COMMENTARY The Journal of Clinical Investigation BAG3 plays a central role in proteostasis in the heart

Wataru Mizushima and Junichi Sadoshima Department of Cell Biology and Molecular Medicine, Cardiovascular Research Institute, Rutgers New Jersey Medical School, Newark, New Jersey, USA.

These mechanisms include aggresome formation through retrograde transport of Proteinopathies are characterized by the accumulation of misfolded misfolded proteins, which depends on the proteins, which ultimately interfere with normal cell function. While interaction of BAG3 with dynein motors neurological diseases, such as Huntington disease and Alzheimer disease, and the microtubule organization center are well-characterized proteinopathies, cardiac diseases have recently been (9), and stimulation of autophagy, which associated with alterations in proteostasis. In this issue of the JCI, Fang and depends on BAG3 interaction with HSPB8. colleagues demonstrate that mice with cardiac-specific deficiency of the Both retrograde transport and autophagy co-chaperone protein BCL2-associated athanogene 3 (BAG3) develop dilated facilitate the sequestration and removal of cardiomyopathy that is associated with a destabilization of small HSPs as irreversibly misfolded proteins. BAG3 is the result of a disrupted interaction between BAG3 and HSP70. Together, also involved in chaperone-assisted selec- the results of this study suggest that strategies to upregulate BAG3 during tive autophagy (CASA). CASA is a cargo-­ cardiac dysfunction may be beneficial. selective form of autophagy that is mediated through the HSC70-BAG3-HSPB8 complex and allows ubiquitination of selected pro- teins recognized by HSC70 via the CHIP Protein aggregates and disease suggest that proteotoxicity is also involved ubiquitin ligase and sequestration by auto- Protein homeostasis (proteostasis) is essen- in the pathogenesis of many heart diseases, phagosomes through the LC3 adapter p62/ tial for maintaining normal cell function (1, including cardiomyopathy and heart failure. SQSTM in the presence of synaptopodin 2 2). Proper protein activity is achieved through Cells possess several protein quality control or DNAJB6 (10). In muscles, BAG3 is partic- rigorous coordination of synthesis, folding, (PQC) mechanisms, comprising chaperones ularly important for maintaining the struc- and posttranslational modification, all of and protein degradation systems, including ture and integrity of sarcomeres through which are prone to malfunction in response the ubiquitin-proteasome system (UPS) and its interaction with HSC70 and an actin-­ to stress. For example, protein folding in the autophagy-lysosome system, that help min- capping sarcomeric protein (actin-capping endoplasmic reticulum (ER) is easily disrupt- imize proteotoxicity. Chaperones function protein β1) (11). In mice, a homozygous null ed in the presence of ER stress and oxidative as the first line of defense by either refolding mutation of Bag3 causes a fulminant form stress. Disruption of proteostasis induces misfolded proteins or by promoting the deg- of dilated cardiomyopathy (DCM) (12). In the accumulation of misfolded proteins, radation of misfolded or damaged proteins addition, a genome-wide association study which then interfere with the normal func- that are not suitable for refolding via the UPS (GWAS) identified several mutations in tion of cells through improper degradation, or autophagy. Accumulation of misfolded BAG3, including an E455K mutation in the gain and/or loss of function, and aggregate proteins occurs through dysregulation of evolutionarily conserved BAG domain, that formation. The adverse effects caused by PQC mechanisms, and the consequent pro- underlie a familial form of DCM (13). How- altered proteostasis are collectively termed teotoxicity develops through many different ever, the molecular mechanism through proteotoxicity, and pathological conditions mechanisms. In vivo animal models have which the loss of BAG3 function in cardio- resulting from proteotoxicity are collectively proven quite useful in elucidating the molec- myocytes leads to the development of DCM termed proteinopathies (3, 4). Proteostasis is ular mechanisms of proteinopathy (6). remains to be