Calcium spark properties in ventricular myocytes are altered in aged mice

This study determined whether whole cell Ca(2+) transients and unitary sarcoplasmic reticulum (SR) Ca(2+) release events are constant throughout adult life or whether Ca(2+) release is altered in aging ventricular myocytes. Myocytes were isolated from young adult (approximately 5 mo old) and aged (approximately 24 mo old) mice. Spontaneous Ca(2+) sparks and Ca(2+) transients initiated by field stimulation were detected with fluo-4. All experiments were conducted at 37 degrees C. Ca(2+) transient amplitudes were reduced, and Ca(2+) transient rise times were abbreviated in aged cells stimulated at 8 Hz compared with young adult myocytes. Furthermore, the incidence and frequency of spontaneous Ca(2+) sparks were markedly higher in aged myocytes compared with young adult cells. Spark amplitudes and spatial widths were similar in young adult and aged myocytes. However, spark half-rise times and half-decay times were abbreviated in aged cells compared with younger cells. Resting cytosolic Ca(2+) levels and SR Ca(2+) stores were assessed by rapid application of caffeine in fura-2-loaded cells. Neither resting Ca(2+) levels nor SR Ca(2+) content differed between young adult and aged cells. Thus increased spark frequency in aging cells was not attributable to increased SR Ca(2+) stores. Furthermore, the decrease in Ca(2+) transient amplitude was not due to a decrease in SR Ca(2+) load. These results demonstrate that alterations in fundamental SR Ca(2+) release units occur in aging ventricular myocytes and raise the possibility that alterations in Ca(2+) release may reflect age-related changes in fundamental release events rather than changes in SR Ca(2+) stores and diastolic Ca(2+) levels.     

Factors shaping the confocal image of the calcium spark in cardiac muscle cells.

The interpretation of confocal line-scan images of local [Ca2+]i transients (such as Ca2+ sparks in cardiac muscle) is complicated by uncertainties in the position of the origin of the Ca2+ spark (relative to the scan line) and by the dynamics of Ca(2+)-dye interactions. An investigation of the effects of these complications modeled the release, diffusion, binding, and uptake of Ca2+ in cardiac cells (producing a theoretical Ca2+ spark) and image formation in a confocal microscope (after measurement of its point-spread function) and simulated line-scan images of a theoretical Ca2+ spark (when it was viewed from all possible positions relative to the scan line). In line-scan images, Ca2+ sparks that arose in a different optical section or with the site of origin displaced laterally from the scan line appeared attenuated, whereas their rise times slowed down only slightly. These results indicate that even if all Ca2+ sparks are perfectly identical events, except for their site of origin, there will be an apparent variation in the amplitude and other characteristics of Ca2+ sparks as measured from confocal line-scan images. The frequency distributions of the kinetic parameters (i.e., peak amplitude, rise time, fall time) of Ca2+ sparks were calculated for repetitive registration of stereotyped Ca2+ sparks in two experimental situations: 1) random position of the scan line relative to possible SR Ca(2+)-release sites and 2) fixed position of the scan line going through a set of possible SR Ca(2+)-release sites. The effects of noise were incorporated into the model, and a visibility function was proposed to account for the subjective factors that may be involved in the evaluation of Ca(2+)-spark image parameters from noisy experimental recordings. The mean value of the resulting amplitude distributions underestimates the brightness of in-focus Ca2+ sparks because large numbers of out-of-focus Ca2+ sparks are detected (as small Ca2+ sparks). The distribution of peak amplitudes may split into more than one subpopulation even when one is viewing stereotyped Ca2+ sparks because of the discrete locations of possible SR Ca(2+)-release sites in mammalian ventricular heart cells.     

Temperature dependence and thermodynamic properties of Ca2+ sparks in rat cardiomyocytes

To elucidate the temperature dependence and underlying thermodynamic determinants of the elementary Ca2+ release from the sarcoplasmic reticulum, we characterized Ca2+ sparks originating from ryanodine receptors (RyRs) in rat cardiomyocytes over a wide range of temperature. From 35 degrees C to 10 degrees C, the normalized fluo-3 fluorescence of Ca2+ sparks decreased monotonically, but the Delta[Ca2+]i were relatively unchanged due to increased resting [Ca2+]i. The time-to-peak of Ca2+ sparks, which represents the RyR Ca2+ release duration, was prolonged by 37% from 35 degrees C to 10 degrees C. An Arrhenius plot of the data identified a jump of apparent activation energy from 5.2 to 14.6 kJ/mol at 24.8 degrees C, which presumably reflects a transition of sarcoplasmic reticulum lipids. Thermodynamic analysis of the decay kinetics showed that active transport plays little role in early recovery but a significant role in late recovery of local Ca2+ concentration. These results provided a basis for quantitative interpretation of intracellular Ca2+ signaling under various thermal conditions. The relative temperature insensitivity above the transitional 25 degrees C led to the notion that Ca2+ sparks measured at a "warm room" temperature are basically acceptable in elucidating mammalian heart function.     

