (Gp 2), or treated IPS (Gp 3). At 4-week of engraftment of IPS cell derived cardiac progenitors (Gp 3) showed reduced apoptosis, extensive survival and myogenesis with attenuation of infarct size and improved heart function as compared to other groups.
Conclusion: This study provides the first evidence that small molecule differentiate IPS to cardiac progenitors and regenerate the ischemic myocardium by targeting downstream targets that regulate the transcription of critical genes and proteins expression to facilitate receptor- tissue- cell signaling.