Abstract-MitochondrialKey Words: calcium uniporter Ⅲ Na ϩ /Ca 2ϩ exchange Ⅲ calcium buffer Ⅲ energy metabolism Ⅲ oxidative phosphorylation Ⅲ Krebs cycle C ardiac excitation-contraction (EC) coupling requires enormous amounts of ATP. 1 Mitochondria, which are spatially interleaved with myofibrils and the sarcoplasmic reticulum (SR), 2 are the primary site of ATP production. Two main regulatory factors match mitochondrial ATP production to the constantly varying energy demand of the cell, ADP and Ca 2ϩ .

Background-Oxidative stress is causally linked to the progression of heart failure, and mitochondria are critical sources of reactive oxygen species in failing myocardium. We previously observed that in heart failure, elevated cytosolic Na ϩ ([Na ϩ ] i ) reduces mitochondrial Ca 2ϩ ([Ca 2ϩ ] m ) by accelerating Ca 2ϩ efflux via the mitochondrial Na ϩ /Ca 2ϩ exchanger. Because the regeneration of antioxidative enzymes requires NADPH, which is indirectly regenerated by the Krebs cycle, and Krebs cycle dehydrogenases are activated by [Ca 2ϩ ] m , we speculated that in failing myocytes, elevated [Na ϩ ] i promotes oxidative stress. Methods and Results-We used a patch-clamp-based approach to simultaneously monitor cytosolic and mitochondrial Ca 2ϩ and, alternatively, mitochondrial H 2 O 2 together with NAD(P)H in guinea pig cardiac myocytes. Cells were depolarized in a voltage-clamp mode (3 Hz), and a transition of workload was induced by ␤-adrenergic stimulation. During this transition, NAD(P)H initially oxidized but recovered when [Ca 2ϩ ] m increased. The transient oxidation of NAD(P)H was closely associated with an increase in mitochondrial H 2 O 2 formation. This reactive oxygen species formation was potentiated when mitochondrial Ca 2ϩ uptake was blocked (by Ru360) or Ca 2ϩ efflux was accelerated (by elevation of [Na ϩ ] i ). In failing myocytes, H 2 O 2 formation was increased, which was prevented by reducing mitochondrial Ca 2ϩ efflux via the mitochondrial Na ϩ /Ca 2ϩ exchanger. Conclusions-Besides matching energy supply and demand, mitochondrial Ca 2ϩ uptake critically regulates mitochondrial reactive oxygen species production. In heart failure, elevated [Na ϩ ] i promotes reactive oxygen species formation by reducing mitochondrial Ca 2ϩ uptake. This novel mechanism, by which defects in ion homeostasis induce oxidative stress, represents a potential drug target to reduce reactive oxygen species production in the failing heart. (Circulation. 2010;121:1606-1613.) Key Words: heart failure Ⅲ sodium Ⅲ calcium Ⅲ free radicals Ⅲ ion channels O xidative stress plays a fundamental role in many cardiovascular diseases and aging. 1,2 In chronic heart failure, oxidative stress is causally linked to the progression of the disease, 1,3,4 and mitochondria were identified as critical sources of reactive oxygen species (ROS) in the heart. 5 ROS impair excitation-contraction (EC) coupling, 6 -8 cause arrhythmias, 9 and contribute to cardiac remodeling by activating signaling pathways that induce hypertrophy, apoptosis, and necrosis. 10 -13 The precise mechanisms that regulate mitochondrial ROS formation, however, are incompletely understood. Clinical Perspective on p 1613In cardiac myocytes, the processes of EC coupling consume large amounts of ATP, which is replenished by oxidative phosphorylation in mitochondria. Because the heart undergoes frequent changes in workload, precise matching of ATP supply and demand is essential to maintain cardiac function. 14 Two key regulators of oxidative phosphorylation are ADP and...

