Hypertrophic Cardiomyopathy-Associated CRYAB(R123W) Activates Calcineurin, Reduces Calcium Sequestration, and Alters the CRYAB Interactome and the Proteomic Response to Pathological Hypertrophy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular condition in the world, affecting around 1 in 500 people. HCM is characterized by ventricular wall thickening, decreased ventricular chamber volume, and diastolic dysfunction. Inherited HCM is most commonly caused by sarcomere gene mutations; however, approximately 50% of patients do not present with a known mutation, highlighting the need for further research into additional pathological mutations. The alpha-B crystallin (CRYAB) mutation CRYAB(R123W) was previously identified as a novel sarcomere-independent mutation causing HCM associated with pathological NFAT signaling in the setting of pressure overload. We generated stable H9C2 cell lines expressing FLAG-tagged wild-type and mutant CRYAB, which demonstrated that CRYAB(R123W) increases calcineurin activity. Using AlphaFold to predict structural and interaction changes, we generated a model where CRYAB(R123W) uniquely binds to the autoinhibitory domain of calcineurin. Co-immunoprecipitation using the CRYAB FLAG tag followed by mass spectrometry showed novel and distinct changes in the protein interaction patterns of CRYAB(R123W). Finally, mouse heart extracts from our wild-type CRYAB and CRYAB(R123W) models with and without pressure overload caused by transverse aortic constriction (TAC) were used in global proteomic and phosphoproteomic mass spectrometry analysis, which showed dysregulation in cytoskeletal, metabolomic, cardiac, and immune function. Our data illustrate how CRYAB(R123W) drives calcineurin activation and exhibits distinct changes in protein interaction and cellular pathways during the development of HCM and pathological cardiac hypertrophy.