HSP70 alleviates sepsis-induced cardiomyopathy by attenuating mitochondrial dysfunction-initiated NLRP3 inflammasome-mediated pyroptosis in cardiomyocytes

Abstract

Background: Sepsis-induced cardiomyopathy (SIC) is an identified serious complication of sepsis that is associated with adverse outcomes and high mortality. Heat shock proteins (HSPs) have been implicated in suppressing septic inflammation. The aim of this study was to investigate whether HSP70 can attenuate cellular mitochondrial dysfunction, exuberated inflammation and inflammasome-mediated pyroptosis for SIC intervention. Methods: Mice with cecal ligation plus perforation (CLP) and lipopolysaccharide (LPS)-treated H9C2 cardiomyocytes were used as models of SIC. The mouse survival rate, gross profile, cardiac function, pathological changes and mitochondrial function were observed by photography, echocardiography, hematoxylin–eosin staining and transmission electron microscopy. In addition, cell proliferation and the levels of cardiac troponin I (cTnI), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were determined by Cell Counting Kit-8, crystal violet staining and enzyme-linked immunosorbent assay. Moreover, mitochondrial membrane potential was assessed by immunofluorescence staining, and dynamin-related protein 1 and pyroptosis-related molecules [nucleotide-binding domain, leucine-rich-repeat containing family pyrin domain-containing 3 (NLRP3), caspase-1, gasdermin-D (GSDMD), gasdermin-D N-terminal (GSDMD-N)] were measured by western blotting, immunoprecipitation and immunoblotting. Finally, hsp70.1 knockout mice with CLP were used to verify the effects of HSP70 on SIC and the underlying mechanism. Results: Models of SIC were successfully established, as reduced consciousness and activity with liparotrichia in CLP mice were observed, and the survival rate and cardiac ejection fraction (EF) were decreased; conversely, the levels of cTnI, TNF-α and IL-1β and myocardial tissue damage were increased in CLP mice. In addition, LPS stimulation resulted in a reduction in cell viability, mitochondrial destabilization and activation of NLRP3-mediated pyroptosis molecules in vitro. HSP70 treatment improved myocardial tissue damage, survival rate and cardiac dysfunction caused by CLP. Additionally, HSP70 intervention reversed LPS-induced mitochondrial destabilization, inhibited activation of the NLRP3 inflammasome, caspase-1, GSDMD and GSDMD-N, and decreased pyroptosis. Finally, knockout of hsp70.1 mice with CLP aggravated cardiac dysfunction and upregulated NLRP3 inflammasome activity, and exogenous HSP70 significantly rescued these changes. It was further confirmed that HSP70 plays a protective role in SIC by attenuating mitochondrial dysfunction and inactivating pyroptotic molecules. Conclusions: Our study demonstrated that mitochondrial destabilization and NLRP3 inflammasome activation-mediated pyroptosis are attributed to SIC. Interestingly, HSP70 ameliorates sepsis-induced myocardial dysfunction by improving mitochondrial dysfunction and inhibiting the activation of NLRP3 inflammasome-mediated pyroptosis, and such a result may provide approaches for novel therapies for SIC.

Keywords

HSP70 / Sepsis-induced cardiomyopathy / Mitochondria destabilization / Inflammation / NLRP3 inflammasome / Pyroptosis

References

[1] Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA. 2016; 315: 801-10.

[2] Zaky A, Deem S, Bendjelid K, Treggiari MM. Characterization of cardiac dysfunction in sepsis: an ongoing challenge. Shock. 2014; 41: 12-24. https://doi.org/10.1097/SHK.0000000000000065.

[3] Kakihana Y, Ito T, Nakahara M, Yamaguchi K, Yasuda T. Sepsis-induced myocardial dysfunction: pathophysiology and management. J Intensive Care. 2016; 4: 22. https://doi.org/10.1186/s40560-016-0148-1.

[4] Zaky A, Deem K, Fau-Bendjelid S, Bendjelid MM, Fau-Treggiari K, Treggiari MM. Characterization of cardiac dysfunction in sepsis: an ongoing challenge. Shock. 2014; 41: 12-24.

[5] Rocha M, Herance S, Fau-Rovira R, Rovira A, Fau-Hernández-Mijares S, Hernández-Mijares VM, et al. Mitochondrial dysfunction and antioxidant therapy in sepsis. Infect Disord Drug Targets. 2012; 12: 161-78.

[6] Kang R, Zeng L, Zhu S, Xie Y, Liu J, Wen Q, et al. Lipid peroxidation drives Gasdermin D-mediated Pyroptosis in lethal Polymicrobial sepsis. Cell Host Microbe. 2018; 24: 97-108.

[7] Yu X, Jia F, Fau-Wang B, Wang X, Fau-Lv F, Lv X, et al. α1 adrenoceptor activation by norepinephrine inhibits LPS-induced cardiomyocyte TNF-α production via modulating ERK1/2 and NF-κB pathway. J Cell Mol Med. 2014; 18: 263-73.

