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In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca2+/calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca2+ alternans. The aim of this study was to test whether the suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca2+ alternans. We studied spontaneous Ca2+ release events and Ca2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca2+ release events in RyR2-R2474S cardiomyocytes exposed to the β-adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca2+ alternans and increased Ca2+ alternans ratio compared with both an inactive analog of KN-93 and with vehicle-treated controls. This increased propensity for Ca2+ alternans was explained by prolongation of Ca2+ release refractoriness. Importantly, the increased propensity for Ca2+ alternans in KN-93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild type. In conclusion, inhibition of CaMKII efficiently reduces spontaneous Ca2+ release but promotes Ca2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca2+ release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca2+ alternans should include investigation of both phenomena.NEW & NOTEWORTHY Genetically increased RyR2 activity promotes arrhythmogenic Ca2+ release. Inhibition of CaMKII suppresses RyR2 activity and arrhythmogenic Ca2+ release. Suppression of RyR2 activity prolongs refractoriness of Ca2+ release. Prolonged refractoriness of Ca2+ release leads to arrhythmogenic Ca2+ alternans. CaMKII inhibition promotes Ca2+ alternans by prolonging Ca2+ release refractoriness.

Previous results from our laboratory showed that phosphorylation of ryanodine receptor 2 (RyR2) by Ca21 calmodulin-dependent kinase II (CaMKII) was a critical but not the unique event responsible for the production of reperfusion-induced arrhythmogenesis, suggesting the existence of other mechanisms cooperating in an additive way to produce these rhythm alterations. Oxidative stress is a prominent feature of ischemia/reperfusion injury. Both CaMKII and RyR2 are proteins susceptible to alteration by redox modifications. This study was designed to elucidate whether CaMKII and RyR2 redox changes occur during reperfusion and whether these changes are involved in the genesis of arrhythmias. Langendorff-perfused hearts from rats or transgenic mice with genetic ablation of CaMKII phosphorylation site on RyR2 (S2814A) were subjected to ischemia-reperfusion in the presence or absence of a free radical scavenger (mercaptopropionylglycine, MPG) or inhibitors of NADPH oxidase and nitric oxide synthase. Left ventricular contractile parameters and monophasic action potentials were recorded. Oxidation and phosphorylation of CaMKII and RyR2 were assessed. Increased oxidation of CaMKII during reperfusion had no consequences on the level of RyR2 phosphorylation. Avoiding the reperfusion-induced thiol oxidation of RyR2 with MPG produced a reduction in the number of arrhythmias and did not modify the contractile recovery. Conversely, selective prevention of S-nitrosylation and S-glutathionylation of RyR2 was associated with higher numbers of arrhythmias and impaired contractility. In S2814A mice, treatment with MPG further reduced the incidence of arrhythmias. Taken together, the results suggest that redox modification of RyR2 synergistically with CaMKII phosphorylation modulates reperfusion arrhythmias., Centro de Investigaciones Cardiovasculares, Comisión de Investigaciones Científicas de la provincia de Buenos Aires, Facultad de Ciencias Médicas

Sudden cardiac death from ventricular arrhythmias is more common in adult patients with with heart failure compared with pediatric patients with heart failure. We identified age-specific differences in arrhythmogenesis using a guinea pig model of acute β-adrenergic stimulation. Young and adult guinea pigs were exposed to the β-adrenergic agonist isoproterenol (ISO; 0.7 mg/kg) for 30 min in the absence or presence of flecainide (20 mg/kg), an antiarrhythmic that blocks Na+ and ryanodine channels. Implanted cardiac monitors (Reveal LINQ, Medtronic) were used to monitor heart rhythm. Alterations in phosphorylation and oxidation of ryanodine receptor 2 (RyR2) were measured in left ventricular tissue. There were age-specific differences in arrhythmogenesis and sudden death associated with acute β-adrenergic stimulation in guinea pigs. Young and adult guinea pigs developed arrhythmias in response to ISO; however, adult animals developed significantly more premature ventricular contractions and experienced higher arrhythmia-related mortality than young guinea pigs treated with ISO. Although there were no significant differences in the phosphorylation of left ventricular RyR2 between young and adult guinea pigs, adult guinea pigs exposed to acute ISO had significantly more oxidation of RyR2. Flecainide treatment significantly improved survival and decreased the number of premature ventricular contractions in young and adult animals in association with lower RyR2 oxidation. Adult guinea pigs had a greater propensity to develop arrhythmias and suffer sudden death than young guinea pigs when acutely exposed to ISO. This was associated with higher oxidation of RyR2. The incidence of sudden death can be rescued with flecainide treatment, which decreases RyR2 oxidation. NEW & NOTEWORTHY Clinically, adult patients with heart failure are more likely to develop arrhythmias and sudden death than pediatric patients with heart failure. In the present study, older guinea pigs also showed a greater propensity to arrhythmias and sudden death than young guinea pigs when acutely exposed to isoproterenol. Although there are well-described age-related cardiac structural changes that predispose patients to arrhythmogenesis, the present data suggest contributions from dynamic changes in cellular signaling also play an important role in arrhythmogenesis.

