Abstract Body (Do not enter title and authors here): 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) 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.