D G Allen

The cellular basis of the length-tension relation in cardiac muscle

The relation between muscle length or sarcomere length and developed tension for lengths up to the optimal for contraction (Lmax) is much steeper in cardiac muscle than in skeletal muscle. The steepness of the cardiac length--tension relation arises because the degree of activation of the cardiac myofibrils by calcium increases as muscle length is increased. Two processes contribute to this length-dependence of activation: (i) the calcium sensitivity of the myofibrils increases with muscle length and (ii) the amount of calcium supplied to the myofibrils during systole increases with muscle length. Of these two, the change in calcium sensitivity is the most clearly defined and is responsible for a large part of the rapid change in developed tension when muscle length is altered. It is likely that this change in calcium sensitivity is due to a change in the affinity of troponin for calcium but the underlying mechanism has not been identified. There is good evidence that changes in the calcium supply to the myofibrils can account for the slow changes in tension that follow an alteration in length; there may also be rapid changes in calcium supply but this is less clearly established at present.

Intracellular calcium and tension during fatigue in isolated single muscle fibres from Xenopus laevis.

1. Single muscle fibres were dissected from Xenopus lumbrical muscles and microinjected with the photoprotein aequorin in order to measure the myoplasmic free calcium concentration ([Ca2+]i). Fatigue was produced by repeated intermittent tetanic stimulation continued until tension had declined to approximately 50% of the initial level. Fibres were then allowed to recover by giving tetani at less frequent intervals. Aequorin light (a measure of [Ca2+]i) and tension were measured during fatiguing stimulation and recovery. 2. During fatiguing stimulation, tetanic tension declined steadily, but peak aequorin light first increased before declining substantially. The largest light signal was about 155% of initial control while at the end of fatiguing stimulation the tetanic light fell to about 14% of control. 3. Fibres showed a characteristic slowing of relaxation in the fatigued state. This was associated with a slowing of the rate of decline of the aequorin light signal. 4. Intracellular acidosis produced by equilibrating the Ringer solution with either 5 or 15% CO2 caused an increase in the light signal associated with a tetanus. Carbon dioxide also caused a reduction of tension and a slowing of relaxation. 5. In vivo pCa‐tension curves were constructed by exposing the fibres to a series of K+ concentrations which produced contractures of different sizes. Light and tension were measured during periods when both were relatively stable and the light signal was subsequently converted to pCa. 6. Exposure of fibres to 5 or 15% CO2 caused the pCa‐tension curve to be shifted to the right of the control curve. This indicates a reduced Ca2+ sensitivity of the contractile proteins, which is in agreement with results from skinned fibre studies. 7. The pCa‐tension points obtained from tetani during the early part of fatiguing stimulation also deviated to the right of the control pCa‐tension curve, suggesting a reduced Ca2+ sensitivity of the contractile proteins. At the end of fatiguing stimulation, however, pCa‐tension points did not differ greatly from the control pCa‐tension curve, suggesting that Ca2+ sensitivity was approximately normal. Thus the reduced [Ca2+]i during tetani at the end of fatiguing stimulation (when tension was reduced to approximately 50%) could explain all of the reduction in tension. 8. After fatiguing stimulation, tension and light recovered monotonically in some fibres; however, in the majority of fibres, tension and light showed a secondary decline followed by a slower recovery (post‐contractile depression). 9. During post‐contractile depression, caffeine contractures or tetani in the presence of caffeine gave increased aequorin light signals and the tension developed was close to that produced in an unfatigued tetanus.(ABSTRACT TRUNCATED AT 400 WORDS)

A nuclear magnetic resonance study of metabolism in the ferret heart during hypoxia and inhibition of glycolysis.

