Time-dependent Effects of BRAF-V600E on Cell Cycling, Metabolism, and Function in Engineered Myocardium

Candidate cardiomyocyte (CM) mitogens such as those affecting ERK signaling pathway represent potential targets for functional heart regeneration. We explored whether activating ERK via a constitutively active mutant of BRAF, BRAF-V600E (caBRAF), can induce pro-proliferative effects in neonatal rat engineered cardiac tissues (ECTs). Sustained CM-specific caBRAF expression induced chronic ERK activation, significant 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 pro-migratory phenotype. Transient, doxycycline-controlled caBRAF expression revealed that the induction of CM cycling is rapid, precedes functional decline, and the effects are reversible only with a 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. Overall design: Bulk RNA-sequencing from engineered cardiac tissues comparing tissues transduced with a control lentivirus (mCherry only) to tissues transduced with a lentivirus expressing BRAF-V600E-2A-mCherry, both viruses were driven by the muscle-specific MHCK7 promoter. Each sequencing sample is the bulk sequencing data from 6-8 tissues pooled from one cell batch such that each treatment group and time point (we assessed at 1wk and 2wk in vitro) is comprised of three biological replicates.