Restoration Of High-energy Phosphates Research Articles

Multiple Causes of Fatigue during Shortening Contractions in Rat Slow Twitch Skeletal Muscle

Fatigue in muscles that shorten might have other causes than fatigue during isometric contractions, since both cross-bridge cycling and energy demand are different in the two exercise modes. While isometric contractions are extensively studied, the causes of fatigue in shortening contractions are poorly mapped. Here, we investigate fatigue mechanisms during shortening contractions in slow twitch skeletal muscle in near physiological conditions. Fatigue was induced in rat soleus muscles with maintained blood supply by in situ shortening contractions at 37°C. Muscles were stimulated repeatedly (1 s on/off at 30 Hz) for 15 min against a constant load, allowing the muscle to shorten and perform work. Fatigue and subsequent recovery was examined at 20 s, 100 s and 15 min exercise. The effects of prior exercise were investigated in a second exercise bout. Fatigue developed in three distinct phases. During the first 20 s the regulatory protein Myosin Light Chain-2 (slow isoform, MLC-2s) was rapidly dephosphorylated in parallel with reduced rate of force development and reduced shortening. In the second phase there was degradation of high-energy phosphates and accumulation of lactate, and these changes were related to slowing of muscle relengthening and relaxation, culminating at 100 s exercise. Slowing of relaxation was also associated with increased leak of calcium from the SR. During the third phase of exercise there was restoration of high-energy phosphates and elimination of lactate, and the slowing of relaxation disappeared, whereas dephosphorylation of MLC-2s and reduced shortening prevailed. Prior exercise improved relaxation parameters in a subsequent exercise bout, and we propose that this effect is a result of less accumulation of lactate due to more rapid onset of oxidative metabolism. The correlation between dephosphorylation of MLC-2s and reduced shortening was confirmed in various experimental settings, and we suggest MLC-2s as an important regulator of muscle shortening.

Nitric oxide synthase mediates the apelin-induced improvement of myocardial postischemic metabolic and functional recovery

The adipocytokine apelin is capable to reduce myocardial ischemia/reperfusion injury in rodents. Cardioprotective activity of apelin may be attributed to upregulation of endothelial nitric oxide synthase (eNOS). This study was designed to examine metabolic and functional effects of a synthesized 12 C-terminal residue of apelin (A-12) and NG-nitro-L-arginine methyl ester (L-NAME), a non-selective eNOS inhibitor, in isolated working rat hearts subjected to global ischemia. Preischemic infusion of A-12 increased recovery of cardiac function during reperfusion compared with control and resulted in enhanced restoration of myocardial ATP, adenine nucleotide pool, phosphocreatine and reduction of myocardial lactate and lactate/pyruvate ratio. Coadministration of A-12 and L-NAME aggravated recovery of coronary flow and cardiac function compared with these indices after A-12 treatment. Cardiac dysfunction was associated with increase in lactate dehydrogenase release in myocardial effluent, reduction of glucose oxidation and abolishment of augmented restoration of high energy phosphates. The results clearly demonstrate involvement of NOS-dependent mechanisms in cardioprotection afforded by apelin.

A role of PKC in the improvement of energy metabolism in preconditioned heart.

A possible link between activation of PKC and improvement of energy metabolism during reperfusion in ischemic preconditioning hearts was examined. Isolated perfused rat hearts were preconditioned by 5-min ischemia and 5-min reperfusion in the presence and absence of a PKC inhibitor polymyxin B (50 microM) and then subjected to 40-min sustained ischemia and subsequent 30-min reperfusion. In another set of experiments, the hearts pretreated with and without a PKC activator PMA (15 pmol/5 min) were subjected to the sustained ischemia and reperfusion. Myocardial high-energy phosphates, glycolytic intermediates and mitochondrial oxygen consumption capacity were determined at appropriate experimental sequences. Preconditioning enhanced the recovery of cardiac function such as left ventricular developed pressure, heart rate and rate-pressure product of the reperfused heart, suppressed the release of creatine kinase, enhanced the reperfusion-induced restoration of myocardial high-energy phosphates, attenuated the reperfusion-induced accumulation in glucose 6-phosphate and fructose 6-phosphate contents, abolished the ischemia-induced increase in tissue lactate content and prevented the ischemia-induced decrease in mitochondrial oxygen consumption capacity. Treatment of the perfused heart with PMA mimicked the effects of preconditioning on post-ischemic contractile function, enzyme release, levels of myocardial energy store, glycolytic intermediates and lactate, and mitochondrial function. Polymyxin B-treatment abolished the preconditioning-induced recovery of post-ischemic contractile function, the suppression of the release of CK, the restoration of myocardial energy store, and the preservation of mitochondrial function, whereas it did not cancel the improvement of glycolytic intermediate levels and the reduction in tissue lactate accumulation. Post-ischemic contractile function was closely related to restoration of high-energy phosphates and mitochondrial oxygen consumption capacity in all hearts subjected to ischemia/reperfusion. The results suggest that activation of PKC and preservation of mitochondrial function are closely linked with each other in the preconditioned heart, which may lead to the improvement of post-ischemic contractile function.

