Cardiac tolerance to ischaemia can be increased by dietary interventions such as fasting, which is associated with significant changes in myocardial gene expression. Among the possible mechanisms of how gene expression may be altered are epigenetic modifications of RNA - epitranscriptomics. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are two of the most prevalent modifications in mRNA. These methylations are reversible and regulated by proteins called writers, erasers, readers, and m6A-repelled proteins. We analysed 33 of these epitranscriptomic regulators in rat hearts after cardioprotective 3-day fasting using RT-qPCR, Western blot, and targeted proteomic analysis. We found that the most of these regulators were changed on mRNA or protein levels in fasting hearts, including up-regulation of both demethylases - FTO and ALKBH5. In accordance, decreased methylation (m6A+m6Am) levels were detected in cardiac total RNA after fasting. We also identified altered methylation levels in Nox4 and Hdac1 transcripts, both of which play a role in the cytoprotective action of ketone bodies produced during fasting. Furthermore, we investigated the impact of inhibiting demethylases ALKBH5 and FTO in adult rat primary cardiomyocytes (AVCMs). Our findings indicate that inhibiting these demethylases reduced the hypoxic tolerance of AVCMs isolated from fasting rats. This study showed that the complex epitranscriptomic machinery around m6A and m6Am modifications is regulated in the fasting hearts and might play an important role in cardiac adaptation to fasting, a well-known cardioprotective intervention.

Department of Pharmaceutical Sciences College of Pharmacy Glendale Midwestern University Glendale Arizona USA

Department of Physiology Faculty of Science Charles University Prague Czech Republic

Institute of Chemistry University of Tartu Tartu Estonia

Laboratory of Developmental Cardiology Institute of Physiology of the Czech Academy of Sciences Prague Czech Republic

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WHO . The Top 10 Causes Of Death. [2020. Nov 24]. Available from: https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death.

Ostadal B. The past, the present and the future of experimental research on myocardial ischemia and protection. Pharmacol Rep. 2009;61(1):3–12. doi: 10.1016/S1734-1140(09)70002-7 PubMed DOI

Wan R, Ahmet I, Brown M, et al. Cardioprotective effect of intermittent fasting is associated with an elevation of adiponectin levels in rats. J Nutr Biochem. 2010;21(5):413–7. doi: 10.1016/j.jnutbio.2009.01.020 PubMed DOI PMC

Snorek M, Hodyc D, Sedivy V, et al. Short-term fasting reduces the extent of myocardial infarction and incidence of reperfusion arrhythmias in rats. Physiol Res. 2012;61(6):567–74. doi: 10.33549/physiolres.932338 PubMed DOI

Longenecker JZ, Gilbert CJ, Golubeva VA, et al. Epitranscriptomics in the heart: a focus on m(6)A. Curr Heart Fail Rep. 2020;17(5):205–212. doi: 10.1007/s11897-020-00473-z PubMed DOI PMC

Desrosiers R, Friderici K, Rottman F. Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc Natl Acad Sci U S A. 1974;71(10):3971–5. doi: 10.1073/pnas.71.10.3971 PubMed DOI PMC

Wei C, Gershowitz A, Moss B. N6, O2’-dimethyladenosine a novel methylated ribonucleoside next to the 5’ terminal of animal cell and virus mRnas. Nature. 1975;257(5523):251–253. doi: 10.1038/257251a0 PubMed DOI

Benak D, Benakova S, Plecita-Hlavata L, et al. The role of m6A and m6Am RNA modifications in the pathogenesis of diabetes mellitus. Front Endocrinol. 2023;14:1223583. doi: 10.3389/fendo.2023.1223583 PubMed DOI PMC

Oerum S, Meynier V, Catala M, et al. A comprehensive review of m6A/m6Am RNA methyltransferase structures. Nucleic Acids Res. 2021;49(13):7239–7255. doi: 10.1093/nar/gkab378 PubMed DOI PMC

Edupuganti RR, Geiger S, Lindeboom RGH, et al. N(6)-methyladenosine (m(6)A) recruits and repels proteins to regulate mRNA homeostasis. Nat Struct Mol Biol. 2017;24(10):870–878. doi: 10.1038/nsmb.3462 PubMed DOI PMC

Dieterich C, Völkers M. Chapter 6 - RNA modifications in cardiovascular disease—An experimental and computational perspective. In: Devaux Y, and Robinson EL, editors Epigenetics in cardiovascular disease. London, UK: Academic Press; 2021. pp. 113–125.

