Ryanodine and dihydropyridine receptor binding in ventricular cardiac muscle of fish with different temperature preferences

Ca(2+)-induced Ca2+ release (CICR) mechanism of cardiac excitation-contraction (e-c) coupling is dependent on the close apposition between the sarcolemmal dihydropyridine receptors (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptors (RyR). In particular, high RyR/DHPR ratio is considered to reflect strong dependence on SR Ca2+ stores for the intracellular Ca2+ transient. To indirectly evaluate the significance of CICR in fish hearts, densities of cardiac DHPRs and RyRs were compared in ventricular homogenates of three fish species (burbot, rainbow trout, and crucian carp) and adult rat by [3H] PN200-110 and [3H] ryanodine binding. The density of RyRs was significantly (P<0.05) higher in the adult rat (124+/-10 channels/microm3 myocyte volume) than in any of the fish species. Among the fish species, cold-acclimated (4 degrees C) trout had more RyRs than burbot, and crucian carp. The density of DHPRs was highest in the trout heart. RyR/DHPR ratio was significantly (P<0.05) higher in rat (4.1+/-0.5) than in the fish hearts (varying from 0.97+/-0.16 to 1.91+/-0.49) suggesting that "mammalian type" CICR is less important during e-c coupling in fish ventricular myocytes. In rainbow trout, acclimation to cold did not affect the RyR/DHPR ratio, while in crucian carp it was depressed in cold-acclimated animals (4 degrees C; 0.97+/-0.16) when compared to warm-acclimated fish (23 degrees C; 1.91+/-0.49). Although RyR/DHPR ratios were relatively low in fish hearts, there was a close correlation (r2=0.78) between the RyR/DHPR ratio and the magnitude of the Ry-sensitive component of contraction in ventricular muscle among the fish species examined in this study.     

Doxorubicin-induced late cardiotoxicity: delayed impairment of Ca2+-handling mechanisms in the sarcoplasmic reticulum in the rat

Doxorubicin treatment causes delayed development of cardiotoxicity. Whether the doxorubicin-induced impairment of cardiac functions reverses or progresses with time after the cessation of the treatment was examined. The rats were injected with doxorubicin (2.5 mg/kg, i.v., once a week for 3 weeks) and sacrificed at 1 (1W), 13 (13W), or 18 (18W) weeks after the final doxorubicin administration. The time to peak of twitch contraction observed at 2-Hz stimulation was not altered in left atrial or ventricular muscle preparations isolated from 1W rats, but it was prolonged in those from 13W and 18W rats. The reduction of the magnitude of postrest contraction and the alteration of force-frequency relationships in left atrial muscle preparations in 1W rats were not significant, but were intensified in the 13W and 18W groups. Alterations in the postrest contraction and the force-frequency relationships in ventricular muscle preparations isolated from doxorubicin-treated rat hearts were weaker, but the pattern of alteration was similar to that observed in left atrial muscle preparations. Caffeine-induced contraction observed in skinned fibers that were isolated from the 1W rats was not altered, but it was reduced in the 18W rats. The Ca2+ sensitivity of contractile proteins was not altered in doxorubicin-treated rat hearts in any of the groups. The K(d) values estimated from a [3H]ryanodine binding study were not altered, but the B(max) values were significantly lower in the 13W and 18W groups than those observed in control rats. These results suggest that the dysfunction of the sarcoplasmic reticulum progresses after the completion of doxorubicin treatment and contributes to the doxorubicin-induced late cardiotoxicity.     

Ryanodine receptors in liver

The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.     

Thyroid hormone-induced overexpression of functional ryanodine receptors in the rabbit heart

