Rick J. Alleman

Redox-dependent increases in glutathione reductase and exercise preconditioning: role of NADPH oxidase and mitochondria

AIMS We have previously shown that exercise leads to sustainable cardioprotection through a mechanism involving improved glutathione replenishment. This study was conducted to determine if redox-dependent modifications in glutathione reductase (GR) were involved in exercise cardioprotection. Furthermore, we sought to determine if reactive oxygen species generated by NADPH oxidase and/or mitochondria during exercise were triggering events for GR modulations. METHODS AND RESULTS Rats were exercised for 10 consecutive days, after which isolated hearts were exposed to ischaemia/reperfusion (25 min/120 min). Exercise protected against infarction and arrhythmia, and preserved coronary flow. The GR inhibitor BCNU abolished the beneficial effects. GR activity was increased following exercise in a redox-dependent manner, with no change in GR protein levels. Because fluorescent labelling of GR protein thiols showed lower amounts of reduced thiols after exercise, we sought to determine the source of intracellular reactive oxygen species that may be activating GR. Subsets of animals were exercised immediately after treatment with either NADPH-oxidase inhibitors apocynin or Vas2870, or with mitoTEMPO or Bendavia, which reduce mitochondrial reactive oxygen species levels. The cardioprotective effects of exercise were abolished if animals exercised in the presence of NADPH oxidase inhibitors, in clear contrast to the mitochondrial reagents. These changes correlated with thiol-dependent modifications of GR. CONCLUSION Adaptive cardioprotective signalling is triggered by reactive oxygen species from NADPH oxidase, and leads to improved glutathione replenishment through redox-dependent modifications in GR.

The "Goldilocks Zone" from a redox perspective-Adaptive vs. deleterious responses to oxidative stress in striated muscle.

Consequences of oxidative stress may be beneficial or detrimental in physiological systems. An organ systems position on the “hormetic curve” is governed by the source and temporality of reactive oxygen species (ROS) production, proximity of ROS to moieties most susceptible to damage, and the capacity of the endogenous cellular ROS scavenging mechanisms. Most importantly, the resilience of the tissue (the capacity to recover from damage) is a decisive factor, and this is reflected in the disparate response to ROS in cardiac and skeletal muscle. In myocytes, a high oxidative capacity invariably results in a significant ROS burden which in homeostasis, is rapidly neutralized by the robust antioxidant network. The up-regulation of key pathways in the antioxidant network is a central component of the hormetic response to ROS. Despite such adaptations, persistent oxidative stress over an extended time-frame (e.g., months to years) inevitably leads to cumulative damages, maladaptation and ultimately the pathogenesis of chronic diseases. Indeed, persistent oxidative stress in heart and skeletal muscle has been repeatedly demonstrated to have causal roles in the etiology of heart disease and insulin resistance, respectively. Deciphering the mechanisms that underlie the divergence between adaptive and maladaptive responses to oxidative stress remains an active area of research for basic scientists and clinicians alike, as this would undoubtedly lead to novel therapeutic approaches. Here, we provide an overview of major types of ROS in striated muscle and the divergent adaptations that occur in response to them. Emphasis is placed on highlighting newly uncovered areas of research on this topic, with particular focus on the mitochondria, and the diverging roles that ROS play in muscle health (e.g., exercise or preconditioning) and disease (e.g., cardiomyopathy, ischemia, metabolic syndrome).

