scholarly journals Reoxygenation-dependent decrease in mitochondrial NADH:CoQ reductase (Complex I) activity in the hypoxic/reoxygenated rat heart

1991 ◽  
Vol 274 (1) ◽  
pp. 133-137 ◽  
Author(s):  
L Hardy ◽  
J B Clark ◽  
V M Darley-Usmar ◽  
D R Smith ◽  
D Stone
Keyword(s):  
Respiratory Function ◽  
Complex I ◽  
Ruthenium Red ◽  
Isolated Cardiomyocytes ◽  
Isolated Mitochondria ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Complex I Activity ◽  
Perfused Hearts ◽  
Total Tissue

Reoxygenation of the hypoxic myocardium results in a number of processes, including an O2-dependent increase in total tissue Ca2+ and cell lysis in which mitochondrial electron transport plays a key role. In the present study we have isolated mitochondria from perfused rat hearts subjected to hypoxia and found no change in their respiratory function relative to controls. In contrast, mitochondria isolated immediately after reoxygenation of hypoxic-perfused hearts exhibited a specific and significant decrease in NADH:CoQ reductase (Complex I; EC 1.6.5.3) activity, as measured both polarographically and spectrophotometrically. Isolated cardiomyocytes subjected to a similar protocol of hypoxia/reoxygenation also exhibited a specific decrease in Complex I activity. Myocardial perfusion with media containing Ruthenium Red protected against the reoxygenation-dependent loss of Complex I activity. These observations taken together suggest that mitochondrial Ca2+ uptake on reoxygenation is implicated in the mechanism of the specific loss of Complex I activity.

scholarly journals Inhibition of the ubiquitous calpains protects complex I activity and enables improved mitophagy in the heart following ischemia-reperfusion

2019 ◽  
Vol 317 (5) ◽  
pp. C910-C921 ◽  
Author(s):  
Qun Chen ◽  
Jeremy Thompson ◽  
Ying Hu ◽  
Joseph Dean ◽  
Edward J. Lesnefsky
Keyword(s):  
Complex I ◽  
Ischemia Reperfusion ◽  
Cardiac Injury ◽  
Beclin 1 ◽  
Rat Hearts ◽  
Heart Perfusion ◽  
Interfibrillar Mitochondria ◽  
Perfused Rat Hearts ◽  
Subsarcolemmal Mitochondria ◽  
Complex I Activity

Activation of calpain 1 (CPN1) and calpain 2 (CPN2) contributes to cardiac injury during ischemia (ISC) and reperfusion (REP). Complex I activity is decreased in heart mitochondria following ISC-REP. CPN1 and CPN2 are ubiquitous calpains that exist in both cytosol (cs)-CPN1 and 2 and mitochondria (mit)-CPN1 and 2. Recent work shows that the complex I subunit (NDUFS7) is a potential substrate of the mit-CPN1. We asked whether ISC-REP led to decreased complex I activity via proteolysis of the NDUFS7 subunit via activation of mit-CPN1 and -2. Activation of cs-CPN1 and -2 decreases mitophagy in hepatocytes following ISC-REP. We asked whether activation of cs-CPN1 and -2 impaired mitophagy in the heart following ISC-REP. Buffer-perfused rat hearts underwent 25 min of global ISC and 30 min of REP. MDL-28170 (MDL; 10 µM) was used to inhibit CPN1 and -2. Cytosol, subsarcolemmal mitochondria (SSM), and interfibrillar mitochondria (IFM) were isolated at the end of heart perfusion. Cardiac ISC-REP led to decreased complex I activity with a decrease in the content of NDUFS7 in both SSM and IFM. ISC-REP also resulted in a decrease in cytosolic beclin-1 content, a key component of the autophagy pathway required to form autophagosomes. MDL treatment protected the contents of cytosolic beclin-1 and mitochondrial NDUFS7 in hearts following ISC-REP. These results support that activation of both cytosolic and mitochondrial calpains impairs mitochondria during cardiac ISC-REP. Mitochondria-localized calpains impair complex I via cleavage of a key subunit. Activation of cytosolic calpains contributes to mitochondrial dysfunction by impairing removal of the impaired mitochondria through depletion of a key component of the mitophagy process.