31P nuclear magnetic resonance was used to measure the relative concentrations of phosphorus‐containing metabolites in Langendorff‐perfused ferret hearts. Intracellular concentrations of inorganic phosphate ([Pi]i), phosphocreatine ([PCr]i), ATP ([ATP]i) and H+ (pHi) were monitored under control conditions and while oxidative phosphorylation and/or glycolysis were prevented. Mechanical performance was assessed by recording the pressure developed in a balloon placed in the left ventricle. Oxidative phosphorylation was prevented either by replacement of O2 with N2 or by addition of cyanide. When the rate of oxidative phosphorylation was reduced by either method, developed pressure fell to a stable level of about 35% of control after 5 min. The pHi (control value 6.98) first increased to a peak of 7.07 after 2 min but then decreased to give a stable acidosis (pH 6.85). [PCr]i decreased rapidly to about 15% of the control value after 5 min whereas [ATP]i declined very slowly, reaching about 90% of the control value after 10 min. Reduction in the rate of glycolysis was achieved either (i) by removal of external glucose and depletion of glycogen stores by a long (1‐2 h) period of stimulation or (ii) by removal of glucose and application of 2‐deoxyglucose (1 mM) for 30‐60 min. These procedures had only a small effect on pressure development, [ATP]i, [PCr]i and pHi. Measurements of lactate production showed that these procedures reduced the rate of glycolysis by a factor of about 10. When oxidative phosphorylation was prevented during periods when the rate of glycolysis was reduced, developed pressure fell to less than 5% of control after 5 min and there was a subsequent increase in resting pressure (hypoxic contracture). pHi (control value 7.03) first increased to a peak of 7.12 and then declined to about pH 7.00, but there was no subsequent acidosis. [PCr]i fell rapidly to about 10% of control after about 5 min while [ATP]i declined to about half of its control value over 10 min. It is concluded that (i) when oxidative phosphorylation alone is prevented, the changes in pHi can account for a substantial part of the changes in developed pressure. The increase in [Pi]i probably also contributes to the decline of developed pressure. (ii) When oxidative phosphorylation was prevented under conditions in which the rate of glycolysis was also reduced, the more pronounced decline in developed pressure which occurs within 5 min cannot be accounted for by pHi changes and is probably not explained by the rise in [Pi]i or by the moderate fall of [ATP]i.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of acidosis on ventricular muscle from adult and neonatal rats.

We compared the response of ventricular muscle from adult and neonatal rats to hypercapnic acidosis. In adult muscle, acidosis caused an initial rapid fall of developed tension to 30 ± 5 % of control (mean ± SEM, n = 6). However, tension recovered slowly to a steady state that was 56 ± 6% of control. In neonatal muscle, acidosis caused a significantly smaller initial fall in tension to 43 ± 3% (n = 8, p<0.05), but the tension then showed a subsequent slower fall to a steady state that was 29 ± 4% of control, significantly less than in the adult (p<0.01). We have attempted to identify the mechanisms underlying these differences in response. In detergent-skinned myofibrils, reducing the pH from 7.0 to 6.5 caused a reduction in the pCa50 of 0.61 units in the adult muscle, but only 0.27 units in the neonatal ventricular muscle. Myofibrillar Ca2+ sensitivity in neonatal ventricular muscle is thus less susceptible to the effects of acidic pH than that of adult muscle. Since intracellular pH decreases rapidly on application of increased external CO2, these results are consistent with the finding that, initially, developed tension in neonatal muscles is less sensitive to the effects of acidosis. Sodium dodecylsulfate gel electrophoresis of myofibrillar preparations from adult and neonatal rats demonstrated differences in thin filament proteins, including troponin I, which may underlie the observed differences in Ca2+ sensitivity. In adult rat ventricular muscles, the slow recovery of tension during acidosis is associated with an increase in the amplitude of the Ca2+ transients to 263 ± 34% of control (n = 4). In neonatal ventricular muscle, however, the Ca2+ transients decreased to 91 ± 3% of control during acidosis (n = 3). This difference in Ca2+ handling probably contributes to the difference in the slow mechanical response and could be related to the smaller amount of sarcoplasmic reticulum in neonatal muscle. Our results thus indicate that the difference in the initial rapid effect of acidosis on neonatal and adult rat heart can be explained by the effects of acidosis on the myofibrils, whereas the difference in the later, slower changes may be caused by differences in Ca2+ transport.

The effects of shortening on myoplasmic calcium concentration and on the action potential in mammalian ventricular muscle.

When cardiac muscle shortens during a contraction, the duration of mechanical activity is abbreviated (shortening deactivation), but the duration of the action potential is prolonged. Neither of these phenomena is fully understood, but both may be related to changes in the myoplasmic free calcium concentration. In these experiments, isolated papillary muscles from cats and ferrets were allowed to contract under various mechanical conditions while myoplasmic calcium was monitored with aequorin, or in parallel experiments the membrane potential was recorded with microelectrodes or a sucrose gap. When shortening occurred, myoplasmic calcium was increased and the membrane potential was more positive than in isometric contractions. The changes in calcium apparently precede the depolarization. We propose that muscle shortening reduces calcium binding to the contractile proteins and leads to a rise in myoplasmic calcium, and that this rise in myoplasmic calcium activates an inward current leading to the observed changes in the action potential. These processes may be important contributory factors in some arrhythmias.

