23Na and flame photometric studies of the NMR visibility of sodium in rat muscle.

23Na nuclear magnetic resonance spectroscopy (NMR) is increasingly being used to study Na+ gradients and fluxes in biological tissues. However, the quantitative aspects of 23Na NMR applied to living systems remain controversial. This paper compares sodium concentrations determined by 23Na NMR in intact rat hindlimb (n = 8) and excised rat gastrocnemius muscle (n = 4) with those obtained by flame photometric methods. In both types of samples, 90% of the sodium measured by flame photometry was found to be NMR-visible. This is much higher than previously reported values. The NMR measurements for intact hindlimb correlated linearly with the flame photometric measurements, implying that one pool of sodium, predominantly extracellular, is 100% visible. From measurements on excised muscle, in which extracellular space is more clearly defined, the NMR visibility of intracellular Na+ was calculated to be 70%, assuming an extracellular space of 12% of the total tissue water volume and an extracellular NMR visibility of 100%. 23Na transverse relaxation measurements were carried out using a Hahn spin echo on both intact hindlimb (n = 1) and excised muscle (n = 2) samples. These showed relaxation curves that could each be described adequately using two relaxation times. The rapidly relaxing component showed a T2 value of 3-4 ms and the slowly relaxing component a T2 of 21-37 ms. A spin lattice relaxation (T1) measurement on intact hindlimb yielded a value of 51 ms. These relatively long relaxation times show that the quadrupolar relaxation effect of Na+ complexing to large macromolecules or being otherwise motionally restricted is relatively weak. This is consistent with the high NMR visibilities reported here.

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.

Cardiac hypothermia: 31P and 1H NMR spectroscopic studies of the effect of buffer on preservation of human heart atrial appendages.

31P and 1H nuclear magnetic resonance spectroscopy has been used to follow noninvasively the time course of energetic metabolite levels in human heart atrial appendages preserved under various temperatures and buffer conditions. From sample harvest up to the normal 5-h time limit for heart preservation, ATP levels in human atrial appendages are much better maintained in 0.9% saline and PIPES-buffered preservation solutions at 12 degrees C than at 4 degrees C. Furthermore, preservation at 12 degrees C can be improved considerably by using high extracellular buffer concentrations. The increased buffer concentration allows better maintenance of the intracellular pH and leads to a faster glycolytic rate as measured by lactate production. At 4 degrees C, ATP levels decline rapidly during the first 5 h but reached a stable plateau, which is well maintained over 15-20 h. At this temperature, the rate of lactate production is similar at all buffer concentrations (20, 60, and 100 mM PIPES). As a consequence of these observations, we postulate that the mechanisms of ATP production and utilization at 4 degrees C and at 12 degrees C are different. At 4 degrees C, the rate of glycolysis is temperature limited whereas at 12 degrees C, low intracellular pH inhibits glycolysis.

An NMR probe to study function and metabolism simultaneously in isolated human cardiac tissue.

Trabeculae isolated from human atrial appendages have been used to study preservation of donor hearts for cardiac transplantation. We have developed a perifusion system equipped with a fiber optic strain gauge to study mechanical performance of human atrial trabeculae (10-20 mg) while simultaneously observing the energetic compounds by 31P NMR spectroscopy. The NMR probe consists of an eight-turn solenoid coil (2.3 mm i.d. x 5 mm length, 24-gauge wire) double tuned to allow observation of 1H and 31P nuclei. The probe and the perifusion system are temperature regulated and permit preservation studies using a variety of small muscles at low temperatures (down to 4 degrees C) as well as at physiological temperature (37 degrees C). 31P NMR spectra suitable for quantification can be obtained from approximately 10 mg of human atrial trabeculae in 15 min. Spectra of 8- to 12-mg mouse extensor digitalis longus or soleus muscle can be obtained in less than 10 min.

Preservation of high-energy phosphates in human myocardium. A phosphorus 31-nuclear magnetic resonance study of the effect of temperature on atrial appendages.

