SUMMARY1. The influence of the interstimulus interval on twitch duration was analysed in isolated heart muscle of the rat. When the muscle was in the steady state at interstimulus intervals at 5 s a test interval was interposed and varied. Duration of twitch and action potential, sarcomere length and peak force of the test beats were measured.2. Twitch force and duration increased when the test interval was increased from 0 4 to 10 s. This effect was abolished by inhibitors of sarcoplasmic reticulum function (ryanodine, caffeine, Sr2"). Hence, the interval dependence is controlled by the sarcoplasmic reticulum. 4. Action potential duration was much shorter than twitch duration and, depending on the intervention, changes were in the same or in opposite direction. Hence, the action potential did not determine twitch duration.5. Small variations in sarcomere length amongst test contractions were observed, but these variations could not account for the effects of the test interval.6. It is proposed that the Ca2+ pump in the sarcoplasmic reticulum is activated during each contraction and inactivates slowly. Thus, after a short interval the pump is still activated and rapidly sequesters much of the released Ca2+ leading to a small twitch and rapid relaxation. This mechanism ensures proper relaxation and diastolic filling of the ventricle. The biochemical basis and implications of the hypothesis are discussed.

SUMMARY1. Intracellular action potentials and isometric force were measured from thin trabeculae of the right ventricle of rat heart. Characteristic for the action potential of rat myocardium is a short plateau and a slow final repolarization phase. We have studied the influence of ionic composition ofthe medium and of stimulation frequency on the slow phase of repolarization and its relation to peak force.2. The results confirmed a positive correlation between peak force and the duration of the slow phase of repolarization, as has been reported for other species.3. An increase of [Ca2+]. caused a shortening of the slow phase of repolarization when peak force was kept constant. 5. In the presence of the Ca2+ entry blocker nifedipine the action potential duration and peak force were reduced. Low [Na+]. caused less shortening of the slow phase of repolarization and a greater increase of peak force. The slow phase of repolarization was prolonged transiently following reperfusion at normal [Na+]O, but only during a few beats.6. These results are in agreement with the hypothesis that the slow phase of repolarization is due to an inward current generated by Na+-Ca2+ exchange, as the latter mechanism is known to be sensitive to the intracellular and extracellular concentrations of both Na+ and Ca2+.

Peak force and membrane potential were recorded from papillary muscles and trabeculae excised from the ventricles of adult rat hearts. Experiments were performed at 2.5 mM Ca2+ and 26 degrees C. In thick preparations (diameter 0.2-1.2 mm) an increase of stimulation frequency caused a reduction of peak force and action potential duration as has been found in many studies previously. In thin preparations (diameter less than 0.2 mm) both peak force and action potential duration were almost independent of stimulation frequency. When the flow of Tyrode solution through the muscle bath was reduced an increase of stimulation frequency caused a reduction of peak force and action potential duration in thin preparations. We conclude that the reduced peak force and action potential duration in papillary muscles at high stimulation frequencies is due to insufficient exchange of metabolites and oxygen between the medium and the core of the muscle. The results indicate that the critical diameter for the preparations is about 0.2 mm.

SUMMARY1. Increased coronary perfusion leads to increased myocardial contraction and oxygen consumption (Gregg's phenomenon) even when oxygen supply is presumably sufficient. Previous studies concerned whole hearts, however, in which local hypoxia may play a role. We developed techniques for internal perfusion of thin papillary muscles from rat heart. The influence of perfusion pressure on muscle contraction was studied. We investigated whether Gregg's phenomenon is due to (a) hypoxia, (b) stretch of the muscle fibres, or (c) increased contractility.2. The effectiveness of the perfusion technique was demonstrated in four ways: (a) the diameter of the capillaries increased with perfusion pressure; (b) 14 + 4 % (mean+ S.D., n = 11) increase in muscle diameter was observed on a change of perfusion pressure from 0 to 50 cmH2O; (c) addition of India ink to the perfusate caused rapid staining of the entire muscle; (d) during internal perfusion and external superfusion peak force was mainly determined by the [Ca2+1 in the internal perfusate.3. An increase of perfusion pressure from 0 to 70 cmH2O induced 74+20 % (mean + S.D., n = 11) increase in peak force of contraction. In the absence of internal perfusion peak force was not affected by approximately 50 % reduction of the Po2 in the bathing solution (from 700 to 350 mmHg). Hence, oxygen supply was not a limiting factor, i.e. the effect of internal perfusion on force was not related to hypoxia.4. Segment length was measured with markers attached to the surface of the muscle. Perfusion-induced changes in segment length were negligible (-0-2 + 1-5 %, n = 11). Force-length relationships at different perfusion pressures show that the perfusion-induced increase in force was generally larger than the maximum increase in force that could be induced by stretch. Furthermore, the time course of stretch and perfusion effects on force was different. We conclude that Gregg's phenomenon is not related to changes in fibre length, i.e. the hypothesis of pressure-induced stretch ('garden hose' effect) does not apply to papillary muscles.5. The pressure-induced changes in the force-length relationship were similar to the changes obtained with interventions that increase contractility, such as increased [Ca21].* To whom reprint requests should be sent.MS 9434 586 V. J. A. SCHOUTEN, C. P. ALLAART AND N. WESTERHOF 6. Since hypoxia and length effects were not involved, and the effect of perfusion pressure was similar to that of inotropic interventions, we conclude that Gregg's phenomenon is a change in contractility. Possible explanations include changes in the ionic composition or volume of the interstitium, and inotropic factors produced by the endothelium or intramyocardial neurons.

The whole-cell patch-clamp technique was used to study the effects of holding potential and frequency on the Ca2+ current in frog ventricular myocytes. INa was blocked by TTX, and ica was activated with depolarizing clamps from different holding potentials. Variation of the holding potential revealed three new effects on ica: (1) At -40 mV iCa declined with a time constant of 15 min, while at -90 mV, this irreversible decline (run down) in iCa did not occur. (2) The decline of iCa at -40 mV was biphasic: run down was preceeded by a slow inactivation with a time constant of 40 s, which was reversible upon returning the holding potential to -90 mV. (3) Increasing the frequency of the clamp pulses from 0.1 to 1 Hz led to a rapid decline of iCa when the holding potential was positive to -60 mV, but at -90 mV had either no effect or increased iCa by 35%, if c-AMP was included in the dialyzing solution. On the other hand, c-AMP did not alter the time course of the run down and the slow inactivation. Replacement of extracellular Ca2+ by Ba2+ markedly slowed iCa kinetics, but did not change the very slow inactivation or the frequency-induced enhancement of iCa. Injection of c-AMP led to a transient increase of iCa. The phosphodiesterase inhibitor theophylline enhanced the amplitude of the transient and slowed its decay. This effect was mimicked by increased frequency. It is concluded that frequency-induced enhancement of iCa is highly dependent on the holding potential, independent of Ca2+, and may involve elevation of the intracellular level of c-AMP via inhibition of phosphodiesterase activity. The new type of very slow inactivation is probably under direct voltage control and independent of Ca2+ and c-AMP.