Normal Sarcoplasmic Reticulum Research Articles

A new cardioprotective agent, JTV519, improves defective channel gating of ryanodine receptor in heart failure.

Defective interaction between FKBP12.6 and ryanodine receptors (RyR) is a possible cause of cardiac dysfunction in heart failure (HF). Here, we assess whether the new cardioprotective agent JTV519 can correct it in tachycardia-induced HF. HF was induced in dogs by 4-wk rapid ventricular pacing, and sarcoplasmic reticulum (SR) was isolated from left ventricular muscles. In failing SR, JTV519 increased the rate of Ca(2+) release and [(3)H]ryanodine binding. RyR were then labeled in a site-directed fashion with the fluorescent conformational probe methylcoumarin acetamide. In failing SR, the polylysine induced a rapid change in methylcoumarin acetamide fluorescence, presumably because the channel opening preceding the Ca(2+) release was smaller than in normal SR (consistent with a decreased rate of Ca(2+) release in failing SR), and JTV519 increased it. In conclusion, JTV519, a new 1,4-benzothiazepine derivative, corrected the defective channel gating in RyR (increase in both the rapid conformational change and the subsequent Ca(2+) release rate) in HF.

Propranolol prevents the development of heart failure by restoring FKBP12.6-mediated stabilization of ryanodine receptor.

In heart failure, protein kinase A-mediated hyperphosphorylation of ryanodine receptors (RyRs) in sarcoplasmic reticulum (SR) causes dissociation of FKBP12.6 from RyRs. This results in an abnormal Ca2+ leak through RyRs, possibly leading to cardiac dysfunction. In the present study, we assess whether beta-blockers can correct this defect in RyR in tachycardia-induced heart failure and thereby improve cardiac function. SRs were isolated from dog left ventricular muscles (normal group, 4 weeks of rapid right ventricular pacing with or without propranolol [P(+) or P(-)]). End-diastolic and end-systolic diameters both increased less in P(+) than P(-), associated with a smaller decrease in fractional shortening in P(+). In SR from P(-), a prominent Ca2+ leak was observed, and FK506 (which dissociates FKBP12.6 from RyR) did not induce an additional Ca2+ leak. However, there was no appreciable Ca2+ leak in SR from P(+), although FK506 induced a Ca2+ leak as in normal SRs. In SR from P(+), an FK506-induced conformational change in RyR, which was virtually absent in SR from P(-), was observed as in normal SRs. Both the stoichiometry of FKBP12.6 versus RyR, assessed by [3H]FK506 and [3H]ryanodine binding assays, and the protein expression of FKBP12.6, assessed by Western blot analysis, were restored by propranolol toward the levels seen in normal SRs. Low-dose propranolol corrects the defective interaction of FKBP12.6 with RyR (restoration of RyR conformational change and prevention of Ca2+ leak from RyR), apparently resulting in an attenuation of intracellular Ca2+ overload and hence preventing the development of left ventricular remodeling in heart failure.

Divergent Effects of the Malignant Hyperthermia-Susceptible Arg 615→Cys Mutation on the Ca 2+ and Mg 2+ Dependence of the RyR1

The sarcoplasmic reticulum (SR) Ca 2+ release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg 2+. This diminished Mg 2+ inhibition has been attributed to a lower Mg 2+ affinity of the inhibition (I) site. To determine whether alterations in the Ca 2+ and Mg 2+ affinity of the activation (A) site contribute to the altered Mg 2+ inhibition, we estimated the Ca 2+ and Mg 2+ affinities of the A- and I-sites of normal and MHS RyR1. Compared with normal SR, MHS SR required less Ca 2+ to half-maximally activate [ 3H]ryanodine binding ( K A,Ca: MHS = 0.17 ± 0.01 μM; normal = 0.29 ± 0.02 μM) and more Ca 2+ to half-maximally inhibit ryanodine binding ( K I,Ca: MHS = 519.3 ± 48.7 μM; normal = 293.3 ± 24.2 μM). The apparent Mg 2+ affinity constants of the MHS RyR1 A- and I-sites were approximately twice those of the A- and I-sites of the normal RyR1 ( K A,Mg: MHS = 44.36 ± 4.54 μM; normal = 21.59 ± 1.66 μM; K I,Mg: MHS = 660.8 ± 53.0 μM; normal = 299.2 ± 24.5 μM). Thus, the reduced Mg 2+ inhibition of the MHS RyR1 compared with the normal RyR1 is due to both an enhanced selectivity of the MHS RyR1 A-site for Ca 2+ over Mg 2+ and a reduced Mg 2+ affinity of the I-site.