Prediction of recovery after ischemia. A microcalorimetric and biochemical study of rat liver tissue

Abstract Alteration of the hepatocellular function following ischemic damage may play a crucial role in the limited recovery after reperfusion. In spite of numerous efforts, finding a simple technique for predicting the recovery of the liver after ischemic damage is still an unresolved problem. During the ischemic storage of isolated rat livers at 25°C, tissue concentrations of high energy phosphates, glycogen and lactate were determined photometrically and, in comparison, the metabolic rate was measured continuously by direct calorimetry. In addition, interstitial pH was measured with microelectrodes. In a second series of experiments these results were compared with functional recovery after ischemia and reperfusion. Following ischemic storage at 25°C for 60, 120 and 240 min, the isolated livers were reperfused for 30 min in a non-recirculating system with a constant flow rate. During reperfusion, functional recovery as assessed by oxygen consumption and bile flow was determined. At the end of reperfusion, tissue samples were taken for biochemical analysis of adenine nucleotides and tissue lactate. Though the ATP concentration drops within 60 min of ischemia to 6.94% of the control value without further significant change, the metabolic rate measured continuously by direct calorimetry decreases in an exponential manner. According to the calorimetrically measured heat output, functional hepatic metabolism, as assessed by oxygen consumption, bile flow and restoration of high energy phosphates during reperfusion, recovered to an extent depending on the duration of ischemia. It is concluded that the metabolic rate of the ischemic rat liver, as determined continuously by direct calorimetry, is a good predictor for recovery after ischemic damage.

Early reversal of acidosis and metabolic recovery following ischemia

Tissue acidosis is believed to be a key element in ischemic injury of neural tissue. The goal of this study was to determine whether persisting postischemic acidosis or the extent of acidosis would affect metabolic recovery following an ischemic event. Intracellular pH (pHi), adenosine triphosphate, phosphocreatine, and lactate levels were measured in the cerebral cortex during the early stages of reperfusion, following either 5 or 10 minutes of global ischemia in both normo- and hyperglycemic gerbils. A total of 130 gerbils were injected with a solution containing 1.5 ml Neutral Red (1%) (+/- 2.5 gm/kg glucose); 30 minutes later, the gerbils were placed under halothane anesthesia, and the carotid arteries were occluded for either 5 or 10 minutes. The brains were frozen in liquid nitrogen at 0, 15, 30, 60, and 120 seconds after reperfusion; they were sectioned and the block face was photographed to determine the pHi by using Neutral Red histophotometry. At the conclusion of the ischemia, the pHi in all groups had decreased significantly from a control value of 7.05 +/- 0.03) (mean +/- standard error of the mean). In normoglycemic brains, the pHi values fell to 6.71 +/- 0.04 and 6.68 +/- 0.11 after 5 and 10 minutes of ischemia, respectively. Hyperglycemic brains were more acidotic; values fell to 6.57 +/- 0.10 and 6.52 +/- 0.24 after 5 and 10 minutes of ischemia, respectively. Lactate levels were approximately fivefold greater than those of control tissue in normoglycemic brains, while lactate levels in hyperglycemic brains were increased eightfold. The adenosine triphosphate and phosphocreatine levels were depleted at the end of ischemia in all groups. After 2 minutes of reflow activity, the pHi levels in both normo- and hyperglycemic brains were restored to those of control values in the '5-minute ischemic group, while the pHi levels remained significantly depressed in the 10-minute ischemic group. Restoration of high-energy phosphates was similar in normoglycemic brains regardless of ischemic duration, recovering to only 20% of the restoration obtained in control tissue at 2 minutes. In hyperglycemic brains, however, there was complete recovery of high-energy phosphates by 2 minutes of reflow activity following 5 minutes of ischemia. Extending the ischemic period to 10 minutes in hyperglycemic brains slowed the rate of metabolic recovery to that observed in normoglycemic brains. The results indicate that the reflow period permits the rapid restoration of pHi levels substantially before the normalization of primary energetic compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of high buffer cardioplegia and secondary cardioplegia on cardiac preservation and postischemic functional recovery: a 31P NMR and functional study in Langendorff perfused pig hearts

