Investigating the Molecular Mechanisms of Cardiac Dysfunction Underlying the Junctophilin-2 E169K Mutation
Junctophilin-2 (JPH2), is a structural membrane protein that plays a critical role in the regulation of calcium in the heart. In humans, mutations in the JPH2 have been associated with a litany of cardiac diseases including hypertrophic cardiomyopathy, dilated cardiomyopathy, arrhythmias, and sudden cardiac death. Of the many mutant variants of interest in JPH2, one particular mutation, E169K, has previously been linked to atrial fibrillation and hypertrophic cardiomyopathies. JPH2 co-localizes and exhibits a binding interaction with a peripheral mitochondrial membrane protein mitofusin-2 (MFN2). However, the precise molecular mechanisms underlying the hypertrophic cardiomyopathy cardiac dysfunction resulting from this variant are still not yet well delineated. With this thesis project, we hoped to investigate the hypothesis that the presence of the E169K variant of junctophilin-2 impairs the mitochondrial function of cardiomyocytes and leads to structural irregularities. This project used CRISPR Cas9 genome editing technology to genetically engineer the E169K mutation into the WTC11 TOM20 human induced pluripotent stem (iPS) cell line. DNA from edited cells was purified then amplified by PCR. Restriction enzyme digests were used to verify the success of editing. The E169K mutant cells would then be differentiated into cardiomyocytes and both qualitatively and quantitatively evaluated to determine the E169K mutation’s impact on cardiomyocyte mitochondrial function and morphology. However, the CRISPR Cas9 genome editing was unsuccessful. As a result, further experiments could not be performed.