Abstract P1030: Alpha Kinase 3 Signaling At The M-band Regulates Sarcomeric Protein Quality Networks

Abstract

Objectives: Striated muscle contraction is driven by molecular machinery composed of specialized cytoskeletal proteins, called the sarcomere. Pathogenic variants in genes that encode these sarcomeric proteins trigger a heart disease called cardiomyopathy, which causes abnormal growth and function of the heart muscle and can cause sudden death. However, there is a growing appreciation that genetic mutations in genes not encoding the traditional sarcomeric proteins also substantially contribute to cardiomyopathy. One such gene is ALPK3 , encoding an atypical alpha kinase. Despite its strong link to cardiomyopathy, there have been no studies to define its molecular function. Therefore, the objective of our study was to understand the genetic basis of ALPK3- induced cardiomyopathy and define its molecular function in health and disease. Methods & Results: We created a suite of gene-edited human pluripotent stem cells (hPSCs) carrying endogenous fusion proteins, loss of function mutations, or disease-causing patient variants, and differentiated these hPSC lines into human ventricular muscle cells. Using reporter hPSCs, we found that ALPK3 is localized to the M-band of the cardiac sarcomere, in contrast to its posited nuclear localization. ALPK3 deficiency impaired muscle cell function and sarcomere organization in both hPSC cardiac organoids and mice harboring a pathogenic ALPK3 variant. We performed multiomic analysis on ALPK3-deficient hPSC cardiomyocytes to define the function of ALPK3 and observed consistent enrichment of sarcomere and protein quality control pathways. Interactomics demonstrated that ALPK3 links the cytoskeleton to protein surveillance networks at the M-Band. Notably, the ubiquitin-binding protein SQSTM1 bound ALPK3, could be phosphorylated by ALPK3, and was mislocalized from the sarcomere in three independent ALPK3 patient lines. Conclusions: Together, our data demonstrate that ALPK3 regulates the link between the sarcomere and protein surveillance system and that its deficiency impairs cardiac function via this mechanism. This work highlights a potential new pathway to target for therapy development and treatment of patients with cardiomyopathy.