Abstract-Mitochondrial ATP production is continually adjusted to energy demand through coordinated increases in oxidative phosphorylation and NADH production mediated by mitochondrial Ca 2ϩ ( Key Words: energy metabolism Ⅲ excitation-contraction coupling Ⅲ heart failure Ⅲ ion transport Ⅲ Na ϩ /Ca 2ϩ exchanger Ⅲ oxidative phosphorylation C ardiac muscle contraction requires continuous matching of ATP supply with a constantly varying workload, yet the mechanism of mitochondrial bioenergetic control is still incompletely understood. The rate of oxidative phosphorylation depends on the protonmotive force across the inner membrane, which is influenced by the balance between the rate of production of reducing equivalents (NADH and FADH 2 ) by the tricarboxylic acid (TCA) cycle and the rate of electron transfer to O 2 by the respiratory chain. When energy demand increases, NADH oxidation is accelerated, requiring a concomitant increase in dehydrogenase activity to maintain NADH/NAD ϩ redox potential and ATP production. Two main lines of evidence support the idea that mitochondrial Ca 2ϩ ([Ca 2ϩ ] m ) homeostasis plays a central role in energy supply and demand matching. First, matrix-free Ca 2ϩ activates several enzymes in the TCA cycle, including pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and NAD ϩ -linked isocitrate dehydrogenase, 1 thereby increasing NADH production. Second, increases in [Ca 2ϩ ] m have been recorded during excitation-contraction coupling and are correlated with changes in metabolism, indicating that mitochondria take up Ca 2ϩ in response to cytosolic Ca 2ϩ on a beat-to-beat basis (reviewed in 2,3 Materials and MethodsAn expanded methods section is included in the online data supplement available at http://circres.ahajournals.org. Briefly, isolated guinea pig cardiomyocytes were subjected to rapid change of workload: from resting state to 4-Hz stimulation and then back to resting state in the presence of 100 nmol/L isoproterenol. NADH autofluorescence was recorded. [Ca 2ϩ ] m was monitored with rhod-2 and [Na ϩ ] i was measured with SBFI. The heart failure model was produced with ascending aortic constriction. Figure 1A-B Figure 1B). Diastolic normalized rhod-2 fluorescence (F/F0) was 1.21Ϯ0.04l in 15 mmol/L [Na ϩ ] i at the end of stimulation and 1.07Ϯ0.04 after 100-second recovery at rest ( Figure 1B), whereas with 5 mmol/L [Na ϩ ] i , it was 1.35Ϯ0.05 at the end of stimulation and 1.16Ϯ0.04 after 100-second recovery at rest ( Figure 1A). The decrease of [Ca 2ϩ ] m accumulation in the cells with 15 mmol/L Na ϩ was associated with net oxidation of NADH. On a rapid increase of workload (4 Hz stimulation for 100 seconds) from rest with 15 mmol/L Na ϩ , NADH gradually decreased from 82.1Ϯ4.1% before stimulation to 63.7Ϯ7.4% at the end of stimulation ( Figure 1D). In contrast, with 5 mmol/L Na ϩ , the NADH levels were maintained during stimulation: NADH was 67.7Ϯ5.8% before stimulation and 66.3Ϯ6.3% at the end of stimulation ( Figure 1C). Figure 1G). Diastolic rhod-2 F/F0 in c...

SUMMARY Impaired mitochondrial respiratory activity contributes to the development of insulin resistance in type 2 diabetes. Metformin, a first-line antidiabetic drug, functions mainly by improving patients’ hyperglycemia and insulin resistance. However, its mechanism of action is still not well understood. We show here that pharmacological metformin concentration increases mitochondrial respiration, membrane potential, and ATP levels in hepatocytes and a clinically relevant metformin dose increases liver mitochondrial density and complex 1 activity along with improved hyperglycemia in high-fat- diet (HFD)-fed mice. Metformin, functioning through 5′ AMP-activated protein kinase (AMPK), promotes mitochondrial fission to improve mitochondrial respiration and restore the mitochondrial life cycle. Furthermore, HFD-fed-mice with liver-specific knockout of AMPKα1/2 subunits exhibit higher blood glucose levels when treated with metformin. Our results demonstrate that activation of AMPK by metformin improves mitochondrial respiration and hyperglycemia in obesity. We also found that supra-pharmacological metformin concentrations reduce adenine nucleotides, resulting in the halt of mitochondrial respiration. These findings suggest a mechanism for metformin’s anti-tumor effects.

