Ca(2+) has been postulated as a cytosolic second messenger in the regulation of cardiac oxidative phosphorylation. This hypothesis draws support from the well-known effects of Ca(2+) on muscle activity, which is stimulated in parallel with the Ca(2+)-sensitive dehydrogenases (CaDH). The effects of Ca(2+) on oxidative phosphorylation were further investigated in isolated porcine heart mitochondria at the level of metabolic driving force (NADH or Deltapsi) and ATP production rates (flow). The resulting force-flow (F-F) relationships permitted the analysis of Ca(2+) effects on several putative control points within oxidative phosphorylation, simultaneously. The F-F relationships resulting from additions of carbon substrates alone provided a model of pure CaDH activation. Comparing this curve with variable Ca(2+) concentration ([Ca(2+)]) effects revealed an approximate twofold higher ATP production rate than could be explained by a simple increase in NADH or Deltapsi via CaDH activation. The half-maximal effect of Ca(2+ )at state 3 was 157 nM and was completely inhibited by ruthenium red (1 microM), indicating matrix dependence of the Ca(2+) effect. Arsenate was used as a probe to differentiate between F(0)/F(1)-ATPase and adenylate translocase activity by a futile recycling of ADP-arsenate within the matrix, catalyzed by the F(0)/F(1)-ATPase. Ca(2+) increased the ADP arsenylation rate more than twofold, suggesting a direct effect on the F(0)/F(1)-ATPase. These results suggest that Ca(2+) activates cardiac aerobic respiration at the level of both the CaDH and F(0)/F(1)-ATPase. This type of parallel control of both intermediary metabolism and ATP synthesis may provide a mechanism of altering ATP production rates with minimal changes in the high-energy intermediates as observed in vivo.

Phosphate (P i ) is a putative cytosolic signaling molecule in the regulation of oxidative phosphorylation. Here, by using a multiparameter monitoring system, we show that P i controls oxidative phosphorylation in a balanced fashion, modulating both the generation of useful potential energy and the formation of ATP by F 1 F 0 -ATPase in heart and skeletal muscle mitochondria. In these studies the effect of P i was determined on the mitochondria [NADH], NADH generating capacity, matrix pH, membrane potential, oxygen consumption, and cytochrome reduction level. P i enhanced NADH generation and was obligatory for electron flow under uncoupled conditions. P i oxidized cytochrome b (cyto-b) and reduced cytochrome c (cyto-c), potentially improving the coupling between the NADH free energy and the proton motive force. The apparent limitation in reducing equivalent flow between cyto-b and cyto-c in the absence of P i was confirmed in the intact heart by using optical spectroscopic techniques under conditions with low cytosolic [P i ]. These results demonstrate that P i signaling results in the balanced modulation of oxidative phosphorylation, by influencing both ⌬G H ؉ generation and ATP production, which may contribute to the energy metabolism homeostasis observed in intact systems. Phosphate (P i )1 is the substrate for the phosphorylation of ADP to ATP in oxidative phosphorylation. Because ADP and P i are generated by ATPases in the cytosol, the potential roles of ADP and P i as cytosolic feedback signaling molecules regulating the rate of ATP production was one of the first models of the cytosolic regulation of mitochondrial ATP production (1, 2).However, over the years it has become apparent that the cellular regulation of oxidative phosphorylation is a very complex control network, with numerous potential rate-limiting steps affected by a variety of signaling molecules, including ADP, P i , Ca 2ϩ , creatine, and Mg 2ϩ (3-7). This network results in the ability of tissues to change significantly the rate of ATP generation without significantly modifying the metabolic intermediates coupled to many other processes in the cell. This has been termed an energy metabolism homeostasis (8). Toward a better understanding of this regulatory network, the effects of each putative signaling molecule on oxidative phosphorylation need to be characterized. The purpose of the current work was to further evaluate the effects of P i on different regulatory sites of oxidative phosphorylation in cardiac mitochondria.Phosphate is believed to enter cardiac mitochondria via a neutral phosphate transporter (P t ) in exchange for OH Ϫ or by co-transport with H ϩ (9). Thus, P i transport is linked to the mitochondrial inner membrane pH gradient (⌬pH m ) and the phosphate concentration gradient but not to the membrane potential (⌬⌿). Although P i transport has not been ascribed as a rate-limiting step for phosphate utilization in oxidative phosphorylation, this particular aspect of phosphate metabolism has not been extensively studied, espec...

Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondria-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondria matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca 2+ ) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis/mass spectrometry of Pro-Q Diamond staining while many more Pro-Q Diamond stained proteins were below mass spectrometry detection. Time dependent 32 P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondria matrix is extensive and dynamic. Ca 2+ has previously been shown to activate various dehydrogenases, promote reactive oxygen species (ROS) generation, and initiate apoptosis via cytochrome c release. To evaluate the Ca 2+ signaling network, the effects of a Ca 2+ challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca 2+ -induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca 2+ dose dependent dephosphorylation of MnSOD was associated with a ∼2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca 2+ . These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca 2+ modification of mitochondrial function at the level of MnSOD.Mitochondria are thought to be the result of an early interaction of two lines of cellular life, the bacterium and eukaryotic cell (1;2). At this point in time, mitochondria play a critical role in energy metabolism, apoptosis and cell signaling pathways in the cell. However, the acute and chronic regulatory mechanisms of this organelle remain poorly defined. One approach to assessing the function and regulation of the mitochondrion is an evaluation of the mitochondrial Address correspondence to: Robert S. Balaban, Laboratory of Cardiac Energetics, National Heart Lung and Blood Institute, National Institutes of Health, 10 Center Drive Room B1D416, Bethesda, MD 20892-1061. Tel. 301 496-3658; Fax. 301 402-2389; E-mail: rsb@nih.gov. proteome. Estimates predict up to 3000 proteins (3;4) in mitochondria, however, recent largescale screening studies by Taylor (5) and Mootha (6) identified only about 600 distinct mitochondrial proteins. Many have used proteomic approaches to evaluate differential protein expr...

Parallel activation of heart mitochondria NADH and ATP production by Ca(2+) has been shown to involve the Ca(2+)-sensitive dehydrogenases and the F(0)F(1)-ATPase. In the current study we hypothesize that the response time of Ca(2+)-activated ATP production is rapid enough to support step changes in myocardial workload ( approximately 100 ms). To test this hypothesis, the rapid kinetics of Ca(2+) activation of mV(O(2)), [NADH], and light scattering were evaluated in isolated porcine heart mitochondria at 37 degrees C using a variety of optical techniques. The addition of Ca(2+) was associated with an initial response time (IRT) of mV(O(2)) that was dose-dependent with a minimum IRT of 0.27 +/- 0.02 s (n = 41) at 535 nm Ca(2+). The IRTs for NADH fluorescence and light scattering in response to Ca(2+) additions were similar to mV(O(2)). The Ca(2+) IRT for mV(O(2)) was significantly shorter than 1.6 mm ADP (2.36 +/- 0.47 s; p < or = 0.001, n = 13), 2.2 mm P(i) (2.32 +/- 0.29, p < or = 0.001, n = 13), or 10 mm creatine (15.6.+/-1.18 s, p < or = 0.001, n = 18) under similar experimental conditions. Calcium effects were inhibited with 8 microm ruthenium red (2.4 +/- 0.31 s; p < or = 0.001, n = 16) and reversed with EGTA (1.6 +/- 0.44; p < or = 0.01, n = 6). Estimates of Ca(2+) uptake into mitochondria using optical Ca(2+) indicators trapped in the matrix revealed a sufficiently rapid uptake to cause the metabolic effects observed. These data are consistent with the notion that extramitochondrial Ca(2+) can modify ATP production, via an increase in matrix Ca(2+) content, rapidly enough to support cardiac work transitions in vivo.

functionality of the mitochondrion is primarily determined by nuclear encoded proteins. The mitochondrial functional requirements of different tissues vary from a significant biosynthetic role (liver) to a primarily energy metabolism-oriented organelle (heart). The purpose of this study was to compare the mitochondrial proteome from four different tissues of the rat, brain, liver, heart, and kidney, to provide insight into the extent of mitochondrial heterogeneity and to further characterize the overall mitochondrial proteome. Mitochondria were isolated, solubilized, digested, and subjected to quantitative liquid chromatography-mass spectroscopy. Of the 16,950 distinct peptides detected, 8,045 proteins were identified. High-confidence identification threshold was reached by 1,162 peptides, which were further analyzed. Of these 1,162 proteins, 1,149 were significantly different in content (P and q values Ͻ 0.05) between at least 2 tissues, whereas 13 were not significantly different between any tissues. Confirmation of the mitochondrial origin of proteins was determined from the literature or via NH2-terminal mitochondrial localization signals. With these criteria, 382 proteins in the significantly different groups were confirmed to be mitochondrial, and 493 could not be confirmed to be mitochondrial but were not definitively localized elsewhere in the cell. A total of 145 proteins were assigned to the rat mitochondrial proteome for the first time via their NH 2-terminal mitochondrial localization signals. Among the proteins that were not significantly different between tissues, three were confirmed to be mitochondrial. Most notable of the significantly different proteins were histone family proteins and several structural proteins, including tubulin and intermediate filaments. The mitochondrial proteome from each tissue had very specific characteristics indicative of different functional emphasis. These data confirm the notion that mitochondria are tuned by the nucleus for specific functions in different tissues. structural proteins; oxidative phosphorylation; liquid chromatography; mass spectrometry; electrophoresis; histone; liver; heart; kidney; brain MITOCHONDRIA ARE semi-self-replicating organelles responsible for several essential functions in mammalian cells, including oxidative phosphorylation, apoptosis, iron-sulfur cluster assembly, and several important catabolic pathways. Each mitochondrion contains 2-10 copies of a circular genome that codes for 13 proteins, 22 tRNAs, and 2 rRNAs (2). The balance of the proteins that are incorporated into mitochondria are coded by the nuclear genome and then incorporated via the mitochondrial import apparatus in a distribution that is specific to the tissue in which the mitochondria reside (3,21,23,33). This differential protein expression leads to mitochondria that are vastly different in their metabolic function and activity level between tissues. A scan of the human genome predicts as many as 3,000 proteins localized to the mitochondria (28, 32). The most current...