Expression silencing of junctophilin-2 (JPH2) in mouse heart leads to ryanodine receptor type 2 (RyR2)-mediated sarcoplasmic reticulum (SR) Ca2+ leak and rapid development of heart failure. The mechanism and physiological significance of JPH2 in regulating RyR2-mediated SR Ca2+ leak remains elusive. We sought to elucidate the role of JPH2 in regulating RyR2-mediated SR Ca2+ release in the setting of cardiac failure. Cardiac myocytes isolated from tamoxifen-inducible conditional knockdown mice of JPH2 (MCM-shJPH2) were subjected to confocal Ca2+ imaging. MCM-shJPH2 cardiomyocytes exhibited an increased spark frequency width with altered spark morphology, which caused increased SR Ca2+ leakage. Single channel studies identified an increased RyR2 open probability in MCM-shJPH2 mice. The increase in spark frequency and width was observed only in MCM-shJPH2 and not found in mice with increased RyR2 open probability with native JPH2 expression. Na+/Ca2+-exchanger (NCX) activity was reduced by 50% in MCM-shJPH2 with no detectable change in NCX expression. Additionally, 50% inhibition of NCX through Cd2+ administration alone was sufficient to increase spark width in myocytes obtained from wild-type mice. Additionally, superresolution analysis of RyR2 and NCX colocalization showed a reduced overlap between RyR2 and NCX in MCM-shJPH2 mice. In conclusion, decreased JPH2 expression causes increased SR Ca2+ leakage by directly increasing open probability of RyR2 and by indirectly reducing junctional NCX activity through increased dyadic cleft Ca2+. This demonstrates two novel and independent cellular mechanisms by which JPH2 regulates RyR2-mediated SR Ca2+ leak and heart failure development.