After prolonged exposure to low temperatures (1 degree and 4 degrees C), human atrial trabeculae show poor recovery of contraction. At somewhat higher temperatures (12 degrees and 20 degrees C), recovery is much better (Keon and associates. Ann Thorac Surg 1988;46:337-41). Although better preservation of adenosine triphosphate and therefore improved contractile recovery might be expected after exposure to lower temperatures, it remained possible that, below a certain temperature, adenosine triphosphate-generating mechanisms could be slowed more than adenosine triphosphate utilization. To investigate this phenomenon further, we followed the time course of metabolic changes in human atrial appendages, harvested during cardiac bypass operations, at 1 degree, 4 degrees, 12 degrees, and 20 degrees C using high-resolution 31P and 1H nuclear magnetic resonance spectroscopy. The results are quantitated by correlation with data obtained from biochemical assays on quick-frozen tissues. Initial adenosine triphosphate levels in myocytes of human atrial appendages are 3.3 to 4.3 mumol.gm-1 tissue wet weight. At 20 degrees C, adenosine triphosphate disappears after 6 hours; at 12 degrees C, about half the initial adenosine triphosphate is still observable at this time; at 4 degrees C or 1 degree C, the decline is still slower. Only a small contribution toward adenosine triphosphate maintenance comes from creatine phosphate, since creatine phosphate, inorganic phosphate, and total creatine levels in the appendage are low (less than 2 mumol.gm-1 tissue wet weight). Glycolysis is active at all temperatures; the rate of glycolysis correlates positively with increasing temperature. Adenosine triphosphate generated by glycolysis falls just short of demand at all temperatures, but the difference is small at 1 degree and 4 degrees C. These studies lead us to conclude that the relatively poor recovery of contractile response of human atrial trabeculae, together with contracture reported previously at lower temperatures (1 degree and 4 degrees C), is not due to a failure to maintain adenosine triphosphate levels.

The effects of temperature and buffer concentration on the metabolism of human atrial appendages measured by 31P and 1H NMR.

In this study we measured changes in intracellular ATP and pH together with lactate production in isolated ischemic human atrial tissue. The measurements were made using 31P and 1H NMR. ATP preservation is improved as temperature is reduced from 20 degrees C to 1 degree C because of a progressive decrease in energy demand. At a constant temperature (12 degrees C), ATP preservation is improved by increasing the extracellular buffer capacity with PIPES buffer at concentrations up to 100 mM. Under these conditions, energy demand appears to increase but the ATP level is kept relatively constant for periods of 10 hours or longer. This appears to be due to a tighter regulation between supply and demand in which glycolysis is driven faster at relatively lower ADP and Pi levels. This tight regulation may be attributed to the better maintenance of intracellular pH.

31P NMR studies of the metabolic status of pig hearts preserved for transplantation.

31P NMR spectroscopy has been used to evaluate the metabolic status of cardioplegically arrested pig hearts. Hearts were stored with Plegisol for up to 12 hours at either 5 degrees C or 12 degrees C. Results indicated that the ATP content of hearts could be maintained (greater than 70% of initial values) for up to 5 hours in the ischemic storage state. The ATP loss was greater at 12 degrees C. PCr was lost exponentially under the same conditions. Functional testing by reperfusing the stored hearts in vitro indicated a good correlation between the ATP content and survivability of the preparations. Twenty-four hour preservation of pig hearts using slow perfusion with a modified cardioplegic solution (Wicomb) allowed for preservation of both PCr and ATP, in all cases, reperfusion of hearts revealed a loss of NMR- visible ATP and PCr.

Cardiac transplantation: the ideal myocardial temperature for graft transport.

The ideal preservation method and cooling temperature for transport of donor hearts are not known. Serious derangements in myocardial relaxation are well described with different methods of cooling. To assess this problem, human right atrial trabeculae contracting isometrically at 34 degrees C in vitro were subjected to hypothermic arrest at 1, 4, 12, and 20 degrees C for 1, 2, 4, 24, and 48 hours. Control conditions were resumed, and myocardial mechanical recovery was assessed over 1 hour. Contraction was 50% depressed after a 1- to 2-hour exposure to 1 degree C and was almost completely arrested following a 4-hour exposure. Muscles cooled to 4 degrees C recovered poorly, whereas those cooled to 12 and 20 degrees C did well. In the latter 2 groups, force development increased rapidly on rewarming and exceeded the precooling contraction force (p less than 0.05). A 100% increase in relative resting force was seen in muscles cooled to 1 and 4 degrees C (p less than 0.05). This finding suggests a failure of calcium homeostasis at very low temperatures. We conclude that atrial preservation is optimal at about 12 degrees C.

The pH dependence of the contractile response of fatigued skeletal muscle.

