Transgenic activation of skeletal muscle Nrf2 protects the heart against ischemia-reperfusion injury in male mice

Physical activity is well-known to protect the heart against ischemia-reperfusion (I/R) injury, yet the underlying mechanisms, particularly those involving interorgan crosstalk, remain incompletely understood. This study investigated whether activation of the skeletal muscle (SkM) Nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant system confers cardioprotection by promoting antioxidant transfer via extracellular vesicles and non-vesicular extracellular nanoparticles (EVs/nvENPs). We utilized iMS-Keap1flox/flox mice, a transgenic model generated in our laboratory (Gao et al., 2020; PMID: 32893883), in which SkM-specific Keap1 knockout (KO) selectively activates Nrf2 and upregulates SkM antioxidant enzymes. Under isoflurane anesthesia, a Millar pressure transducer was inserted into the left ventricular cavity to continuously assess real-time cardiac function, quantified by the cardiac contractility index (CCI), throughout the I/R protocol (60-min ischemia/60-min reperfusion with 20-min baseline). Following functional assessment, hearts were collected for TTC infarct staining; soleus muscle, myocardium, and plasma EVs/nvENPs were analyzed by Western blot for antioxidant enzymes; and left ventricular myocardium was subjected to mass spectrometry-based proteomics and pathway enrichment analysis. We found that, while ischemia depressed cardiac function similarly in both WT and KO mice, the reperfusion response diverged markedly. WT mice exhibited further deterioration during reperfusion (CCI: baseline 156.98 ± 14.97 1/s; ischemia 86.43 ± 10.33 1/s; reperfusion 44.15 ± 9.13 1/s; p < 0.01 between the three phases, n = 6), consistent with additional reperfusion-induced injury. In contrast, KO mice demonstrated significant functional improvement during reperfusion (CCI: baseline 165.44 ± 18.65 1/s; ischemia 98.86 ± 13.62 1/s; reperfusion 121.44 ± 11.73 1/s; p < 0.01 between the three phases, n = 6), suggesting partial rescue of ischemic myocardium. TTC staining confirmed reduced infarct size in KO versus WT mice (24.05 ± 3.37% vs. 34.56 ± 2.59%, p < 0.001, n = 6/group). Western blot analysis showed markedly elevated antioxidant enzymes (NQO1 and GSTA2) in the soleus muscle, plasma EVs/nvENPs, and myocardium of KO mice, supporting a mechanism of systemic antioxidant transfer. On the other hand, the upregulated Nrf2 protein was detected only in soleus muscle of KO mice, excluding an off-target effect of SkM Nrf2 activation. Proteomics of KO mouse myocardium revealed enrichment of additional cardioprotective pathways, including TCA cycle II, integrin-linked kinase signaling, integrin signaling, and paxillin signaling. These findings demonstrate that selective activation of the SkM Nrf2-antioxidant system confers substantial protection against myocardial I/R injury, potentially through circulating EV/nvENP-mediated interorgan antioxidant delivery. Targeting skeletal muscle Nrf2-antioxidant pathways may represent a promising therapeutic strategy to enhance cardiac resilience to I/R injury. Supported by NIH R01HL160820. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.