Mitochondria play a key role in energy production in various organs of the human body via the process of oxidative phosphorylation (OXPHOS) (Lima et al., 2022; Vercellino and Sazanov, 2022). Some organs, such as the brain, heart, and skeletal muscle, are highly dependent on energy supplies in the form of ATP produced via OXPHOS at rest and during moderate-intensity exercise (Dunker et al., 2019; Hargreaves and Spriet, 2020). It is well documented that physical training increases mitochondrial protein biogenesis in rat limb skeletal muscle (Holloszy, 1967) as well as in human leg skeletal muscle (Morgan et al., 1971; Wibom et al., 1992; Zoladz et al., 2022), but its effects on mitochondrial biogenesis and function in other tissues, such as the heart, brain, lungs, or liver, are much less known. It has long been known that mitochondria produce two main oxidants (Loschen et al., 1971; Boveris and Chance, 1973): superoxide anion radicals (O₂ ^•−) and hydrogen peroxide (H₂O₂) (Brand et al., 2004; Kowaltowski et al., 2009; Jarmuszkiewicz et al., 2015). Davies et al. were the first to demonstrate that intense physical exercise increases the production of reactive oxygen species (ROS) in skeletal muscle and liver (Davies et al., 1982), which has since been confirmed by others (Powers and Jackson, 2008; Powers et al., 2024b). It is widely accepted that excessive ROS production leads to oxidative stress (Zhou et al., 2024), resulting in the dysfunction of many body organs (Nathan and Cunningham-Bussel, 2013; Ma et al., 2017; Chen et al., 2018).

Opposite to inactivity, physical activity improves the functioning of many body organs and significantly reduces the risk of many chronic medical conditions (Pedersen and Saltin, 2015; Warburton and Bredin, 2017; 2019). Increasing evidence suggests that exercise-induced increases in ROS (especially H₂O₂) can trigger a range of adaptive responses via redox signaling that beneficially affect the function of various organs (Shadel and Horvath, 2015; Sies and Jones, 2020; Powers et al., 2024b; Sies, 2024; Zhou et al., 2024). Therefore, exercise-induced ROS production is believed to be essential to achieve the full benefits of exercise-induced skeletal muscle adaptation (Powers et al., 2024b). Nonetheless, little is still known about the effect of endurance training on mitochondrial H₂O₂ production in different organs, such as the lung, brain, liver, heart, and skeletal muscle, as well as on the antioxidant defense status in these organs after physical training.

It is well-documented that muscles have a potent antioxidant system (Powers et al., 2024a; Zhou et al., 2024), which effectively protects them from oxidative stress in many conditions. Indeed, exercise increases the levels of several antioxidant enzymes, such as superoxide dismutase 1 (SOD1), glutathione peroxidase, glutathione reductase, and catalase (Powers et al., 2024a). In recent years, the primary focus of antioxidant defense research has been nuclear factor erythroid 2-related factor 2 (NRF2), considered the master regulator of antioxidant responses (He et al., 2020; Gao et al., 2021; Esteras and Abramov, 2022; Powers et al., 2024a; Zhou et al., 2024). It should be noted, however, that most studies to date on the effects of physical activity or training on redox control have focused primarily on skeletal muscle (Powers and Jackson, 2008; Powers et al., 2024a; Zhou et al., 2024). In recent years, knowledge about the effects of physical exercise on ROS production in some vital body organs such as the brain, heart and liver has also increased (Davies et al., 1982; Radak et al., 2016; German et al., 2017; Quan et al., 2020; Dominiak et al., 2022), but knowledge about the effects of exercise on redox regulation and the functioning of these organs is still very limited. Surprisingly, little is known about the effects of physical training on mitochondrial H₂O₂ production in the lung, brain, liver, heart, and hind limb skeletal muscle in studies using the same animals. Furthermore, no data have been published on the effects of physical training on NRF2 expression in these organs.

