Effects of cardiac work on electrical potential gradient across mitochondrial membrane in perfused rat hearts

1993 ◽  
Vol 265 (2) ◽  
pp. H453-H460 ◽  
Author(s):  
B. Wan ◽  
C. Doumen ◽  
J. Duszynski ◽  
G. Salama ◽  
T. C. Vary ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Mitochondrial Membrane ◽  
Atp Synthesis ◽  
High Energy ◽  
Kinetic Properties ◽  
O2 Consumption ◽  
Cardiac Work ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Total Tissue

The myocardium responds to alterations in cardiac work by changing its rate of O2 consumption. This reflects an increase in the oxidative synthesis of ATP to meet the contractile demand for ATP. However, the biochemical mechanisms responsible for increased ATP synthesis are not fully understood. To localize the flux-controlling reaction(s) in the pathway of ATP synthesis, the effects of substrates and cardiac work on mitochondrial membrane potential (delta psi m), total tissue NADH-to-NAD+ ratio, and high-energy phosphate metabolites were examined in perfused rat hearts. Delta psi m was measured using the equilibrium distribution of tetraphenylphosphonium (33). Cytosolic phosphorylation potential, total tissue NADH-to-NAD+ ratio, and delta psi m were higher in hearts perfused with pyruvate than in those perfused with glucose. Increasing cardiac work induced a four-fold increase in O2 consumption, which was accompanied by 1) decreased or unaltered cytosolic ADP concentration, 2) increased tissue NADH-to-NAD+ ratio, and 3) decreased delta psi m. The results indicate that both NADH-generating reactions and the ATP synthase-catalyzed reaction are important in causing the increase in respiration that accompanies increased work. Because the activation of ATP synthase by cardiac work occurred in the absence of increases in delta psi m, ADP, and Pi, it is possible that the work-related acceleration in ATP synthesis may be due to modification of the kinetic properties of the ATP synthase.

Effect of BDM, verapamil, and cardiac work on mitochondrial membrane potential in perfused rat hearts

1995 ◽  
Vol 269 (2) ◽  
pp. H515-H523 ◽  
Author(s):  
C. Doumen ◽  
B. Wan ◽  
O. Ondrejickova
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Atp Synthase ◽  
Mitochondrial Membrane ◽  
Cardiac Work ◽  
Beta Adrenergic ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Butanedione Monoxime ◽  
Adrenergic Agent

The biochemical link providing effective coordination between the mitochondrial ATP synthetic machinery and the contractile apparatus following transitions in cardiac work remains enigmatic. Studies were designed to determine whether activation of the actomyosin adenosinetriphosphatase (ATPase) is a necessary part of the signaling mechanism to the mitochondrial ATP synthase or whether a rise in cytosolic free Ca2+ is sufficient to activate the synthase. With the use of Langendorff-perfused rat hearts, cardiac work was varied via changes in perfusion pressure and by the inclusion of a beta-adrenergic agent. Furthermore, 2,3-butanedione monoxime and verapamil were used to vary independently either the activity of the actomyosin ATPase or the level of cytosolic free Ca2+. Determinations of the in vivo mitochondrial membrane potential [delta psi m; see Wan et al. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H445-H452, 1993] and its vectorial displacement during work transitions provide valuable information concerning direct activation of the ATP synthase and proton movement through the membrane domain of the synthase. Increased cardiac work in the presence of the beta-adrenergic agent resulted in a decrease in delta psi m. Addition of 2,3-butanedione monoxime decreased cardiac work but did not change delta psi m. The inclusion of verapamil resulted in similar decreases in cardiac work. However, delta psi m reversed back to a value observed under control, low-work conditions. These results in conjunction with data regarding levels of high-energy phosphates, free Mg2+, and adenosine 3',5'-cyclic monophosphate suggest a Ca(2+)-mediated increase in the activity of the ATP synthase.

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.