The cardiac ryanodine receptor (RyR2) is inhibited by calmodulin (CaM) and S100A1. Simultaneous substitution of three amino acid residues (W3587A, L3591D, F3603A; RyR2ADA) in the CaM binding domain of RyR2 results in loss of CaM inhibition at submicromolar (diastolic) and micromolar (systolic) Ca2+, cardiac hypertrophy, and heart failure in Ryr2ADA/ADAmice. To address whether cardiac hypertrophy results from the elimination of CaM and S100A1 inhibition at diastolic or systolic Ca2+, a mutant mouse was generated with a single RyR2 amino acid substitution (L3591D; RyR2D). Here we report that in single-channel measurements RyR2-L3591D isolated from Ryr2D/Dhearts lost CaM inhibition at diastolic Ca2+only, whereas S100A1 regulation was eliminated at both diastolic and systolic Ca2+. In contrast to the ∼2-wk life span of Ryr2ADA/ADAmice, Ryr2D/Dmice lived longer than 1 yr. Six-month-old Ryr2D/Dmice showed a 9% increase in heart weight-to-body weight ratio, modest changes in cardiac morphology, and a twofold increase in atrial natriuretic peptide mRNA levels compared with wild type. After 4-wk pressure overload with transverse aortic constriction, heart weight-to-body weight ratio and atrial natriuretic peptide mRNA levels increased and echocardiography showed changes in heart morphology of Ryr2D/Dmice compared with sham-operated mice. Collectively, the findings indicate that the single RyR2-L3591D mutation, which distinguishes the effects of diastolic and systolic Ca2+, alters heart size and cardiac function to a lesser extent in Ryr2D/Dmice than the triple mutation in Ryr2ADA/ADAmice. They further suggest that CaM inhibition of RyR2 at systolic Ca2+is important for maintaining normal cardiac function.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant arrhythmogenic disorder linked to mutations in the cardiac ryanodine receptor (RyR2) and calsequestrin, predisposing the young to syncope and cardiac arrest. To define the role of β-adrenergic stimulation (BAS) and to identify potential therapeutic targeted sites relating to intracellular calcium cycling, we used a Luo-Rudy dynamic ventricular myocyte model incorporated with interacting Markov models of the L-type Ca2+ channel ( ICa,L) and RyR2 to simulate the heterozygous state of mouse RyR2 R4496C mutation (RyR2R4496C+/−) comparable with CPVT patients with RyR2 R4497C mutation. Characteristically, in simulated cells, pacing at 4 Hz or faster or pacing at 2 Hz under BAS with effects equivalent to those of isoproterenol at ≥0.1 μM could readily induce delayed afterdepolarizations (DADs) and DAD-mediated triggered activity (TA) in RyR2R4496C+/− but not in the wild-type via enhancing both ICa,L and sarcoplasmic reticulum (SR) Ca2+ ATPase ( IUP). Moreover, with the use of steady state values of isolated endocardial (Endo), mid-myocardial (M), and epicardial (Epi) cells as initial data for conducting single cell and one-dimensional strand studies, the M cell was more vulnerable for developing DADs and DAD-mediated TA than Endo and Epi cells, and the gap junction coupling represented by diffusion coefficient ( D) of ≤0.000766*98 cm2/ms was required for generating DAD-mediated TA in RyR2R4496C+/−. Whereas individual reduction of Ca2+ release channel of SR and Na-Ca exchanger up to 50% was ineffective, 30% or more reduction of either ICa,L or IUP could totally suppress the inducibility of arrhythmia under BAS. Of note, 15% reduction of both ICa,L and IUP exerted a synergistic antiarrhythmic efficacy. Findings of this model study confirm that BAS facilitates induction of ventricular tachyarrhythmias via its action on intracellular Ca2+ cycling and a pharmacological regimen capable of reducing ICa,L could be an effective adjunctive to β-adrenergic blockers for suppressing ventricular tachyarrhythmias during CPVT.

The present study was undertaken to assess the effects of exercise training (ExT) initiated after the onset of diabetes on cardiac ryanodine receptor expression and function. Type 1 diabetes was induced in male Sprague-Dawley rats using streptozotocin (STZ). Three weeks after STZ injection, diabetic rats were divided into two groups. One group underwent ExT for 4 wk while the other group remained sedentary. After 7 wk of sedentary diabetes, cardiac fractional shortening, rate of rise of left ventricular pressure, and myocyte contractile velocity were reduced by 14, 36, 44%, respectively. Spontaneous Ca2+spark frequency increased threefold, and evoked Ca2+release was dyssynchronous with diastolic Ca2+releases. Steady-state type 2 ryanodine receptor (RyR2) protein did not change, but its response to Ca2+was altered. RyR2 also exhibited 1.8- and 1.5-fold increases in phosphorylation at Ser2808and Ser2814. PKA activity was reduced by 75%, but CaMKII activity was increased by 50%. Four weeks of ExT initiated 3 wk after the onset of diabetes blunted decreases in cardiac fractional shortening and rate of left ventricular pressure development, increased the responsiveness of the myocardium to isoproterenol stimulation, attenuated the increase in Ca2+spark frequency, and minimized dyssynchronous and diastolic Ca2+releases. ExT also normalized the responsiveness of RyR2 to Ca2+activation, attenuated increases in RyR2 phosphorylation at Ser2808and Ser2814, and normalized CaMKII and PKA activities. These data are the first to show that ExT during diabetes normalizes RyR2 function and Ca2+release from the sarcoplasmic reticulum, providing insights into mechanisms by which ExT during diabetes improves cardiac function.