Sweaad WK, Stefanizzi FM, Chamorro-Jorganes A, et al. Relevance of N6-methyladenosine regulators for transcriptome: Implications for development and the cardiovascular system. J Mol Cell Cardiol. 2021;160:56–70. doi: 10.1016/j.yjmcc.2021.05.006 PubMed DOI

Semenovykh D, Benak D, Holzerova K, et al. Myocardial m6A regulators in postnatal development: effect of sex. Physiol Res. 2022;71(6):877–882. doi: 10.33549/physiolres.934970 PubMed DOI PMC

Benak D, Kolar F, Zhang L, et al. RNA modification m6Am: the role in cardiac biology. Epigenetics. 2023;18(1):2218771. doi: 10.1080/15592294.2023.2218771 PubMed DOI PMC

Song H, Feng X, Zhang H, et al. METTL3 and ALKBH5 oppositely regulate m6a modification of TFEB mRNA, which dictates the fate of hypoxia/reoxygenation-treated cardiomyocytes. Autophagy. 2019;15(8):1419–1437. doi: 10.1080/15548627.2019.1586246 PubMed DOI PMC

Mathiyalagan P, Adamiak M, Mayourian J, et al. FTO-Dependent N6-methyladenosine regulates cardiac function during remodeling and repair. Circulation. 2019;139(4):518–532. doi: 10.1161/CIRCULATIONAHA.118.033794 PubMed DOI PMC

Kmietczyk V, Riechert E, Kalinski L, et al. m6A-mRNA methylation regulates cardiac gene expression and cellular growth. Life Sci Alliance. 2019;2(2):e201800233. doi: 10.26508/lsa.201800233 PubMed DOI PMC

Deng W, Jin Q, Li L. Protective mechanism of demethylase fat mass and obesity-associated protein in energy metabolism disorder of hypoxia-reoxygenation-induced cardiomyocytes. Exp Physiol. 2021;106(12):2423–2433. doi: 10.1113/EP089901 PubMed DOI

Shen W, Li H, Su H, et al. FTO overexpression inhibits apoptosis of hypoxia/reoxygenation-treated myocardial cells by regulating m6A modification of Mhrt. Mol Cell Biochem. 2021;476(5):2171–2179. doi: 10.1007/s11010-021-04069-6 PubMed DOI

Ke WL, Huang Z-W, Peng C-L, et al. M6a demethylase FTO regulates the apoptosis and inflammation of cardiomyocytes via YAP1 in ischemia-reperfusion injury. Bioengineered. 2022;13(3):5443–5452. doi: 10.1080/21655979.2022.2030572 PubMed DOI PMC

Zhang X, Li F, Ma J, et al. ALKBH5 alleviates hypoxia postconditioning injury in D-galactose–induced senescent cardiomyocytes by regulating STAT3. Shock. 2023;59(1):91–98. doi: 10.1097/SHK.0000000000002031 PubMed DOI

Xu Z, Qin Y, Lv B, et al. Intermittent fasting improves high-fat diet-induced obesity cardiomyopathy via alleviating lipid deposition and apoptosis and decreasing m6A methylation in the heart. Nutrients. 2022;14(2):251. doi: 10.3390/nu14020251 PubMed DOI PMC

Hrdlicka J, Neckar J, Papousek F, et al. Epoxyeicosatrienoic acid-based therapy attenuates the progression of postischemic heart failure in Normotensive Sprague-Dawley but not in Hypertensive Ren-2 Transgenic Rats. Front Pharmacol. 2019;10:159. doi: 10.3389/fphar.2019.00159 PubMed DOI PMC

Lee TM, Lin MS, Chang NC. Effect of ATP-sensitive potassium channel agonists on ventricular remodeling in healed rat infarcts. J Am Coll Cardiol. 2008;51(13):1309–18. doi: 10.1016/j.jacc.2007.11.067 PubMed DOI

Alanova P, Chytilova A, Neckar J, et al. Myocardial ischemic tolerance in rats subjected to endurance exercise training during adaptation to chronic hypoxia. J Appl Physiol. 2017;122(6):1452–1461. doi: 10.1152/japplphysiol.00671.2016 PubMed DOI

