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Cardiomyocyte Contractile Impairment in Heart Failure Results from Reduced BAG3-Mediated Sarcomeric Protein Turnover

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Cardiomyocyte Contractile Impairment in Heart Failure Results from Reduced BAG3-Mediated Sarcomeric Protein Turnover ARTICLE https://doi.org/10.1038/s41467-021-23272-z OPEN Cardiomyocyte contractile impairment in heart failure results from reduced BAG3-mediated sarcomeric protein turnover Thomas G. Martin1, Valerie D. Myers2, Praveen Dubey2, Shubham Dubey2, Edith Perez 1, ✉ Christine S. Moravec3, Monte S. Willis4, Arthur M. Feldman2 & Jonathan A. Kirk 1 fi 1234567890():,; The association between reduced myo lament force-generating capacity (Fmax) and heart failure (HF) is clear, however the underlying molecular mechanisms are poorly understood. Here, we show impaired Fmax arises from reduced BAG3-mediated sarcomere turnover. Myofilament BAG3 expression decreases in human HF and positively correlates with Fmax. We confirm this relationship using BAG3 haploinsufficient mice, which display reduced Fmax and increased myofilament ubiquitination, suggesting impaired protein turnover. We show cardiac BAG3 operates via chaperone-assisted selective autophagy (CASA), conserved from skeletal muscle, and confirm sarcomeric CASA complex localization is BAG3/proteotoxic stress-dependent. Using mass spectrometry, we characterize the myofilament CASA inter- actome in the human heart and identify eight clients of BAG3-mediated turnover. To determine if increasing BAG3 expression in HF can restore sarcomere proteostasis/Fmax,HF mice were treated with rAAV9-BAG3. Gene therapy fully rescued Fmax and CASA protein turnover after four weeks. Our findings indicate BAG3-mediated sarcomere turnover is fundamental for myofilament functional maintenance. 1 Department of Cell and Molecular Physiology, Loyola University Stritch School of Medicine, Maywood, IL, USA. 2 Department of Medicine, Temple University Lewis Katz School of Medicine, Philadelphia, PA, USA. 3 Department of Medicine, Cleveland Clinic Lerner College of Medicine, Cleveland, OH, ✉ USA. 4 Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN, USA. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:2942 | https://doi.org/10.1038/s41467-021-23272-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-23272-z eart failure is the leading cause of morbidity and mortality Table 1 Clinical characteristics of the human heart tissue in the industrialized world and is characterized by samples. H 1 impaired contractility and decreased cardiac output .At the cellular level, several factors have been implicated in the n = n = P development of heart failure, including defective calcium hand- Characteristic Nonfailing ( 9) DCM ( 21) -value ling, neurohormonal imbalance, and functional decline of the Age, (years), Mean ± SD 53.3 ± 8.9 55.5 ± 11.5 0.66 — 2–4 Female, n, (%) 4 (44.4) 8 (38.1) sarcomere the fundamental molecular unit of contraction . n The sarcomere is a highly organized protein complex that med- Ethnicity, , (%) fi White 8 (88.9) 16 (76.2) iates contraction through myosin and actin laments, which Black 1 (11.1) 4 (19.1) engage in the calcium-dependent crossbridge cycle. Increased fi Hispanic 0 (0.0) 1 (4.8) myo lament calcium sensitivity and decreased maximum force- LVEF, (%), Mean ± SD 57.5 ± 3.2 20.3 ± 2.2 <0.001 generating capacity (Fmax) are well-documented in heart failure5–10. However, while altered calcium sensitivity is uni- formly attributed to changes in site-specific post-translational effect of heart failure on these mechanisms are unknown. How- 11 modifications, most commonly phosphorylation of troponin I , ever, given the near universally observed depression of Fmax in the molecular mechanisms for decreased Fmax are incompletely systolic heart failure, sarcomeric PQC represents an attractive understood. therapeutic target. The sarcomere is under constant mechanical strain, which Here, we show that sarcomere protein turnover is impaired in predisposes its members to denaturation, and yet must maintain human heart failure samples with reduced Fmax. We verify the optimal mechanical function for decades in the terminally dif- presence of the CASA complex at the sarcomere in the human ferentiated adult cardiomyocyte. To preserve function, stress- heart, confirm its role in filamin-C turnover therein, and identify denatured sarcomere proteins must be refolded or targeted to seven novel clients of BAG3-mediated protein turnover. We degradation pathways by molecular chaperones so that new further show myofilament BAG3 expression decreases in human proteins can be incorporated12. However, sarcomeric protein DCM and correlates with functional decline, where lowest BAG3 quality control (PQC) is frequently impaired in heart failure, expression corresponds to weakest force generation. BAG3 hap- leading to toxic