As a critical step toward understanding the role of abnormal intracellular Ca2+release via the ryanodine receptor (RyR2) during the development of hypertension-induced cardiac hypertrophy and heart failure, this study examines two questions: 1) At what stage, if ever, in the development of hypertrophy and heart failure is RyR2hyperphosphorylated at Ser2808? 2) Does the spatial distribution of RyR2clusters change in failing hearts? Using a newly developed semiquantitative immunohistochemistry method and Western blotting, we measured phosphorylation of RyR2at Ser2808in the spontaneously hypertensive rat (SHR) at four distinct disease stages. A major finding is that hyperphosphorylation of RyR2at Ser2808occurred only at late-stage heart failure in SHR, but not in age-matched controls. Furthermore, the spacing between RyR2clusters was shortened in failing hearts, as predicted by quantitative model simulation to increase spontaneous Ca2+wave generation and arrhythmias.

Calcium malfunction plays a central role in heart failure. Here, we provide evidence that adenylyl cyclase type VI restores sarco(endo)plasmic reticulum 2a (SERCA2a) affinity for calcium and maximum velocity of cardiac calcium uptake by sarcoplasmic reticulum in murine dilated cardiomyopathy. Restoration of normal SERCA2a affinity for calcium is associated not only with decreased phospholamban protein expression but also with increased phospholamban phosphorylation by PKA activation. The ratio of phosphorylated ryanodine receptor 2 (RyR2) to RyR2 protein was increased, but the amount of phosphorylated RyR2 was unaffected. These data provide a possible mechanism by which adenylyl cyclase type VI (in contrast to other signaling elements associated with increased cAMP generation) has a salutary effect in the failing heart.

The cardiac sarcoplasmic reticulum calcium-ATPase (SERCA2a), Na+/Ca2+exchanger (NCX1), and ryanodine receptor (RyR2) are proteins involved in the regulation of myocyte calcium. We tested whether exercise training (ET) alters those proteins during development of chronic heart failure (CHF). Ten dogs were chronically instrumented to permit hemodynamic measurements. Five dogs underwent 4 wk of cardiac pacing (210 beats/min for 3 wk and 240 beats/min for the 4th wk), whereas five dogs underwent the same pacing regimen plus daily ET (5.1 ± 0.3 km/h, 2 h/day). Paced animals developed CHF characterized by hemodynamic abnormalities and reduced ejection fraction. ET preserved resting hemodynamics and ejection fraction. Left ventricular samples were obtained from all dogs and another five normal dogs for mRNA (Northern analysis, band intensities normalized to glyceraldehyde-3-phosphate dehydrogenase) and protein level (Western analysis, band intensities normalized to tubulin) measurements. In failing hearts, SERCA2a was decreased by 33% ( P < 0.05) and 65% ( P < 0.05) in mRNA and protein level, respectively, compared with normal hearts; there was only an 8.6% reduction in mRNA and a 32% reduction in protein in exercised animals ( P < 0.05 from CHF). mRNA expression of NCX1 increased by 44% in paced-only dogs compared with normal ( P < 0.05) but only by 22% in trained dogs ( P < 0.05 vs. CHF); protein level of NCX1 was elevated in paced-only dogs (71%, P < 0.05) but partially normalized by ET (33%, P < 0.05 from CHF). RyR2 was not altered in any of the dogs. In conclusion, long-term ET may ameliorate cardiac deterioration during development of CHF, in part via normalization of myocardial calcium-handling proteins.

Sarcoplasmic reticulum (SR)-mediated Ca2+sequestration and release are important determinants of cardiac contractility. In end-stage heart failure SR dysfunction has been proposed to contribute to the impaired cardiac performance. In this study we tested the hypothesis that a targeted interference with SR function can be a primary cause of contractile impairment that in turn might alter cardiac gene expression and induce cardiac hypertrophy. To study this we developed a novel animal model in which ryanodine, a substance that alters SR Ca2+release, was added to the drinking water of mice. After 1 wk of treatment, in vivo hemodynamic measurements showed a 28% reduction in the maximum speed of contraction (+dP/d tmax) and a 24% reduction in the maximum speed of relaxation (−dP/d tmax). The slowing of cardiac relaxation was confirmed in isolated papillary muscles. The late phase of relaxation expressed as the time from 50% to 90% relaxation was prolonged by 22%. After 4 wk of ryanodine administration the animals had developed a significant cardiac hypertrophy that was most prominent in both atria (right artrium +115%, left atrium +100%, right ventricle +23%, and left ventricle +13%). This was accompanied by molecular changes including a threefold increase in atrial natriuretic factor mRNA and a sixfold increase in β-myosin heavy chain mRNA. Sarcoplasmic endoplasmic reticulum Ca2+mRNA was reduced by 18%. These data suggest that selective impairment of SR function in vivo can induce changes in cardiac gene expression and promote cardiac growth.

