Inhibition of Ca(2+) sparks by ruthenium red in permeabilized rat ventricular myocytes

We have compared the effects of the sarcoplasmic reticulum (SR) Ca(2+) release inhibitor, ruthenium red (RR), on single ryanodine receptor (RyR) channels in lipid bilayers, and on Ca(2+) sparks in permeabilized rat ventricular myocytes. Ruthenium red at 5 microM inhibited the open probability (P(o)) of RyRs approximately 20-50-fold, without significantly affecting the conductance or mean open time of the channel. At the same concentration, RR inhibited the frequency of Ca(2+) sparks in permeabilized myocytes by approximately 10-fold, and reduced the amplitude of large amplitude events (with most probable localization on the line scan) by approximately 3-fold. According to our theoretical simulations, performed with a numerical model of Ca(2+) spark formation, this reduction in Ca(2+) spark amplitude corresponds to an approximately 4-fold decrease in Ca(2+) release flux underlying Ca(2+) sparks. Ruthenium red (5 microM) increased the SR Ca(2+) content by approximately 2-fold (from 151 to 312 micromol/l cytosol). Considering the degree of inhibition of local Ca(2+) release events, the increase in SR Ca(2+) load by RR, and the lack of effects of RR on single RyR open time and conductance, we have estimated that Ca(2+) sparks under normal conditions are generated by openings of at least 10 single RyRs.     

Modification of smooth muscle Ca2+-sparks by tetracaine: evidence for sequential RyR activation

Spontaneous Ca(2+)-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 microM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 microM tetracaine, as expected if the spark was propagated across the cell but the average P(o) for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR(2) in arteriole smooth muscle but not RyR(1). We conclude that Ca(2+)-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several microm(2), rather than from tightly clustered channels as in striated muscle.     

Regulation of cardiac excitation-contraction coupling by sorcin, a novel modulator of ryanodine receptors

Activation of Ca2+ release channels/ryanodine receptors (RyR) by the inward Ca2+ current (I(Ca)) gives rise to Ca(2+)-induced Ca2+ release (CICR), the amplifying Ca2+ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. Sorcin, a 21.6 kDa Ca(2+)-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca2+ sparks) and the I(Ca)-triggered activity of RyRs that gives rise to [Ca2+]i transients. Since sorcin decreases the amplitude of the [Ca2+]i transient without affecting the amplitude of I(Ca), the overall effect of sorcin is to reduce the "gain" of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca2+ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca2+] required for the latter process (ED50 = approximately 200 microM) appears to be reached only within the dyadic space. Thus, sorcin is a potent inhibitor of both spontaneous and I(Ca)-triggered RyR activity and may play a role in helping terminate the positive feedback loop of CICR.     

Altered Ca2+ sparks and gating properties of ryanodine receptors in aging cardiomyocytes

To investigate the cellular mechanisms for altered cardiac function in senescence, we measured Ca(2+) transients and Ca(2+) sparks in ventricular cardiomyocytes from 6- to 24-month-old Fisher 344 (F344) rat hearts. The single channel properties of ryanodine receptors from adult and senescent hearts were also studied. In senescent myocytes, we observed a decreased peak [Ca(2+)](i) amplitude and an increased time constant for decay (tau), both of which correlated with a reduced Ca(2+) content of the sarcoplasmic reticulum (SR). Our studies also revealed that senescent cardiomyocytes had an increased frequency of Ca(2+) sparks and a slight but statistically significant decrease in average amplitude, full-width-at-half-maximum (FWHM) and full-duration-at-half-maximum (FDHM). Single channel recordings of ryanodine receptors (RyR2) demonstrated that in aging hearts, the open probability (P(o)) of RyR2 was increased but the mean open time was shorter, providing a molecular correlate for the increased frequency of Ca(2+) sparks and decreased size of sparks, respectively. Thus, modifications of normal RyR2 gating properties may play a role in the altered Ca(2+) homeostasis observed in senescent myocytes.     

Cell culture modifies Ca2+ 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.     

Dynamic regulation of sarcoplasmic reticulum Ca(2+) content and release by luminal Ca(2+)-sensitive leak in rat ventricular myocytes.

In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.     

