Regulation of mitochondrial contact sites in neonatal,juvenile and diabetic hearts

Mitochondrial contact sites (MiCS) are dynamic structures involved in high capacity transport of energy from mitochondria into the cytosole. Previous studies revealed that in normal conditions the actual number of MiCS is in correlation with the energy requirements of the heart, particularly with those for its contractile work. Although the detailed mechanisms of signalling between the processes of energy utilisation and MiCS formation in the heart are not yet elucidated, it is known that intracellular Ca²⁺ transients are intimately involved in this crosstalk. The present study is devoted to investigation of Ca²⁺-linked MiCS formation in healthy adult hearts and in hearts with modified Ca²⁺-handling such as in developing, in juvenile and diabetic myocardium. Experiments were performed on hearts of healthy rats on the 22nd embryonal day, 1st, 4th, 7th and 14th postnatal days as well as on adult hearts. Diabetic hearts were investigated on the 8th day after streptozotocin injection (45 mg.kg^–1 i.v.) to adult rats. Intracellular Ca²⁺ movements were affected by modulation of Ca²⁺ concentration in perfusion solution (1.6 or 2.2 mmol.l^–1) in isolated, Langendorff-perfused hearts, by calcium paradox (CaP) or by replacing of Ca²⁺ by Cd²⁺ ions. Elevation of extracellular Ca²⁺ was reflected by 30.1, 10.4 and 24.1% increase in intracellular free Ca²⁺ concentration in healthy adult, diabetic and 14-day old hearts respectively. In developing hearts the amount of MiCS was culminating on the 4th postnatal day. In adult hearts, elevated calcium in the perfusion solution, CaP as well as diabetes led to a significant increase in the amounts of MiCS formed (58.1, 77.2 and 86.5% respectively; p < 0.05). Diabetic and 14-day old hearts naturally exhibited amounts of MiCS comparable to those obtained by Ca²⁺-stimulation of MiCS formation in adult healthy hearts. In contrast to healthy controls, perfusion of diabetic and 14-day old hearts with elevated Ca²⁺ as well as induction of CaP exerted little influence on MiCS formation (4.4 and 8.2% for elevated Ca²⁺; 2.9 and 10.7% for CaP; p > 0.05). A replacement of Ca²⁺ by Cd²⁺ ions lowered the amount of MiCS in healthy adult and diabetic hearts (61 and 52.2%; p < 0.05). In conclusion, during development, the formation of MiCS may be influenced by both, permanent stimulation by Ca²⁺-signalling and the availability of mCPK. In healthy adult hearts the amount of MiCS is modulated by intracellular Ca²⁺ transients in response to changes in extracellular Ca²⁺ concentration. In diabetic hearts the modulation of MiCS formation is naturally attenuated, apparently as a consequence of persisting alterations in Ca²⁺-handling.     

A computational model integrating electrophysiology, contraction, and mitochondrial bioenergetics in the ventricular myocyte

An intricate network of reactions is involved in matching energy supply with demand in the heart. This complexity arises because energy production both modulates and is modulated by the electrophysiological and contractile activity of the cardiac myocyte. Here, we present an integrated mathematical model of the cardiac cell that links excitation-contraction coupling with mitochondrial energy generation. The dynamics of the model are described by a system of 50 ordinary differential equations. The formulation explicitly incorporates cytoplasmic ATP-consuming processes associated with force generation and ion transport, as well as the creatine kinase reaction. Changes in the electrical and contractile activity of the myocyte are coupled to mitochondrial energetics through the ATP, Ca2+, and Na+ concentrations in the myoplasmic and mitochondrial matrix compartments. The pseudo steady-state relationship between force and oxygen consumption at various stimulus frequencies and external Ca2+ concentrations is reproduced in both model simulations and direct experiments in cardiac trabeculae under normoxic conditions, recapitulating the linearity between cardiac work and respiration in the heart. Importantly, the model can also reproduce the rapid time-dependent changes in mitochondrial NADH and Ca2+ in response to abrupt changes in workload. The steady-state and dynamic responses of the model were conferred by ADP-dependent stimulation of mitochondrial oxidative phosphorylation and Ca2+ -dependent regulation of Krebs cycle dehydrogenases, illustrating how the model can be used as a tool for investigating mechanisms underlying metabolic control in the heart.     