Arg 615Cys Substitution in Pig Skeletal Ryanodine Receptors Increases Activation of Single Channels by a Segment of the Skeletal DHPR II-III Loop

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca 2+ release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr 671-Leu 690; 36 μM) enhanced the rate of Ca 2+ release from normal SR (SR N) and from SR of MH-susceptible muscle (SR MH) by 10 ± 3.2 nmole/mg/min and 76 ± 9.7 nmole/mg/min, respectively. Ca 2+ release from SR N or SR MH was not increased by control peptide NB (Gly 689-Lys 708). AS (scrambled A sequence; 36 μM) did not alter Ca 2+ release from SR N, but increased release from SR MH by 29 ± 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR MH) were up to about fourfold more strongly activated by peptide A (≥1 nM) than normal RyR channels (RyR N) at −40 mV. Neither NB or AS activated RyR N. RyR MH showed an ∼1.8-fold increase in mean current with 30 μM AS. Inhibition at +40 mV was stronger in RyR MH and seen with peptide A (≥0.6 μM) and AS (≥0.6 μM), but not NB. These results show that the Arg 615Cys substitution in RyR MH has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca 2+ release from SR in MH-susceptible muscle.

Altered stoichiometry of FKBP12.6 versus ryanodine receptor as a cause of abnormal Ca(2+) leak through ryanodine receptor in heart failure.

In the pathogenesis of cardiac dysfunction in heart failure, a decrease in the activity of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase is believed to be a major determinant. Here, we report a novel mechanism of cardiac dysfunction revealed by assessing the functional interaction of FK506-binding protein (FKBP12.6) with the cardiac ryanodine receptor (RyR) in a canine model of pacing-induced heart failure. SR vesicles were isolated from left ventricular muscles (normal and heart failure). The stoichiometry of FKBP12.6 per RyR was significantly decreased in failing SR, as assessed by the ratio of the B(max) values for [(3)H]dihydro-FK506 to those for [(3)H]ryanodine binding. In normal SR, the molar ratio was 3.6 ( approximately 1 FKBP12.6 for each RyR monomer), whereas it was 1.6 in failing SR. In normal SR, FK506 caused a dose-dependent Ca(2+) leak that showed a close parallelism with the conformational change in RyR. In failing SR, a prominent Ca(2+) leak was observed even in the absence of FK506, and FK506 produced little or no further increase in Ca(2+) leak and only a slight conformational change in RyR. The level of protein expression of FKBP12.6 was indeed found to be significantly decreased in failing SR. An abnormal Ca(2+) leak through the RyR is present in heart failure, and this leak is presumably caused by a partial loss of RyR-bound FKBP12.6 and the resultant conformational change in RyR. This abnormal Ca(2+) leak might possibly cause Ca(2+) overload and consequent diastolic dysfunction, as well as systolic dysfunction.

Activation and Inhibition of Skeletal RyR Channels by a Part of the Skeletal DHPR II-III Loop: Effects of DHPR Ser687 and FKBP12

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2>A1>A1S>NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by ∼30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at −40mV, with 10μM A1 or 50nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100nM cis Ca2+. Inhibition by A1 was greatest at +40mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100μM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.

The action of perchlorate on malignant-hyperthermia-susceptible muscle.

