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Aims Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been shown to induce aberrant Ca2+ release from the cardiac ryanodine receptor (RyR2) in various diseased hearts. However, the precise pathogenic mechanism remains to be elucidated. Here, we investigated the effect of dantrolene (DAN): a RyR2 stabilizer on local Ca2+ release, cardiac function, and lethal arrhythmia in CaMKIIδc transgenic (TG) mice. Methods and results The TG mice showed an increase in left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) with a reduction in LV fractional shortening (LVFS). The phosphorylation levels of Ser2814 in RyR2 and Thr287 in CaMKII increased in TG mice. In TG cardiomyocytes, peak cell shortening (CS) decreased, and the frequency of spontaneous Ca2+ transients (sCaTs) increased. Endogenous RyR2-associated calmodulin (CaM) markedly decreased in TG cardiomyocytes. After chronic DAN treatment for 1 month, LVESD (but not LVEDD) decreased with an increase in LVFS. In the chronic DAN-treated cardiomyocytes, CS increased, sCaTs decreased, and the endogenous CaM binding to RyR2 normally restored. The phosphorylation levels of Ser2814 in RyR2 and Thr287 in CaMKII remained elevated even after DAN treatment. Moreover, in TG mice, chronic DAN treatment prevented sustained ventricular tachycardia induced by epinephrine. Conclusions Defective association of CaM with RyR2 is most likely to be involved in the pathogenesis of CaMKII-mediated cardiac dysfunction and lethal arrhythmia.

Aims Cardiac Troponin T (TnT) mutation-linked familial hypertrophic cardiomyopathy (FHC) is known to cause sudden cardiac death at a young age. Here, we investigated the role of the Ca2+ release channel of the cardiac sarcoplasmic reticulum (SR), ryanodine receptor (RyR2), in the pathogenic mechanism of lethal arrhythmia in FHC-related TnT-mutated transgenic mice (TG; TnT-delta160E). Methods and results In TG cardiomyocytes, the Ca2+ spark frequency (SpF) was much higher than that in non-TG cardiomyocytes. These differences were more pronounced in the presence of isoproterenol (ISO; 10 nM). This increase in SpF was largely reversed by a CaMKII inhibitor (KN-93), but not by a protein kinase A inhibitor (H89). CaMKII phosphorylation at Ser2814 in RyR2 was increased significantly in TG. Spontaneous Ca2+ transients (sCaTs) after cessation of a 1–5 Hz pacing, frequently observed in ISO-treated TG cardiomyocytes, were also attenuated by KN-93, but not by H89. The RyR2 stabilizer dantrolene attenuated Ca2+ sparks and sCaTs in ISO-treated TG cardiomyocytes, indicating that the mutation-linked aberrant Ca2+ release is mediated by destabilized RyR2. Conclusions In FHC-linked TnT-mutated hearts, RyR2 is susceptible to CaMKII-mediated phosphorylation, presumably because of a mutation-linked increase in diastolic [Ca2+]i, causing aberrant Ca2+ release leading to lethal arrhythmia.

Aberrant Ca2+ release from cardiac ryanodine receptors (RyR2) has been shown to be one of the most important causes of lethal arrhythmia in various types of failing hearts. We previously showed that dantrolene, a specific agent for the treatment of malignant hyperthermia, inhibits Ca2+ leakage from the RyR2 by correcting the defective inter-domain interaction between the N-terminal (1-619 amino acids) and central (2000-2500 amino acids) domains of the RyR2 and allosterically enhancing the binding affinity of calmodulin to the RyR2 in diseased hearts. In this study, we examined whether dantrolene inhibits this Ca2+ leakage, thereby preventing the pharmacologically inducible ventricular tachycardia in ventricular pressure-overloaded failing hearts. Ventricular tachycardia (VT) was easily induced after an injection of epinephrine in mice after 8 weeks of transverse aortic constriction-induced pressure-overload. Pretreatment with dantrolene almost completely inhibited the pharmacologically inducible VT. In the presence of dantrolene, the occurrence of both Ca2+ sparks and spontaneous Ca2+ transients was inhibited, which was associated with enhanced calmodulin binding affinity to the RyR2. These results suggest that dantrolene could be a new potent agent in the treatment of lethal arrhythmia in cases of acquired heart failure.

The heart LIM protein (HLP) is a LIM-only protein family member that mediates protein-protein interactions. To date, no studies have yet been conducted regarding its function in the heart. In the present study, we have identified that HLP binds the cytosolic region of RyR2 in the heart using a bacterial two-hybrid system, LC-MS/MS, co-immunoprecipitation, and GST-pull down assays. Microscopy revealed that HLP forms a triple complex with RyR2 and caveolin-3. siRNA and adenovirus-mediated KD of HLP decreased the electrically evoked Ca(2+) release from the sarcoplasmic reticulum without directly affecting SERCA2 and RyR2 activities. Collectively, the HLP-RyR2 interaction in the cell surface caveolae region may be essential for efficient excitation-contraction coupling in the heart.

Ryanodine is a cell permeant plant alkaloid that binds selectively and with high affinity to ryanodine receptor (RyR) Ca(2+) release channels. Sub-micromolar ryanodine concentrations activate RyR channels while micromolar concentrations are inhibitory. Several reports indicate that neuronal synaptic plasticity, learning and memory require RyR-mediated Ca(2+)-release, which is essential for muscle contraction. The use of micromolar (inhibitory) ryanodine represents a common strategy to suppress RyR activity in neuronal cells: however, micromolar ryanodine promotes RyR-mediated Ca(2+) release and endoplasmic reticulum Ca(2+) depletion in muscle cells. Information is lacking in this regard in neuronal cells; hence, we examined here if addition of inhibitory ryanodine elicited Ca(2+) release in primary hippocampal neurons, and if prolonged incubation of primary hippocampal cultures with inhibitory ryanodine affected neuronal ER calcium content. Our results indicate that inhibitory ryanodine does not cause Ca(2+) release from the ER in primary hippocampal neurons, even though ryanodine diffusion should produce initially low intracellular concentrations, within the RyR activation range. Moreover, neurons treated for 1 h with inhibitory ryanodine had comparable Ca(2+) levels as control neurons. These combined findings imply that prolonged incubation with inhibitory ryanodine, which effectively abolishes RyR-mediated Ca(2+) release, preserves ER Ca(2+) levels and thus constitutes a sound strategy to suppress neuronal RyR function.

Ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of the L-type Ca(2+) channel (LTCC). The aim was to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-Specific dominant-negative suppression of Rad. Transgenic (TG) mice overexpressing dominant-negative mutant Rad (S105N Rad TG) were generated. To measure intracellular Ca(2+) concentration ([Ca(2+)]i), we recorded [Ca(2+)]i transients and Ca(2+) sparks from isolated cardiomyocytes using confocal microscopy. The mean [Ca(2+)]i transient amplitude was significantly increased in S105N Rad TG cardiomyocytes, compared with control littermate mouse cells. The frequency of Ca(2+) sparks was also significantly higher in TG cells than in control cells, although there were no significant differences in amplitude. The sarcoplasmic reticulum Ca(2+) content was not altered in the S105N Rad TG cells, as assessed by measuring caffeine-induced [Ca(2+)]i transient. In contrast, phosphorylation of Ser(2809) on the cardiac ryanodine receptor (RyR2) was significantly enhanced in TG mouse hearts compared with controls. Additionally, the Rad-mediated RyR2 phosphorylation was regulated via a direct interaction of Rad with protein kinase A (PKA).

Cardiac ryanodine receptor gene (RyR2) mutations cause fatal arrhythmogenic diseases such as catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy. The N-terminal region of RyR2 is one of the hot spots for mutations. In this study, we investigated cardiac phenotypes of a knock-in mouse model carrying R420W mutation of RyR2. The N-terminal R420W mutation has already been found in juvenile sudden death cadavers of unrelated families. The depolarization-induced Ca(2+) transient amplitude was significantly lower in cardiomyocytes from RyR2(R420W/R420W) mice compared with wild-type mice. The time to peak of the Ca(2+) transient was significantly increased in RyR2(R420W/R420W) mice. Furthermore, the prolonged decay time from the peak of the Ca(2+) transient was detected in RyR2(R420W/R420W) mice. ECG telemetry revealed that various types of arrhythmias were induced in RyR2(R420W/R420W) mice in response to administration of caffeine and adrenaline. The mutant mice showed high occurrences of arrhythmias in response to heart stimulants compared with wild-type mice. These findings suggest that R420W mutation impairs depolarization-induced Ca(2+) oscillation in cardiomyocytes, which possibly results in sudden death due to stress-induced arrhythmias.

Cyclic ADP-ribose (cADPR), a potent Ca 2+ mobilizing intracellular messenger synthesized by CD38, regulates the opening of ryanodine receptors (RyRs). Increases in intracellular Ca 2+ concentrations in pancreatic islets, resulting from Ca 2+ mobilization from RyRs as well as Ca 2+ influx from extracellular sources, are important in insulin secretion by glucose. In the present study, by screening a rat islet cDNA library, we isolated a novel RyR cDNA (the islet-type RyR), which is generated from the RyR2 gene by alternative splicing of exons 4 and 75. When the expression vectors for the islet-type and the authentic RyRs were transfected into HEK293 cells, the islet-type RyR2 as well as the authentic one showed high affinity [ 3 H]ryanodine binding. Intracellular Ca 2+ release in the islet-type RyR2-transfected cells was enhanced in the presence of cADPR but not in the authentic RyR2-transfected cells. The islet-type RyR2 mRNA was expressed in a variety of tissues such as in pancreatic islets, cerebrum, and cerebellum, whereas the authentic RyR2 mRNA was predominantly expressed in heart and aorta. These results suggest that the islet-type RyR2 may be an intracellular target for cADPR signaling.

We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 [3]). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca(2+)/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca(2+) transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca(2+) transients results in the appearance of trains of Ca(2+) spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca(2+) events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program.

The discovery of the actomyosin system provided for the first time a model system that enabled the study of the role of the muscle protein components in the contraction and relaxation cycle to be undertaken. It soon became apparent that ATP was essential for both processes but progress really began when it became clear that components both in the myofibrillar and sarcoplasmic fractions were involved in relaxation. After it was apparent that a trace of calcium was required for the activation of the MgATPase of the myofibrils it was shown that an active calcium pump was located in the sarcoplasmic reticulum. The report by Ebashi in 1963 that a new myofibrillar protein, troponin, was the target for calcium opened up the investigation of the calcium control of the MgATPase. Troponin was shown to be a complex of troponin C, I and T, each protein being under individual genetic control and existing in isoforms specific for the muscle type. The unique forms of troponin I and T in cardiac muscle make them the biomarkers of choice for cardiac injury.

