Rat heart papillary muscles: action potentials and mechanical response to paired stimuli.

Electrical reexcitation of rat papillary muscle after a short interval (50 to 80 milliseconds) results in action potentials with no significant mechanical counterpart. The mechanical response recovers as the interval is increased beyond 80 milliseconds. The rate of recovery is slowed at low external calcium levels. It appears that the coupling mechanism passes through a refractory stage owing to the depletion of an intracellular "releasable calcium" fraction.

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.

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 relationship between surface potentials and the number of active motor units.

One of the assumptions inherent in a technique recently devised for enumerating motor units in human muscles is that the surface potentials from active motor units summate in a linear fashion. We present an electrical model of a muscle which predicts that a linear relationship between the number of active units and the electrical response recorded at the surface overlying the muscle would not be expected. The extent of the non-linearity, and hence the error in the calculation of the number of motor units in a given muscle, depends upon the ratio between the mean conductance of the motor units themselves and that of the external conduction pathway through which the electrical signal is fed (Gu/Ge). The extent of non-linearity is assessed experimentally in human hypothenar muscles using a "collision" technique. The average underestimate introduced into the calculation of the number of motor units in this particular case was concluded to be 26%. The value of Gu/Ge derived from these experiments, in 2 subjects, was checked by simulating an intramuscular action potential and determining the attenuation at the surface. The 2 independently obtained values were sufficiently close to suggest that the model may be a valid one. We conclude that caution should be employed in the interpretation of experiments which purport to determine the number of motor units in a muscle by means of surface recordings.

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.

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.

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.

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.

The effect of bicarbonate concentration on fatigue and recovery in isolated rat diaphragm muscle.

Diaphragm strips from young rats (45--60 g) about 0.3 mm thick were fatigued by tetanic stimulation at a train repetition rate of 2 HZ for 3 min. The isometric tension developed was measured during fatigue and recovery in solutions containing 25, 10, or 2 mM bicarbonate at both 37 and 30 degrees C. Tension fell during fatigue to between 20 and 30% of the initial value and this was not significantly influenced by external bicarbonate concentration or temperature over the range considered. Recovery of tension was complete and rapid (t1/2 < 1 min) in 25 mM bicarbonate at both temperatures. In 10 and 2 mM bicarbonate recovery was slowed (t1/2 3.5 and 7 min, respectively, at 30 degrees C, 1.6 and 4.5 min at 37 degrees C) and incomplete (85 and 72% at 30 degrees C, 82 and 61% at 37 degrees C). Muscle creatine phosphate fell during fatigue but was completely restored within 4 min at 30 degrees C in either 2 or 25 mM HCO3. Lactate increased less in muscles fatigued in 2 mM HCO3 and fell at a slower rate during recovery. The results seem to exclude intracellular creatine phosphate concentration as a major determinant in recovery. The evidence suggests that external bicarbonate can affect the recovery of tension following fatigue by altering intracellular acid-base balance.

The effects of extracellular pH and buffer concentration on the efflux of lactate from frog sartorius muscle.

1. The rate of efflux of lactate from isolated frog sartorius muscles is measured with a superfusion technique. Efflux curves are followed after raising the internal lactate level of the muscles by repetitive electrical stimulation over a 200 sec period.2. With an external pH of 7.0 or below the measured efflux rates following stimulation reach 100-150 n-mole/g.min. Increasing the pH of the superfusion fluid to 8.0 results in a two or threefold increase in the peak efflux rate. The effect is independent of the buffer system used and occurs fairly rapidly when the pH of the superfusion fluid is changed. This suggests that the effect of pH on lactate efflux is extracellular.3. The increase in efflux rate due to an increase in pH is dependent on buffer concentration. This fact together with measurements of surface pH changes in muscles following arrest of superfusion indicates that a pH gradient exists through the muscle thickness during lactate efflux.4. The low lactate efflux rate seen at a low buffer concentration (1 mM) is reduced to an even lower level by depolarization with potassium sulphate suggesting a membrane potential dependent component. At pH 8.0 with a high buffer concentration (25 mM) potassium sulphate only reduces efflux rate slightly.The observations are interpreted as indicating that a fraction of lactate lost is in the form of undissociated acid and that this fraction increases with increasing external pH.5. Conditions which favour loss of hydrogen ions and lactate from muscle are also associated with improved recovery of twitch tension.

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.

A model for intracellular energy transport.

A model for oxygen transfer to cells from capillaries is considered in which mitochondria are either clustered at the cell periphery around capillaries or homogeneously distributed through the cytosol. The capillary Po2 required to supply cells utilizing oxygen at the same rate is much less when mitochondria cluster around capillaries. Two alternative mechanisms are considered for distributing energy from peripheral mitochondria to the rest of the cell; i.e., diffusion of ATP or creatine phosphate with enough creatine kinase to ensure equilibrium between the approximately P carriers. The latter has clear advantages and would appear to be adequate to supply a fairly large mitochondria-free cell core (e.g., 24-micrometer diameter) with very little change in ADP levels or in the free energy of ATP hydrolysis at maximum work rates. Thus, a viable alternative to the traditional Krogh model is presented which takes into account the inhomogeneity of the diffusion pathway as a result of mitochondrial clustering.

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.

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.

A square-pulse flow method for measuring characteristics of the arterial bed.

A device was designed to provide a "square" pulse of blood flow into the arterial system. Pulses were injected into the carotid artery of the rabbit during transient cardiac arrest. Analysis of pressure response curves generated by the flow provides information as to the state of the arterial tree. With certain assumptions it is possible to estimate from these curves lumped values of peripheral resistance, critical closing pressure, and arterial compliance. In a series of 12 rabbits the mean value of peripheral resistance was found to be 0.21 +/- 0.7 mmHg-ml-1-min and critical closing pressure was estimated to be 23.6 +/- 3.8 mmHg. This method gives two possible values for arterial compliance 0.036 +/- 0.010 and 0.055 +/- 0.010 ml-mm-1 based, respectively, on the rise and decay curves of the pressure response. The theory and limitations of the method are discussed. The use of the method is illustrated in following the response to increased PCO2 and hemorrhage.

Carbon dioxide and acid base balance in the isolated rat diaphragm.

A method for measuring the net acid base exchange in an isolated rat diaphragm preparation is described. Particular attention is paid to monitoring the functional status and maintaining optimal diffusion conditions. A steady net acid efflux of the order of 250 n mole/g-min is found in the resting state. This increases following a series of isometric contractions. In the resting state the total measured lactate + pyruvate efflux was found to be less than the net acid efflux. The net acid efflux increases following a sudden decrease in pCO2 and decreases or reverses following a sudden increase in pCO2 or a decrease in external bicarbonate. The net base loss during a period of 1 h following the exposure to high (20%) CO2 represents a large fraction of the predicted total bicarbonate generated within the fibres by non-bicarbonate buffers. This indicates that the effects of intracellular non-bicarbonate buffers can be transmitted to the external solution following a change in pCO2. The most plausible explanation is that passive bicarbonate ion movements are responsible. Values of the 'apparent PHCO3' have been calculated and vary under different conditions from a value of 1.3 X 10(-7) to 1.9 X 10(-6) cm-s-1.

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)

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.