Following a period of intense repetitive stimulation (e.g., brief tetanic stimuli every second for 3 min), muscle isometric tension development is reduced by about 80%. This suppression is reversible at a high external pH (8.0) with a half time of 15-20 min, but if the external pH is low (6.4) or the buffer concentration is low, recovery is prevented. Inhibition of recovery is associated with a slowed rate of lactate loss, which may suggest that intracellular lactacidosis is the cause of the inhibition. Alternatively, a low external pH may affect recovery from fatigue quite independently of its effect on lactate efflux. The possibility that surface membrane properties are changed by fatigue in a pH-dependent fashion was examined by measuring the cable properties and action potentials of fatigued fibres at different external pH values. A low external pH during recovery from fatigue was shown to result in a prolonged membrane depolarization of 10-12 mV, an increased transmembrane resistance, and a prolonged action potential. At a high external pH transmembrane resistance is lowered by fatigue, the depolarization lasts only about 10-15 min, and there is a smaller effect on the action potential. While the fatigued fibre membrane does show a changed response that is dependent on external pH, it is not clear that this could be related to the suppression of contraction. Direct measurements of intracellular pH show a fall of about 0.4 to 0.5 pH units in the surface fibres following fatigue. This results from the lactic acid generated during activity. It is now clear that lactate crosses the membrane in association with protons and at least part of this flux is mediated by a specific carrier mechanism. Efflux is limited by the transmembrane pH gradient, which in turn depends on the extracellular buffer concentration in the diffusion limited space around the fibres. Intracellular lactacidosis in resting muscles can be generated by a reversal of the normal flux. Fibres can be loaded with lactate (L) by increasing the extracellular [H+][L-] product with a resultant fall in intracellular pH. Lactate loads similar to those seen in fatigued muscle simulate some but not all of the responses seen in the postfatigue state. The twitch is prolonged with a slow relaxation phase, an increased time to peak tension but with an increase in peak tension. The effects are reversible but usually result in a reduced contractile response following the washout.(ABSTRACT TRUNCATED AT 400 WORDS)

Is the change in intracellular pH during fatigue large enough to be the main cause of fatigue?

The intracellular pH of frog sartorius muscles exposed to an extracellular pH 8.0 (25 mM HCO3-, 1% CO2) was 6.9-7.1. Following a fatiguing stimulation period (one tetanic contraction per second for 3 min), the intracellular pH was 6.5-6.7. When similar experiments were repeated with frog sartorius muscles exposed to pH 6.4 (2mM HCO3-, 1% CO2), the intracellular pH was 6.8-6.9 at rest and 6.3-6.4 following fatigue. So, in both experiments the intracellular pH decreased by 0.4-0.5 pH unit during fatigue. When the CO2 concentration of the bathing solution was increased from 1 to 30%, the intracellular pH of resting muscles decreased from 7.0 to 6.2-6.3. Although the effect of CO2 on the intracellular pH was greater than the fatigue effect, the decrease in tetanic force with CO2 was less than 40%, while during fatigue the tetanic force decreased by at least 70%. Therefore in frog sartorius muscle the decrease in tetanic force during fatigue exceeds the decrease that is expected from just a change in intracellular pH.

The influence of extracellular buffer concentration and propionate on lactate efflux from frog muscle.

Lactate efflux from frog sartorius muscles was measured following a lactate load of about 18 mumol X g-1 induced by a 4-min period of stimulation. Lactate efflux rate was buffer concentration dependent. The initial efflux rate increased from about 150 nmol X g-1 X min-1 in 1 mM MOPS buffer to 400 nmol X g-1 X min-1 in 25 mM MOPS buffer. The addition of 20 mM propionate reduced mean intracellular pH by about 0.2 units and increased lactate efflux rate by 70% at the highest buffer concentration and 400% at the lowest buffer concentration. The observed results are in reasonable agreement with predictions based on a model in which net efflux is limited by diffusion of both buffer and lactate in the extracellular space. Transmembrane lactate efflux appears to consist of two components, one of which is proton linked and carried either by undissociated lactic acid or coupled proton-lactate transport, the other being carried by independent lactate ions.

The interactive effects of fatigue and pH on the ionic conductance of frog sartorius muscle fibers.

