Oxygen therapy attenuates neuroinflammation after spinal cord injury.

Acute hyperbaric O2 (HBO) therapy after spinal cord injury (SCI) can reduce inflammation and increase neuronal survival. To our knowledge, it is unknown if these benefits of HBO require hyperbaric vs. normobaric hyperoxia. We used a C4 lateralized contusion SCI in adult male and female rats to test the hypothesis that the combination of hyperbaria and 100% O2 (i.e. HBO) more effectively mitigates spinal inflammation and neuronal loss, and enhances respiratory recovery, as compared to normobaric 100% O2. Experimental groups included spinal intact, SCI no O2 therapy, and SCI + 100% O2 delivered at normobaric pressure (1 atmosphere, ATA), or at 2- or 3 ATA. O2 treatments lasted 1-h, commenced within 2-h of SCI, and were repeated for 10 days. The spinal inflammatory response was assessed with transcriptomics (RNAseq) and immunohistochemistry. Gene co-expression network analysis showed that the strong inflammatory response to SCI was dramatically diminished by both hyper- and normobaric O2 therapy. Similarly, both HBO and normobaric O2 treatments reduced the prevalence of immunohistological markers for astrocytes (glial fibrillary acidic protein) and microglia (ionized calcium binding adaptor molecule) in the injured spinal cord. However, HBO treatment also had unique impacts not detected in the normobaric group including upregulation of an anti-inflammatory cytokine (interleukin-4) in the plasma, and larger inspiratory tidal volumes at 10-days (whole body-plethysmography measurements). We conclude that normobaric O2 treatment can reduce the spinal inflammatory response after SCI, but pressured O2 (i.e., HBO) provides further benefit.

Exercise and Doxorubicin Modify Markers of Iron Overload and Cardiolipin Deficiency in Cardiac Mitochondria.

Doxorubicin (DOX) is a chemotherapeutic agent highly effective at limiting cancer progression. Despite the efficacy of this anticancer drug, the clinical use of DOX is limited due to cardiotoxicity. The cardiac mitochondria are implicated as the primary target of DOX, resulting in inactivation of electron transport system complexes, oxidative stress, and iron overload. However, it is established that the cardiac mitochondrial subpopulations reveal differential responses to DOX exposure, with subsarcolemmal (SS) mitochondria demonstrating redox imbalance and the intermyofibrillar (IMF) mitochondria showing reduced respiration. In this regard, exercise training is an effective intervention to prevent DOX-induced cardiac dysfunction. Although it is clear that exercise confers mitochondrial protection, it is currently unknown if exercise training mitigates DOX cardiac mitochondrial toxicity by promoting beneficial adaptations to both the SS and IMF mitochondria. To test this, SS and IMF mitochondria were isolated from sedentary and exercise-preconditioned female Sprague Dawley rats exposed to acute DOX treatment. Our findings reveal a greater effect of exercise preconditioning on redox balance and iron handling in the SS mitochondria of DOX-treated rats compared to IMF, with rescue of cardiolipin synthase 1 expression in both subpopulations. These results demonstrate that exercise preconditioning improves mitochondrial homeostasis when combined with DOX treatment, and that the SS mitochondria display greater protection compared to the IMF mitochondria. These data provide important insights into the molecular mechanisms that are in part responsible for exercise-induced protection against DOX toxicity.

Doxorubicin-induced oxidative stress differentially regulates proteolytic signaling in cardiac and skeletal muscle.

Doxorubicin (DOX) is a highly effective antineoplastic agent used in cancer treatment. Unfortunately, clinical use of DOX is limited due to the development of dose-dependent toxicity to cardiac and respiratory (i.e., diaphragm) muscles. After administration, DOX preferentially localizes to the inner mitochondrial membrane, where it promotes cellular toxicity via enhanced mitochondrial reactive oxygen species (ROS) production. Although recent evidence suggests that amelioration of mitochondrial ROS emission preserves cardiorespiratory muscle function following DOX treatment, the mechanisms responsible for this protection remain unknown. Therefore, we tested the hypothesis that DOX-induced mitochondrial ROS production is required to stimulate pathological signaling by the autophagy/lysosomal system (ALS), the ubiquitin-proteasome pathway (UPP), and the unfolded protein response (UPR). Cause and effect were determined by administration of the mitochondria-targeted peptide SS-31 to DOX-treated animals. Interestingly, while SS-31 abrogated aberrant ROS emission in cardiorespiratory muscles of DOX-treated animals, our results revealed muscle-specific regulation of effector pathways. In the heart, SS-31 prevented DOX-induced proteolytic signaling through the ALS and UPP. In contrast, ALS signaling was inhibited by SS-31 in the diaphragm, but the UPP was not affected. UPR signaling was activated in both muscles at eukaryotic translation initiation factor 2α (eIF2α) S51 in the heart and diaphragm of DOX-treated animals and was attenuated with SS-31 treatment in both tissues. However, downstream signaling of eIF2α (activating transcription factor 4 and CCAAT/enhancer-binding protein homologous protein) was diminished in the heart but upregulated in the diaphragm with DOX. Collectively, these results show that DOX-induced ROS production plays distinct roles in the regulation of cardiac and diaphragm muscle proteolysis.

