Intrinsic Modifier Effect of cTnT Isoform Switching in Sarcomeric Cardiomyopathies

Abstract

Reduced contractility, caused either by dysfunction or loss of cardiomyocytes, is the leading cause of systolic heart failure (HF). We have demonstrated that deoxy-ATP (dATP) improves systolic cardiac contractility by activating myosin without affecting the diastolic function, suggesting a promising candidate to mitigate cardiac dysfunction. Endogenously, Ribonucleotide Reductase (RNR), a rate-limiting enzyme for de novo synthesis of deoxynucleotides, produces dATP. The goal is to overexpress RNR in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to create cells with elevated dATP levels for use as a novel therapeutic strategy to improve contractility and regeneration in hearts following myocardial infarct. We used CRISPR-Cas9 gene editing to integrate an RNR expression cassette at the AAVS1 locus in hiPSCs WTC11 cell line (#GM25256, Coriell Institute). A double mutant form (RNR-DM) was generated by site-directed mutagenesis to limit proteolytic degradation of RNR in post-mitotic CMs. RNR subunits-R1 and R2-DMintervened by a self-cleavage peptide P2A were expressed under a strong constitutively expressed CAG promoter. The integration of RNR cassette at the AAVS1 locus in hiRNR clones were confirmed by PCR genotyping, sequencing, transgene transcript, and protein expression. The stability and function of RNR-DM transgene were assessed in hiRNR-CMs, differentiated from the hiRNR clone. hiRNR-CMs expressed elevated levels of R1 and R2 subunits as compared to control WTC11-CMs (N=3, where N is an independent run of cardiac differentiation) by immunoblotting. This resulted in an elevation of dATP/ATP ratio (3.451.9 fold) (N=2) for hiRNR-CMs vs. WTC11-CMs, as detected by Mass Spectrometry. Cardiomyocyte shortening measurements by video-based IonOptix were increased (2.0350.7 fold) in hiRNR CMs as compared to WTC11-CMs (N=2). Currently, we are characterizing the hiRNR-CMs, including DNA stability, apoptosis, cell division, calcium cycling, hypertrophy, and structural maturation for future studies of transplantation into models of HF.