Evidence that a Ca2+ sparks/STOCs coupling mechanism is responsible for the inhibitory effect of caffeine on electro-mechanical coupling in guinea pig ureteric smooth muscle

Recent studies have highlighted the role of the sarcoplasmic reticulum (SR) in controlling excitability, Ca2+ signalling and contractility in smooth muscle. Caffeine, an agonist of ryanodine receptors (RyRs) on the SR has been previously shown to effect Ca2+ signalling but its effects on excitability and contractility are not so clear. We have studied the effects of low concentration of caffeine (1 mM) on Ca2+ signalling, action potential and contractility of guinea pig ureteric smooth muscle. Caffeine produced reversible inhibition of the action potentials, Ca2+ transients and phasic contractions evoked by electrical stimulation. It had no effect on the inward Ca2+ current or Ca2+ transient but increased the amplitude and the frequency of spontaneous transient outward currents (STOCs) in voltage clamped ureteric myocytes, suggesting Ca2+-activated K+ channels (BK) are affected by it. In isolated cells and cells in situ caffeine produced an increase in the frequency and the amplitude of Ca2+ sparks as well the number of spark discharging sites per cell. Inhibition of Ca2+ sparks by ryanodine (50 microM) or SR Ca2+-ATPase (SERCA) cyclopiazonic acid (CPA, 20 microM) or BKCa channels by iberiotoxin (200 nM) or TEA (1 mM), fully reversed the inhibitory effect of caffeine on Ca2+ transients and force evoked by electrical field stimulation (EFS). These data suggest that the inhibitory effect of caffeine on the action potential, Ca2+ transients and force in ureteric smooth muscle is caused by activation of Ca2+ sparks/STOCs coupling mechanism.     

Oxidative stress-induced leaky sarcoplasmic reticulum underlying acute heart failure in severe burn trauma

Burn trauma causes cardiac dysfunction. However, much of the underlying cellular and molecular mechanisms remain elusive. In the present study, we demonstrate the roles of excessive sarcoplasmic reticulum (SR) Ca(2+) leakage and oxidative stress in burn-associated acute heart failure. In cardiomyocytes from failing rat hearts 12 h after full-thickness cutaneous burn of about 40% of the total body surface area, we found that Ca(2+) transients and contractility were impaired, but the triggering L-type Ca(2+) channel current density was unaltered, giving rise to a significantly reduced gain of excitation-contraction coupling. This deficiency in SR Ca(2+) release was accompanied by a reduction in Ca(2+) content in the SR. Surprisingly, the frequency of spontaneous Ca(2+) sparks was increased by 1.4-fold; Ca(2+) tolerance test (10 mM extracellular Ca(2+)) further showed 2.0- and 1.5-fold more frequent Ca(2+) waves and Ca(2+) sparks, respectively. Myofilament sensitivity to Ca(2+), however, seemed to be unaffected. These results suggest hyperactivity of the ryanodine receptor (RyR) Ca(2+) release channel and a leaky SR in burn. Importantly, pretreatment with antioxidant vitamins C and E seemed to prevent burn-induced RyR hypersensitivity and SR leakage and thereby normalize Ca(2+) transients and contractility. Concomitantly, the in vivo cardiac functions were also more tolerant of traumatic burn. Collectively, our findings suggest that SR leakage due to oxidative stress is likely a major candidate mechanism underlying burn-associated acute heart failure. Antioxidant therapy in burn trauma provides cardioprotection, at least in part, by protecting RyR's from oxidative stress-induced hypersensitivity.     

Effects of tetracaine on voltage-activated calcium sparks in frog intact skeletal muscle fibers

The properties of Ca(2+) sparks in frog intact skeletal muscle fibers depolarized with 13 mM [K(+)] Ringer's are well described by a computational model with a Ca(2+) source flux of amplitude 2.5 pA (units of current) and duration 4.6 ms (18 degrees C; Model 2 of Baylor et al., 2002). This result, in combination with the values of single-channel Ca(2+) current reported for ryanodine receptors (RyRs) in bilayers under physiological ion conditions, 0.5 pA (Kettlun et al., 2003) to 2 pA (Tinker et al., 1993), suggests that 1-5 RyR Ca(2+) release channels open during a voltage-activated Ca(2+) spark in an intact fiber. To distinguish between one and greater than one channel per spark, sparks were measured in 8 mM [K(+)] Ringer's in the absence and presence of tetracaine, an inhibitor of RyR channel openings in bilayers. The most prominent effect of 75-100 microM tetracaine was an approximately sixfold reduction in spark frequency. The remaining sparks showed significant reductions in the mean values of peak amplitude, decay time constant, full duration at half maximum (FDHM), full width at half maximum (FWHM), and mass, but not in the mean value of rise time. Spark properties in tetracaine were simulated with an updated spark model that differed in minor ways from our previous model. The simulations show that (a) the properties of sparks in tetracaine are those expected if tetracaine reduces the number of active RyR Ca(2+) channels per spark, and (b) the single-channel Ca(2+) current of an RyR channel is 相似文献   