elucidated. particularly important for postmitotic cells, BCL2-associated athanogene 3 (BAG3) such as neurons and adult cardiomyocytes, is a co-chaperone protein that regulates the Loss of BAG3 function alters that have negligible regenerative poten- ATPase activity of the HSP70 family and is PQC in murine DCM tial, because in these mature cells, proteo- predominantly expressed in striated mus- In this issue, Fang and colleagues used toxicity is not readily diluted through cell cles (7, 8). BAG3 has been studied exten- mice with cardiac-specific deletion of division. The concept of proteinopathy was sively during the past decade, because, Bag3 (herein referred to as cKO mice) initially used to explain the pathogenesis together with small HSPB8, it directs the and mice with cardiac-specific expres- of neurodegenerative diseases, including HSP70 chaperone complex to other pro- sion of BAG3E455K (herein referred to as Huntington disease and Alzheimer disease teins involved in major cellular functions, cMUT mice) and demonstrated that loss (5); however, several lines of evidence now thereby mediating key PQC mechanisms. of function of BAG3 induces the develop- ment of DCM by attenuating PQC over Related Article: p. 3189 a specific subset of proteins involved in metabolic and contractile functions in Conflict of interest: The authors have declared that no conflict of interest exists. cardiomyocytes (14). Downregulation of Reference information: J Clin Invest. 2017;127(8):2900–2903. https://doi.org/10.1172/JCI95839. BAG3 or expression of BAG3E455K disrupt-

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Figure 1. Putative functions of BAG3 in the heart and the underlying mechanisms of cardiomyopathy caused by loss of BAG3 function. BAG3 plays a critical role in the PQC system in the heart. WT BAG3 forms multi-chaperone complexes with HSP70/HSC70 and sHSPs via its IPV motif and BAG3 domain. The BAG3-HSPB8-HSC70 complex binds to misfolded proteins and either promotes refolding or degradation of these misfolded proteins. Degradation is mediated primarily through general autophagy. The BAG3-HSPB8-HSC70 complex also maintains the structure and integrity of sarcomeres via CASA, a substrate-specific form of autophagy. In addition, BAG3 binds to dynein via the proline-rich motif (PXXP) and segregates misfolded proteins into aggresomes located in the perinuclear space through retrograde transport. The BAG3E455K mutation, which is located in the middle of the BAG domain, attenuates the interaction between BAG3 and HSP70/HSC70, leading to destabilization of sHSPs, including HSPB8. This disturbance of the chaperone complexes induces dysregulation of the refolding and degradation processes (autophagy and CASA), resulting in accumulation of BAG3 complex substrate proteins involved in metabolism and cardiac contraction in the detergent-insoluble fraction. Interestingly, aggresomes are not obvious in the BAG3 loss-of-function mouse model, possibly because of the defect in retrograde transport. Despite the absence of large protein aggregates, BAG3 loss-of-function mice develop DCM.

ed the interaction between BAG3 and dysfunction. This proposed contribution thy, overexpression of BAG3P209L induces HSP70 and consequently downregulated of small aggregates to cardiac dysfunction the formation of myofibrillar aggregates several small HSPs (sHSPs), including in cKO and cMUT mice is similar to a study that sequester functional WT BAG3, HSPB5, HSPB6, and HSPB8, through pro- that demonstrated that the presence of sol- thereby acting in a toxic gain-of-func- tein destabilization. As a result, both pro- uble preamyloid oligomers (PAOs) is suffi- tion manner (17). More extensive ultra- tein refolding and degradation of selected cient to induce cardiomyocyte dysfunction structural and biochemical analyses of HSP70-BAG3-HSPB8 complex–interact- in desmin cardiomyopathy mouse models the proteotoxicity associated with the ing proteins, many of which are involved (16). It is possible that aggresome forma- different BAG3 mutants and of the pres- in metabolism and cardiac contraction, tion through BAG3-mediated retrograde ence of preamyloid oligomers would be were suppressed, resulting in accumula- transport may