Previous studies have determined that mice with a homozygous deletion in the adapter protein p66shc have an extended life span and that cells derived from these mice exhibit lower levels of reactive oxygen species. Here we demonstrate that a fraction of p66 shc localizes to the mitochondria and that p66 shc؊/؊ fibroblasts have altered mitochondrial energetics. In particular, despite similar cytochrome content, under basal conditions, the oxygen consumption of spontaneously immortalized p66shc؊/؊ mouse embryonic fibroblasts were lower than similarly maintained wild type cells. shc may extend life span by repartitioning metabolic energy conversion away from oxidative and toward glycolytic pathways.Generation of ATP in the mitochondria represents the most efficient pathway to meet the energetic needs of a cell. This process, however, requires the consumption of molecular oxygen with a corresponding production of reactive oxygen species (ROS).2 Generation of ROS appears to be one of the central mechanisms that contribute to aging in a wide range of organisms (1-3). In contrast, under aerobic conditions, energy generation can also be achieved through glycolytic pathways present in the cytosol. These cytosolic pathways are inherently less efficient but do not produce ROS. Each cell employs a different relative balance between these two major energetic pathways, although relatively little is known about how this partition is established or maintained.In lower organisms, such as Caenorhabditis elegans and Drosophila, a number of longevity-associated genes have been isolated. One prominent and well characterized aging pathway regulates the activity of the transcription factor DAF-16, a member of the Forkhead family of transcriptional regulators. Evidence suggests that DAF-16 is involved in responding to numerous environmental stresses (4). A rise in intracellular ROS is one particular stress that may be relevant to life span determination, and in this regard, it is of interest that both DAF-16 and its closest mammalian ortholog Foxo3a appear to regulate a number of cellular antioxidant proteins (5-9).In addition to the DAF-16 pathway, RNAi screens performed in C. elegans has identified a number of other putative longevity genes (10, 11). Interestingly, functional characterization of these longevity-associated genes have determined that a number of them appear to be important for mitochondrial function. Similarly, direct knockdown of components of the electron transport chain has also been shown to extend the life span of the worm (12). Analysis of these long lived mitochondrial mutants, as well as in depth energetic analysis of the previously characterized DAF-16-related mutants, has led to the proposal that many life span-extending mutants in C. elegans slow aging by decreasing mitochondrial metabolism (13). This hypothesis suggests that a shift away from trichloroacetic acid-based mitochondrial metabolism might extend life by a reduction in oxidative stress. Nonetheless, it should be mentioned that the relationship bet...

Using iTRAQ labeling and mass spectrometry on an LTQ-Orbitrap with HCD capability, we assessed relative changes in protein phosphorylation in the mitochondria upon physiological perturbation. As a reference reaction, we monitored the well-characterized regulation of pyruvate dehydrogenase (PDH) activity via phosphorylation/dephosphorylation by pyruvate dehydrogenase kinase/pyruvate dehydrogenase phosphatase in response to dichloroacetate, de-energization and Ca 2+ . Relative quantification of phosphopeptides of PDH-E1α subunit from porcine heart revealed dephosphorylation at three serine sites (Ser231, Ser292 and Ser299). Dephosphorylation at Ser292 (i.e., the inhibitory site) with DCA correlated with an activation of PDH activity as previously reported, consistent with our de-energization data. Calcium also dephosphorylated (i.e., activated) PDH thus confirming calcium activation of PDP. With this approach, we successfully monitored other phosphorylation sites of mitochondrial proteins including adenine nucleotide translocase, malate dehydrogenase and mitochondrial creatine kinase, etc. Among them four proteins exhibited phosphorylation changes with these physiological stimuli: (1) BCKDH-E1α subunit increased phosphorylation at Ser337 with DCA and de-energization; (2) apoptosis-inducing factor phosphorylation was elevated at Ser345 with calcium; (3) ATP synthase F1 complex α subunit and (4) mitofilin dephosphorylated at Ser65 and Ser264 upon de-energization. This screening validated the iTRAQ/HCD technology as a method for functional quantitation of mitochondrial protein phosphorylation as well as providing insights into the regulation of mitochondria via phosphorylation.

We have optimized a method to directly measure oxygen consumption in acutely isolated, ex vivo mouse retina and demonstrate that photoreceptors have low mitochondrial reserve capacity. Our data provide a plausible explanation for the high vulnerability of photoreceptors to altered energy homeostasis caused by mutations or metabolic challenges.