aggregation of misfolded/dysfunctional loinsufficient mice also exhibit this functional deficit and display proteins12. An arrested removal of dysfunctional sarcomere increased myofilament protein ubiquitination and reduced CASA proteins in heart failure may thus serve as a plausible mechanism protein expression. Finally, increasing BAG3 using adeno- for the observed decrease in Fmax. However, the distinct associated virus gene therapy in mice with heart failure restores fi fi mechanisms of sarcomere protein turnover in the adult heart are myo lament Fmax and sarcomere protein turnover. Our ndings poorly characterized, and the functional relevance of sarcomere indicate that impaired sarcomere protein turnover contributes to PQC has not been determined. decreased Fmax in heart failure and highlight BAG3 as an essential Bcl2-associated athanogene 3 (BAG3) is a heat shock protein factor for sarcomere functional maintenance. (HSP) co-chaperone that mediates protein degradation through autophagy13. Evidence in recent years indicates an essential role for BAG3 in the heart with numerous clinical studies finding that Results BAG3 mutations are associated with dilated cardiomyopathy Human DCM cardiomyocytes have reduced sarcomere con- (DCM) and myofibrillar myopathy14–20. Due to limitations with tractile function and impaired myofilament protein turnover. long-term culture of adult cardiomyocytes, mechanistic studies of Decreased contractility in heart failure has been linked to sarco- BAG3 have primarily been performed in immature neonatal mere dysfunction3. In cases of genetic heart failure, decreased myocytes, where BAG3 was shown to stabilize sarcomere struc- Fmax may be explained by structural alterations to proteins ture through maintenance of the actin-capping protein CapZβ21. involved in the crossbridge cycle, which are detrimental to their However, the role of BAG3 at the sarcomere in adult myocytes function29,30. However, functional deficits have also been under mechanical load is less understood and the association with observed in nongenetic heart failure6,7,9,29, which makes up the CapZβ does not appear to be conserved22. Moreover, sarcomere majority of cases. We therefore sought to determine the under- structure is unimpaired at the neonatal stage with germline BAG3 lying mechanism for myofilament dysfunction in heart failure knockout (KO), but rapidly disintegrates postnatum23. These using left ventricle (LV) samples from patients with idiopathic findings suggest separate or additional sarcomeric roles for BAG3 dilated cardiomyopathy (DCM) and heart failure (Table 1). in the adult. Using skinned myocytes from DCM and nonfailing (NF) fi fi An important study of BAG3 in the adult heart came from samples, we rst showed that myo lament Fmax decreased Fang et al. who showed BAG3 KO in mice caused DCM, desta- significantly in DCM (Fig. 1a, b). Consistent with prior studies bilized the small HSPs (HSPBs), and resulted in protein aggre- discussed above, the EC50 (calcium concentration required to gation, indicating impaired protein turnover24. Notably, BAG3 elicit half maximal force) also decreased in DCM, indicating KO in iPSC-derived cardiomyocytes caused reduced contractility increased myofilament calcium sensitivity (Fig. 1c). Since and altered expression of HSPs, suggesting sarcomeric functional impairment to PQC mechanisms is a common feature of heart relevance for BAG3-dependent PQC25. However, iPSC- failure31, we hypothesized that disrupted removal of misfolded/ cardiomyocytes also have immature morphology and are not dysfunctional sarcomere proteins might underly the observed under mechanical load, making functional inferences to the adult decrease in Fmax. With functioning PQC, misfolded proteins are problematic, especially at the sarcomere, which only fully matures tagged with ubiquitin and then removed through various protein under chronic load. While little is known regarding BAG3’s role degradation mechanisms12. Thus, increased ubiquitin levels may in the mature cardiac sarcomere, studies in mature skeletal serve as a readout for disrupted protein turnover, where proteins muscle identified BAG3 assembles with HSP70 and HSPB8, are effectively marked for degradation but fail to be turned over. forming the chaperone-assisted selective autophagy (CASA) To determine if sarcomere protein turnover was impaired, we complex, which mediates the turnover of the actin crosslinking assessed ubiquitination in myofilament-enriched fractions from protein filamin-C26–28. Whether CASA operates at the cardiac the DCM and NF tissue by western blot. As expected, we found sarcomere, the myofilament functional impact of BAG3, and the significantly increased ubiquitin at the myofilament in DCM, 2 NATURE COMMUNICATIONS | (2021) 12:2942 | https://doi.org/10.1038/s41467-021-23272-z | www.nature.com/naturecommunications NATURE COMMUNICATIONS
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