Effect of inotropic stimulation on mitochondrial calcium in cardiac muscle.

Ca(2+)-dependent activation of citric acid cycle enzymes has been demonstrated in isolated cardiac mitochondria. These observations led to the hypothesis that Ca2+ is the signal coupling myofibrillar energy use to mitochondrial energy production in vivo. To test this hypothesis we have measured mitochondrial Ca2+ content during increased energy demand, using electron probe microanalysis. Mitochondrial Ca2+ was measured in hamster papillary muscles rapidly frozen at the peak rate of tension rise under control conditions and after stimulation with the beta-adrenergic agonist isoproterenol (10(-6) M). A third group of muscles was frozen after incubation in low (46.5 mM) Na+ solution to Ca2+ load the cells. Pyruvate dehydrogenase activity was measured in each of the muscles. Isoproterenol caused a 39% increase in force and a 43% increase in pyruvate dehydrogenase activity but no change in mitochondrial Ca2+ (0.46 +/- 0.19 (S.E.) mmol of Ca2+/kg, dry weight) compared with control (0.54 +/- 0.12). In contrast, low Na+ increased pyruvate dehydrogenase activity by 56% and also elevated mitochondrial Ca2+ to 1.28 +/- 0.31 (p less than 0.02). These results demonstrate that mitochondrial Ca2+ is not elevated after inotropic stimulation of cardiac muscle by beta-adrenergic agonists although pyruvate dehydrogenase activity is increased. We conclude that Ca2+ uptake by mitochondria is not a requirement for activation of mitochondrial respiration after increased energy demand.     

Activation of pyruvate dehydrogenase by electrical stimulation, and low-Na+ perfusion of guinea-pig heart

(1) Electrical stimulation (2 Hz) of guinea-pig hearts, perfused with medium containing 11 mM D-glucose plus 0.1 mM octanoate as substrate, resulted in an increase in the percentage of pyruvate dehydrogenase in the active form (PDHa) from 16 to 68%. (2) Rapid isolation of mitochondria by a technique designed to minimize net loss or gain of Ca2+ revealed an increase in mitochondrial Ca2+ content of the stimulated hearts, as measured with 45Ca (2.74 +/- 0.27 versus 1.37 +/- 0.11 nmol/mg protein; stimulated versus rested). (3) Perfusion of rested hearts with a medium containing a reduced Na+ concentration (20 mM, with the remainder replaced with Li+) also gave increased values of PDHa content (30.9% versus 16% for the normal, physiological medium). This procedure is known to raise cytosol Ca2+ concentrations and would be expected to give mitochondrial Ca2+ loading. (4) These results are consistent with a role of mitochondrial Ca2+ in activating pyruvate dehydrogenase in the intact heart.     

Aldehyde stress and up-regulation of Nrf2-mediated antioxidant systems accompany functional adaptations in cardiac mitochondria from mice fed n-3 polyunsaturated fatty acids

Diets replete with n-3 PUFAs (polyunsaturated fatty acids) are known to have therapeutic potential for the heart, although a specifically defined duration of the n-3 PUFA diet required to achieve these effects remains unknown, as does their mechanism of action. The present study was undertaken to establish whether adaptations in mitochondrial function and stress tolerance in the heart is evident following short- (3?weeks) and long- (14?weeks) term dietary intervention of n-3 PUFAs, and to identify novel mechanisms by which these adaptations occur. Mitochondrial respiration [mO2 (mitochondrial O2)], H2O2 emission [mH2O2 (mitochondrial H2O2)] and Ca2+-retention capacity [mCa2+ (mitochondrial Ca2+)] were assessed in mouse hearts following dietary intervention. Mice fed n-3 PUFAs for 14?weeks showed significantly lower mH2O2 and greater mCa2+ compared with all other groups. However, no significant differences were observed after 3?weeks of the n-3 PUFA diet, or in mice fed on an HFC (high-fat control) diet enriched with vegetable shortening, containing almost no n-3 PUFAs, for 14?weeks. Interestingly, expression and activity of key enzymes involved in antioxidant and phase II detoxification pathways, all mediated by Nrf2 (nuclear factor E2-related factor 2), were elevated in hearts from mice fed the n-3 PUFA diet, but not hearts from mice fed the HFC diet, even at 3?weeks. This increase in antioxidant systems in hearts from mice fed the n-3 PUFA diet was paralleled by increased levels of 4-hydroxyhexenal protein adducts, an aldehyde formed from peroxidation of n-3 PUFAs. The findings of the present study demonstrate distinct time-dependent effects of n-3 PUFAs on mitochondrial function and antioxidant response systems in the heart. In addition, they are the first to provide direct evidence that non-enzymatic oxidation products of n-3 PUFAs may be driving mitochondrial and redox-mediated adaptations, thereby revealing a novel mechanism for n-3 PUFA action in the heart.     

Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using 31P n.m.r. and ruthenium red.

1. The concentrations of free ATP, phosphocreatine (PCr), Pi, H+ and ADP (calculated) were monitored in perfused rat hearts by 31P n.m.r. before and during positive inotropic stimulation. Data were accumulated in 20 s blocks. 2. Administration of 0.1 microM-(-)-isoprenaline resulted in no significant changes in ATP, transient decreases in PCr, and transient increases in ADP and Pi. However, the concentrations of all of these metabolites returned to pre-stimulated values within 1 min, whereas cardiac work and O2 uptake remained elevated. 3. In contrast, in hearts perfused continuously with Ruthenium Red (2.5 micrograms/ml), a potent inhibitor of mitochondrial Ca2+ uptake, administration of isoprenaline caused significant decreases in ATP, and also much larger and more prolonged changes in the concentrations of ADP, PCr and Pi. In this instance values did not fully return to pre-stimulated concentrations. Administration of Ruthenium Red alone to unstimulated hearts had minor effects. 4. It is proposed that, in the absence of Ruthenium Red, the transmission of changes in cytoplasmic Ca2+ across the mitochondrial inner membrane is able to maintain the phosphorylation potential of the heart during positive inotropic stimulation, through activation of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) leading to enhanced NADH production. 5. This mechanism is unavailable in the presence of Ruthenium Red, and oxidative phosphorylation must be stimulated primarily by a fall in phosphorylation potential, in accordance with the classical concept of respiratory control. However, the full oxidative response of the heart to stimulation may not be achievable under such circumstances.     

Mechanisms of mitochondrial response to variations in energy demand in eukaryotic cells

This review focuses on the different mechanisms involved in the adjustment of mitochondrial ATP production to cellular energy demand. The oxidative phosphorylation steady state at constant mitochondrial enzyme content can vary in response to energy demand. However, such an adaptation is tightly linked to a modification in both oxidative phosphorylation yield and phosphate potential and is obviously very limited in eukaryotic cells. We describe the three main mechanisms involved in mitochondrial response to energy demand. In heart cells, a short-term adjustment can be reached mainly through metabolic signaling via phosphotransfer networks by the compartmentalized energy transfer and signal transmission. In such a complex regulatory mechanism, Ca²⁺ signaling participates in activation of matricial dehydrogenases as well as mitochondrial ATP synthase. These processes allow a large increase in ATP production rate without an important modification in thermodynamic forces. For a long-term adaptation, two main mechanisms are involved: modulation of the mitochondrial enzyme content as a function of energy demand and/or kinetic regulation by covalent modifications (phosphorylations) of some respiratory chain complex subunits. Regardless of the mechanism involved (kinetic regulation by covalent modification or adjustment of mitochondrial enzyme content), the cAMP signaling pathway plays a major role in molecular signaling, leading to the mitochondrial response. We discuss the energetic advantages of these mechanisms. yeast; C6 glioma cells; muscle; kinetic regulation     

Energy requirements for the Na+ gradient in the oxygenated isolated heart: effect of changing the free energy of ATP hydrolysis

