Membrane-mediated mechanisms of NSAID-induced cardiotoxicity independent of cyclooxygenase selectivity: Evidence from rat and zebrafish models.

Non-steroidal anti-inflammatory drugs (NSAIDs) are associated with cardiovascular adverse events that are not fully explained by cyclooxygenase (COX) inhibition. Here, we investigated the contribution of membrane-mediated mechanisms to NSAID-induced toxicity using integrated in vivo, developmental, and biophysical approaches. Ketoprofen, indomethacin, and celecoxib were evaluated in rats (serum biomarkers and histopathology) and zebrafish embryos (cardiac function), alongside mechanistic studies in cardiac-mimicking lipid membranes using differential scanning calorimetry, NMR spectroscopy, and molecular dynamics simulations. Ketoprofen significantly increased serum creatine kinase and lactate dehydrogenase levels and induced moderate myocardial damage. Indomethacin produced minimal changes in circulating biomarkers but caused mild-to-moderate histological alterations. Celecoxib showed limited toxicity in adult tissues but significantly reduced zebrafish heart rate, indicating pronounced developmental cardiotoxicity. In membrane systems, ketoprofen increased transition temperature and enthalpy, consistent with bilayer condensation and enhanced lipid order. Celecoxib decreased transition enthalpy and increased vesicle heterogeneity, indicating membrane destabilization. Indomethacin reduced transition cooperativity with modest effects on bilayer structure. NMR and simulation data supported differential drug-insertion and lipid-interaction profiles. These findings demonstrate that NSAIDs exhibit compound-specific toxicity profiles that depend on biological context and are linked to distinct drug-membrane interactions. Membrane perturbation represents a COX-independent mechanism contributing to NSAID-associated cardiotoxicity and provides a framework for integrating molecular and organism-level toxicological responses.