Calumenin is a multiple EF-hand Ca 2+ -binding protein located in endo/sarcoplasmic reticulum of mammalian tissues. In the present study, we cloned two rabbit calumenin isoforms (rabbit calumenin-1 and -2, GenBank Accession Nos. AY225335 and AY225336 , respectively) by RT-PCR. Both isoforms contain a 19 aa N-terminal signal sequence, 6 EF-hand domains, and a C-terminal ER/SR retrieval signal, HDEF. Both calumenin isoforms exist in rabbit cardiac and skeletal muscles, but calumenin-2 is the main isoform in skeletal muscle. Presence of calumenin in rabbit sarcoplasmic reticulum (SR) was identified by Western blot analysis. GST-pull down and co-immunoprecipitation experiments showed that ryanodine receptor 1 (RyR1) interacted with calumenin-2 in millimolar Ca 2+ concentration range. Experiments of gradual EF-hand deletions suggest that the second EF-hand domain is essential for calumenin binding to RyR1. Adenovirus-mediated overexpression of calumenin-2 in C2C12 myotubes led to increased caffeine-induced Ca 2+ release, but decreased depolarization-induced Ca 2+ release. Taken together, we propose that calumenin-2 in the SR lumen can directly regulate the RyR1 activity in Ca 2+ -dependent manner.

In this study, we have investigated block of potassium (K(+)) current by neomycin, a large polycation, from the luminal face of the type 3 ryanodine receptor (RyR3). Previous studies have shown that neomycin is an open channel blocker of RyR2 that interacts with negatively charged residues in the mouth of the conduction pathway to partially occlude it. In the current study, we have used neomycin as a probe to investigate proposed negatively charged regions in the luminal pore mouth of RyR3. Luminal neomycin induces concentration- and voltage-dependent partial block to a subconductance state in RyR3. Blocking parameters calculated in this study show that neomycin has a higher affinity for RyR3 than RyR2, but block may occur at the same site within the pore mouth. The change in affinity may be due to altered negative charge density at the site of interaction.

Ryanodine receptors (RyR) are the Ca2+ release channels of sarcoplasmic reticulum that provide the majority of the [Ca2+] necessary to induce contraction of cardiac and skeletal muscle cells. In their cellular environment, RyRs are exquisitely regulated by a variety of cytosolic factors and accessory proteins so that their output signal (Ca2+) induces cell contraction without igniting signaling pathways that eventually lead to contractile dysfunction or pathological cellular remodeling. Here we review how dysfunction of RyRs, most commonly expressed as enhanced Ca2+ release at rest (skeletal muscle) or during diastole (cardiac muscle), appears to be the fundamental mechanism underlying several genetic or acquired syndromes. In skeletal muscle, malignant hyperthermia and central core disease result from point mutations in RYR1, the skeletal isoform of RyRs. In cardiac muscle, RYR2 mutations lead to catecholaminergic polymorphic ventricular tachycardia and other cardiac arrhythmias. Lastly, an altered phosphorylation of the RyR2 protein may be involved in some forms of congestive heart failure.

Aims Calmodulin (CaM) plays a key role in modulating channel gating in ryanodine receptor (RyR2). Here, we investigated (a) the pathogenic role of CaM in the channel disorder in CPVT and (b) the possibility of correcting the CPVT-linked channel disorder, using knock-in (KI) mouse model with CPVT-associated RyR2 mutation (R2474S). Methods and results Transmembrane potentials were recorded in whole cell current mode before and after pacing (1–5 Hz) in isolated ventricular myocytes. CaM binding was assessed by incorporation of exogenous CaM fluorescently labeled with HiLyte Fluor® in saponin-permeabilized myocytes. In the presence of cAMP (1 μM) the apparent affinity of CaM binding to the RyR decreased in KI cells (Kd: 140–400 nM), but not in WT cells (Kd: 110–120 nM). Gly-Ser-His-CaM (GSH-CaM that has much higher RyR-binding than CaM) restored normal binding to the RyR of cAMP-treated KI cells (140 nM). Neither delayed afterdepolarization (DAD) nor triggered activity (TA) were observed in WT cells even at 5 Hz pacing, whereas both DAD and TA were observed in 20% and 12% of KI cells, respectively. In response to 10 nM isoproterenol, only DAD (but not TA) was observed in 11% of WT cells, whereas in KI cells the incidence of DAD and TA further increased to 60% and 38% of cells, respectively. Addition of GSH-CaM (100 nM) to KI cells decreased both DADs and TA (DAD: 38% of cells; TA: 10% of cells), whereas CaM (100 nM) had no appreciable effect. Addition of GSH-CaM to saponin-permeabilized KI cells decreased Ca 2+ spark frequency (+33% of WT cells), which otherwise markedly increased without GSH-CaM (+100% of WT cells), whereas CaM revealed much less effect on the Ca 2+ spark frequency (+76% of WT cells). Then, by incorporating CaM or GSH-CaM to intact cells (with protein delivery kit), we assessed the in situ effect of GSH-CaM (cytosolic [CaM] = ∼240 nM, cytosolic [GSH-CaM] = ∼230 nM) on the frequency of spontaneous Ca 2+ transient (sCaT, % of total cells). Addition of 10 nM isoproterenol to KI cells increased sCaT after transient 5 Hz pacing (37%), whereas it was much more attenuated by GSH-CaM (9%) than by CaM (26%) ( P Conclusions Several disorders in the RyR channel function characteristic of the CPVT-mutant cells (increased spontaneous Ca 2+ leak, delayed afterdepolarization, triggered activity, Ca 2+ spark frequency, spontaneous Ca 2+ transients) can be corrected to a normal function by increasing the affinity of CaM binding to the RyR.

We found that glutathione transport across endo/sarcoplasmic reticulum membranes correlates with the abundance of ryanodine receptor type 1 (RyR1). The transport was the fastest in muscle terminal cisternae, fast in muscle microsomes and slow in liver, heart, and brain microsomes. Glutathione influx could be inhibited by RyR1 blockers and the inhibitory effect was counteracted by RyR1 agonists. The effect of blockers was specific to glutathione, as the transport of other small molecules was not hindered. Therefore, the glutathione transport activity seems to be associated with RyR1 in sarcoplasmic reticulum.

