Ryanodine Receptor Density Research Articles

What Is Required for Neuronal Calcium Waves? A Numerical Parameter Study

Neuronal calcium signals propagating by simple diffusion and reaction with mobile and stationary buffers are limited to cellular microdomains. The distance intracellular calcium signals can travel may be significantly increased by means of calcium-induced calcium release from internal calcium stores, notably the endoplasmic reticulum. The organelle, which can be thought of as a cell-within-a-cell, is able to sequester large amounts of cytosolic calcium ions via SERCA pumps and selectively release them into the cytosol through ryanodine receptor channels leading to the formation of calcium waves. In this study, we set out to investigate the basic properties of such dendritic calcium waves and how they depend on the three parameters dendrite radius, ER radius and ryanodine receptor density in the endoplasmic membrane. We demonstrate that there are stable and abortive regimes for calcium waves, depending on the above morphological and physiological parameters. In stable regimes, calcium waves can travel across long dendritic distances, similar to electrical action potentials. We further observe that abortive regimes exist, which could be relevant for spike-timing dependent plasticity, as travel distances and wave velocities vary with changing intracellular architecture. For some of these regimes, analytic functions could be derived that fit the simulation data. In parameter spaces, that are non-trivially influenced by the three-dimensional calcium concentration profile, we were not able to derive such a functional description, demonstrating the mathematical requirement to model and simulate biochemical signaling in three-dimensional space.

Cardiac Resynchronization Therapy Reduces Subcellular Heterogeneity of Ryanodine Receptors, T-Tubules, and Ca2+ Sparks Produced by Dyssynchronous Heart Failure.

Cardiac resynchronization therapy (CRT) is a major advance for treatment of patients with dyssynchronous heart failure (DHF). However, our understanding of DHF-associated remodeling of subcellular structure and function and their restoration after CRT remains incomplete. We investigated subcellular heterogeneity of remodeling of structures and proteins associated with excitation-contraction coupling in cardiomyocytes in DHF and after CRT. Three-dimensional confocal microscopy revealed subcellular heterogeneity of ryanodine receptor (RyR) density and the transverse tubular system (t-system) in a canine model of DHF. RyR density at the ends of lateral left ventricular cardiomyocytes was higher than that in cell centers, whereas the t-system was depleted at cell ends. In anterior left ventricular cardiomyocytes, however, we found a similar degree of heterogeneous RyR remodeling, despite preserved t-system. Synchronous heart failure was associated with marginal heterogeneity of RyR density. We used rapid scanning confocal microscopy to investigate effects of heterogeneous structural remodeling on calcium signaling. In DHF, diastolic Ca(2+) spark density was smaller at cell ends versus centers. After CRT, subcellular heterogeneity of structures and function was reduced. RyR density exhibits remarkable subcellular heterogeneity in DHF. RyR remodeling occurred in lateral and anterior cardiomyocytes, but remodeling of t-system was confined to lateral myocytes. These findings indicate that different mechanisms underlie remodeling of RyRs and t-system. Furthermore, we suggest that ventricular dyssynchrony exacerbates subcellular remodeling in heart failure. CRT efficiently reduced subcellular heterogeneity. These results will help to explain remodeling of excitation-contraction coupling in disease and restoration after CRT.

Slow Ca2 + sparks de-synchronize Ca2 + release in failing cardiomyocytes: Evidence for altered configuration of Ca2 + release units?

In heart failure, cardiomyocytes exhibit slowing of the rising phase of the Ca2+ transient which contributes to the impaired contractility observed in this condition. We investigated whether alterations in ryanodine receptor function promote slowing of Ca2+ release in a murine model of congestive heart failure (CHF). Myocardial infarction was induced by left coronary artery ligation. When chronic CHF had developed (10weeks post-infarction), cardiomyocytes were isolated from viable regions of the septum. Septal myocytes from SHAM-operated mice served as controls. Ca2+ transients rose markedly slower in CHF than SHAM myocytes with longer time to peak (CHF=152±12% of SHAM, P<0.05). The rise time of Ca2+ sparks was also increased in CHF (SHAM=9.6±0.6ms, CHF=13.2±0.7ms, P<0.05), due to a sub-population of sparks (≈20%) with markedly slowed kinetics. Regions of the cell associated with these slow spontaneous sparks also exhibited slowed Ca2+ release during the action potential. Thus, greater variability in spark kinetics in CHF promoted less uniform Ca2+ release across the cell. Dyssynchronous Ca2+ transients in CHF additionally resulted from T-tubule disorganization, as indicated by fast Fourier transforms, but slow sparks were not associated with orphaned ryanodine receptors. Rather, mathematical modeling suggested that slow sparks could result from an altered composition of Ca2+ release units, including a reduction in ryanodine receptor density and/or distribution of ryanodine receptors into sub-clusters. In conclusion, our findings indicate that slowed, dyssynchronous Ca2+ transients in CHF result from alterations in Ca2+ sparks, consistent with rearrangement of ryanodine receptors within Ca2+ release units. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.