Cardiac contraction is initiated by Ca2+ leaving the sarcoplasmic reticulum through the ryanodine receptor (RyR). Although opening of the RyR can be modified by various ligands, these have no maintained effect on contraction. We conclude that modulation of the RyR controls sarcoplasmic reticulum Ca2+ content rather than cytoplasmic Ca2+ concentration.

Cyclic ADP-ribose (cADPR), an endogenous metabolite of beta-NAD+, activates Ca2+ release from endoplasmic reticulum in sea urchin eggs via the ryanodine receptor (RyR) pathway. A similar role has been proposed in cardiac sarcoplasmic reticulum (SR), although this remains controversial. We therefore investigated the ability of cADPR to induce Ca2+ release from canine cardiac SR microsomes using fluo 3 to monitor extravesicular Ca2+ concentration. We found that cADPR induced Ca2+ release in a concentration-dependent manner, whereas neither its precursor, NAD+, nor its metabolite, ADP-ribose, elicited a consistent effect. In addition, an additive effect on calcium release between cADPR and 9-Me-7-Br-eudistomin-D (MBED), an activator of RyR, was found as well as no cross-desensitization between cADPR and MBED. Specific blockers of the RyR did not abolish the cADPR-induced Ca2+ release. These results provide evidence for cADPR-induced Ca2+ release from dog cardiac SR via a novel mechanism which is independent of RyR activation.

The ryanodine receptor (RyR) is a high-conductance Ca2+ channel of the sarcoplasmic reticulum in muscle and of the endoplasmic reticulum in other cells. In striated muscle fibers, RyRs are responsible for the rapid release of Ca2+ that activates contraction. Ryanodine receptors are complex molecules, with unusually large cytoplasmic domains containing numerous binding sites for agents that control the state of activity of the channel-forming domain of the molecule. Structural considerations indicate that long-range interactions between cytoplasmic and intramembrane domains control channel function. Ryanodine receptors are located in specialized regions of the SR, where they are structurally and functionally associated with other intrinsic proteins and, indirectly, also with the luminal Ca2(+)-binding protein calsequestrin. Activation of RyRs during the early part of the excitation-contraction coupling cascade is initiated by the activity of surface-membrane Ca2+ channels, the dihydropyridine receptors (DHPRs). Skeletal and cardiac muscles contain different RyR and DHPR isoforms and both contribute to the diversity in cardiac and skeletal excitation-contraction coupling mechanisms. The architecture of the sarcoplasmic reticulum-surface junctions determines the types of RyR-DHPR interactions in the two muscle types.

Individuals working in commercial hog confinement facilities have elevated incidences of headaches, depression, nausea, skeletal muscle weakness, fatigue, gastrointestinal disorders, and cardiovascular diseases, and the molecular mechanisms for these nonrespiratory ailments remain incompletely undefined. A common element underlying these diverse pathophysiologies is perturbation of intracellular Ca2+homeostasis. This study assessed whether the dust generated inside hog confinement facilities contains compounds that alter Ca2+mobilization via ryanodine receptors (RyRs), key intracellular channels responsible for mobilizing Ca2+from internal stores to elicit an array of physiologic functions. Hog barn dust (HBD) was extracted with phosphate-buffered saline, sterile-filtered (0.22 μm), and size-separated using Sephadex G-100 resin. Fractions (F) 1 through 9 (Mw>10,000 Da) had no measurable effects on RyR isoforms. However, F10 through F17, which contained compounds of Mw≤2,000 Da, modulated the [3H]ryanodine binding to RyR1, RyR2, and RyR3 in a biphasic (Gaussian) manner. The Kivalues for F13, the most potent fraction, were 3.8 ± 0.2 μg/ml for RyR1, 0.2 ± 0.01 μg/ml and 19.1 ± 2.8 μg/ml for RyR2 (two binding sites), and 44.9 ± 2.8 μg/ml and 501.6 ± 9.2 μg/ml for RyR3 (two binding sites). In lipid bilayer assays, F13 dose-dependently decreased the open probabilities of RyR1, RyR2, and RyR3. Pretreating differentiated mouse skeletal myotubes (C2C12 cells) with F13 blunted the amplitudes of ryanodine- and K+-induced Ca2+transients. Because RyRs are present in many cell types, impairment in Ca2+mobilization from internal stores via these channels is a possible mechanism by which HBD may trigger these seemingly unrelated pathophysiologies.