Spatial gradients of intracellular calcium in skeletal muscle during fatigue

We have measured the distribution of intracellular calcium concentration in isolated single muscle fibres fromXenopus laevis using the fluorescent calcium indicator fura-2 with digital imaging fluorescence microscopy. Under control conditions, resting and tetanic calcium were uniform throughout a fibre. When fatigue was produced using a prolonged, high-frequency tetanus, the distribution of calcium within muscle fibres became non-uniform, with greater levels near the outer parts of a fibre than near the centre. This non-uniform distribution of calcium was rapidly abolished by lowering the stimulation frequency. When fatigue was produced using a series of repeated intermittent tetani, tetanic calcium showed an initial small increase, followed by a decrease as stimulation was continued. The distribution of calcium remained uniform under these conditions. Calcium distribution was also uniform during recovery from intermittent tetanic stimulation. Although fibres varied considerably in their fatigue resistance, the time for tension to fall to 50% was correlated with the reduction in tetanic calcium seen at this time. These results indicate that there are at least two patterns of reduced calcium release that can contribute to the development of fatigue. The appearance of a calcium gradient is consistent with impaired t-tubular conduction, while a uniform reduction of calcium is likely to be due to the action of metabolic factors on systems controlling calcium homeostasis within the cell.

Characterization of oscillations of intracellular calcium concentration in ferret ventricular muscle.

The photoprotein aequorin was injected into superficial cells of ferret papillary muscles. Tension and aequorin light (a function of intracellular [Ca2+]) were monitored. Increasing intracellular Ca concentration ([Ca2+]i), either by decreasing extracellular Na, or by inhibiting the Na pump with strophanthidin, produced spontaneous oscillations of [Ca2+]i and tension. Fourier analysis showed that these oscillations had frequencies of up to 3‐4 Hz. If the muscle was stimulated in these conditions the Ca transient associated with the twitch was followed by a series of damped oscillations of [Ca2+]i which were accompanied by after‐contractions. Under a given set of conditions the frequency of the stimulated oscillations was similar to that of the spontaneous oscillations. Manoeuvres which increase [Ca2+]i increased the frequency of both spontaneous and stimulated oscillations. Drugs which inhibit the function of the sarcoplasmic reticulum (caffeine and ryanodine) abolished both stimulated and spontaneous oscillations. The spontaneous oscillations during a Na‐free contracture were unaffected by the Ca channel blocker D‐600. When repetitive stimulation was begun the frequency and magnitude of the stimulated oscillations increased over several minutes. Increasing the frequency of stimulation increased the magnitude of the stimulated oscillations. It is concluded that the spontaneous oscillations of [Ca2+]i may be due to oscillatory Ca release from the sarcoplasmic reticulum. The similar properties of the spontaneous and stimulated oscillations suggest that the latter may be due to a synchronization of the former.

The effects of low sodium solutions on intracellular calcium concentration and tension in ferret ventricular muscle.

Papillary muscles from the right ventricles of ferrets were micro‐injected with the photoprotein aequorin. Both tension and the light emitted by the aequorin, which is a measure of the free intracellular Ca concentration [( Ca2+]i), were monitored. Exposure of the papillary muscle to a solution in which all the Na had been replaced by K (0 Na(K) solution) resulted in an increase in tension which subsequently slowly decreased. This contracture was associated with a large increase in [Ca2+]i followed by a decrease to a steady‐state‐level which was often significantly greater than that in Na‐containing solutions. If choline, Li or Tris was used instead of K as a substitute for Na, both the contracture and the associated increase of [Ca2+]i were reduced. The effects of depolarization alone (by raising external K at constant Na concentration) were compared with those of Na removal alone (at constant external K concentration). Na removal contributes more than depolarization to the effects of a Na‐free, K‐containing solution on the contracture and rise of [Ca2+]i. Increasing intracellular Na concentration [( Na+]i), by exposure to strophanthidin (10 mumol/l), increased the magnitude of both the contracture and [Ca2+]i in 0 Na(K) solutions. Conversely, decreasing [Na+]i by exposure to a solution containing a decreased extracellular Na concentration [( Na+]o), decreased the contracture and [Ca2+]i. When contractures were produced by solutions with various [Na+]o, the size of the resulting contracture and [Ca2+]i were inversely related to [Na+]o. No contracture was seen unless [Na+]o was reduced to below 70 mmol/l. A decrease in the extracellular Ca concentration [( Ca2+]o) from 2 to 0.5 mmol/l or an increase to 8 mmol/l produced, respectively, large decreases and increases of the twitch and accompanying Ca transient. However, if [Ca2+]o was changed at the same time as Na was replaced by K there was little effect on either the contracture or the rise of [Ca2+]i. If [Ca2+]o was changed before replacing Na by K then increasing [Ca2+]o from 2 to 8 mmol/l decreased, and decreasing [Ca2+]o from 2 to 0.5 mmol/l increased, the rise of [Ca2+]i produced by replacing Na by K. The difference between this result and that obtained when [Ca2+]o was changed at the same time as Na was removed may be due to changes of [Na+]i produced by prolonged exposure to an altered [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous measurements of action potential duration and intracellular ATP in isolated ferret hearts exposed to cyanide.