Lessons from calsequestrin-1 ablation in vivo: much more than a Ca2+ buffer after all

Calsequestrin type-1 (CASQ1), the main sarcoplasmic reticulum (SR) Ca(2+) binding protein, plays a dual role in skeletal fibers: a) it provides a large pool of rapidly-releasable Ca(2+) during excitation-contraction (EC) coupling; and b) it modulates the activity of ryanodine receptors (RYRs), the SR Ca(2+) release channels. We have generated a mouse lacking CASQ1 in order to further characterize the role of CASQ1 in skeletal muscle. Contrary to initial expectations, CASQ1 ablation is compatible with normal motor activity, in spite of moderate muscle atrophy. However, CASQ1 deficiency results in profound remodeling of the EC coupling apparatus: shrinkage of junctional SR lumen; proliferation of SR/transverse-tubule contacts; and increased density of RYRs. While force development during a twitch is preserved, it is nevertheless characterized by a prolonged time course, likely reflecting impaired Ca(2+) re-uptake by the SR. Finally, lack of CASQ1 also results in increased rate of SR Ca(2+) depletion and inability of muscle to sustain tension during a prolonged tetani. All modifications are more pronounced (or only found) in fast-twitch extensor digitorum longus muscle compared to slow-twitch soleus muscle, likely because the latter expresses higher amounts of calsequestrin type-2 (CASQ2). Surprisingly, male CASQ1-null mice also exhibit a marked increased rate of spontaneous mortality suggestive of a stress-induced phenotype. Consistent with this idea, CASQ1-null mice exhibit an increased susceptibility to undergo a hypermetabolic syndrome characterized by whole body contractures, rhabdomyolysis, hyperthermia and sudden death in response to halothane- and heat-exposure, a phenotype remarkably similar to human malignant hyperthermia and environmental heat-stroke. The latter findings validate the CASQ1 gene as a candidate for linkage analysis in human muscle disorders.

Contribution of Sarcoplasmic Reticulum Ca2+ Release and Ca2+ Transporters on Sarcolemmal Channels to Ca2+ Transient in Fetal Mouse Heart

Sarcoplasmic reticulum (SR) Ca release has been shown not to be the predominant mechanism responsible for excitation-contraction (E-C) coupling in fetal myocytes. However, most of the studies have been conducted either on primary cultures or acutely isolated cells, in which an apparent reduction of ryanodine receptor density have been reported. We aimed to elucidate the contribution of SR Ca release and Ca transporters on sarcolemmal channels to Ca transients in fetal mouse whole hearts. On embryonic day 13.5, ryanodine significantly reduced the amplitude of the Ca transient to 27.2 ± 4.4% of the control, and both nickel and SEA0400 significantly prolonged the time to peak from 84 ± 2 ms to 140 ± 5 ms and 129 ± 6 ms, respectively, whereas nifedipine did not alter it. Therefore, at early fetal stages, SR Ca release should be an important component of E-C coupling, and T-type Ca channel and reverse mode sodium-calcium exchanger (NCX)-mediated SR Ca release could be the predominant contributors. Using embryonic mouse cultured cardiomyocytes, we showed that both nifedipine and nickel inhibited the ability of NCX to extrude Ca from the cytosol. From these results, we propose a novel idea concerning E-C coupling in immature heart.

