Dysregulated Autophagy and Sarcomere Dysfunction in Patients With Heart Failure With Co-Occurrence of P63A and P380S BAG3 Variants

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 BAG(363/380) to determine how these mutations impact protein quality control and cardiomyocyte contractile function. We found dysregulated autophagy and increased protein ubiquitination in patients with BAG(363/380) 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 BAG(363/380). Interestingly, myofilament calcium sensitivity was increased in DCM but not with BAG(363/380), 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.