Therefore, this study primarily aimed to determine − for the first time, to the best of our knowledge − the effect of forced endurance training (performed on a treadmill) on NRF2 expression in the lung, brain, liver, heart, and hind limb skeletal muscle in rats. It also aimed to determine the impact of physical training on (i) mitochondrial H₂O₂ production under phosphorylating and non-phosphorylating conditions, (iii) the level of malondialdehyde (MDA, a marker of lipid peroxidation), SOD1 and glutathione reductase (markers of antioxidant defense), (iv) mitochondrial biogenesis, and (v) the maximal activity of cytochrome c oxidase (COX) and citrate synthase (CS, markers of mitochondrial oxidative capacity) in the homogenates of the studied tissues.

Our study showed that 8 weeks of endurance training elicited numerous adaptive responses in vital organs, including the brain, heart, lungs, liver, and skeletal muscle. This finding is consistent with recent reports indicating that the beneficial effects of physical activity/endurance training are not limited to skeletal muscle but extend to almost all body organs (Chow et al., 2022).

The applied endurance training increased the expression of factors that promote mitochondrial biogenesis, including TFAM and NRF2 (Figures 1, 4). Interestingly, endurance training did not increase TFAM levels only in the heart. Similarly, among the organs studied, only the heart did not show any increase in COX levels and activity after training (Figures 2A,E). The increase in VDAC1 expression in the heart without a corresponding increase in TFAM levels (Figure 1) may reflect adaptations related to increased metabolic activity and mitochondrial membrane transport induced by endurance training rather than a direct stimulation of TFAM-mediated mitochondrial biogenesis. These results indicate that the applied endurance training affected mitochondrial biogenesis differently in different organs. Specifically, the results indicate that mitochondrial biogenesis in the heart was more resistant to endurance training than in the other studied organs (skeletal muscle, lung, liver, and brain). Our results are consistent with recent studies by Khetarpal et al. (2025), who indicated that the mechanism of cardiac muscle adaptation to physical training appears to be different from that of skeletal muscle adaptation (Khetarpal et al., 2025). The authors demonstrated that cardiac adaptation to endurance training requires inhibition of growth differentiation factor 15 (GDF15), the expression of which is regulated by peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α). They highlighted the fact that, according to their studies, the adaptation of cardiomyocytes to physical training depends to a greater extent on PGC-1α than the adaptation of skeletal muscle (Khetarpal et al., 2025). These observations clearly indicate that the mechanisms of mitochondrial biogenesis during endurance training in different body organs may differ from those occurring in skeletal muscle. This may at least partially explain the differences in the regulation of mitochondrial biogenesis in skeletal muscle and heart that we observed in our study.

Among the organs studied, only the heart did not show any increase in COX levels and activity after training (Figures 2A,E). Regarding markers of tissue oxidative capacity, it has been argued that COX activity is the most reliable indirect marker of OXPHOS activity in tissues (Larsen et al., 2012; Zoladz et al., 2014). In our study, the maximal COX activity was similar in the lung, brain, and liver in the sedentary control group. Notably, that COX activity was significantly higher in the skeletal muscle and especially in the heart than in the other studied organs in the sedentary control group (Figure 2A). The applied endurance training significantly increased oxidative capacity in the hind limb muscles, as evidenced by the increase in maximal COX activity (∼70%; Figure 2A). This result is consistent with the previously described effects of endurance training on COX activity in hind limb skeletal muscles in rats (Holloszy, 1967) and in leg muscles in humans (Morgan et al., 1971; Wibom et al., 1992; Zoladz et al., 2022). Interestingly, in our study, we also observed that the applied endurance training significantly increased oxidative capacity in most studied organs (lungs, liver and brain) except, surprisingly, the heart (Figure 2A). Notably, the endurance training-induced increase in COX activity was considerable and similar in the liver and lung (∼50%) but much smaller in the brain (∼8%; Figure 2A). Our results indicate that in young animals, organs in which endurance training has no or minimal effect on oxidative capacity (brain and heart) have sufficient reserves of oxidative capacity to withstand the load of endurance training without the need to increase its level, unlike the other studied organs (lungs, liver and skeletal muscle).

