Identifying the Role of HSP70 Co-Chaperones in Modulating Sarcomeric Proteostasis

Branscom G,1 Wagner, M.J.,1 and Day, S.M.1

1Dept. Of Medicine, Cardiovascular Division, University of Pennsylvania

Abstract

Background: Hypertrophic cardiomyopathy (HCM) is the most common genetically inherited cardiovascular disease that affects one in 500 people. Variants in MYBPC3 result in premature termination codons, ultimately causing haploinsufficiency in MyBP-C protein–a hallmark of familial HCM pathophysiology. Recent GWAS reports identify risk alleles for HCM that are concordant with left ventricular (LV) functional traits of decreased left ventricular chamber volumes and increased ejection fraction, and in contrast are protective alleles for dilated cardiomyopathy. Several of the top HCM risk alleles encode for co-chaperones of HSP70. However, how these co-chaperones directly modulate sarcomeric protein turnover and HCM pathophysiology is unknown.

Hypothesis: We hypothesize that HSP70 co-chaperones identified by recent GWAS regulate contractility by affecting sarcomeric and Z-disc steady state expression.

Methods and Results: Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) were transduced with GFP-tagged adenovirus (AdV) expressing shRNA targeted against BAG3, DNAJC18, HSPB7, or scrambled shRNA (control) and transduction efficiency was assessed by flow cytometry for GFP expression. A >70% transduction efficiency was achieved with each viral construct (p<0.0001). Viral transduction and knockdown of HSP70 co-chaperone did not induce cellular toxicity. BAG3, DNAJC18, and HSPB7 were reduced by ≥50% at both the transcript and protein levels as measured via RT-qPCR and western blot. BAG3 KD reduced MyBP-C protein levels (p<0.0001) by 53%, along with most other profiled sarcomeric and Z-disk proteins (p<0.05). DNAJC18 had no significant effect on MyBP-C protein levels, but caused a decrease in tropomyosin (p<0.0001) and myosin light chain 3 (p<0.0001). HSPB7 KD increased MyBP-C protein levels (p<0.01) by 46%. Myosin (p<0.01), tropomyosin (p<0.05), and myosin light chain 2 (p<0.05) also showed increased expression upon HSPB7 knockdown.

Conclusion: GWAS-identified HSP70 co-chaperones BAG3, DNAJC18, and HSPB7 regulate the steady state expression of sarcomeric and Z-disc proteins. Further studies will be needed to elucidate whether regulation is occurring at the transcription, translational, or posttranslational level, and whether therapeutic modulation of these co-chaperones can be leveraged to stabilize MyBP-C and other sarcomeric and Z-disc proteins in patients with familial HCM.