To better understand the altered skeletal muscle excitation-contraction (E-C) coupling that occurs in malignant hyperthermia, we have examined the potentiating actions of perchlorate in intact muscle fiber bundles, isolated sarcoplasmic reticulum (SR) vesicles, and the purified ryanodine receptor/Ca2+ release channel (RyR) isolated from malignant-hyperthermia-susceptible (MHS) and normal porcine muscle. The concentration of perchlorate that half-maximally potentiated twitch tension (2.5-3.5 mM) was not significantly different for MHS and normal muscles. The effect of perchlorate on fractional twitch force was significantly greater for normal than for MHS muscle, although the absolute twitch potentiation was similar for both muscle types. The K-contracture threshold of MHS muscle bundles is significantly lower than that of normal bundles; perchlorate shifted the K-contraction activation curves of both MHS and normal muscle bundles to lower K+ concentrations. Perchlorate both increased ryanodine binding to MHS and normal SR vesicles and increased single-channel open probability of the purified MHS and normal RyR. In both cases, the percentage increase was greater for normal than for MHS preparations; however, the absolute increase in activity was not different for MHS and normal RyR indicating that there is no difference in the perchlorate sensitivity of MHS and normal SR Ca2+ release channels. Thus, the greater absolute responses of the MHS Ca2+ release channel in the presence of perchlorate is likely to be due to the greater basal activity of the MHS release channel and does not reflect an underlying defect in the site of action of perchlorate on the MHS skeletal muscle Ca2+ release channel.

Downregulation of sarcoplasmic reticulum Ca(2+)-ATPase during progression of left ventricular hypertrophy.

To determine whether reduced sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase) (SERCA2) activity contributes to delayed myocardial relaxation during chronic left ventricular hypertrophy (LVH) progression, LVH was produced in rats by abdominal aortic coarctation. Systolic and diastolic functions were assessed in vivo 8 and 16 wk after surgery, and compositional alterations in LV myocardium [SERCA2 concentration, myosin heavy chain (MHC) isoenzymes, and tissue collagen] were correlated with the development of prolonged isovolumic relaxation and impaired cardiac performance over time. Myocardial relaxation was prolonged in 8-wk banded rats, despite normal isovolumic systolic function and LV end-diastolic pressure (LVEDP). No significant alterations in SERCA2 protein, beta-MHC, or fibrillar collagen levels were observed at this early time point. In contrast, LV SERCA2, beta-MHC, and fibrillar collagen concentrations were all significantly altered in 16-wk banded rats. These late compositional changes were associated with reduced cardiac performance, as manifested by a significant elevation in LVEDP (14 +/- 2 mmHg). The 34% decrease in SERCA2 protein was associated with reduced SR Ca2+ uptake and an even greater reduction (76%) in SERCA2 mRNA. SERCA2 mRNA levels were also significantly reduced to 43 +/- 10% of sham-operated rats 8 wk after banding, despite unchanged SERCA2 protein levels and normal SR Ca2+ uptake. These results argue against a significant contribution of SERCA2 downregulation to the subtle alterations in myocardial relaxation observed in compensated LVH. However, the early reduction in SERCA2 mRNA levels may serve as a molecular marker for impaired cardiac performance during the transition from compensated LVH to heart failure.

Effects of azumolene on Ryanodine binding to sarcoplasmic reticulum of normal and malignant hyperthermia susceptible swine skeletal muscles

Dantrolene is a primary specific therapeutic drug for prevention and treatment of malignant hyperthermia symptoms. The mechanisms underlying the therapeutic effects of the drug are not well understood. The present study aimed at the characterization of the effects of azumolene, a water soluble dantrolene analogue, on ryanodine binding to sarcoplasmic reticulum (SR) from normal and malignant hyperthermia susceptible (MHS) swine muscles. Characteristics of [3H]ryanodine binding were clearly different between the two types of SR. Kinetic analysis of [3H]ryanodine binding to SR in the presence of 2 μM Ca2+ showed that association constant (Kryanodine) is significantly higher in MHS than normal muscle SR (2.83 vs. 1.32×107 M‐1), whereas the maximal ryanodine binding capacity (Bmax) is similar between the two types of SR. Addition of azumolene (e.g. 400μM) did not significantly alter both Kryanodine and Bmax of [3H]ryanodine binding in both types of SR, indicating that the azumolene effect was not on the ryanod...