FK506 binding protein (FKBP) is a cytosolic receptor for the immunosuppressive drug FK-506. The common isoform, FKBP12, was found to be associated with the calcium release channel (ryanodine receptor 1) of different species of vertebrate skeletal muscle, whereas 12.6, a novel FKBP isoform was found to be associated with canine cardiac ryanodine receptor (ryanodine receptor 2). Until recently, canine cardiac sarcoplasmic reticulum was considered to be the prototype for studying heart RyR2 and its interactions with FKBP. In this study, cardiac microsomes were isolated from diverse vertebrates: human, rabbit, rat, mice, dog, chicken, frog, and fish and were analyzed for their ability to bind or exchange with FKBP isoforms 12 and 12.6. Our studies indicate that RyR2 from seven out of the eight animals contain both FKBP12 and 12.6. Dog is the exception. It can now be concluded that the association of FKBP isoforms with RyR2 is widely conserved in the hearts of different species of vertebrates.

Unloading of skeletal muscles by hindlimb unweighting is known to induce muscle atrophy and a shift toward faster contractile properties associated with an increase in the expression of fast contractile proteins, particularly in slow soleus muscles. Contractile properties suggest that slow soleus muscles acquire SR properties close to those of a faster one. We studied the expression and properties of the sarcoplasmic reticulum calcium release (RyR) channels in soleus and gastrocnemius muscles of rats submitted to hindlimb unloading (HU). An increase in RyR1 and a slight decrease in RyR3 expression was detected in atrophied soleus muscles only after 4 weeks of HU. No variation appeared in fast muscles. [(3)H]Ryanodine binding experiments showed that HU neither increased the affinity of the receptors for [(3)H]ryanodine nor changed the caffeine sensitivity of [(3)H]ryanodine binding. Our results suggested that not only RyR1 but also RyR3 expression can be regulated by muscle activity and innervation in soleus muscle. The changes in the RyR expression in slow fibers suggested a transformation of the SR from a slow to a fast phenotype.

Ca2+ influx via L-type voltage-gated Ca2+ channels supports the plateau phase of ventricular action potentials and is the trigger for excitation–contraction (EC) coupling in the myocardium. Rad, a member of the RGK ( R em, R em2, R ad, G em/ K ir) family of monomeric G proteins, regulates ventricular action potential duration and EC coupling gain through its ability to inhibit cardiac L-type channel activity. In this study, we have investigated the potential dysfunction of a naturally occurring Rad variant (Q66P) that has been associated with congestive heart failure in humans. Specifically, we have tested whether Rad Q66P limits, or even eliminates, the inhibitory actions of Rad on CaV1.2 and CaV1.3, the two L-type channel isoforms known to be expressed in the heart. We have found that mouse Rad Q65P (the murine equivalent of human Rad Q66P) inhibits L-type currents conducted by CaV1.2 or CaV1.3 channels as potently as wild-type Rad (>95% inhibition of both channels). In addition, Rad Q65P attenuates the gating movement of both channels as effectively as wild-type Rad, indicating that the Q65P substitution does not differentially impair any of the three described modes of L-type channel inhibition by RGK proteins. Thus, we conclude that if Rad Q66P contributes to cardiomyopathy, it does so via a mechanism that is not related to its ability to inhibit L-type channel-dependent processes per se. However, our results do not rule out the possibility that decreased expression, mistargeting or altered regulation of Rad Q66P may reduce the RGK protein’s efficacy in vivo.

Functional studies on vascular smooth muscle suggest the presence of ryanodine receptors (RyRs) with differing properties. In an attempt to understand such differences we investigated, using reverse transcription-polymerase chain reaction (RT-PCR), the possibility that smooth muscle cells express multiple types of RyRs. RNA was extracted from rat aorta, superior and small mesenteric arterial vessels, purified aortic smooth muscle media, or cultured aortic smooth muscle cells for cDNA synthesis. The cDNAs encoding RyR1, RyR2, and RyR3 were amplified using PCR primers based on sequences close to the 3' coding region of the RyR genes and PCR products verified by restriction endonuclease analysis. All three members of the RyR gene family were found to be present in vascular smooth muscle. This finding of multiple types of RyRs expressed in the same cell type indicates a complex mechanism of RyR Ca2+ channel regulation involving the formation of homo- and/or heterotetrameric complexes.

The ryanodine receptor (RyR) is a large, intracellular calcium (Ca 2+ ) channel that is associated with several accessory proteins and is an important component of a cell’s ability to respond to changes in the environment. Three isoforms of the RyR exist and are well documented for skeletal and cardiac muscle and the brain, but the isoforms in non-excitable cells are poorly understood. The aggressiveness of breast cancers in women has been positively correlated with the expression of the RyR in breast tumor tissue, but it is unknown if this is limited to specific isoforms. Identification and characterization of RyRs in cancer models is important in understanding the role of the RyR channel complex in cancer and as a potential therapeutic target. The objective of this report was to identify the RyR isoforms expressed in widely used prostate cancer cell lines, DU-145 and LNCaP, and the non-tumorigenic prostate cell line, PWR-1E. Oligonucleotide primers specific for each isoform were used in semi-quantitative and real-time PCR to determine the identification and expression levels of the RyR isoforms. RyR1 was expressed in the highest amount in DU-145 tumor cells, expression was 0.48-fold in the non-tumor cell line PWR-1E compared to DU-145 cells, and no expression was observed in LNCaP tumor cells. DU-145 cells had the lowest expression of RyR2. The expression was 26- and 15-fold higher in LNCaP and PWR-1E cells, respectively. RyR3 expression was not observed in any of the cell lines. All cell types released Ca 2+ in response to caffeine showing they had functional RyRs. Total cellular RyR-associated Ca 2+ release is determined by both the number of activated RyRs and its accessory proteins which modulate the receptor. Our results suggest that the correlation between the expression of the RyR and tumor aggression is not related to specific RyR isoforms, but may be related to the activity and number of receptors.