This study tests the hypothesis that a decrease of the free energy of ATP hydrolysis (Delta GATP) below a threshold value will inhibit Na+-K+-ATPase (Na+ pump) activity and result in an increase of intracellular Na+ concentration ([Na+]i) in the heart. Conditions were designed in which hearts were solely dependent on ATP derived from oxidative phosphorylation. The only substrate supplied was the fatty acid butyrate (Bu) at either low, 0.1 mM (LowBu), or high, 4 mM (HighBu), concentrations. Escalating work demand reduced the Delta GATP of the LowBu hearts. 31P, 23Na, and 87Rb NMR spectroscopy measured high-energy phosphate metabolites, [Na+]i, and Rb+ uptake. Rb+ uptake was used to estimate Na+ pump activity. To measure [Na+]i using a shift reagent for cations, extracellular Ca2+ was reduced to 0.85 mM, which eliminated work demand Delta GATP reductions. Increasing extracellular Na+ (Nae+) to 200 mM restored work demand Delta GATP reductions. In response to higher [Na+]e, [Na+]i increased equally in LowBu and HighBu hearts to approximately 8.6 mM, but Delta GATP decreased only in LowBu hearts. At lowest work demand the LowBu heart Delta GATP was -53 kJ/mol, Rb+ uptake was similar to that of HighBu hearts, and [Na+]i was constant. At highest work demand the LowBu heart Delta GATP decreased to -48 kJ/mol, the [Na+]i increased to 25 mM, and Rb+ uptake was 56% of that in HighBu hearts. At the highest work demand the HighBu heart Delta GATP was -54 kJ/mol and [Na+]i increased only approximately 10%. We conclude that a Delta GATP below -50 kJ/mol limits the Na+ pump and prevents maintenance of [Na+]i homeostasis.     

Identification of the Ca2+-release activity and ryanodine receptor in sarcoplasmic-reticulum membranes during cardiac myogenesis.

Ca2+-induced Ca2+ release and pH-induced Ca2+ release activities were identified in sarcoplasmic-reticulum (SR) vesicles isolated from adult- and fetal-sheep hearts. Ca2+-induced Ca2+ release and pH-induced Ca2+ release appear to proceed via the same channels, since both phenomena are similarly inhibited by Ruthenium Red. Ca2+ release from fetal SR vesicles is inhibited by higher concentrations of Ruthenium Red than is that from adult membranes. Both fetal and adult SR vesicles bind ryanodine. Fetal SR shows higher ryanodine-binding capacity than adult SR vesicles. Scatchard analysis of ryanodine binding revealed only one high-affinity binding site (Kd 6.7 nM) in fetal SR vesicles compared with two distinct binding sites (Kd 6.6 and 81.5 nM) in the adult SR vesicles. SR vesicles isolated from fetal and adult hearts were separated on discontinuous sucrose gradients into light (free) and heavy (junctional) SR vesicles. Heavy SR vesicles isolated from adult hearts exhibited most of the Ca2+ release activities. In contrast, Ca2+-induced Ca2+ release, pH-induced Ca2+ release and ryanodine receptors were detected in both light and heavy fetal SR. These results suggest that fetal SR may not be morphologically and functionally as well differentiated as that of adult cardiac muscle and that it may contain a greater number of Ca2+-release channels than that present in adult SR membranes.     

心肌细胞发育过程中胞浆内钙稳态的调控

Ca^2＋信号是细胞和各器官生长发育、行使其生理功能的基础,维持心肌细胞的钙稳态是保持正常心脏功能的先决条件。作为在胚胎发育过程中最早出现并行使功能的器官,胚胎期心脏的形态结构发生了明显的变化,泵血功能不断增强,以适应不断增强的机体的生理需求。从胚胎到成年,心肌细胞的功能有非常大的改变,各钙离子通道的表达也发生明显变化。因此,发育早期心肌细胞的钙稳态调控与成熟心肌细胞有明显的不同,在发育过程中引起细胞收缩的Ca^2＋来源也有明显的变化。随着分子和细胞生物学研究的发展,以及胚胎干细胞体外分化模型的应用,人们对心肌细胞发育过程中钙稳态的调控有了进一步的认识。本文综述了早期心肌细胞发育过程中胞浆内钙稳态的变化,总结了早期心肌细胞钙稳态调控机制的最新研究进展。     

Hormonal effects on mitochondrial respiration: potential role of endogenous lipolytic activities