Decreased inotropic response to beta-adrenergic stimulation and normal sarcoplasmic reticulum calcium stores in the spontaneously hypertensive rat heart

Cardiac hypertrophy in the spontaneously hypertensive rat has been shown to be accompanied by a diminished inotropic response to beta-adrenergic stimulation. This diminished response has been attributed to abnormalities in various components of the beta-adrenergic signaling system. There is also evidence that regulation of intracellular Ca2+ cycling may be altered in the hypertrophied heart of the spontaneously hypertensive rat. We proposed that the diminished response to beta-adrenergic stimulation may reflect abnormalities in Ca2+ cycling, specifically alterations in the ability of the sarcoplasmic reticulum to effectively release and resequester Ca2+. We have used the unique combination of functional measurements on isolated, isometrically contracting papillary muscles from hearts of 26-week-old spontaneously hypertensive rats and their Wistar-Kyoto controls, together with electron probe microanalysis measurements of sarcoplasmic reticulum Ca2+ content in the same muscles after rapid freezing, to determine the availability of Ca2+ for activation of contraction, following beta-adrenergic stimulation. We observed a significant decrease in the inotropic response to beta-adrenergic stimulation in papillary muscles from the spontaneously hypertensive rats, as compared with Wistar-Kyoto controls, however in these same muscles, frozen during relaxation, there was no evidence of an accompanying decrease in the size of the sarcoplasmic reticulum Ca2+ store. In an additional group of muscles which were frozen during contraction, the amount of Ca2+ remaining in the sarcoplasmic reticulum after stimulated release was also not different in the two strains. These results indicate that the decreased inotropic response to beta-adrenergic stimulation in hypertrophied hearts of the spontaneously hypertensive rat is unlikely to be due to decreased availability of Ca2+ for activation of contraction. Additionally, to determine whether there is intracellular Ca2+ overload in the cardiac muscle cells of hearts of spontaneously hypertensive rats, we measured the amount of Ca2+ in mitochondrial and at the level of the myofilaments by electron probe microanalysis. These results indicate that intracellular Ca2+ overload does not accompany cardiac hypertrophy in the spontaneously hypertensive rat. This study therefore shows no correlation between altered intracellular Ca2+ cycling and the decreased inotropic response to isoproterenol in the spontaneously hypertensive rat at 26 weeks of age.

Kinetics of thapsigargin-Ca(2+)-ATPase (sarcoplasmic reticulum) interaction reveals a two-step binding mechanism and picomolar inhibition.

Thapsigargin is a high affinity inhibitor of sarco- and endoplasmic reticulum (SERCA) type ATPases. We have used kinetics to determine the dissociation constant of thapsigargin-sarcoplasmic reticulum Ca(2+)-ATPase interaction in the absence and presence of non-ionic detergent. The observed "off" rate constant was measured as 0.0052 s-1 at 26 degrees C by the kinetics of inhibition of ATPase activity following transfer from an inactivated thapsigargin-ATPase complex to native ATPase. Inactive ATPase was produced by cross-linking the active site with glutaraldehyde. The observed dissociation rate constant was increased 7-fold by 0.1% Triton X-100, indicating that perturbation of the transmembrane and stalk region by detergent altered the binding parameters of the inhibitor. In addition, thapsigargin stabilized the ATPase against inactivation caused by detergent in the absence of Ca2+. The observed "on" rate constant of thapsigargin was measured at 26 degrees C as 25 s-1 irrespective of thapsigargin concentration, by the kinetics of thapsigargin- induced change in intrinsic fluorescence. An Arrhenius plot showed a temperature dependence of this rate constant, indicative of a conformational change in the protein with an activation energy of 9.5 kcal/mol for thapsigargin binding. The affinity of the Ca(2+)-ATPase for thapsigargin was calculated to be greater than 2 pM at pH 7.0 and 26 degrees C.

Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes

The release of sarcoplasmic reticulum (SR) Ca in cardiac muscle during excitation-contraction coupling is known to be graded by the amount of activating Ca outside the SR (i.e., Ca-induced Ca release). However, little is known about how intra-SR Ca affects the release process. In this study we assessed how the fractional SR Ca release as described by Bassani et al. [Am. J. Physiol. 265 (Cell Physiol. 34): C533-C540, 1993] is affected by alteration of trigger Ca and of SR Ca content. Experiments were done with isolated ferret ventricular myocytes using indo 1 to measure Ca concentration, perforated patch to measure Ca current (ICa), caffeine application to release SR Ca, and thapsigargin to completely block SR Ca uptake. For what we consider a Normal SR Ca load and trigger Ca [action potential at 0.5 Hz with 2 mM extracellular Ca concentration ([Ca]o)], 35 +/- 3% of the SR Ca content was released at a twitch. Changing trigger Ca by altering [Ca]o (to 0.5 and 8 mM) at a test twitch changed this fractional SR Ca release to 10 +/- 2 and 59 +/- 6%, with the same SR Ca load (and peak ICa changed in a parallel manner in separate voltage-clamp experiments). Three different levels of SR Ca load were studied (Low, Normal, and High; by action potential stimulation at different frequencies from 0.05 to 0.8 Hz) using the same standard test trigger Ca (2 mM). Surprisingly, the High-load condition only increased SR Ca content by approximately 4% but appeared to be very close to the limiting SR Ca capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