even be an adaptive process helpful to address this issue. Alternative- tion of these proteins in the detergent-in- to protect cardiomyocytes from the toxici- ly, the depletion of some proteins in the soluble fraction (Figure 1). ty caused by small protein aggregates. soluble fraction may be more important Interestingly, large protein aggregates and directly cause the functional defi- were not observed in cKO or cMUT mice, Remaining questions and cit in the BAG3 loss-of-function mouse despite a drastic increase in the amount future directions models. Fang et al. show that a limited of proteins in the detergent-insoluble Although the study by Fang et al. clearly number of proteins involved in muscle fraction. As BAG3 and HSPB8 are critical suggests that proper coordination of the contraction, actin filament capping, pro- regulators of dynein-mediated retrograde actions of HSP70, BAG3, and HSPB8 is tein folding, mitochondrial organization, transport (15), aggresome formation may essential for the maintenance of PQC and cell junction assembly are shifted to be actively suppressed in these loss-of- in the adult heart at baseline, several the insoluble fraction as a result of BAG3 function mouse models. As mentioned important issues remain to be addressed. deficiency. Interestingly, there does not by Fang et al., the lack of aggresomes in For example, it remains unclear wheth- appear to be any compensation for the BAG3-deficient mice strongly suggests er the selected proteins that accumulate decreased protein level within the sol- that small and invisible protein aggregates, in the detergent-insoluble fraction act uble faction. Although BAG3 is capable rather than large protein aggregates, are as toxic molecules on their own. In a of compensating for the loss of proteins sufficient for the development of cardiac zebrafish model of myofibrillar myopa- degraded through autophagy by activat-

jci.org Volume 127 Number 8 August 2017 2901 COMMENTARY The Journal of Clinical Investigation ing mTOR or YAP/TAZ (18), these com- or isoleucine, have been shown to alter protein degradation and heart disease. J Mol Cell Cardiol. 2014;71:16–24. M81 pensatory mechanisms are apparently BAG3 function. Specifically, BAG3 has 4. Willis MS, Patterson C. Proteotoxicity and car- missing in the BAG3 loss-of-function been shown to have improved binding to diac dysfunction — Alzheimer’s disease of the mouse models. Future studies should test HSPB8 and to increase autophagy in limb heart? N Engl J Med. 2013;368(5):455–464. whether restoring the level of selected muscles in response to ischemia com- 5. Ross CA, Poirier MA. Protein aggregation proteins in the soluble fraction can res- pared with BAG3I81 (23). These results are and neurodegenerative disease. Nat Med. 2004;10(Suppl):S10–S17. cue cardiac dysfunction in the cKO and consistent with the notion that the PQC 6. Sanbe A, et al. Desmin-related cardiomyopathy cMUT mouse models. mechanisms controlled by endogenous in transgenic mice: a cardiac amyloidosis. Proc BAG3 has pleiotropic functions in BAG3 are involved in protection against Natl Acad Sci U S A. 2004;101(27):10132–10136. cardiomyocytes due to the presence of myocardial stress. In fact, upregulation 7. Behl C. Breaking BAG: The co-chaperone BAG3 multiple functional domains, including of BAG3 improves cardiac function in in health and disease. Trends Pharmacol Sci. 2016;37(8):672–688. the WW domain, the IPV (Ile-Pro-Val) a mouse model of chronic myocardi- 8. Knezevic T, et al. BAG3: a new player in motif, the proline-rich motif, and the BAG al infarction and reduces cardiomyo- the heart failure paradigm. Heart Fail Rev. domain, that interact with distinct sets of cyte death in response to ischemia and 2015;20(4):423–434. proteins. It is not clear how or which of reperfusion (8). Thus, interventions to 9. Gamerdinger M, Kaya AM, Wolfrum U, the different BAG3 domains contribute upregulate the level of endogenous BAG3 Clement AM, Behl C. BAG3 mediates chaper- to the overall cardiac phenotype observed should be further considered for treating one-based aggresome-targeting and selective autophagy of misfolded proteins. EMBO Rep. in the BAG3 loss-of-function mouse mod- diseases associated with cardiac stress. 