The effects of fatigue on the membrane conductance of frog sartorius muscle at the resting potential and during an action potential were studied. When muscles were exposed to an extracellular pH of 8.0 the membrane conductance at the resting potential increased during fatigue by about 20% and returned to prefatigue level in about 20 min. The membrane conductance of muscle fibers exposed to pH 6.4 was about three times less than that of pH 8.0 and decreased further during fatigue. Furthermore, the recovery of a normal membrane conductance was slow at pH 6.4. Both the inward, depolarizing and the outward, repolarizing currents during the action potential are reduced in fatigue. In each case the effect is greater at pH 6.4 than at 8.0 and recovery towards normal values is slower at pH 6.4. It is concluded that the ionic conductance of the sarcolemmal membrane at the resting potential and during an action potential are modified by fatigue and that these changes are modulated by pHo.

The effects of pH on the kinetics of fatigue and recovery in frog sartorius muscle.

The effects of pH on the kinetics of fatigue and recovery in frog sartorius muscle were studied to establish whether the pH to which muscles are exposed (extracellular pH) has an effect on both the rate of fatigue development and recovery from fatigue. When frog sartorius muscles were stimulated with short tetanic stimuli at rates varying from 0.2 to 2.0 trains/s, a time- and frequency-dependent decrease in force development was observed, but extracellular pH had comparatively little effect. The recovery of tetanic force was dependent on the extracellular pH. This effect was characterized by a rapid recovery in force at pH 8.0 and an inhibition of recovery at pH 6.4 even when force decreased by only 25% during stimulation. Even when muscles were fatigued at pH 8.0 the rate of force recovery was still very small at pH 6.4. A model is proposed in which a step of the contraction cycle changes from a normal to a fatigued state. The rate of this transition is a function of the stimulation frequency and not pH. The reverse transition, from a fatigued to normal state is pH dependent; i.e., it is inhibited by H+. Measurements of resting and action potentials show that extracellular pH influences these parameters in the fatigue state, but there is no evidence that these changes are directly responsible for the pH-dependent step in the reversal of fatigue.

Post-tetanic responses in creatine-depleted rat EDL muscle.

The total creatine content of rat extensor digitorum longus (EDL) muscles is reduced from 26.7 mumol X g-1 to 5.2 mumol X g-1 after 4 weeks on a diet containing beta-guanidinopropionate. The resting muscles of these animals contain only 5% of the normal creatine phosphate and about 30% of the normal adenosine triphosphate (ATP). Isometric twitch and brief tetanic responses are not significantly different from normal. However, following a 1-second tetanus, the normal twitch potentiation (+80%) is reversed in depleted muscles (-40%). The initial suppression lasts 1-2 seconds and is followed by a small delayed potentiation (+25%). Relaxation rate, which is normally increased following a tetanus, is considerably reduced in depleted muscles. Reversal of potentiation appears to be due to a suppression of twitch activation. The suppression may result from a transient fall in delta GATP, which is thought to be a critical parameter in the uptake of Ca++ by the sarcoplasmic reticulum (SR).

The effect of acid-base balance on fatigue of skeletal muscle.

H+ ions are generated rapidly when muscles are maximally activated. This results in an intracellular proton load. Typical proton loads in active muscles reach a level of 20-25 mumol X g-1, resulting in a fall in intracellular pH of 0.3-0.5 units in mammalian muscle and 0.6-0.8 units in frog muscle. In isolated frog muscles stimulated to fatigue a proton load of this magnitude is developed, and at the same time maximum isometric force is suppressed by 70-80%. Proton loss is slowed when external pH is kept low. This is paralleled by a slow recovery of contractile tension and seems to support the idea that suppression results from intracellular acidosis. Nonfatigued muscles subjected to similar intracellular proton loads by high CO2 levels show a suppression of maximal tension by only about 30%. This indicates that only a part of the suppression during fatigue is normally due to the direct effect of intracellular acidosis. Further evidence for a component of fatigue that is not due to intracellular acidosis is provided by the fact that some muscle preparations (rat diaphragm) can be fatigued with very little lactate accumulation and very low proton loads. Even under these conditions, a low external pH (6.2) can slow recovery of tension development 10-fold compared with normal pH (7.4). We must conclude that there are at least two components to fatigue. One, due to a direct effect of intracellular acidosis, acting directly on the myofibrils, accounts for a part of the suppression of contractile force. A second, which in many cases may be the major component, is not dependent on intracellular acidosis. This component seems to be due to a change of state in one or more of the steps of the excitation-contraction coupling process. Reversal of this state is sensitive to external pH which suggests that this component is accessible from the outside of the cell.

Human atrial trabeculae: an experimental preparation for studying myocardial response to perioperative manipulations.

