Retinoic acid-related orphan receptors regulate autophagy and cell survival in cardiac myocytes during hypoxic stress

Abstract

Introduction: Autophagy is a highly conserved evolutionary process that regulates cell quality control through protein degradation, organelle turnover, and recycling of cellular components by fusing with lysosomes. Defects in autophagy can lead to increased reactive oxygen species (ROS) and oxidative stress from impaired mitochondrial clearance by mitophagy. These defects are commonly associated with chronic human diseases such as cancer, myocardial infarction, neurodegenerative diseases, and aging.

Aim: Herein, we show that the gene Retinoic Acid-Related Orphan Receptors α (Rora) is cardioprotective through modulation of autophagy and clearance of damaged ROS-producing mitochondria in cardiac myocytes.

Methods and results: We show that RORα is downregulated during hypoxia, leading to increased death of cardiac cells and enhanced mitochondrial perturbations. We demonstrate that the small molecule Nobiletin, a polymethoxy flavonoid, can induce RORα activation and downregulate the aging-associated marker p16, coincident with reduced ROS-producing mitochondria. We further show that Nobiletin binds directly to the Rora gene promoter, leading to activation of autophagic function and increased cell survival of cardiac myocytes during hypoxia. Interestingly, loss of RORα activity during hypoxia resulted in the failure of Nobiletin to rescue autophagy and inhibits its capacity for cardiac protection. Furthermore, the inactivation of autophagy by ATG7 knockdown abrogated the cytoprotective effects of Nobiletin on autophagic activation.

Conclusion: Collectively, these results demonstrate that RORα regulates autophagic processes linked to aging upon activation with Nobiletin. Interventions that activate RORα may prove beneficial in reducing hypoxia-induced mitochondrial ROS associated with cardiac aging.

Keywords

RORα / nobiletin / autophagy / mitochondria / cell death

References

[1] Rabinovich-Nikitin I,Martino TA.Circadian-regulated cell death in cardiovascular diseases.Circulation2019;139:965-80

[2] Rabinovich-Nikitin I,Kirshenbaum LA.Intersection of autophagy regulation and circadian rhythms in the heart.Biochim Biophys Acta Mol Basis Dis2022;1868:166354

[3] Sciarretta S,Zablocki D.The role of autophagy in the heart.Annu Rev Physiol2018;80:1-26

[4] Rabinovich-Nikitin I,Reitz CJ.Mitochondrial autophagy and cell survival is regulated by the circadian clock gene in cardiac myocytes during ischemic stress.Autophagy2021;17:3794-812 PMCID:PMC8632283

[5] Wang EY,Aviv Y,Margulets V.p53 mediates autophagy and cell death by a mechanism contingent on Bnip3.Hypertension2013;62:70-7

[6] Cassidy LD.Autophagy at the intersection of aging, senescence, and cancer.Mol Oncol2022;16:3259-75 PMCID:PMC9490138

[7] Ito H,Kono J.Effectiveness and safety of a mixture of nobiletin and tangeretin in nocturia patients: a randomized, placebo-controlled, double-blind, crossover study.J Clin Med2023;12:2757 PMCID:PMC10143228

[8] Kim E,Ji Z.ROR activation by nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer.Cell Death Dis2022;13:374 PMCID:PMC9018867

[9] Dusabimana T,Kim HJ,Kim H.Nobiletin ameliorates hepatic ischemia and reperfusion injury through the activation of SIRT-1/FOXO3a-mediated autophagy and mitochondrial biogenesis.Exp Mol Med2019;51:1-16 PMCID:PMC6486618

[10] Moon JY.Nobiletin induces protective autophagy accompanied by ER-stress mediated apoptosis in human gastric cancer SNU-16 cells.Molecules2016;21:914 PMCID:PMC6272843

[11] Lee JM,Baek SH.Unraveling the physiological roles of retinoic acid receptor-related orphan receptor α.Exp Mol Med2021;53:1278-86 PMCID:PMC8492739

[12] Hariharan N,Sadoshima J.Oxidative stress stimulates autophagic flux during ischemia/reperfusion.Antioxid Redox Signal2011;14:2179-90 PMCID:PMC3085947

[13] Cui H,Zhang H.Oxidative stress, mitochondrial dysfunction, and aging.J Signal Transduct2012;2012:646354 PMCID:PMC3184498

[14] Katayama H,Mizushima N,Miyawaki A.A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.Chem Biol2011;18:1042-52

[15] Biala AK.The interplay between cell death signaling pathways in the heart.Trends Cardiovasc Med2014;24:325-31

[16] Kirshenbaum LA.Regulation of autophagy in the heart in health and disease.J Cardiovasc Pharmacol2012;60:109

[17] Rubinsztein DC,Kroemer G.Autophagy and aging.Cell2011;146:682-95

[18] Wang HH,Qu TQ.Nobiletin prevents D-galactose-induced C2C12 cell aging by improving mitochondrial function.Int J Mol Sci2022;23:11963 PMCID:PMC9569543

[19] Dhingra R.Regulation of mitochondrial dynamics and cell fate.Circ J2014;78:803-10

[20] Huang H,Shi W.The multifunctional effects of nobiletin and its metabolites in vivo and in vitro.Evid Based Complement Alternat Med2016;2016:2918796 PMCID:PMC5059563