Abstract Body: Direct cardiac reprogramming offers a promising strategy to overcome the heart’s limited regenerative capacity by converting resident cardiac fibroblasts within injured myocardium into cardiomyocyte-like cells. Although initially achieved using the transcription factor trio of Mef2c, Gata4, and Tbx5 (MGT), clinical translation has been limited by low efficiency and delivery challenges arising from multi-factor expression. Our lab recently addressed this by downscaling MGT to a minimalistic combination of the basic helix-loop-helix pioneer factor Ascl1 and Mef2c (A+M), which more robustly induces cardiac reprogramming. However, A+M retains residual neuronal activity driven by Ascl1’s proneuronal properties.

Here, we aim to enhance minimalist A+M reprogramming through structure-guided engineering of Ascl1 while maintaining compatibility with Mef2c. We pursue two complementary strategies: (i) single-residue mutagenesis to strengthen cooperative interactions between Ascl1 and Mef2c for increased redirection of Ascl1 toward cardiac genomic targets, and (ii) domain swapping between Ascl1 and cardiac bHLH factor Hand2 to generate chimeric proteins that preserve pioneer activity while conferring cardiac specificity.

We developed a rigorous computational framework for the in silico design and evaluation of these Ascl1 variants prior to experimental incorporation. Our modeling has identified candidate residues and chimeric architectures predicted to favor cardiac targeting while minimizing neuronal engagement. Ongoing studies are validating these predictions in vitro. Collectively, this work establishes a generalizable computational pipeline for rational transcription factor engineering to guide protein design in cardiac reprogramming.