Trabeculae taken from discarded human right atrial specimens during cardiac surgery provide a useful preparation for studies in myocardial physiology and pharmacology. Three extrinsic measurements that have a marked effect on contraction of this preparation are temperature, stimulation frequency and calcium ion (Ca2+) concentration. There is a large decline in developed force below 30 degrees C. The optimal stimulation frequency (temperature 34 degrees C, Ca2+ concentration 2.5 mM) is 1 Hz. The Ca2+ level required to give half maximal force development is 2.0 mM. In a series of 46 atrial trabeculae (approximately 1 mm in diameter) from 33 patients, the authors found a mean contraction tension of 1.37 +/- 0.09 g/mm2 (+/- standard error) (temperature 34 degrees C, stimulation frequency 1 Hz, pH 7.4, Ca2+ concentration 2.5 mM) at maximum force. The preparation appears to have great potential for the study of perioperative manipulation on myocardial function.

Lactate and pyruvate production in isolated thiamine-deficient rat diaphragm strips.

Rats were maintained on a thiamine-deficient diet to deplete skeletal muscle of thiamine pyrophosphate, and thus decrease the oxidative metabolism of pyruvate. The blood lactate concentration was significantly elevated in thiamine-deficient (TD) animals when compared with pair-fed (TP) controls. Analysis of diaphragm strips from these animals revealed that tissue lactate and pyruvate concentrations were not affected by any of the treatments employed. The rate of lactate efflux from TD tissues was, however, twice that from TP and 4.5 times that from weight-control (WC) tissues. The H+ efflux rate was also much greater in the TD muscle preparation than either of the control groups. Following 3 min of stimulation (150-Hz, 200-ms pulse train every 0.5 s), the degree of fatigue of tissues from each of these three treatment groups was not different. The observation in this study that glycolysis becomes the predominant metabolic pathway in thiamine deficiency without increasing the intracellular level of products, indicates that this treatment also has other effects which increase the effective lactate permeability of the fibre membranes.

Evidence for an extracellular mechanism for the action of H+ on recovery of muscles following fatigue.

Isolated rat diaphragm strips were fatigued by repetitive trains of tetanic stimulation for 3 min. Maximum isometric tension fell to approximately 20% of the normal level. Tension recovered to the prefatigue level a few minutes after the fatiguing stimulation stopped. In normal, bicarbonate-buffered Tyrode solution at pH 7.4, the half recovery time (t1/2) is typically 1-2 min. This is not significantly changed by presoaking in Tyrode at low pH (6.2), amiloride (10(-4) M), which blocks H+--Na+ exchange, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (10(-4) M), which blocks HCO-3--Cl- exchange, or a combination of these treatments. When the extracellular pH is low (pH 6.2) during recovery the recovery rate falls to approximately 10% of its normal level (t1/2 = 15 min). In the absence of external bicarbonate, using morpholinopropanesulfonic acid buffers, recovery can still be rapid (t1/2 = 2.6 min) if the pH is high but is very slow (t1/2 = 30 min) at pH 6.25. Sites on the external muscle fibre surface thus appear to influence the recovery process. These sites are pH dependent in the range of pH 6.2--7.4 which suggests that they contain a chemical group with a pK in this range.

The effect of glucose loading on steady-state lactate levels and contraction in the isolated rat diaphragm.

Isolated rat diaphragm strips were used to investigate two questions: (a) can the steady-state level of lactate in contracting muscles be increased by glucose loading resulting from the addition of increased external glucose (50 mM) in the presence of insulin (10 mU/mL)? (b) is the isometric tension developed by muscles contracting at different frequencies (0.125 to 1.0 Hz) affected by glucose loading? The results show that lactate levels in contracting muscles are increased by glucose loading over the whole range of contraction frequencies studied. Suppression of isometric contraction increases with contraction frequency but the extent of the suppression is not influenced by glucose loading. Steady-state lactate levels are well correlated with suppression in glucose-loaded muscles (r = 0.89) but not well correlated with suppression in normal muscles (r = 0.46). Isometric tension in the steady-state condition is well correlated with creatine phosphate levels (r = 0.98, 0.92 in glucose-loaded and normal muscles) and reasonably well with ATP (r = 0.88, 0.86, glucose loaded and normal). The increase in resting tension seen during development of steady-state conditions is reduced by glucose loading. It is concluded that several factors may contribute to the suppression of tension in contracting muscle but metabolic product inhibition, at least by products of glycolysis, does not normally play an important part in the isolated rat diaphragm preparation.