We find that the purified ryanodine receptor/calcium release channel (CRC) isolated from canine heart sarcoplasmic reticulum (SR) is tightly associated with an FK-506 binding protein which is distinct from human recombinant FKBP-12. Fractions from both skeletal and heart muscle were probed by Western Blot analysis using sequence specific antibody raised against the N-terminus of human recombinant FKBP-12. Only FKBP-12 was detected in rabbit and dog skeletal muscle SR as well as in dog heart cytosol. Canine heart SR and the purified canine heart ryanodine receptor contained an immunoreactive band with a somewhat slower relative mobility than that of human recombinant FKBP-12. These observations indicate that the association between the cardiac CRC and this novel FK-506 binding protein is specific.

Endothelial outgrowth cells (EOCs) derived from blood mononuclear cells can differentiate to an endothelial-like phenotype. There are deficits in understanding of the biology of these cells, particularly detailed characterisation of their Ca(2+) signalling mechanisms. In the current study, it was found that human EOCs express two forms of ryanodine receptor (RyR1 and RyR2) Ca(2+) release channel in their endoplasmic reticulum. Individual EOCs display heterogeneous Ca(2+) responses to physiologically relevant regulators fibrinogen and collagen. Some EOCs showed distinctive, multiphasic Ca(2+) responses to fibrinogen consisting of rapid decreases, transient increases then a gradual return to the resting levels. Transient elevations in Ca(2+) required both L-type voltage gated calcium channels and RyRs. Decreases in Ca(2+) stimulated by fibrinogen depended on plasma membrane Ca(2+) ATPase pumps, but did not require thapsigargin-sensitive Ca(2+) ATPases. These results indicate that EOCs possess sophisticated Ca(2+) signalling mechanisms, capable of generating distinct Ca(2+) waveforms in response to different physiologically relevant cues.

Calmodulin (CaM), one of the accessory proteins of the cardiac ryanodine receptor (RyR2), is known to play a significant role in the channel regulation of the RyR2. However, the possible involvement of calmodulin in the pathogenic process of catecholaminergic polymorphic ventricular tachycardia (CPVT) has not been investigated. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions using a knock-in (KI) mouse model with CPVT-linked RyR2 mutation (R2474S). Without added effectors, the affinity of CaM binding to the RyR2 was indistinguishable between KI and WT hearts. In response to cAMP (1 μmol/L), the RyR2 phosphorylation at Ser2808 increased in both WT and KI hearts to the same extent. However, cAMP caused a significant decrease of the CaM-binding affinity in KI hearts, but the affinity was unchanged in WT. Dantrolene restored a normal level of CaM-binding affinity in the cAMP-treated KI hearts, suggesting that defective inter-domain interaction between the N-terminal domain and the central domain of the RyR2 (the target of therapeutic effect of dantrolene) is involved in the cAMP-induced reduction of the CaM-binding affinity. In saponin-permeabilized cardiomyocytes, the addition of cAMP increased the frequency of spontaneous Ca2+ sparks to a significantly larger extent in KI cardiomyocytes than in WT cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca2+ sparks. In conclusion, CPVT mutation causes defective inter-domain interaction, significant reduction in the ability of CaM binding to the RyR2, spontaneous Ca2+ leak, and then lethal arrhythmia.

S-Adenosyl-l-methionine (SAM) is the biological methyl-group donor for the enzymatic methylation of numerous substrates including proteins. SAM has been reported to activate smooth muscle derived ryanodine receptor calcium release channels. Therefore, we examined the effects of SAM on the cardiac isoform of the ryanodine receptor (RyR2). SAM increased cardiac sarcoplasmic reticulum [(3)H]ryanodine binding in a concentration-dependent manner by increasing the affinity of RyR2 for ryanodine. Activation occurred at physiologically relevant concentrations. SAM, which contains an adenosine moiety, enhanced ryanodine binding in the absence but not in the presence of an ATP analogue. S-Adenosyl-l-homocysteine (SAH) is the product of the loss of the methyl-group from SAM and inhibits methylation reactions. SAH did not activate RyR2 but did inhibit SAM-induced RyR2 activation. SAH did not alter adenine nucleotide activation of RyR2. These data suggest SAM activates RyR2 via a site that interacts with, but is distinct from, the adenine nucleotide binding site.

The intracellular Ca 2+ release channels are indispensable molecular machinery in practically all eukaryotic cells of multicellular animals. They serve a key role in cell signaling by way of Ca 2+ as a second messenger. In response to a signaling event, the channels release Ca 2+ from intracellular stores. The resulting rise in cytoplasmic Ca 2+ concentration triggers the cell to carry out its specialized role, after which the intracellular Ca 2+ concentration must be reduced so that the signaling event can again be repeated. There are two types of intracellular Ca 2+ release channels, i.e., the ryanodine receptors and the inositol triphosphate receptors. My focus in this minireview is to present a personal account, from the vantage point our laboratory, of the discovery, isolation, and characterization of the ryanodine receptors from mammalian muscle. There are three isoforms: ryanodine receptor 1 (RyR1), first isolated from rabbit fast twitch skeletal muscle; ryanodine receptor 2 (RyR2), first isolated from dog heart; and ryanodine receptor 3 (RyR3), first isolated from bovine diaphragm muscle. The ryanodine receptors are the largest channel structures known. The RyR isoforms are very similar albeit with important differences. Natural mutations in humans in these receptors have already been associated with a number of muscle diseases.

