Kisuk Min

Mitochondria-targeted antioxidants protect against mechanical ventilation-induced diaphragm weakness*

Background:Mechanical ventilation is a life-saving intervention used to provide adequate pulmonary ventilation in patients suffering from respiratory failure. However, prolonged mechanical ventilation is associated with significant diaphragmatic weakness resulting from both myofiber atrophy and contractile dysfunction. Although several signaling pathways contribute to diaphragm weakness during mechanical ventilation, it is established that oxidative stress is required for diaphragmatic weakness to occur. Therefore, identifying the site(s) of mechanical ventilation- induced reactive oxygen species production in the diaphragm is important. Objective:These experiments tested the hypothesis that elevated mitochondrial reactive oxygen species emission is required for mechanical ventilation-induced oxidative stress, atrophy, and contractile dysfunction in the diaphragm. Design:Cause and effect was determined by preventing mechanical ventilation-induced mitochondrial reactive oxygen species emission in the diaphragm of rats using a novel mitochondria-targeted antioxidant (SS-31). Interventions:None. Measurements and Main Results:Compared to mechanically ventilated animals treated with saline, animals treated with SS-31 were protected against mechanical ventilation-induced mitochondrial dysfunction, oxidative stress, and protease activation in the diaphragm. Importantly, treatment of animals with the mitochondrial antioxidant also protected the diaphragm against mechanical ventilation-induced myofiber atrophy and contractile dysfunction. Conclusions:These results reveal that prevention of mechanical ventilation-induced increases in diaphragmatic mitochondrial reactive oxygen species emission protects the diaphragm from mechanical ventilation-induced diaphragmatic weakness. This important new finding indicates that mitochondria are a primary source of reactive oxygen species production in the diaphragm during prolonged mechanical ventilation. These results could lead to the development of a therapeutic intervention to impede mechanical ventilation-induced diaphragmatic weakness.

Mitochondrial-targeted antioxidants protect skeletal muscle against immobilization-induced muscle atrophy

Prolonged periods of muscular inactivity (e.g., limb immobilization) result in skeletal muscle atrophy. Although it is established that reactive oxygen species (ROS) play a role in inactivity-induced skeletal muscle atrophy, the cellular pathway(s) responsible for inactivity-induced ROS production remain(s) unclear. To investigate this important issue, we tested the hypothesis that elevated mitochondrial ROS production contributes to immobilization-induced increases in oxidative stress, protease activation, and myofiber atrophy in skeletal muscle. Cause-and-effect was determined by administration of a novel mitochondrial-targeted antioxidant (SS-31) to prevent immobilization-induced mitochondrial ROS production in skeletal muscle fibers. Compared with ambulatory controls, 14 days of muscle immobilization resulted in significant muscle atrophy, along with increased mitochondrial ROS production, muscle oxidative damage, and protease activation. Importantly, treatment with a mitochondrial-targeted antioxidant attenuated the inactivity-induced increase in mitochondrial ROS production and prevented oxidative stress, protease activation, and myofiber atrophy. These results support the hypothesis that redox disturbances contribute to immobilization-induced skeletal muscle atrophy and that mitochondria are an important source of ROS production in muscle fibers during prolonged periods of inactivity.

Exercise protects against doxorubicin-induced oxidative stress and proteolysis in skeletal muscle

Doxorubicin (Dox) is a potent antitumor agent used in cancer treatment. Unfortunately, Dox is myotoxic and results in significant reductions in skeletal muscle mass and function. Complete knowledge of the mechanism(s) by which Dox induces toxicity in skeletal muscle is incomplete, but it is established that Dox-induced toxicity is associated with increased generation of reactive oxygen species and oxidative damage within muscle fibers. Since muscular exercise promotes the expression of numerous cytoprotective proteins (e.g., antioxidant enzymes, heat shock protein 72), we hypothesized that muscular exercise will attenuate Dox-induced damage in exercise-trained muscle fibers. To test this postulate, Sprague-Dawley rats were randomly assigned to the following groups: sedentary, exercise, sedentary with Dox, or exercise with Dox. Our results show increased oxidative stress and activation of cellular proteases (calpain and caspase-3) in skeletal muscle of animals treated with Dox. Importantly, our findings reveal that exercise can prevent the Dox-induced oxidative damage and protease activation in the trained muscle. This exercise-induced protection against Dox-induced toxicity may be due, at least in part, to an exercise-induced increase in muscle levels of antioxidant enzymes and heat shock protein 72. Together, these novel results demonstrate that muscular exercise is a useful countermeasure that can protect skeletal muscle against Dox treatment-induced oxidative stress and protease activation in skeletal muscles.