Hormonal effects on heart mitochondrial metabolism are investigated by comparing respiratory rates, Ca2+ uptake capacity, and lipolytic activities of mitochondria isolated from control rats to those of mitochondria isolated from thyroparathyroidectomized animals. Two biochemically and morphologically distinct populations of heart mitochondria are prepared--one derived from the region of the cell directly beneath the sarcolemma (subsarcolemmal mitochondria), the other originally between the myofibrils (interfibrillar mitochondria). Subsarcolemmal mitochondria isolated from normal rat cardiac tissue have both lower respiratory rates and Ca2+ uptake capacity than do interfibrillar mitochondria. However, when these mitochondrial populations are isolated from hearts from thyroparathyroidectomized rats, there is a selective increase in the maximal ability of the subsarcolemmal mitochondria to accumulate Ca2+, which is accompanied by a proportionate increase in their maximal respiratory rates. Neither Ca2+ uptake capacity nor respiratory rates are similarly increased in the interfibrillar mitochondria. Cytochrome contents and mitochondrial protein recoveries are not significantly changed in either of these mitochondrial preparations. The relationship between these selective increases in respiratory properties of the subsarcolemmal mitochondria to endogenous lipolytic activities is also investigated. It was previously demonstrated that, in the absence of Ca2+, both the rate and extent of formation of free fatty acids from endogenous phospholipids is greater in subsarcolemmal than interfibrillar mitochondria (J. W. Palmer et al. (1981) Arch. Biochem. Biophys. 211, 674-682). In this study it is shown that lipolysis is also more sustained in the subsarcolemmal mitochondria when Ca2+ is added. In the subsarcolemmal mitochondria isolated from thyroparathyroidectomized rats, however, the rates of release of stearic acid and oleic acid are reduced in both the presence and absence of Ca2+. In the presence of added Ca2+, the rate of release of arachidonic acid is also decreased compared to control subsarcolemmal mitochondria, suggesting that the expressed activity of Ca2+-activated phospholipase A2 is lower in those mitochondria isolated from the thyroparathyroidectomized animals, in which respiratory rates and Ca2+ uptake capacity are increased.     

Effects of calcium ions and adenosine diphosphate on the activities of NAD+-linked isocitrate dehydrogenase from the radular muscles of the whelk and flight muscles of insects.

1. The activity of NAD+-linked isocitrate dehydrogenase from the radular muscle of the whelk is higher than those in many vertebrate muscles and only slightly lower than in the flight muscles of insects. The enzyme activity from the whelk (Buccinum undatum) is stable for several hours after homogenization of the radular muscle, whereas that from insect flight muscle is very unstable. Consequently, the enzyme from the whelk muscle is suitable for a systematic investigation of the effects of Ca2+ and ADP. 2. The sigmoid response of the enzyme activity to isocitrate concentration is markedly increased by raising the Ca2+ concentration from 0.001 to 10 muM, but it is decreased by ADP. The inhibitory effect of Ca2+ is most pronounced at pH7.1; it is not observed at pH 6.5. Similar effects are observed for the enzyme from the flight muscle of the locust (Schistocerca gregaria) and the water bug (Lethocerus cordofanus). The percentage activation by ADP of the enzyme from either the whelk or the insects is greater at 10 muM-Ca2+, and 50% of the maximum activation is obtained at 0.10 and 0.16 mM-ADP for the enzyme from whelk and locust respectively at this Ca2+ concentration. At 10 muM-Ca2+ in the absence of added ADP, the apparent Km for isocitrate is markedly higher than in other conditions. Ca2+ concentrations of 0.01, 0.1 and 0.2 muM cause 50% inhibition of maximum activity of the enzyme from the muscles of the whelk, locust and water bug respectively. 3. Recent work has indicated that mitochondria may play a complementary role to the sarcoplasmic reticulum in the control of the distribution of Ca2+ in muscle. The opposite effects of Ca2+ on the activities of isocitrate dehydrogenase and mitochondrial glycerol phosphate dehydrogenase from muscle tissue are consistent with the hypothesis that changes in the intracellular distribution of Ca2+ control the activities of these two enzymes in order to stimulate energy production for the contraction process in the muscle. Although both enzymes are mitochondrial, glycerol phosphate dehydrogenase resides on the outer surface of the inner membrane and responds to sarcoplasmic changes in Ca2+ concentration (i.e. an increase during contraction), whereas the isocitrate dehydrogenase resides in the matrix of the mitochondria and responds to intramitochondrial concentrations of Ca2+ (i.e. a decrease during contraction). It is suggested that changes in intramitochondrial Ca2+ concentrations are primarily responsible for regulation of the activity of NAD+-isocitrate dehydrogenase in order to control energy formation for the contractile process. However, when the muscle is at rest, changes in intramitochondrial concentrations of ADP may regulate energy formation for non-contractile processes.     

