In Vitro Engineered Tissue Screens for Cardiac Regeneration

Cell and gene therapies for heart regeneration represent a promising approach to remuscularize the heart following myocardial injury. These regenerative therapies can benefit from <i>in vitro</i> chemical and genetic screens in engineered heart tissues to identify and study novel inducers of cardiomyocyte (CM) proliferation. Our recent screens in neonatal rat and human iPSC-derived CMs have identified activations of the mitogen activated protein kinase (MAPK) pathway and pentose phosphate pathway (PPP) as two promising strategies with pro-proliferative effects in engineered cardiac tissues (ECTs). Specifically, a doxycyclin-inducible, CM-specific expression of constitutively-active mutant of BRAF (BRAF-V600E), a major kinase in the MAPK pathway, was sufficient to induce CM cycling in ECTs, but resulted in functional decline and tissue stiffening. These adverse functional effects were associated with broad transcriptional changes, a shift to glycolytic metabolism, and induction of a pro-migratory CM phenotype. While ERK activation was required for the initial establishment of the caBRAF-induced phenotype, inhibition of ERK after the phenotype had been established was insufficient to restore the loss of tissue function. Transient caBRAF expression in ECTs rapidly induced CM cycling that preceded functional decline, which was reversible only if MAPK activation was brief (&lt;3 days). Notably, the increase in ECT stiffness resulting from sustained caBRAF expression was dispensable for induction of CM cycling, as transient caBRAF expression yielded increased CM cycling without altered stiffness. Furthermore, in an independent in vitro CRISPR/Cas9 knockout screen, we identified adenosine deaminase knockout (ADA-KO) as the most effective inducer of CM cycle activity in neonatal rat ECTs and long-term cultured hiPSC-CMs. RNA sequencing of ADA-KO vs. control ECTs and measurements of metabolite abundance and enzyme activity suggested lactate generation and PPP upregulation as potential drivers of CM proliferation. Inhibition of the PPP rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD) via the small molecule inhibitor 6-aminonicotinamide prevented ADA-KO induced CM cycling, while increased PPP activity <i>via</i> doxycycline-inducible lentiviral G6PD overexpression yielded a significant increase in CM cycling. Overall, our studies demonstrate the utility of <i>in vitro</i> engineered cardiac tissues as a testbed for identification of novel CM mitogens with potential for use in cardiac regenerative therapies.