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Journal of Geriatric Cardiology 2023, 20(1): 68-82 https://doi.org/10.26599/1671-5411.2023.01.001
Research Article | Open Access | Issue | Published: 06 February 2023

Crocetin protects cardiomyocytes against hypoxia/reoxygenation injury by attenuating Drp1-mediated mitochondrial fission via PGC-1α

Show Author's Information You-Ling MOU1,*,  Rui ZHAO1,*,  Shi-Ying LYU1,  Zi-Yi ZHANG1,  Mei-Fei ZHU2( ),  Qian LIU1( )
School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, China
Department of Critical Care Medicine, the First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China

*The authors contributed equally to this manuscript

Cite this article:
MOU Y-L, ZHAO R, LYU S-Y, et al. Crocetin protects cardiomyocytes against hypoxia/reoxygenation injury by attenuating Drp1-mediated mitochondrial fission via PGC-1α. Journal of Geriatric Cardiology, 2023, 20(1): 68-82. https://doi.org/10.26599/1671-5411.2023.01.001
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Abstract Full text About this article
BACKGROUND

Saffron (Crocus sativus L.) has been traditionally used as food, spice, and medicine. Crocetin (CRT), as main bioactive component of saffron, has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion (I/R) injury. However, the mechanisms are poorly explored. This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation (H/R) and elucidated the possible underlying mechanism.

METHODS

H/R attack was performed on H9c2 cells. Cell counting kit-8 was used to detect the cell viability. Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and cellular adenosine triphosphate (ATP) content. Various fluorescent probes were used to detect cell apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) content, mitochondrial morphology, mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (mPTP) opening. Proteins were evaluated via Western Blot.

RESULTS

H/R exposure severely reduced cell viability and increased LDH leakage. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) suppression and dynamin-related protein 1 (Drp1) activation were coincided with excessive mitochondrial fission, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse in H9c2 cells treated with H/R. Mitochondria fragmentation under H/R injury induced ROS over-production, oxidative stress, and cell apoptosis. Notably, CRT treatment significantly prevented mitochondrial fission, mPTP opening, MMP loss, and cell apoptosis. Moreover, CRT sufficiently activated PGC-1α and inactivated Drp1. Interestingly, mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction, oxidative stress and cell apoptosis. However, silencing PGC-1α with small interfering RNA (siRNA) abolished the beneficial effects of CRT on H9c2 cells under H/R injury, accompanied with increased Drp1 and p-Drp1ser616 levels. Furthermore, over-expression of PGC-1α with adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells.

CONCLUSIONS

Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission. We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury. Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack, and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.


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About this article

Crocetin protects cardiomyocytes against hypoxia/reoxygenation injury by attenuating Drp1-mediated mitochondrial fission via PGC-1α

Show Author's information You-Ling MOU1,*, Rui ZHAO1,*, Shi-Ying LYU1, Zi-Yi ZHANG1, Mei-Fei ZHU2( ), Qian LIU1( )
School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, China
Department of Critical Care Medicine, the First Affiliated Hospital, Zhejiang Chinese Medical University, Hangzhou, China

*The authors contributed equally to this manuscript

Abstract

BACKGROUND

Saffron (Crocus sativus L.) has been traditionally used as food, spice, and medicine. Crocetin (CRT), as main bioactive component of saffron, has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion (I/R) injury. However, the mechanisms are poorly explored. This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation (H/R) and elucidated the possible underlying mechanism.

METHODS

H/R attack was performed on H9c2 cells. Cell counting kit-8 was used to detect the cell viability. Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and cellular adenosine triphosphate (ATP) content. Various fluorescent probes were used to detect cell apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) content, mitochondrial morphology, mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (mPTP) opening. Proteins were evaluated via Western Blot.

RESULTS

H/R exposure severely reduced cell viability and increased LDH leakage. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) suppression and dynamin-related protein 1 (Drp1) activation were coincided with excessive mitochondrial fission, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse in H9c2 cells treated with H/R. Mitochondria fragmentation under H/R injury induced ROS over-production, oxidative stress, and cell apoptosis. Notably, CRT treatment significantly prevented mitochondrial fission, mPTP opening, MMP loss, and cell apoptosis. Moreover, CRT sufficiently activated PGC-1α and inactivated Drp1. Interestingly, mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction, oxidative stress and cell apoptosis. However, silencing PGC-1α with small interfering RNA (siRNA) abolished the beneficial effects of CRT on H9c2 cells under H/R injury, accompanied with increased Drp1 and p-Drp1ser616 levels. Furthermore, over-expression of PGC-1α with adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells.