Substrate dependence of metabolic state and coronary flow in perfused rat heart

1985 ◽  
Vol 249 (4) ◽  
pp. H799-H806 ◽  
Author(s):  
J. W. Starnes ◽  
D. F. Wilson ◽  
M. Erecinska
Keyword(s):  
Energy Metabolism ◽  
Coronary Flow ◽  
Work Load ◽  
Creatine Phosphate ◽  
High Energy ◽  
Cardiac Cells ◽  
O2 Consumption ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Perfused Hearts

The effect of substrate source on the regulation of energy metabolism and coronary flow was studied in isolated perfused rat hearts. Compared with glucose-perfused hearts, those perfused at the same work load with palmitate or acetate demonstrated increases (P less than 0.01) in O2 consumption of 16 and 18%, respectively, and increases (P less than 0.01) in coronary flow of 30 and 32%, respectively. Parallel substrate-related changes occurred in the levels of high-energy phosphate compounds: tissue creatine, ADP free, and inorganic phosphate (Pi) were significantly decreased, leading to increases (P less than 0.01) in [creatine phosphate]/[creatine] and [ATP]free/[ADP]free[Pi]. These changes were accompanied by increased reduction of intramitochondrial pyridine nucleotides. Omitting orthophosphate from perfusate lowered intracellular Pi and modified cardiac function, but substrate-related differences were similar to those in Pi containing media. Differences in intracellular pH among substrates were observed, which may contribute in some instances to differences in energy metabolism and coronary flow. When work load was altered in glucose- and acetate-perfused hearts, both O2 consumption and coronary flow were linearly related to cytosolic [ATP]free/[ADP]free[Pi], and slopes of regression lines were similar for both substrates. These correlations support the view that [ATP]free/[ADP]free[Pi] is a major determinant of O2 consumption by cardiac cells and of coronary flow.

Fasudil prevents KATP channel-induced improvement in postischemic functional recovery

2005 ◽  
Vol 288 (6) ◽  
pp. H3011-H3015 ◽  
Author(s):  
Kenya Nishizawa ◽  
Paul E. Wolkowicz ◽  
Tadashi Yamagishi ◽  
Ling-Ling Guo ◽  
Martin M. Pike
Keyword(s):  
Diastolic Pressure ◽  
Rho Kinase ◽  
High Energy ◽  
Left Ventricular ◽  
High Energy Phosphate ◽  
Cellular Processes ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Rock Activity ◽  
Mechanical Recovery

Whereas activation of ATP-dependent potassium (KATP) channels greatly improves postischemic myocardial recovery, the final effector mechanism for KATP channel-induced cardioprotection remains elusive. RhoA is a GTPase that regulates a variety of cellular processes known to be involved with KATP channel cardioprotection. Our goal was to determine whether the activity of a key rhoA effector, rho kinase (ROCK), is required for KATP channel-induced cardioprotection. Four groups of perfused rat hearts were subjected to 36 min of zero-flow ischemia and 44 min of reperfusion with continuous measurements of mechanical function and 31P NMR high-energy phosphate data: 1) untreated, 2) pinacidil (10 μM) to activate KATP channels, 3) fasudil (15 μM) to inhibit ROCK, and 4) both fasudil and pinacidil. Pinacidil significantly improved postischemic mechanical recovery [39 ± 16 vs. 108 ± 4 mmHg left ventricular diastolic pressure (LVDP), untreated and pinacidil, respectively]. Fasudil did not affect reperfusion LVDP (41 ± 13 mmHg) but completely blocked the marked improvement in mechanical recovery that occurred with pinacidil treatment (54 ± 15 mmHg). Substantial attenuation of the postischemic energetic recovery was also observed. These data support the hypothesis that ROCK activity plays a role in KATP channel-induced cardioprotection.

A method of determining electrical potential gradient across mitochondrial membrane in perfused rat hearts

1993 ◽  
Vol 265 (2) ◽  
pp. H445-H452 ◽  
Author(s):  
B. Wan ◽  
C. Doumen ◽  
J. Duszynski ◽  
G. Salama ◽  
K. F. LaNoue
Keyword(s):  
Mitochondrial Membrane ◽  
Electrical Potential ◽  
Potential Gradient ◽  
Order Rate Constant ◽  
Isolated Rat Heart ◽  
First Order ◽  
Order Rate ◽  
Rat Hearts ◽  
Perfused Rat Hearts ◽  
Electrical Potential Gradient