Compromised myocardial energetics in hypertrophied mouse hearts diminish the beneficial effect of overexpressing SERCA2a

The sarcoplasmic reticulum calcium ATPase (SERCA) plays a central role in regulating intracellular Ca(2+) homeostasis and myocardial contractility. Several studies show that improving Ca(2+) handling in hypertrophied rodent hearts by increasing SERCA activity results in enhanced contractile function. This suggests that SERCA is a potential target for gene therapy in cardiac hypertrophy and failure. However, it raises the issue of increased energy cost resulting from a higher ATPase activity. In this study, we determined whether SERCA overexpression alters the energy cost of increasing myocardial contraction in mouse hearts with pressure-overload hypertrophy using (31)P NMR spectroscopy. We isolated and perfused mouse hearts from wild-type (WT) and transgenic (TG) mice overexpressing the cardiac isoform of SERCA (SERCA2a) 8 weeks after ascending aortic constriction (left ventricular hypertrophy (LVH)) or sham operation. We found that overexpressing SERCA2a enhances myocardial contraction and relaxation in normal mouse hearts during inotropic stimulation with isoproterenol. Energy consumption was proportionate to the increase in contractile function. Thus, increasing SERCA2a expression in the normal heart allows an enhanced inotropic response with no compromise in energy supply and demand. However, this advantage was not sustained in LVH hearts in which the energetic status was compromised. Although the overexpression of SERCA2a prevented the down-regulation of SERCA protein in LVH hearts, TG-LVH hearts showed no increase in inotropic response when compared with WT-LVH hearts. Our results suggest that energy supply may be a limiting factor for the benefit of SERCA overexpression in hypertrophied hearts. Thus, strategies combining energetic support with increasing SERCA activity may improve the therapeutic effectiveness for heart failure.     

Structural and functional changes in heart mitochondria from sucrose-fed hypertriglyceridemic rats

In the heart of sugar-induced hypertriglyceridemic (HTG) rats, cardiac performance is impaired with glucose as fuel, but not with fatty acids. Accordingly, the glycolytic flux and the transfer of energy diminish in the HTG heart, in comparison to control heart. To further explore the biochemical nature of such alteration in the HTG heart, the components of the non-glycolytic energy systems involved were evaluated. Total creatine kinase (CK) activity in the myocardial tissue was depressed by 30% in the HTG heart whereas the activity of the mitochondrial CK (mitCK) isoenzyme fraction that is functionally associated with oxidative phosphorylation decreased in isolated HTG heart mitochondria by 45%. Adenylate kinase (AK) was 20% lower in the HTG heart. In contrast, respiratory rates with 2-oxoglutarate (2-OG) and pyruvate/malate (pyr) were significantly higher in HTG heart mitochondria than in control mitochondria. 2-OG dehydrogenase activity was also higher in HTG mitochondria. Respiration with succinate was similar in both groups. Content of cytochromes b, c + c1 and a + a3, and cytochrome c oxidase activity, were also similar in the two kinds of mitochondria. A larger content of saturated and monounsaturated fatty acids was found in the HTG mitochondrial membranes with no changes in phospholipids composition or cholesterol content. Mitochondrial membranes from HTG hearts were more rigid, which correlated with the generation of higher membrane potentials. As the mitochondrial function was preserved or even enhanced in the HTG heart, these results indicated that deficiency in energy transfer was associated with impairment in mitCK and AK. This situation brought about uncoupling between the site of ATP production and the site of ATP consumption (contractile machinery), in spite of compensatory increase in mitochondrial oxidative capacity and membrane potential generation.     