770-4 Single Channel Characteristics of Sarcoplasmic Reticulum Calcium Release Channels from Normal and Failed Human Myocardium

Ryanodine receptors or sarcoplasmic reticulum (SR) Ca-release channels playa key role in normal cardiac muscle excitation-contraction coupling. The single channel behavior of the Ca++-release channel in failing human hearts has not been investigated. We compared characteristics of normal and heart failure cardiac SR calcium channels, using the planar lipid bilayer technique. Membrane vesicles were prepared from freshly explanted human hearts either prior to transplantation, or from organ donors with normal hearts not accepted for transplantation (because of the age of the donor). 1.0 μg/ml of membrane protein was used in asymmetric bath solutions: CIS (125 mM TRIS; 250 mM HEPES; 2 uM free calcium) TRANS (250 mM HEPES; 50 mM BaCI2). Lipid bilayers were formed with 5:3 ratio of phosphatidylethanolamine and phosphatidylserine. Data were digitized and analyzed with PCLAMP6 software. Normal heart SR Ca release channels exhibited a slope conductance of 153 pS over the range of voltages from 0 to -60 mV SR Ca release channels from failing hearts exhibited multiple conductance states. the main state had a slope conductance of 500 pS, and a subconductance of 300 pS over the same voltage range. Normal heart open probability range was 0.05–01. In heart failure open state probability was increased. ranging from 0.1–0.5. Single channel properties of SR Ca release channels from the failing hearts are significantly different than normal hearts. This work is the first attempt to investigate the possible role of SR Ca channel derangements in human heart failure.

Sodium-calcium exchange-mediated contractions in feline ventricular myocytes.

The hypothesis that Ca entry by the sarcolemmal Na-Ca exchange mechanism induces sarcoplasmic reticulum (SR) Ca release, loads the SR with Ca, and/or directly induces contractions by elevating cytosolic free Ca was tested in voltage-clamped feline ventricular myocytes. Intracellular Na concentration was increased by cellular dialysis to enhance Ca influx via "reverse-mode" Na-Ca exchange at positive membrane potentials, at which the "L-type" Ca current (ICa) should be small. Contractions were induced in the presence of Ca channel antagonists by depolarization to these potentials, suggesting that Ca influx via reverse-mode Na-Ca exchange was involved. These contractions had both phasic (SR related) and tonic components of shortening. They were smaller and began with more delay after depolarization than contractions which involved ICa. The magnitude of shortening was graded by the amount and duration of depolarization, suggesting that Ca influx via reverse-mode Na-Ca exchange has the capacity to induce and grade SR Ca release. Small slow contractions could be evoked in the presence of ryanodine (to impair SR function) and verapamil (to block ICa), supporting the idea that Ca influx via Na-Ca exchange is sufficient to directly activate the contractile proteins. Contractions induced by voltage steps to +10 mV, which were usually small when ICa was blocked, were potentiated if preceded by a voltage step to strongly positive potentials. This potentiation was inhibited by ryanodine, suggesting that Ca entry that occurs by Na-Ca exchange may be important for normal SR Ca loading.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of volatile anesthetics on calcium regulation by malignant hyperthermia-susceptible sarcoplasmic reticulum.