Invited review: significance of spatial and temporal heterogeneity of calcium transients in smooth muscle.

The multiplicity of mechanisms involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in smooth muscle results in both intra- and intercellular heterogeneities in [Ca(2+)](i). Heterogeneity in [Ca(2+)](i) regulation is reflected by the presence of spontaneous, localized [Ca(2+)](i) transients (Ca(2+) sparks) representing Ca(2+) release through ryanodine receptor (RyR) channels. Ca(2+) sparks display variable spatial Ca(2+) distributions with every occurrence within and across cellular regions. Individual sparks are often grouped, and fusion of sparks produces large local elevations in [Ca(2+)](i) that occasionally trigger propagating [Ca(2+)](i) waves. Ca(2+) sparks may modulate membrane potential and thus smooth muscle contractility. Sparks may also be the target of other regulatory factors in smooth muscle. Agonists induce propagating [Ca(2+)](i) oscillations that originate from foci with high spark incidence and also represent Ca(2+) release through RyR channels. With increasing agonist concentration, the peak of regional [Ca(2+)](i) oscillations remains relatively constant, whereas both frequency and propagation velocity increase. In contrast, the global cellular response appears as a concentration-dependent increase in peak as well as mean cellular [Ca(2+)](i), representing a spatial and temporal integration of the oscillations. The significance of agonist-induced [Ca(2+)](i) oscillations lies in the establishment of a global [Ca(2+)](i) level for slower Ca(2+)-dependent physiological processes.     

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.     

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.     

cADP ribose and [Ca(2+)](i) regulation in rat cardiac myocytes

cADP ribose (cADPR)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1-10 microM) induced sustained, concentration-dependent [Ca(2+)](i) responses ranging from 50 to 500 nM, which were completely inhibited by 20 microM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca(2+)](i) waves, increasing concentrations of cADPR increased wave frequency up to approximately 250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca(2+)](i) transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca(2+)](i) responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca(2+)](i) responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca(2+)](i) response. These results indicate that in cardiac myocytes, cADPR induces Ca(2+) release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.     

Contribution of ryanodine receptor type 3 to Ca(2+) sparks in embryonic mouse skeletal muscle.

The kinetic behavior of Ca(2+) sparks in knockout mice lacking a specific ryanodine receptor (RyR) isoform should provide molecular information on function and assembly of clusters of RyRs. We examined resting Ca(2+) sparks in RyR type 3-null intercostal myotubes from embryonic day 18 (E18) mice and compared them to Ca(2+) sparks in wild-type (wt) mice of the same age and to Ca(2+) sparks in fast-twitch muscle cells from the foot of wt adult mice. Sparks from RyR type 3-null embryonic cells (368 events) were significantly smaller, briefer, and had a faster time to peak than sparks from wt cells (280 events) of the same age. Sparks in adult cells (220 events) were infrequent, yet they were highly reproducible with population means smaller than those in embryonic RyR type 3-null cells but similar to those reported in adult amphibian skeletal muscle fibers. Three-dimensional representations of the spark peak intensity (DeltaF/Fo) vs. full width at half-maximal intensity (FWHM) vs. full duration at half-maximal intensity (FTHM) showed that wt embryonic sparks were considerably more variable in size and kinetics than sparks in adult muscle. In all cases, tetracaine (0.2 mM) abolished Ca(2+) spark activity, whereas caffeine (0.1 mM) lengthened the spark duration in wt embryonic and adult cells but not in RyR type 3-null cells. These results confirmed that sparks arose from RyRs. The low caffeine sensitivity of RyR type 3-null cells is entirely consistent with observations by other investigators. There are three conclusions from this study: i) RyR type-1 engages in Ca(2+) spark activity in the absence of other RyR isoforms in RyR type 3-null myotubes; ii) Ca(2+) sparks with parameters similar to those reported in adult amphibian skeletal muscle can be detected, albeit at a low frequency, in adult mammalian skeletal muscle cells; and iii) a major contributor to the unusually large Ca(2+) sparks observed in normal (wt) embryonic muscle is RyR type 3. To explain the reduction in the size of sparks in adult compared to embryonic skeletal muscle, we suggest that in embryonic muscle, RyR type 1 and RyR type 3 channels co-contribute to Ca(2+) release during the same spark and that Ca(2+) sparks undergo a maturation process which involves a decrease in RyR type 3.     

