Optimization of basic helix-loop-helix (bHLH) Transcription Factor Function in Direct Cardiac Reprogramming
Description: The heart’s limited regenerative capacity underlies the persistent global burden of heart disease. Direct cardiac reprogramming offers a promising solution by converting fibroblasts into cardiomyocyte (CM)-like cells through the expression of cardiac transcription factors. Initially established using three factors (MEF2C, GATA4, TBX5), our lab developed a two-factor cocktail, consisting of proneuronal basic helix-loop-helix (bHLH) factor ASCL1 and MEF2C (A+M). This two-factor cocktail is more efficient than the canonical MGT cocktail, and its minimal composition makes it better suited for future clinical translation. However, A+M induces residual neuronal reprogramming, presenting challenges for optimization. This proposal aims to use protein engineering to refine A+M by (1) residue-based modulation to enhance ASCL1’s interactions with MEF2C, and (2) through domain swapping with a cardiac bHLH factor to enhance ASCL1’s cardiac potency. To prioritize protein constructs for experimental design, I will first use computational protein modeling (AlphaFold3, Rosetta, Schrödinger) to predict how structural changes affect DNA- and protein-binding interactions. Preliminary modeling has identified ASCL1’s basic domain as critical for DNA-binding, and suggests that combining this with a cardiac-specific N-terminal and ASCL1’s C-terminal may enhance its cardiac potency. Aim 1 uses alanine scanning to identify critical residues that mediate ASCL1’s interactions with MEF2C, and thus its redirection to cardiogenic sites during reprogramming. Constructs prioritized through computational modeling will be co-expressed with MEF2C in cardiac fibroblasts and evaluated in vitro using flow cytometry, ChIP-qPCR, and co-immunoprecipitation. Aim 2 focuses on domain-swapping between ASCL1 and cardiac bHLH factor HAND2 to create a chimeric protein that can drive cardiac reprogramming while minimizing neuronal effects. HAND2 is a suitable protein for domain swapping as it shares structural homology with ASCL1 in addition to having established roles in cardiac reprogramming and CM proliferation. Constructs will undergo computational modeling, followed by in vitro and in vivo reprogramming validation. This work will not only establish a computational-experimental pipeline for protein-DNA interaction studies, but will more importantly help advance cardiac reprogramming by optimizing this two-factor system, with the ultimate goal of working towards a single chimeric factor.
Funder: American Heart Association
Amount: $70,680
Funding type: training
Funder scheme: Predoctoral Fellowship
Start date: 2026-07-01
End date: 2028-06-29
Investigators: Rachelle Ambroise, Rachelle Ambroise
Institution: The University of North Carolina at Chapel Hill
Funded outputs count: 0
Funder award ID: 26PRE1557536
OpenAlex ID: G1112569113