Interaction between the sarcoplasmic reticulum and mitochondria in the control of contractile calcium in guinea-pig atria--does it exist?

The inhibitory action of caffeine on calcium (Ca2+) release from the sarcoplasmic reticulum (SR) and interference with mitochondrial (Ca2+) fluxes by a mitochondrial uncoupler protonfore CCCP were utilized to define a calcium pool responsible for potentiation of post-rest twitch tension in guinea-pig atria. The Ca2+ fluxes were assessed by means of 45Ca2+. Caffeine and CCCP when applied separately did not affect post-rat 45Ca2+ content. Yet, when they were applied together it was markedly reduced to the resting level. It is concluded that a possible source of contractile Ca2+ may be located in mitochondria and an eventual shift of Ca2+ between mitochondria and the SR seems to be a plausible assumption.     

Effects of rapid application of caffeine on intracellular calcium concentration in ferret papillary muscles

In this paper we investigate the effects of caffeine (5-20 mM) on ferret papillary muscle. The intracellular Ca2+ concentration ( [Ca2+]i) was measured from the light emitted by the photoprotein aequorin, which had previously been microinjected into superficial cells. Isometric tension was measured simultaneously. The rapid application of caffeine produced a transient increase of [Ca2+]i, which decayed spontaneously within 2-3 s and was accompanied by a transient contracture. The removal of extracellular Na+ or an increase in the concentration of intracellular Na+ (produced by strophanthidin) increased the magnitude of the caffeine response. Cessation of stimulation for several minutes or stimulation at low rates decreased the magnitude of the stimulated twitch and Ca2+ transient. These maneuvers also decreased the size of the caffeine response. These results are consistent with the hypothesis that the caffeine-releasable pool of Ca2+ (sarcoplasmic reticulum) is modulated by maneuvers that affect contraction. Ryanodine (10 microM) decreased the magnitude of the caffeine response as well as that of the stimulated twitch. In contrast, the rapid removal of external Ca2+ abolished the systolic Ca2+ transient within 5 s, but had no effect on the caffeine response. From this we conclude that the abolition of twitch by Ca2+-free solutions is not due to depletion of the sarcoplasmic reticulum of Ca2+, but may be due to a requirement of Ca2+ entry into the cell to trigger Ca2+ release from the sarcoplasmic reticulum.     

Spontaneous Ca2+ release from the sarcoplasmic reticulum limits Ca2+- dependent twitch potentiation in individual cardiac myocytes. A mechanism for maximum inotropy in the myocardium

We hypothesized that the occurrence of spontaneous Ca2+ release from the sarcoplasmic reticulum (SR), in diastole, might be a mechanism for the saturation of twitch potentiation common to a variety of inotropic perturbations that increase the total cell Ca. We used a videomicroscopic technique in single cardiac myocytes to quantify the amplitude of electrically stimulated twitches and to monitor the occurrence of the mechanical manifestation of spontaneous SR Ca2+ release, i.e., the spontaneous contractile wave. In rat myocytes exposed to increasing bathing [Ca2+] (Cao) from 0.25 to 10 mM, the Cao at which the peak twitch amplitude occurred in a given cell was not unique but varied with the rate of stimulation or the presence of drugs: in cells stimulated at 0.2 Hz in the absence of drugs, the maximum twitch amplitude occurred in 2 mM Cao; a brief exposure to 50 nM ryanodine before stimulation at 0.2 Hz shifted the Cao of the maximum twitch amplitude to 7 mM. In cells stimulated at 1 Hz in the absence of drugs, the maximum twitch amplitude occurred in 4 mM Cao; 1 microM isoproterenol shifted the Cao of the maximum twitch amplitude to 3 mM. Regardless of the drug or the stimulation frequency, the Cao at which the twitch amplitude saturated varied linearly with the Cao at which spontaneous Ca2+ release first occurred, and this relationship conformed to a line of identity (r = 0.90, p = less than 0.001, n = 25). The average peak twitch amplitude did not differ among these groups of cells. In other experiments, (a) the extent of rest potentiation of the twitch amplitude in rat myocytes was also limited by the occurrence of spontaneous Ca2+ release, and (b) in both rat and rabbit myocytes continuously stimulated in a given Cao, the twitch amplitude after the addition of ouabain saturated when spontaneous contractile waves first appeared between stimulated twitches. A mathematical model that incorporates this interaction between action potential-mediated SR Ca2+ release and the occurrence of spontaneous Ca2+ release in individual cells predicted the shape of the Cao-twitch relationship observed in other studies in intact muscle. Thus, the occurrence of spontaneous SR Ca2+ release is a plausible mechanism for the saturation of the inotropic response to Ca2+ in the intact myocardium.     