CONCLUSIONS

Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission. We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury. Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack, and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.

References(55)

[1]

Roe MT, Messenger JC, Weintraub WS, et al. Treatments, trends, and outcomes of acute myocardial infarction and percutaneous coronary intervention. J Am Coll Cardiol 2010; 56: 254−263.
DOI Google Scholar
[2]

Reed GW, Rossi JE, Cannon CP. Acute myocardial infarction. Lancet 2017; 389: 197−210.
DOI Google Scholar
[3]

Mozaffarian D, Benjamin EJ, Go AS, et al. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation 2016; 133: e38−360.
Google Scholar
[4]

Bochaton T, Ovize M. Circadian rhythm and ischaemia-reperfusion injury. Lancet 2018; 391: 8−9.
DOI Google Scholar
[5]

Zhao L, Cheng G, Choksi K, et al. Transplantation of human umbilical cord blood-derived cellular fraction improves left ventricular function and remodeling after myocardial ischemia/reperfusion. Circ Res 2019; 125: 759−772.
DOI Google Scholar
[6]

Hausenloy DJ, Yellon DM. Targeting myocardial reperfusion injury-the search continues. N Engl J Med 2015; 373: 1073−1075.
DOI Google Scholar
[7]

Li Y, Chen B, Yang X, et al. S100a8/a9 signaling causes mitochondrial dysfunction and cardiomyocyte death in response to ischemic/reperfusion injury. Circulation 2019; 140: 751−764.
DOI Google Scholar
[8]

Zhou H, Zhang Y, Hu S, et al. Melatonin protects cardiac microvasculature against ischemia/reperfusion injury via suppression of mitochondrial fission-VDAC1-HK2-mPTP-mitophagy axis. J Pineal Res 2017: e12413.
DOI Google Scholar
[9]

Hausenloy DJ, Botker HE, Engstrom T, et al. Targeting reperfusion injury in patients with ST-segment elevation myocardial infarction: trials and tribulations. Eur Heart J 2017; 38: 935−941.
DOI Google Scholar
[10]

Petz A, Grandoch M, Gorski DJ, et al. Cardiac hyaluronan synthesis is critically involved in the cardiac macrophage response and promotes healing after ischemia reperfusion injury. Circ Res 2019; 124: 1433−1447.
DOI Google Scholar
[11]

Hausenloy DJ, Yellon DM. Combination therapy to target reperfusion injury after ST-segment-elevation myocardial infarction: a more effective approach to cardioprotection. Circulation 2017; 136: 904−906.
DOI Google Scholar
[12]

Ziegler M, Hohmann JD, Searle AK, et al. A single-chain antibody-CD39 fusion protein targeting activated platelets protects from cardiac ischaemia/reperfusion injury. Eur Heart J 2018; 39: 111−116.
DOI Google Scholar
[13]

Bi X, Zhang G, Wang X, et al. Endoplasmic reticulum chaperone GRP78 protects heart from ischemia/reperfusion injury through Akt activation. Circ Res 2018; 122: 1545−1554.
DOI Google Scholar
[14]

Zhang Y, Wang Y, Xu J, et al. Melatonin attenuates myocardial ischemia-reperfusion injury via improving mitochondrial fusion/mitophagy and activating the AMPK-OPA1 signaling pathways. J Pineal Res 2019; 66: e12542.
DOI Google Scholar
[15]

Zhou H, Toan S, Zhu P, et al. DNA-PKcs promotes cardiac ischemia reperfusion injury through mitigating BI-1-governed mitochondrial homeostasis. Basic Res Cardiol 2020; 115: 11.
DOI Google Scholar
[16]

Heusch G, Gersh BJ. The pathophysiology of acute myocardial infarction and strategies of protection beyond reperfusion: a continual challenge. Eur Heart J 2017; 38: 774−784.
DOI Google Scholar
[17]

Heusch G, Rassaf T. Time to give up on cardioprotection? A critical appraisal of clinical studies on ischemic pre-, post-, and remote conditioning. Circ Res 2016; 119: 676−695.
DOI Google Scholar
[18]

Umigai N, Murakami K, Ulit MV, et al. The pharmacokinetic profile of crocetin in healthy adult human volunteers after a single oral administration. Phytomedicine 2011; 18: 575−578.
DOI Google Scholar
[19]

Colapietro A, Mancini A, Vitale F, et al. Crocetin extracted from saffron shows antitumor effects in models of human Glioblastoma. Int J Mol Sci 2020: 21.
DOI Google Scholar
[20]

