Increased calpain-1 in mitochondria induces dilated heart failure in mice: role of mitochondrial superoxide anion

  • Ting Cao
  • Shuai Fan
  • Dong Zheng
  • Grace Wang
  • Yong Yu
  • Ruizhen Chen
  • Long-Sheng Song
  • Guo-Chang Fan
  • Zhuxu Zhang
  • Tianqing PengEmail author
Original Contribution


We and others have reported that calpain-1 was increased in myocardial mitochondria from various animal models of heart disease. This study investigated whether constitutive up-regulation of calpain-1 restricted to mitochondria induced myocardial injury and heart failure and, if so, whether these phenotypes could be rescued by selective inhibition of mitochondrial superoxide production. Transgenic mice with human CAPN1 up-regulation restricted to mitochondria in cardiomyocytes (Tg-mtCapn1/tTA) were generated and characterized with low and high over-expression of transgenic human CAPN1 restricted to mitochondria, respectively. Transgenic up-regulation of mitochondria-targeted CAPN1 dose-dependently induced cardiac cell death, adverse myocardial remodeling, heart failure, and early death in mice, the changes of which were associated with mitochondrial dysfunction and mitochondrial superoxide generation. Importantly, a daily injection of mitochondria-targeted superoxide dismutase mimetics mito-TEMPO for 1 month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodeling and heart failure, and reduced mortality in Tg-mtCapn1/tTA mice. In contrast, administration of TEMPO did not achieve similar cardiac protection in transgenic mice. Furthermore, transgenic up-regulation of mitochondria-targeted CAPN1 induced a reduction of ATP5A1 protein and ATP synthase activity in hearts. In cultured cardiomyocytes, increased calpain-1 in mitochondria promoted mitochondrial permeability transition pore (mPTP) opening and induced cell death, which were prevented by over-expression of ATP5A1, mito-TEMPO or cyclosporin A, an inhibitor of mPTP opening. In conclusion, this study has provided direct evidence demonstrating that increased mitochondrial calpain-1 is an important mechanism contributing to myocardial injury and heart failure by disrupting ATP synthase, and promoting mitochondrial superoxide generation and mPTP opening.


Calpain Heart failure Mitochondria Superoxide onion ATP synthase 



This study was supported by operating grants from the National Natural Science Foundation of China (81,470,499 to T.P., 81521001 and 31570904 to R.C.), Natural Science Foundation of Jiangsu Province (BK20171216 to T.P.), and the Canadian Institutes of Health Research (MOP-133657 to T.P.).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

395_2019_726_MOESM1_ESM.pptx (282 kb)
Supplementary material 1 (PPTX 282 kb)