Dissociation of changes in apparent myofibrillar Ca2+ sensitivity and twitch relaxation induced by adrenergic and cholinergic stimulation in isolated ferret cardiac muscle

In isolated, aequorin-injected ferret cardiac muscle we measured the apparent myofilament Ca2+ sensitivity and its relationship to twitch relaxation time in the presence of autonomic perturbations. The Ca2+-tension relation was determined from the peak aequorin luminescence and peak twitch tension measured in muscles across a broad range of bathing [Ca2+] in the presence and absence of acetylcholine (ACh) (1 microM) or isoproterenol (ISN) (1 microM), or both drugs. ACh shifted the relationship of peak tension to (peak) aequorin light leftward, which suggests an increase in myofilament Ca2+ sensitivity, but it did not alter relaxation, which was measured as the time for peak tension to decay by 50% (t 1/2 R). ISN produced its previously documented effects, i.e., a rightward shift of the relationship of peak tension to peak aequorin light and a decrease in t1/2R. ACh abolished the ISN effect on the peak tension-aequorin light relationship but did not reverse the effect of ISN to decrease t1/2R. The effects of ACh and ISN of modulating the apparent myofilament Ca2+ sensitivity in intact muscles, corroborate findings of previous studies in isolated myofibrillar preparations. However, these perturbations of myofilament Ca2+ sensitivity in the intact muscle do not relate to twitch relaxation, measured as t1/2R, since (a) ACh affects the former but not the later and (b) the effect of ISN on the Ca2+-tension relationship is abolished by ACh, while the relaxant effect persists.     

Effects of quinidine and lidocaine on action potential and membrane currents of frog ventricles

The effects of quinidine and lidocaine on frog ventricle were studied by using a single sucrose gap voltage clamp technique. In Ca2+-Ringer, quinidine (80 microM) caused slight prolongation of action potential duration (APD50) and significant inhibition of twitch tension. Lidocaine (40 microM) shortened APD50 without significant effect on twitch tension. In tetrodotoxin (TTX)-treated preparations, quinidine caused significant prolongation of APD50 from 529 +/- 19 msec to 597 +/- 11 msec, (n = 9) and inhibition of twitch tension, but lidocaine did not affect APD50 and twitch tension. Under voltage clamp condition, quinidine reduced peak inward current in the absence of TTX, but enhanced peak inward current in the presence of TTX. The steady state outward current was increased by quinidine. Lidocaine didn't affect peak inward current in the absence or in the presence of TTX. Membrane current through the inward rectifier (IK1) was slightly increased by lidocaine, but significantly inhibited by quinidine. The enhancement of peak inward current by quinidine was retarded or reversed in preparation bathed with Sr2+-Ringer. When Ni2+ was added to a preparation bathed in Ca2+-Ringer, an inhibition of calcium inward current and action potential plateau was observed. The spike amplitude of the action potential was, however, unaffected by Ni2+. In this Ni2+-treated preparation, lidocaine (20 microM) caused significant shortening of APD50 without significant effect on action potential amplitude. The shortening of APD50 was associated with a slight increase of steady state outward current. The increase of steady state outward current by lidocaine was absent in the TTX-treated preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of agonists and antagonists of rhyanodine receptors on potassium contractures in twitch and tonic frog skeletal muscle fibers

