Substrain-Dependent Differences in Doxorubicin-Induced Cardiotoxicity in Adult C57BL/6 Mice

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.