Exercise protects against doxorubicin-induced markers of autophagy signaling in skeletal muscle

Doxorubicin (DOX) is an effective antitumor agent used in cancer treatment. Unfortunately, DOX is also toxic to skeletal muscle and can result in significant muscle wasting. The cellular mechanism(s) by which DOX induces toxicity in skeletal muscle fibers remains unclear. Nonetheless, DOX-induced toxicity is associated with increased generation of reactive oxygen species, oxidative damage, and activation of the calpain and caspase-3 proteolytic systems within muscle fibers. It is currently unknown if autophagy, a proteolytic system that can be triggered by oxidative stress, is activated in skeletal muscles following DOX treatment. Therefore, we tested the hypothesis that systemic administration of DOX leads to increased expression of autophagy markers in the rat soleus muscle. Our results reveal that DOX administration results in increased muscle mRNA levels and/or protein abundance of several important autophagy proteins, including: Beclin-1, Atg12, Atg7, LC3, LC3II-to-LCI ratio, and cathepsin L. Furthermore, given that endurance exercise increases skeletal muscle antioxidant capacity and protects muscle against DOX-induced oxidative stress, we performed additional experiments to determine whether exercise training before DOX administration would attenuate DOX-induced increases in expression of autophagy genes. Our results clearly show that exercise can protect skeletal muscle from DOX-induced expression of autophagy genes. Collectively, our findings indicate that DOX administration increases the expression of autophagy genes in skeletal muscle, and that exercise can protect skeletal muscle against DOX-induced activation of autophagy.

Short-term exercise training protects against doxorubicin-induced cardiac mitochondrial damage independent of HSP72

Doxorubicin (Dox) is an antitumor agent used in cancer treatment, but its clinical use is limited due to cardiotoxicity. Although exercise training can defend against Dox-mediated cardiac damage, the means for this cardioprotection remain unknown. To investigate the mechanism(s) responsible for exercise training-induced cardioprotection against Dox-mediated cardiotoxicity, we tested a two-pronged hypothesis: 1) exercise training protects against Dox-induced cardiotoxicity by preventing Dox-mediated mitochondrial damage/dysfunction and increased oxidative stress and 2) exercise training-induced cardiac expression of the inducible isoform of the 70-kDa heat shock protein 72 (HSP72) is essential to achieve exercise training-induced cardioprotection against Dox toxicity. Animals were randomly assigned to sedentary or exercise groups and paired with either placebo or Dox treatment (i.e., 20 mg/kg body wt ip Dox hydrochloride 24 h before euthanasia). Dox administration resulted in cardiac mitochondrial dysfunction, activation of proteases, and apoptosis. Exercise training increased cardiac antioxidant enzymes and HSP72 protein abundance and protected cardiac myocytes against Dox-induced mitochondrial damage, protease activation, and apoptosis. To determine whether exercise-induced expression of HSP72 in the heart is required for this cardioprotection, we utilized an innovative experimental strategy that successfully prevented exercise-induced increases in myocardial HSP72 levels. However, prevention of exercise-induced increases in myocardial HSP72 did not eliminate the exercise-induced cardioprotective phenotype that is resistant to Dox-mediated injury. Our results indicate that exercise training protects against the detrimental side effects of Dox in cardiac myocytes, in part, by protecting mitochondria against Dox-mediated damage. However, this exercise-induced cardioprotection is independent of myocardial HSP72 levels. Finally, our data are consistent with the concept that increases in cardiac mitochondrial antioxidant enzymes may contribute to exercise-induced cardioprotection.

Immobilization-induced activation of key proteolytic systems in skeletal muscles is prevented by a mitochondria-targeted antioxidant

Long periods of skeletal muscle disuse result in muscle fiber atrophy, and mitochondrial production of reactive oxygen species (ROS) appears to be a required signal for the increase in protein degradation that occurs during disuse muscle atrophy. The experiments detailed here demonstrate for the first time in limb muscle that the inactivity-induced increases in E3 ligase expression and autophagy biomarkers result from increases in mitochondrial ROS emission. Treatment of animals with a mitochondrial-targeted antioxidant also prevented the disuse-induced decrease in anabolic signaling (Akt/mammalian target of rapamycin signaling) that is normally associated with prolonged inactivity in skeletal muscles. Additionally, our results confirm previous findings that treatment with a mitochondrial-targeted antioxidant is sufficient to prevent casting-induced skeletal muscle atrophy, mitochondrial dysfunction, and activation of the proteases calpain and caspase-3. Collectively, these data reveal that inactivity-induced increases in mitochondrial ROS emission play a required role in activation of key proteolytic systems and the downregulation of important anabolic signaling molecules in muscle fibers exposed to prolonged inactivity.