The electrical potential gradient across the mitochondrial membrane (delta psi m) in perfused rat hearts was estimated by calculating the equilibrium distribution of the lipophilic cation tetraphenylphosphonium (TPP+), using measured kinetic constants of uptake and release of TPP+. First-order rate constants of TPP+ uptake were measured during 30-min perfusions of intact rat hearts with tracer amounts (5.0 nM) of tritium-labeled TPP+ ([3H]TPP+) in the perfusate. This was followed by a 30-min washout, during which the first-order rate constant of efflux was estimated. Values of [3H]TPP+ outside the heart and total [3H]TPP+ inside the heart at equilibrium were calculated. From this information and separately estimated time-averaged plasma membrane potentials (delta psi c) it was possible to calculate free cytosolic [3H]TPP+ at equilibrium. It was also possible to calculate free intramitochondrial [3H]TPP+ at equilibrium as the difference between total tissue [3H]TPP+ minus free cytosolic TPP+ and the sum of all the bound [3H]TPP+. Bound [3H]TPP+ was determined from [3H]TPP+ binding constants measured in separate experiments, using both isolated mitochondria and isolated cardiac myocytes under conditions where both delta psi m and delta psi c were zero. Delta psi m was calculated from the intramitochondrial and cytosolic free TPP+ concentrations using the Nernst equation. Values of delta psi m were 144.9 +/- 2.0 mV in hearts perfused with 5 mM pyruvate and 118.2 +/- 1.4 mV in hearts perfused with 11 mM glucose, in good agreement with delta psi m obtained from isolated rat heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Monitoring of mitochondrial membrane potential in isolated perfused rat heart

1984 ◽  
Vol 247 (4) ◽  
pp. H508-H516
Author(s):  
R. A. Kauppinen ◽  
I. E. Hassinen
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Optical Methods ◽  
Perfused Heart ◽  
Rat Hearts ◽  
Carbonyl Cyanide ◽  
Perfused Rat Hearts ◽  
Beating Rate ◽  
Isolated Perfused Rat Hearts

Optical methods were tested for measuring the membrane potential changes of mitochondria in isolated perfused rat hearts. Safranin was found to be rapidly taken up by the Langendorff-perfused heart, and after loading with the dye there was practically no washout of the stain during perfusion with Krebs-Ringer bicarbonate solution. Staining with safranin induced the appearance of an intense absorption band in the reflectance spectrum of the heart, but the absorbance spectrum changes were not useful for monitoring the mitochondrial membrane potential changes because of interference by endogenous hemoproteins. The fluorescence intensity, however, responded in a manner which indicated that its changes originated from dye attached to the mitochondria. A decrease of the fluorescence was found on energizing the mitochondria by decreasing the cellular energy consumption by arrest induced by 18 mM K+ or by decreasing the beating rate of an electrically paced heart from 5 Hz to the endogenous ventricular frequency of 1.5 Hz. In hearts arrested by Ca2+ depletion, 18 mM K+ did not affect the safranin fluorescence. This was taken to indicate that under these conditions the safranin fluorescence was not sensitive to the plasma membrane potential. The uncoupler carbonyl cyanide m-chlorophenylhydrazone induced an intense enhancement of safranin fluorescence in the perfused heart, demonstrating that the probe is sensitive to mitochondrial membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak

2001 ◽  
Vol 280 (4) ◽  
pp. H1762-H1769 ◽  
Author(s):  
Masayuki Taniguchi ◽  
Craig Wilson ◽  
Charlene A. Hunter ◽  
Daniel J. Pehowich ◽  
Alexander S. Clanachan ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Glucose Metabolism ◽  
Pyruvate Dehydrogenase ◽  
Mitochondrial Membrane ◽  
Glucose Oxidation ◽  
Proton Leak ◽  
Cardiac Efficiency ◽  
Cardiac Work ◽  
Atp Production ◽  
Rat Hearts

Dichloroacetate (DCA) is a pyruvate dehydrogenase activator that increases cardiac efficiency during reperfusion of ischemic hearts. We determined whether DCA increases efficiency of mitochondrial ATP production by measuring proton leak in mitochondria from isolated working rat hearts subjected to 30 min of ischemia and 60 min of reperfusion. In untreated hearts, cardiac work and efficiency decreased during reperfusion to 26% and 40% of preischemic values, respectively. Membrane potential was significantly lower in mitochondria from reperfused (175.6 ± 2.2 mV) versus aerobic (185.8 ± 3.1 mV) hearts. DCA (1 mM added at reperfusion) improved recovery of cardiac work (1.9-fold) and efficiency (1.5-fold) but had no effect on mitochondrial membrane potential (170.6 ± 2.9 mV). At the maximal attainable membrane potential, O2consumption (nmol O2 · mg−1 · min−1) did not differ between untreated or DCA-treated hearts (128.3 ± 7.5 and 120.6 ± 7.6, respectively) but was significantly greater than aerobic hearts (76.6 ± 7.6). During reperfusion, DCA increased glucose oxidation 2.5-fold and decreased H+production from glucose metabolism to 53% of untreated hearts. Because H+ production decreases cardiac efficiency, we suggest that DCA increases cardiac efficiency during reperfusion of ischemic hearts by increasing the efficiency of ATP use and not by increasing the efficiency of ATP production.

