Estimates of beat to beat handling of activator calcium using measurements of [Ca2+]i in aequorin loaded ferret cardiac muscle.

OBJECTIVE: The aims were (1) to measure simultaneously and on a beat to beat basis intracellular calcium concentration ([Ca2+]i) transients and force transients in isolated ferret cardiac trabeculae; (2) to obtain and compare independent estimates of the recirculating fraction of Ca2+ using the [Ca2+]i data and the force data (recirculating fraction is the fraction of activator Ca2+ taken up by the sarcoplasmic reticulum in each beat and, in the steady state twitch, the fraction of activator Ca2+ released by the sarcoplasmic reticulum); and (3) to estimate the amount of Ca2+ that returns to the sarcoplasmic reticulum and the amount that, during steady state contractions, enters the cytosol, presumably from the extracellular compartment, with each beat. METHODS: Eight trabeculae were mounted in the myograph. The servo-controlled muscle length was 98% of the length at which developed force was maximal. A modified technique was used for chemical loading of aequorin, and a new method for computer controlled low level photon counting, storage, calibration, and analysis. [Ca2+]i transients and force transients were simultaneously recorded during potentiated beats, together with their respective decays toward control steady state [Ca2+]i transients and force transients. A modified test of postextrasystolic potentiation achieved with a brief train of rapid pacing followed by a pause was used to evoke the potentiated beats. RESULTS: At 2.0 mM extracellular Ca2+ ([Ca2+]o), resting [Ca2+]i was 283(SD 77) nM. The resting tension was 1.6(0.3) g.mm-2. The steady state [Ca2+]i transient and the peak potentiated [Ca2+]i transient averaged 992(165) and 1290(154) nM respectively. The corresponding tensions were 4.0(1.9) and 8.7(3.1) g.mm-2 respectively. The recirculating fraction of Ca2+ calculated from the dissipation of the potentiated [Ca2+]i transient averaged 45(4)%. This recirculating fraction was indistinguishable from the one calculated with another method from the decay of the force potentiation. CONCLUSIONS: This is the first study to estimate the recirculating fraction of activator Ca2+ using measurements of [Ca2+]i. The results indicate that over a wide range of [Ca2+]i and tensions the Ca(2+)-force relationship is well approximated by a straight line. At 2.0 mM [Ca2+]o it appears that some 450 nM of Ca2+ recirculates and that a similar amount per steady state beat enters the cytosol, probably from the extracellular compartment.

Beat-to-beat measurements of [Ca2+]i and force in ferret cardiac muscle after chemical loading of aequorin.

This communication reports the development of a modified procedure for chemical loading of aequorin in small multicellular cardiac preparations, with special emphasis directed toward the implementation of a new method for computer-controlled low-photon counting and digital processing and analysis of the data to obtain intracellular Ca2+ concentration ([Ca2+]i). In eight ferret right ventricular trabeculae, we measured the mechanical performance and found that, at 1.25 mM extracellular Ca2+ concentration ([Ca2+]o), resting tension, developed tension, and time to peak tension were unchanged by the loading procedure. Estimated resting and peak systolic [Ca2+]i were 299 +/- 65 and 766 +/- 131 nM, respectively. Thirty minutes after raising the [Ca2+]o to 5 mM, there was a robust increase in mechanical performance, with peak systolic [Ca2+]i averaging 1,218 +/- 222 nM. The diastolic [Ca2+]i remained unchanged. In four other trabeculae, exposure to a low-Na(+)-containing superfusate demonstrated a remarkable beat-to-beat correspondence of increases in diastolic [Ca2+]i and resting tensions. The same beat-to-beat concordance was also observed between the rapidly changing amplitudes of peak [Ca2+]i and developed tension. In additional experiments, simultaneous recordings of [Ca2+]i and force transients were obtained during rapid pace pause maneuvers. These studies showed distinct and quantifiable fluctuations of [Ca2+]i in a 1:1 relation to the mechanical record to a frequency of at approximately 300 beats/min. These results demonstrate that beat-to-beat measurements of [Ca2+]i and tension transients can be obtained with good resolution in multicellular cardiac preparations.