Fetal and Neonatal Development of Ca2+ Transients and Functional Sarcoplasmic Reticulum in Beating Mouse Hearts

It is generally accepted that Ca(2+)-induced Ca(2+) release is not the predominant mechanism during embryonic stages. Most studies have been conducted either on primary cultures or acutely isolated cells, in which an apparent reduction of ryanodine receptor density and alterations in the cell shape have been reported. The aim of the present study was to investigate developmental changes in Ca(2+) transients using whole hearts of mouse embryos and neonates. Fluo-3 fluorescence signals from stimulated whole hearts were detected using a photomultiplier and stored as Ca(2+) transients. The upstroke and decay of Ca(2+) transients became more rapid from the late embryonic stages to the neonatal stage. After thapsigargin application (an inhibitor of the sarcoplasmic Ca(2+)-ATPase [SERCA]), time to 50% relaxation (T(50)) of Ca(2+) transients was significantly prolonged. There were no significant changes in T(50) after Ru360 application (an inhibitor of mitochondrial Ca(2+) uniporter). The rate of increase in the amplitude of Ca(2+) transients after caffeine application became larger during developmental stages. Ca(2+) homeostasis developmentally changes from a slow rise and decay of Ca(2+) transients to rapid kinetics after the mid-embryonic stage. SERCA began to contribute significantly to Ca(2+) homeostasis at early embryonic stages and sarcoplasmic reticulum Ca(2+) contents increased from embryonic to neonatal stages, whereas mitochondrial Ca(2+) uptake did not contribute to Ca(2+) transients on a beat-to-beat basis.

Cell culture modifies Ca 2+ signaling during excitation–contraction coupling in neonate cardiac myocytes

In heart, the excitation–contraction coupling (ECC) mechanism changes during development. Primary cell culture has been used to study Ca 2+ signaling in newborn (NB) rat heart. In this work, the effects of cell culture on the action potential (AP) and ECC Ca 2+ signaling during development were investigated. Specifically, AP, Ca 2+ currents ( I Ca), and ryanodine receptor (RyR) properties (i.e. density, distribution, and contribution to Ca 2+ transients and Ca 2+ sparks) were defined in cultured myocytes (CM) from 0-day-old NB rat at different times in culture (1–4 days). Compared with acutely dissociated myocytes (ADM) from NB of equivalent ages (1–4 days), CM showed lower RyR density (50% at 1 day, 25% at 4 days), but larger RyR contribution to the Ca 2+ transient (25% at 1 day, 57% at 4 days). Additionally, Ca 2+ sparks were larger, longer, wider, and more frequent in CM than in ADM. RyR cellular distribution also showed different arrangement. While in CM, RyRs were located peripherally, in ADM of equivalent ages a sarcomeric arrangement was predominant. Finally, CM showed a two-fold increase in sarcolemmal Ca 2+ entry during the AP. These results indicated that primary culture is a feasible model to study Ca 2+ signaling in heart; however, it does not precisely reproduce what occurs in ECC during development.

Sarcoplasmic Reticulum Calcium Release Channels in Ventricles of Older Adult Hamsters

Whether the density of sarcoplasmic reticulum (SR) calcium release channels/ryanodine receptors in the heart declines with age is not clear. We investigated age-related changes in the density of [3H]-ryanodine receptors in crude ventricular homogenates, which contained all ligand binding sites in heart and in isolated junctional SR membranes. Experiments utilized young (120 days) and older adult (300 days) hamsters. [3H]-ryanodine binding site density did not change with age in crude homogenate preparations, although total heart protein concentration increased significantly with age. In contrast, the density of [3H]-ryanodine binding sites decreased markedly in heavy SR membranes purified from older hearts. These results show that demonstration of age-related changes in cardiac ryanodine receptor density depends upon the preparation used. Furthermore, the increase in total ventricular protein with age suggests that normalization of data by membrane protein should be used with caution in studies of aging heart.