  1. 1.
    Barth E, Stammler G, Speiser B, Schaper J (1992) Ultrastructural quantitation of mitochondria and myofilaments in cardiac muscle from 10 different animal species including man. J Mol Cell Cardiol 24:669–681. CrossRefPubMedGoogle Scholar
  2. 2.
    Botker HE, Hausenloy D, Andreadou I, Antonucci S, Boengler K, Davidson SM, Deshwal S, Devaux Y, Di Lisa F, Di Sante M, Efentakis P, Femmino S, Garcia-Dorado D, Giricz Z, Ibanez B, Iliodromitis E, Kaludercic N, Kleinbongard P, Neuhauser M, Ovize M, Pagliaro P, Rahbek-Schmidt M, Ruiz-Meana M, Schluter KD, Schulz R, Skyschally A, Wilder C, Yellon DM, Ferdinandy P, Heusch G (2018) Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection. Basic Res Cardiol 113:39. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Brown DI, Griendling KK (2015) Regulation of signal transduction by reactive oxygen species in the cardiovascular system. Circ Res 116:531–549. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Bukowska A, Lendeckel U, Bode-Boger SM, Goette A (2012) Physiologic and pathophysiologic role of calpain: implications for the occurrence of atrial fibrillation. Cardiovasc Ther 30:e115–e127. CrossRefPubMedGoogle Scholar
  5. 5.
    Chen Q, Lesnefsky EJ (2015) Heart mitochondria and calpain 1: location, function, and targets. Biochim Biophys Acta 1852:2372–2378. CrossRefPubMedGoogle Scholar
  6. 6.
    Chen Q, Paillard M, Gomez L, Ross T, Hu Y, Xu A, Lesnefsky EJ (2011) Activation of mitochondrial mu-calpain increases AIF cleavage in cardiac mitochondria during ischemia-reperfusion. Biochem Biophys Res Commun 415:533–538. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Crompton M, Ellinger H, Costi A (1988) Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress. Biochem J 255:357–360. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Dai DF, Chen T, Szeto H, Nieves-Cintron M, Kutyavin V, Santana LF, Rabinovitch PS (2011) Mitochondrial targeted antioxidant peptide ameliorates hypertensive cardiomyopathy. J Am Col Cardiol 58:73–82. CrossRefGoogle Scholar
  9. 9.
    Dai DF, Hsieh EJ, Chen T, Menendez LG, Basisty NB, Tsai L, Beyer RP, Crispin DA, Shulman NJ, Szeto HH, Tian R, MacCoss MJ, Rabinovitch PS (2013) Global proteomics and pathway analysis of pressure-overload-induced heart failure and its attenuation by mitochondrial-targeted peptides. Circ Heart Fail 6:1067–1076. CrossRefPubMedGoogle Scholar
  10. 10.
    Dai DF, Johnson SC, Villarin JJ, Chin MT, Nieves-Cintron M, Chen T, Marcinek DJ, Dorn GW 2nd, Kang YJ, Prolla TA, Santana LF, Rabinovitch PS (2011) Mitochondrial oxidative stress mediates angiotensin II-induced cardiac hypertrophy and Galphaq overexpression-induced heart failure. Circ Res 108:837–846. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Dromparis P, Michelakis ED (2013) Mitochondria in vascular health and disease. Annu Rev Physiol 75:95–126. CrossRefPubMedGoogle Scholar
  12. 12.
    Fernandez-Sanz C, Ruiz-Meana M, Castellano J, Miro-Casas E, Nunez E, Inserte J, Vazquez J, Garcia-Dorado D (2015) Altered FoF1 ATP synthase and susceptibility to mitochondrial permeability transition pore during ischaemia and reperfusion in aging cardiomyocytes. Thromb Haemost 113:441–451. CrossRefPubMedGoogle Scholar
  13. 13.
    Gadicherla AK, Wang N, Bulic M, Agullo-Pascual E, Lissoni A, De Smet M, Delmar M, Bultynck G, Krysko DV, Camara A, Schluter KD, Schulz R, Kwok WM, Leybaert L (2017) Mitochondrial Cx43 hemichannels contribute to mitochondrial calcium entry and cell death in the heart. Basic Res Cardiol 112:27. CrossRefPubMedGoogle Scholar
  14. 14.
    Galvez AS, Diwan A, Odley AM, Hahn HS, Osinska H, Melendez JG, Robbins J, Lynch RA, Marreez Y, Dorn GW 2nd (2007) Cardiomyocyte degeneration with calpain deficiency reveals a critical role in protein homeostasis. Circ Res 100:1071–1078. CrossRefPubMedGoogle Scholar
  15. 15.
    Goll DE, Thompson VF, Li H, Wei W, Cong J (2003) The calpain system. Physiol Rev 83:731–801. CrossRefPubMedGoogle Scholar
  16. 16.