Ischemic preconditioning does not protect via blockade of electron transport

2007 ◽  
Vol 103 (2) ◽  
pp. 623-628 ◽  
Author(s):  
Christine Tanaka-Esposito ◽  
Qun Chen ◽  
Shadi Moghaddas ◽  
Edward J. Lesnefsky
Keyword(s):  
Electron Transport ◽  
Infarct Size ◽  
Ischemic Preconditioning ◽  
Complex I ◽  
Myocardial Infarct ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Myocardial Infarct Size ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Complex I Activity

Ischemic preconditioning (IPC) before sustained ischemia decreases myocardial infarct size mediated in part via protection of cardiac mitochondria. Reversible blockade of electron transport at complex I immediately before sustained ischemia also preserves mitochondrial respiration and decreases infarct size. We proposed that IPC would attenuate electron transport from complex I as a potential effector mechanism of cardioprotection. Isolated, Langendorff-perfused rat hearts underwent IPC (3 cycles of 5-min 37°C global ischemia and 5-min reperfusion) or were perfused for 40 min without ischemia as controls. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated. IPC did not decrease ADP-stimulated respiration measured in intact mitochondria using substrates that donate reducing equivalents to complex I. Maximally expressed complex I activity measured as rotenone-sensitive NADH:ubiquinone oxidoreductase in detergent-solubilized mitochondria was also unaffected by IPC. Thus the protection of IPC does not occur as a consequence of a partial decrease in complex I activity leading to a decrease in integrated respiration through complex I. IPC and blockade of electron transport both converge on mitochondria as effectors of cardioprotection; however, each modulates mitochondrial metabolism during ischemia by different mechanisms to achieve cardioprotection.

scholarly journals The α-adrenergic-mediated activation of the cardiac mitochondrial Ca2+ uniporter and its role in the control of intramitochondrial Ca2+in vivo

1983 ◽  
Vol 216 (2) ◽  
pp. 333-342 ◽  
Author(s):  
M Crompton ◽  
P Kessar ◽  
I Al-Nasser
Keyword(s):  
Oxidative Metabolism ◽  
Ruthenium Red ◽  
Isolated Mitochondria ◽  
Control Heart ◽  
Rat Hearts ◽  
Adrenergic Control ◽  
Perfused Rat Hearts

Administration of methoxamine (10 microM, 2 min) to perfused rat hearts increased the rate at which subsequently isolated mitochondria accumulated Ca2+. Methoxamine did not change significantly the development of delta phi with time or the basal rates of Ca2+ flux on inhibition of the uniporter with Ruthenium Red. With 200 microM-Pi, the rates of Ca2+ uptake at constant delta phi were unaffected by the small variations in endogenous [Pi] between mitochondrial preparations, and were also unaffected by changes in internal Ca2+ over the approximate range 8-43 nmol of Ca2+/mg. At low internal Ca2+ (about 8 nmol/mg of protein) the rates of Ca2+ uptake at constant delta phi were unaffected by addition of 200 microM-Pi. Under these conditions, the uniporter activity and the uniporter conductance were increased by 38-40% by methoxamine pretreatment. The endogenous Ca2+ content of mitochondria from control heart was about 1.8 nmol of Ca2+/mg of protein. Perfusion with agonist increased the Ca2+ content as follows: 10 microM-methoxamine (2 min), 48%; 1 microM-isoprenaline (2 min), 100%; 1 microM-adrenaline (2 min), 140%. The implications of the data for the adrenergic control of oxidative metabolism by intramitochondrial Ca2+ is discussed.

scholarly journals Development of a New Assay for Complex I of the Respiratory Chain

2000 ◽  
Vol 46 (3) ◽  
pp. 345-350 ◽  
Author(s):  
Hilary Brooks ◽  
Stephan Krähenbühl
Keyword(s):  
Skeletal Muscle ◽  
Complex I ◽  
Human Skeletal Muscle ◽  
Specific Activity ◽  
Mitochondrial Diseases ◽  
Spectrophotometric Analysis ◽  
Isolated Mitochondria ◽  
Tissue Homogenates ◽  
Multiple Samples ◽  
Complex I Activity

Abstract Background: Measurement of complex I activity has been hampered by the large amounts of tissue required and the resulting turbidity of the assay solution, which makes spectrophotometric analysis difficult. We have developed a new assay for measuring the activity of complex I in isolated mitochondria that is also applicable to skeletal muscle homogenate in patients with suspected mitochondrial diseases. Methods: The method was a radioenzymatic assay based on the preferential oxidation of the 4B hydrogen of NADH by complex I. We prepared tritiated isoforms of NADH for both the respective 4A-3H and 4B-3H positions. Enzyme in the form of purified mitochondria or homogenate was prepared from rat or human skeletal muscle and incubated with the respective radioisotopes. The product (3H2O) was collected after charcoal adsorption of unreacted NADH and taken as an indicator of NADH oxidation. Sensitivity to rotenone was used as a measure of complex I specific activity. Results: The assay was linear with time and protein for isolated mitochondria and tissue homogenates from rats and humans. The Vmax and Km values obtained for 4B-NADH with isolated rat skeletal muscle mitochondria were 35 μmol/L and 90 μmol · min−1 · mg protein−1, respectively. The assay was reproducible and useable for routine measurements in human skeletal muscle. The sensitivity was >10-fold higher than the sensitivities of spectrophotometric techniques. Conclusions: The results of our studies demonstrate the successful development of a new assay for complex I that is rapid, easy to perform, and that enables the processing of multiple samples at one time.