Exercise Training Increases Beta Adrenergic Responsiveness In Hypertension

Cardiac responses to beta-adrenergic receptor (BAR) stimulation are depressed in chronic hypertension. We tested whether exercise training (ET) could improve BAR responsiveness in spontaneous hypertensive rats (SHR) and whether BAR kinase (GRK2) plays a role after exercise in SHR. Methods Female, SHR (age: 4mo) were placed into a treadmill running (SHR-TRD, n=12; 20m/min, 1hr/day, 5d/wk, 12 wks) or sedentary group (SHR-SED, n=12). Age-matched WKY rats (n=12) were controls. Tail cuff systolic and diastolic blood pressures were determined. Isovolumic, Langendorff performance was measured (4.5 Hz, LVEDP = 12mmHg, coronary flow=16 ml/min, 2mM Ca2+). Following baseline, 5-minute infusions of isoproterenol (ISO) were administered (10−10 - 10−7 mol/L). Western blot analysis, myocardial morphometry, and histomorphometry were performed. Results Mean blood pressure was higher in SHR vs. WKY (P < 0.05) and unaltered with ET. Myocardial wall thickness and cell surface area were greater in SHR than WKY (P < 0.05) and augmented with exercise training (P < 0.05). At baseline, LV developed pressure (LVDevP) was greater in SHR vs. WKY (WKY: 154 ± 23; SHR-SED: 173 ± 10; SHR-TRD: 175 ± 5 mmHg). The peak response to ISO was shifted rightward 100-fold in both SHR groups relative to WKY. The peak ISO LVDevP response was similar between WKY and SHR-TRD, while the peak response of SHR-SED was blunted (WKY: 38 ± 11%; SHR-SED: 19 ± 9%; SHR-TRD: 49 ± 7%, P < 0.05) (Figure 1A). L type Ca2+ channel (LTCC) and the Na+/Ca2+ exchanger (NCX) abundance were increased in both SHR groups. Training increased ryanodine phosphorylation (RyRp) with no difference in ryanodine receptor density (RyRt) among groups. GRK2 levels were increased in SHR-SED vs. SHR-TRD relative to WKY (SED: 97.65 ± 38%; TRD: 17.53 ± 19%, P < 0.05) (Figure 1B.).Figure 1AFigure 1BConclusion BAR responsiveness is improved with exercise training in SHR, an effect at least in part, secondary to a downregulation of GRK2. Supported by the American Heart Association, Mid-Atlantic Affiliate (JRL), and the National Institute of Health SRH (HL33921) and WJK (HL61690 and HL56205).

Ryanodine receptor function in newborn rat heart

The role of ryanodine receptor (RyR) in cardiac excitation-contraction (E-C) coupling in newborns (NB) is not completely understood. To determine whether RyR functional properties change during development, we evaluated cellular distribution and functionality of sarcoplasmic reticulum (SR) in NB rats. Sarcomeric arrangement of immunostained SR Ca(2+)-ATPase (SERCA2a) and the presence of sizeable caffeine-induced Ca2+ transients demonstrated that functional SR exists in NB. E-C coupling properties were then defined in NB and compared with those in adult rats (AD). Ca2+ transients in NB reflected predominantly sarcolemmal Ca2+ entry, whereas the RyR-mediated component was approximately 13%. Finally, the RyR density and functional properties at the single-channel level in NB were compared with those in AD. Ligand binding assays revealed that in NB, RyR density can be up to 36% of that found in AD, suggesting that some RyRs do not contribute to the Ca2+ transient. To test the hypothesis that RyR functional properties change during development, we incorporated single RyRs into lipid bilayers. Our results show that permeation and gating kinetics of NB RyRs are identical to those of AD. Also, endogenous ligands had similar effects on NB and AD RyRs: sigmoidal Ca2+ dependence, stronger Mg(2+)-induced inhibition at low cytoplasmic Ca2+ concentrations, comparable ATP-activating potency, and caffeine sensitivity. These observations indicate that NB rat heart contains fully functional RyRs and that the smaller contribution of RyR-mediated Ca2+ release to the intracellular Ca2+ transient in NB is not due to different single RyR channel properties or to the absence of functional intracellular Ca2+ stores.

Age-dependent biochemical and contractile properties in atrium of transgenic mice overexpressing junctin.

