The effect of acidosis on the relationship between Ca2+ and force in isolated ferret cardiac muscle.

1. The relationship between force and intracellular [Ca2+] (monitored using the protein aequorin) has been investigated in papillary muscles isolated from ferret hearts, under control conditions (superfusate pH (pHo) 7.3) and during acidosis (pHo 6.8). 2. At pHo 7.3, increasing bathing [Ca2+] from 0.5 mmol l-1 to 8 mmol l-1 led to an increase in the size of the intracellular calcium transient. At the lower [Ca2+] this was accompanied by an increase in developed force; however, at the higher bathing [Ca2+] developed force reached a plateau. 3. Acidosis (produced by increasing the [CO2] of the gas with which the muscle superfusate was equilibrated) decreased maximum force and shifted the curve relating peak developed force to peak intracellular [Ca2+] to the right. 4. The mechanisms underlying the apparent decrease in the sensitivity of the contractile proteins to Ca2+ were investigated by applying rapid length changes to papillary muscles at control pHo, during acidosis, and after bathing [Ca2+] had been increased to match force during acidosis to that in control. 5. Acidosis decreased the change in force produced in response to a given length change (i.e. decreased muscle stiffness) but when bathing [Ca2+] was increased during acidosis, muscle stiffness returned to control. 6. Acidosis had no effect on muscle stiffness after the induction of rigor in the muscle (produced by metabolic inhibition). 7. It is suggested that in intact cardiac muscle the major effect of a mild acidosis is to decrease the sensitivity of the contractile proteins to Ca2+, hence decreasing the number of bound cross-bridges.

An improved apparatus for the optical recording of contraction of single heart cells.

An optical system for measuring changes in cell length during unloaded contractions of cardiac myocytes is described. A one-dimensional video "image" of a cell is obtained every 4 ms with a linear photodiode array, which is aligned with the longitudinal axis of the cell. The circuit used to process the image from the photodiode array has a variety of features to aid in the accurate determination of the distance between the ends of the cell, i.e. the cell length. First, the video image of the cell is divided into two "windows", one encompassing the "front" edge of the cell, the other encompassing the "rear" edge. Other cells or debris beyond the cell edges are excluded. Changes in the general light level, for example as a result of debris floating above the cell, have little effect because within the windows the "background light level" is subtracted from the signals before they are processed further. To detect the cell edges, the system determines when the signals within the windows exceed (front edge) or drop below (rear edge) chosen thresholds, which are different for the front and rear edges. The system has "memory" and it identifies the rear edge of the cell as the last time the signal falls below the threshold; because of this "bright spots" within the cell are not mistaken for the end of the cell. The system has "hysteresis", which enables it to ignore small fluctuations in brightness around the threshold. The system is easy to use, accurate, readily calibrated, and it has good spatial and time resolution (about 0.25 micron and 4 ms respectively).

The Anrep effect: an intrinsic myocardial mechanism.

In cat papillary muscles contracting physiologically, increasing the afterload caused a biphasic change in contractility. In response to an increase in afterload, contractility (as measured by peak shortening, peak developed force, or peak dF/dt) initially decreased (antihomeometric autoregulation) over the first few beats and then increased slowly with t 1/2 of about 3 min at 30 degrees C and about 1 min at 37 degrees C (homeometric autoregulation). The antihomeometric autoregulation is due to decreased active shortening when the afterload is increased, since it also occurs in response to increased afterload in isotonic contractions. The secondary slow increase in contractility is primarily due to the increase in mean diastolic length that occurs as a result of increased afterload. The time course and the magnitude of the biphasic change in contractility are very similar to those observed in response to afterload increase in intact hearts; we suggest that the secondary slow increase in contractility that we observed is a contributory mechanism to homeometric autoregulation (or the Anrep effect), as it is observed in the whole heart.

Effects of inotropic interventions on end systolic length-force curve of cat ventricular muscle.

A computer controlled servosystem was used to generate physiological contractions in which cat papillary muscles went through a cycle of force and length changes similar to that undergone by muscle fibres in the heart during systole and diastole, net external work being done during each cardiac cycle. End systolic length-force curves were obtained by varying the setpoint force against which the muscle shortened. The end systolic length-force curve was very similar to the isometric length-force curve obtained under comparable experimental conditions. It was altered in the same way by inotropic interventions (increase in stimulus frequency, increasing the bathing calcium concentration, and exposure to isoprenaline), each of which increased the slope of the curve and decreased its (extrapolated) intercept on the length axis. As these results were obtained from studies of muscles producing physiological contractions at 37 degrees C, they provide evidence for length dependence of activation in the working myocardium.

Effects of physiological beating on the contractility of cat ventricular muscle.