Inhibition of mitochondrial Na+-Ca2+ exchange restores agonist-induced ATP production and Ca2+ handling in human complex I deficiency

Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca2+ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca2+ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca2+ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca2+ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.     

Inhibition of the mitochondrial calcium uniporter by the oxo-bridged dinuclear ruthenium amine complex (Ru360) prevents from irreversible injury in postischemic rat heart

Mitochondrial calcium overload has been implicated in the irreversible damage of reperfused heart. Accordingly, we studied the effect of an oxygen-bridged dinuclear ruthenium amine complex (Ru360), which is a selective and potent mitochondrial calcium uniporter blocker, on mitochondrial dysfunction and on the matrix free-calcium concentration in mitochondria isolated from reperfused rat hearts. The perfusion of Ru360 maintained oxidative phosphorylation and prevented opening of the mitochondrial permeability transition pore in mitochondria isolated from reperfused hearts. We found that Ru360 perfusion only partially inhibited the mitochondrial calcium uniporter, maintaining the mitochondrial matrix free-calcium concentration at basal levels, despite high concentrations of cytosolic calcium. Additionally, we observed that perfused Ru360 neither inhibited Ca2+ cycling in the sarcoplasmic reticulum nor blocked ryanodine receptors, implying that the inhibition of ryanodine receptors cannot explain the protective effect of Ru360 in isolated hearts. We conclude that the maintenance of postischemic myocardial function correlates with an incomplete inhibition of the mitochondrial calcium uniporter. Thus, the chemical inhibition by this molecule could be an approach used to prevent heart injury during reperfusion.     

Role of cytosolic vs. mitochondrial Ca2+ accumulation in burn injury-related myocardial inflammation and function

This study was designed to examine the role of mitochondrial Ca2+ homeostasis in burn-related myocardial inflammation. We hypothesized that mitochondrial Ca2+ is a primary modulator of cardiomyocyte TNF-alpha, IL-1beta, and IL-6 responses to injury and infection. Ventricular myocytes were prepared by Langendorff perfusion of hearts from adult rats subjected to sham burn or burn injury over 40% of total body surface area to produce enzymatic (collagenase) digestion. Isolated cardiomyocytes were suspended in MEM, cell number was determined, and aliquots of myocytes from each experimental group were loaded with fura 2-AM (2 microg/ml) for 1) 45 min at room temperature to measure total cellular Ca2+, 2) 45 min at 30 degrees C followed by incubation at 37 degrees C for 2 h to eliminate cytosolic fluorescence, and 3) 20 min at 37 degrees C in MnCl2 (200 microM)-containing buffer to quench cytosolic fura 2-AM signal. In vitro studies included preparation of myocytes from control hearts and challenge of myocytes with LPS or burn serum (BS), which have been shown to increase cytosolic Ca2+. Additional aliquots of myocytes were challenged with LPS or BS with or without a selective inhibitor of mitochondrial Ca2+, ruthenium red (RR). All cells were examined on a stage-inverted microscope that was interfaced with the InCyt Im2 fluorescence imaging system. Heat treatment or MnCl2 challenge eliminated myocyte cytosolic fluorescence, whereas cells maintained at room temperature retained 95% of their initial fluorescence. Compared with Ca2+ levels measured in sham myocytes, burn trauma increased cytosolic Ca2+ from 90 +/- 3 to 293 +/- 6 nM (P < 0.05) and mitochondrial Ca2+ from 24 +/- 1 to 75 +/- 2 nM (P < 0.05). LPS (25 microg/5 x 10(4) cells) or BS (10% by volume) challenge for 18 h increased cardiomyocyte cytosolic and mitochondrial Ca2+ and promoted myocyte secretion of TNF-alpha, IL-1beta, and IL-6. RR pretreatment decreased LPS- and BS-related rise in mitochondrial Ca2+ and cytokine secretion but had no effect on cytosolic Ca2+. BS challenge in perfused control hearts impaired myocardial contraction/relaxation, and RR pretreatment of hearts prevented BS-related myocardial contractile dysfunction. Our data suggest that a rise in mitochondrial Ca2+ is one modulator of myocardial inflammation and dysfunction in injury states such as sepsis and burn trauma.     

