Regulation of BAG3 localization and sarcomeric protein turnover in the cardiac myocyte.

Abstract

Bcl2-associated athanogene-3 (BAG3) is a pro-autophagy co-chaperone that we have previously shown localizes to the cardiac sarcomere and is critical for proteostasis and maintenance of normal sarcomeric function. Indeed, BAG3 loss in heart failure (HF) results in accumulation of ubiquitinated sarcomeric proteins, and depressed maximum force generating capacity (Fmax). However, how BAG3 is regulated in the cell and how it coordinates sarcomeric protein turnover is unknown. During our analysis of human heart tissue, BAG3 appears as a “doublet”, with one band at 74 kDa (BAG3-L) and a second at a higher 84 kDa (BAG3-H). Previous studies hypothesized the BAG3-H band was due to phosphorylation. To test this, we treated human myocardial tissue with 10 units of alkaline phosphatase for 1 hour, which decreased troponin I phosphorylation by ∼60%, but had no impact on the intensity of BAG3-H or BAG3-L bands, suggesting these two bands are not due to phosphorylation. When we ran purified human BAG3 protein, it ran near the same molecular weight as the BAG3-H band, suggesting the lighter BAG3-L band is the modified version. These results suggest the BAG3-L band has been cleaved at either the N- or C-termini, which would be around 90 amino acids to cause the observed mass shift. Mass spectrometry analysis is being performed to determine the cleavage site and the domains being interrupted by this cleavage. Importantly, the BAG3-H/BAG3-L is higher in the myofilament compared to the cytoplasm of cardiomyocytes, suggesting the heavier form of BAG3 binds with sarcomere with increased affinity. We are also using high resolution live-cell imaging reveal the kinetics of the sarcomeric protein quality control system orchestrated by BAG3. These findings reveal novel regulation of the critical BAG3 protein and provides potential for BAG3-based HF therapies.