[8] Wang Y, Wang Y, Yang D, Yu X, Li H, Lv X, et al. β1-adrenoceptor stimulation promotes LPS-induced cardiomyocyte apoptosis through activating PKA and enhancing CaMKII and IκBα phosphorylation. Crit Care. 2015; 19: 76. https://doi.org/10.1186/s13054-015-0820-1.

[9] Yi RF, Lin JZ, Cui L, Zhang Q, Jia JZ, Lyu YL, et al. Role of hexokinase II in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro. Zhonghua Shao Shang Za Zhi. 2019; 35: 116-24.

[10] Shi J, Gao W, Shao F. Pyroptosis: Gasdermin-mediated programmed necrotic cell death. 2017; 42: 245-54.

[11] Xiang F, Xue DD, Luo J, Hu JH, Yuan LL, Jia JZ, et al. Effects and mechanism of mitochondrial transcription factor a and cytochrome c oxidase pathway in the energy production of hypoxic cardiomyocytes of rats regulated by tumor necrosis factor receptor associated protein 1. Zhonghua Shao Shang Za Zhi. 2020; 36: 651-7.

[12] Zhou R, Yazdi AS, Menu P, Tschopp J. A role for mitochondria in NLRP3 inflammasome activation. Nature. 2011; 469: 221-5. https://doi.org/10.1038/nature09663.

[13] Kelley N, Jeltema D, Duan Y, He Y. The NLRP3 Inflammasome: an overview of mechanisms of activation and regulation. Int J Mol Sci. 2019; 20: 3328. https://doi.org/10.3390/ijms20133328.

[14] Sun L, Ma W, Gao W, Xing Y, Chen L, Xia Z, et al. Propofol directly induces caspase-1-dependent macrophage pyroptosis through the NLRP3-ASC inflammasome. Cell Death Dis. 2019; 10: 542. https://doi.org/10.1038/s41419-019-1761-4.

[15] Qiu Z, Lei S, Zhao B, Wu Y, Su W, Liu M, et al. NLRP3 Inflammasome activation-mediated Pyroptosis aggravates myocardial ischemia/reperfusion injury in diabetic rats. Oxidative Med Cell Longev. 2017; 2017: 9743280. https://doi.org/10.1155/2017/9743280.

[16] Fernández-Fernández MR, Valpuesta JM. Hsp70 chaperone: a master player in protein homeostasis. F1000Res. 2018; 7:F1000. Faculty Rev-1497. https://doi.org/10.12688/f1000research.15528.1.

[17] Nillegoda NB, Kirstein J, Szlachcic A, Berynskyy M, Stank A, Stengel F, et al. Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature. 2015; 524: 247-51. https://doi.org/10.1038/nature14884.

[18] Leu JI, Barnoud T, Zhang G, Tian T, Wei Z, Herlyn M, et al. Inhibition of stress-inducible HSP70 impairs mitochondrial proteostasis and function. Oncotarget. 2017; 8, 28: 45656-69. https://doi.org/10.18632/oncotarget.17321.

[19] Yuan ZQ, Li XL, Peng YZ, Wang P, Huang YS, Yang ZC. [Influence of HSP70 on function and energy metabolism of mitochondria in intestinal epithelial cells after hypoxia/reoxygenation]. Zhonghua Shao Shang Za Zhi. 2008; 24(3): 203-6.

[20] Stankiewicz AR, Lachapelle G, Foo CP, Radicioni SM, Mosser DD. Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation. J Biol Chem. 2005; 280(46): 38729-39.

[21] Rumora L, Hlapčić I, Hulina-Tomašković A, Somborac-Bačura A, Bosnar M, Rajković MG. Pathogen-associated molecular patterns and extracellular Hsp70 interplay in NLRP3 inflammasome activation in monocytic and bronchial epithelial cellular models of COPD exacerbations. APMIS. 2021; 129: 80-90.

[22] Hawiger J, Veach RA, Zienkiewicz J. New paradigms in sepsis: from prevention to protection of failing microcirculation. J Thromb Haemost. 2015; 13: 1743-56.

[23] Wang X, Zhu Y, Zhou Q, Yan Y, Qu J, Ye H. Heat shock protein 70 expression protects against sepsis-associated cardiomyopathy by inhibiting autophagy. Hum Exp Toxicol. 2021; 40: 735-41.

[24] Vicencio JM, Yellon DM, Sivaraman V, Das D, Boi-Doku C, Arjun S, et al. Plasma exosomes protect the myocardium from ischemia-reperfusion injury. J Am Coll Cardiol. 2015; 65: 1525-36.

[25] Wick G, Knoflach M, Xu Q. Autoimmune and inflammatory mechanisms in atherosclerosis. Annu Rev Immunol. 2004; 22: 361-403. https://doi.org/10.1146/annurev.immunol.22.012703.104644.

[26] Dybdahl B, Slørdahl SA, Waage A, Kierulf P, Espevik T, Sundan A. Myocardial ischaemia and the inflammatory response: release of heat shock protein 70 after myocardial infarction. Heart. 2005; 91: 299-304.