Modifications in the Ca(2+)-uptake and -release functions of the sarcoplasmic reticulum (SR) may be a major component of the mechanisms underlying thyroid state-dependent alterations in heart rate, myocardial contractility, and metabolism. We investigated the influence of hyperthyroid state on the expression and functional properties of the ryanodine receptor (RyR), a major protein in the junctional SR (JSR), which mediates Ca(2+) release to trigger muscle contraction. Experiments were performed using homogenates and JSR vesicles derived from ventricular myocardium of euthyroid and hyperthyroid rabbits. Hyperthyroidism, with attendant cardiac hypertrophy, was induced by the injection of L-thyroxine (200 microg/kg body wt) daily for 7 days. Western blotting analysis using cardiac RyR-specific antibody revealed a significant increase (>50%) in the relative amount of RyR in the hyperthyroid compared with euthyroid rabbits. Ca(2+)-dependent, high-affinity [(3)H]ryanodine binding was also significantly greater ( approximately 40%) in JSR from hyperthyroid rabbits. The Ca(2+ )sensitivity of [(3)H]ryanodine binding and the dissociation constant for [(3)H]ryanodine did not differ significantly between euthyroid and hyperthyroid hearts. Measurement of Ca(2+)-release rates from passively Ca(2+)-preloaded JSR vesicles and assessment of the effect of RyR-Ca(2+)-release channel (CRC) blockade on active Ca(2+)-uptake rates revealed significantly enhanced (>2-fold) CRC activity in the hyperthyroid, compared with euthyroid, JSR. These results demonstrate overexpression of functional RyR in thyroid hormone-induced cardiac hypertrophy. Relative abundance of RyR may be responsible, in part, for the changes in SR Ca(2+) release, cytosolic Ca(2+) transient, and cardiac systolic function associated with thyroid hormone-induced cardiac hypertrophy.     

Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium

During development fast-contracting atrial and ventricular chambers develop from a peristaltic-contracting heart tube. This study addresses the question of whether chamber formation is paralleled by a matching expression of the sarcoplasmic reticulum (SR) Ca(2+) pump. We studied indo-1 Ca(2+) transients elicited by field stimulation of linear heart tube stages and of explants from atria and outflow tracts of the prototypical preseptational E13 rat heart. Ca(2+) transients of H/H 11+ chicken hearts, which constitute the prototypic linear heart tube stage, were sensitive to verapamil only, indicating a minor contribution of Ca(2+)-triggered SR Ca(2+) release. Outflow tract transients displayed sensitivity to the inhibitors similar to that of the linear heart tube stages. Atrial Ca(2+) transients disappeared upon addition of ryanodine, tetracaine, or verapamil, indicating the presence of Ca(2+)-triggered SR Ca(2+) release. Quantitative radioactive in situ hybridization on sections of E13 rat hearts showed approximately 10-fold higher SERCA2a mRNA levels in the atria compared to nonmyocardial tissue and approximately 5-fold higher expression in compact ventricular myocardium. The myocardium of atrioventricular canal, outflow tract, inner curvature, and ventricular trabecules displayed weak expression. Immunohistochemistry on sections of rat and human embryos showed a similar pattern. The significance of these findings is threefold. (i) A functional SR is present long before birth. (ii) SR development is concomitant with cardiac chamber development, explaining regional differences in cardiac function. (iii) The pattern of SERCA2a expression underscores a manner of chamber development by differentiation at the outer curvature, rather than by segmentation of the linear heart tube.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Temperature and Ca2+ dependence of [3H]ryanodine binding in the burbot (Lota lota L.) heart