Propagation of ryanodine receptor (RyR2)-derived Ca2+signals to the mitochondrial matrix supports oxidative ATP production or facilitates mitochondrial apoptosis in cardiac muscle. Ca2+transfer likely occurs locally at focal associations of the sarcoplasmic reticulum (SR) and mitochondria, which are secured by tethers. The outer mitochondrial membrane and inner mitochondrial membrane (OMM and IMM, respectively) also form tight focal contacts (contact points) that are enriched in voltage-dependent anion channels, the gates of OMM for Ca2+. Contact points could offer the shortest Ca2+transfer route to the matrix; however, their alignment with the SR-OMM associations remains unclear. Here, in rat heart we have studied the distribution of mitochondria-associated SR in submitochondrial membrane fractions and evaluated the colocalization of SR-OMM associations with contact points using transmission electron microscopy. In a sucrose gradient designed for OMM purification, biochemical assays revealed lighter fractions enriched in OMM only and heavier fractions containing OMM, IMM, and SR markers. Pure OMM fractions were enriched in mitofusin 2, an ∼80 kDa mitochondrial fusion protein and SR-mitochondrial tether candidate, whereas in fractions of OMM + IMM + SR, a lighter (∼50 kDa) band detected by antibodies raised against the NH2terminus of mitofusin 2 was dominating. Transmission electron microscopy revealed mandatory presence of contact points at the junctional SR-mitochondrial interface versus a random presence along matching SR-free OMM segments. For each SR-mitochondrial junction at least one tether was attached to contact points. These data establish the contact points as anchorage sites for the SR-mitochondrial physical coupling. Close coupling of the SR, OMM, and IMM is likely to provide a favorable spatial arrangement for local ryanodine receptor-mitochondrial Ca2+signaling.

Mutations in the skeletal muscle RyR1 isoform of the ryanodine receptor (RyR) Ca2+-release channel confer susceptibility to malignant hyperthermia, which may be triggered by inhalational anesthetics such as halothane. Using immunoblotting, we show here that the ryanodine receptor, calmodulin, junctin, calsequestrin, sarcalumenin, calreticulin, annexin-VI, sarco(endo)plasmic reticulum Ca2+-ATPase, and the dihydropyridine receptor exhibit no major changes in their expression level between normal human skeletal muscle and biopsies from individuals susceptible to malignant hyperthermia. In contrast, protein gel-shift studies with halothane-treated sarcoplasmic reticulum vesicles from normal and susceptible specimens showed a clear difference. Although the α2-dihydropyridine receptor and calsequestrin were not affected, clustering of the Ca2+-ATPase was induced at comparable halothane concentrations. In the concentration range of 0.014–0.35 mM halothane, anesthetic-induced oligomerization of the RyR1 complex was observed at a lower threshold concentration in the sarcoplasmic reticulum from patients with malignant hyperthermia. Thus the previously described decreased Ca2+-loading ability of the sarcoplasmic reticulum from susceptible muscle fibers is probably not due to a modified expression of Ca2+-handling elements, but more likely a feature of altered quaternary receptor structure or modified functional dynamics within the Ca2+-regulatory apparatus. Possibly increased RyR1 complex formation, in conjunction with decreased Ca2+uptake, is of central importance to the development of a metabolic crisis in malignant hyperthermia.

