BAG3 Localizes to the Mature Sarcomere and Maintains Myofilament Function

Abstract

Bcl-2-associated athanogene 3 (BAG3) is a co-chaperone canonically involved in protein quality control (PQC). Decreased levels and mutations to BAG3 are linked with heart failure, though the mechanisms remain unclear. Neonatal myocyte studies showed BAG3 localizes to the Z-disk, however, the functional role of BAG3 at the sarcomere is unknown. First, we confirmed BAG3 co-localization with Z-disk α-actinin in the mature myofilament using immunofluorescence in human left ventricular (LV) cardiomyocytes. Furthermore, by western blot this myofilament-fraction of BAG3 decreased by ∼20% in human heart failure. To test the hypothesis that decreased myofilament-bound BAG3 is detrimental to function, we used a mouse model of myocardial infarction (MI)-induced heart failure. Eight weeks post-MI, BAG3 was overexpressed via associated adenovirus-9 for three weeks. Myofilament function was assessed in LV cardiomyocytes. Compared with sham, MI decreased maximal calcium-induced force (Fmax). However, BAG3 overexpression restored Fmax to sham levels. To explore the mechanism responsible, we identified BAG3's myofilament interactome using immunoprecipitation and mass spectrometry. Interestingly, Hsp70 and HspB8, which together with BAG3 promote PQC in other cell types, were among the top hits. Association of this complex with the myofilament was confirmed by co-immunoprecipitation and immunofluorescence showing colocalization of HspB8 and Hsp70 at the Z-disk, suggesting a role for the complex in myofilament PQC. To support the functional significance of this complex, myofilament function was assessed in mice expressing the BAG3 P209L mutant, previously shown to impair client processing by the BAG3-Hsp complex. P209L mice displayed age-dependent depression of myofilament function. This work is the first to show BAG3 localizes to the mature sarcomere and modulates myofilament function in cardiomyocytes, which is disrupted by MI and mutation. Additionally, our data suggests that the maintenance of function following cardiac stress may be due to myofilament PQC.