A comparative analysis of the effects of the concentrations of Ca2+ in external medium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of rhyanodine-sensitive Ca2+ channels of carcoplasmic reticulum on the characteristics of potassium contracture in frog twitch and tonic skeletal muscles has been performed. It was shown that the duration of contracture in tonic muscles is not restricted by the presence of Ca2+, as distinct from twitch muscles. Dandrolene does not practically affect the contractile responses of tonic fibres, and the concentration of cresol eliciting the contracture for tonic fibres is substantially higher (1 mM) than for twitch fibers (0.25 mM). In twitch fibers, the potassium contracture activated in the presence of cresol is comparable in amplitude and dynamics with the contracture under control conditions, and in tonic fibers a summing of responses without relaxation after the washing of excessive potassium is observed. This suggests that, in twitch fibers, the influx of Ca2+ can directly create the concentration sufficient for the maintenance of contraction, and in tonic fibers its involvement is mediated through the Ca(2+)-dependent activation of the beta-isoform of rhyanodine-sensitive channels.     

Energy metabolism of spermatozoa. IV. Effect of calcium on respiration of mature epididymal sperm of the rabbit.

The effect of calcium (Ca+2) on the respiration rate of mature rab bit epididymal sperm was studied. The addition of Ca+2 did not further stimulate the respiration rate of sperm already stimulated by glucose or pyruvate. Oligomycin, which inhibits mitochondrial ATP synthesis and slows respiration, did not inhibit the uptake of mitochond rial Ca+2. The addition of the ionophore A23187, which promotes selective permeability of cell membranes to Ca+2, caused a marked stimulation of respiration when Ca+2 was added, indicating that the sperm cell membrane is not permeable to Ca+2. The stimulation of the respiration rate by pyruvate, but not glucose, was enhanced by the addition of 45 mM HCO3, which did not affect the response to added Ca+2. With or without Ca+2, cyclic AMP and dibutyl cyclic AMP did not stimulate respiration in the presence of pyruvate or glucose. The results suggest that mature rabbit sperm from the cauda epididymis are intrinsically motile, and not dependent on Ca+2.     

The interaction of electrically stimulated twitches and spontaneous contractile waves in single cardiac myocytes

Spontaneous myofilament motion that propagates within cells as a contractile wave is a manifestation of localized Ca2+ release from sarcoplasmic reticulum (SR). At 37 degrees C, when bathing [Ca2+] (Cao) is 1.0 mM, rat myocytes exhibit contractile waves at rest and the interwave interval averages 9.1 +/- 1.5 s (n = 6). We determined whether there was an interaction between this type of SR Ca2+ release and that induced by electrical stimulation to cause a twitch, and whether such an interaction had functional significance. Progressive decreases in SR Ca2+ loading effected by graded concentrations of caffeine produced proportional decreases in the mechanical amplitude of the twitch and of the spontaneous contractile wave. Regular electrical stimulation in physiologic Cao abolished the waves and, after stimulation, waves did not reappear for a period of time (delay interval). Over a range of stimulation frequencies (6-72 min-1), the delay interval ranged from 11.4 +/- 3.6 to 12.4 +/- 1.7 s and was usually greater than the interwave interval at rest. The delay interval for a wave to occur after a twitch was reduced in the presence of increased Cao, glycosides, or catecholamines. When the interstimulus interval exceeded the delay interval, waves could appear between twitches and had a marked effect of shortening the duration of the action potential and decreasing the amplitude of the subsequent twitch. The magnitude of this effect varied inversely with time (up to 2 s) between the onset of the spontaneous diastolic wave and the subsequent stimulated twitch. A reduction of the interstimulus interval to less than the delay interval prevented the occurrence of diastolic waves. These results demonstrate the presence of an interaction between spontaneous and action potential-mediated Ca2+ release, which can be interpreted on the basis of a common Ca2+ pool and perhaps common release mechanisms. This interaction can explain many of the known effects of electrical stimulation on phenomena that are thought to result from spontaneous Ca2+ oscillations in intact tissue.     

