Abstract Body: Cardiomyocytes (CMs) lost due to ischemic cardiac injury cannot be replaced because adult CMs have limited proliferation capacity. Unlike adult CMs, neonatal CMs retain the ability to regenerate with sarcomere disassembly observed during proliferation. Recently we demonstrated that overexpression of 4 cell cycle genes (Cdk1, Cdk4, Ccnb, Ccnd; 4F) induces adult CM proliferation in vitro and in vivo with sarcomere disassembly also occurring during anaphase and cytokinesis. We aim to identify the protein that controls sarcomere disassembly during forced CM proliferation.
To achieve that, we conducted temporal proteomics and ubiquitin-linked proteomics on 60-day-old hiPS-CMs transduced with control or 4F adenovirus for 24h (early mitosis), 48h (mitotic peak), and 72h (final division stage). Csrp3, which interacts with T-Cap and α-actinin at Z-disks, was the most downregulated sarcomere protein at 24 and 48h, returning to baseline at 72h. The Csrp3 ubiquitination sites, K119 and K113, were significantly increased only at 24h, suggesting that Csrp3 is actively degraded during mitosis and replenished post-division.
To confirm Csrp3's role in forced CM proliferation, we knocked down Csrp3 (KD) in P7 mouse neonatal CMs (NMCMs) with/without 4F. In 4F-transduced NMCMs, Csrp3 KD significantly increased PHH3-positive CMs (G2/M marker) and mono-nucleated CMs without affecting EDU-positive CMs (G1/S marker), indicating enhanced G2/M entry without altering G1/S.
In vivo, P21 Csrp3^KO hearts exhibited disrupted sarcomere structure, increased HW/BW ratio with no change in CM size, and a significant increase in PHH3⁺ and AurkB⁺ CMs compared to WT, suggesting increased cell number rather than hypertrophy. RNA sequencing revealed upregulation of cell cycle genes, confirmed by RT-PCR. Similarly, Csrp3 KD in human heart slices showed a significantly higher number of PHH3⁺ CMs and increased cell cycle gene expression compared to control.
These findings suggest Csrp3 is crucial for sarcomere disassembly during CM proliferation. Genetic deletion of Csrp3 disrupts CM sarcomere structure and enhances CM mitotic cell cycle entry.