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca2+ concentrations, whereas at micromolar Ca2+ concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1D/D) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1D/D mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1D/D mice had depressed Ca2+ transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca2+ transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1D/D mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1D/D fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca2+ release flux, consistent with increased summation of the Ca2+ transient and contractile force. Peak Ca2+ release flux was suppressed at all voltages in voltage-clamped Ryr1D/D fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca2+ release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

Reactive oxygen intermediates (ROI) have been implicated in a variety of pathophysiological conditions, and vascular smooth muscle may be a site of damage in such oxygen toxicity. Mechanisms of the effects of these intermediates on vascular smooth muscle at the cellular level, however, have not been well studied. We have previously shown that xanthine oxidase (XO)-generated superoxide radicals (O2-.) inhibited the Ca(2+)-adenosine triphosphatase of vascular smooth muscle sarcoplasmic reticulum (SR) through mechanisms that do not involve H2O2 or hydroxyl radicals. In the present study, we report that the D-myo-inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release from bovine aortic SR was also affected by O2-(.). Hypoxanthine (100 microM) plus XO (10 mU/ml) in the presence of catalase (100 U/ml) stimulated the IP3-induced Ca2+ release from SR monitored using arsenazo III. At 10 microM IP3, the release was doubled by O2-. treatment. As a consequence of using the higher SR protein concentrations required to observe the Ca2+ release, this effect was independent of Ca2+ uptake inhibition induced by O2-(.). Since the effect of O2-. was not seen when a nonhydrolyzable analogue of IP3 was used to induce Ca2+ release, O-2. may be inhibiting the degradation processes of IP3.

it has long been known that release of intracellular stored Ca [presumably in the sarcoplasmic reticulum (SR)] plays a major role in initiating contraction in airway smooth muscle (ASM) from a variety of species ([6][1]) including human ([17][2]). Therefore, the issue of how extracellular calcium

to the editor: We were delighted to note the concurrent publication of two studies linking decreased expression and function of sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) in human airway (bronchial) smooth muscle (ASM) to asthma. The first study, published in the July 2009 issue of American

The hypothesis of a Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is supported by experiments done in skinned cardiac cells (sarcolemma removed by microdissection). According to this hypothesis, the transsarcolemmal Ca2+ influx does not activate the myofilaments directly but through the induction of a Ca2+ release from the SR. The stimulus gating CICR is not a small change in free Ca2+ concentration (delta[free Ca2+]) outside the SR but a function of the rate of this change (delta[free Ca2+/delta t]). The initial relatively fast component of the transsarcolemmal Ca2+ current would trigger Ca2+ release; the subsequent slow component, perhaps corresponding to noninactivating Ca2+ channels, would load the SR with an amount of Ca2+ available for release during subsequent beats. Inactivation of CICR is caused by the large increase of [free Ca2+] outside the SR resulting from Ca2+ release, which inhibits further release. This negative feedback helps to explain that CICR is not all or none. During relaxation the Ca2+ reaccumulation in the SR is backed up by the Ca2+ efflux across the sarcolemma through Na+-Ca2+ exchange and the sarcolemmal Ca2+ pump. Computations of the Ca2+ buffering in the mammalian ventricular cell and of the systolic transsarcolemmal Ca2+ influx do not support the alternative hypothesis that this influx of Ca2+ is large enough to activate the myofilaments directly. Yet the hypothesis of a CICR can be challenged because of many problems and uncertainties related to the preparations and methods used for skinned cardiac cell experiments.

To study effects of physiologic hypertrophy on contractile protein ATPases and sarcoplasmic reticulum, hypertrophy was caused in female Wistar rats by a chronic swimming program. Nonhypertrophied hearts of female control sedentary rats and rats made to run on a treadmill program were also examined. The swimming program, but not the running program, resulted in a significant increase in heart weight. Actomyosin ATPase activity was also increased by 15% in the hearts of swimmers but not runners. Similar increases were observed for Ca2+-activated myosin ATPase activity and actin-activated ATPase of myosin. Sarcoplasmic reticulum from the hearts of swimmers showed increased calcium binding and calcium uptake as a function of time and of calcium concentration. Sarcoplasmic reticulum ATPase activities were not altered by hypertrophy. These findings in physiologic hypertrophy contrast with those of pathologic hypertrophy in which ATPase activity of contractile proteins and calcium binding and uptake of sarcoplasmic reticulum have generally been found to be depressed.