Excitation-contraction coupling model to estimate the recirculating fraction of activator calcium in intact cardiac muscle.

Potentiated contractions were evoked with rapid pace pause maneuver in 14 length-clamped ferret papillary muscles paced 12 times/min at 25 degrees C. At 1.25 mM [Ca2+]o the average steady-state force was 2.94 +/- 1.08 g/mm2 and the potentiated contraction averaged 10.96 +/- 1.61 g/mm2. At 5.0 mM [Ca2+]o the steady-state force increased to 6.18 +/- 1.23 g/mm2 and the potentiated contraction averaged 12.08 +/- 1.15 g/mm2. Under the conditions of these experiments the potentiated contraction obtained at 5.0 mM [Ca2+]o is equal to the maximum twitch tension (Fmax) these muscles can generate. We have previously shown that Fmax is an equivalent of maximal calcium activated force. Since there is a beat to beat nearly exponential decay of the evoked potentiation, the fraction (= fraction x) of the potentiation that is not dissipated with each beat is nearly constant. Using an excitation-contraction coupling model we have previously found that x reflects a measure of the recirculating fraction of activator calcium. Because the tension-calcium relationship is better characterized by a sigmoidal curve, we have now incorporated the Hill equation in the model. To account for the inverse relationship between [Ca2+]i and the magnitude of the slow inward current, a term for negative feedback (h) was also included. We have determined the quantity (x-h) because x and h could not be determined separately. The quantity (x-h) was denoted as x'. The average values of x' at 1.25 and 5.0 mM [Ca2+]o were significantly different (p less than 0.0001), approximately 20% at the lower [Ca2+]o and about 50% at the higher [Ca2+]o.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ryanodine on contractile performance of intact length-clamped papillary muscle.

Extent, time course, and underlying mechanisms of the negative inotropic effect of ryanodine were examined in 22 length-clamped ferret right ventricular papillary muscles paced 12/min at 25 degrees C. After 60 minutes of exposure to 5 microM ryanodine a new steady state was attained with developed forces averaging 10-15% of maximum twitch force. Ryanodine does not pharmacologically excise the sarcoplasmic reticulum (SR) in this preparation. Ryanodine does not appreciably inhibit the ability of the SR to take up Ca2+ as evidenced by the potentiated beats obtained after a short pause that are nearly as large after ryanodine as before. On comparing equipotent beats before and after ryanodine, we found that ryanodine actually increases the rate at which Ca2+ is released during the twitch if the SR Ca2+ stores are equal or similar. The evidence for this conclusion is a larger maximum rate of tension rise and briefer time to peak tension after ryanodine. Since ryanodine increases the time that SR Ca2+ release channels are open and decreases their conductivity, it must follow that the former effect predominates over the latter in our experiments. Ryanodine increases the leakiness of the SR during diastole probably by inhibiting closure of SR Ca2+ release channels. The evidence for this conclusion is as follows: the early peak of the restitution curves after ryanodine, the brevity of the time required for a rested state contraction after ryanodine, and the small amplitude of the steady-state contraction at a rate of 12/min. The SR leaks even in the absence of ryanodine, but if external Ca2+ is so high that Ca2+ loss from the cell is slowed or a Ca2+ leak into the cell through the sarcolemma cancels the SR leak, then the effects of the SR leak are minimized. The evidence for this conclusion is the time required for rested-state contraction to occur or the slope of the descending limb of restitution curve; however, in presence of ryanodine even high external Ca2+ cannot prevent rapid depletion of SR Ca2+ stores. Even though we have presented evidence for a mechanism whereby ryanodine increases the number of open SR Ca2+ release channels in both systole and diastole, we do not mean to imply that most of them stay open in diastole; the SR would leak too fast to accumulate any Ca2+ for the potentiated beat. Thus, probably most channels close after being open a certain length of time, even in the presence of ryanodine.

Maximal twitch tension in intact length-clamped ferret papillary muscles evoked by modified postextrasystolic potentiation.

