Abstract Body: Background
Human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) technology has advanced translational investigations of heart disease. However, terminal differentiation into adult-like left ventricular cardiomyocytes (LV-CMs) remains incomplete. We previously identified PR/SET Domain Containing 16 (PRDM16) through a massively multiplexed CRISPR-Cas9 activation (CRISPRa) screen covering all cardiac transcription factors as a driver of the Myosin Light Chain 7 (MYL7) to Myosin Light Chain 2 (MYL2) paralog switch in hPSC-CMs. PRDM16 is known to facilitate white-to-brown adipose tissue conversion and enhance metabolic function in adipocytes.
Hypothesis
We hypothesized that PRDM16 promotes hPSC-CM commitment toward left ventricular lineage and augments cardiomyocyte metabolic function.
Methods
PRDM16 expression was elevated via CRISPRa or transgene overexpression in hPSC-CMs. Maturation toward LV fate was assessed across transcriptomics, metabolomics, metabolic functional assays, and physiological functions in engineered heart tissues (EHTs). Genomic occupancy of PRDM16 was characterized by CUT&RUN-seq.
Results
PRDM16 activation transcriptionally upregulated LV-CM markers (MYL3, MYH7, NPPB) while suppressing immature and atrial markers (MYL7, MYL4, MYH6). MYL2 protein upregulation was further confirmed by immunofluorescence. Flux pathway analysis showed that the fatty acid metabolism pathway was upregulated, and untargeted metabolite screening demonstrated fatty acid metabolites were enriched in PRDM16-activated CMs, which also exhibited increased respiratory capacity as shown in Seahorse assays. CUT&RUN-seq revealed no dominant consensus binding motif for PRDM16; however, PRDM16-bound loci were enriched for cardiac transcription factor binding sites, including GATA and MEF2. Gene ontology analysis of PRDM16-bound, cardiomyocyte cis-regulatory elements highlighted heart development and metabolic pathways as significantly enriched terms. Functionally, PRDM16 activation in EHTs also increased contractile force.
Conclusion
PRDM16 promotes hPSC-CM commitment toward left ventricular lineage and augments cardiomyocyte metabolic function, as evidenced by transcriptional, metabolic, and contractile improvements. These findings support PRDM16 as a viable target for enhancing the maturity and fidelity of hPSC-CM models in disease modeling and regenerative applications.