Background-Cardiac resynchronization therapy (CRT) is widely applied in patients with heart failure and dyssynchronous contraction (DHF), but the electrophysiological consequences of CRT in heart failure remain largely unexplored. Methods and Results-Adult dogs underwent left bundle-branch ablation and either right atrial pacing (190 to 200 bpm) for 6 weeks (DHF) or 3 weeks of right atrial pacing followed by 3 weeks of resynchronization by biventricular pacing at the same pacing rate (CRT). Isolated left ventricular anterior and lateral myocytes from nonfailing (control), DHF, and CRT dogs were studied with the whole-cell patch clamp. Quantitative polymerase chain reaction and Western blots were performed to measure steady state mRNA and protein levels. DHF significantly reduced the inward rectifier K ϩ current (I K1 ), delayed rectifier K ϩ current (I K ), and transient outward K ϩ current (I to ) in both anterior and lateral cells. CRT partially restored the DHF-induced reduction of I K1 and I K but not I to , consistent with trends in the changes in steady state K ϩ channel mRNA and protein levels. DHF reduced the peak inward Ca 2ϩ current (I Ca ) density and slowed I Ca decay in lateral compared with anterior cells, whereas CRT restored peak I Ca amplitude but did not hasten decay in lateral cells. Calcium transient amplitudes were depressed and the decay was slowed in DHF, especially in lateral myocytes. CRT hastened the decay in both regions and increased the calcium transient amplitude in lateral but not anterior cells. No difference was found in Ca V 1.2 (␣1C) mRNA or protein expression, but reduced Ca V ␤2 mRNA was found in DHF cells. DHF reduced phospholamban, ryanodine receptor, and sarcoplasmic reticulum Ca 2ϩ ATPase and increased Na ϩ -Ca 2ϩ exchanger mRNA and protein. CRT did not restore the DHF-induced molecular remodeling, except for sarcoplasmic reticulum Ca 2ϩ ATPase. Action potential durations were significantly prolonged in DHF, especially in lateral cells, and CRT abbreviated action potential duration in lateral but not anterior cells. Early afterdepolarizations were more frequent in DHF than in control cells and were reduced with CRT. Conclusions-CRT partially restores DHF-induced ion channel remodeling and abnormal Ca 2ϩ homeostasis and attenuates the regional heterogeneity of action potential duration. The electrophysiological changes induced by CRT may suppress ventricular arrhythmias, contribute to the survival benefit of this therapy, and improve the mechanical performance of the heart.

Significance Cardiac hypertrophy and dysfunction in response to sustained hormonal and mechanical stress are sentinel features of most forms of heart disease. Activation of non–voltage-gated transient receptor potential canonical channels TRPC3 and TRPC6 may contribute to this pathophysiology and provide a therapeutic target. Effects from combined selective inhibition have not been tested previously. Here we report the capability of highly selective TRPC3/6 inhibitors to block pathological hypertrophic signaling in several cell types, including adult cardiac myocytes. We show in vivo redundancy of each channel; individual gene deletion was not protective against sustained pressure overload, whereas combined deletion ameliorated the response. These data strongly support a role for both channels in cardiac disease and the utility of selective combined inhibition.

Rationale In cardiomyocytes from failing hearts, insufficient mitochondrial Ca2+ ([Ca2+]m) accumulation secondary to cytoplasmic Na+ overload decreases NAD(P)H/NAD(P)+ redox potential and increases oxidative stress when workload increases. These effects are abolished by enhancing [Ca2+]m with acute treatment with CGP-37157 (CGP), an inhibitor of the mitochondrial Na+/Ca2+ exchanger. Objective To determine if chronic CGP treatment mitigates contractile dysfunction and arrhythmias in an animal model of heart failure (HF) and sudden cardiac death (SCD). Methods and Results Here, we describe a novel guinea-pig HF/SCD model employing aortic constriction combined with daily β-adrenergic receptor stimulation (ACi) and show that chronic CGP treatment (ACi+CGP) attenuates cardiac hypertrophic remodeling, pulmonary edema, and interstitial fibrosis and prevents cardiac dysfunction and SCD. In the ACi group 4 weeks after pressure-overload, fractional shortening and the rate of left ventricular pressure development decreased by 36% and 32%, respectively, compared to sham-operated controls; in contrast, cardiac function was completely preserved in the ACi+CGP group. CGP treatment also significantly reduced the incidence of premature ventricular beats and prevented fatal episodes of ventricular fibrillation, but did not prevent QT prolongation. Without CGP treatment, mortality was 61% in the ACi group within 4 weeks of aortic constriction, while the death rate in the ACi+CGP group was not different from sham-operated animals. Conclusions The findings demonstrate the critical role played by altered mitochondrial Ca2+ dynamics in the development of HF and HF-associated SCD; moreover, they reveal a novel strategy for treating SCD and cardiac decompensation in HF.

Background: Mitochondrial protein O-GlcNAcylation is not well understood.Results: Eighty eight mitochondrial proteins, involved in diverse pathways, are O-GlcNAcylated, and an overall increased O-GlcNAcylation leads to altered mitochondrial function. Conclusion: O-GlcNAcylation is on many mitochondrial proteins within the oxidative phosphorylation system, modulating cardiac mitochondrial function. Significance: O-GlcNAc cycles on many proteins within mitochondria, leading to altered function.