2011;12(2):149–156. els, especially because these domains are Transcription of BAG3 is regulated by 10. Ulbricht A, et al. Cellular mechanotrans- involved in protein refolding, aggresome NF-κB and miR-371a-5p and in response duction relies on tension-induced and formation, protection against apoptosis, to physical exercise (8). Interestingly, chaperone-assisted autophagy. Curr Biol. and stimulation of both macroautophagy although both exercise and overexpres- 2013;23(5):430–435. 11. Hishiya A, Kitazawa T, Takayama S. BAG3 and CASA. For example, BAG3 inhibits sion of ATG7 attenuate intracellular and Hsc70 interact with actin capping apoptosis by interacting with the antia- protein aggregates by inducing autoph- protein CapZ to maintain myofibrillar poptotic protein BCL-2 through the BAG agy in desmin-related cardiomyopathy, integrity under mechanical stress. Circ Res. domain (19). Additional experiments are exercise and ATG7 overexpression have 2010;107(10):1220–1231. needed to determine whether cardiac the additive effects of improving cardiac 12. Homma S, Iwasaki M, Shelton GD, Engvall E, Reed JC, Takayama S. BAG3 deficiency results apoptosis contributes to the cardiac sys- function in another model of proteinopa- in fulminant myopathy and early lethality. Am J tolic dysfunction and heart failure found thy (24). Thus, combining multiple meth- Pathol. 2006;169(3):761–773. in BAG3-deficient mice. BAG3-mediat- ods to enhance autophagy may allow for 13. Villard E, et al. A genome-wide association study ed activation of general autophagy and better control of cardiomyopathy caused identifies two loci associated with heart failure CASA are expected to degrade different by loss of function of BAG3. due to dilated cardiomyopathy. Eur Heart J. 2011;32(9):1065–1076. sets of cargo targets. In addition, a loss 14. Fang X, et al. Loss-of-function mutations in of BAG3 function is often compensated Acknowledgments co-chaperone BAG3 destabilize small HSPs for by BAG1, which activates degradation The authors thank Daniela Zablocki (Rut- and cause cardiomyopathy. J Clin Invest. through the UPS (20). It would be inter- gers New Jersey Medical School, Newark, 2017;127(8):3189–3200. esting to elucidate how the loss of general NJ, USA) for critical reading of the manu- 15. Zhang X, Qian SB. Chaperone-mediated autophagy and CASA contributes to and script. This work was supported in part by hierarchical control in targeting misfold- ed proteins to aggresomes. Mol Biol Cell. how the compensatory activation of the US Public Health Service grants HL67724, 2011;22(18):3277–3288. UPS affects overall protein degradation in HL91469, HL102738, HL112330, and 16. Pattison JS, Sanbe A, Maloyan A, Osinska the BAG3 loss-of-function mice. AG23039 (to JS) and by the Leducq Foun- H, Klevitsky R, Robbins J. Cardiomyocyte Finally, BAG3 is downregulated in dation Transatlantic Network of Excel- expression of a polyglutamine preamyloid response to heart failure in both mice and lence (to JS). oligomer causes heart failure. Circulation. 2008;117(21):2743–2751. humans, as well as in a subset of patients 17. Ruparelia AA, Oorschot V, Vaz R, Ramm G, with idiopathic DCM (14, 21). Although Address correspondence to: Junichi Bryson-Richardson RJ. Zebrafish models of heart failure is often accompanied by Sadoshima, Rutgers New Jersey Medical BAG3 myofibrillar myopathy suggest a toxic gain attenuation of the PQC mechanisms and School, Department of Cell Biology and of function leading to BAG3 insufficiency. Acta increased protein misfolding, it remains Molecular Medicine, 185 South Orange Neuropathol. 2014;128(6):821–833. 18. Kathage B, et al. The cochaperone BAG3 to be determined whether downregula- Ave, MSB G-609, Newark, New Jersey coordinates protein synthesis and autopha- tion of BAG3 substantially contributes 07103, USA. Phone: 973.972.8920; Email: gy under mechanical strain through spatial to decreased PQC during heart failure. [email protected]. regulation of mTORC1. Biochim Biophys Acta. While BAG3 is a stress-responsive pro- 2017;1864(1):62–75. tein, its expression is reduced in epineph- 1. McLendon PM, Robbins J. Proteotoxicity and car- 19. Takayama S, Xie Z, Reed JC. 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