A modified test of postextrasystolic potentiation achieved with a brief episode of rapid pacing followed by a 6-second pause (RPP maneuver) was used to evoke maximal force in isolated intact ferret right ventricular papillary muscles. Maximal RPP tensions were examined under length-clamped conditions and compared with the steady-state forces obtained when further increases in [Ca2+]o, did not further increase force and to the tensions recorded at the point of saturation of force when similarly length-clamped muscles were subjected to caffeine-induced tetanization. The results show that the calculated maximal twitch tension achieved with RPP is comparable to the 25-35 g/mm2 observed in intact single skeletal muscle fibers. The study also shows that the beat-to-beat decay of the potentiated contraction is exponential. While the amount of the constant fractional beat-to-beat decay is a function of [Ca2+]o, it is not influenced by length. During the decay of potentiation, the ratio of the potentiation of any beat divided by that of the previous beat is a constant, called (X). With certain assumptions, it is shown that (X) is a measure of the fraction of activator calcium taken up by the sarcoplasmic reticulum in each beat and, in the steady state, the fraction of activator calcium that comes from the sarcoplasmic reticulum. The (X) amounted to 33%, 50%, and 65% when [Ca2+]o was 1.25, 2.50, and 5.0 mM, respectively. Thus, at 1.25 mM [Ca2+]o, some two thirds of the total calcium required to activate the myofilaments comes from the extracellular compartment during excitation and only one third is contributed via release from the sarcoplasmic reticulum. In the region of optimal myofilament overlap, RPP force-length curves are remarkably shallow and almost indistinguishable from the sarcomere length-tension relation observed in skinned single cardiac cells. Tetanus plateau tensions are significantly smaller than RPP forces at any length, and the slope of the tetanus force-length curves is greater than that obtained with RPP. Thus, and by exclusion, we also suggest that caffeine may exert significant downstream inhibitory effects.

Mechanism of additive effects of digoxin and quinidine on contractility in isolated cardiac muscle.

To evaluate the mechanism of the effect of the interaction of digoxin and quinidine on myocardial contractility, ferret right ventricular papillary muscles were isolated and the effects of digoxin, 4 x 10(-7) M, quinidine, 1 x 10(5) M and atropine, 1.5 x 10(-6) M, on peak developed force, peak rate of development of force (dF/dt) and time to peak tension were determined. The addition of quinidine to muscles treated with digoxin increased developed force 18 percent (p = 0.006) and dF/dt 35 percent (p = 0.001) without significantly changing time to peak tension. This effect was abolished by pretreatment with atropine. Quinidine alone increased developed force 35 percent (p less than 0.001) and dF/dt 70 percent (p less than 0.001) and decreased time to peak tension 22 percent (p less than 0.001) from pretreatment control values. Atropine alone increased developed force 17 percent (p = 0.02) and dF/dt 32 percent (p = 0.001) and decreased time to peak tension 13 percent (p = 0.003) from pretreatment control values. The addition of quinidine to muscles treated with atropine or of atropine to muscles treated with quinidine did not significantly change developed force, dF/dt or time to peak tension from values with either drug alone. It is concluded that digoxin and quinidine in these doses have additive effects of myocardial contractility, and that this interaction is at least partially mediated through antagonism of cholinergic influences by quinidine.

Effect of damaged ends in papillary muscle preparations.

The mechanical characteristics of the central segment of isolated cat papillary muscle were determined with recently developed equipment. Two small sharpened stainless steel pins, inserted transversely through the muscle, were used to mark the ends of a segment not damaged by attachments. Installation of the pins did not affect the performance of the muscle. The distance between the pins was measured and controlled to produce isometric and afterloaded isotonic contractions of the segment of the muscle between the pins. Data from such contractions were compared with traditional whole muscle measurements made on the same preparation. The isometric length-tension curve of the central segment was significantly higher than that of the whole muscle, and there was no plateau of developed force at long lengths in five of six muscles studied. In the resting state, the segment was more compliant than the whole muscle for physiologic lengths and much stiffer for longer lengths. Segment velocity and shortening were significantly higher than whole muscle velocity and shortening at comparable loads.

Left ventricular force-length relations of isovolumic and ejecting contractions.