Neckar J, Hsu A, Hye Khan MA, et al. Infarct size-limiting effect of epoxyeicosatrienoic acid analog EET-B is mediated by hypoxia-inducible factor-1α via downregulation of prolyl hydroxylase 3. Am J Physiol Heart Circ Physiol. 2018;315(5):H1148–h1158. doi: 10.1152/ajpheart.00726.2017 PubMed DOI PMC

Benak D, Sotakova-Kasparova D, Neckar J, et al. Selection of optimal reference genes for gene expression studies in chronically hypoxic rat heart. Mol Cell Biochem. 2019;461(1–2):15–22. doi: 10.1007/s11010-019-03584-x PubMed DOI

Sander H, Wallace S, Plouse R, et al. Ponceau S waste: Ponceau S staining for total protein normalization. Anal Biochem. 2019;575:44–53. doi: 10.1016/j.ab.2019.03.010 PubMed DOI PMC

Cajka T, Hricko J, Rudl Kulhava L, et al. Optimization of Mobile phase modifiers for fast LC-MS-Based untargeted metabolomics and Lipidomics. Int J Mol Sci. 2023;24(3):1987. doi: 10.3390/ijms24031987 PubMed DOI PMC

Hricko J, Rudl Kulhava L, Paucova M, et al. Short-term stability of serum and liver extracts for untargeted metabolomics and Lipidomics. Antioxidants. 2023;12(5):986. doi: 10.3390/antiox12050986 PubMed DOI PMC

Kolb H, Kempf K, Röhling M, et al. Ketone bodies: from enemy to friend and guardian angel. BMC Med. 2021;19(1):313. doi: 10.1186/s12916-021-02185-0 PubMed DOI PMC

Hlavackova M, Kardami E, Fandrich R, et al. Do different nuclei in a binucleated cardiomyocyte have different rates of nuclear protein import? J Mol Cell Cardiol. 2019;126:140–142. doi: 10.1016/j.yjmcc.2018.08.030 PubMed DOI

Zheng G, Cox T, Tribbey L, et al. Synthesis of a FTO inhibitor with anticonvulsant activity. ACS Chem Neurosci. 2014;5(8):658–65. doi: 10.1021/cn500042t PubMed DOI PMC

Selberg S. Rational design of novel anticancer small-molecule RNA m6A demethylase ALKBH5 inhibitors. ACS Omega. 2021;6(20):13310–13320. doi: 10.1021/acsomega.1c01289 PubMed DOI PMC

Pokorna Z, Jirkovsky E, Hlavackova M, et al. In vitro and in vivo investigation of cardiotoxicity associated with anticancer proteasome inhibitors and their combination with anthracycline. Clin Sci (Lond). 2019;133(16):1827–1844. doi: 10.1042/CS20190139 PubMed DOI

Snytnikova O, Tsentalovich Y, Sagdeev R, et al. Quantitative Metabolomic Analysis of Changes in the rat blood serum during autophagy modulation: a focus on accelerated senescence. Int J Mol Sci. 2022;23(21):23(21. doi: 10.3390/ijms232112720 PubMed DOI PMC

Peng L, Long T, Li F, et al. Emerging role of m6A modification in cardiovascular diseases. Cell Biol Int. 2022;46(5):711–722. doi: 10.1002/cbin.11773 PubMed DOI

Hlavackova M. Fat mass and obesity-associated protein in chronically hypoxic myocardium. High Altitude Med Bio. 2018;19(4):A–443. doi: 10.1089/ham.2018.29015.abstracts DOI

Sepich-Poore C, Zheng Z, Schmitt E, et al. The METTL5-TRMT112 N(6)-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation. J Biol Chem. 2022;298(3):101590. doi: 10.1016/j.jbc.2022.101590 PubMed DOI PMC

Han Y, Du T, Guo S, et al. Loss of m(6)A methyltransferase METTL5 promotes cardiac hypertrophy through epitranscriptomic control of SUZ12 expression. Front Cardiovasc Med. 2022;9:852775. doi: 10.3389/fcvm.2022.852775 PubMed DOI PMC

Ge M, Bai X, Liu A, et al. An eIf3a gene mutation dysregulates myocardium growth with left ventricular noncompaction via the p-ERK1/2 pathway. Genes Dis. 2021;8(4):545–554. doi: 10.1016/j.gendis.2020.02.003 PubMed DOI PMC

Li B, Chen H, Yang X, et al. Knockdown of eIf3a ameliorates cardiac fibrosis by inhibiting the TGF-β1/Smad3 signaling pathway. Cell Mol Biol (Noisy-le-Grand). 2016;62(7):97–101. PubMed