Dantrolene prevents arrhythmogenic Ca2+ release in heart failure

In heart failure (HF), arrhythmogenic Ca(2+) release and chronic Ca(2+) depletion of the sarcoplasmic reticulum (SR) arise due to altered function of the ryanodine receptor (RyR) SR Ca(2+)-release channel. Dantrolene, a therapeutic agent used to treat malignant hyperthermia associated with mutations of the skeletal muscle type 1 RyR (RyR1), has recently been suggested to have effects on the cardiac type 2 RyR (RyR2). In this investigation, we tested the hypothesis that dantrolene exerts antiarrhythmic and inotropic effects on HF ventricular myocytes by examining multiple aspects of intracellular Ca(2+) handling. In normal rabbit myocytes, dantrolene (1 μM) had no effect on SR Ca(2+) load, postrest decay of SR Ca(2+) content, the threshold for spontaneous Ca(2+) wave initiation (i.e., the SR Ca(2+) content at which spontaneous waves initiate) and Ca(2+) spark frequency. In cardiomyocytes from failing rabbit hearts, SR Ca(2+) load and the wave initiation threshold were decreased compared with normal myocytes, Ca(2+) spark frequency was increased, and the postrest decay was potentiated. Using a novel approach of measuring cytosolic and intra-SR Ca(2+) concentration (using the low-affinity Ca(2+) indicator fluo-5N entrapped within the SR), we showed that treatment of HF cardiomyocytes with dantrolene rescued postrest decay and increased the wave initiation threshold. Additionally, dantrolene decreased Ca(2+) spark frequency while increasing the SR Ca(2+) content in HF myocytes. These data suggest that dantrolene exerts antiarrhythmic effects and preserves inotropy in HF cardiomyocytes by decreasing the incidence of diastolic Ca(2+) sparks, increasing the intra-SR Ca(2+) threshold at which spontaneous Ca(2+) waves occur, and decreasing the loss of Ca(2+) from the SR. Furthermore, the observation that dantrolene reduces arrhythmogenicity while at the same time preserves inotropy suggests that dantrolene is a potentially useful drug in the treatment of arrhythmia associated with HF.     

Early and delayed afterdepolarizations in rabbit heart Purkinje cells viewed by confocal microscopy

We investigated action potentials and Ca(2+) transients in rabbit Purkinje myocytes using whole cell patch clamp recordings and a confocal microscope. Purkinje cells were loaded with 5 microM Fluo-3/AM for 30min. Action potentials were elicited by application of a stimulus delivered through the recording pipettes. When Purkinje cells were stimulated in 2.0mM Ca(2+), transverse XT line scans revealed a symmetrical 'U'-shaped Ca(2+) transient demonstrating that the transient was initiated at the cell periphery. When Purkinje cells were superfused with 1 microM isoprenaline, both early and delayed afterdepolarizations were induced. XT line scans of cells exhibiting early afterdepolarizations showed a second symmetrical 'U'-shaped transient. This Ca(2+) transient was initiated at the cell periphery suggesting reactivation of the Ca(2+) current. In contrast, in Purkinje cells exhibiting delayed afterdepolarizations and a corresponding transient inward current, XT line scans revealed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell. Immunofluorescence staining of Purkinje cells with an antibody to ryanodine receptors (RyRs) revealed that RyRs are located at regularly spaced intervals throughout the interior of Purkinje cells. These results suggest that, although RyRs are located throughout Purkinje cells, only peripheral RyRs are activated to produce transients, sparks and early afterdepolarizations. During delayed afterdepolarizations, we observed a heterogenous rise in Ca(2+) at both peripheral and central regions of the cell as well as large central increases in Ca(2+). Although the latter may result from central release, we cannot exclude the possibility that it reflects Ca(2+) diffusion from subsarcolemmal sites.     

