Abstract 4368641: PRDM16 Drives iPSC-Derived Cardiomyocyte Maturation Towards Left Ventricular Commitment

Backround: 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) has not been fully attained. In particular, myofilament paralog switching that occurs during human cardiac maturation, such as the switch from myosin light chain 7 (MYL7) to myosin light chain 2 (MYL2) expression, only partially occurs in hPSC-CMs, even with advanced maturation protocols. Hypothesis: We hypothesized that hPSC-CM maturation with current differentiation protocols towards an LV phenotype reaches a transcriptional bottleneck that can be surpassed by activation of specific transcription factor(s). Methods: We performed a massively multiplexed CRISPR-Cas9 activation (CRISPRa) screen covering all cardiac transcription factors at 15X gRNA coverage. A lentiviral pooled CRISPRa library with these gRNAs (N=12,750) was transduced into reporter ventricular-differentiated and metabolically-matured iPSC-CMs expressing MYL2-GFP. hPSC-CMs were sorted into GFP-positive and -negative populations and Illumina sequencing was used to identify enriched gRNAs in the positive population. Validation and functional assays in engineered heart tissues (EHTs) were performed. Results: PRDM16 was identified as the top enriched gene in the multiplexed assay. Validation studies demonstrated an increase in MYL2 with PRDM16 CRISPRa by qRT-PCR and immunofluorescence staining. RNA sequencing showed PRDM16 overexpression also upregulated other LV-CM markers (MYL3, MYH7, NPPB) and downregulated immature/atrial markers (MYL7, MYL4, MYH6). Functionally, PRDM16 activation in EHTs increased contractile forces and decreased contraction frequency. Metabolically, PRDM16 overexpression improved the ATP:ADP ratio under increased workload in hPSC-CMs. Although PRDM16 has been reported to act as a histone methyltransferase, overexpression of a mutant PRDM16 without histone methyltransferase activity also activated MYL2 expression. Conclusion: We identified PRDM16 as a key regulator driving hPSC-CM maturation towards a terminally differentiated LV fate. Activation PRDM16 promotes LV-specific paralog switching and improves contractile function and metabolic reserve in hPSC-CMs.