Arrhythmogenic point mutations in RyR2 result in abnormal Ca(2+) release following cardiac stimulation, leading to sudden cardiac death (SCD). Recently, we have demonstrated that significant functional differences exist between SCD-linked RyR2 mutations. Here, we investigated the molecular basis of this heterogeneity and determined the sensitivity of mutant RyR2 channels to cytoplasmic [Ca(2+)] ([Ca(2+)](c)) in living cells. Using streptolysin-O permeabilised human embryonic kidney cells, [Ca(2+)](c) was clamped in cells expressing GFP-tagged wild-type (WT) or SCD-linked RyR2 mutants (L(433)P, N(2386)I, and R(176)Q/T(2504)M). Although resting [Ca(2+)](c) was comparable in all cells, RyR2 mutants were characterised by a profound loss of Ca(2+)-dependent inhibition following caffeine stimulation when compared with WT channels. The ER Ca(2+) store was not perturbed in these experiments. Our findings support the hypothesis that SCD-linked mutational loci may be an important mechanistic determinant of RyR2 dysfunction and indicate that there is unlikely to be a unifying mechanism for channel dysfunction in SCD.

Altered cardiac ryanodine receptor (RyR2) function has an important role in heart failure and genetic forms of arrhythmias. RyR2 constitutes the major intracellular Ca2+ release channel in the cardiac sarcoplasmic reticulum (SR). The peptidyl-prolyl isomerase calstabin2 (FKBP12.6) is a component of the RyR2 macromolecular signaling complex. Calstabin2 binding to RyR2 is regulated by PKA phosphorylation of Ser2809 in RyR2. PKA phosphorylation of RyR2 decreases the binding affinity for calstabin2 and increases RyR2 open probability and sensitivity to Ca2+-dependent activation. In heart failure, a majority of studies have found that RyR2 becomes chronically PKA hyper-phosphorylated which depletes calstabin2 from the channel complex. Calstabin2 dissociation causes a diastolic SR Ca2+ leak contributing to depressed intracellular Ca2+ cycling and decreased cardiac contractility. Missense mutations linked to genetic forms of exercise-induced arrhythmias and sudden cardiac death also cause decreased calstabin2-binding affinity and leaky RyR2 channels. We review the importance of calstabin2 for RyR2 function and excitation-contraction coupling, and discuss new observations that implicate dysregulation of calstabin2 binding as a central mechanism for abnormal calcium cycling in heart failure and triggered arrhythmias.

Ryanodine receptor (RyR), a homotetrameric Ca2+ release channel, is one of the main actors in the generation of Ca2+ signals that trigger muscle contraction. Three genes encode three isoforms of RyRs, which have tissue-restricted distribution. RyR1 and RyR2 are typical of muscle cells, with RyR1 originally considered the skeletal muscle type and RyR2 the cardiac type. However, RyR1 and RyR2 have recently been found in numerous other cell types, including, for instance, peripheral B and T lymphocytes. In contrast, RyR3 is widely distributed among cells. RyR1 and RyR2 are localized in a specialized portion of the sarcoplasmic reticulum (SR), the terminal cisternae, which is the portion of the SR Ca2+ store that releases Ca2+ to control the process of muscle contraction. A specific role for RyR3 has not yet been established: probably, its co-expression with the other RyR isoforms contributes to qualitatively modulate Ca2+-dependent processes in muscle cells and in neurons. Several mutations in the genes encoding RyR1 and RyR2 have been identified in autosomal dominant diseases of skeletal and cardiac muscle, such as malignant hyperthermia (MH), central core disease (CCD), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). More recently, CCD cases with recessive inheritance have also been described. MH is a pharmacogenetic disease, but the others manifest as congenital myopathies. Even if their clinical phenotypes are well established, particularly in skeletal muscle, the molecular mechanisms that generate the conditions are not clear. A number of studies on cellular models have attempted to elucidate the molecular defects associated with the different mutations, but the problem of understanding how mutations in the same gene generate such an array of diverse pathological traits and diseases of widely different degrees of severity is still open. This review will consider the molecular and cellular effects of RyR mutations, summarizing recent data in the literature on Ca2+ dysregulation, which may lead to a better understanding of the functioning of RyRs.

The recently devised domain peptide probe technique was used to identify and characterize critical domains of the cardiac ryanodine receptor (RyR2). A synthetic peptide corresponding to the Gly(2460)-Pro(2495) domain of the RyR2, designated DPc10, enhanced the ryanodine binding activity and increased the sensitivity of the RyR2 to activating Ca(2+): the effects that resemble the typical phenotypes of cardiac diseases. A single Arg-to-Ser mutation made in DPc10, mimicking the recently reported Arg(2474)-to-Ser(2474) human mutation, abolished all of these effects that would have been produced by DPc10. On the basis of the principle of the domain peptide probe approach (see Model 1), these results indicate that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. This domain peptide approach opens the new possibility in the studies of the regulatory and pathogenic mechanisms of the cardiac Ca(2+) channel.

Although it is well established that voltage-sensing of the alpha(1)-dihydropyridine receptor triggers Ca(2+)-release via the ryanodine receptor during excitation-contraction coupling in skeletal muscle fibers, it remains to be determined which junctional components are responsible for the assembly, maintenance, and stabilization of triads. Here, we analyzed the expression pattern and neighborhood relationship of a novel 90-kDa sarcoplasmic reticulum protein. This protein is highly enriched in the triad fraction and is predominantly expressed in fast-twitching muscle fibers. Chronic low-frequency electro-stimulation induced a drastic decrease in the relative abundance of this protein. Chemical crosslinking showed a potential overlap between the 90-kDa junctional face membrane protein and the ryanodine receptor Ca(2+)-release channel, suggesting tight protein-protein interactions between these two triad components. Hence, Ca(2+)-regulatory muscle proteins have a strong tendency to oligomerize and the triad region of skeletal muscle fibers forms supramolecular membrane complexes involved in the regulation of Ca(2+)-homeostasis and signal transduction.

