Project Summary/Abstract Desmoplakin is an elongated protein that links the desmosomal components to the cytoskeleton through intermediate filaments. Heterozygous truncation mutations in the DSP gene, which encodes desmoplakin, cause arrhythmogenic cardiomyopathy (AC) impacting the left ventricle. A striking feature associated with DSP truncation variants (DSPtv) is a prominent inflammatory component, and this inflammation can be well visualized through cardiac imaging using magnetic resonance or positron emission tomography. The effect of DSP truncations in mediating cardiac inflammation is not well understood. The nature of DSP truncations implies that haploinsufficiency or reduction of desmoplakin protein is contributory feature of left ventricular cardiomyopathy with inflammation. To begin to characterize the transcriptional program associated with DSP- cardiomyopathy, I analyzed RNA-sequencing from neonatal rat ventricular cardiomyocytes with siRNA to reduce expression of DSP. Analysis of the differentially expressed genes in siRNA-treated cardiomyocytes identified immune chemotactic pathways as having increased expression. Of particular note were several genes connected to pyroptosis, an immunologically active form of cell death. Taken together, these data suggest that innate immune pathways may contribute significantly to DSP-associated cardiomyopathy. I hypothesize that reduction of desmoplakin promotes excessive inflammasome activation in cardiomyocytes that results in aberrant calcium handling and pro-arrhythmogenic heterocellular junctions. In Aim 1 of this proposal, I will assess this inflammatory component of DSP-cardiomyopathy in human-induced pluripotent stem cell (hiPSC) lines with distinct DSP truncations derived from individuals with inflammatory-arrhythmogenic cardiomyopathy. To characterize 2D- and 3D-hiPSC models, I will assess whether DSP haploinsufficiency is sufficient to drive expression of inflammasome components. I will then examine DSPtvs for their effects on contractility and calcium transients in response to pathogen- and damage-derived activators of the inflammasome. I will conduct coculture assays of hiPSC-derived cardiomyocytes and fibroblasts to evaluate the inflammatory modulation of heterocellular junctions and cardiac conduction in DSP-cardiomyopathy. In Aim 2 of the proposal, I will modulate innate immune activation in hiPSC-derived cardiomyocyte models using siRNA, small molecule inhibition, and epigenetic modulation strategies. These studies will target Never in Mitosis A-related Kinase 7 (NEK7), an inflammasome component with differential gene expression in heart failure. The proposed studies will expand from observations in patients with DSPtvs and define the inflammatory triggers refining the distinct pathology related to DSP-mediated disease. In particular, the findings from this study have implications for other forms of cardiomyopathy driven by fibrotic transformation such as dilated cardiomyopathy an...