Mori K, Torii H, Fujimoto S, et al. The effect of dietary supplementation of crocetin for myopia control in children: a randomized clinical trial. J Clin Med 2019; 8: 1179.
DOI Google Scholar
[21]

Farkhondeh T, Samarghandian S, Samini F, et al. Protective effects of crocetin on depression-like behavior induced by immobilization in rat. CNS Neurol Disord Drug Targets 2018; 17: 361−369.
DOI Google Scholar
[22]

Mizuma H, Tanaka M, Nozaki S, et al. Daily oral administration of crocetin attenuates physical fatigue in human subjects. Nutr Res 2009; 29: 145−150.
DOI Google Scholar
[23]

Song L, Kang C, Sun Y, et al. Crocetin inhibits lipopolysaccharide-induced inflammatory response in human umbilical vein endothelial cells. Cell Physiol Biochem 2016; 40: 443−452.
DOI Google Scholar
[24]

Armellini R, Peinado I, Pittia P, et al. Effect of saffron (Crocus sativus L.) enrichment on antioxidant and sensorial properties of wheat flour pasta. Food Chem 2018; 254: 55−63.
DOI Google Scholar
[25]

Abedimanesh S, Bathaie SZ, Ostadrahimi A, et al. The effect of crocetin supplementation on markers of atherogenic risk in patients with coronary artery disease: a pilot, randomized, double-blind, placebo-controlled clinical trial. Food Funct 2019; 10: 7461−7475.
DOI Google Scholar
[26]

Zhang W, Li Y, Ge Z. Cardiaprotective effect of crocetin by attenuating apoptosis in isoproterenol induced myocardial infarction rat model. Biomed Pharmacother 2017; 93: 376−382.
DOI Google Scholar
[27]

Zhang Y, Geng J, Hong Y, et al. Orally administered crocin protects against cerebral ischemia/reperfusion injury through the metabolic transformation of crocetin by gut microbiota. Front Pharmacol 2019; 10: 440.
DOI Google Scholar
[28]

Chang G, Chen Y, Zhang H, et al. Trans sodium crocetinate alleviates ischemia/reperfusion-induced myocardial oxidative stress and apoptosis via the SIRT3/FOXO3a/SOD2 signaling pathway. Int Immunopharmacol 2019; 71: 361−371.
DOI Google Scholar
[29]

Yang M, Mao G, Ouyang L, et al. Crocetin alleviates myocardial ischemia/reperfusion injury by regulating inflammation and the unfolded protein response. Mol Med Rep 2020; 21: 641−648.
DOI Google Scholar
[30]

Hou T, Zhang R, Jian C, et al. NDUFAB1 confers cardio-protection by enhancing mitochondrial bioenergetics through coordination of respiratory complex and supercomplex assembly. Cell Res 2019; 29: 754−766.
DOI Google Scholar
[31]

Giacomello M, Pyakurel A, Glytsou C, et al. The cell biology of mitochondrial membrane dynamics. Nat Rev Mol Cell Biol 2020; 21: 204−224.
DOI Google Scholar
[32]

Ong SB, Subrayan S, Lim SY, et al. Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury. Circulation 2010; 121: 2012−2022.
DOI Google Scholar
[33]

Zhu H, Tan Y, Du W, et al. Phosphoglycerate mutase 5 exacerbates cardiac ischemia-reperfusion injury through disrupting mitochondrial quality control. Redox Biol 2021; 38: 101777.
DOI Google Scholar
[34]

Tsushima K, Bugger H, Wende AR, et al. Mitochondrial reactive oxygen species in lipotoxic hearts induce post-translational modifications of AKAP121, DRP1, and OPA1 that promote mitochondrial fission. Circ Res 2018; 122: 58−73.
DOI Google Scholar
[35]

Livingston MJ, Wang J, Zhou J, et al. Clearance of damaged mitochondria via mitophagy is important to the protective effect of ischemic preconditioning in kidneys. Autophagy 2019; 15: 2142−2162.
DOI Google Scholar
[36]

Bi J, Zhang J, Ren Y, et al. Irisin alleviates liver ischemia-reperfusion injury by inhibiting excessive mitochondrial fission, promoting mitochondrial biogenesis and decreasing oxidative stress. Redox Biol 2019; 20: 296−306.
DOI Google Scholar
[37]

Rutkai I, Merdzo I, Wunnava SV, et al. Cerebrovascular function and mitochondrial bioenergetics after ischemia-reperfusion in male rats. J Cereb Blood Flow Metab 2019; 39: 1056−1068.
DOI Google Scholar
[38]

Corbalan JJ, Kitsis RN. RCAN1-calcineurin axis and the set-point for myocardial damage during ischemia-reperfusion. Circ Res 2018; 122: 796−798.
DOI Google Scholar
[39]