Insulin stimulates mitochondrial protein synthesis and respiration in isolated perfused rat heart.

1990 ◽  
Vol 259 (3) ◽  
pp. E413
Author(s):  
E E McKee ◽  
B L Grier
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Control Level ◽  
Respiratory Control ◽  
Mitochondrial Proteins ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Perfused Hearts

The rates of synthesis of mitochondrial proteins by both the cytoplasmic and mitochondrial protein synthetic systems, as well as parameters of respiration, were measured and compared in mitochondria isolated from fresh, control perfused, and insulin-perfused rat hearts. The respiratory control ratio (RCR) in mitochondria from fresh hearts was 8.1 +/- 0.4 and decreased to 6.0 +/- 0.2 (P less than 0.001 vs. fresh) in mitochondria from control perfused hearts and to 6.7 +/- 0.2 (P less than 0.005 vs. fresh and P less than 0.02 vs. control perfused) for mitochondria from hearts perfused in the presence of insulin. A positive correlation between the RCR and the rate of mitochondrial translation was demonstrated in mitochondria from fresh hearts. In mitochondria isolated from control perfused hearts, the rate of protein synthesis decreased to 84 +/- 3% of the fresh rate after 30 min of perfusion and fell further to 64 +/- 3% after 3 h of perfusion. The inclusion of insulin in the perfusion buffer stimulated mitochondrial protein synthesis 1.2-fold by 1 h (P less than 0.005) and 1.34-fold by 3 h of perfusion (P less than 0.001). The addition of insulin to 1-h control perfused hearts shifted the rate of mitochondrial protein synthesis from the control level to the insulin-perfused level within 30 min of additional perfusion, whereas 1 h was required to shift the RCR values of these mitochondria from control levels to insulin-perfused levels. Thus, whereas RCR was a useful predictor of mitochondrial translation rates, it did not account for the effects of insulin on mitochondrial translation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using31P n.m.r. and ruthenium red

1989 ◽  
Vol 262 (1) ◽  
pp. 293-301 ◽  
Author(s):  
J F Unitt ◽  
J G McCormack ◽  
D Reid ◽  
L K MacLachlan ◽  
P J England
Keyword(s):  
Oxidative Phosphorylation ◽  
Direct Evidence ◽  
Respiratory Control ◽  
Ruthenium Red ◽  
Phosphorylation Potential ◽  
Mitochondrial Inner Membrane ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Inotropic Stimulation

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.

scholarly journals Protein synthesis by perfused hearts from normal and insulin-deficient rats. Effect of insulin in the presence of glucose and after depletion of glucose, glucose 6-phosphate and glycogen

1973 ◽  
Vol 132 (3) ◽  
pp. 593-601 ◽  
Author(s):  
Ernst B. Chain ◽  
Peter M. Sender
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Glucose Metabolism ◽  
Glucose Utilization ◽  
Amino Acid Incorporation ◽  
Perfused Heart ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Streptozotocin Induced Diabetes ◽  
Perfused Hearts

In the absence of glucose, insulin stimulated the incorporation of 14C-labelled amino acids into protein by perfused rat hearts that had been previously substantially depleted of endogenous glucose, glucose 6-phosphate and glycogen by substrate-free perfusion. This stimulation was also demonstrated in hearts perfused with buffer containing 2-deoxy-d-glucose, an inhibitor of glucose utilization. It is concluded that insulin exerts an effect on protein synthesis independent of its action on glucose metabolism. Streptozotocin-induced diabetes was found to have no effect either on 14C-labelled amino acid incorporation by the perfused heart or on the polyribosome profile and amino acid-incorporating activity of polyribosomes prepared from the non-perfused hearts of these insulin-deficient rats, which show marked abnormalities in glucose metabolism. Protein synthesis was not diminished in the perfused hearts from rats treated with anti-insulin antiserum. The significance of these findings is discussed in relation to the reported effects of insulin deficiency on protein synthesis in skeletal muscle.