High buffer cardioplegia may provide protection against ischemic damage by reducing the extent of intracellular acidosis. Secondary cardioplegia may improve postischemic recovery by restoration of high energy phosphates, ionic gradients, and intracellular pH. To test these hypotheses, pig hearts were arrested with high buffer (150 mM MOPS) cardioplegia or modified St. Thomas' solution II and then kept ischemic at 12 degrees C for 8 h. High energy phosphates and intracellular pH were followed during the period of ischemia, using 31P nuclear magnetic resonance spectroscopy, and functional recovery was followed during reperfusion. The hearts arrested by high buffer cardioplegia showed significantly higher intracellular pH than hearts preserved with St. Thomas' solution, but there were no significant differences in high energy phosphates. There were no significant differences in functional recovery. We found, however, that secondary cardioplegia abolished ventricular fibrillation, and resulted in improved functional recovery after 8 h of ischemic preservation compared with the hearts reperfused with Krebs-Henseleit solution alone. Our results suggest that despite attenuating the decreases in intracellular pH, high buffer cardioplegia does not improve recovery following 8 h of preservation at 12 degrees C. Secondary cardioplegia reduces the incidence of ventricular fibrillation and improves postischemic functional recovery of the myocardium.

Effects of accumulation of phosphocreatine on utilization and restoration of high-energy phosphates during anoxia and recovery in thin hippocampal slices from the guinea pig

Effects of accumulation of phosphocreatine (P-creatine) on utilization and restoration of high-energy phosphates during anoxia and recovery were studied using thin hippocampal slices from the guinea pig. Incubation of slices with creatine (5 to 30 m m) increased the content of tissue P-creatine, in a linear fashion, and these increases depended on the concentration of creatine and the incubation period to as long as 3 h. Concentration of ATP in the slices was not altered. A high concentration of tissue P-creatine (40 to 100 mmol/kg protein) accumulated by incubation with creatine reduced the rate of ATP exhaustion during anoxia whereas P-creatine itself was rapidly decreased. During reoxygenation after anoxia, concentrations of ATP and P-creatine rapidly recovered in slices preincubated with creatine. Concentrations of P-creatine previously exhausted during anoxia increased to 100 mmol/kg protein after 25 min of reoxygenation whereas the concentration was 35 mmol/kg in the control slice. Elevation of the concentration of P-creatine may therefore prolong the survival time of brain tissue during anoxia and facilitate recovery during reoxygenation.

Significance of enzyme release from ischemic isolated rat heart.

Whole-heart ischemia has been induced in isolated working rat heart. The distribution of the reduced coronary flow was even, as judged by 3H-antipyrine autoradiographs. Reducing the coronary flow resulted in myocardial ischemia, as indicated by a lowered tissue content of glycogen, ATP and creatine phosphate and accumulation of lactate. After a reperfusion period of 30 min there was a restoration of glycogen, ATP and creatine phosphate for hearts that were ischemic for 5 and 10 min, with a concomitant normalization of tissue lactate. Hearts that were ischemic for 30 min did not show restoration of high energy phosphates and glycogen. There was a leakage of ASAT, CK and LD in all groups of hearts, suggesting that a release of these enzymes does not necessarily indicate an irreversibly damaged myocardial cell.

Clinical Physiology: Skeletal Muscle Metabolites in Patients with Cardiogenic Shock or Severe Congestive Heart Failure

Skeletal muscle metabolite concentrations were determined in 19 patients in either cardiogenic shock or severe left ventricular failure by obtaining a needle biopsy specimen of lateral thigh muscle. Evidence of anaerobic skeletal muscle metabolism was found in both patient groups with the greatest lactate accumulation and most severe high-energy phosphate depletion present in the patients in cardiogenic shock. The skeletal muscle lactate accumulation was most pronounced in the patients that died. Blood lactate values did not absolutely predict skeletal muscle lactate concentrations in those patients in whom skeletal muscle lactate concentrations were the highest. The patients in cardiogenic shock and severe left ventricular failure who survived demonstrated a reduction in skeletal muscle lactate levels and a restoration of high-energy phosphates over several days which correlated with clinical and hemodynamic improvement.