Protection against Doxorubicin-Induced Cardiac Dysfunction Is Not Maintained Following Prolonged Autophagy Inhibition.

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of various cancer types. Nevertheless, it is well known that DOX promotes the development of severe cardiovascular complications. Therefore, investigation into the underlying mechanisms that drive DOX-induced cardiotoxicity is necessary to develop therapeutic countermeasures. In this regard, autophagy is a complex catabolic process that is increased in the heart following DOX exposure. However, conflicting evidence exists regarding the role of autophagy dysregulation in the etiology of DOX-induced cardiac dysfunction. This study aimed to clarify the contribution of autophagy to DOX-induced cardiotoxicity by specifically inhibiting autophagosome formation using a dominant negative autophagy gene 5 (ATG5) adeno-associated virus construct (rAAV-dnATG5). Acute (2-day) and delayed (9-day) effects of DOX (20 mg/kg intraperitoneal injection (i.p.)) on the hearts of female Sprague-Dawley rats were assessed. Our data confirm established detrimental effects of DOX on left ventricular function, redox balance and mitochondrial function. Interestingly, targeted inhibition of autophagy in the heart via rAAV-dnATG5 in DOX-treated rats ameliorated the increase in mitochondrial reactive oxygen species emission and the attenuation of cardiac and mitochondrial function, but only at the acute timepoint. Deviation in the effects of autophagy inhibition at the 2- and 9-day timepoints appeared related to differences in ATG5-ATG12 conjugation, as this marker of autophagosome formation was significantly elevated 2 days following DOX exposure but returned to baseline at day 9. DOX exposure may transiently upregulate autophagy signaling in the rat heart; thus, long-term inhibition of autophagy may result in pathological consequences.

Effects of exercise and doxorubicin on acute diaphragm neuromuscular transmission failure.

Doxorubicin (DOX) is a highly effective anthracycline antibiotic used to treat a wide variety of cancers including breast cancer, leukemia and lymphoma. Unfortunately, clinical use of DOX is limited due to adverse off-target effects resulting in fatigue, respiratory muscle weakness and dyspnea. The diaphragm is the primary muscle of inspiration and respiratory insufficiency is likely the result of both muscle weakness and neural impairment. However, the contribution of neuropathology to DOX-induced respiratory muscle dysfunction is unclear. We hypothesized that diaphragm weakness following acute DOX exposure is associated with neurotoxicity and that exercise preconditioning is sufficient to improve diaphragm muscle contractility by maintaining neuromuscular integrity. Adult female Sprague-Dawley rats were randomized into four experimental groups: 1) sedentary-saline, 2) sedentary-DOX, 3) exercise-saline or 4) exercise-DOX. Endurance exercise preconditioning consisted of treadmill running for 1 h/day at 30 m/min for 10 days. Twenty-four hours after the last bout of exercise, animals were treated with DOX (20 mg/kg, I.P.) or saline (equal volume). Our results demonstrate that 48-h following DOX administration diaphragm muscle specific force is reduced in sedentary-DOX rats in response to both phrenic nerve and direct diaphragm stimulation. Importantly, endurance exercise preconditioning in DOX-treated rats attenuated the decrease in diaphragm contractile function, reduced neuromuscular transmission failure and altered phrenic nerve morphology. These changes were associated with an exercise-induced reduction in circulating biomarkers of inflammation, nerve injury and reformation. Therefore, the results are consistent with exercise preconditioning as an effective way of reducing respiratory impairment via preservation of phrenic-diaphragm neuromuscular conduction.

Effects of Hyperbaric Oxygen Preconditioning on Doxorubicin Cardiorespiratory Toxicity.