In our study, we observed systematically lower ROS production in all tissues (lung, brain, liver, skeletal muscle, and heart) under phosphorylating conditions (in the presence of ADP) than under non-phosphorylating conditions (in the absence of ADP; Figures 3A,B). These findings are consistent with previous studies that showed that H₂O₂ production in isolated skeletal muscle mitochondria was several times higher in the non-phosphorylating state (state 4) than in the phosphorylating state (state 3) (Jarmuszkiewicz et al., 2015; Zoladz et al., 2016). Another study also addressed this issue (Korshunov et al., 1997), postulating that the high proton force in state 4 is potentially dangerous for the cell due to the increased probability of superoxide formation. Assuming, as a rough approximation, that state 3 respiration is closer to the functioning of muscle mitochondria during muscular activity and state 4 respiration is closer to the functioning of mitochondria at rest (Kavazis et al., 2009; Goncalves et al., 2015), this observation suggests that tissues such as skeletal muscle and heart, which dramatically increase their metabolism during exercise (Zoladz et al., 2016), are exposed to lower ROS production by mitochondria during exercise than at rest. Based on our results (Figures 3A,B), it appears that mitochondrial ROS production is much lower under phosphorylating conditions than under non-phosphorylating conditions also in other tissues, including the lung, brain, and liver. They are consistent with the view that mitochondria are not the primary sources of H₂O₂ in working muscles (Powers et al., 2024b; 2024a). Therefore, NADPH oxidase 2 (NOX2) is considered the primary source of ROS during physical exercise (Henríquez-Olguin et al., 2019).

It is also worth noting that in our study, mitochondrial H₂O₂ production in the heart, measured under both phosphorylating and non-phosphorylating conditions, was several times higher than in skeletal muscle, regardless of training status (sedentary vs. endurance training group) (Figures 3A,B). Similarly, the MDA level in the heart was also several times higher than in skeletal muscle (Figure 3C). These findings are consistent with our previous report, which showed that glutathione disulfide content in the heart at basal state was significantly higher than in skeletal muscle (Majerczak et al., 2022). Together, these results suggest that the heart, even at rest, is exposed to greater oxidative stress than resting skeletal muscle. This may be explained by the fact that the metabolic rate of the heart at rest, when expressed per unit of tissue mass, is several times higher than that of resting skeletal muscle (Zoladz et al., 2015).

Endurance training has previously been shown to significantly increase H₂O₂ production in isolated rat skeletal muscle mitochondria under non-phosphorylating conditions and to decrease their H₂O₂ production under phosphorylating conditions (Zoladz et al., 2016). In our study, we found that the applied endurance training had almost no effect on ROS production under phosphorylating conditions in the studied tissues, except in the heart, where H₂O₂ production was higher after training (Figure 3A). Interestingly, H₂O₂ production under non-phosphorylating conditions in the heart and liver was higher in the endurance-trained group than in the sedentary control group (Figure 3B). The observed endurance training-induced increases in H₂O₂ production in the heart (both under phosphorylating and non-phosphorylating conditions) and liver (only under non-phosphorylating conditions) could be traditionally interpreted as a potentially harmful effect of endurance training on these tissues (Davies et al., 1982). However, given the increasing evidence, this response (training-induced increase in mitochondrial H₂O₂ emission) should instead be interpreted as training-induced activation of redox signaling pathways, which seem to play a key role in tissue adaptation to endurance training (Powers et al., 2024b; Powers et al., 2024a).