Opening and closing of the cardiac ryanodine (Ry) receptor (RyR) are coordinated by the free intracellular Ca2+ concentration, thus making the Ca2+ binding properties of the RyR important for excitation-contraction coupling. Unlike mammalian cardiac RyRs, which lose their normal function at low temperatures, RyRs of ectothermic vertebrates remain operative at 2-4 degrees C, as indicated by Ry sensitivity of contractile force. To investigate the mechanisms of low temperature adaptation of ectothermic RyRs, we compared Ca2+-dependent kinetics of [3H]ryanodine binding in cardiac preparations of a fish (burbot, Lota lota) and a mammal (rat). The number of ventricular [3H]ryanodine binding sites determined at 20 degrees C was 1.54 times higher in rat than burbot heart (0.401 +/- 0.039 and 0.264 +/- 0.019 pmol/mg protein, respectively) (P < 0.02), while the binding affinity (Kd) for [3H]ryanodine was similar (3.38 +/- 0.63 and 4.38 +/- 1.14 nM for rat and burbot, respectively) (P = 0.47). The high-affinity [3H]ryanodine binding to burbot and rat cardiac preparations was tightly coordinated by the free Ca2+ concentration at both 20 degrees C and 2 degrees C and did not differ between the two species. Half-maximal [3H]ryanodine binding occurred at 0.191 +/- 0.027 microM and 0.164 +/- 0.034 microM Ca2+ for rat and at 0.212 +/- 0.035 microM and 0.188 +/- 0.039 microM Ca2+ for burbot (P = 0.65), at 2 degrees C and 20 degrees C, respectively. In two other fish species, rainbow trout (Oncorhynchus mykiss) and crucian carp (Carassius carassius), the Ca2+-binding affinity at 20 degrees C was 4.4 and 5.9 times lower, respectively, than in the burbot. At 20 degrees C, the rate of [3H]ryanodine binding to the high-affinity binding site was similar in rat and burbot but was drastically slowed in rat at 2 degrees C. At 2 degrees C, [3H]ryanodine failed to dissociate from rat cardiac RyRs, and at 10 degrees C and 20 degrees C, the rate of dissociation was two to three times slower in rat than burbot preparations. The latter finding is compatible with a channel gating mechanism, where the closing of the Ca2+ release channel is impaired or severely retarded by low temperature in rat but less so in burbot preparations. The stronger effect of low temperature on association and dissociation rate of [3H]ryanodine binding in rat compared with burbot suggests that RyRs of the ectothermic fish, unlike those of endothermic rat, are better able to open and close at low temperatures.     

Ultrastructural morphometry of the myocardium of Thunnus alalunga

The common ventricle in the heart of the Thunnus alalunga was studied. The ventricular myocardium consists of an outer compact layer and a thick inner spongy layer. The compact layer has slightly larger cells (4-6 microns diameter) than the spongy layer (2.5-5 microns diameter). Ultrastructurally the myocardium displays normal arrangements of myofibrils and mitochondria. The sarcoplasmic reticulum is poorly developed. The intercalated discs are simple with the fascia adherens being the most frequent junctional type observed; occasionally a desmosome was seen. Nexus type junctions are present but are unassociated with the intercalated discs. There are no t-tubules evident but the plasmalemma exhibits numerous caveolae which rarely form couplings with the sarcoplasmic reticulum. A morphometric analysis of the volume percent of mitochondria and myofibrils showed that the myocardial cells in the spongy layer of the heart have a significantly greater volume percentage of mitochondria than the compact layer. No significant differences were found between myocardial regions when the volume percentages of myofibrils were compared. The physiological studies revealed that the albacore tuna has heart rates (120 bpm) and ventricular blood pressures (100 mmHg) that are among the highest reported for fish.     

Thyroid hormone downregulates the expression and function of sarcoplasmic reticulum-associated CaM kinase II in the rabbit heart

Phosphorylation of sarcoplasmic reticulum (SR) Ca2+-cycling proteins by a membrane-associated Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is a well-documented physiological mechanism for regulation of transmembrane Ca2+ fluxes and the cardiomyocyte contraction-relaxation cycle. The present study investigated the effects of L-thyroxine-induced hyperthyroidism on protein expression of SR CaM kinase II and its substrates, endogenous CaM kinase II-mediated SR protein phosphorylation, and SR Ca2+ pump function in the rabbit heart. Membrane vesicles enriched in junctional SR (JSR) or longitudinal SR (LSR) isolated from euthyroid and hyperthyroid rabbit hearts were utilized. Endogenous CaM kinase II-mediated phosphorylation of ryanodine receptor-Ca2+ release channel (RyR-CRC), Ca2+-ATPase, and phospholamban (PLN) was significantly lower (30-70%) in JSR and LSR vesicles from hyperthyroid than from euthyroid rabbit heart. Western immunoblotting analysis revealed significantly higher (approximately 40%) levels of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) in JSR, but not in LSR, from hyperthyroid than from euthyroid rabbit heart. Maximal velocity of Ca2+ uptake was significantly increased in JSR (130%) and LSR (50%) from hyperthyroid compared with euthyroid rabbit hearts. Apparent affinity of the Ca2+-ATPase for Ca2+ did not differ between the two groups. Protein levels of PLN and CaM kinase II were significantly lower (30-40%) in JSR, LSR, and ventricular tissue homogenates from hyperthyroid rabbit heart. These findings demonstrate selective downregulation of expression and function of CaM kinase II in hyperthyroid rabbit heart in the face of upregulated expression and function of SERCA2 predominantly in the JSR compartment.     

