Abstract 289: Improved Disease Modeling Reveals 9p21 Risk Allele Regulates Connexins to Induce Arrhythmic Phenotypes

Abstract

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the non-coding 9p21 gene locus associated with increased risk of coronary artery disease (CAD) and myocardial infarction (MI). In addition, SNP correlations with sudden and arrhythmic death, even after accounting for patient and family history for CAD and MI, suggest an altered cardiac remodeling response. However, little is known about a possible cardiac phenotype as studies have largely focused on its effect on CAD and have trouble describing regulation with non-coding loci. Using induced pluripotent stem cell-derived CMs from patients that are homozygous risk/risk (R/R) and non-risk/non-risk (N/N) for 9p21 SNPs, we assessed cardiomyocyte (CM) function when cultured on hydrogels capable of mimicking the fibrotic stiffening associated with disease post-heart attack, i.e. stiffening from 10 kiloPascals (kPa) to 50 kPa. While all CMs independent of genotype beat synchronously on soft matrices, R/R CMs cultured on dynamically stiffened hydrogels exhibited asynchronous contractions versus N/N CMs in the same conditions. Dynamic stiffening reduced connexin 43 expression and gap junction assembly in R/R CMs but not N/N CMs. To eliminate patient-to-patient variability, we created an isogenic line by deleting the 9p21 locus from a R/R patient, i.e. R/R KO. R/R KO CMs maintained synchronous contractions and organized connexin 43 junctions after stiffening. The 9p21 locus suppresses the activity of the cell cycle regulator CDKN2A. p16, a protein produced by CDKN2A, prevents JNK phosphorylation (p-JNK), which in turn reduces gap junction expression in CMs and contributes to the development of arrhythmias in rabbit myocardium in response to stress. We observed that treatment with the p-JNK antagonist SP600125 after stiffening restored synchronous contractions and organized gap junction assembly to R/R CM. As a non-coding locus, 9p21 appears to repress connexin transcription, but only when the niche is stiffened as in disease. These data are the first to demonstrate that disease-specific niche remodeling can differentially affect CM function depending on SNPs within a non-coding locus.