Targeting co-chaperones of HSP70 to modulate Myosin Binding Protein C turnover in hypertrophic cardiomyopathy.

Abstract

Rationale: Hypertrophic Cardiomyopathy (HCM) is an inherited cardiovascular disease that affects 1 in 500 people. Over half of familial HCM cases arise from MyBP-C haploinsufficiency, thus stabilizing MyBP-C protein expression is an appealing therapeutic approach. Recently, two GWAS studies identified multiple HCM risk alleles. Three of the top 10 are localized near or in gene loci that code for HSP70 co-chaperones, highlighting the important role of the HSP70 system in HCM. Hypothesis: Co-chaperones of HSP70, (BAG3, DNAJC18, and HSPB7) regulate the expression and stability of sarcomeric and z-disc proteins, specifically MyBP-C. Methods/Results: Proximity labeling was performed to identify which components of the HSP70 co-chaperone network interact with or lie in close proximity to MyBP-C. Human iPSC-CMs (hiPSC-CMs) were transduced with a Turbo-ID-MYBPC3 adenoviral (AdV) construct, treated with biotin, and biotinylated proteins were captured by streptavidin beads. HSPA1A (i.e. HSP70), BAG3, DNAJC13, HSPB7, HSPB1, and CRYAB were identified by mass spectrometry as proximity proteins to wild-type MyBP-C but were absent in controls (AdV-MyBP-C without Turbo). hiPSC-CMs were transfected with AdV-shRNA to knock down HSP70 co-chaperone expression (BAG3, DNAJC18, or HSPB7). DNAJC18 was included because of the risk allele identified in GWAS, even though DNAJC18 was not identified as a direct interactor with MyBP-C during proximity labeling. Adv transfection efficiency was > 70% (p<0.0001) and resulted in a >75% decrease (p<0.01) in co-chaperone transcript expression and a >50% decrease (p<0.001) in protein. BAG3 knockdown decreased the steady state protein expression of MyBP-C (p<0.0001) and a majority of other sarcomeric proteins. Utilizing a cycloheximide (CHX) chase assay to measure protein ½ life, MyBP-C protein degradation was greatly accelerated by BAG3 knockdown compared to scrambled shRNA 24 hours post-CHX (p<0.0001). DNAJC18 knockdown had no significant effects on steady state protein expression of MyBP-C or other sarcomeric proteins, while HSPB7 knockdown increased MyBP-C (p<0.01), myosin (p<0.01), tropomyosin (p<0.05), and myosin light chain 2 (p<0.05) protein compared to scrambled shRNA. DNAJC18 and HSPB7 knockdown each increased BAG3 protein expression (p<0.01), while BAG3 knockdown reduced DNAJC18 protein (p<0.0001) but had no effect on HSPB7. Conclusion: MyBP-C interacts with and is stabilized by BAG3. MyBP-C may also be a direct client for HSPB7, but further studies are needed. Knockdown experiments of DNAJC13, HSBP1 and CRYAB, identified as proximity proteins of MyBP-C by Turbo-ID, are also underway. The HSP70 co-chaperone network may serve a therapeutic target to normalize MyBP-C levels in the context of MyBP-C haploinsufficiency. Institutional Start Up Funds This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.