The changes in Ca2+ sparks associated with measured modifications of intra-store Ca2+ concentration in skeletal muscle

In cardiac muscle and amphibian skeletal muscle, the intracellular Ca2+ release that signals contractile activation proceeds by discrete local packets, which result in Ca2+ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca2+ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca2+ sparks while reversibly changing and measuring [Ca2+] in the SR ([Ca2+]SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca2+]SR reversibly between a low value (299 microM on average in 10 experiments) and a high value (433 microM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak amplitude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and amplitude were statistically significant. The "strength" of the effect of [Ca2+]SR on frequency, quantified by decomposition of variance, was <6%. While the average change in [Ca2+]SR was limited, it reached up to 200% in individual fibers, without causing massive Ca2+ release or an increase of >3.5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca2+ sparks or release elicited by an action potential, the mild effects of [Ca2+]SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca2+ release at the global level in frog skeletal muscle.     

Overexpression of human beta2-adrenergic receptors increases gain of excitation-contraction coupling in mouse ventricular myocytes

This study investigated cardiac excitation-contraction coupling at 37 degrees C in transgenic mice with cardiac-specific overexpression of human beta2-adrenergic receptors (TG4 mice). In field-stimulated myocytes, contraction was significantly greater in TG4 compared with wild-type (WT) ventricular myocytes. In contrast, when duration of depolarization was controlled with rectangular voltage clamp steps, contraction amplitudes initiated by test steps were the same in WT and TG4 myocytes. When cells were voltage clamped with action potentials simulating TG4 and WT action potential configurations, contractions were greater with long TG4 action potentials and smaller with shorter WT action potentials, which suggests an important role for action potential configuration. Interestingly, peak amplitude of L-type Ca2+ current (I(Ca-L)) initiated by rectangular test steps was reduced, although the voltage dependencies of contractions and currents were not altered. To explore the basis for the altered relation between contraction and I(Ca-L), Ca2+ concentrations were measured in myocytes loaded with fura 2. Diastolic concentrations of free Ca2+ and amplitudes of Ca2+ transients were similar in voltage-clamped myocytes from WT and TG4 mice. However, sarcoplasmic reticulum (SR) Ca2+ content assessed with the rapid application of caffeine was elevated in TG4 cells. Increased SR Ca2+ was accompanied by increased frequency and amplitudes of spontaneous Ca2+ sparks measured at 37 degrees C with fluo 3. These observations suggest that the gain of Ca(2+)-induced Ca2+ release is increased in TG4 myocytes. Increased gain counteracts the effects of decreased amplitude of I(Ca-L) in voltage-clamped myocytes and likely contributes to increased contraction amplitudes in field-stimulated TG4 myocytes.     

Regulation of Ca2+ Sparks by Ca2+ and Mg2+ in Mammalian and Amphibian Muscle. An RyR Isoform-specific Role in Excitation–Contraction Coupling?

Ca2+ and Mg2+ are important mediators and regulators of intracellular Ca2+ signaling in muscle. The effects of changes of cytosolic [Ca2+] or [Mg2+] on elementary Ca2+ release events were determined, as functions of concentration and time, in single fast-twitch permeabilized fibers of rat and frog. Ca2+ sparks were identified and their parameters measured in confocal images of fluo-4 fluorescence. Solutions with different [Ca2+] or [Mg2+] were rapidly exchanged while imaging. Faster and spatially homogeneous changes of [Ca2+] (reaching peaks >100 microM) were achieved by photolysing Ca NP-EGTA with laser flashes. In both species, incrementing cytosolic [Ca2+] caused a steady, nearly proportional increase in spark frequency, reversible upon [Ca2+] reduction. A greater change in spark frequency, usually transient, followed sudden increases in [Ca2+] after a lag of 100 ms or more. The nonlinearity, lag, and other features of this delayed effect suggest that it requires increase of [Ca2+] inside the SR. In the frog only, increases in cytosolic [Ca2+] often resulted, after a lag, in sparks that propagated transversally. An increase in [Mg2+] caused a fall of spark frequency, but with striking species differences. In the rat, but not the frog, sparks were observed at 4-40 mM [Mg2+]. Reducing [Mg2+] below 2 mM, which should enable the RyR channel's activation (CICR) site to bind Ca2+, caused progressive increase in spark frequency in the frog, but had no effect in the rat. Spark propagation and enhancement by sub-mM Mg2+ are hallmarks of CICR. Their absence in the rat suggests that CICR requires RyR3 para-junctional clusters, present only in the frog. The observed frequency of sparks corresponds to a channel open probability of 10(-7) in the frog or 10(-8) in the rat. Together with the failure of photorelease to induce activation directly, this indicates a basal inhibition of channels in situ. It is proposed that relief of this inhibition could be the mechanism by which increased SR load increases spark frequency.     