    Goncalves RL, Quinlan CL, Perevoshchikova IV, Hey-Mogensen M, Brand MD (2015) Sites of superoxide and hydrogen peroxide production by muscle mitochondria assessed ex vivo under conditions mimicking rest and exercise. J Biol Chem 290:209–227. CrossRefPubMedGoogle Scholar
  17. 17.
    Heusch G, Libby P, Gersh B, Yellon D, Bohm M, Lopaschuk G, Opie L (2014) Cardiovascular remodelling in coronary artery disease and heart failure. Lancet 383:1933–1943. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kang PT, Chen CL, Lin P, Chilian WM, Chen YR (2017) Impairment of pH gradient and membrane potential mediates redox dysfunction in the mitochondria of the post-ischemic heart. Basic Res Cardiol 112:36. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kar P, Samanta K, Shaikh S, Chowdhury A, Chakraborti T, Chakraborti S (2010) Mitochondrial calpain system: an overview. Arch Biochem Biophys 495:1–7. CrossRefPubMedGoogle Scholar
  20. 20.
    Li J, Zhu H, Shen E, Wan L, Arnold JM, Peng T (2010) Deficiency of rac1 blocks NADPH oxidase activation, inhibits endoplasmic reticulum stress, and reduces myocardial remodeling in a mouse model of type 1 diabetes. Diabetes 59:2033–2042. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Li S, Zhang L, Ni R, Cao T, Zheng D, Xiong S, Greer PA, Fan GC, Peng T (2016) Disruption of calpain reduces lipotoxicity-induced cardiac injury by preventing endoplasmic reticulum stress. Biochim Biophys Acta 1862:2023–2033. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Li X, Li Y, Shan L, Shen E, Chen R, Peng T (2009) Over-expression of calpastatin inhibits calpain activation and attenuates myocardial dysfunction during endotoxaemia. Cardiovasc Res 83:72–79. CrossRefPubMedGoogle Scholar
  23. 23.
    Li Y, Ma J, Zhu H, Singh M, Hill D, Greer PA, Arnold JM, Abel ED, Peng T (2011) Targeted inhibition of calpain reduces myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes. Diabetes 60:2985–2994. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ma J, Wei M, Wang Q, Li J, Wang H, Liu W, Lacefield JC, Greer PA, Karmazyn M, Fan GC, Peng T (2012) Deficiency of Capn4 gene inhibits nuclear factor-kappaB (NF-kappaB) protein signaling/inflammation and reduces remodeling after myocardial infarction. J Biol Chem 287:27480–27489. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mann PJ, Quastel JH (1946) Toxic effects of oxygen and of hydrogen peroxide on brain metabolism. Biochem J 40:139–144. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Merlo M, Caiffa T, Gobbo M, Adamo L, Sinagra G (2018) Reverse remodeling in dilated cardiomyopathy: insights and future perspectives. Int J Cardiol Heart Vasc 18:52–57. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Merlo M, Cannata A, Gobbo M, Stolfo D, Elliott PM, Sinagra G (2018) Evolving concepts in dilated cardiomyopathy. Eur J Heart Fail 20:228–239. CrossRefPubMedGoogle Scholar
  28. 28.
    Murphy MP (2009) How mitochondria produce reactive oxygen species. Biochem J 417:1–13. CrossRefPubMedGoogle Scholar
  29. 29.
    Ni R, Cao T, Xiong S, Ma J, Fan GC, Lacefield JC, Lu Y, Le Tissier S, Peng T (2016) Therapeutic inhibition of mitochondrial reactive oxygen species with mito-TEMPO reduces diabetic cardiomyopathy. Free Radic Biol Med 90:12–23. CrossRefPubMedGoogle Scholar
  30. 30.
    Ni R, Zheng D, Wang Q, Yu Y, Chen R, Sun T, Wang W, Fan GC, Greer PA, Gardiner RB, Peng T (2015) Deletion of capn4 protects the heart against endotoxemic injury by preventing ATP synthase disruption and inhibiting mitochondrial superoxide generation. Circ Heart Fail 8:988–996. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Ni R, Zheng D, Xiong S, Hill DJ, Sun T, Gardiner RB, Fan GC, Lu Y, Abel ED, Greer PA, Peng T (2016) Mitochondrial calpain-1 disrupts atp synthase and induces superoxide generation in type 1 diabetic hearts: a novel mechanism contributing to diabetic cardiomyopathy. Diabetes 65:255–268. CrossRefPubMedGoogle Scholar
  32. 32.
