pcij-2846-1788265678 20260901122758 PCIJ bmpublisher@bmpublisher.net Pathophysiology of Cell Injury Journal Pathophysiology of Cell Injury Journal 2378-5225 11 1 2022 2 22 Daniel R. Whitmore MD PhD Emily J. Carter FRCP Michael A. Reynolds MD Sophia L. Bennett PhD Background: Myocardial ischemia–reperfusion (I/R) injury remains an important determinant of cardiomyocyte loss and impaired ventricular recovery despite successful restoration of coronary blood flow. Activation of the NLRP3 inflammasome may couple reperfusion-associated cellular stress to caspase-1-dependent gasdermin D (GSDMD) cleavage and pyroptotic cell injury. This study investigated the contribution of the NLRP3–caspase-1–GSDMD axis to myocardial I/R injury and evaluated the cardioprotective effects of pharmacological NLRP3 inhibition. Methods: Adult male C57BL/6J mice were randomized to Sham, Sham + MCC950, I/R, or I/R + MCC950 groups (n = 12/group). Myocardial I/R was induced by 30 min of left anterior descending coronary artery occlusion followed by 24 h of reperfusion. MCC950 (10 mg/kg, intraperitoneally) was used to inhibit NLRP3 activation. The primary outcome was infarct size relative to the area at risk (AAR), assessed by Evans blue/2,3,5-triphenyltetrazolium chloride staining. Cardiac function was evaluated by echocardiography, and myocardial injury was assessed using cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and histopathology. NLRP3, ASC, cleaved caspase-1, and GSDMD-N were evaluated as components of inflammasome/pyroptotic signaling. Myocardial IL-1β and IL-18, oxidative-stress indices, and TUNEL positivity were additionally assessed. Results: The AAR was comparable between I/R and I/R + MCC950 groups (46.8 ± 5.7% vs 45.9 ± 5.3%; P = 0.71), whereas MCC950 reduced infarct size from 45.7 ± 6.8% to 27.4 ± 5.9% of the AAR (P < 0.001). I/R markedly impaired left ventricular systolic function, reducing left ventricular ejection fraction (LVEF) to 43.6 ± 6.1%, compared with 72.1 ± 4.3% in Sham animals; MCC950 preserved LVEF at 58.9 ± 5.4% (P < 0.001 vs I/R). MCC950 also reduced cTnI (8.74 ± 1.62 to 4.31 ± 1.18 ng/mL) and CK-MB (38.6 ± 6.7 to 21.4 ± 4.8 ng/mL; both P < 0.001) and attenuated histopathological myocardial injury. Mechanistically, I/R produced coordinated increases in NLRP3 and ASC expression, caspase-1 cleavage, GSDMD-N formation, and IL-1β and IL-18 concentrations. MCC950 significantly suppressed these changes and was additionally associated with reduced lipid peroxidation, restoration of endogenous antioxidant defenses, and decreased TUNEL-positive myocardial nuclei. NLRP3 and GSDMD-N expression correlated positively with infarct size and inversely with ventricular systolic function. Conclusion: Myocardial I/R induces coordinated activation of the NLRP3–ASC–caspase-1–GSDMD pathway, linking inflammasome signaling to pyroptotic/inflammatory myocardial injury, infarct expansion, and acute ventricular dysfunction. Pharmacological NLRP3 inhibition attenuated downstream pyroptotic signaling and produced concordant molecular, biochemical, structural, and functional cardioprotection. These findings identify the NLRP3–caspase-1–GSDMD axis as a potentially actionable therapeutic pathway for limiting myocardial reperfusion injury. Keywords: NLRP3 inflammasome; pyroptosis; myocardial ischemia–reperfusion injury; MCC950; gasdermin D; caspase-1; myocardial infarction. 2022 2 22 10.18081/2378-5225/11.1 https://pcij.net/archives/2846