It is well established that SODs are the first line of defense against free oxygen radicals, and most organisms living in the presence of oxygen express at least one SOD isoform (Wang et al., 2018). Since SODs are the only enzymes that interact specifically with superoxide anion and thus control ROS and reactive nitrogen species levels, they also act as key regulators of cell signaling. Mammals possess three SOD isoforms (SOD1, SOD2, and SOD3), of which SOD1, acting in the largest compartment of the cell, including the cytoplasm and the intermembrane space of mitochondria, appears to play a key role in the antioxidant system. In our study, we found that the applied endurance training had different effects on SOD1 expression in the studied tissues. Specifically, we observed increased SOD1 levels in the lung, brain, and skeletal muscle, where the increase was greatest (Figure 4). However, no changes in the liver, and even a reduction in SOD1 levels in the heart, were observed after endurance training. These results indicate that endurance training differentially affects the SOD1-related antioxidant defense mechanism in different organs. Notably, both the heart and liver displayed higher ROS production than the other organs studied, both before and after training. This observation suggests that these organs are under greater oxidative load and may therefore rely on organ-specific regulatory strategies to maintain redox balance. In the heart, the reduction in SOD1 levels may be offset by increased activity of other antioxidant enzymes, such as glutathione reductase (Figure 4), while the liver may sustain its defenses without altering SOD1. Therefore, the observed decrease in SOD1 levels in the heart does not necessarily indicate reduced protection but may reflect compensatory adjustments tailored to ROS management in this organ. Interestingly, the applied endurance training did not increase lipid peroxidation in the studied tissues, as assessed by MDA levels in their homogenates (Figure 3C). In the heart and liver, the increase in H₂O₂ production was not accompanied by elevated MDA levels, which may indicate the involvement of compensatory antioxidant pathways such as catalase, peroxiredoxins, or the glutathione antioxidant system (as shown by glutathione reductase, Figure 4). Another possibility is that ROS production occurred in specific subcellular compartments, where localized increases in H₂O₂ did not translate into detectable systemic oxidative damage. However, our results indicate that endurance training sufficiently increased antioxidant defense activity in all studied tissues (lung, brain, liver, skeletal muscle, and heart), as assessed by the observed increases in NRF2 levels in all studied tissues (Figure 4). The intensification of systemic or organ antioxidant activity after training is a well-document physiological response (Powers and Jackson, 2008; Powers et al., 2024b; Powers et al., 2024a). Indeed, numerous studies have shown an upregulation of antioxidant activity in various organs, including skeletal muscle, brain, liver, and heart (for reviews see (Goto and Radák, 2009; Powers et al., 2024b; Powers et al., 2024a). This physiological adaptive response is considered to be beneficial to the body because it attenuates the potentially harmful effects of exercise-induced ROS production. On the other hand, supplementation with antioxidants (e.g., vitamins C and E) has been shown to attenuate the beneficial effects of training, including preventing exercise-induced antioxidant expression and attenuating/preventing enhanced mitochondrial biogenesis in the time course of training (for reviews see (Hamilton et al., 2003; Gomez-Cabrera et al., 2008; Goto and Radák, 2009; Ristow et al., 2009; Powers et al., 2024b; Powers et al., 2024a). These studies have led to the conclusion that mild oxidative stress induced by exercise is actually beneficial to the body because it stimulates a number of adaptive responses, including upregulation of the antioxidant system, resulting in reduced muscle damage and increased mitochondrial biogenesis. This concept (the hormetic effect of ROS) revealed the other side of exercise-induced ROS production (Goto and Radák, 2009; Powers et al., 2024b; Powers et al.,2024a). However, the effect of exercise on redox balance in various organs is still poorly understood.