31P-NMR of high-energy phosphates in perfused rat heart during metabolic acidosis

1992 ◽  
Vol 263 (3) ◽  
pp. H903-H909
Author(s):  
L. A. Jelicks ◽  
R. Gupta
Keyword(s):  
Metabolic Acidosis ◽  
Atp Synthesis ◽  
High Energy ◽  
Magnesium Concentration ◽  
High Energy Phosphates ◽  
Solution Ph ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Creatine Kinase Equilibrium ◽  
Perfused Hearts

Intracellular pH (pHi), intracellular free magnesium concentration ([Mg2+]i), and high-energy phosphates in Langendorff perfused rat hearts were evaluated by 31P-nuclear magnetic resonance (NMR) during metabolic acidosis. During acidosis, cardiac pHi approached that of the perfusing solution (pH approximately 6.7) and [Mg2+]i increased. In hearts perfused with glucose as the sole carbon source, the ratio of [phosphocreatine] to [ATP] decreased during acidosis. In contrast, in hearts supplemented with pyruvate (either 2.8 or 10 mM) this ratio increased during acidosis. Oxygen consumption decreased in hearts perfused with glucose only and with pyruvate-glucose. Using the creatine kinase equilibrium constant, we find that [MgADP] is significantly decreased in pyruvate-perfused hearts but is not significantly altered in glucose-perfused hearts during metabolic acidosis. These data indicate that [MgADP] may be the regulator of cardiac oxidative phosphorylation in the presence of excess pyruvate; however, during metabolic acidosis in hearts perfused with glucose only, ATP synthesis appears limited by the availability of pyruvate via glycolysis.

scholarly journals The effects of lactate, acetate, glucose, insulin, starvation and alloxan-diabetes on protein synthesis in perfused rat hearts

1986 ◽  
Vol 236 (2) ◽  
pp. 543-547 ◽  
Author(s):  
D M Smith ◽  
S J Fuller ◽  
P H Sugden
Keyword(s):  
Protein Synthesis ◽  
Time Course ◽  
Diabetic Rats ◽  
Perfused Heart ◽  
Translational Activity ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Percentage Basis ◽  
Perfused Hearts ◽  
Stimulation Of

Compared with glucose, lactate + acetate stimulated ventricular protein synthesis in anterogradely perfused hearts from fed or 72 h-starved rats. Stimulation was greater on a percentage basis in starved rats. Atrial protein synthesis was not detectably stimulated by lactate + acetate. Insulin stimulated protein synthesis in atria and ventricles. The stimulation of protein synthesis by lactate + acetate and insulin was not additive, the percentage stimulation by insulin being less in the ventricles of lactate + acetate-perfused hearts than in glucose-perfused hearts. Perfusion of hearts from 72 h-starved or alloxan-diabetic rats with glucose + lactate + acetate + insulin did not increase protein-synthesis rates or efficiencies (protein synthesis expressed relative to total RNA) to values for fed rats, implying there is a decrease in translational activity in these hearts. In the perfused heart, inhibition of protein synthesis by starvation and its reversal by re-feeding followed a relatively prolonged time course. Synthesis was still decreasing after 3 days of starvation and did not return to normal until after 2 days of re-feeding.

Pantothenate kinase and control of CoA synthesis in heart

1984 ◽  
Vol 246 (4) ◽  
pp. H532-H541 ◽  
Author(s):  
J. D. Robishaw ◽  
J. R. Neely
Keyword(s):  
Pantothenic Acid ◽  
Cysteine Synthase ◽  
Pantothenate Kinase ◽  
Isolated Hearts ◽  
Low Concentrations ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
And Control ◽  
Perfused Hearts ◽  
Oxidation Of Glucose

Control of coenzyme A (CoA) synthesis was studied in isolated perfused rat hearts. The data obtained support the hypothesis that phosphorylation of pantothenic acid by pantothenate kinase is the flux-generating reaction in the pathway of CoA synthesis. This reaction operated in the cell far removed from its thermodynamic equilibrium; it was saturated with substrates under all conditions studied; and the concentration of substrate changed in the opposite direction to flux when flux was altered. The reaction was subject to control by external factors associated with oxidation of glucose, pyruvate, or palmitate. CoA synthesis from 4'-phosphopantothenic acid was not inhibited by glucose and pyruvate, suggesting that pantothenate kinase is the only reaction in the pathway that is controlled in isolated hearts. Maximum rates of CoA synthesis in perfused hearts with pantothenate kinase stimulation were dependent on a supply of exogenous cysteine. Perfusate [14C]cysteine was incorporated into intermediates of this pathway and CoA. When protected from oxidation to cystine by low concentrations of dithiothreitol, 0.1 mM cysteine in the perfusate resulted in maximum rates of CoA synthesis. Evidence was obtained that indicates that addition of cysteine relieves a substrate limitation at the 4'-phosphopantothenyl cysteine synthase reaction.

Sign in / Sign up

Export Citation Format

Share Document