Force and length changes corresponding to those undergone by muscle fibers in the ventricular wall of the heart during systole and diastole were generated in isolated cat ventricular muscles contracting at 30 degrees C. The effect on contractility of such cycles of force and length changes ("physiological" contractions) was assessed by measuring force production in isometric test beats. Contractility rose over the 1st min of physiological beating but then fell over the next 7-10 min. These changes in contractility were compared with those occurring during a series of isotonic contractions or modified isotonic contractions in which muscles were held at the short length during relaxation (reversed relaxation contractions). In all cases contractility increased to the same extent over the 1st min, but over the next 10 min it fell significantly more when the contractions were physiological than when they were isotonic (normal or reversed relaxation). This difference could be accounted for experimentally by the diastolic length changes that occur in physiological contractions. This study demonstrates 1) that contractility depends on length changes occurring in the muscle during both systole and diastole, and 2) that length-dependent changes in contractility contain both positive and negative components.

Some characteristics of Ca2+- regulated force production in EGTA-treated muscles from rat heart.

McClellan and Winegrad (1980, J. Gen. Physiol., 75:283-295) have reported that in rat ventricular muscles that have reportedly been made "hyperpermeable" to small ions such as Ca2+, CaEGTA2-, and MgATP2- by a soak in EGTA, the maximum Ca2+-regulated force can be permanently increased by a short exposure to positively inotropic drugs, such as epinephrine or cAMP plus theophylline, in the presence of the detergent Triton X-100. The experiments reported here were begun as an attempt to repeat and extend this important observation. However, no evidence could be found for a potentiation of force that was not merely produced by Triton alone. In addition, the thickest muscles used (250-440 microns diameter) exhibited very low values for force per unit cross-sectional area, which suggested that either Ca2+ reached only a fraction of the myofibrils or the myofibrils were in a state of low contractility. The results of further experiments that were designed to test the permeability characteristics of these EGTA-treated muscles indicated that the movement of certain ions into these preparations was restricted, even in thin muscles (80-200 microns diameter). The rate of development of Ca2+-regulated force was slow (t1/2 approximately equal to 1-3 min), but was greatly accelerated after the muscles had been superfused with Triton X-100 (t1/2 approximately equal to 10-20 s). Removal of creatine phosphate (CP) in the presence of MgATP produced a partial rigor contracture in the EGTA-treated muscles. The results were consistent with the suggestion that the EGTA-treated muscles were permeable to some extent to Ca2+ and HCP2- ions but not to CaEGTA2- and MgATP2-. Thus, it would seem unlikely that the [Ca2+], [MgATP2-], and [Mg2+] in the immediate vicinity of the myofibrils in these preparations can be adequately controlled by the solution bathing the muscles.

Calcium- and length-dependent force production in rat ventricular muscle.

1. Trabeculae from the right ventricles of rat hearts were ;skinned' by immersion for 30 min in a solution containing the non-ionic detergent Brij-58 at a concentration of 1%.2. The average sarcomere length in the central region of the relaxed preparation was estimated by laser diffraction and set at pre-determined values within the range of 1.9-2.4 mum by adjustment of muscle length. Isometric contractions were then induced by raising the Ca(2+) concentration under carefully controlled chemical conditions.3. The dependence of Ca(2+)-activated force production on sarcomere length over the ascending limb of the length-force relation was examined at Ca(2+) concentrations giving partial and full activation of the contractile system of the muscle.4. The dependence of Ca(2+)-activated force on Ca(2+) concentration was compared at sarcomere lengths on the ascending limb and plateau of the length-force relation.5. The results obtained from both kinds of experiment showed that the sensitivity of the contractile system to Ca(2+) increases with sarcomere length over the ascending limb of the length-force relation.6. Possible explanations for this observation have been discussed.

Activation of contraction in cardiac muscle.

This paper begins with consideration of a qualitative model of excitation-contraction coupling in cardiac muscle which is based to a large extent on experimental work carried out on isolated preparations of cardiac muscle by Earl Wood during sabbatical years in Europe in 1966-67 and 1972-73. It goes on to consider newer evidence about excitation-contraction coupling that has been obtained from studies of "skinned" fiber preparations (that is, muscles in which the cell membranes have been disrupted or are absent) and from studied of isolated preparations in which the photoprotein aequorin was used to monitor changes in sarcoplasmic Ca2+ concentration. The results obtained from these two kinds of study are then combined in a qualitative way to illustrate current ideas about the mode of action of inotropic interventions and to provide possible explanations of the Frank-Starling effect as it appears in isolated preparations of cardiac muscle.

A study of the factors responsible for rate-dependent shortening of the action potential in mammalian ventricular muscle.

