Cardiac ischemia/reperfusion, aging, and redox-dependent alterations in mitochondrial function

Alexandrium affine is a global harmful algal bloom (HAB)-forming dinoflagellate. In this study, the effect of non-toxin-producing A. affine on the gill and liver tissues of red seabream, Pagrus major, was analyzed over 24 h exposure and 2 h depuration phases. After exposure to three concentrations of A. affine (4,000, 6,000, and 7,000 cells mL⁻¹), survival rates, respiration rates, immunities (lysozyme, total Ig), hepatic biomarkers (alanine aminotransferase, ALT; aspartate aminotransferase, AST; and alkaline phosphatase, ALP), lipid peroxidation (malondialdehyde, MDA), and antioxidant defense systems (glutathione, GSH; catalase, CAT; superoxide dismutase, SOD; glutathione peroxidases, GPx; and glutathione reductase, GR) were analyzed in gill and liver tissues. Dose-dependent decreases in survival and respiration rates were detected in red seabream. A. affine levels of to 6,000 and 7,000 cells mL⁻¹ induced immunosuppression and hepatic impairment in both tissues, as measured by significant decreases in lysozyme activity, total Ig level, ALT, AST, and ALP content. The levels of GSH, CAT, SOD, GPx, and GR were significantly decreased in the gills and liver in response to 7,000 cells mL⁻¹ of A. affine at 24 h, and MDA was elevated. However, different response patterns were observed between tissues in response to 4,000 cells mL⁻¹. Activity of antioxidant defense enzymes was significantly elevated in the liver but decreased in the gills. This suggests that the gills were more vulnerable than the liver. In the case of 6,000 and 7,000 cells mL⁻¹ treatments, higher susceptibility was also detected at 3 h in the gill compared to the overall responses of each parameter measured in liver. Taken together, direct attachment of A. affine to the gill tissue strongly affects immunity and antioxidant capacity of red seabream even after a short exposure period. These results could be helpful for understanding HAB-mediated effects in marine fish.

Reversible glutathionylation plays a critical role in protecting protein function under conditions of oxidative stress generally and for endothelial nitric-oxide synthase (eNOS) specifically. Glutathione-dependent glutaredoxin-mediated deglutathionylation of eNOS has been shown to confer protection in a model of heart damage termed ischemia-reperfusion injury, motivating further study of eNOS deglutathionylation in general. In this report, we present evidence for an alternative mechanism of deglutathionylation. In this pathway thioredoxin (Trx), a small cellular redox protein, is shown to rescue eNOS from glutathionylation during ischemia-reperfusion in a GSH-independent manner. By comparing mice with global overexpression of Trx and mice with cardiomyocyte-specific overexpression of Trx, we demonstrate that vascular Trx-mediated deglutathionylation of eNOS protects against ischemia-reperfusion-mediated myocardial infarction. Trx deficiency in endothelial cells promoted eNOS glutathionylation and reduced its enzymatic activity, whereas increased levels of Trx led to deglutathionylated eNOS. Thioredoxin-mediated deglutathionylation of eNOS in the coronary artery in vivo protected against reperfusion injury, even in the presence of normal levels of GSH. We further show that Trx directly interacts with eNOS, and we confirmed that Cys-691 and Cys-910 are the glutathionylated sites, as mutation of these cysteines partially rescued the decrease in eNOS activity, whereas mutation of a distal site, Cys-384, did not. Collectively, this study shows for the first time that Trx is a potent deglutathionylating protein in vivo and in vitro that can deglutathionylate proteins in the presence of high levels of GSSG in conditions of oxidative stress.

Resveratrol is known to exert a cardioprotective effect against hypoxia/reoxygenation (H/R) injury. HS-1793 is a novel, more stable resveratrol analog, but its cardioprotective effects were unknown. The present study aimed to test the cardioprotective effect of HS-1793 against H/R injury and investigate the role of mitochondria in Sprague Dawley rat heart damage using an ex vivo Langendorff system. HS-1793 ameliorated H/R-induced mitochondrial dysfunction by reducing mitochondrial reactive oxygen species production, improving mitochondrial oxygen consumption and suppressing mitochondrial calcium (Ca²⁺) overload during reperfusion. Moreover, HS-1793-treated rat heart showed reduced infarct size. Our data suggest that HS-1793 can protect cardiac against mitochondrial damage following H/R, thereby suppressing injury.