Shortening of the cardiac action potential during ischemia and anoxia is likely to contribute to the decline in contractility that occurs under such conditions. It has been hypothesized that a decrease in the intracellular ATP concentration ([ATP]1) underlies the changes in the action potential. The recently discovered potassium channel activated at low ATP concentrations might provide the link between action potential shortening and low [ATP]1. However, it has yet to be shown that [ATP]1 falls to the range required for channel activation at the time when action potential shortening occurs. We have measured action potentials and [ATP]1 simultaneously in isolated ferret hearts during ininbition of both oxidative phosphorylation and anaerobic glycolysis (metabolic blockade). Metabolic blockade caused a rapid decline in cardiac contractility, accompanied by a rapid fall in action potential duration. [ATP]1 fell only slightly and remained well above the range where activation of the ATP-sensitive K+ channel would be expected to occur. Moreover, rentroduction of glucose to the perfusate led to a substantial recovery in both contraction and in action potential duration, again in the absence of any great change in [ATP]1. These results suggest that the action potential shortening observed in metabolic blockade cannot be explained by the simple hypothesis of K+ channel opening as a consequence of a decrease in bulk [ATP]1 unless the K± for suppression of channel activity by ATP is very much ingher in intact cells than in any of the patch configurations studied. An alternative explanation is that the channel may be regulated under these conditions by mechanisms other than a change in [ATP]1.

Changes in tetanic and resting [Ca2+i during fatigue and recovery of single muscle fibres from Xenopus laevis

1. Single muscle fibres were dissected from the toe muscles of Xenopus laevis and microinjected with Fura‐2 to measure myoplasmic calcium concentration ([Ca2+]i). Injected fibres were illuminated at 340 and 380 nm and the ratio of the resulting fluorescence at 505 nm (the Fura‐2 ratio) was taken as a measure of [Ca2+]i. Fibres were fatigued at 21 degrees C by repeated tetani until developed tension had fallen to 50% of control. 2. Tetanic tension declined monotonically during fatiguing stimulation, whereas the tetanic Fura‐2 ratio first increased and then declined. At the 10th tetanus, tension was 87% of control whereas the Fura‐2 ratio was 106% of control. At the end of fatiguing stimulation, where tension was around 50% of control, the tetanic Fura‐2 ratio was reduced to 71%. The rate of decline of both tension and the Fura‐2 ratio after a tetanus slowed during fatigue. During recovery, the tension and the tetanic Fura‐2 ratio recovered in parallel. 3. The resting Fura‐2 ratio increased throughout fatigue reaching 237% of control when tension had declined to 50%. There was a rapid phase of recovery, complete within 1 min, by which time the resting Fura‐2 ratio was 198% of control. Subsequent recovery was slower and took 20‐30 min to reach a stable level which was 121% of control. 4. The resting Fura‐2 ratio towards the end of fatiguing stimulation was greater than the tetanic Fura‐2 ratio in the early part of recovery although there was no detectable increase of resting tension during fatiguing stimulation. This observation suggests that the Ca2+ sensitivity of the contractile proteins was reduced at the end of fatiguing stimulation. 5. Plots of the tetanic tension against tetanic Fura‐2 ratios throughout fatiguing stimulation and recovery also suggested that Ca2+ sensitivity was reduced during fatiguing stimulation when compared to recovery. 6. The increases in resting [Ca2+]i caused by raised [K+]o (from 2.5 to 10 mM) and/or by application of 15% CO2 were much less than those produced by fatiguing stimulation. Much of the elevated [Ca2+]i in fatigue could be reversed by application of dantrolene (25 microM). 7. The results suggest that both reduced tetanic [Ca2+]i and reduced Ca2+ sensitivity contribute to the decline of tension during fatigue.(ABSTRACT TRUNCATED AT 400 WORDS)