Abstract Body (Do not enter title and authors here): Background:
Myocardial ischemia-reperfusion (IR) injury impairs cardiac function after coronary revascularization. Cuproptosis, copper-dependent cell death, is implicated in cardiac injury. METTL3 mediates m6A RNA methylation, influencing cardiovascular pathology, but its role in cuproptosis during IR is unknown. We hypothesized METTL3 stabilizes ferredoxin-1 (FDX1) mRNA via m6A, amplifying cuproptosis and worsening IR injury.
Methods:
180 adults undergoing cardiopulmonary bypass (CPB)-assisted cardiac surgery were prospectively enrolled to measure serum copper, FDX1, high-sensitivity troponin T (hs-TnT), left ventricular ejection fraction (LVEF), and analyze their correlations. IR injury studies were conducted using in vitro hypoxia-reoxygenation (HR) in H9c2 cardiomyocytes and in vivo myocardial IR in mice. Interventions included FDX1 silencing (via siRNA in vitro and AAV9-cTnT-mediated knockdown in vivo), copper chelation with tetrathiomolybdate (TTM), and METTL3 inhibition with STM2457. Assessments included measurements of copper levels, oxidative stress markers, mitochondrial function, cardiac function and infarct size. Molecular analyses included qRT-PCR, Western blot, and m6A RIP-qPCR.
Results:
Postoperative serum copper and FDX1 were markedly elevated, concurrent with increased hs-TnT and reduced LVEF; copper correlated with preoperative/postoperative hs-TnT and cardiovascular complications, and FDX1 correlated with postoperative hs-TnT. Both HR-treated cardiomyocytes and IR-injured hearts showed increased copper accumulation, oxidative stress, and cuproptosis markers, alongside decreased cell viability, mitochondrial dysfunction . IR hearts exhibited increased infarct size and impaired cardiac function. Treatments of TTM, FDX1 silencing, or STM2457 mitigated these pathological changes, with reduced oxidative stress and better preserved mitochondrial function. Those treatments also attenuated infarct size and improved cardiac function in IR hearts. Molecular analyses showed that STM2457 decreased FDX1 expression and cuproptosis activation, indicating that METTL3-mediated m6A methylation stabilized FDX1 mRNA.
Conclusions:
METTL3-mediated m6A methylation stabilizes FDX1 mRNA, thereby promoting cuproptosis and exacerbating myocardial IR injury. Targeting the METTL3-m6A-FDX1 axis may represent a novel therapeutic strategy for perioperative cardioprotection in patients undergoing CPB.