The co-chaperone Bcl2-associated athanogene-3 (BAG3) maintains cellular protein quality control through the regulation of heat shock protein 70 (HSP70). Cancer cells manipulate BAG3-HSP70-regulated pathways for tumor initiation and proliferation, which has led to the development of promising small molecule therapies, such as JG-98, which inhibit the BAG3-HSP70 interaction and mitigate tumor growth. However, it is not known how these broad therapies impact cardiomyocytes, where the BAG3-HSP70 complex is a key regulator of protein turnover and contractility. Here, we show that JG-98 exposure is toxic in neonatal rat ventricular myocytes (NRVMs). Using immunofluorescence microscopy to assess cell death, we found that apoptosis increased in NRVMs treated with JG-98 doses as low as 10 nM. JG-98 treatment also reduced autophagy flux and altered expression of BAG3 and several binding partners involved in BAG3-dependent autophagy, including SYNPO2 and HSPB8. We next assessed protein half-life with disruption of the BAG3-HSP70 complex by treating with JG-98 in the presence of cycloheximide and found BAG3, HSPB5, and HSPB8 half-lives were reduced, indicating that complex formation with HSP70 is important for their stability. Next, we assessed sarcomere structure using super-resolution microscopy and found that disrupting the interaction with HSP70 leads to sarcomere structural disintegration. To determine whether the effects of JG-98 could be mitigated by pharmacological autophagy induction, we cotreated NRVMs with rapamycin, which partially reduced the extent of apoptosis and sarcomere disarray. Finally, we investigated whether the effects of JG-98 extended to skeletal myocytes using C2C12 myotubes and found again increased apoptosis and reduced autophagic flux. Together, our data suggest that nonspecific targeting of the BAG3-HSP70 complex to treat cancer may be detrimental for cardiac and skeletal myocytes.

中文翻译：

---

拟议的癌症治疗剂 JG-98 对 BAG3-HSP70 的药理学抑制对心肌细胞有毒

共伴侣 Bcl2 相关 athanogene-3 (BAG3) 通过调节热休克蛋白 70 (HSP70) 维持细胞蛋白质质量控​​制。癌细胞操纵 BAG3-HSP70 调节的肿瘤起始和增殖途径，这导致了有前途的小分子疗法的发展，例如 JG-98，它抑制 BAG3-HSP70 相互作用并减缓肿瘤生长。然而，尚不清楚这些广泛的疗法如何影响心肌细胞，其中 BAG3-HSP70 复合物是蛋白质周转和收缩力的关键调节因子。在这里，我们表明 JG-98 暴露对新生大鼠心室肌细胞 (NRVM) 有毒。使用免疫荧光显微镜评估细胞死亡，我们发现用低至 10 nM 的 JG-98 剂量处理的 NRVM 细胞凋亡增加。JG-98 治疗还降低了自噬通量并改变了 BAG3 和参与 BAG3 依赖性自噬的几种结合伙伴的表达，包括 SYNPO2 和 HSPB8。接下来，我们通过在环己酰亚胺存在下用 JG-98 处理来评估 BAG3-HSP70 复合物破坏的蛋白质半衰期，发现 BAG3、HSPB5 和 HSPB8 半衰期缩短，表明与 HSP70 形成复合物对它们很重要稳定。接下来，我们使用超分辨率显微镜评估了肌节结构，发现破坏与 HSP70 的相互作用会导致肌节结构崩解。为了确定 JG-98 的作用是否可以通过药理学自噬诱导来减轻，我们用雷帕霉素共同处理 NRVM，这部分减少了细胞凋亡和肌节紊乱的程度。最后，我们使用 C2C12 肌管研究了 JG-98 的作用是否扩展到骨骼肌细胞，并再次发现细胞凋亡增加和自噬通量减少。总之，我们的数据表明，非特异性靶向 BAG3-HSP70 复合物来治疗癌症可能对心脏和骨骼肌细胞有害。