Effects of ryanodine on intracellular Ca2+ transients in mammalian cardiac muscle

We observed the effects of ryanodine on the aequorin luminescence, membrane potential, and contraction of canine cardiac Purkinje fibers and ferret ventricular muscle. In canine Purkinje fibers, ryanodine (10 nM to 1 microM) abolished the spontaneous spatiotemporal fluctuations in [Ca2+] that occur as a result of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) during exposure to low-Na+ solutions. Ryanodine strongly reduced the twitch and both components of the intracellular aequorin luminescence signal (L1 and L2), which normally accompanies contraction. The small luminescence signals that remained in ryanodine could be abolished by a Ca2+ channel blocker (nitrendipine, 10 microM). The plateau phase of the action potential was reduced by nitrendipine in the presence of ryanodine, which suggests that Ca2+ current was not blocked by ryanodine. In ferret ventricular tissue, ryanodine (1 microM) prolonged the action potential and reduced the peak amplitudes of both the aequorin transient and the twitch, while greatly prolonging the time-to-peak of both signals. Increases in extracellular [Ca2+] restored the peak amplitudes of the twitch and the aequorin luminescence, but did not restore the normal time-to-peak. The results show that in both tissues, the negative inotropic effect of ryanodine is due to the reduction of the intracellular [Ca2+] transient. Inasmuch as neither Ca2+ entry via surface membrane Ca2+ channels nor Na+-Ca2+ exchange appears to be blocked by ryanodine, the most probable cause of reduction of the [Ca2+] transient is an inhibition of Ca2+ release by the SR.     

Verapamil and zero Ca2+ alter responses of cat muscle to halothane and caffeine

Strips of soleus (slow twitch, oxidative) and gracilis (fast-twitch, glycolytic) muscle were obtained from 27 anesthetized cats and mounted in organ baths filled with oxygenated Krebs-Ringer solution (37 degrees C). The responses to caffeine, halothane (1%), caffeine in the presence of halothane, and electrical stimulation in the presence of halothane were examined in the two fiber types. These responses were compared with those observed in paired strips of muscle that had been treated with verapamil (10 or 28 microM), a slow calcium (Ca2+) channel blocker, with zero Ca2+, or with zero Ca2+ where magnesium (3.7 mM Ca2+) was added to replace the Ca2+. Halothane-induced contractures in the soleus were blocked by verapamil and zero Ca2+. Caffeine-induced contractures and tetanic contractions were attenuated in zero Ca2+ and by verapamil in both fiber types. Halothane overcame verapamil-induced reductions of caffeine contractures and tetanic contractions in both fiber types. In contrast, halothane did not overcome zero Ca2+-induced reductions in caffeine contractures or tetanic contractions in either fiber type. Furthermore, the addition of Mg2+ to the zero Ca2+ did not restore the responses. The findings with verapamil indicate that in cat muscle, both halothane- and caffeine-induced contractures and tetanic contractions are dependent on the influx of extracellular Ca2+. This extracellular Ca2+ may enter through the slow Ca2+ channels. However, because halothane in combination with caffeine or electrical stimulation overcame the effects of verapamil, there may be other sites involved.     