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.     

Simulation of calcium sparks in cut skeletal muscle fibers of the frog

Spark mass, the volume integral of Delta F/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 microM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5-10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3-5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers--and, perhaps, in other disrupted preparations--than in intact fibers.     

Estimation of the sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks

Using a combination of experimental and numerical approaches, we have tested two different approaches to calculating the sarcoplasmic reticulum (SR) Ca2+ release flux, which gives rise to cardiac muscle Ca2+ sparks. By using two-photon excited spot photolysis of DM-Nitrophen, known Ca2+ release flux time courses were generated to provide the first experimental validation of spark flux reconstruction algorithms. These artificial Ca2+ sparks show that it is possible to calculate the SR Ca2+ release waveform with reasonable accuracy, provided the flux equations reasonably reflect the properties of the experimental system. Within cardiac muscle cells, we show that Ca2+ flux reconstruction is complicated by the substantial dye binding to proteins, a factor that has not been adequately addressed in previous flux reconstruction algorithms. Furthermore, our numerical experiments suggest that the calculated time course of release flux inactivation based on conventional flux reconstruction algorithms is likely to be in error. We therefore developed novel algorithms based on an explicit dye binding scheme. When these algorithm were applied to evoked Ca2+ sparks in rat cardiac ventricular myocytes, the reconstructed Ca2+ release waveform peaked in ~5 ms and decayed with a halftime of approximately 5 ms. The peak flux magnitude was 7-12 pA, suggesting that sparks must arise from clusters of >15 ryanodine receptors.     

Temporal and spatial properties of cellular Ca2+ flux in trout ventricular myocytes

Confocal microscopy was used to investigate the temporal and spatial properties of Ca(2+) transients and Ca(2+) sparks in ventricular myocytes of the rainbow trout (Oncorhynchus mykiss). Confocal imaging confirmed the absence of T tubules and the long ( approximately 160 microm), thin ( approximately 8 microm) morphology of trout myocytes. Line scan imaging of Ca(2+) transients evoked by electrical stimulation in cells loaded with fluo 4 revealed spatial inhomogeneities in the temporal properties of Ca(2+) transients across the width of the myocytes. The Ca(2+) wavefront initiated faster, rose faster, and reached larger peak amplitudes in the periphery of the myocyte compared with the center. These differences were exacerbated by stimulation with the L-type Ca(2+) channel agonist (-)BAY K 8644 or by sarcoplasmic reticulum (SR) inhibition with ryanodine and thapsigargin. Results reveal that the shape of the trout myocyte allows for rapid diffusion of Ca(2+) from the cell periphery to the cell center, with SR Ca(2+) release contributing to the cytosolic Ca(2+) rise in a time-dependent manner. Spontaneous Ca(2+) sparks were exceedingly rare in trout myocytes under control conditions (1 sparking cell from 238 cells examined). This is in marked contrast to the rat where a total of 56 spontaneous Ca(2+) sparks were observed in 9 of 11 myocytes examined. Ca(2+) sparklike events were observed in a very small number of trout myocytes (15 sparks from 9 of 378 cells examined) after stimulation with either (-)BAY K 8644 or high Ca(2+) (6 mM). Reducing temperature to 15 degrees C in intact myocytes or permeabilizing myocytes to adjust intracellular conditions to favor Ca(2+) spark detection was without significant effects. Possible reasons for the rarity of Ca(2+) sparks in a cardiac myocyte with an active SR are discussed.     