Sorcin inhibits calcium release and modulates excitation-contraction coupling in the heart

Activation of Ca2+ release channels/ryanodine receptors (RyR) by the inward Ca2+ current (ICa) gives rise to Ca2+-induced Ca2+ release (CICR), the amplifying Ca2+ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. We reported previously (Lokuta, A. J., Meyers, M. B., Sander, P. R., Fishman, G. I., and Valdivia, H. H. (1997) J. Biol. Chem. 272, 25333-25338) that sorcin, a 22-kDa Ca2+-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Here we show that sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca2+ sparks) and the ICa-triggered activity of RyRs that gives rise to [Ca2+]i transients. Because sorcin decreased the amplitude of the [Ca2+]i transient without affecting the amplitude or kinetics of ICa, the overall effect of sorcin was to reduce the "gain" of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca2+ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca2+] required for the latter process (ED50 = approximately 200 microM) appears to be reached only within the dyadic space. Rapid injection of 5 microM sorcin onto the cytosolic face of RyRs reconstituted in lipid bilayers resulted in complete inhibition of channel activity in < or = 20 ms. Thus, sorcin is a potent inhibitor of both spontaneous and ICa-triggered RyR activity and is kinetically capable of playing a role in terminating the positive feedback loop of CICR.     

Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca2+ stores in pulmonary arterial smooth muscle cells

Ryanodine receptors (RyRs) of pulmonary arterial smooth muscle cells (PASMCs) play important roles in major physiological processes such as hypoxic pulmonary vasoconstriction and perinatal pulmonary vasodilatation. Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca2+ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, approximately 15-20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca2+ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca2+ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) = 193.3 +/- 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca2+ sparks (FDHM = 44.6 +/- 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca2+ stores; the molecular complexity and spatial heterogeneity of RyRs may facilitate specific Ca2+ regulation of cellular functions in PASMCs.     

Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca2+ release and contraction in pulmonary artery myocytes

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca(2+) release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP(3) receptors (IP(3)Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca(2+)](i) and Ca(2+)-activated Cl(-) and K(+) currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca(2+)](i). FK506-induced [Ca(2+)](i) increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP(3)R antagonist heparin. Hypoxic Ca(2+) response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca(2+)](i), but did not alter caffeine-induced Ca(2+) release (SR Ca(2+) content) in PASMCs. Norepinephrine-induced Ca(2+) release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca(2+) and contractile responses by regulating the activity of RyRs in PASMCs.     

CaMKIIδC slows [Ca]i decline in cardiac myocytes by promoting Ca sparks

Acute activation of calcium/calmodulin-dependent protein kinase (CaMKII) in permeabilized phospholamban knockout (PLN-KO) mouse myocytes phosphorylates ryanodine receptors (RyRs) and activates spontaneous local sarcoplasmic reticulum (SR) Ca release events (Ca sparks) even at constant SR Ca load. To assess how CaMKII regulates SR Ca release in intact myocytes (independent of SR Ca content changes or PLN effects), we compared Ca sparks in PLN-KO versus mice, which also have transgenic cardiac overexpression of CaMKIIδC in the PLN-KO background (KO/TG). Compared with PLN-KO mice, these KO/TG cardiomyocytes exhibited 1), increased twitch Ca transient and fractional release (both by ～35%), but unaltered SR Ca load; 2), increased resting Ca spark frequency (300%) despite a lower diastolic [Ca]i, which also slowed twitch [Ca]i decline (suggesting CaMKII-dependent RyR Ca sensitization); 3), elevated Ca spark amplitude and rate of Ca release (which might indicate that more RyR channels participate in a single spark); 4), prolonged Ca spark rise time (which implies that CaMKII either delays RyR closure or prolongs the time when openings can occur); 5), more frequent repetitive sparks at single release sites. Analysis of repetitive sparks from individual Ca release sites indicates that CaMKII enhanced RyR Ca sensitivity, but did not change the time course of SR Ca refilling. These results demonstrate that there are dramatic CaMKII-mediated effects on RyR Ca release that occur via regulation of both RyR activation and termination processes.