Cysteine and Glycine Rich Protein 3 (Csrp3) modulates cardiomyocyte mitotic cell cycle entry through Ring Finger Protein 5 (Rnf5)/p38 MAPK dependent pathway

Adult cardiomyocyte (CM) has limited proliferative capacity, which is correlated with rigid sarcomere structures. Our previous studies showed that overexpression of Cdk1, Cdk4, Ccnb, and Ccnd (collectively 4F) induces adult CM proliferation with observed sarcomere disassembly during mitosis. However, it is unclear how the sacrcomeres disassemble during mitosis and whether sarcomere disassembly alone could enhance mitotic cell cycle entry in CMs. In this study we identified CSRP3 that is highly expressed in adult CM, as a structural checkpoint that links sarcomere integrity to ubiquitination-dependent regulation of CM cell cycle progression. Temporal proteomics and ubiquitin linked proteomics in CMs transduced with control or 4F adenovirus for 24h (early cell cycle entery), 48h (mitosis dominant), and 72h ( end of cell cycle) revealed that CSRP3, a Z-disk–associated protein interacting with T-Cap and α-actinin, was the most downregulated sarcomeric protein during mitosis, returning to baseline at the end of the cell cycle. Csrp3 Knockdown (KD) results in sarcomere disassembly and promotes CM mitotic entry in adult human heart slices, evidenced by a significant increase in PHH3-positive nuclei, a marker for G2/M phase, with no major impact on DNA synthesis. In MADM mice, Csrp3 KD results in a significant increase in single coloured cells which indicates complete CM cell division confirming the role of Csrp3 in modulating mitosis in CMs. In vivo, 21 day old (P21) Csrp3KO mice hearts showed a significant increase in mononucleation, proliferation markers and cell cycle gene expression. Mechanistically, RNAseq from P21 Csrp3KO mice hearts demonstrated increased expression of Rnf5, an E3 ubiquitin ligase. The upregulated Rnf5 resulted in significant reduction in p38 MAPK protein expression, hence promotes mitotic entry. Moreover, Rnf5 overexpression in human heart slices results in activation of CM cell cycle while Rnf5 KD in naturally proliferating neonatal P1 CM reduced cell cycle activity. In conclusion, our findings identify CSRP3 as a structural checkpoint that couples sarcomere integrity to Ring Finger Protein 5 (RNF5)-mediated ubiquitination of p38 MAPK, thereby controlling CM mitotic entery and uncovering a previously unrecognized mechanism governing cardiac regenerative potential.