Abstract Thu163: S-nitrosoglutathione Reductase Deficiency Impairs Systolic Calcium In Human Induced Pluripotent Stem Cell-derived Cardiomyocytes

Introduction: Females are cardioprotected from myocardial infarction (MI) compared to males. However, female mice deficient in S-nitrosoglutathione reductase (GSNOR –/– ), a denitrosylase regulating protein S-nitrosylation (S-NO), exhibit impaired cardiac function following MI compared to WT mice. To investigate the impact of GSNOR deficiency on cardiomyocyte (CM) function, we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from female donor. Hypothesis: GSNOR deficiency alters Ca 2+ handling and oxidative phosphorylation-related gene expression in CMs. Methods: Female hiPSCs lacking GSNOR (GSNOR KO ) were generated using CRISPR-Cas9 gene editing. Sanger sequencing and GSNOR activity were performed to confirm successful knockout. The percentage of cardiac troponin-positive (cTnT + ) cells was quantified using flow cytometry. Calcium handling in response to increasing pacing rate was assessed using the Ca 2+ -sensitive probe Fura-2-AM and an IonOptix system. RNA sequencing was performed to compare parental and mutated iPSC-CMs. Results: Sanger sequencing of isolated clones (N=36) showed that several had an adenine (A) insertion, resulting in a frame shift mutation. Clones (N=6) showed an absence of GSNOR expression and activity, whereas the parental line exhibited normal GSNOR activity. More than 90% of control and GSNOR KO female hiPSC-CMs were cTnT + . Female GSNOR KO hiPSC-CMs (n=3) exhibited reduced levels of Ca 2+ compared with their corresponding controls (N=6 and N=3, respectively) which resembled the pattern observed in CMs isolated from female GSNOR –/– mice (N=3). Particularly, female GSNOR KO hiPSC-CMs showed a significant reduction in systolic Ca 2+ (p<0.001) compared to controls. RNA-seq data, validated by KEGG, Reactome and WIKI Pathway analyses (control N=2, GSNOR KO N=3), revealed upregulation of mitochondrial complex I and IV enzymes in female GSNOR KO hiPSC-CMs (P<0.01), suggesting increased expression of genes related to oxidative phosphorylation. Conclusions: GSNOR deficiency reduced systolic calcium and increased the expression of oxidative phosphorylation genes, which may contribute to the impaired ability of the GSNOR KO female cardiomyocytes to respond to stress.