Effect of fatty acid oxidation on efficiency of energy production in rat heart

1985 ◽  
Vol 249 (4) ◽  
pp. H723-H728 ◽  
Author(s):  
J. F. Hutter ◽  
H. M. Piper ◽  
P. G. Spieckerman
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Acid Oxidation ◽  
O2 Consumption ◽  
Mitochondrial Fatty Acid Oxidation ◽  
Rat Hearts ◽  
Mitochondrial Fatty Acid ◽  
Perfused Rat Hearts ◽  
On Line ◽  
Exact Amount

Myocardial fatty acid oxidation has been reported to be accompanied by an elevated O2 consumption compared with carbohydrate oxidation. The exact amount of this additional O2 consumption is controversial. Different investigators have observed an O2 wasting effect that is too large to be explained by the different ATP-to-O2 ratios of these substrates. With the use of isolated perfused rat hearts, O2 consumption and hemodynamic measurements were computer analyzed to provide on-line estimates of the ratio between O2 consumption and demand (EQ). Increasing palmitate or octanoate concentrations decreased the respiratory quotient, which was accompanied by a disproportionate increase of EQ. Inhibition of fatty acid oxidation by an inhibitor of acylcarnitine transferase or a blockade of mitochondrial thiolase caused a drastic reduction of fatty acid oxidation. The fatty acid-induced enhancement of O2 consumption was decreased to a much smaller extent, indicating that there are two different mechanisms responsible for the O2-wasting effect, one that depends on mitochondrial fatty acid oxidation and another that is not affected by an inhibition of this pathway.

NMR-visible ATP and Pi in normoxic and reperfused rat hearts: a quantitative study

1991 ◽  
Vol 260 (1) ◽  
pp. H6-H12 ◽  
Author(s):  
S. M. Humphrey ◽  
P. B. Garlick
Keyword(s):  
Chemical Analysis ◽  
31P Nmr ◽  
Ischemia Reperfusion ◽  
High Energy ◽  
High Energy Phosphates ◽  
Rat Hearts ◽  
External Standard ◽  
Perfused Rat Hearts ◽  
Krebs Buffer ◽  
Isolated Perfused Rat Hearts

Nuclear magnetic resonance (NMR) spectroscopy detects only free, unbound metabolites. We have therefore compared the free high-energy phosphate content of isolated perfused rat hearts (determined by 31P-NMR) with the total high-energy phosphates of the same hearts (determined by chemical analysis) to determine the fractions, if any, that are NMR invisible. Aerobic perfusion (40 min at 37 degrees C, Pi-free Krebs buffer) was followed by 10, 14, or 18 min total global ischemia and 30 min reperfusion (n = 6 in each group). Fully relaxed 31P-NMR spectra (40 scans using 90 degrees pulses at 15-s intervals) were collected at various times throughout the protocol, and the signal intensities of the beta-phosphate of ATP, phosphocreatine (PCr), and Pi were quantified using methylenediphosphonate as an external standard. Hearts were freeze clamped either before ischemia or at the end of reperfusion and were chemically assayed for ATP, PCr, and Pi. After 40 min of normoxia, the ATP and PCr contents determined by NMR were almost identical to the values determined by chemical analysis. However, only 39 +/- 8% of the total Pi was NMR visible. After reperfusion, after 14 or 18 min of ischemia, the proportion of NMR-visible ATP had decreased to 64 +/- 9% (P less than 0.005). After reperfusion after 18 min ischemia, the proportion of NMR-visible Pi had increased to 76 +/- 10% (P less than 0.05). In conclusion, whereas the total cellular content of PCr is always NMR visible, ischemia-reperfusion can alter the fraction of NMR-visible ATP and Pi.

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