To determine the interrelationships between ejecting and isovolumic force-length relations and the extent to which the left ventricle will shorten, data obtained in 27 isolated, servo-regulated hearts were examined. For each heart a series of contractions, variably loaded (delta L) were derived for a thickwalled sphere and normalized by the cross-sectional area of muscle and length at zero end-diastolic pressure. It was found that within the physiological range examined total and active force were essentially a linear function of initial L with respective increments or reductions in slope produced by positive or negative shifts in contractile state. The force-L relations obtained isovolumically and at end ejection were virtually identical. For a given ejection pressure, end-systolic L was constant, despite variations in filling and therefore independent of initial L and deltaL; moreover, the L to which the ventricle shortened was determined by the course of the systolic force L-relation. Thus, irrespective of loading, delta L occurs within the confines of the contractile state-dependent isovolumic force-L relation and where the latter is equivalent to the end-systolic force-length relation.

Afterload-induced homeometric autoregulation in isolated cardiac muscle.

Pressure-induced homeometric autoregulation (HAR) has been demonstrated by many investigators in the mammalian ventricle; In isolated cardiac muscle, however, several investigators have reported the opposite effect (anti-HAR); namely, that the first beat after a transition from isotonic to isometric contraction is the most forceful, with a decline over several beats to a steady state. In the present study we find that trabeculae from the canine right ventricle demonstrate either HAR or anti-HAR, depending on the rate of stimulation, the calcium level, and the temperature. Higher calcium, higher temperature, and lower rate of stimulation produce either less HAR or more anti-HAR. When only temperature and rate of stimulation are varied, in each muscle there is a unique rate for each temperature above which HAR occurs and below which anti-HAR occurs.

Factors influencing left ventricular shortening in isolated canine heart.

To assess in the intact ventricle the steady-state influence of several mechanical variables on the extent of left ventricular midwall circumferential shortening, a pressure servo system was utilized in isolated canine hearts. The system permits continuous monitoring of ventricular volume and control of diastolic and systolic pressures. After determination of the diastolic volume at zero filling pressure (V0), a series of variably preloaded or afterloaded contractions were generated including the isovolumic state. Contractile state was manipulated in a positive (calcium, 12-18 mg/100 ml; norepinephrine, 0.4-1.4 mug/min) or negative (propranolol, 0.12-0.50 mg/min) direction. Force and length terms derived for a thick-walled sphere were expressed per cross-sectional area of muscle and length, respectively, calculated at V0. For any preload, an inverse linear (r greater than 0.96) force-shortening relation was obtained, and each line was identified by its slope and isovolumic load (sigma0). Both slope and sigma0 increased with positive inotropic agents (vis-a-vis propranolol) or increments in preload. Thus, in the intact ventricle an inverse linear relation characterizes the force-shortening relation with the amount of shortening determined by initial fiber length, afterload, and the contractile state of the myocardium.

Comparison of contractile performance of canine atrial and ventricular muscles.

This study compared the contractile performance of a canine right atrial trabecula with that of a macroscopically indistinguishable trabecula isolated from the right ventricular apex. The heart was removed from nine mongrel puppies weighing 6-8 kg and placed in Krebs-Ringer's bicarbonate solution. The bathing solution contained only 1.25 mmoles of Ca2+ and was bubbled with a 95% O2-5% CO2 gas mixture. Each atrial trabecula was specially selected from the right atrial appendage. Histologically, these trabeculae showed a remarkable longitudinal orientation of the fibers. At Lmax (the length of the muscle at which developed tension was maximum) under identical conditions of temperature, rate of stimulation, ionic milieu, pH, and O2 and CO2 supply, right atrial trabeculae achieved the same developed and total tensions but in a much shorter time than did ventricular trabeculae. In both muscle groups the maximum developed tension averaged about 2.5 g/mm2. Since Lo (expressed as a fraction of Lmax) was less in atrial muscle than it was in ventribular muscle, we concluded that atrial muscle can be stretched considerably more than can ventricular muscle before optimum length is reached. At any given initial muscle length, the maximum of tension rise for atrial trabeculae amounted to at least twice that for ventricular trabeculae. At any given load up to 1.5 g/mm2, the maximum velocity of shortening of an atrial trabecula was about three to four times that of a ventricular trabecula. These results collectively indicate that the contractile performance of the right atrial muscle is in many respects superior to that of the right ventricle, at least under the conditions of these experiments.