Youle RJ, Karbowski M. Mitochondrial fission in apoptosis. Nat Rev Mol Cell Biol 2005; 6: 657−663.
DOI Google Scholar
[40]

Ding M, Ning J, Feng N, et al. Dynamin-related protein 1-mediated mitochondrial fission contributes to post-traumatic cardiac dysfunction in rats and the protective effect of melatonin. J Pineal Res 2018; 64: e12447.
DOI Google Scholar
[41]

Shah MS, Brownlee M. Molecular and cellular mechanisms of cardiovascular disorders in diabetes. Circ Res 2016; 118: 1808−1829.
DOI Google Scholar
[42]
Andrzejewski S, Klimcakova E, Johnson RM, et al. PGC-1 alpha promotes breast cancer metastasis and confers bioenergetic flexibility against metabolic drugs. Cell Metab 2017; 26: 778-787 e775.
DOI
[43]

Ding M, Feng N, Tang D, et al. Melatonin prevents Drp1-mediated mitochondrial fission in diabetic hearts through SIRT1-PGC1alpha pathway. J Pineal Res 2018; 65: e12491.
DOI Google Scholar
[44]

Du J, Hang P, Pan Y, et al. Inhibition of miR-23a attenuates doxorubicin-induced mitochondria-dependent cardiomyocyte apoptosis by targeting the PGC-1alpha/Drp1 pathway. Toxicol Appl Pharmacol 2019; 369: 73−81.
DOI Google Scholar
[45]

Ruiz-Meana M, Inserte J, Fernandez-Sanz C, et al. The role of mitochondrial permeability transition in reperfusion-induced cardiomyocyte death depends on the duration of ischemia. Basic Res Cardiol 2011; 106: 1259−1268.
DOI Google Scholar
[46]

Wang Y, Sun J, Liu C, et al. Protective effects of crocetin pretreatment on myocardial injury in an ischemia/reperfusion rat model. Eur J Pharmacol 2014; 741: 290−296.
DOI Google Scholar
[47]

DiMauro S, Schon EA. Mitochondrial respiratory-chain diseases. N Engl J Med 2003; 348: 2656−2668.
DOI Google Scholar
[48]
Civenni G, Bosotti R, Timpanaro A, et al. Epigenetic control of mitochondrial fission enables self-renewal of stem-like tumor cells in human prostate cancer. Cell Metab 2019; 30: 303-318 e306.
DOI
[49]

Mukherjee UA, Ong SB, Ong SG, et al. Parkinson’s disease proteins: Novel mitochondrial targets for cardioprotection. Pharmacol Ther 2015; 156: 34−43.
DOI Google Scholar
[50]

Ji W, Wei S, Hao P, et al. Aldehyde dehydrogenase 2 has cardioprotective effects on myocardial ischaemia/reperfusion injury via suppressing mitophagy. Front Pharmacol 2016; 7: 101.
DOI Google Scholar
[51]

Zhou H, Ren J, Toan S, et al. Role of mitochondrial quality surveillance in myocardial infarction: From bench to bedside. Ageing Res Rev 2021; 66: 101250.
DOI Google Scholar
[52]

Kalia R, Wang RY, Yusuf A, et al. Structural basis of mitochondrial receptor binding and constriction by DRP1. Nature 2018; 558: 401−405.
DOI Google Scholar
[53]

Wang J, Toan S, Zhou H. New insights into the role of mitochondria in cardiac microvascular ischemia/reperfusion injury. Angiogenesis 2020; 23: 299−314.
DOI Google Scholar
[54]

Tan Y, Mui D, Toan S, et al. SERCA overexpression improves mitochondrial quality control and attenuates cardiac microvascular ischemia-reperfusion injury. Mol Ther Nucleic Acids 2020; 22: 696−707.
DOI Google Scholar
[55]

Chang X, Lochner A, Wang HH, et al. Coronary microvascular injury in myocardial infarction: perception and knowledge for mitochondrial quality control. Theranostics 2021; 11: 6766−6785.
DOI Google Scholar
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Published: 06 February 2023
Issue date: January 2023

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© 2023 JGC All rights reserved

Acknowledgements

ACKNOWLEDGMENTS

This work was supported by the grants from National Natural Science Foundation of China (No.81903830), the Natural Science Foundation of Zhejiang Province (No.LY21H280005), the Research Project of Zhejiang Chinese Medical University (No.2021JKZKTS023B), the 2021 Innovation and Entrepreneurship Training Program for College Students (No.S202110344009). All authors had no conflicts of interest to disclose.

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