Competitive interaction between ATP and GTP regulates mitochondrial ATP-sensitive potassium channels.

Mitochondrial ATP-sensitive K+ channels (mitoKATP) have been recently characterized structurally, and possess a protein through which K+ enters mitochondria (MitoKIR), and a regulatory subunit (mitoSUR). The mitoSUR regulatory subunit is an ATP-binding cassette (ABC) protein isoform 8 (ABCB8). Opening these channels is known to be cardioprotective, but the molecular and physiological mechanisms that activate them are not fully known. Here, to better understand the molecular and physiological mechanisms of activators (GTP) and inhibitors (ATP) on the activity of mitoKATP, we exposed isolated mitochondria to both nucleotides. We also used molecular docking directed to the nucleotide-binding domain of human ABCB8/mitoSUR to test a comparative model of ATP and GTP effects. As expected, we find that ATP dose-dependently inhibits mitoKATP activity (IC50 = 21.24 ± 1.4 µM). However, simultaneous exposure of mitochondria to GTP dose-dependently (EC50 = 13.19 ± 1.33 µM) reversed ATP inhibition. Pharmacological and computational studies suggest that GTP reverses ATP activity competitively. Docking directed to the site of crystallized ADP reveals that both nucleotides bind to mitoSUR with high affinity, with their phosphates directed to the Mg2+ ion and the walker A motif of the protein (SGGGKTT). These effects, when combined, result in GTP binding, ATP displacement, mitochondrial ATP-sensitive K+ transport, and lower formation of reactive oxygen species. Overall, our findings demonstrate the basis for ATP and GTP binding in mitoSUR using a combination of biochemical, pharmacological, and computational experiments. Future studies may reveal the extent to which the balance between ATP and GTP actions contributes toward cardioprotection against ischemic events.

Calorie restriction changes lipidomic profiles and maintains mitochondrial function and redox balance during isoproterenol-induced cardiac hypertrophy

Typically, healthy cardiac tissue utilizes more fat than any other organ. Cardiac hypertrophy induces a metabolic shift leading to a preferential consumption of glucose over fatty acids to support the high energetic demand. Calorie restriction is a dietary procedure that induces health benefits and lifespan extension in many organisms. Given the beneficial effects of calorie restriction, we hypothesized that calorie restriction prevents cardiac hypertrophy, lipid content changes, mitochondrial and redox dysregulation. Strikingly, calorie restriction reversed isoproterenol-induced cardiac hypertrophy. Isolated mitochondria from hypertrophic hearts produced significantly higher levels of succinate-driven H2O2 production, which was blocked by calorie restriction. Cardiac hypertrophy lowered mitochondrial respiratory control ratios, and decreased superoxide dismutase and glutathione peroxidase levels. These effects were also prevented by calorie restriction. We performed lipidomic profiling to gain insights into how calorie restriction could interfere with the metabolic changes induced by cardiac hypertrophy. Calorie restriction protected against the consumption of several triglycerides (TGs) linked to unsaturated fatty acids. Also, this dietary procedure protected against the accumulation of TGs containing saturated fatty acids observed in hypertrophic samples. Cardiac hypertrophy induced an increase in ceramides, phosphoethanolamines, and acylcarnitines (12:0, 14:0, 16:0, and 18:0). These were all reversed by calorie restriction. Altogether, our data demonstrate that hypertrophy changes the cardiac lipidome, causes mitochondrial disturbances, and oxidative stress. These changes are prevented (at least partially) by calorie restriction intervention in vivo. This study uncovers the potential for calorie restriction to become a new therapeutic intervention against cardiac hypertrophy, and mechanisms in which it acts. (AU)

Calorie restriction changes lipidomic profiles and maintains mitochondrial function and redox balance during isoproterenol-induced cardiac hypertrophy.

Typically, healthy cardiac tissue utilizes more fat than any other organ. Cardiac hypertrophy induces a metabolic shift leading to a preferential consumption of glucose over fatty acids to support the high energetic demand. Calorie restriction is a dietary procedure that induces health benefits and lifespan extension in many organisms. Given the beneficial effects of calorie restriction, we hypothesized that calorie restriction prevents cardiac hypertrophy, lipid content changes, mitochondrial and redox dysregulation. Strikingly, calorie restriction reversed isoproterenol-induced cardiac hypertrophy. Isolated mitochondria from hypertrophic hearts produced significantly higher levels of succinate-driven H2O2 production, which was blocked by calorie restriction. Cardiac hypertrophy lowered mitochondrial respiratory control ratios, and decreased superoxide dismutase and glutathione peroxidase levels. These effects were also prevented by calorie restriction. We performed lipidomic profiling to gain insights into how calorie restriction could interfere with the metabolic changes induced by cardiac hypertrophy. Calorie restriction protected against the consumption of several triglycerides (TGs) linked to unsaturated fatty acids. Also, this dietary procedure protected against the accumulation of TGs containing saturated fatty acids observed in hypertrophic samples. Cardiac hypertrophy induced an increase in ceramides, phosphoethanolamines, and acylcarnitines (12:0, 14:0, 16:0, and 18:0). These were all reversed by calorie restriction. Altogether, our data demonstrate that hypertrophy changes the cardiac lipidome, causes mitochondrial disturbances, and oxidative stress. These changes are prevented (at least partially) by calorie restriction intervention in vivo. This study uncovers the potential for calorie restriction to become a new therapeutic intervention against cardiac hypertrophy, and mechanisms in which it acts.

Quercetin treatment increases H2O2 removal by restoration of endogenous antioxidant activity and blocks isoproterenol-induced cardiac hypertrophy.

Oxidative stress, characterized by the accumulation of reactive oxygen species (ROS), is implicated in the pathogenesis of several diseases, including cardiac hypertrophy. The flavonoid quercetin is a potent ROS scavenger, with several beneficial effects for the cardiovascular system, including antihypertrophic effects. Oxidative imbalance has been implicated in cardiac hypertrophy and heart failure. In this work, we tested whether quercetin could attenuate cardiac hypertrophy by improving redox balance and mitochondrial homeostasis. To test this hypothesis, we treated a group of mice with isoproterenol (30 mg/kg/day) for 4 or 8 consecutive days. Another group received quercetin (10 mg/kg/day) from day 5th of isoproterenol treatment. We carried out the following assays in cardiac tissue: measurement of cardiac hypertrophy, protein sulfhydryl, catalase, Cu/Zn and Mn-superoxide dismutase (SOD) activity, detection of H2O2, and opening of the mitochondrial permeability transition pore. The animals treated with isoproterenol for the initial 4 days showed increased cardiac weight/tibia length ratio, decreased protein sulfhydryl content, compromised SOD and catalase activity, and high H2O2 levels. Quercetin was able to attenuate cardiac hypertrophy, restore protein sulfhydryl, and antioxidant activity, in addition to efficiently blocking the H2O2. We also observed that isoproterenol decreases mitochondrial SOD activity, while quercetin reverses it. Strikingly, quercetin also protects mitochondria against the opening of mitochondrial permeability transition pore. Taken together, these results suggest that quercetin is capable of reversing established isoproterenol-induced cardiac hypertrophy through the restoration of cellular redox balance and protecting mitochondria.