    Ono Y, Saido TC, Sorimachi H (2016) Calpain research for drug discovery: challenges and potential. Nat Rev Drug Discov 15:854–876. CrossRefPubMedGoogle Scholar
  33. 33.
    Poncelas M, Inserte J, Aluja D, Hernando V, Vilardosa U, Garcia-Dorado D (2017) Delayed, oral pharmacological inhibition of calpains attenuates adverse post-infarction remodelling. Cardiovasc Res 113:950–961. CrossRefPubMedGoogle Scholar
  34. 34.
    Sanbe A, Gulick J, Hanks MC, Liang Q, Osinska H, Robbins J (2003) Reengineering inducible cardiac-specific transgenesis with an attenuated myosin heavy chain promoter. Circ Res 92:609–616. CrossRefPubMedGoogle Scholar
  35. 35.
    Seidlmayer LK, Juettner VV, Kettlewell S, Pavlov EV, Blatter LA, Dedkova EN (2015) Distinct mPTP activation mechanisms in ischaemia–reperfusion: contributions of Ca2+, ROS, pH, and inorganic polyphosphate. Cardiovasc Res 106:237–248. CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Sheeran FL, Pepe S (2006) Energy deficiency in the failing heart: linking increased reactive oxygen species and disruption of oxidative phosphorylation rate. Biochim Biophys Acta 1757:543–552. CrossRefPubMedGoogle Scholar
  37. 37.
    Shintani-Ishida K, Yoshida K (2015) Mitochondrial m-calpain opens the mitochondrial permeability transition pore in ischemia–reperfusion. Int J Cardiol 197:26–32. CrossRefPubMedGoogle Scholar
  38. 38.
    Sorescu D, Griendling KK (2002) Reactive oxygen species, mitochondria, and NAD(P)H oxidases in the development and progression of heart failure. Congest Heart Fail 8:132–140. CrossRefPubMedGoogle Scholar
  39. 39.
    Teshima Y, Takahashi N, Nishio S, Saito S, Kondo H, Fukui A, Aoki K, Yufu K, Nakagawa M, Saikawa T (2014) Production of reactive oxygen species in the diabetic heart. Roles of mitochondria and NADPH oxidase. Circ J 78:300–306. CrossRefPubMedGoogle Scholar
  40. 40.
    Thompson J, Hu Y, Lesnefsky EJ, Chen Q (2016) Activation of mitochondrial calpain and increased cardiac injury: beyond AIF release. Am J Physiol Heart Circ Physiol 310:H376–H384. CrossRefPubMedGoogle Scholar
  41. 41.
    Xu T, Ding W, Ao X, Chu X, Wan Q, Wang Y, Xiao D, Yu W, Li M, Yu F, Wang J (2019) ARC regulates programmed necrosis and myocardial ischemia/reperfusion injury through the inhibition of mPTP opening. Redox Biol 20:414–426. CrossRefPubMedGoogle Scholar
  42. 42.
    Yang D, Ma S, Tan Y, Li D, Tang B, Zhang X, Sun M, Yang Y (2010) Increased expression of calpain and elevated activity of calcineurin in the myocardium of patients with congestive heart failure. Int J Mol Med 26:159–164. CrossRefPubMedGoogle Scholar
  43. 43.
    Zhu H, Shan L, Schiller PW, Mai A, Peng T (2010) Histone deacetylase-3 activation promotes tumor necrosis factor-alpha (TNF-alpha) expression in cardiomyocytes during lipopolysaccharide stimulation. J Biol Chem 285:9429–9436. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Ting Cao
    • 1
  • Shuai Fan
    • 1
  • Dong Zheng
    • 1
    • 2
    • 3
    • 8
  • Grace Wang
    • 4
  • Yong Yu
    • 5
  • Ruizhen Chen
    • 5
  • Long-Sheng Song
    • 6
  • Guo-Chang Fan
    • 7
  • Zhuxu Zhang
    • 3
    • 8
  • Tianqing Peng
    • 1
    • 2
    • 3
    • 8
    Email author
  1. 1.Institutes of Biology and Medical SciencesSoochow UniversitySuzhouChina
  2. 2.Critical Illness ResearchLawson Health Research Institute, London Health Sciences CentreLondonCanada
  3. 3.Department of MedicineUniversity of Western OntarioLondonCanada
  4. 4.Faculty of MedicineUniversity of TorontoTorontoCanada
  5. 5.Shanghai Institute of Cardiovascular Diseases, Shanghai Zhongshan HospitalFudan UniversityShanghaiChina
  6. 6.Division of Cardiovascular Medicine, Department of Internal Medicine, François M. Abboud Cardiovascular Research CenterUniversity of Iowa Carver College of MedicineIowa CityUSA
  7. 7.Department of Pharmacology and Systems PhysiologyUniversity of Cincinnati College of MedicineCincinnatiUSA
  8. 8.Department of Pathology and Laboratory MedicineUniversity of Western OntarioLondonCanada

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