1. An intracellular micro-electrode was used to record action potentials from superficial cells of a cat papillary muscle during isometric contractions. The muscle was stimulated regularly and test stimuli were interpolated at various times between regular (control) responses. 2. The duration of test action potentials (measured at 80% repolarization) increases exponentially with time as the interval between the test stimulus and the preceeding stimulus is increased and a curve drawn through the data reaches a plateau at test intervals of 1.0-1.5 s. This curve is considered to reflect the time course with which membrane conductances return to their pre-stimulus values after a control response, and it is known as the 'electrical restitution curve'. 3. At much longer test intervals the action potential duration duration increases again and it approaches the rested state value of about 0.5 s when the interval between stimuli is 200-300 s. 4. Interventions that raise the peak tension developed in isometric contractions, such as an increase in the rate of stimulation or in the bathing calcium concentration, displace the electrical restitution curve downwards (to shorter action potential durations) and to the left (to shorter stimulus intervals). This shift in the curve is accompanied by a reduction in its magnitude (i.e. the difference in duration between the earliest possible action potential and the plateau value), but the interventions differ in their effects on the time course of electrical restitution: an increase in stimulus frequency causes a marked slowing, whereas an increase in bathing calcium concentration produces a slight speeding up of its time course. 5. The reduction in action potential duration produced by an increase in stimulus frequency (rate-dependent shortening) can be separated into two components, one resulting from the downward displacement of the electrical restitution curve and the other depending on the time available between consecutive responses for membrane recovery. The second component becomes increasingly important at stimulus frequencies above 100 min-1. 6. Changes in action potential duration observed during the tension staircases produced by regular stimulation of a rested preparation and by paired pulse stimulation can also be accounted for by interaction of downward displacement of the electrical restitution curve and variations in the degree of recovery of the membrane between consecutive responses. 7. Downward displacement of the electrical restitution curve is thought to result from intracellular accumulation of calcium and/or extracellular accumulation of potassium, and the available evidence is considered to favour the former mechanism.

Length-dependent activation: its effect on the length-tension relation in cat ventricular muscle.

In cat papillary muscles at 30 degrees C, bathed with Tyrode's solution containing 2.25 mM Ca2+, the effect of various inotropic interventions (varying the stimulus frequency and continual paired stimulation) on the shape of the steady state length-tension relation was examined at lengths from Lmax, where tension production is maximal, to 0.87 Lmax. The relative steepness of the length-tension curves for peak tension developed (DT) and for maximum rate of tension development (dT/dT) varied inversely with the degree of potentiation. Thus, during paired pulse stimulation the relative decline in DT and dT/dT for a given change in muscle length was significantly less than the decline observed during stimulation at 5 min-1. When a muscle was stretched DT did not reach its final steady level for several minutes, and this slow increase in DT contributed significantly to the steepness of the steady state length-tension relation. The half-time of the slow increase in DT exhibited beat-dependency, and conditions that reduce the transsarcolemmal influx of calcium (reduction in bathing [Ca2+] or the presence of verapamil) significantly prolonged the time course of the slow increase and reduced its magnitude. These results support the hypothesis (1) that there is length-dependence of the excitation-contraction coupling process, such that an increase in muscle length is accompanied by greater activation of the contractile system; and (2) that this is due at least in part to an increased influx of calcium into the muscle cells. The implication of this hypothesis is that the influence of muscle length on myocardial performance (the Frank-Starling relation) should not be regarded as fundamentally different in character from other inotropic interventions.

Studies of the contractility of mammalian myocardium at low rates of stimulation.

1. Measurements have been made of tension development in papillary muscles isolated from the right ventricles of young cats. In some cases membrane potentials have also been recorded, using micro electrodes. 2. Regular contractions at a stimulation rate of 20 min(-1) (the 'standard' rate used in this study) had the following characteristics (30 degrees C): peak tension developed, about 43mN mm(-2); time to peak tension and time to 80% repolarization of the cell membrane, about 400 ms. 3. The corresponding figures for the first contraction after a rest of several minutes (rested state contraction) were: tension developed, about 4mN mm(-2); time to peak tension and time to 80% repolarization of the cell membrane, about 560 ms. Sometimes there was also an early peak in the mechanical response, about 250 ms after stimulation. 4. The time course with which tension development declined when the muscle was allowed to rest was examined under various conditions. It was found to decline more slowly when the muscle was potentiated by raising the bathing Ca2+ concentration and by stimulation at rates above 20 min(-1). 5. Tension development in rested state contractions was found to depend on the Ca2+ and Na+ concentrations in the bathing solution. The full effect of a change in either could be produced by exposing the resting muscle to the altered ionic conditions. 6. These experimental findings have been interpreted in terms of a simple model of the calcium movements involved in excitation-contraction coupling in the myocardial cell.