Abstract 10321: Disrupting the Bag3-hsp70 Complex with the Proposed Cancer Therapeutic Jg-98 is Detrimental for Cardiomyocytes

Abstract

The heat shock protein 70 (HSP70) co-chaperone Bcl-2-associated athanogene-3 (BAG3) is critical for cell viability through facilitating HSP70-dependent protein quality control (PQC). Unfortunately, the same vital functions of BAG3-HSP70 in healthy cells are known to promote cell survival and proliferation in various cancers. Recent promising studies found that disrupting BAG3-HSP70 with the small molecule JG-98 has potent anti-cancer effects in cultured cells and in vivo . However, it is unknown what impact this therapy has on other cell types such as cardiomyocytes, where BAG3-mediated PQC is important for function. Using neonatal rat ventricular myocytes (NRVMs), we assessed the impact of JG-98 on cardiomyocyte viability and found apoptosis increased, even at doses 10-100-fold lower than those used with cancer cells. Next, we measured autophagic flux using ammonium chloride to block lysosomal degradation and found flux was reduced with JG-98. To explore mechanism, we assessed expression of BAG3, HSP70, and three other proteins that assemble with the complex during autophagy (HSPB5, HSPB8, SYNPO2). We found BAG3, HSPB8, HSPB5, and SYNPO2 expression decreased at the protein and transcript level with JG-98, while HSP70 expression was not changed. Next, we tested whether the BAG3-HSP70 interaction was important for protein stability of the complex members by treating NRVMs with JG-98 in the presence of cycloheximide to block translation. JG-98 decreased the protein half-life of BAG3, HSPB8, and HSPB5, indicating that protein stability was reduced, while SYNPO2 and HSP70 half-lives were unaffected. As we previously showed BAG3-HSP70 was important for maintaining sarcomere PQC, we next assessed the impact of JG-98 on sarcomere structure. In agreement, we found here that JG-98 caused sarcomere structural disintegration, which was exacerbated with stress. Finally, the detrimental effects of JG-98 on cell viability and sarcomere structure could be partially ameliorated with autophagy induction by rapamycin. Taken together, our findings indicate the importance of BAG3-HSP70 in cardiomyocytes and caution against broadly targeting this complex therapeutically.