To clarify the mechanism by which volatile anesthetics initiate malignant hyperthermia (MH), we examined the effect of halothane, isoflurane, and enflurane on Ca2+ uptake and release by sarcoplasmic reticulum vesicles isolated from MH-susceptible (MHS) and normal pig muscle. Clinical concentrations of these anesthetics (0.1-0.5 mM) stimulated sarcoplasmic reticulum ATP-dependent Ca2+ uptake (maximal at approximately 4 mM), whereas 10-20 times the clinical anesthetic concentration inhibited Ca2+ uptake. There was no significant difference between MHS and normal sarcoplasmic reticulum in any aspect of Ca2+ uptake. Ca2+ release from 45Ca(2+)-filled sarcoplasmic reticulum vesicles in a 10(-8) M Ca(2+)-containing medium (pH 7.0) was significantly stimulated at clinical concentrations of all three volatile anesthetics (anesthetic concentration for the 50% stimulation of Ca2+ release = 0.096-0.22 mM); however, the rate constant for Ca2+ release from MHS sarcoplasmic reticulum was in all cases significantly greater than that from normal sarcoplasmic reticulum. Furthermore, 0.5 mM halothane had no effect on Ca2+ release from normal sarcoplasmic reticulum at pH values less than 6.8, although it could still significantly stimulate Ca2+ release from MHS sarcoplasmic reticulum even at pH 6.4; similar results were obtained for isoflurane and enflurane. These studies thus demonstrate that the interaction of volatile anesthetics with the sarcoplasmic reticulum Ca(2+)-release channel is altered in MHS porcine muscle such that the channel may be activated even at a Ca2+ concentration or pH that would be expected to maintain the channel in the closed state.

Excitation-contraction coupling in pigs heterozygous for malignant hyperthermia.

A defect in the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel of malignant hyperthermia-susceptible (MHS) pigs greatly enhances SR calcium release in pigs homozygous for the malignant hyperthermia (MH) gene. In pigs heterozygous at this locus, rates of calcium release from isolated SR stimulated by Ca2+, ATP, or caffeine are intermediate to those of both MHS and normal SR [Mickelson et al. Am. J. Physiol. 257 (Cell Physiol. 26): C787-C794, 1989]. In this study bundles of intact muscle cells dissected from pigs of various genotypes were used to examine the effects of the MH gene on contractile responses to caffeine (direct stimulation of the SR) or to surface membrane (sarcolemma) depolarization (i.e., stimulation by way of the steps in excitation-contraction coupling). The caffeine threshold for contractures in the heterozygous muscles (5 mM) was intermediate to both types of homozygous muscles (2 mM for MHS and 10 mM for normal) as is the case with direct stimulation of calcium release from SR vesicles [Mickelson et al. Am. J. Physiol. 257 (Cell Physiol. 26): C787-C794, 1989]. Sarcolemmal depolarization was elicited by electrical stimuli or elevated extracellular potassium. Control twitch tension for MHS and heterozygous muscles did not differ and was significantly greater in both than in homozygous normal muscles. Potassium-induced contractures were significantly larger in MHS and heterozygous than in normal muscles. Thus, in heterozygous muscles, force production via sarcolemmal depolarization (twitches and potassium contractures) was enhanced as much as in homozygous MHS muscles. This could be the result of feedback from abnormal SR calcium channels producing altered (enhanced) transverse tubule to SR signal transduction.

Ryanodine receptor in different malignant hyperthermia-susceptible porcine muscles

The sarcoplasmic reticulum (SR) ryanodine receptor was studied in SR vesicles isolated from the vastus intermedius skeletal muscle and cardiac muscle of malignant hyperthermia-susceptible (MHS) and normal pigs. MHS and normal heavy SR preparations isolated from the vastus intermedius muscle had similar yields, polyacrylamide gel electrophoretic patterns, Ca2(+)-ATPase activities, mitochondrial enzyme activities, calsequestrin contents, and maximal [3H]ryanodine-binding activities. However, while half-maximal calcium concentrations (Ca0.5) for stimulation of MHS and normal vastus intermedius SR [3H]ryanodine binding were not significantly different, the Ca0.5 for inhibition of [3H]ryanodine binding to MHS vastus intermedius SR (76 +/- 17 microM) was significantly greater than to normal SR (16 +/- 9 microM). MHS vastus intermedius SR also exhibited a significantly lower Kd value (62 +/- 15 nM) for [3H]ryanodine binding compared with normal SR (Kd = 284 +/- 102 nM). These values for MHS and normal vastus intermedius SR are similar to those reported using SR isolated from a muscle composed of predominantly fast-twitch fibers, indicating the similarity of the ryanodine receptor in fast- and slow-twitch skeletal muscles. In contrast, there were no differences in the properties of the ryanodine receptor of porcine cardiac SR isolated from MHS and normal pigs. We therefore conclude that there is a defect in the SR ryanodine receptor of both slow- and fast-twitch skeletal muscle fiber types but not in cardiac muscle of MHS individuals.

