Hypertrophic Cardiomyopathy-Associated CRYABR123W Activates Calcineurin, Reduces Calcium Sequestration, 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 1:500 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 pathologic mutations. CRYABR123W was previously identified as a novel sarcomere-independent mutation causing HCM associated with pathologic 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 CRYABR123W has increased calcineurin activity. Using Alphafold to predict structural and interaction changes we generated a model where CRYABR123W 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 CRYABR123W. Finally, mouse hearts extracted from our wild type and CRYABR123W model 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 illustrates how CRYABR123W drives calcineurin activation and exhibits distinct changes in protein interaction and cellular pathways during the development of HCM and pathological cardiac hypertrophy.