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

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.