Cardiorespiratory dysfunction resulting from doxorubicin (DOX) chemotherapy treatment is a debilitating condition affecting cancer patient outcomes and quality of life. DOX treatment promotes cardiac and respiratory muscle pathology due to enhanced reactive oxygen species (ROS) production, mitochondrial dysfunction and impaired muscle contractility. In contrast, hyperbaric oxygen (HBO) therapy is considered a controlled oxidative stress that can evoke a substantial and sustained increase in muscle antioxidant expression. This HBO-induced increase in antioxidant capacity has the potential to improve cardiac and respiratory (i.e., diaphragm) muscle redox balance, preserving mitochondrial function and preventing muscle dysfunction. Therefore, we determined whether HBO therapy prior to DOX treatment is sufficient to enhance muscle antioxidant expression and preserve muscle redox balance and cardiorespiratory muscle function. To test this, adult female Sprague Dawley rats received HBO therapy (2 or 3 atmospheres absolute (ATA), 100% O2, 1 h/day) for 5 consecutive days prior to acute DOX treatment (20 mg/kg i.p.). Our data demonstrate that 3 ATA HBO elicits a greater antioxidant response compared to 2 ATA HBO. However, these effects did not correspond with beneficial adaptations to cardiac systolic and diastolic function or diaphragm muscle force production in DOX treated rats. These findings suggest that modulating muscle antioxidant expression with HBO therapy is not sufficient to prevent DOX-induced cardiorespiratory dysfunction.

Consideration of Sex as a Biological Variable in the Development of Doxorubicin Myotoxicity and the Efficacy of Exercise as a Therapeutic Intervention.

Doxorubicin (DOX) is an anthracycline antibiotic used to treat a wide variety of hematological and solid tumor cancers. While DOX is highly effective at reducing tumor burden, its clinical use is limited by the development of adverse effects to both cardiac and skeletal muscle. The detrimental effects of DOX to muscle tissue are associated with the increased incidence of heart failure, dyspnea, exercise intolerance, and reduced quality of life, which have been reported in both patients actively receiving chemotherapy and cancer survivors. A variety of factors elevate the probability of DOX-related morbidity in patients; however, the role of sex as a biological variable to calculate patient risk remains unclear. Uncertainty regarding sexual dimorphism in the presentation of DOX myotoxicity stems from inadequate study design to address this issue. Currently, the majority of clinical data on DOX myotoxicity come from studies where the ratio of males to females is unbalanced, one sex is omitted, and/or the patient cohort include a broad age range. Furthermore, lack of consensus on standard outcome measures, difficulties in long-term evaluation of patient outcomes, and other confounding factors (i.e., cancer type, drug combinations, adjuvant therapies, etc.) preclude a definitive answer as to whether differences exist in the incidence of DOX myotoxicity between sexes. This review summarizes the current clinical and preclinical literature relevant to sex differences in the incidence and severity of DOX myotoxicity, the proposed mechanisms for DOX sexual dimorphism, and the potential for exercise training to serve as an effective therapeutic countermeasure to preserve muscle strength and function in males and females.

Prevention of Doxorubicin-Induced Autophagy Attenuates Oxidative Stress and Skeletal Muscle Dysfunction.

Clinical use of the chemotherapeutic doxorubicin (DOX) promotes skeletal muscle atrophy and weakness, adversely affecting patient mobility and strength. Although the mechanisms responsible for DOX-induced skeletal muscle dysfunction remain unclear, studies implicate the significant production of reactive oxygen species (ROS) in this pathology. Supraphysiological ROS levels can enhance protein degradation via autophagy, and it is established that DOX upregulates autophagic signaling in skeletal muscle. To determine the precise contribution of accelerated autophagy to DOX-induced skeletal muscle dysfunction, we inhibited autophagy in the soleus via transduction of a dominant negative mutation of the autophagy related 5 (ATG5) protein. Targeted inhibition of autophagy prevented soleus muscle atrophy and contractile dysfunction acutely following DOX administration, which was associated with a reduction in mitochondrial ROS and maintenance of mitochondrial respiratory capacity. These beneficial modifications were potentially the result of enhanced transcription of antioxidant response element-related genes and increased antioxidant capacity. Specifically, our results showed significant upregulation of peroxisome proliferator-activated receptor gamma co-activator 1-alpha, nuclear respiratory factor-1, nuclear factor erythroid-2-related factor-2, nicotinamide-adenine dinucleotide phosphate quinone dehydrogenase-1, and catalase in the soleus with DOX treatment when autophagy was inhibited. These findings establish a significant role of autophagy in the development of oxidative stress and skeletal muscle weakness following DOX administration.