There are convincing reports that NRF2 plays a key role as a transcription factor that regulates cellular defense against toxic and oxidative attacks by modulating the expression of genes involved in oxidative stress response and drug detoxification (He et al., 2020; Gao et al., 2021; Esteras and Abramov, 2022; Powers et al., 2024a; Zhou et al., 2024). Therefore, through its involvement in metabolic reprogramming, unfolded protein response, proteostasis, autophagy, mitochondrial biogenesis, inflammation, and immunity (He et al., 2020; Gao et al., 2021; Esteras and Abramov, 2022; Powers et al., 2024a), NRF2 seems to play a key role in adaptation to physical exercise. Our study is the first to our knowledge to demonstrate that endurance training increases NRF2 expression in the lung, brain, liver, skeletal muscle, and heart (Figure 4). It is intriguing that, as demonstrated in the present study, physical training effectively increased NRF2 expression simultaneously in multiple organs, including the lungs, brain, liver, skeletal muscle, and heart. It is not clear whether and how the signal informing about oxidative stress occurring in a specific organ of the body, e.g., in skeletal muscles during exercise, is transmitted to another organ in order to build an adaptive stress response (Sies, 2024). We cannot exclude the possibility that the multi-organ upregulation of NRF2 expression in response to physical training, as observed in our study, is the result of intercellular or inter-organ communication, a mechanism recently postulated by Sies et al. (Sies, 2024; Sies et al., 2024). We postulate that the endurance training-induced increase in NRF2 levels in various organs may play a key role in the observed beneficial effects of physical activity on the functioning of various organs in the human body (Pedersen and Saltin, 2015; Warburton and Bredin, 2017; Warburton and Bredin, 2019).

In this study, we examined for the first time the effects of endurance training on mitochondrial H₂O₂ production under phosphorylating and non-phosphorylating conditions, as well as on NRF2 levels in multiple vital organs (lungs, brain, liver, heart, and hind limb skeletal muscle) within the same animals. A major strength of this work lies in its comparative approach, which revealed tissue-specific differences in ROS production and antioxidant responses to endurance training. By integrating measurements of ROS generation, oxidative damage, and antioxidant enzyme expression, the study provides an organ-level overview of redox regulation under physiological stress. Nevertheless, several limitations should be acknowledged. The study was conducted exclusively on young adult male rats, limiting extrapolation to females or older animals. Further studies are needed to assess sex-dependent differences and to explore the impact of aging on mitochondrial ROS production and NRF2-modulated antioxidant defenses in different organs. Although five key tissues were analyzed, other organs with high oxidative susceptibility, such as the kidneys and intestine, were not included. NRF2 activation was assessed indirectly, based on protein levels of selected downstream enzymes (SOD1, glutathione reductase), without assessment of NRF2 nuclear translocation or a broader set of NRF2 signaling targets, which are central to the antioxidant response (e.g., heme oxygenase 1 (HO1), NAD(P)H quinone dehydrogenase 1 (NQO1), catalase, or peroxiredoxins). Moreover, compartment-specific ROS production and direct measurements of mitochondrial oxidative capacity or OXPHOS protein expression were not included. Future studies should expand the range of tissues, include both sexes and older animals, and apply more detailed analyses of NRF2 signaling, compartmental ROS dynamics, mitochondrial function, and total antioxidant capacity. Such work would provide deeper mechanistic insight into organ-specific redox responses to endurance training.

Our study showed that endurance training increased oxidative potential in skeletal muscle and in all studied organs (lung, liver, and brain) except the heart. These results indicate that, compared to other studied tissues, the heart of a young animal, which is already characterized by the highest oxidative activity before training, has sufficient ATP production capacity to maintain endurance without upregulating the oxidative ATP supply system. However, endurance training significantly increased mitochondrial H₂O₂ production in the heart (both under phosphorylating and non-phosphorylating conditions) but not in the other studied tissues, except for the liver, where it was increased under non-phosphorylating conditions after endurance training. This observation indicates that physical exercise induced greater activation of H₂O₂-related cell signaling in the heart than in the other studied tissues. The most interesting and, to our knowledge, original finding of our study was the observed endurance training-induced increase in NRF2 levels in all studied tissues (brain, heart, lung, liver, and hind limb skeletal muscle). Given the cytoprotective properties of NRF2 discussed above, we believe that this multi-organ adaptive response plays a key role in the widely described beneficial effects of physical activity on various body organs and organismal health.