The effects of an inhibitor of ADP/ATP translocase (AAT) mainly expressed in the mitochondria inner membrane, atractyloside (ATR), on the gating property of the Ca2+ channels in the sarcoplasmic reticulum (SR) vesicles from the rabbit skeletal muscle were investigated using ion flux measurement and single channel recording. At 10 microM of cytoplasmic Ca2+, ATR decreased the rate constant of choline+ influx through the Ca2+ channels up to about 60% and perfectly inhibited about half the population of single Ca2+ channels incorporated into planar bilayers. Furthermore, the inhibition of the Ca2+ channels by ATR was effective at lower Ca2+. These results support the previous results that AAT exists in the skeletal muscle SR and plays a key role in the Ca2+ mobilization of the skeletal muscle cell [Yamaguchi, N., and Kasai, M. (1998) Biochem. J. 335, 541-547], and the number of Ca2+ channels regulated by AAT is thought to depend on the cytoplasmic Ca2+ concentration.

Two types of ADP-ribosyl cyclase activity were distinguished in dog and rat cardiac muscles by measuring the enzymatic conversion of NGD (as an NAD analog) into the fluorescent product cyclic GDP-ribose in cardiac muscle subcellular fractions. Both types of activity were confined to membrane fractions isolated from microsomes by sucrose gradient centrifugation. One of the activities co-purified with fractions that were enriched in sarcolemma (SLM), as evidenced by immunodetection of the dihydropyridine receptor, while the other activity was found to co-precipitate with the sarcoplasmic reticulum (SR), that was identified on the basis of its immuno-staining with a ryanodine receptor monoclonal antibody. In certain aspects, the plasma membrane-bound ADP-ribosyl cyclase activity resembled the characteristics of CD38 or CD38-like proteins: it was sensitive to thiols and lectins and was recognized by a monoclonal anti CD38 antibody. The SR enzyme had apparently distinct properties, as it was insensitive to both thiols and lectins and was not recognized by the CD38 antibody. In addition, the SR-associated ADP-ribosyl cyclase was inhibited by endogenous protein kinase C (PKC)-dependent phosphorylation in both dog and rat cardiac SR. The PKC-modulated SR ADP-ribosyl cyclase we describe here might be a principal component of the signal transduction machinery that is responsible for regulation of the intracellular levels of cADPR.

The endogenous calmodulin-protein kinase system of sarcoplasmic reticulum terminal cisternae of rabbit fast-twitch muscle was studied. Investigation of a single Ca 2+ -channel in terminal cisternae fused to planar lipid bilayers demonstrated that the endogenous kinase inhibits the channel, although it remained unclear whether the phosphorylation sites are on the channel protein or on other junctional sarcoplasmic reticulum specific proteins [Hain et al., (1994) Biophys. J. 67 , 1823-1833]. Our results, which show that two junctional sarcoplasmic reticulum specific proteins,i.e., triadin and histidine-rich, Ca 2+ -binding protein, but not the ryanodine receptor/Ca 2+ -channel protein, are phosphorylated by membrane-bound 60 kDa protein kinase, seem to be able to resolve this ambiguity. Furthermore,such aprobably specific protein isoform of calmodulin-protein kinase, by its substrate specificity and exposure to the cytoplasmic side of terminal cisternae at the junctional membrane domain and based on protease sensitivity, also seems to possess some of the potential requirements for a regulatory role in the functional state of the Ca 2+ -channel.

The ryanodine receptor has been widely demonstrated to be a Ca2+ release channel in both skeletal and cardiac muscle. Ryanodine also functionally affects Ca2+ release from an intracellular pool of smooth muscle. In the present study, both high and low affinity ryanodine binding sites are identified in dog mesenteric artery (with Kd 5 nM and 269 nM, respectively). [3H]ryanodine binding is not correlated with the density of innervation in different smooth muscles. This study provides direct evidence of the presence of ryanodine binding sites in dog mesentery artery. These ryanodine binding sites are of smooth muscle origin.

Summary The effect of clinical concentrations of volatile anesthetics on ryanodine receptors of cardiac and skeletal muscle sarcoplasmic reticulum was evaluated using [ 3 H]ryanodine binding. At 2 volume percent, halothane and enflurane stimulated binding to cardiac SR by 238% and 204%, respectively, while isoflurane had no effect. In contrast, halothane and enflurane had no effect on [ 3 H]ryanodine binding to skeletal ryanodine receptors, while isoflurane produced a significant stimulation. These results suggest that volatile anesthetics interact in a site-specific manner with ryanodine receptors of cardiac or skeletal muscle to effect Ca 2+ release-channel gating.

Summary Suramin is shown to be a powerful inhibitor of the calcium uptake and the ATPase activity of sarcoplasmic reticulum. It would appear that suramin is a stoichiometric inhibitor of calcium transport.

Effects of the lethal fraction (MD-9) from the venom of the Mojave rattlesnake, Crotalus scutulatus , on sarcoplasmic reticulum were investigated. The calcium sequestering activity of the vesicles was reduced by the lethal fraction and subsequent release of calcium was enhanced. These effects were observed to be dependent upon MD-9 concentration and the length of preincubation time with the vesicles. An enhanced ATPase activity that was affected by concentration and MD-9 preincubation time was also observed. Both calcium uptake and ATPase activity effects may be due to a phospholipase activity associated with the fraction.

