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.
- MeSH
- adenosin * genetika metabolismus MeSH
- krysa rodu rattus MeSH
- messenger RNA genetika MeSH
- omezení příjmu potravy MeSH
- proteomika * MeSH
- RNA metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Epitranscriptomic modifications have recently emerged into the spotlight of researchers due to their vast regulatory effects on gene expression and thereby cellular physiology and pathophysiology. N6,2'-O-dimethyladenosine (m6Am) is one of the most prevalent chemical marks on RNA and is dynamically regulated by writers (PCIF1, METTL4) and erasers (FTO). The presence or absence of m6Am in RNA affects mRNA stability, regulates transcription, and modulates pre-mRNA splicing. Nevertheless, its functions in the heart are poorly known. This review summarizes the current knowledge and gaps about m6Am modification and its regulators in cardiac biology. It also points out technical challenges and lists the currently available techniques to measure m6Am. A better understanding of epitranscriptomic modifications is needed to improve our knowledge of the molecular regulations in the heart which may lead to novel cardioprotective strategies.
The rapidly developing research field of epitranscriptomics has recently emerged into the spotlight of researchers due to its vast regulatory effects on gene expression and thereby cellular physiology and pathophysiology. N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) are among the most prevalent and well-characterized modified nucleosides in eukaryotic RNA. Both of these modifications are dynamically regulated by a complex set of epitranscriptomic regulators called writers, readers, and erasers. Altered levels of m6A and also several regulatory proteins were already associated with diabetic tissues. This review summarizes the current knowledge and gaps about m6A and m6Am modifications and their respective regulators in the pathophysiology of diabetes mellitus. It focuses mainly on the more prevalent type 2 diabetes mellitus (T2DM) and its treatment by metformin, the first-line antidiabetic agent. A better understanding of epitranscriptomic modifications in this highly prevalent disease deserves further investigation and might reveal clinically relevant discoveries in the future.
N6-methyladenosine (m6A) is an abundant mRNA modification affecting mRNA stability and protein expression. It is a highly dynamic process, and its outcomes during postnatal heart development are poorly understood. Here we studied m6A machinery in the left ventricular (LV) myocardium of Fisher344 male and female rats (postnatal days one to ninety; P1-P90) using Western Blot. A downward pattern of target protein levels (demethylases FTO and ALKBH5, methyltransferase METTL3, reader YTHDF2) was revealed in male and female rat LVs during postnatal development. On P1, the FTO protein level was significantly higher in male LVs compared to females.
- MeSH
- krysa rodu rattus MeSH
- myokard * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Adaptation to chronic hypoxia renders the heart more tolerant to ischemia/reperfusion injury. To evaluate changes in gene expression after adaptation to chronic hypoxia by RT-qPCR, it is essential to select suitable reference genes. In a chronically hypoxic rat model, no specific reference genes have been identified in the myocardium. This study aimed to select the best reference genes in the left (LV) and right (RV) ventricles of chronically hypoxic and normoxic rats. Sprague-Dawley rats were adapted to continuous normobaric hypoxia (CNH; 12% O2 or 10% O2) for 3 weeks. The expression levels of candidate genes were assessed by RT-qPCR. The stability of genes was evaluated by NormFinder, geNorm and BestKeeper algorithms. The best five reference genes in the LV were Top1, Nupl2, Rplp1, Ywhaz, Hprt1 for the milder CNH and Top1, Ywhaz, Sdha, Nupl2, Tomm22 for the stronger CNH. In the RV, the top five genes were Hprt1, Nupl2, Gapdh, Top1, Rplp1 for the milder CNH and Tomm22, Gapdh, Hprt1, Nupl2, Top1 for the stronger CNH. This study provides validation of reference genes in LV and RV of CNH rats and shows that suitable reference genes differ in the two ventricles and depend on experimental protocol.
- MeSH
- chronická nemoc MeSH
- hypoxie genetika MeSH
- myokard metabolismus patologie MeSH
- potkani Sprague-Dawley MeSH
- referenční standardy MeSH
- regulace genové exprese * MeSH
- srdeční komory metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Epoxyeicosatrienoic acids (EETs) decrease cardiac ischemia-reperfusion injury; however, the mechanism of their protective effect remains elusive. Here, we investigated the cardioprotective action of a novel EET analog, EET-B, in reperfusion and the role of hypoxia-inducible factor (HIF)-1α in such action of EET-B. Adult male rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion. Administration of 14,15-EET (2.5 mg/kg) or EET-B (2.5 mg/kg) 5 min before reperfusion reduced infarct size expressed as a percentage of the area at risk from 64.3 ± 1.3% in control to 42.6 ± 1.9% and 46.0 ± 1.6%, respectively, and their coadministration did not provide any stronger effect. The 14,15-EET antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (2.5 mg/kg) inhibited the infarct size-limiting effect of EET-B (62.5 ± 1.1%). Similarly, the HIF-1α inhibitors 2-methoxyestradiol (2.5 mg/kg) and acriflavine (2 mg/kg) completely abolished the cardioprotective effect of EET-B. In a separate set of experiments, the immunoreactivity of HIF-1α and its degrading enzyme prolyl hydroxylase domain protein 3 (PHD3) were analyzed in the ischemic areas and nonischemic septa. At the end of ischemia, the HIF-1α immunogenic signal markedly increased in the ischemic area compared with the septum (10.31 ± 0.78% vs. 0.34 ± 0.08%). After 20 min and 2 h of reperfusion, HIF-1α immunoreactivity decreased to 2.40 ± 0.48% and 1.85 ± 0.43%, respectively, in the controls. EET-B blunted the decrease of HIF-1α immunoreactivity (7.80 ± 0.69% and 6.44 ± 1.37%, respectively) and significantly reduced PHD3 immunogenic signal in ischemic tissue after reperfusion. In conclusion, EET-B provides an infarct size-limiting effect at reperfusion that is mediated by HIF-1α and downregulation of its degrading enzyme PHD3. NEW & NOTEWORTHY The present study shows that EET-B is an effective agonistic 14,15-epoxyeicosatrienoic acid analog, and its administration before reperfusion markedly reduced myocardial infarction in rats. Most importantly, we demonstrate that increased hypoxia-inducible factor-1α levels play a role in cardioprotection mediated by EET-B in reperfusion likely by mechanisms including downregulation of the hypoxia-inducible factor -1α-degrading enzyme prolyl hydroxylase domain protein 3.