An integrated model of cardiac mitochondrial energy metabolism and calcium dynamics

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.     

Specific enhancement of sarcomeric response to Ca2+ protects murine myocardium against ischemia-reperfusion dysfunction

Alteration in myofilament response to Ca2+ is a major mechanism for depressed cardiac function after ischemia-reperfusion (I/R) dysfunction. We tested the hypothesis that hearts with increased myofilament response to Ca2+ are less susceptible to I/R. In one approach, we studied transgenic (TG) mice with a constitutive increase in myofilament Ca2+ sensitivity in which the adult form of cardiac troponin I (cTnI) is stoichiometrically replaced with the embryonic/neonatal isoform, slow skeletal TnI (ssTnI). We also studied mouse hearts with EMD-57033, which acts specifically to enhance myofilament response to Ca2+. We subjected isolated, perfused hearts to an I/R protocol consisting of 25 min of no-flow ischemia followed by 30 min of reperfusion. After I/R, developed pressure and rates of pressure change were significantly depressed and end-diastolic pressure was significantly elevated in nontransgenic (NTG) control hearts. These changes were significantly blunted in TG hearts and in NTG hearts perfused with EMD-57033 during reperfusion, with function returning to nearly baseline levels. Ca2+- and cross bridge-dependent activation, protein breakdown, and phosphorylation in detergent-extracted fiber bundles were also investigated. After I/R NTG fiber bundles exhibited a significant depression of cross bridge-dependent activation and Ca2+-activated tension and length dependence of activation that were not evident in TG preparations. Only NTG hearts demonstrated a significant increase in cTnI phosphorylation. Our results support the hypothesis that specific increases in myofilament Ca2+ sensitivity are able to diminish the effect of I/R on cardiac function.     

Proteomic analyses of transgenic LQT1 and LQT2 rabbit hearts elucidate an increase in expression and activity of energy producing enzymes

Various biochemical and genomic mechanisms are considered to be a hallmark of metabolic remodeling in the stressed heart, including the hypertrophied and failing heart. In this study, we used quantitative proteomic 2-D Fluorescence Difference In-Gel Electrophoresis (2-D DIGE) in conjunction with mass spectrometry to demonstrate differential protein expression in the hearts of transgenic rabbit models of Long QT Syndrome 1 (LQT1) and Long QT Syndrome 2 (LQT2) as compared to littermate controls (LMC). The results of our proteomic analysis revealed upregulation of key metabolic enzymes involved in all pathways associated with ATP generation, including creatine kinase in both LQT1 and LQT2 rabbit hearts. Additionally, the expression of lamin-A protein was increased in both LQT1 and LQT2 rabbit hearts as was the expression of mitochondrial aldehyde dehydrogenase and desmoplakin in LQT1 and LQT 2 rabbit hearts, respectively. Results of the proteomic analysis also demonstrated down regulation in the expression of protein disulfide-isomerase A3 precuorsor and dynamin-like 120 kDa protein (mitochondrial) in LQT1, and of alpha-actinin 2 in LQT2 rabbit hearts. Up regulation of the expression of the enzymes associated with ATP generation was substantiated by the results of selective enzyme assays in LQT1 and LQT2 hearts, as compared to LMC, which revealed increases in the activities of glycogen phosphorylase (+50%, +65%, respectively), lactate dehydrogenase (+25%, +25%) pyruvate dehydrogenase (+31%, +22%), and succinate dehydrogenase (+32%, +60%). The activity of cytochrome c-oxidase, a marker for the mitochondrial function was also found to be significantly elevated (+80%) in LQT1 rabbit hearts as compared with LMC. Western blot analysis in LQT1 and LQT2 hearts compared to LMC revealed an increase in the expression of very-long chain-specific acyl-CoA dehydrogenase (+35%, +33%), a rate-limiting enzymes in β-oxidation of fatty acids. Collectively, our results demonstrate similar increases in the expression and activities of key ATP-generating enzymes in LQT1 and LQT2 rabbit hearts, suggesting an increased demand, and in turn, increased energy supply across the entire metabolic pathway by virtue of the upregulation of enzymes involved in energy generation.