Inhibition of Zn2+-induced potentiation of twitch tension by Ni2+ in frog muscle

Effect of Ni2+ on Zn2+-induced potentiation of twitch tension was studied electrophysiologically in the toe muscle fibers of Rana catesbeiana. The major findings of this investigation are as follows. When 2 mM Ni2+ was applied to fibers in a normal Ringer's solution containing 50 microM Zn2+ (Zn2+ solution), the Zn2+-potentiated twitch tension decreased remarkably to about one-third of that before Ni2+ treatment. This concentration of Ni2+ caused a 23% decrease in the duration of action potential which had been prolonged by Zn2+ (6.61-5.09 ms). Ni2+ (2 mM) added to normal Ringer's solution led to increases of about 30 and 42% in twitch tension and in the duration of action potential, respectively. A slight increase in the mechanical threshold was induced by 2 mM Ni2+. The inhibitory action of Ni2+ on the twitch tension in Zn2+ solution was larger than that in the case of tetanus tension. Diltiazem (40 microM), a Ca2+ channel blocker, did not inhibit the twitch tension potentiated in Zn2+ solution. These results suggest that the decrease in Zn2+-potentiated twitch tension by Ni2+ may possibly derive from impairment of the propagation of action potential along the T tubules.     

The effect of dantrolene on skeletal-muscle sarcoplasmic-reticulum function in malignant hyperpyrexia in pigs.

The effect of the muscle relaxant dantrolene on isolated sarcoplasmic reticulum was studied in control and malignant-hyperpyrexia-susceptible Landrace pigs. The membranes prepared from both sources showed similar Ca2+-dependent ATPase activities, had comparable phospholipid/protein ratios, and their sodium dodecyl sulphate/polyacrylamide-gel patterns were indistinguishable. Membranes from both sources appeared to bind similar amounts of dantrolene. The drug did not stimulate Ca2+-dependent ATPase activity in preparations from either source. The rates of Ca2+ exchange and Ca2+ efflux appeared to be similar in sarcoplasmic reticulum of control and malignant-hyperpyrexia-susceptible pigs. Dantrolene did not affect either the rates or the amount of Ca2+ lost from the vesicles. These results suggest that dantrolene does not directly affect the movement of Ca2+ across the sarcoplasmic-reticulum membrane.     

Failure of Leiurus quinquestriatus venom to affect potassium movements in pancreatic islets

The venom from the Israeli scorpion Leiurus quinquestriatus failed to affect 86Rb and 45Ca outflow from rat pancreatic islets perifused in the presence of tetrodotoxin and stimulated by the Ca2+-ionophore A23187 or the hypoglycaemic sulfonylurea tolbutamide. In non-stimulated islets, the venom components whose effects are insensitive to tetrodotoxin did not affect 45Ca and 86Rb outflow. Last, the venom did not alter 86Rb inflow. These findings suggest that 86Rb, 45Ca fluxes and more specifically the Ca2+-activated K+ permeability in the pancreatic B-cell are insensitive to the venom.     

Partial inhibition of skeletal muscle contraction by dantrolene sodium and its modification with perchlorate and Bay K 8644.

The effects of dantrolene sodium (DAN) on the dihydropyridine receptor (DHPR) of the transverse (T) tubule voltage sensor (Ca2+ channel) was studied with single fibers from bullfrog toe muscle. Perchlorate (ClO4-), which acts selectively on the DHPR, overcame DAN-induced inhibition of twitch tension. Bay K 8644, a DHPR agonist, slowed the rate of twitch inhibition by DAN. DAN inhibited twitch tension to a greater extent in Ca(2+)-free solution than in Ringer solution or solution containing Zn2+, whereas twitch inhibition by DAN was less in caffeine-containing solution than in the control. The effects of DAN on Zn(2+)- and caffeine-treated fibers and on fibers in Ca(2+)-free solution suggest that DAN must act near the voltage sensor of the T tubule. However, differences in net twitch inhibition by DAN between control fibers and fibers potentiated by ClO4- or Bay K 8644 suggest that DAN does not bind to the same site as these potentiating agents do. The role of myoplasmic Ca2+ in DAN-induced inhibition of twitch and the effects of DAN on the mechanical threshold and membrane potential in skeletal muscle are discussed.     

Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle

Intact cardiac cells from the adult rat or rabbit ventricle were isolated by enzymatic digestion with a progressive increase of the [free Ca2+] in the solution. These cells were electrically stimulated in the presence of 2.50 mM free Ca2+, and a twitch of maximum amplitude was elicited by the positive inotropic interventions that were found to be optimum. Then the cells were chemically skinned, and the maximum tension induced by a saturating [free Ca2+] was used as a reference to express the tension developed during the twitch of the intact cells. The myoplasmic [free Ca2+] reached during the twitch was inferred from the tension-pCa curve. In mechanically skinned cells of the same animal species, the myoplasmic [free Ca2+] reached during Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR) was inferred by two methods using (a) the tension-pCa curve and (b) a direct calibration of the transients of aequorin bioluminescence. The induction of a maximum Ca2+ release from the SR required a larger Ca2+ preload of the SR and a higher [free Ca2+] trigger in the rabbit than in the rat skinned cells. However, the results obtained with the two methods of inference of the myoplasmic [free Ca2+] suggest that in both animal species a maximum myoplasmic [free Ca2+] of pCa approximately 5.40 was reached during both the optimum Ca2+-induced release of Ca2+ from the SR of the skinned cells and the optimum twitch of the intact cells. This was much lower than the [free Ca2+] necessary for the full activation of the myofilaments (pCa approximately 4.90).     

The kinetics relating calcium and force in skeletal muscle.

The kinetics relating Ca2+ transients and muscle force were examined using data obtained with the photoprotein aequorin in skeletal muscles of the rat, barnacle, and frog. These data were fitted by various models using nonlinear methods for minimizing the least mean square errors. Models in which Ca2+ binding to troponin was rate limiting for force production did not produce good agreement with the observed data, except for a small twitch of the barnacle muscle. Models in which cross-bridge kinetics were rate limiting also did not produce good agreement with the observed data, unless the detachment rate constant was allowed to increase sharply on the falling phase of tension production. Increasing the number of cross-bridge states did not dramatically improve the agreement between predicted and observed force. We conclude that the dynamic relationship between Ca2+ transients and force production in intact muscle fibers under physiological conditions can be approximated by a model in which (a) two Ca2+ ions bind rapidly to each troponin molecule, (b) force production is limited by the rate of formation of tightly bound cross-bridges, and (c) the rate of cross-bridge detachment increases rapidly once tension begins to decline and free Ca2+ levels have fallen to low values after the last stimulus. Such a model can account not only for the pattern of force production during a twitch and tetanus, but also the complex, nonlinear pattern of summation which is observed during an unfused tetanus at intermediate rates of stimulation.     

Cyclic AMP enhances inositol trisphosphate-induced mobilization of intracellular Ca2+ in cultured aortic smooth muscle cells

The effect of cAMP on ATP-induced intracellular Ca+ mobilization in cultured rat aortic smooth muscle cells was investigated. Treatment of cells for 3 min at 37 degrees C with dibutyryl cAMP, a membrane-permeable analogue of cAMP, at concentration up to 500 microM resulted in 1.5- to 1.7-fold increase in the peak cytosolic Ca2+ concentration when cells were stimulated with 3 to 200 microM ATP either in the presence or absence of extracellular Ca2+. Similar results were obtained when 0.5 mM 8-Br-cAMP or 10 microM forskolin was used instead of dibutyryl cAMP. In contrast to the Ca2+ response, dibutyryl cAMP did not affect ATP-induced formation of inositol trisphosphate (IP3). Furthermore, the dibutyryl cAMP treatment did not affect the size of the Ca2+ response elicited by 10 microM ionomycin. These results suggest that intracellular cAMP potentiates the ATP-induced Ca2+ response by enhancing Ca2+ release from the intracellular Ca2+ store(s), rather than by increasing the ATP-induced production of IP3 or by increasing the size of the intracellular Ca2+ store. Using saponin-permeabilized cells, we have shown directly that cAMP enhances Ca2+ mobilization by potentiating the Ca2+-releasing effect of IP3 from the intracellular Ca2+ store.     

