Jonathan A. Kirk

Background Mutations to the co‐chaperone protein BAG3 (B‐cell lymphoma‐2–associated athanogene‐3) are a leading cause of dilated cardiomyopathy (DCM). These mutations often impact the C‐terminal BAG domain (residues 420–499), which regulates heat shock protein 70‐dependent protein turnover via autophagy. While mutations in other regions are less common, previous studies in patients with DCM found that co‐occurrence of 2 BAG3 variants (P63A, P380S) led to worse prognosis. However, the underlying mechanism for dysfunction is not fully understood. Methods and Results In this study, we used proteomics, Western blots, and myofilament functional assays on left ventricular tissue from patients with nonfailing, DCM, and DCM with BAG3 63/380 to determine how these mutations impact protein quality control and cardiomyocyte contractile function. We found dysregulated autophagy and increased protein ubiquitination in patients with BAG3 63/380 compared with nonfailing and DCM, suggesting impaired protein turnover. Expression and myofilament localization of BAG3‐binding proteins were also uniquely altered in the BAG3, 63/380 including abolished localization of the small heat shock protein CRYAB (alpha‐crystallin B chain) to the sarcomere. To determine whether these variants impacted sarcomere function, we used cardiomyocyte force‐calcium assays and found reduced maximal calcium‐activated force in DCM and BAG3 63/380 . Interestingly, myofilament calcium sensitivity was increased in DCM but not with BAG3 63/380 , which was not explained by differences in troponin I phosphorylation. Conclusions Together, our data support that the disease‐enhancing mechanism for BAG3 variants outside of the BAG domain is through disrupted protein turnover leading to compromised sarcomere function. These findings suggest a shared mechanism of disease among pathogenic BAG3 variants, regardless of location.