Abstract
Necroptosis is crucially involved in severe cardiac pathological conditions. However, whether necroptosis contributes to age‐related intolerance to ischemia/reperfusion (I/R) injury remains elusive. In addition, metformin as a potential anti‐aging related injury drug, how it interacts with myocardial necroptosis is not yet clear. Male C57BL/6 mice at 3–4‐ (young) and 22–24 months of age (aged) and RIPK3‐deficient (Ripk3−/−) mice were used to investigate aging‐related I/R injury in vivo. Metformin (125 μg/kg, i.p.), necrostatin‐1 (3.5 mg/kg), and adenovirus vector encoding p62‐shRNAs (Ad‐sh‐p62) were used to treat aging mice. I/R‐induced myocardial necroptosis was exaggerated in aged mice, which correlated with autophagy defects characterized by p62 accumulation in aged hearts or aged human myocardium. Functionally, blocking autophagic flux promoted H/R‐evoked cardiomyocyte necroptosis in vitro. We further revealed that p62 forms a complex with RIP1‐RIP3 (necrosome) and promotes the binding of RIP1 and RIP3. In mice, necrostatin‐1 treatment (a RIP1 inhibitor), RIP3 deficiency, and cardiac p62 knockdown in vivo demonstrated that p62‐RIP1‐RIP3‐dependent myocardial necroptosis contributes to aging‐related myocardial vulnerability to I/R injury. Notably, metformin treatment disrupted p62‐RIP1‐RIP3 complexes and effectively repressed I/R‐induced necroptosis in aged hearts, ultimately reducing mortality in this model. These findings highlight previously unknown mechanisms of aging‐related myocardial ischemic vulnerability: p62‐necrosome‐dependent necroptosis. Metformin acts as a cardioprotective agent that inhibits this unfavorable chain mechanism of aging‐related I/R susceptibility.
Highlights
Aging hearts work on the edge of disease (Dai, Chen, Johnson, Szeto, & Rabinovitch, 2012; Xu et al, 2018)
We found that MI/R-induced cardiac necrosis was markedly enhanced in aged mice, as evidenced by enhanced Evans blue dye (EBD) penetration (Figure 1f) and lactate dehydrogenase (LDH) release (Figure 1g) in aged hearts
We demonstrated that metformin treatment repressed myocardial necroptosis in aging
Summary
Aging hearts work on the edge of disease (Dai, Chen, Johnson, Szeto, & Rabinovitch, 2012; Xu et al, 2018). Nec-1 treatment reduced the MI/R-induced RIP3 and MLKL phosphorylation in aged hearts (Figure 3b) and induced resistance to I/R-induced myocardial necrosis, as evidenced by reductions in EBD-positive staining (Figure 3c,d), LDH release (Figure 3e), and cardiac HMGB1 levels (Figure 3f) and release (Figure 3g) These results suggest that necroptosis is, at least in part, responsible for MI/R injury aggravation in aged hearts. Immunofluorescence in vivo results showed that metformin treatment effectively prevented the I/R-induced membrane translocation of p-MLKL (Figure S8) Consistent with these biochemical data, we found that metformin treatment protects the aged heart from MI/R-induced necrosis (as assessed by EBD-positive staining and LDH release, Figure S9A,B), reduces cardiac contractile dysfunction (as indicated by increases in the ejection fraction and fractional shortening, Figure S9C), and reduces the myocardial infarct size (Figure S9D). Metformin treatment significantly decreased mortality in I/R-injured aged mice (Figure S9E)
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