Exercise protects cardiac mitochondria against ischemia-reperfusion injury

PURPOSE Three to five consecutive days of endurance exercise can protect the heart against an ischemia-reperfusion (IR) insult. However, the mechanisms responsible for this exercise-mediated cardioprotection remain unknown. Given the important role that mitochondria play in IR-induced cardiac myocyte injury, we hypothesized that exercise training promotes cardioprotection, at least in part, by increasing mitochondrial antioxidants, preventing mitochondrial release of reactive oxygen species, and protecting cardiac mitochondria against IR-induced oxidative damage and functional impairment. METHODS To test our hypothesis, Sprague-Dawley rats were assigned to either sedentary (n = 16) or exercise-trained (n = 16) groups. Exercise-trained animals performed 5 d of treadmill running for 60 min·d(-1) at 30 m·s(-1). Hearts were excised from sedentary and exercised-trained animals and were either perfused for 80 min or exposed to 40 min of global ischemia followed by 45 min of reperfusion by using an ex vivo isolated working heart model. After the protocol, cardiac subsarcolemmal and intermyofibrillar mitochondria were isolated and used to determine respiratory control ratio, reactive oxygen species emission, and indices of oxidative stress and apoptosis. RESULTS Our results support our hypothesis because exercise training protected both cardiac subsarcolemmal and intermyofibrillar mitochondria from IR-induced uncoupling and oxidative damage. Specifically, the levels of cardiac mitochondrial 4-hydroxynonenal-conjugated proteins were elevated in hearts from sedentary animals exposed to IR compared with cardiac mitochondria isolated from exercise-trained animals. Exercise also resulted in an increase in mitochondrial antioxidant enzymes (copper-zinc superoxide dismutase, manganese superoxide dismutase, and glutathione peroxidase) and prevented the IR-induced release of proapoptotic proteins from the mitochondria. CONCLUSIONS Collectively, these novel findings reveal that exercise-induced cardioprotection is mediated, at least in part, through mitochondrial adaptations resulting in a mitochondrial phenotype that resists IR-induced damage.

Calpain and caspase-3 play required roles in immobilization-induced limb muscle atrophy

Prolonged skeletal muscle inactivity results in a rapid decrease in fiber size, primarily due to accelerated proteolysis. Although several proteases are known to contribute to disuse muscle atrophy, the ubiquitin proteasome system is often considered the most important proteolytic system during many conditions that promote muscle wasting. Emerging evidence suggests that calpain and caspase-3 may also play key roles in inactivity-induced atrophy of respiratory muscles, but it remains unknown if these proteases are essential for disuse atrophy in limb skeletal muscles. Therefore, we tested the hypothesis that activation of both calpain and caspase-3 is required for locomotor muscle atrophy induced by hindlimb immobilization. Seven days of immobilization (i.e., limb casting) promoted significant atrophy in type I muscle fibers of the rat soleus muscle. Independent pharmacological inhibition of calpain or caspase-3 prevented this casting-induced atrophy. Interestingly, inhibition of calpain activity also prevented caspase-3 activation, and, conversely, inhibition of caspase-3 prevented calpain activation. These findings indicate that a regulatory cross talk exists between these proteases and provide the first evidence that the activation of calpain and caspase-3 is required for inactivity-induced limb muscle atrophy.

Doxorubicin-induced markers of myocardial autophagic signaling in sedentary and exercise trained animals

Doxorubicin (DOX) is an effective antitumor agent used in cancer treatment. However, its clinical use is limited due to cardiotoxicity. Indeed, the side effects of DOX are irreversible and include the development of cardiomyopathy and ultimately congestive heart failure. Although many studies have investigated the events leading to DOX-induced cardiotoxicity, the mechanisms responsible for DOX-induced cardiotoxicity remain unknown. In general, evidence suggests that DOX-induced cardiotoxicity is associated with an increased generation of reactive oxygen species and oxidative damage, leading to the activation of cellular proteolytic systems. In this regard, the autophagy/lysosomal proteolytic system is a constitutively active catabolic process that is responsible for the degradation of both organelles and cytosolic proteins. We tested the hypothesis that systemic DOX administration results in altered cardiac gene and protein expression of mediators of the autophagy/lysosomal system. Our results support this hypothesis, as DOX treatment increased both the mRNA and protein levels of numerous key autophagy genes. Because exercise training has been shown to be cardioprotective against DOX-induced damage, we also determined whether exercise training before DOX administration alters the expression of important components of the autophagy/lysosomal system in cardiac muscle. Our findings show that exercise training inhibits DOX-induced cardiac increases in autophagy signaling. Collectively, our results reveal that DOX administration promotes activation of the autophagy/lysosomal system pathway in the heart, and that endurance exercise training can be a cardioprotective intervention against myocardial DOX-induced toxicity.

Endurance exercise attenuates ventilator-induced diaphragm dysfunction

Controlled mechanical ventilation (MV) is a life-saving measure for patients in respiratory failure. However, MV renders the diaphragm inactive leading to diaphragm weakness due to both atrophy and contractile dysfunction. It is now established that oxidative stress is a requirement for MV-induced diaphragmatic proteolysis, atrophy, and contractile dysfunction to occur. Given that endurance exercise can elevate diaphragmatic antioxidant capacity and the levels of the cellular stress protein heat shock protein 72 (HSP72), we hypothesized that endurance exercise training before MV would protect the diaphragm against MV-induced oxidative stress, atrophy, and contractile dysfunction in female Sprague-Dawley rats. Our results confirm that endurance exercise training before MV increased both HSP72 and the antioxidant capacity in the diaphragm. Importantly, compared with sedentary animals, exercise training before MV protected the diaphragm against MV-induced oxidative damage, protease activation, myofiber atrophy, and contractile dysfunction. Further, exercise protected diaphragm mitochondria against MV-induced oxidative damage and uncoupling of oxidative phosphorylation. These results provide the first evidence that exercise can provide protection against MV-induced diaphragm weakness. These findings are important and establish the need for future experiments to determine the mechanism(s) responsible for exercise-induced diaphragm protection.