HSP70 co-chaperones regulate proteostasis of sarcomeric and z-disc proteins in hypertrophic cardiomyopathy

Objective: Recent Genome Wide Association Studies (GWAS) identified non-sarcomeric gene variants that are concordant with functional traits of Hypertrophic Cardiomyopathy (HCM), yet discordant with functional traits of Dilated Cardiomyopathy (DCM). Three of the top 10 risk alleles localize to genes in or near loci that encode co-chaperones of HSP70, specifically BAG3, DNAJC18, and HSPB7. Since these variants have opposite direction of effect in HCM and DCM, this led us to hypothesize that GWAS variants in HSP70 co-chaperones can affect the proteostasis of contractile proteins in the HCM heart. As a first step, we determined the effects each HSP70 co-chaperone knockdown (KD) has on sarcomeric and Z-disc protein expression and the poly-ubiquitinated state of cardiomyocyte-specific proteins. We further explored leveraging BAG3 and HSPB7 expression as a regulator of sarcomeric proteostasis.Methods and Results: Transduction of human iPSC-CMs (iPSC-CMs) with adenoviral vectors (Adv) driving shRNA expression against BAG3, DNAJC18, HSPB7, or scramble (control) yielded transduction efficiencies >75% and >80% reduction in target transcript 4 days post-transduction. Immunofluorescence (IF) confirmed BAG3 localization enriched in the myofilament, particularly in the Z-disc region. BAG3 KD increased poly-ubiquitinated proteins by 38+/-9% (p<0.001) and reduced steady state expression of most sarcomeric and Z-disc proteins, including: MyBP-C (p<0.0001), actin (p<0.0001), cardiac troponin I (p<0.05), tropomyosin (TPM1) (p<0.01), myosin light chain 3 (MYL3) (p<0.0001), filamin C (p<0.05), and alpha-actinin-2 (p<0.0001). IF showed diffuse cytosolic localization of DNAJC18 as opposed tothe myofilament. DNAJC18 KD increased poly-ubiquitinated protein by 66+/-17% (p<0.01) but did not impact sarcomeric or Z-disc protein expression. Conversely, HSPB7 KD reduced poly-ubiquitinated proteins by 21+/-7% (p<0.01) and increased protein expression of MyBP-C (p<0.01), TPM1 (p<0.05), and myosin light chain 2 (p<0.05). IF confirmed HSPB7 colocalizing with MyBP-C in the myofilament. Given BAG3 had the largest magnitude of effect on MyBP-C expression, we aimed to determine whether overexpression (OE) of BAG3 could be used to correct haploinsufficiency of MyBP-C, a main pathophysiological mechanism of patients with familial HCM due to truncating variants in MYBPC3. An AAV9 vector driving BAG3 expression was transduced into iPSC-CMs and yielded a transduction efficiency of 46% (p<0.0001) without inducing cellular toxicity. Transcript and protein were increased by 10 and 4.9 fold, respectively (p<0.001). BAG3 OE increased MyBP-C by 1.9 fold (p<0.01). Using a iPSC-CM line haploinsufficient for MYBPC3, BAG3 OE increased BAG3 levels by 3.8 fold (p<0.0001), and resulted in a 14.8% increase in MyBP-C. HSPB7 OE via AAV9 transduction resulted in a 54% transduction efficiency (p<0.0001) and increased HSPB7 protein expression by 17 fold (p<0.01) and transcript expression by 3.9 fold (p<0.001), without inducing cellular toxicity. Contrary to BAG3 OE, the OE of HSPB7 in MYBPC3 haploinsufficient CMs reduced BAG3 levels by 21%, with a concomitant reduction in MyBP-C levels of 26%. Conclusions: HSP70 co-chaperones have an established genetic association with human HCM and are important in regulating cardiomyocyte proteostasis. BAG3 has the largest magnitude effect as KD of BAG3 reduces MyBP-C expression, while OE of BAG3 increases MyBP-C. Viewing HCM as a polygenic disease could implicate sarcomere proteostasis as key therapeutic target. Disclosures: Dr. Day is supported by the NIH and Dr. Wagner was supported by the Brody Family Medical Trust Post-Doctoral Fellowship. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.