Molecular Dissection of Hand2 During the Formation of Pacemaker-Like Myocytes During Direct Reprogramming

Direct reprogramming of one cell type into another has great promise for regenerative medicine, disease modeling, and lineage specification. Currently, the conversion of fibroblasts into induced cardiomyocytes (iCM) by Gata4, Mef2c, and Tbx5 (GMT) represents an important avenue for generating de novo cardiac myocytes. Recent evidence has shown that iCM formation and diversity can be enhanced by the addition of Hand2 to GMT (GHMT). These four transcription factors give rise to a heterogenous CM population, consisting of atrial (iAM), ventricular (iVM), and pacemaker myocytes (iPM). However, the molecular mechanisms that drive this plastic fate conversion remain poorly understood. Although chromatin and single-cell studies in GMT-iCM have shown the existence of a set of temporal steps that orchestrate iCM formation, little is known about how Hand2 enhances this process. In the present study, we seek to characterize these Hand2-dependent mechanisms. We hypothesize that Hand2 regulates a discrete pacemaker regulatory network that becomes active during GHMT-iCM reprogramming. To test this, we compared the transcriptional and genomic profiles of fibroblasts, GMT, GHMT, and endogenous mouse Pacemaker cells. We observe similar chromatin landscape and gene expression profiles between Hand2-iPM and endogenous sinoatrial node (SAN), however several known key PM pathways are not active. Activation of these networks further enhances iCM-iPM fo Moreover, we show that Hand2 enhances chromatin accessibility in regions related to sarcomere function and electrical coupling, as well as promoting the closing of regions related to alternative fates. Utilizing integrative genomics between ATAC-seq and RNA-seq datasets, we identify the desmosome machinery as an important feature of iPM formation. In parallel, we define a novel Hand2 domain region that regulates cardiac subtype diversity. Taken together, our results showcase Hand2-dependent mechanisms for iPM formation and gives insight into the improvement of future iPM engineering.