Liu T, Wei Q, Jin J, et al. The m6A reader YTHDF1 promotes ovarian cancer progression via augmenting EIF3C translation. Nucleic Acids Res. 2020;48(7):3816–3831. doi: 10.1093/nar/gkaa048 PubMed DOI PMC

Ge Y, Jin J, Li J, et al. The roles of G3BP1 in human diseases (review). Gene. 2022;821:146294. doi: 10.1016/j.gene.2022.146294 PubMed DOI

Jin G, Zhang Z, Wan J, et al. G3BP2: structure and function. Pharmacol Res. 2022;186:106548. doi: 10.1016/j.phrs.2022.106548 PubMed DOI

Hong HQ, Lu J, Fang X-L, et al. G3BP2 is involved in isoproterenol-induced cardiac hypertrophy through activating the NF-κB signaling pathway. Acta Pharmacol Sin. 2018;39(2):184–194. doi: 10.1038/aps.2017.58 PubMed DOI PMC

Li T, Safitri M, Zhang K, et al. Downregulation of G3BP2 reduces atherosclerotic lesions in ApoE(-/-) mice. Atherosclerosis. 2020;310:64–74. doi: 10.1016/j.atherosclerosis.2020.08.003 PubMed DOI

Xiao X, He Z, Tong S, et al., lncRNA XIST knockdown suppresses hypoxia/reoxygenation (H/R)-induced apoptosis of H9C2 cells by regulating miR-545-3p/G3BP2. Life IUBMB. 2021;73(9):1103–1114. doi: 10.1002/iub.2512 PubMed DOI

Masuda K, Abdelmohsen K, Gorospe M. RNA-binding proteins implicated in the hypoxic response. J Cell Mol Med. 2009;13(9a):2759–69. doi: 10.1111/j.1582-4934.2009.00842.x PubMed DOI PMC

Chen H-Y, Xiao Z-Z, Ling X, et al. ELAVL1 is transcriptionally activated by FOXC1 and promotes ferroptosis in myocardial ischemia/reperfusion injury by regulating autophagy. Mol Med. 2021;27(1):14. doi: 10.1186/s10020-021-00271-w PubMed DOI PMC

Krishnamurthy P, Lambers E, Verma S, et al. Myocardial knockdown of mRNA-stabilizing protein HuR attenuates post-MI inflammatory response and left ventricular dysfunction in IL-10-null mice. FASEB J. 2010;24(7):2484–94. doi: 10.1096/fj.09-149815 PubMed DOI PMC

Zhang DH, Zhang J-L, Huang Z, et al. Deubiquitinase Ubiquitin-Specific Protease 10 Deficiency Regulates Sirt6 signaling and Exacerbates Cardiac Hypertrophy. J Am Heart Assoc. 2020;9(22):e017751. doi: 10.1161/JAHA.120.017751 PubMed DOI PMC

Liu LB, Huang S-H, Qiu H-L, et al. Limonin stabilises sirtuin 6 (SIRT6) by activating ubiquitin specific peptidase 10 (USP10) in cardiac hypertrophy. Br J Pharmacol. 2022;179(18):4516–4533. doi: 10.1111/bph.15899 PubMed DOI

Huang J, Liu Y, Wang M, et al. FoxO4 negatively modulates USP10 transcription to aggravate the apoptosis and oxidative stress of hypoxia/reoxygenation-induced cardiomyocytes by regulating the Hippo/YAP pathway. J Bioenerg Biomembr. 2021;53(5):541–551. doi: 10.1007/s10863-021-09910-7 PubMed DOI

Gray SP, Shah AM, Smyrnias I. NADPH oxidase 4 and its role in the cardiovascular system. Vasc Biol. 2019;1(1):H59–h66. doi: 10.1530/VB-19-0014 PubMed DOI PMC

Varga ZV, Pipicz M, Baán JA, et al. Alternative splicing of NOX4 in the failing human heart. Front Physiol. 2017;8:935. doi: 10.3389/fphys.2017.00935 PubMed DOI PMC

Li Y, Zhang Z, Zhou X, et al. Histone Deacetylase 1 inhibition protects against hypoxia-induced swelling in H9c2 cardiomyocytes through regulating cell stiffness. Circ J. 2017;82(1):192–202. doi: 10.1253/circj.CJ-17-0022 PubMed DOI

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