Abnormal fluidity state in membranes of malignant hyperthermia pig skeletal muscle

The fluidity state was analyzed on sarcoplasmic reticulum membranes and phospholipid vesicles prepared from normal and malignant hyperthermia susceptible pig muscle. Electron spin resonance studies were performed to determine the fluidity state at the region near the polar headgroups and in the central core of the bilayer using 5-nitroxide (5-NS) and 16-nitroxide stearic acid (16-NS), respectively. With the 5-NS label, no differences were found between normal and malignant hyperthermia sarcoplasmic reticulum (MH SR) membranes whereas with the 16-NS label, a significant increase of the activation energy was shown with MH membranes. Lower values of fluorescence anisotropy observed with DPH-labeled MH membranes as compared with normal ones, confirmed the higher abnormal fluidity state of these membranes. The fluidizing effect of halothane, a triggering agent of malignant hyperthermia syndrome, was also studied in these membranes. We show that a relatively low concentration of the drug destabilized not only the diseased sarcoplasmic reticulum membranes but also the vesicles made of total phospholipids extracted from MH skeletal muscle. Together, these findings strongly suggest that an overall increase in membrane fluidity may be implied in the MH disease, improving the general membrane defect hypothesis for this syndrome.

Stimulation and inhibition of [ 3H]ryanodine binding to sarcoplasmic reticulum from malignant hyperthermia susceptible pigs

When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [ 3H]ryanodine-binding activity in the presence of μ m Ca 2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg 2+) of the SR calcium release channel were examined for effects on MHS and normal SR [ 3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 m m ATP ( K d = 6.0 nM) or 10 m m caffeine ( K d = 28 nM) was significantly greater than that of the normal SR ( K d = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively); the B max (12–16 pmol/mg) was similar in all cases. The Ca 0.5 2+ for inhibition of [ 3H]ryanodine binding in the presence of 5 m m AMPPNP (238 vs 74 μ m for MHS and normal SR, respectively) and the Ca 0.5 2+ for stimulation of [[su3H]ryanodine binding in the presence of 5 m m caffeine (0.049 vs 0.070 μ m for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca 2+, MHS SR [ 3H]ryanodine binding was more sensitive to caffeine stimulation ( C 0.5 of 1.7 vs 3.4 m m) and was less sensitive to ruthenium red ( C 0.5 of 1.9 vs 1.2 μ m) or Mg 2+ inhibition ( C 0.5 of 0.34 vs 0.21 m m) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.

Abnormal ryanodine receptor channels in malignant hyperthermia

Previous studies have demonstrated a defect associated with the calcium release mechanism of sarcoplasmic reticulum (SR) from individuals susceptible to malignant hyperthermia (MH). To examine whether SR calcium release channels were indeed altered in MH, SR vesicles were purified from normal and MH susceptible (MHS) porcine muscle. The Ca2+ dependence of calcium efflux rates from 45Ca2(+)-filled SR vesicles was then compared with the Ca2+ dependence of single-channel recordings of SR vesicles incorporated into planar lipid bilayers. The rate constants of 45Ca2+ efflux from MHS SR were two to threefold larger than from normal SR over a wide range of myoplasmic Ca2+. Normal and MHS single channels were progressively activated in a similar fashion by cis Ca2+ from pCa 7 to 4. However, below pCa 4, normal channels were inactivated by cis Ca2+, whereas MHS channels remained open for significantly longer times. The altered Ca2+ dependence of channel inactivation in MHS SR was also evident when Ca2+ was increased on the trans side while cis Ca2+ was held constant. We propose that a defect in a low-affinity Ca2+ binding site is responsible for the altered gating of MHS SR channels. Such a defect could logically result from a mutation in the gene encoding the calcium release channel, providing a testable hypothesis for the molecular basis of this inherited disorder.