A High‐Content RNAi Screen to Elucidate Novel Effectors of Cardiac Proteotoxicity

Rationale Proteotoxicity has emerged as an important underlying pathologic mechanism in cardiac disease. Many heart failure phenotypes, including ischemia/reperfusion, pressure overload‐induced hypertrophy and desmin‐related cardiomyopathy accumulate misfolded protein within the cytoplasm of cardiomyocytes, potentially leading to cell death and dysfunction. While recent success in treating these phenotypes has been achieved by inducing the protein degradation mechanisms that mediate protein quality control in cardiomyocytes, more study is needed to understand the players and processes involved in pathologic aggregation. Objective To uncover, in a non‐biased way, novel gene targets that reduce protein accumulation in a cellular model of proteotoxic heart disease. Methods and Results To address this, we developed a cellular model of cardiac protein aggregation in primary mouse cardiomyocytes by expressing a fluorescent aggregation‐prone protein, a mutated αλπηα−B‐crystallin (CryAB R120G ). The cells developed large intracellular aggregates, consistent with data from mice and humans. We subjected this cell model to a high‐content, genome‐wide RNAi knockdown screen using a lentiviral shRNA library (~ 16,000 genes). The effect of gene knockdown was calculated by measuring the quantity of punctate aggregate structures within cardiomyocytes. Using shRNA knockdown, we identified 236 genes in the mouse genome that could substantially ( > 50%) reduce aggregate content in cardiomyocytes. The hits span diverse and interesting cellular processes including mitochondrial and apoptotic cell death, calcium signaling, post‐translational modifications and metabolism and suggest overlapping therapeutic mechanisms between proteotoxicity and other cardiovascular pathologies. Conclusions While rigorous validation of our candidate list is ongoing, we have confirmed several candidates which can lead to reduction of proteotoxic inclusions in cardiomyocytes. We are confident that further interrogation of our candidate genes will contribute vital details on the molecular mechanisms of pathologic protein aggregation, as well as yield new and interesting therapeutic targets. Support or Funding Information This work is supported by funding from the National Institutes of Health (to PMM and JR) and by Le Fondation Leducq (to JR)