Reactive oxygen species (ROS) and/or Ca 2؉ overload can trigger depolarization of mitochondrial inner membrane potential (⌬⌿ m ) and cell injury. Little is known about how loss of ⌬⌿ m in a small number of mitochondria might influence the overall function of the cell. Here we employ the narrow focal excitation volume of the two-photon microscope to examine the effect of local mitochondrial depolarization in guinea pig ventricular myocytes. Remarkably, a single local laser flash triggered synchronized and self-sustained oscillations in ⌬⌿ m , NADH, and ROS after a delay of ϳ40s, in more than 70% of the mitochondrial population. Oscillations were initiated only after a specific threshold level of mitochondrially produced ROS was exceeded, and did not involve the classical permeability transition pore or intracellular Ca 2؉ overload. The synchronized transitions were abolished by several respiratory inhibitors or a superoxide dismutase mimetic. Anion channel inhibitors potentiated matrix ROS accumulation in the flashed region, but blocked propagation to the rest of the myocyte, suggesting that an inner membrane, superoxide-permeable, anion channel opens in response to free radicals. The transitions in mitochondrial energetics were tightly coupled to activation of sarcolemmal K ATP currents, causing oscillations in action potential duration, and thus might contribute to catastrophic arrhythmias during ischemia-reperfusion injury.Mitochondria play a multifunctional role as key arbiters of cell life and death. In addition to oxidative phosphorylation, mitochondria are involved in thermogenesis, free radical production, and intracellular Ca 2ϩ homeostasis. Impairment of mitochondrial function during and after ischemia causes rapid energy depletion, contractile failure, and loss of cellular integrity, which may lead to necrotic or apoptotic cell death (1-3). ROS 1 have been implicated in ischemic dysfunction; however, they play a dual role as determinants of cell survival, on the one hand contributing to Ca 2ϩ overload and the induction of a mitochondrial permeability transition, but on the other acting as second messengers that protect cells against injury (4 -6). Mitochondria are a major site of physiological ROS production in the cardiomyocyte, with ϳ1-5% of the electrons flowing through the electron transport chain leaking into the production of ROS (7, 8). The negative effects of ROS on metabolism are evident in several studies showing rapid and spatiotemporally heterogeneous discharge of ⌬⌿ m in response to oxidative stress (1, 2) and/or Ca 2ϩ overload (9), including protocols employing laser-induced photooxidation (10, 11) and mitochondrial ROS-induced ROS release (12). In light of our previous work showing that substrate deprivation can initiate synchronized oscillations of mitochondrial redox and membrane potential (13), and that a diffusible cytoplasmic messenger may be involved (14), the present study tests whether similar global self-organizing behavior can be triggered by a highly localized perturbation of ...

We present an integrated thermokinetic model describing control of cardiac mitochondrial bioenergetics. The model describes the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial Ca(2+) handling. The kinetic component of the model includes effectors of the TCA cycle enzymes regulating production of NADH and FADH(2), which in turn are used by the electron transport chain to establish a proton motive force (Delta mu(H)), driving the F(1)F(0)-ATPase. In addition, mitochondrial matrix Ca(2+), determined by Ca(2+) uniporter and Na(+)/Ca(2+) exchanger activities, regulates activity of the TCA cycle enzymes isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase. The model is described by twelve ordinary differential equations for the time rate of change of mitochondrial membrane potential (Delta Psi(m)), and matrix concentrations of Ca(2+), NADH, ADP, and TCA cycle intermediates. The model is used to predict the response of mitochondria to changes in substrate delivery, metabolic inhibition, the rate of adenine nucleotide exchange, and Ca(2+). The model is able to reproduce, qualitatively and semiquantitatively, experimental data concerning mitochondrial bioenergetics, Ca(2+) dynamics, and respiratory control. Significant increases in oxygen consumption (V(O(2))), proton efflux, NADH, and ATP synthesis, in response to an increase in cytoplasmic Ca(2+), are obtained when the Ca(2+)-sensitive dehydrogenases are the main rate-controlling steps of respiratory flux. These responses diminished when control is shifted downstream (e.g., the respiratory chain or adenine nucleotide translocator). The time-dependent behavior of the model, under conditions simulating an increase in workload, closely reproduces experimentally observed mitochondrial NADH dynamics in heart trabeculae subjected to changes in pacing frequency. The steady-state and time-dependent behavior of the model support the hypothesis that mitochondrial matrix Ca(2+) plays an important role in matching energy supply with demand in cardiac myocytes.

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 .

Calorie restriction (CR) is the most robust non-genetic intervention to delay aging. However, there are a number of emerging experimental variables that alter CR responses. We investigated the role of sex, strain, and level of CR on health and survival in mice. CR did not always correlated with lifespan extension, though it consistently improved health across strains and sexes. Transcriptional and metabolomics changes driven by CR in liver indicated anaplerotic filling of the Krebs cycle together with fatty acid fueling of mitochondria. CR prevented age-associated decline in the liver proteostasis network while increasing mitochondrial number, preserving mitochondrial ultrastructure and function with age. Abrogation of mitochondrial function negated life-prolonging effects of CR in yeast and worms. Our data illustrate the complexity of CR in the context of aging, with a clear separation of outcomes related to health and survival, highlighting complexities of translation of CR into human interventions.