Rationale Mitochondrial Ca2+ uptake is essential for the bioenergetic feedback response through stimulation of Krebs cycle dehydrogenases. Close association of mitochondria to the sarcoplasmic reticulum (SR) may explain efficient mitochondrial Ca2+ uptake despite low Ca2+ affinity of the mitochondrial Ca2+ uniporter. However, the existence of such mitochondrial Ca2+ microdomains and their functional role are presently unresolved. Mitofusin (Mfn) 1 and 2 mediate mitochondrial outer membrane fusion, while Mfn2, but not Mfn1, tethers endoplasmic reticulum to mitochondria in non-cardiac cells. Objective To elucidate roles for Mfn1 and 2 in SR-mitochondrial tethering, Ca2+ signaling and bioenergetic regulation in cardiac myocytes. Methods and Results Fruit fly heart tubes deficient of the Drosophila Mfn ortholog, MARF, had increased contraction-associated and caffeine-sensitive Ca2+ release, suggesting a role for Mfn in SR Ca2+ handling. While cardiac-specific Mfn1 ablation had no effects on murine heart function or Ca2+ cycling, Mfn2 deficiency decreased cardiomyocyte SR-mitochondrial contact length by 30% and reduced the content of SR-associated proteins in mitochondria-associated membranes. This was associated with decreased mitochondrial Ca2+ uptake (despite unchanged mitochondrial membrane potential) but increased steady-state and caffeine-induced SR Ca2+ release. Accordingly, Ca2+-induced stimulation of Krebs cycle dehydrogenases during β-adrenergic stimulation was hampered in Mfn2-, but not Mfn1-KO myocytes, evidenced by oxidation of the redox states of NAD(P)H/NAD(P)+ and FADH2/FAD. Conclusions Physical tethering of SR and mitochondria via Mfn2 is essential for normal inter-organelle Ca2+ signaling in the myocardium, consistent with a requirement for SR-mitochondrial Ca2+ signaling through microdomains in the cardiomyocyte bioenergetic feedback response to physiological stress.

Mitochondrial reactive oxygen species (ROS) play a central role in most aging-related diseases. ROS are produced at the respiratory chain that demands NADH for electron transport and are eliminated by enzymes that require NADPH. The nicotinamide nucleotide transhydrogenase (Nnt) is considered a key antioxidative enzyme based on its ability to regenerate NADPH from NADH. Here, we show that pathological metabolic demand reverses the direction of the Nnt, consuming NADPH to support NADH and ATP production, but at the cost of NADPH-linked antioxidative capacity. In heart, reverse-mode Nnt is the dominant source for ROS during pressure overload. Due to a mutation of the Nnt gene, the inbred mouse strain C57BL/6J is protected from oxidative stress, heart failure, and death, making its use in cardiovascular research problematic. Targeting Nnt-mediated ROS with the tetrapeptide SS-31 rescued mortality in pressure overload-induced heart failure and could therefore have therapeutic potential in patients with this syndrome.

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...

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