Exercise-induced protection against reperfusion arrhythmia involves stabilization of mitochondrial energetics

Mitochondria influence cardiac electrophysiology through energy- and redox-sensitive ion channels in the sarcolemma, with the collapse of energetics believed to be centrally involved in arrhythmogenesis. This study was conducted to determine if preservation of mitochondrial membrane potential (ΔΨm) contributes to the antiarrhythmic effect of exercise. We utilized perfused hearts, isolated myocytes, and isolated mitochondria exposed to metabolic challenge to determine the effects of exercise on cardiac mitochondria. Hearts from sedentary (Sed) and exercised (Ex; 10 days of treadmill running) Sprague-Dawley rats were perfused on a two-photon microscope stage for simultaneous measurement of ΔΨm and ECG. After ischemia-reperfusion, the collapse of ΔΨm was commensurate with the onset of arrhythmia. Exercise preserved ΔΨm and decreased the incidence of fibrillation/tachycardia (P < 0.05). Our findings in intact hearts were corroborated in isolated myocytes exposed to in vitro hypoxia-reoxygenation, with Ex rats demonstrating enhanced redox control and sustained ΔΨm during reoxygenation. Finally, we induced anoxia-reoxygenation in isolated mitochondria using high-resolution respirometry with simultaneous measurement of respiration and H2O2 Mitochondria from Ex rats sustained respiration with lower rates of H2O2 emission than Sed rats. Exercise helps sustain postischemic mitochondrial bioenergetics and redox homeostasis, which is associated with preserved ΔΨm and protection against reperfusion arrhythmia. The reduction of fatal ventricular arrhythmias through exercise-induced mitochondrial adaptations indicates that mitochondrial therapeutics may be an effective target for the treatment of heart disease.

Increasing mitochondrial membrane phospholipid content lowers the enzymatic activity of electron transport complexes.

Activities of the enzymes involved in cellular respiration are markedly influenced by the composition of the phospholipid environment of the inner mitochondrial membrane. Contrary to previous suppositions, we show that fusion of mitochondria isolated from healthy cardiac muscle with cardiolipin or dioleoylphosphatidylcholine results in a 2-6-fold reduction in the activity of complexes I, II, and IV. The activity of complex III was unaffected by increased phospholipid levels. Phospholipid content had an indiscriminate yet detrimental effect on the combined activities of complexes I+III and II+III. These results have strong implications for therapeutic lipid replacement strategies, in which phospholipid modification of the mitochondria is proposed to enhance mitochondrial function.

Why Does Exercise "Trigger" Adaptive Protective Responses in the Heart?

Numerous epidemiological studies suggest that individuals who exercise have decreased cardiac morbidity and mortality. Pre-clinical studies in animal models also find clear cardioprotective phenotypes in animals that exercise, specifically characterized by lower myocardial infarction and arrhythmia. Despite the clear benefits, the underlying cellular and molecular mechanisms that are responsible for exercise preconditioning are not fully understood. In particular, the adaptive signaling events that occur during exercise to “trigger” cardioprotection represent emerging paradigms. In this review, we discuss recent studies that have identified several different factors that appear to initiate exercise preconditioning. We summarize the evidence for and against specific cellular factors in triggering exercise adaptations and identify areas for future study.

Mitochondrial therapy improves limb perfusion and myopathy following hindlimb ischemia.

Critical limb ischemia is a devastating manifestation of peripheral arterial disease with no effective strategies for improving morbidity and mortality outcomes. We tested the hypothesis that cellular mitochondrial function is a key component of limb pathology and that improving mitochondrial function represents a novel paradigm for therapy. BALB/c mice were treated with a therapeutic mitochondrial-targeting peptide (MTP-131) and subjected to limb ischemia (HLI). Compared to vehicle control, MTP-131 rescued limb muscle capillary density and blood flow (64.7±11% of contralateral vs. 39.9±4%), and improved muscle regeneration. MTP-131 also increased electron transport system flux across all conditions at HLI day-7. In vitro, primary muscle cells exposed to experimental ischemia demonstrated markedly reduced (~75%) cellular respiration, which was rescued by MTP-131 during a recovery period. Compared to muscle cells, endothelial cell (HUVEC) respiration was inherently protected from ischemia (~30% reduction), but was also enhanced by MTP-131. These findings demonstrate an important link between ischemic tissue bioenergetics and limb blood flow and indicate that the mitochondria may be a pharmaceutical target for therapeutic intervention during critical limb ischemia.