Shortening and optimization of MEF2C and GATA4 promote cardiac reprogramming

Direct cardiac reprogramming is a ground-breaking approach in regenerative medicine. Overexpression of three cardiac transcription factors (TFs), MEF2C, GATA4, and TBX5 (MGT), reprograms fibroblasts into induced cardiomyocytes (iCMs). However, to simultaneously express MGT using clinically available, size-constrained adeno-associated viral vectors, the polycistronic MGT gene sequence must be shortened and optimized. TFs consist of DNA-binding and effector domains; however, the effect of MGT effector domains on target gene expression is unclear. Furthermore, whether shortening of MGT by the deletion of effector domains affects cardiac reprogramming is unknown. Here, we systematically generated and analyzed 28 MGT deletion mutants (del muts), in which effector domain of the polycistronic MGT was successively deleted by 150 base pairs (bp) to shorten and optimize MGT for cardiac reprogramming. Our unbiased screening of individual del muts revealed that two MEF2C mutants significantly improved cardiac reprogramming, whereas four reduced it. GATA4 and TBX5 del muts showed minimal or negative effects on cardiac reprogramming. We subsequently removed the effector domains from MGT that were either dispensable or inhibitory to reprogramming. Finally, we generated MΔGΔT, which was 900 bp shorter than MGT, by deleting multiple effector domains in MEF2C and GATA4. Notably, MΔGΔT caused a seven-fold enhancement of cardiac reprogramming and generated more iCMs with well-defined sarcomeric structures than those with MGT. RNA sequencing, ChIP-Atlas, and gene set enrichment analyses revealed that MΔGΔT activated cardiac programs while suppressing fibroblast signatures, likely through activation of MEF2C target genes. Thus, shortened and optimized MGT improves cardiac reprogramming, which may facilitate clinical applications.