The calcium-ryanodine receptor complex of skeletal and cardiac muscle

[3H]Ryanodine binds with high affinity to saturable and Ca2+-dependent sites in heavy sarcoplasmic reticulum (SR) preparations from rabbit skeletal and cardiac muscle. Ruthenium red, known to interfere with Ca2+-induced Ca2+ release from SR vesicles, inhibits [3H]ryanodine specific binding in both skeletal and cardiac preparations whereas Mg2+, Ba2+, Cd2+ and La3+ selectively inhibit the skeletal preparation. The toxicological relevance of the [3H]ryanodine binding site is established by the correlation of binding inhibition with toxicity for seven ryanoids including two botanical insecticides. These findings provide direct evidence for Ca2+-ryanodine receptor complexes that may play a role in excitation-contraction coupling.     

Alterations of contractility and sarcoplasmic reticulum function of rat heart in experimental hypo- and hyperthyroidism

Myocardial contractility and Ca2+-pump function of sarcoplasmic reticulum (SR) were studied on hearts of untreated, thyroidectomized and thyroxine-treated rats. In hypothyroid rats the contractile force, the maximum velocity of tension development and relaxation significantly decreased (by 73.2%, 68.2%; and 67.8%, respectively), while the time to peak tension was prolonged (by 25.9%) as compared with the control group. In hyperthyroidism opposite changes were found. Since the transport of calcium opposite changes were found. Since the transport of calcium by SR plays an important role in controlling contraction and, first of all, relaxation of muscle, function of the sarcoplasmic reticulum was also investigated under the above experimental conditions. In thyroidectomized rats the rate of Ca2+-uptake and Ca2+-activated ATPase activity of SR significantly decreased (by 31.7% and 61.0%, respectively), while Ca2+-binding remained unchanged. After thyroxine treatment both the Ca2+-uptake and binding capacity of SR were even decreased (by 25.6% and 12.9%, respectively), in spite of an increase in Ca2+-activated ATPase activity (by 67.3%). These changes in Ca2+ transport function of cardiac SR may only partially be responsible for the abnormalities in contraction and relaxation observed in hearts from hypo- and hyperthyroid rats.     

Excitation-contraction coupling in isolated adult ventricular myocytes from the rat, dog, and rabbit: effects of various inotropic interventions in the presence of ryanodine

Enzymatically isolated ventricular cells from rats, dogs, and rabbits were electrically stimulated and their membrane potentials were recorded simultaneously with their contractions. Specific pharmacological interventions were used to assess the relative roles of transsarcolemmal Ca2+ entry and the Ca2+ release by the sarcoplasmic reticulum in activating contractions, in these myocytes. We used ryanodine and caffeine to influence Ca2+ release by the sarcoplasmic reticulum, BAY K 8644 and epinephrine to increase Ca2+ entry through Ca2+ channels, and veratridine, ouabain, and monensin to increase Ca2+ entry through Na+-Ca2+ exchange. Ryanodine (1 microM) completely inhibited the shortenings in rat and dog myocytes, but the contractions in rabbit myocytes were much less sensitive to this alkaloid. Similar inhibitory effects of ryanodine were observed in the presence of various inotropic agents with two exceptions: caffeine's effect on the dog myocytes was relatively insensitive to ryanodine and the long-lasting tonic contractions that veratridine triggered in the myocytes of all three species remained completely unaffected by ryanodine. The data indicate that contractile activation in rat and dog ventricular cells is strongly dependent on Ca2+ release from the sarcoplasmic reticulum, while contractility in rabbit myocytes seems to be more dependent on Ca2+ entry through the sarcolemma. The ryanodine-resistant tonic contractions triggered in the myocytes of all three species in the presence of veratridine may be activated by an increased Ca2+ entry via Na+-Ca2+ exchange.     

