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However, the molecular mechanisms of hidden cardiotoxicity are not always known. Therefore, unbiased multi\u2010omics approaches could assist in revealing regulatory pathways. The most notable representative of hidden cardiotoxic drugs is the cyclooxygenase\u20102\u2010inhibitor, rofecoxib. We previously reported increased mortality in rats because of proarrhythmic effects of rofecoxib in ischaemic hearts. Here, we aimed to identify molecular mechanisms of hidden cardiotoxicity exemplified by rofecoxib that present prior to comorbidities.<\/jats:p><\/jats:sec><jats:sec><jats:title>Experimental Approach<\/jats:title><jats:p>Rats were treated with rofecoxib or its vehicle for 4\u2009weeks. RNA sequencing and proteomic datasets of heart samples were used for differential expression and pathway reconstruction analyses.<\/jats:p><\/jats:sec><jats:sec><jats:title>Key Results<\/jats:title><jats:p>In this model, mechanisms of hidden cardiotoxicity could not be revealed by transcriptomic analyses. However, mass\u2010spectrometry\u2010based proteomics showed conspicuous changes, revealing 132 proteins that were dysregulated in expression or on phosphorylation sites. Importantly, the phospho\u2010proteomics allowed us to identify two kinases that may mediate cardiotoxicity. Finally, pathway reconstruction maps a complex molecular machinery whose clustered proteins regulate processes involving cytoskeleton binding, mRNA processing, proteolysis, translation, citrate acid cycle and calcium ion signalling.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion and Implications<\/jats:title><jats:p>This is the first demonstration that multi\u2010omics characterisation can reveal underlying regulatory pathways of hidden cardiotoxicity. Importantly, our study shows that transcriptomics gives limited information on the hidden cardiotoxic effects of rofecoxib, which are mainly mediated by changes in posttranslational modifications and protein expression. These changes, among other mechanisms, may disturb the cardiac calcium handling, which could explain the fatal arrhythmias following ischaemia\/reperfusion observed with rofecoxib.<\/jats:p><\/jats:sec>","DOI":"10.1111\/bph.70106","type":"journal-article","created":{"date-parts":[[2025,6,18]],"date-time":"2025-06-18T18:40:15Z","timestamp":1750272015000},"page":"4668-4692","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Assessment of the molecular mechanisms of drug\u2010induced hidden cardiotoxicity by a multi\u2010omics approach: The example of rofecoxib"],"prefix":"10.1111","volume":"182","author":[{"given":"Bennet Y.","family":"Weber","sequence":"first","affiliation":[{"name":"Center for Pharmacology and Drug Research and Development, Department of Pharmacology and Pharmacotherapy Semmelweis University  Budapest Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"G\u00e1bor 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