Junctin is a transmembrane protein of the cardiac junctional sarcoplasmic reticulum (SR) that binds to the ryanodine receptor, calsequestrin, and triadin 1. This quaternary protein complex is thought to facilitate SR Ca2+ release. To improve our understanding of the contribution of junctin to the regulation of SR function, we examined the age-dependent effects of junctin overexpression in the atrium of 3-, 6-, and 18-wk-old transgenic mice. The ratio of atrial weight and body weight was unchanged between junctin-overexpressing (JCN) and wild-type (WT) mice at all ages investigated (n=6-8). The protein expression of triadin 1 was decreased starting in 3-wk-old JCN atria (by 69%), whereas the expression of the ryanodine receptor was diminished in 6- (by 48%) and 18-wk-old (by 57%) JCN atria compared with age-matched WT atria. Force of contraction was decreased by 35% in 18-wk-old JCN compared with age-matched WT left atrial muscle strips, which was accompanied by a prolonged time of relaxation (48.1 +/- 0.9 vs. 44.2 +/- 0.8 ms, respectively, n=6-8, P <0.05). The spontaneous beating rate of isolated right atria was higher in 18-wk-old JCN mice compared with age-matched WT mice (389 +/- 10 vs. 357 +/- 6 beats/min, respectively, n=6-8, P <0.05). Heart rate was lower by 9% in telemetric ECG recordings in 18-wk-old JCN mice during stress tests. Three-week-old JCN atria exhibited a higher potentiation of force of contraction at rest pauses of 30 s (by 13%) and of 300 s (by 35%), suggesting increased SR Ca2+ content. This was consistent with the higher force of contraction in 3-wk-old JCN atria (by 29%) compared with age-matched WT atria (by 10%) under the administration of caffeine. We conclude that in 3-wk-old atria, junctin overexpression was associated with a reduced expression of triadin 1 resulting in a higher SR Ca2+ load without changes in contractility or heart rate. In 6-wk-old JCN atria, the compensatory downregulation of the ryanodine receptor may offset the effects of junctin overexpression. Finally, the progressive decrease in ryanodine receptor density may contribute to the decreased atrial contractility and lower heart rate during stress in 18-wk-old JCN mice.

Arrhythmogenic mechanisms in left ventricular hypertrophy.

An important mechanism contributing to the high mortality and sudden death in patients with left ventricular hypertrophy (LVH) is ventricular arrhythmia. Part of the risk is associated with the pro-arrhythmic electrophysiological phenotype of the hypertrophied myocardium. The most consistently observed abnormality is prolongation of the action potential duration and refractoriness, which sets the stage for arrhythmias based on early or delayed afterdepolarizations and triggered activity. In addition, non-uniform prolongation of the action potential in LVH may be pro-arrhythmic by leading to increased dispersion of repolarization or refractoriness and favouring re-entry. The occurrence of delayed after depolarization-induced triggered activity and other ventricular arrhythmias are also related to the impaired ability to handle intracellular calcium due to changes in the density of ryanodine receptors and the Ca2+-ATPase of the sarcoplasmic reticulum. Slowing and fractionation of ventricular conduction, creating the conditions for micro-reentry and arrhythmogenesis, are characteristic of severe LVH, as is the expression of the I(f) current (which may be a source of increased automaticity). The pro-arrhythmic potential of LVH is also related to the presence of coexisting 'extrinsic' factors. The most important and pro-arrhythmic association of LVH is that with myocardial ischaemia. Other conditions include neuroendocrine factors, ventricular wall stress or electrolyte disturbances. The electrophysiological mechanisms of the interactions between these 'extrinsic' factors and LVH have not been fully elucidated. Further research into these mechanisms is required and may have important implications for our understanding of the mechanisms of cardiac arrhythmias in LVH and the appropriate use of antiarrhythmic drug therapy.

Cellular mechanisms of altered contractility in the hypertrophied heart: big hearts, big sparks.