Ca2+-sparks constitute elementary building blocks for global Ca2+-signals in myocytes of retinal arterioles

Spontaneous Ca2+-events were imaged in myocytes within intact retinal arterioles (diameter <40 microm) freshly isolated from rat eyes. Ca2+-sparks were often observed to spread across the width of these small cells, and could summate to produce prolonged Ca2+-oscillations and contraction. Application of cyclopiazonic acid (20 microM) transiently increased spark frequency and oscillation amplitude, but inhibited both sparks and oscillations within 60s. Both ryanodine (100 microM) and tetracaine (100 microM) reduced the frequency of sparks and oscillations, while tetracaine also reduced oscillation amplitude. None of these interventions affected spark amplitude. Nifedipine, which blocks store filling independently of any action on L-type Ca2+-channels in these cells, reduced the frequency and amplitude of both sparks and oscillations. Removal of external [Ca2+] (1mM EGTA) also reduced the frequency of sparks and oscillations but these reductions were slower in onset than those in the presence of tetracaine or cyclopiazonic acid. Cyclopiazonic acid, nifedipine and low external [Ca2+] all reduced SR loading, as indicated by the amplitude of caffeine evoked Ca2+-transients. This study demonstrates for the first time that spontaneous Ca2+-events in small arterioles of the eye result from activation of ryanodine receptors in the SR and suggests that this activation is not tightly coupled to Ca2+-influx. The data also supports a model in which Ca2+-sparks act as building blocks for more prolonged, global Ca2+-signals.     

Ca2+ sparks and embers of mammalian muscle. Properties of the sources

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were "lone embers." Average spatial width was approximately 2 microm in the rat and 1.5 microm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37-160 channels opened in the spark. In simulations, 25-60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 microm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35-260 channels. Not infrequently, a few of those remain open and produce the trailing ember.     

Hypothermia increases the gain of excitation-contraction coupling in guinea pig ventricular myocytes

Components of excitation-contraction (EC)-coupling were compared at 37 degrees C and 22 degrees C to determine whether hypothermia altered the gain of EC coupling in guinea pig ventricular myocytes. Ca(2+) concentration (fura-2) and cell shortening (edge detector) were measured simultaneously. Hypothermia increased fractional shortening (8.3 +/- 1.7 vs. 2.6 +/- 0.3% at 37 degrees C), Ca(2+) transients (157 +/- 33 vs. 35 +/- 5 nM at 37 degrees C), and diastolic Ca(2+) (100 +/- 9 vs. 60 +/- 6 nM at 37 degrees C) in field-stimulated myocytes (2 Hz). In experiments with high-resistance microelectrodes, the increase in contractions and Ca(2+) transients was accompanied by a twofold increase in action potential duration (APD). When voltage-clamp steps eliminated changes in APD, cooling still increased contractions and Ca(2+) transients. Hypothermia increased sarcoplasmic reticulum (SR) Ca(2+) stores (83 +/- 17 at 37 degrees C to 212 +/- 50 nM, assessed with caffeine) and increased fractional SR Ca(2+) release twofold. In contrast, peak Ca(2+) current was much smaller at 22 degrees C than at 37 degrees C (1.3 +/- 0.4 and 3.5 +/- 0.7 pA/pF, respectively). In cells dialyzed with sodium-free pipette solutions to inhibit Ca(2+) influx via reverse-mode Na(+)/Ca(2+) exchange, hypothermia still increased contractions, Ca(2+) transients, SR stores, and fractional release but decreased the amplitude of Ca(2+) current. The rate of SR Ca(2+) release per unit Ca(2+) current, a measure of EC-coupling gain, was increased sixfold by hypothermia. This increase in gain occurred regardless of whether cells were dialyzed with sodium-free solutions. Thus an increase in EC-coupling gain contributes importantly to positive inotropic effects of hypothermia in the heart.     

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.     

Effects of temperature and calcium availability on cardiac contractility in Synbranchus marmoratus, a neotropical teleost

An isometric muscle preparation was used to investigate the importance of the ventricular sarcoplasmic reticulum (SR) and extracellular Ca2+ (1.25 up to 11.25 mM) to force generation at 25 degrees C (acclimation temperature), 15 and 35 degrees C. The post-rest tension and force-frequency relationship were conducted with and without 10 microM ryanodine in the bathing medium. Increments in extracellular Ca2+ resulted in increases in twitch force development only at 35 degrees C. A significant post-rest potentiation was recorded for the control preparations at 25 degrees C (100% to 119.8+/-4.1%). However, this post-rest potentiation was inhibited by ryanodine only at 25 degrees C (100% to 97.6+/-1.5%). At 35 degrees C, force remained unchanged in the control preparations, but a significant post-rest decay was recorded in the presence of ryanodine (100% to 76.6+/-4.6%) while at 15 degrees C, ryanodine was not able to preventing the post-rest potentiation observed in the control preparations. The increases in the imposed contraction frequency caused a decline of the force at 25 and 35 degrees C and ryanodine decreased significantly peak tension at both temperatures. The findings suggest a high or medium calcium turnover, possibly related to the presence of a functional SR, whose functionality is diminished when temperature is decreased.     