Thyroid hormone markedly increases the mRNA coding for sarcoplasmic reticulum Ca2+-ATPase in the rat heart

Previous findings have shown that thyroid hormone markedly increases the speed of diastolic relaxation in the heart. This thyroid hormone-dependent change is also accompanied by an increased Ca2+ pumping ability in the sarcoplasmic reticulum. In an effort to determine the underlying cause of improved Ca2+ transport, mRNA levels of the slow Ca2+-ATPase of the sarcoplasmic reticulum were quantified on Northern blots. In hypothyroid rat hearts, the steady state level of Ca2+-ATPase mRNA was only 36% of control levels, whereas hyperthyroid rat heart mRNA levels were 136% of control. Ca2+-ATPase mRNA responded rapidly to T3, as the mRNA level was significantly increased by 2 h and normalized by 5 h after T3 injection into hypothyroid rats. The well established effect of thyroid hormone on improved myocardial contractility and increased speed of diastolic relaxation may in part relate to specific alterations in the level of the mRNA coding for Ca2+-ATPase, resulting in increased pump units.     

Overexpression of SERCA2b in the heart leads to an increase in sarcoplasmic reticulum calcium transport function and increased cardiac contractility

The sarcoplasmic reticulum calcium ATPase SERCA2b is an alternate isoform encoded by the SERCA2 gene. SERCA2b is expressed ubiquitously and has a higher Ca(2+) affinity compared with SERCA2a. We made transgenic mice that overexpress the rat SERCA2b cDNA in the heart. SERCA2b mRNA level was approximately approximately 20-fold higher than endogenous SERCA2b mRNA in transgenic hearts. SERCA2b protein was increased 8-10-fold in the heart, whereas SERCA2a mRNA/protein level remained unchanged. Confocal microscopy showed that SERCA2b is localized preferentially around the T-tubules of the SR, whereas SERCA2a isoform is distributed both transversely and longitudinally in the SR membrane. Calcium-dependent calcium uptake measurements showed that the maximal velocity of Ca(2+) uptake was not changed, but the apparent pump affinity for Ca(2+) (K(0.5)) was increased in SERCA2b transgenic mice (0.199 +/- 0.011 micrometer) compared with wild-type control mice (0.269 +/- 0.012 micrometer, p < 0.01). Work-performing heart preparations showed that SERCA2b transgenic hearts had a higher rates of contraction and relaxation, shorter time to peak pressure and half-time for relaxation than wild-type hearts. These data show that SERCA2b is associated in a subcompartment within the sarcoplasmic reticulum of cardiac myocytes. Overexpression of SERCA2b leads to an increase in SR calcium transport function and increased cardiac contractility, suggesting that SERCA2b plays a highly specialized role in regulating the beat-to-beat contraction of the heart.     

Identification and characterization of the high affinity [3H]ryanodine receptor of the junctional sarcoplasmic reticulum Ca2+ release channel

The high affinity ryanodine receptor of the Ca2+ release channel from junctional sarcoplasmic reticulum of rabbit skeletal muscle has been identified and characterized using monoclonal antibodies. Anti-ryanodine receptor monoclonal antibody XA7 specifically immunoprecipitated [3H]ryanodine-labeled receptor from digitonin-solubilized triads in a dose-dependent manner. [3H]Ryanodine binding to the immunoprecipitated receptor from unlabeled digitonin-solubilized triads was specific, Ca2+-dependent, stimulated by millimolar ATP, and inhibited by micromolar ruthenium red. Indirect immunoperoxidase staining of nitrocellulose blots of various skeletal muscle membrane fractions has demonstrated that anti-ryanodine receptor monoclonal antibody XA7 recognizes a high molecular weight protein (approximately 350,000 Da) which is enriched in isolated triads but absent from light sarcoplasmic reticulum vesicles and transverse tubular membrane vesicles. Thus, our results demonstrate that monoclonal antibodies to the approximately 350,000-Da junctional sarcoplasmic reticulum protein immunoprecipitated the ryanodine receptor with properties identical to those expected for the ryanodine receptor of the Ca2+ release channel.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

Characterization of junctional and longitudinal sarcoplasmic reticulum from heart muscle

