ABSTRACT
Background: Ferroptosis has been proven to contribute to the progression of myocardial ischemia/reperfusion (I/R) injury and can be inhibited or promoted by ATF3. Short-chain fatty acids (SCFAs) have shown benefits in various cardiovascular diseases with anti-inflammatory and antioxidant effects. However, the impact of SCFAs on ferroptosis in ischemic-stimulated cardiomyocytes remains unknown. This study aimed to investigate the effect of SCFAs on cardiomyocyte ferroptosis, the expression of ATF3, and its potential upstream regulators. Methods and results: The expression of ATF3, ferroptosis pathway geneset (FPG), and geneset of potential regulators for ATF3 (GPRA, predicted by the PROMO database) was explored in the public human myocardial infarction single-cell RNA-seq (sma) dataset. Cardiomyocyte data was extracted from the dataset and re-clustered to explore the FPG, ATF3, and GPRA expression patterns in cardiomyocyte subclusters. A dose-dependent toxic experiment was run to detect the suitable dose for SCFA treatment. The erastin-induced ferroptosis model and hypoxia-reoxygenation (H/R) model (10 h of hypoxia followed by 6 h of reoxygenation) were adopted to assess the effect of SCFAs via the CCK8 assay. Gene expression was examined via RT-PCR and western blot. Ferroptosis markers, including lipid peroxides and Fe2+, were detected using the liperfluo and ferroOrange probes, respectively. In the sma dataset, upregulated ferroptosis pathway genes were mainly found in the infarction-stimulated cardiac cells (border zone and fibrotic zone), particularly the cardiomyocytes and adipocytes. The ATF3 and some of its potential transcription factors (VDR, EGR3, PAX5, and SP1) can be regulated by SCFA. SCFA can attenuate erastin-induced lipid peroxidation in cardiomyocytes. SCFA treatment can also reverse erastin-induced Fe2+ increase but may strengthen the Fe2+ in the H/R model. We also precisely defined a ferroptosis subcluster of cardiomyocytes (CM09) that highly expressed FPG, ATF3, and GPRA. Conclusion: The ATF3 and the ferroptosis pathway are elevated in cardiomyocytes of injury-related cardiac regions (border zone, ischemic zone, and fibrotic zone). SCFA can attenuate cardiomyocyte ferroptosis and regulate the expression of ATF3. Our study offers novel insights into the potential targets of SCFAs in the cardiovascular system.
ABSTRACT
Nitroxyl (HNO), the 1-electron reduction product of nitric oxide, improves myocardial contraction in normal and failing hearts. Here we test whether the HNO donor Angeli's salt (AS) will change myocyte action potential (AP) waveform by altering the L-type Ca2+ current (ICa) and contrast the contractile effects of HNO with that of the hydroxyl radical (.OH) and nitrite (NO2-), two potential breakdown products of AS. We confirmed the positive effect of AS/HNO on basal cardiomyocyte function, as opposed to the detrimental effect of .OH and the negligible effect of NO2-. Upon examination of the myocyte AP, we observed no change in resting membrane potential or AP duration to 20 per cent repolarization with AS/HNO, whereas AP duration to 90 per cent repolarization was slightly prolonged. However, perfusion with AS/HNO did not elicit a change in basal ICa, but did hasten ICa inactivation. Upon further examination of the SR, the AS/HNO-induced increase in cardiomyocyte Ca2+ transients was abolished with inhibition of SR Ca2+-cycling. Therefore, the HNO-induced increase in Ca2+ transients results exclusively from changes in SR Ca2+-cycling, and not from ICa.