- MeSH
- down regulace MeSH
- faktor 1 indukovatelný hypoxií - podjednotka alfa genetika metabolismus MeSH
- funkce levé komory srdeční účinky léků MeSH
- infarkt myokardu enzymologie patologie patofyziologie prevence a kontrola MeSH
- kyselina 8,11,14-eikosatrienová analogy a deriváty farmakologie MeSH
- modely nemocí na zvířatech MeSH
- myokard enzymologie patologie MeSH
- potkani Sprague-Dawley MeSH
- prolyl-4-hydroxylasy HIF genetika metabolismus MeSH
- proteolýza MeSH
- remodelace komor účinky léků MeSH
- reperfuzní poškození myokardu enzymologie patologie patofyziologie prevence a kontrola MeSH
- signální transdukce účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Chronic hypoxia and exercise are natural stimuli that confer sustainable cardioprotection against ischemia-reperfusion (I/R) injury, but it is unknown whether they can act in synergy to enhance ischemic resistance. Inflammatory response mediated by tumor necrosis factor-α (TNF-α) plays a role in the infarct size limitation by continuous normobaric hypoxia (CNH), whereas exercise is associated with anti-inflammatory effects. This study was conducted to determine if exercise training performed under conditions of CNH (12% O2) affects myocardial ischemic resistance with respect to inflammatory and redox status. Adult male Wistar rats were assigned to one of the following groups: normoxic sedentary, normoxic trained, hypoxic sedentary, and hypoxic trained. ELISA and Western blot analysis, respectively, were used to quantify myocardial cytokines and the expression of TNF-α receptors, nuclear factor-κB (NF-κB), and selected components of related signaling pathways. Infarct size and arrhythmias were assessed in open-chest rats subjected to I/R. CNH increased TNF-α and interleukin-6 levels and the expression of TNF-α type 2 receptor, NF-κB, inducible nitric oxide synthase (iNOS), cytosolic phospholipase A2α, cyclooxygenase-2, manganese superoxide dismutase (MnSOD), and catalase. None of these effects occurred in the normoxic trained group, whereas exercise in hypoxia abolished or significantly attenuated CNH-induced responses, except for NF-κB, iNOS, and MnSOD. Both CNH and exercise reduced infarct size, but their combination provided the same degree of protection as CNH alone. In conclusion, exercise training does not amplify the cardioprotection conferred by CNH. High ischemic tolerance of the CNH hearts persists after exercise, possibly by maintaining the increased antioxidant capacity despite attenuating TNF-α-dependent protective signaling.NEW & NOTEWORTHYChronic hypoxia and regular exercise are natural stimuli that confer sustainable myocardial protection against acute ischemia-reperfusion injury. Signaling mediated by TNF-α via its type 2 receptor plays a role in the cardioprotective mechanism of chronic hypoxia. In the present study, we found that exercise training of rats during adaptation to hypoxia does not amplify the infarct size-limiting effect. Ischemia-resistant phenotype is maintained in the combined hypoxia-exercise setting despite exercise-induced attenuation of TNF-α-dependent protective signaling.
- MeSH
- cyklooxygenasa 2 metabolismus MeSH
- fyzická vytrvalost fyziologie MeSH
- fyziologická adaptace fyziologie MeSH
- hypoxie metabolismus patologie MeSH
- interleukin-6 metabolismus MeSH
- kondiční příprava zvířat fyziologie MeSH
- krysa rodu rattus MeSH
- myokard metabolismus patologie MeSH
- NF-kappa B metabolismus MeSH
- potkani Wistar MeSH
- receptory TNF - typ II metabolismus MeSH
- reperfuzní poškození myokardu metabolismus patofyziologie MeSH
- superoxiddismutasa metabolismus MeSH
- synthasa oxidu dusnatého, typ II metabolismus MeSH
- TNF-alfa metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