Contraction in voltage-clamped, internally perfused single heart cells

We studied contraction in single voltage-clamped, internally perfused myocytes isolated from guinea pig ventricles. The microscopic appearance of the cell was observed and recorded with a television system, while contractile shortening was measured 1,000 times/s using a linear photodiode array. Uniform, synchronous sarcomere shortening occurred in response to depolarizations that triggered a slow inward current (Isi). Changes in Isi caused by altering the amplitude of the voltage step, the extracellular [Ca2+], or the holding potential were accompanied by immediate parallel changes in the extent and velocity of shortening. In particular, twitch shortening during depolarization was immediately decreased when large voltage steps decreased Isi, and was eliminated by depolarizations that exceeded +75 mV, the apparent reversal potential for Ca2+. In these cases, shortening was associated with the tail current during repolarization. Increases in the amplitude, duration, and the rate of the depolarizing step increased the extent and speed of sarcomere shortening over the course of four to five contractions without a simultaneous parallel increase of Isi. Large prolonged depolarizations caused an asynchronous, nonuniform, oscillatory shortening of the cell and potentiated future twitch contractions. Increases in the duration of the depolarizing step immediately prolonged contraction; otherwise, interventions that altered the extent, velocity, and time course of shortening in intact, nonperfused cells did not affect the time course of the contraction in the internally perfused single cells. Our results provide direct support for the hypothesis that Isi both induces and grades the size of the Ca2+ release from the sarcoplasmic reticulum of intact cardiac muscle. In addition, a separate, depolarization-dependent process unrelated to Isi grades the size of contraction, presumably by modulating Ca2+ accumulation in the intracellular stores, and affects its time course.     

Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog

Inactivation of slow Ca2+ channels was studied in intact twitch skeletal muscle fibers of the frog by using the three-microelectrode voltage-clamp technique. Hypertonic sucrose solutions were used to abolish contraction. The rate constant of decay of the slow Ca2+ current (ICa) remained practically unchanged when the recording solution containing 10 mM Ca2+ was replaced by a Ca2+-buffered solution (126 mM Ca-maleate). The rate constant of decay of ICa monotonically increased with depolarization although the corresponding time integral of ICa followed a bell-shaped function. The replacement of Ca2+ by Ba2+ did not result in a slowing of the rate of decay of the inward current nor did it reduce the degree of steady-state inactivation. The voltage dependence of the steady-state inactivation curve was steeper in the presence of Ba2+. In two-pulse experiments with large conditioning depolarizations ICa inactivation remained unchanged although Ca2+ influx during the prepulse greatly decreased. Dantrolene (12 microM) increased mechanical threshold at all pulse durations tested, the effect being more prominent for short pulses. Dantrolene did not significantly modify ICa decay and the voltage dependence of inactivation. These results indicate that in intact muscle fibers Ca2+ channels inactivate in a voltage-dependent manner through a mechanism that does not require Ca2+ entry into the cell.     

Losartan prevents contractile dysfunction in rat myocardium after left ventricular myocardial infarction

We studied the effects of chronic losartan (Los) treatment on contractile function of isolated right ventricular (RV) trabeculae from rat hearts 12 wk after left ventricular (LV) myocardial infarction (MI) had been induced by ligation of the left anterior descending artery at 4 wk of age. After recovery, one-half of the animals were started on Los treatment (MI+Los; 30 mg x kg(-1) x day(-1) per os); the remaining animals were not treated (MI group). Rats without infarction or Los treatment served as controls (Con group). MI resulted in increases in LV and RV weight and unstressed LV cavity diameter; these were partially prevented by Los treatment. The active peak twitch force-sarcomere length relation was depressed in MI compared with either Con or MI+Los. Likewise, maximum Ca2+ saturated twitch force was depressed in MI, whereas twitch relaxation and twitch duration were prolonged. Myofilament function, as measured in skinned trabeculae, was not significantly different among the Con, MI, and MI+Los groups. We conclude that Los prevents contractile dysfunction in rat RV trabeculae after LV MI. Our results suggest that the beneficiary effect of Los treatment results not from improved myofilament function but rather from improved myocyte Ca2+ homeostasis.