Longitudinal tubules and junctional sarcoplasmic reticulum (SR) were prepared from heart muscle microsomes by Ca2+-phosphate loading followed by sucrose density gradient centrifugation. The longitudinal SR had a high Ca2+ loading rate (0.93 +/- 0.08 mumol.mg-1.min) which was unchanged by addition of ruthenium red. Junctional SR had a low Ca2+ loading rate (0.16 +/- 0.02 mumol.mg-1.min) which was enhanced about 5-fold by ruthenium red. Junctional SR had feet structures observed by electron microscopy and a high molecular weight protein with Mr of 340,000, whereas longitudinal SR was essentially devoid of both. Thus, these subfractions have similar characteristics to longitudinal and junctional terminal cisternae of SR from fast twitch skeletal muscle. Ryanodine binding was localized to junctional cardiac SR as determined by [3H]ryanodine binding. Scatchard analysis of the binding data showed two types of binding (high affinity, Kd approximately 7.9 nM; low affinity, Kd approximately 1 microM), contrasting with skeletal junctional terminal cisternae where only one site with Kd of approximately 50 nM was observed. The ruthenium red enhancement of Ca2+ loading rate in junctional cardiac SR was blocked by pretreatment with low concentrations of ryanodine as reported for junctional terminal cisternae of skeletal muscle SR. The Ca2+ loading rate of junctional cardiac SR was enhanced by preincubation with high concentrations of ryanodine. The apparent inhibition constant (Ki approximately 7 nM) and stimulation constant (Km approximately 1.1 microM) for ryanodine on junctional SR corresponded to the Kd for high affinity binding (Kd approximately 7.9 nM) and low affinity binding (Kd approximately 1.1 microM), respectively. These results suggest that high affinity ryanodine binding locks the Ca2+ release channels in the open state and that low affinity binding closes the Ca2+ release channels of the junctional cardiac SR. The characteristics of the Ca2+ release channels of junctional cardiac SR appear to be similar to that of skeletal muscle SR, but the Ca2+ release channels of cardiac SR are more sensitive to ryanodine.     

Excitation-contraction coupling in heart muscle

We have investigated the links between electrical excitation and contraction in mammalian heart muscle. Using isolated single cells from adult rat ventricle, a whole-cell voltage-clamp technique and quantitative fluorescence microscopy, we have measured simultaneously calcium current (I_ca) and [Ca²⁺]_i (with fura-2). We find that the voltage-dependence of Ica and the [Ca ²⁺]_i-transient and the dependence of [Ca²⁺]_i-transient on depolarization-duration cannot both be readily explained by a simple calcium-induced Ca-release (CICR) mechanism. Additionally, we find that when [Ca²⁺]_i and [Na⁺]_i are at their diastolic levels, activation of the Na-Ca exchange mechanism by depolarization does not measurably trigger the release of Ca²⁺i. Finally, measuring I_ca in adult and neonatal rat heart cells and using the alkaloid ryanodine, we have carried out complementary experiments. These experiments show that there may be an action of ryanodine on I_ca that is independent of [Ca²⁺]_i and independent of a direct action of the alkaloid on the calcium channel itself. Along with experiments of others showing that ryanodine binds to the sarcoplasmic reticulum calcium-release channel/spanning protein complex, our data suggests a model to explain our findings. The model links the calcium channels responsible for Ica to the sarcoplasmic reticulum by means of one or more of the spanning protein(s). Information from the calcium channel can be communitated to the sarcoplasmic reticulum by this route and, presumably, information can move in the opposite direction from the sarcoplasmic reticulum to the calcium channel.     

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.     

Cold adaptation suppresses the contractility of both atrial and ventricular muscle of the crucian carp heart

Adaptation to low temperature in crucian carp Carasius carassius increases twitch duration both in atrial and ventricular muscle and the response is largely explained by a temperatureinduced reduction in myofibrillar ATPase activity. The prolonged twitch increases the refractoriness of both atrial and ventricular muscle, i.e. the optimal force development is achieved at longer diastolic intervals after acclimation to cold. The contractions of atrial and ventricular muscle are insensitive to 10 μ ryanodine, an inhibitor of SR release Ca²+ channel but sensitive to 20 μ verapamil, a blocker of L-type Ca²+ channels. This suggests that E-C-coupling in the crucian carp heart is exclusively based on extracellular Ca²⁺ sources. The present findings indicate that cold-acclimation slows the contraction kinetics of the crucian carp heart and thereby preconditions the cardiac muscle for a low energy supply during winter anoxic conditions.