Introduction: Hypertrophic cardiomyopathy (HCM) is a cardiovascular disease that affects one in 500 people. A known cause of heart failure and sudden cardiac death, HCM is characterized by left ventricular (LV) hypertrophy (>13 mm LV end diastolic wall thickness). HCM is a genetically inherited disease in which 50% of HCM cases arise from allelic variances.  Approximately, 50% of familial HCM cases arise from variants in sarcomeric proteins.  MYBPC3, the gene that codes for MyBP-C protein, is the leading sarcomeric gene that harbors pathogenic variants. The majority of MYBPC3 variants result in premature termination codons, triggering nonsense-mediated mRNA decay or degradation of truncated protein through the ubiquitin-proteasome system (UPS). Accordingly, a reduction in transcript leads to reduced MyBP-C levels in hearts from patients with, suggesting haploinsufficiency as a pathogenic mechanism. Heat shock protein 70 kDa (HSP70) directs client proteins including MyBP-C toward stabilization or UPS-mediated degradation depending on the presence of certain co-chaperone proteins that bind to HSP70. Common variants in 3 of these co-chaperones have been shown to be among the top risk alleles associated with HCM: BAG3, DNAJC18, and HSPB7.

Aim and Hypothesis: I aim to identify whether co-chaperones of HSP70 (BAG3, DNAJC18, and HSPB7) modulate sarcomeric protein expression, specifically MyBP-C. I hypothesize that the knockdown (KD) of HSP70 co-chaperones BAG3 and HSPB7 compromise MyBP-C expression, while DNAJC18 KD will preserve MyBP-C expression.

Methods: Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) were transduced with GFP-tagged adenovirus (AdV) expressing shRNA targeted against BAG3, DNAJC18, HSPB7, with scrambled shRNA as a negative control at an MOI5. Transduction efficiency was measured via fluorescence microscopy and flow cytometry for GFP+ expression. Cellular toxicity following viral transduction was assessed via the CyQUANTTM LDH Cytotoxicity Assay per manufacturer’s protocol. Protein from the hiPC-CMs was isolated 4 days post viral transduction and quantified using the Bio-Rad DCTM Protein Assay. Protein expression was assessed via western blots to GAPDH.  Fold change was determined by comparing expression under knockdown conditions compared to scramble. Student’s t-test was used to determine statistical significance with a p≤0.05 deemed significant.

Key Results: KD of HSP70 co-chaperone BAG3 markedly reduced expression of multiple sarcomeric and Z-disc proteins, most markedly MyBP-C. HSPB7 KD increased MyBP-C and myosin expression while DNAJC18 had minimal to no effects on sarcomere protein content. I also observed that the co-chaperones regulated each other’s expression.

Key Words: Cardiology; Cardiovascular disease; Hypertrophic cardiomyopathy; Chaperones.