Calmodulin in adult mammalian skeletal muscle: localization and effect on sarcoplasmic reticulum Ca2+ release

Calmodulin is a ubiquitous Ca2+ binding protein that binds to ryanodine rectors (RyR) and is thought to modulate its activity. Here we evaluated the effects of recombinant calmodulin on the rate of occurrence and spatial properties of Ca2+ sparks as an assay of activation in saponin-permeabilized mouse myofibers. Control myofibers exhibited a time-dependent increase and subsequent decrease in spark frequency. Recombinant wild-type calmodulin prevented the time-dependent appearance of Ca2+ sparks and decreased the derived Ca2+ flux from the sarcoplasmic reticulum during a spark by approximately 37%. A recombinant Ca2+-insensitive form of calmodulin resulted in an instantaneous increase in spark frequency as well as an increase in the derived Ca2+ flux by approximately 24%. Endogenous calmodulin was found to primarily localize to the Z-line. Surprisingly, removal of endogenous calmodulin did not alter the time dependence of Ca2+ spark appearance. These results indicate that calmodulin may not be essential for RyR1-dependent Ca2+ release in adult mammalian skeletal 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.     

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.     

Large currents generate cardiac Ca2+ sparks

Previous models of cardiac Ca2+ sparks have assumed that Ca2+ currents through the Ca2+ release units (CRUs) were approximately 1-2 pA, producing sparks with peak fluorescence ratio (F/F(0)) of approximately 2.0 and a full-width at half maximum (FWHM) of approximately 1 microm. Here, we present actual Ca2+ sparks with peak F/F(0) of >6 and a FWHM of approximately 2 microm, and a mathematical model of such sparks, the main feature of which is a much larger underlying Ca2+ current. Assuming infinite reaction rates and no endogenous buffers, we obtain a lower bound of approximately 11 pA needed to generate a Ca2+ spark with FWHM of 2 microm. Under realistic conditions, the CRU current must be approximately 20 pA to generate a 2- microm Ca2+)spark. For currents > or =5 pA, the computed spark amplitudes (F/F(0)) are large (approximately 6-12 depending on buffer model). We considered several factors that might produce sparks with FWHM approximately 2 microm without using large currents. Possible protein-dye interactions increased the FWHM slightly. Hypothetical Ca2+ "quarks" had little effect, as did blurring of sparks by the confocal microscope. A clusters of CRUs, each producing 10 pA simultaneously, can produce sparks with FWHM approximately 2 microm. We conclude that cardiac Ca2+ sparks are significantly larger in peak amplitude than previously thought, that such large Ca2+ sparks are consistent with the measured FWHM of approximately 2 microm, and that the underlying Ca2+ current is in the range of 10-20 pA.     

Dual effects of cyclic ADP-ribose on sarcoplasmic reticulum Ca2+ release and storage in cardiac myocytes isolated from guinea-pig and rat ventricle

The actions of cyclic ADP-ribose (cADPR), a regulator of Ca2+-induced Ca2+ release (CICR), were investigated on Ca2+ release and sarcoplasmic reticulum (SR) Ca2+ loading in cardiac myocytes at physiological temperature. In guinea-pig ventricular cells, cADPR, applied via patch pipette or from photorelease of its caged derivative, increased contraction amplitude and whole-cell Ca2+ transients, without affecting SR Ca2+ load (measured in response to rapid caffeine application). Under voltage-clamp conditions, photorelease of caged cADPR enhanced Ca2+ transient magnitude without affecting the peak amplitude of L-type Ca2+ current or its rate of decay, indicative of an increase in CICR gain. In rat permeabilised ventricular myocytes, rapid application of cADPR increased Ca2+ spark frequency within 30 s, and this effect was maintained over a 10 min exposure. Enhancement of spark frequency was not associated with changes in SR Ca2+ load at 30 s and 3 min of exposure to cADPR; however, prolonged exposure (10 min) was associated with an increased SR Ca2+ load (32+/-7%). The observations are consistent with dual actions of cADPR: a rapid effect on CICR that does not depend on an increased SR Ca2+ load, and an additional slower effect that is associated with enhanced SR Ca2+ levels.