Calpains activate during myocardial ischemia-reperfusion and contribute to reperfusion injury. Studies in transgenic animals with altered calpain/calpastatin system subjected to permanent ischemia suggest that calpains are also involved in post-infarction remodelling and heart failure. To determine whether delayed oral administration of the calpain inhibitor SNJ-1945 reduces adverse myocardial remodelling and dysfunction following transient coronary occlusion. Male Sprague–Dawley rats were subjected to 30 min of ischemia followed by 21 days of reperfusion and received the calpain inhibitor SNJ-1945 intraperitoneally at the onset of reperfusion (Acute group), orally starting after 24 h of reperfusion and for 14 days (Chronic group), or the combination of both treatments. Calpain-1 and calpain-2 protein content increased and correlated with higher calpain activity in control hearts. Administration of SNJ-1945 attenuated calpain activation, and reduced scar expansion, ventricular dilation and dysfunction in both acute and chronic groups. Acute treatment reduced infarct size in hearts reperfused for 24 h and inflammation measured after 3 days. Delayed, chronic oral administration of SNJ-1945 attenuated inflammation, cardiomyocyte hypertrophy and collagen infiltration in the non-infarcted myocardium at 21 days in correlation with increased levels of IĸB and reduced NF-ĸB activation. In cultured fibroblasts, SNJ-1945 attenuated TGF-β1-induced fibroblast activation. Our data demonstrate for the first time that long-term calpain inhibition is possible with delayed oral treatment, attenuates adverse post-infarction remodelling, likely through prevention of NF-ĸB activation, and may be a promising therapeutic intervention to prevent adverse remodelling and heart failure in patients with acute myocardial infarction.

• In patients with STEMI treated with primary percutaneous coronary intervention, iron deficiency is associated with larger infarcts, more extensive microvascular obstruction, and a higher frequency of adverse left ventricular remodeling. • An iron-deficient diet reduces the tolerance to ischemia/reperfusion in mice at least in part by interfering with the cardioprotective pathway eNOS/soluble guanylate cyclase/protein kinase G. • An iron-deficient diet reduces eNOS activity by increasing oxidative/nitrosative stress and its proteasome-dependent degradation. • Not only iron excess but also iron deficiency may have deleterious effects in the context of acute myocardial ischemia.

Myocardial ischemia/reperfusion (I/R) injury causes cardiomyocyte death and exacerbates inflammation. Emerging evidence implicates extracellular RNA (eRNA) and tumor necrosis factor-α (TNF-α) as key mediators. We hypothesize that eRNA released from ischemic cardiomyocytes amplifies I/R injury via TNF-α/TNF-receptor-1 (TNF-R1) signaling, and that hydrolysis of eRNA by RNase1 can attenuate I/R injury by disrupting this pathway. Here, we investigated the mechanistic role of eRNA and its interplay with TNF-α signaling in cardiac I/R injury, and evaluated the therapeutic potential of RNase1 and cyclosporine-A (CsA). In ST-segment elevation myocardial infarction patients, plasma eRNA levels were significantly elevated 2 h post-percutaneous coronary intervention (PCI), correlating positively with Creatine Kinase (CK). In murine I/R and hypoxia/reoxygenation models, eRNA released from stressed cardiomyocytes acted as a damage-associated molecular pattern, triggering TNF-α shedding via TACE/ADAM17 and activating TNF-R1-mediated inflammation, mPTP opening, and cell death. Genetic deletion of TNF-α or TNF-R1 abrogated eRNA-induced cytotoxicity, while TNF-receptor- 2 (TNF-R2) deficiency exacerbated injury. Pharmacological inhibition of TACE with TAPI suppressed TNF-α release and preserved cell viability. RNase1 effectively degraded eRNA, blocking upstream pro-inflammatory signaling, whereas CsA preserved mitochondrial integrity by preventing mPTP opening. Notably, RNase1 and CsA showed synergistic protection in vivo when administered at reperfusion, significantly reducing myocardial infarct size. These findings identify eRNA as both a biomarker and pathogenic mediator of myocardial I/R injury, and support a dual-targeted strategy using RNase1 and CsA to interrupt the TNF-α/TNF-R1-driven inflammatory and mitochondrial death pathways. Targeting both upstream inflammatory and downstream mitochondrial mechanisms represents a promising cardioprotective intervention for acute myocardial infarction.