To investigate the cellular mechanisms for altered Ca2+ homeostasis and contractility in cardiac hypertrophy, we measured whole-cell L-type Ca2+ currents (ICa,L), whole-cell Ca2+ transients ([Ca2+]i), and Ca2+ sparks in ventricular cells from 6-month-old spontaneously hypertensive rats (SHRs) and from age- and sex-matched Wistar-Kyoto and Sprague-Dawley control rats. By echocardiography, SHR hearts had cardiac hypertrophy and enhanced contractility (increased fractional shortening) and no signs of heart failure. SHR cells had a voltage-dependent increase in peak [Ca2+]i amplitude (at 0 mV, 1330+/-62 nmol/L [SHRs] versus 836+/-48 nmol/L [controls], P<0.05) that was not associated with changes in ICa,L density or kinetics, resting [Ca2+]i, or Ca2+ content of the sarcoplasmic reticulum (SR). SHR cells had increased time of relaxation. Ca2+ sparks from SHR cells had larger average amplitudes (173+/-192 nmol/L [SHRs] versus 109+/-64 nmol/L [control]; P<0.05), which was due to redistribution of Ca2+ sparks to a larger amplitude population. This change in Ca2+ spark amplitude distribution was not associated with any change in the density of ryanodine receptors, calsequestrin, junctin, triadin 1, Ca2+-ATPase, or phospholamban. Therefore, SHRs with cardiac hypertrophy have increased contractility, [Ca2+]i amplitude, time to relaxation, and average Ca2+ spark amplitude ("big sparks"). Importantly, big sparks occurred without alteration in the trigger for SR Ca2+ release (ICa,L), SR Ca2+ content, or the expression of several SR Ca2+-cycling proteins. Thus, cardiac hypertrophy in SHRs is linked with an alteration in the coupling of Ca2+ entry through L-type Ca2+ channels and the release of Ca2+ from the SR, leading to big sparks and enhanced contractility. Alterations in the microdomain between L-type Ca2+ channels and SR Ca2+ release channels may underlie the changes in Ca2+ homeostasis observed in cardiac hypertrophy. Modulation of SR Ca2+ release may provide a new therapeutic strategy for cardiac hypertrophy and for its progression to heart failure and sudden death.

Effects of long‐term treatment with trandolapril on sarcoplasmic reticulum function of cardiac muscle in rats with chronic heart failure following myocardial infarction

1 Calcium transport activity of isolated cardiac sarcoplasmic reticulum (SR) including Ca2+ uptake and release is decreased in animals with chronic heart failure (CHF) following myocardial infarction. The present study was undertaken to determine whether an angiotensin converting enzyme (ACE) inhibitor, trandolapril, improves cardiac sarcoplasmic reticular function in animals with CHF following myocardial infarction. 2 CHF was induced by left coronary artery ligation in rats, which resulted in an infarction of approximately 45% of the left ventricle. Aortic flow and cardiac output index were decreased, and left ventricular end-diastolic pressure was increased 8 weeks after the operation, suggesting the development of CHF. 3 The developed force transients of cardiac skinned fibres of the rats with CHF were decreased when the skinned fibre was preloaded for 0.25-1 min with 10(-5) M Ca2+ (48-88%) and when preloaded with 10(-6) M Ca2+ and then exposed to 0.1-1 mM caffeine (45-93%). 4 The [3H]-ryanodine-binding activity in SR-enriched fractions was reduced by 23% in the CHF group. These results suggest that the amount of Ca2+ released from SR is decreased due to a reduced rate of SR Ca2+ uptake and a downregulation of the SR Ca2+-release channel. 5 Rats were treated orally with 3 mg kg(-1) day(-1) trandolapril from the 2nd to the 8th week after the coronary artery ligation. Treatment with trandolapril attenuated the reduction in aortic flow and cardiac output index and the increase in left ventricular end-diastolic pressure, and improved the developed force transients of the skinned fibre of the animal with CHF without causing a reduction of infarct size. Treatment with trandolapril also attenuated the reduction in ryanodine receptor density in the viable left ventricle of the rat with CHF. 6 It is concluded that long-term treatment with trandolapril attenuates cardiac SR dysfunction in rats with CHF and that the mechanism underlying this effect is, at least in part, attributed to prevention of downregulation of Ca2+ release channel.

Temporal differences in the induction of dihydropyridine receptor subunits and ryanodine receptors during skeletal muscle development.

