High-throughput screening in hiPSC-cardiac models reveals cardiomyocyte-specific cell cycle regulatory mechanisms

<jats:sec> <jats:title>Introductory Paragraph</jats:title> <jats:p> Multiple regulatory mechanisms govern cardiomyocyte proliferation including epigenetic modifications, metabolism and mechanical load. However, it is unclear whether such mechanisms can be pharmacologically targeted to induce cardiomyocyte proliferation without affecting other cell types. Here, we develop a dual-reporter ( <jats:italic> TNNT2 <jats:sup>eGFP</jats:sup> </jats:italic> ; <jats:italic> PCNA <jats:sup>mScarlet-I</jats:sup> </jats:italic> ) and a high-throughput image-based pipeline in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, with counter screening in non-myocytes, to identify compounds that selectively promote cardiomyocyte proliferation without affecting other cell types. We identify the PIM kinase inhibitor GDC-0339 as a cardiomyocyte-selective pro-proliferative compound. GDC-0339 induced proliferation of hiPSC-derived cardiomyocytes without activity in non-myocytes, non-cardiac fibroblasts or epithelial cells. Phosphoproteomic profiling of GDC-0339 in cardiomyocytes and non-cardiac fibroblasts revealed a cardiomyocyte-specific mechanism of action involving sarcomere disassembly via remodelling of the F-actin cytoskeleton and metabolic reprogramming to anaerobic metabolism via Pyruvate Dehydrogenase Kinases (PDKs). Thus, we uncover cardiomyocyte-specific mechanisms governing the cell cycle that are potentially druggable. </jats:p> </jats:sec>