Time-dependent effects of BRAF-V600E on cell cycling, metabolism, and function in engineered myocardium.

Candidate cardiomyocyte (CM) mitogens such as those affecting the extracellular signal-regulated kinase (ERK) signaling pathway represent potential targets for functional heart regeneration. We explored whether activating ERK via a constitutively active mutant of B-raf proto-oncogene (BRAF), BRAF-V600E (caBRAF), can induce proproliferative effects in neonatal rat engineered cardiac tissues (ECTs). Sustained CM-specific caBRAF expression induced chronic ERK activation, substantial tissue growth, deficit in sarcomeres and contractile function, and tissue stiffening, all of which persisted for at least 4 weeks of culture. caBRAF-expressing CMs in ECTs exhibited broad transcriptomic changes, shift to glycolytic metabolism, loss of connexin-43, and a promigratory phenotype. Transient, doxycycline-controlled caBRAF expression revealed that the induction of CM cycling is rapid and precedes functional decline, and the effects are reversible only with short-lived ERK activation. Together, direct activation of the BRAF kinase is sufficient to modulate CM cycling and functional phenotype, offering mechanistic insights into roles of ERK signaling in the context of cardiac development and regeneration.

Human Erbb2-induced Erk activity robustly stimulates cycling and functional remodeling of rat and human cardiomyocytes.

Multiple mitogenic pathways capable of promoting mammalian cardiomyocyte (CM) proliferation have been identified as potential candidates for functional heart repair following myocardial infarction. However, it is unclear whether the effects of these mitogens are species-specific and how they directly compare in the same cardiac setting. Here, we examined how CM-specific lentiviral expression of various candidate mitogens affects human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and neonatal rat ventricular myocytes (NRVMs) in vitro. In 2D-cultured CMs from both species, and in highly mature 3D-engineered cardiac tissues generated from NRVMs, a constitutively active mutant form of the human gene Erbb2 (cahErbb2) was the most potent tested mitogen. Persistent expression of cahErbb2 induced CM proliferation, sarcomere loss, and remodeling of tissue structure and function, which were attenuated by small molecule inhibitors of Erk signaling. These results suggest transient activation of Erbb2/Erk axis in CMs as a potential strategy for regenerative heart repair.