[27] Gromadzka G, Zielińska J, Ryglewicz D, Fiszer U, Członkowska A. Elevated levels of anti-heat shock protein antibodies in patients with cerebral ischemia. Cerebrovasc Dis. 2001; 12: 235-9.

[28] Konstantinova EV, Chipigina NS, Shurdumova MH, Kovalenko EI, Sapozhnikov AM. Heat shock protein 70 kDa as a target for diagnostics and therapy of cardiovascular and cerebrovascular diseases. Curr Pharm Des. 2019; 25: 710-4.

[29] Wei S, Gao Y, Dai X, Fu W, Cai S, Fang H, et al. SIRT1-mediated HMGB1 deacetylation suppresses sepsis-associated acute kidney injury. Am J Physiol Renal Physiol. 2019; 316: F20-f31.

[30] Haileselassie B, Mukherjee R, Joshi AU, Napier BA, Massis LM, Ostberg NP, et al. Drp1/Fis1 interaction mediates mitochondrial dysfunction in septic cardiomyopathy. J Mol Cell Cardiol. 2019; 130: 160-9.

[31] Piquereau J, Godin R, Deschênes S, Bessi VL, Mofarrahi M, Hussain SN, et al. Protective role of PARK2/parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction. Autophagy. 2013; 9: 1837-51.

[32] Fang X, Wang J. Role of mitochondrial dysfunction in the pathogenesis of septic cardiomyopathy. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2018; 30: 189-92.

[33] Bock FJ, Tait SWG. Mitochondria as multifaceted regulators of cell death. Nat Rev Mol Cell Biol. 2020; 21: 85-100.

[34] Lin Y, Xu Y, Zhang Z. Sepsis-induced myocardial dysfunction (SIMD): the pathophysiological mechanisms and therapeutic strategies targeting mitochondria. Inflammation. 2020; 43: 1184-200.

[35] Paillard M, Tubbs E, Thiebaut PA, Gomez L, Fauconnier J, Da Silva CC, et al. Depressing mitochondria-reticulum interactions protects cardiomyocytes from lethal hypoxia-reoxygenation injury. Circulation. 2013; 128: 1555-65.

[36] Martine P, Chevriaux A, Derangère V, Apetoh L, Garrido C, Ghiringhelli F, et al. HSP70 is a negative regulator of NLRP3 inflammasome activation. Cell Death Dis. 2019; 10: 256. https://doi.org/10.1038/s41419-019-1491-7.

[37] Ullah M, Liu DD, Rai S, Concepcion W, Thakor AS. HSP70-mediated NLRP3 Inflammasome suppression underlies reversal of acute kidney injury following extracellular vesicle and focused ultrasound combination therapy. Int J Mol Sci. 2020; 21: 4085. https://doi.org/10.3390/ijms21114085.

[38] Qu J, Tao XY, Teng P, Zhang Y, Guo CL, Hu L, et al. Blocking ATP-sensitive potassium channel alleviates morphine tolerance by inhibiting HSP70-TLR4-NLRP3-mediated neuroinflammation. J Neuroinflammation. 2017; 14: 228. https://doi.org/10.1186/s12974-017-0997-0.

[39] Wang K, Sun Q, Zhong X, Zeng M, Zeng H, Shi X, et al. Structural mechanism for GSDMD targeting by autoprocessed caspases in Pyroptosis. Cell. 2020; 180: 941-955.e20.

[40] Komada T, Muruve DA. The role of inflammasomes in kidney disease. Nat Rev Nephrol. 2019; 15: 501-20.

[41] Neudorf H, Durrer C, Myette-Cote E, Makins C, O’Malley T, Little JP. Oral ketone supplementation acutely increases markers of NLRP3 Inflammasome activation in human monocytes. Mol Nutr Food Res. 2019; 63: e1801171. https://doi.org/10.1002/mnfr.201801171.

[42] Hollenberg SM, Singer M.Pathophysiology of sepsis-induced cardiomyopathy. Nat Rev Cardiol. 2021; 18: 424-34.

[43] Vinokurov M, Ostrov V, Yurinskaya M, Garbuz D, Murashev A, Antonova O, et al. Recombinant human Hsp70 protects against lipoteichoic acid-induced inflammation manifestations at the cellular and organismal levels. Cell Stress Chaperones. 2012; 17: 89-101.

[44] Dokladny K, Lobb W, Fau-Wharton R, Wharton TY, Fau-Ma W, Ma PL, et al. LPS-induced cytokine levels are repressed by elevated expression of HSP70 in rats: possible role of NF-kappaB. Cell Stress Chaperones. 2010; 15: 153-63.

[45] Zhang C, Liu X, Miao J, Wang S, Wu L, Yan D, et al. Heat shock protein 70 protects cardiomyocytes through suppressing SUMOylation and nucleus translocation of phosphorylated eukaryotic elongation factor 2 during myocardial ischemia and reperfusion. Apoptosis. 2017; 22: 608-25.