Cardiac sarcoplasmic reticulum is phosphorylated by a cytosolic Ca 2+ -activated, phospholipid-dependent protein kinase. This phosphorylation is independent of cyclic nucleotides and enhanced by unsaturated diacylglycerols; saturated diacylglycerols, mono- and tri-glycerides are ineffective. Diacylglycerol stimulation is due to increased Ca 2+ sensitivity of the kinase reaction. Protein kinase catalyzed phosphorylation results in enhanced Ca 2+ -transport ATPase activity and may be an important determinant of cardiac sarcoplasmic reticulum function.

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

A microsomal fraction enriched in sarcoplasmic reticulum membranes has been isolated from bovine aorta smooth muscle. The properties of the Ca-pumping ATPase were compared to those of the enzymes of skeletal and cardiac sarcoplasmic reticulum. The kinetic (Km and turnover rate) and structural (tryptic digestion pattern) properties of the three ATPases were strikingly similar. The three enzymes, however, displayed (almost) no immunological cross-reactivity. Skeletal muscle sarcoplasmic reticulum and aorta microsomes did not contain phospholamban: their Ca-pumping activity was not regulated by either a cAMP-dependent or a calmodulin-dependent pathway.

Junctional sarcoplasmic reticulum (SR) has been identified in microsomes from canine ventricular muscle by the presence of calsequestrin and ryanodinesensitive Ca2+ release channels. These properties, however, are not common to cardiac cells from all species. Seiler et al (1) have recently described a high Mr polypeptide in canine junctional SR similar to the spanning protein subunits of skeletal muscle triads. We now report the existence of a polypeptide with the same mobility in SR from rabbit ventricular muscle and show that those cardiac membranes can associate with transverse (T−) tubules from rabbit skeletal muscle in K cacodylate medium. We propose that this polypeptide and the reaction with T-tubules be considered as criteria for the identification of cardiac junctional SR.

Incubation of cardiac sarcoplasmic reticulum (SR) in the presence of S-adenosyl-L-methionine, a methyl donor for the enzymatic N-methylation of phosphatidylethanolamine, increased Ca2+-stimulated ATPase activity. The increase in Ca2+-ATPase activity was not due to changes in the affinity for Ca2+ and was prevented by methyl acetimidate, an inhibitor of phospholipid N-methylation. The results suggest a possible regulatory role of phospholipid N-methylation in SR Ca2+-pump mechanism.

The purified ryanodine receptor of heart sarcoplasmic reticulum (SR) has been reconstituted into planar phospholipid bilayers and found to form Ca2+-specific channels. The channels are strongly activated by Ca2+ (10 nM) in the presence of ATP (1 mM) and ryanodine, and inactivated by Mg2+ (3 mM) or ruthenium red (30 microM). These characteristics are diagnostic of calcium release from heart SR. The cardiac ryanodine receptor, which has previously been identified as the foot structure, is now identified as the calcium release channel. A similar identity of the calcium release channel has recently been reported for skeletal muscle. The characteristics of the calcium release channel from skeletal muscle and heart are similar in that they: 1) consist of an oligomer of a single high molecular weight polypeptide (Mr 360,000 for skeletal muscle and 340,000 for heart); 2) exist morphologically as the foot structure; 3) are activated (ATP, Ca2+, ryanodine) and inhibited (ruthenium red and Mg2+) by a number of the same ligands. Important differences include: 1) Ca2+ activation at lower concentration of Ca2+ for the heart; 2) more dramatic stabilization by ryanodine of the open state for the skeletal muscle channel; and 3) different relative permeabilities (PCa/PK).

Single K channels from skeletal muscle sarcoplasmic reticulum were incorporated into artificial membranes. Ryanodine applied to either side of the membrane did not affect the gating nor the conductance properties of those channels. These results suggest that the site of action of ryanodine is limited only to the calcium channels present in the membrane of sarcoplasmic reticulum ( 1 ).

Regulation of many cell systems has been shown to be mediated by inositol 1,4,5-trisphosphate which causes a release of calcium from intracellular sites. We have shown that release of Ca2+ from sarcoplasmic reticulum microsomes was not stimulated by IP3. The phorbol ester, TPA, also had no effect on Ca2+ release or Ca2+ ATPase activity. Thus, it is unlikely that the breakdown of polyphosphatidylinositides serves as a second messenger to mediate release of Ca2+ in skeletal muscle.

We have isolated two proteolipid fractions from canine cardiac sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B, and Sephadex LH-60. One, “fraction B”, is phosphorylated by cyclic AMP-dependent protein kinase and was identified as phospholamban, the activator of cardiac sarcoplasmic reticulum. The other, “fraction A”, is not phosphorylated and has an amino acid composition very similar to those of proteolipids we previously isolated from (Na,K)-ATPase.

Phosphatidate (PA) inhibits calcium accumulation by cardiac sarcoplasmic reticulum (SR) and enhances its Ca ++ ATPase activity. These effects seem to be related to a phosphatidate-induced increase in the calcium permeability of the SR membrane with resultant calcium release. The amount of calcium released by phosphatidate is dependent both on the calcium concentration outside the SR vesicles and the internal calcium concentration. The ionophoric effects of phosphatidate on the sarcoplasmic membrane provide a novel pathway for controlling Ca ++ transport in the cardiac cell.

Cyclic AMP-dependent autophosphorylation of canine cardiac sarcoplasmic reticulum (SR) vesicles occurred on a 22,000 Mr protein and it was maximum at pH 6.0. At pH 6.0 cAMP-dependent phosphorylation of cardiac SR resulted in stimulation of the depressed initial rate of formation and the levels of the acid stable phosphorylated intermediate of the Ca2+ ATPase (E∼P). At pH 6.8 phosphorylation of cardiac SR had no effect on either the initial rate or levels of E∼P formed. These findings indicate that cAMP-dependent phosphorylation of cardiac SR can compensate for the effect of acidosis on SR function.