The expression of the dihydropyridine (DHP) and ryanodine receptors in skeletal muscle was investigated during development of rat myotubes in culture as well as during embryonic and postnatal development in the rat. Through the use of specific gene probes, antibodies and radioligand binding ([3H]PN 200-110 (DHP) and [3H]ryanodine), we identified a significant difference between the time course of appearance of the DHP receptor and the ryanodine receptor during muscle development. Although the number of DHP receptors dramatically increased at early stages of development (up to day 7 in tissue culture and day 20 postnatal), increase in the ryanodine receptor density occurred comparatively later at day 10 in culture and day 30 postnatal. This process was associated with parallel changes in the expression of the mRNA encoding the alpha 1, alpha 2, and beta subunits of the DHP receptor and the skeletal muscle ryanodine receptor. The genes encoding the DHP receptor subunits were activated in a temporally distinct transcript appeared and plateaued first, at the onset of myoblast fusion and day 16 embryonic. This was followed closely by an increase in expression of the mRNAs for alpha 1 and alpha 2 subunits which coincided with the sharp rise in the DHP receptor density. Ryanodine receptor gene expression was induced well after the DHP receptor gene expression had plateaued. The temporal appearance of the polypeptides comprising the DHP receptor subunits and the ryanodine receptor paralleled the induction of the genes encoding these receptors. These results imply that gene expression is a major mechanism that contributes to the regulation of DHP and ryanodine receptor numbers during muscle development. The temporal differences in the induction of the genes encoding the DHP receptor subunits and the ryanodine receptor suggests that these genes are under the control of distinct endogenous factors. These differences in expression of the DHP receptor and the ryanodine receptor may contribute to the different mechanisms of excitation-contraction coupling in immature versus adult skeletal muscle.

Density of Ryanodine Receptors is Increased in Sarcoplasmic Reticulum from Prehypertrophic Cardiomyopathic Hamster Heart

Calcium overload has been linked to the development of cardiomyopathy in the cardiomyopathic (CM) hamster, but the site or sites of the lesion remain obscure. To determine whether the number of sarcoplasmic reticulum (SR) calcium release channels (ryanodine receptors) changes in the CM heart, we compared the density ( B max) and affinity ( K d) of [ 3H]-ryanodine binding sites in heavy SR fractions from 40-65 day-old normal and CM hamster hearts. Results showed that the B max was significantly increased in CM heart when compared to normal ( B max = 2489 ± 159 fmol/mg protein in normal heart and 3360 ±223 fmol/mg protein in CM heart, mean ± S.E., P = 0.01). [ 3H]-Ryanodine bound to a single, high affinity site in SR from both normal and CM hearts; values for K d were similar in both groups. Sensitivity of [ 3H]-ryanodine binding to Ca 2+ was unchanged, but the density of binding was increased at all Ca 2+ concentrations which potentiated binding in CM heart. Similarly, potentiation of [ 3H]-ryanodine binding by ATP and inhibition of binding by Mg 2+ were intact in membranes from CM heart. Results demonstrate that the density of [ 3H]-ryanodine receptors is increased in SR from CM hearts early in the development of cardiomyopathy, although the properties of these receptors are unchanged. This suggests an increase in the amount or velocity of Ca 2+ release from SR may contribute to the development of Ca 2+ overload in this model of cardiomyopathy.

The density of ryanodine receptors decreases with pressure overload-induced rat cardiac hypertrophy

We investigate the possibility that alterations in the calcium movements of the hypertrophied rat heart might involve sarcoplasmic reticulum (SR) ryanodine receptors. A decreased receptor density was observed with severe hypertrophy (0.26±0.05 and 0.35±0.06 pmol/mg protein and 170 and 366 receptors/μm 2 of SR in 50–80% hypertrophy and control, respectively); however, the total number of receptors per left ventricle was unchanged. The dissociation constant (0.7 nM) was similar in both hypertrophied and control left ventricles. Thus the decreased density of the ryanodine receptors may participate in altered calcium movements in hypertrophied rat heart.

Solubilization and separation of Ca 2+-ATPase from the Ca 2+-ryanodine receptor complex

Heavy sarcoplasmic reticulum (SR) preparations of rabbit skeletal muscle, which are enriched in Ca2+-release vesicles from the terminal cisternae (TC) and [3H]ryanodine receptor density, exhibit 60% of the Ca2+-ATPase activity, 58% of the EP level, and 30% of the steady state Ca2+ loading compared to membrane vesicles from the longitudinal SR. The Ca2+-ATPase of TC SR is solubilized and separated from the Ca2+-ryanodine receptor complex in the insoluble fraction on treatment with the detergent C12E9. However, a 50% decrease in receptor density is observed upon removal of the Ca2+-ATPase, suggesting a significant contribution of this protein to maintaining optimal receptor complex density.