INTRODUCTION: Ozone exposure induces pulmonary injury and systemic inflammation and is implicated in the pathogenesis of respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma; however, its effects on respiratory muscle bioenergetics have not yet been fully elucidated. Recent evidence indicates that acute ozone exposure alters the expression of genes involved in oxidative phosphorylation and mitochondrial respiration, and pathway-based analyses identify estrogen receptor alpha (ERα) as a key upstream regulator of these gene networks. Despite this potential mechanistic insight, the direct functional and molecular consequences of ERα deficiency on mitochondrial function and oxidative stress, particularly in respiratory skeletal muscle, remain unexplored. Therefore, this study aimed to examine the effects of acute ozone exposure on diaphragmatic mitochondrial respiratory function and redox balance in ERα–deficient male and female mice, with particular attention to sex-specific and post-exposure time–dependent responses (24 and 72 h). Additionally, given that lipid peroxidation is a hallmark of ferroptosis, we also examined whether acute ozone exposure modulates ferroptosis-related signaling in the diaphragm. METHODS: Male and female wild-type (WT) and estrogen receptor α–deficient (ERα−/−) mice (11–13 weeks old) were randomly assigned to air control or acute ozone exposure (0.8 ppm for 3 h) and studied at post-exposure time points (24 or 72 h): WT air control, WT 24 h post-ozone, WT 72 h post-ozone, ERα−/− air control, ERα−/− 24 h post-ozone, and ERα−/− 72 h post-ozone. Analyses were conducted separately for males and females. Mitochondrial respiratory function was assessed in permeabilized diaphragm muscle using high-resolution respirometry (Oroboros O2k). Diaphragm muscle samples were analyzed by Western blotting for lipid peroxidation (4-hydroxynonenal; 4-HNE), mitochondrial fission protein (DRP1), and ferroptosis-related proteins (Nrf2, GPX4, and FSP1). RESULTS: Mitochondrial respiration did not differ among male groups, and female WT mice showed no ozone-induced changes. In contrast, female ERα−/− mice at 24 h post-ozone exhibited a significantly higher maximal respiratory capacity (MAX; ~55.3% increase) and respiratory control ratio (RCR; ~46.2% increase) compared with WT females at the same time point (P < 0.05). This was accompanied by elevated 4-HNE levels relative to WT 24 h post-ozone and ERα−/− air controls (P < 0.05), indicating increased lipid peroxidation. These responses were transient and resolved by 72 h post-ozone exposure. Despite elevated lipid peroxidation, GPX4, FSP1, and Nrf2 were unchanged in all groups, suggesting that ferroptotic signaling was not activated at these time points. CONCLUSION: This study demonstrates that, in female ERα-deficient mice, acute ozone exposure increases lipid peroxidation in the diaphragm, suggesting increased oxidative stress in respiratory skeletal muscle. This increase in oxidative stress may, in turn, elicit an adaptive bioenergetic response, accompanied by a transient elevation in mitochondrial respiratory capacity that normalizes over time. The absence of changes in canonical ferroptosis markers indicates that ERα primarily modulates mitochondrial redox and respiratory adaptations rather than ferroptotic signaling. Collectively, these findings identify that ERα may play a modulatory role in influencing sex- and time-dependent oxidative stress and mitochondrial responses to acute ozone exposure. 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.
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