Novel strategies to stimulate cardiomyocyte regeneration via inhibition of the TGF-beta signalling pathway

Myocardial infarction causes cardiomyocyte death, and the limited proliferative capacity of these cells cannot compensate for post-injury loss. Transforming growth factor-beta (TGF-β) ligands are central mediators of fibrosis and pathological remodeling after cardiac injury, but their direct impact on cardiomyocyte regeneration remains poorly understood. Here, we investigated strategies to promote endogenous cardiomyocyte proliferation, focusing on TGF-β signaling. Neonatal primary cardiomyocytes were treated with TGF-β1, TGF-β2, or TGF-β3. While TGF-β2 had no effect, TGF-β1 and TGF-β3 markedly suppressed cell-cycle progression. In cardiomyocyte-enriched cultures, these ligands similarly inhibited proliferation, demonstrating a direct effect on cardiomyocytes independent of other cardiac cells. TGF-β1/3 also suppressed the proliferative effects of regenerative growth factors and cytokines. Silencing of the type 1 TGF-β receptor (TGFβR1) or co-receptors betaglycan and endoglin abolished TGF-β1/3 effects, while downstream analysis indicated that SMAD2/3 activation mediates TGFβ-induced cell cycle arrest. Pharmacological inhibition of TGFβR1 fully prevented TGF-β1/3–mediated suppression and dose-dependently enhanced neonatal cardiomyocyte proliferation. We further tested combinatorial strategies by pairing TGF-β inhibition with modulation of pathways controlling cell cycle and cytoskeletal dynamics, including p38 MAPK, the G2/M checkpoint regulator WEE1, RhoA/ROCK, and Wnt/β-catenin via GSK3β inhibition. Some combinations yielded promising proliferative effects in both neonatal and post-mitotic cardiomyocytes. Collectively, our results identify TGF-β as a direct negative regulator of cardiomyocyte proliferation and highlight combinatorial approaches in which TGF-β antagonism enhances regenerative signaling. Targeting TGF-β alongside other pathways represents a promising strategy to promote cardiomyocyte proliferation and cardiac repair after injury.