Hypertrophic cardiomyopathy (HCM) is the most common genetically inherited cardiovascular disease that affects one in 500 of the general population.1* HCM is marked by left ventricle muscle thickening, or hypertrophy, and it is a cause of heart failure and sudden cardiac death.2 Among the roughly 900 pathogenic HCM variants,3 variants in MYBPC3–which encodes for the sarcomere protein myosin binding protein-C (MyBP-C)–are present in 50% of familial HCM cases.4 In hearts from patients with a MYBPC3 truncating allele, levels of MyBP-C are reduced, suggesting haploinsufficiency as the pathogenic mechanism.6 The 70-kDa heat shock protein (HSP70) co-chaperone network directs truncated MyBP-C towards degradation mediated by the ubiquitin proteasome system,5 making HSP70 a potential therapeutic target to restore levels of WT MyBP-C.

This research used a chemical probe called JG-231 in vitro and in vivo to allosterically inhibit a protein-protein interaction (PPI) between HSP70 and the co-chaperone bcl-2 associated athanogene (BAG) proteins.9Through this inhibition, JG-231 results in a marked decrease in MyBP-C levels, which provides valuable insight into the mechanisms of the HSP70 co-chaperone network. After applying JG-231 to wild-type (WT) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM), I measured levels of sarcomere proteins at varying concentrations of JG-231. Proteins that were significantly increased or decreased were considered as additional client proteins of HSP70. I also assessed the efficacy of JG-231 in vivo by injecting it into WT and MYBPC3 truncating heterozygous mice and measuring MyBP-C levels, which are known to be decreased by JG-231 in vitro but have never been tested before in vivo.

Before testing JG-231, I identified certain antibodies that yielded a strong concentration-dependent signal to quantify proteins of interest via the AlphaLISA immunoassay in untreated WT iPSC-CMs. I then measured the levels of those proteins with JG-231 on WT iPSC-CMs, and found that cardiac troponin T (TNNT2) displayed a JG-231 concentration-dependent reduction, suggesting that TNNT2 may be an additional client of HSP70. However, filamin-C and myosin levels increased with increasing concentrations of JG-231, suggesting that they may be clients of HSP70 that were stabilized by JG-231 instead of being degraded. To test the efficacy of JG-231 in vivo, I injected 12 MYBPC3 WT and heterozygous truncating mice with the same dosage of JG-231 or a vehicle control (4 mg/kg 3 days a week) over four weeks and found that there was no significant difference in MyBP-C levels between the JG-231-treated and the vehicle-treated mice, indicating a need for further testing of alternative JG-231 dosages in vivo.

These findings provide valuable insight into the HSP70 co-chaperone network, guiding future research into restoring MyBP-C levels in HCM patients.

As my first research experience, PURM has given me valuable experience in both wet lab techniques, data analysis and visualization, and communicating my research to a variety of audiences. PURM also sparked my newfound curiosity in research, which I plan on continuing throughout my academic career.

Lastly, I would like to thank my faculty mentor, Dr. Sharlene Day and lab manager Jaime Yob for their continued support throughout my PURM research fellowship this summer.

*See references in poster.