Irreversible cardiotoxicity limits the clinical application of doxorubicin (DOX). DOX-induced cardiotoxicity has been associated with induction of senescence and activation of the p38 MAPK pathway. Losmapimod (LOSM), an orally active p38 MAPK inhibitor, is an anti-inflammatory agent with cardioprotective effects. Nevertheless, the effect of LOSM against DOX-induced cardiotoxicity has not been reported. In this study, we determined the effects of LOSM on DOX-induced chronic cardiotoxicity in C57BL/6 N mice. Five-week-old C57BL/6 N mice were fed diet containing LOSM (estimated daily intake 12 mg/kg/day) or a control diet for four days. Thereafter, mice were randomized to receive six weekly intraperitoneal injections of either DOX (4 mg/kg) or saline. Three days after the last injection, cardiac function was assessed by trans-thoracic echocardiography. Activation of p38, JNK, and ERK1/2 MAPKs were assessed by immunoblotting in the heart and liver. Gene expressions of senescence, inflammatory, oxidative stress, and mitochondrial function markers were quantified using real-time PCR and serum inflammatory markers were assessed by Luminex. Our results demonstrated that LOSM attenuated p38 MAPK activation, ameliorated DOX-induced cardiac dysfunction, and abrogated DOX-induced expression of the senescence marker p21^Cip1. Additionally, LOSM demonstrated anti-inflammatory effects, with reduced cardiac Il-1α and Il-6 gene expression in DOX-treated mice. Systemic inflammation, assessed by serum cytokine levels, showed decreased IL-6 and CXCL1 in both DOX-treated mice and mice on LOSM diet. LOSM significantly increased mitofusin2 gene expression, which may enhance mitochondrial fusion. These findings underscore the potential therapeutic efficacy of p38 MAPK inhibition, exemplified by LOSM, in ameliorating DOX-induced cardiotoxicity, senescence, and inflammation.

Doxorubicin (DOX)-induced cardiotoxicity is widely studied using C57BL/6 mice. However, potential substrain differences between C57BL/6J (6J) and C57BL/6N (6N) remain unexamined. Comparative studies are needed to improve preclinical reproducibility and translation. Fifteen-week-old male 6N and 6J mice received either DOX (8 mg/kg/week for 3 weeks) or saline. Body weight was monitored weekly, followed by echocardiography to assess cardiac function. Histopathology evaluated cardiac fibrosis, and bulk RNA sequencing identified differentially expressed genes, which were then validated by qPCR. DOX caused significant weight loss in 6N constituted by loss of both fat and fat-free mass, while 6J showed subtle changes. Echocardiography showed a significant ejection fraction decline only in 6J, while cardiac output, stroke volume, and heart weight were reduced in both. Gene expression analysis revealed significant upregulation of atrial natriuretic peptide and myosin heavy chains in both substrains, with B-type natriuretic peptide upregulation in 6N only. Transcriptomic analysis identified 518 upregulated and 203 downregulated genes in 6N, and 406 upregulated and 445 downregulated genes in 6J, with 113 and 115 commonly upregulated and downregulated genes, respectively. Gene set enrichment analysis revealed that DOX-treated 6N mice showed enrichment of TGF-beta signaling pathways, and suppression of IL-6–JAK–STAT3, epithelial-mesenchymal transition, and myogenesis pathways. In contrast, 6J exhibited enrichment of oxidative phosphorylation, p53 pathway, and reactive oxygen species pathways. Both substrains showed enrichment of apoptosis, unfolded protein response, TNFα signaling via NF-κB, and heme metabolism pathways and downregulation of Hedgehog Signaling and Interferon Gamma Response pathways. This study demonstrates substrain-dependent differences in DOX-induced cardiotoxicity, highlighting how genetic background affects cardiac function, gene expression, and pathway enrichment. Graphical Abstract Substrain-specific differences in doxorubicin-induced cardiotoxicity

Background/Objectives: Doxorubicin (DOX) is an effective chemotherapeutic agent whose clinical utility is limited by cardiotoxicity. To investigate underlying mechanisms, we employed a multi-omics approach integrating transcriptomics and proteomics, leveraging established mouse models of chronic DOX-induced cardiotoxicity. Methods: Five-week-old male mice received weekly DOX (4 mg/kg) or saline injections for six weeks, with heart tissues harvested 4 days post-treatment. Differentially expressed genes (DEGs) and proteins (DEPs) were identified by bulk RNA-seq and proteomics, validated via qPCR and Western blot, respectively. Key DEPs were validated in plasma samples from DOX-treated breast cancer patients. Additionally, temporal comparison was conducted between DEPs in the mice hearts 4 days and 6 weeks post-DOX. Results: RNA-seq revealed upregulation of stress-responsive genes (Phlda3, Trp53inp1) and circadian regulators (Nr1d1), with downregulation of Apelin and Cd74. Proteomics identified upregulation of serpina3n, thrombospondin-1, and epoxide hydrolase 1. Plasma SERPINA3 concentrations were significantly elevated in breast cancer patients 24 h post-DOX. Gene set enrichment analysis (GSEA) revealed upregulated pathways, including p53 signaling, apoptosis, and unfolded protein response. Integrated omics analysis revealed 2089 gene–protein pairs. GSEA of concordant gene–protein pairs implicated p53 signaling, apoptosis, and epithelial–mesenchymal transition in upregulated pathways, while oxidative phosphorylation and metabolic pathways were downregulated. Temporal comparison with a delayed timepoint (6 weeks post-DOX) uncovered dynamic remodeling of cardiac signaling, with early response dominated by inflammatory and apoptotic responses, and delayed response marked by cell cycle and DNA repair pathway activation. Conclusions: This integrated omics study reveals key molecular pathways and temporal changes in DOX-induced cardiotoxicity, identifying potential biomarkers for future cardioprotective strategies.

Psychosocial stress is an established cardiovascular risk factor, and is a huge burden challenging cancer patients undergoing chemotherapy treatment. Doxor