Reactive oxygen species (ROS) play an important role in physiological and pathological processes. In recent years, a feed-forward regulation of the ROS sources has been reported. The interactions between the main cellular sources of ROS, such as mitochondria and NADPH oxidases, however, remain obscure. This work summarizes the latest findings on the role of cross talk between mitochondria and NADPH oxidases in pathophysiological processes. Mitochondria have the highest levels of antioxidants in the cell and play an important role in the maintenance of cellular redox status, thereby acting as an ROS and redox sink and limiting NADPH oxidase activity. Mitochondria, however, are not only a target for ROS produced by NADPH oxidase but also a significant source of ROS, which under certain conditions may stimulate NADPH oxidases. This cross talk between mitochondria and NADPH oxidases, therefore, may represent a feed-forward vicious cycle of ROS production, which can be pharmacologically targeted under conditions of oxidative stress. It has been demonstrated that mitochondria-targeted antioxidants break this vicious cycle, inhibiting ROS production by mitochondria and reducing NADPH oxidase activity. This may provide a novel strategy for treatment of many pathological conditions including aging, atherosclerosis, diabetes, hypertension, and degenerative neurological disorders in which mitochondrial oxidative stress seems to play a role. It is conceivable that the use of mitochondria-targeted treatments would be effective in these conditions.

Although arterial limb tourniquet is one of the first-line treatments to prevent exsanguinating hemorrhage in both civilian pre-hospital and battlefield casualty care, prolonged application of a limb tourniquet can lead to serious ischemia–reperfusion injury. However, the underlying pathomechanisms of tourniquet-induced ischemia–reperfusion injury are still poorly understood. Using a murine model of acute limb ischemia–reperfusion, we investigated if acute limb ischemia–reperfusion injury is mediated by superoxide overproduction and mitochondrial dysfunction. Hind limbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Approximately 40% of the gastrocnemius muscle suffered infarction in this model. Activities of mitochondrial electron transport chain complexes including complex I, II, III, and IV in the gastrocnemius muscle were decreased in the ischemia–reperfusion group compared to sham. Superoxide production was increased while activity of manganese superoxide dismutase (MnSOD, the mitochondria-targeted SOD isoform) was decreased in the ischemia–reperfusion group compared to the sham group. Pretreatment with tempol (a SOD mimetic, 50 mg/kg) or co-enzyme Q₁₀ (50 mg/kg) not only decreased the superoxide production, but also reduced the infarct size and normalized mitochondrial dysfunction in the gastrocnemius muscle. Our results suggest that tourniquet-induced skeletal muscle ischemia–reperfusion injuries including infarct size and mitochondrial dysfunction may be mediated via superoxide overproduction and reduced antioxidant activity. In the future, this murine ischemia–reperfusion model can be adapted to mechanistically evaluate anti-ischemic molecules in tourniquet-induced skeletal muscle injury.

The fruit fly Drosophila melanogaster has emerged as a useful model for cardiac diseases, both developmental abnormalities and adult functional impairment. Using the tools of both classical and molecular genetics, the study of the developing fly heart has been instrumental in identifying the major signaling events of cardiac field formation, cardiomyocyte specification, and the formation of the functioning heart tube. The larval stage of fly cardiac development has become an important model system for testing isolated preparations of living hearts for the effects of biological and pharmacological compounds on cardiac activity. Meanwhile, the recent development of effective techniques to study adult cardiac performance in the fly has opened new uses for the Drosophila model system. The fly system is now being used to study long-term alterations in adult performance caused by factors such as diet, exercise, and normal aging. The fly is a unique and valuable system for the study of such complex, long-term interactions, as it is the only invertebrate genetic model system with a working heart developmentally homologous to the vertebrate heart. Thus, the fly model combines the advantages of invertebrate genetics (such as large populations, facile molecular genetic techniques, and short lifespan) with physiological measurement techniques that allow meaningful comparisons with data from vertebrate model systems. As such, the fly model is well situated to make important contributions to the understanding of complicated interactions between environmental factors and genetics in the long-term regulation of cardiac performance.