Predicting Local SR Ca Dynamics during Ca Wave Propagation in Ventricular Myocytes

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca²⁺) wave propagation, and 2), speed of Ca²⁺ diffusion within the SR. Ca²⁺ waves are initiated when a spontaneous local SR Ca²⁺ release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca²⁺ diffusion constant in the SR (D_Ca,SR) severely complicates our understanding of whether dynamic local changes in SR [Ca²⁺] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca²⁺] during Ca²⁺ waves. Simulations have investigated how dynamic local changes in SR [Ca²⁺] are influenced by 1), D_Ca,SR; 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca²⁺ pumps; 4), SR Ca²⁺ pump dependence on cytosolic [Ca²⁺]; and 5), the rate of transfer between network and junctional SR. Of these factors, D_Ca,SR is the primary determinant of how release from one RyR cluster alters SR [Ca²⁺] in nearby regions. Specifically, our results show that local increases in SR [Ca²⁺] ahead of the wave can potentially facilitate Ca²⁺ wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca²⁺] are possible during SR Ca²⁺release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca²⁺]_SR.     

Mechanistic insights into store-operated Ca2+ entry during excitation-contraction coupling in skeletal muscle

Skeletal muscle fibres support store-operated Ca²⁺-entry (SOCE) across the t-tubular membrane upon exhaustive depletion of Ca²⁺ from the sarcoplasmic reticulum (SR). Recently we demonstrated the presence of a novel mode of SOCE activated under conditions of maintained [Ca²⁺]_SR. This phasic SOCE manifested in a fast and transient manner in synchrony with excitation contraction (EC)-coupling mediated SR Ca²⁺-release (Communications Biology 1:31, doi: https://doi.org/10.1038/s42003-018-0033-7). Stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel 1 (ORAI1), positioned at the SR and t-system membranes, respectively, are the considered molecular correlate of SOCE. The evidence suggests that at the triads, where the terminal cisternae of the SR sandwich a t-tubule, STIM1 and ORAI1 proteins pre-position to allow for enhanced SOCE transduction.Here we show that phasic SOCE is not only shaped by global [Ca²⁺]_SR but provide evidence for a local activation within nanodomains at the terminal cisternae of the SR. This feature may allow SOCE to modulate [Ca²⁺]_SR during EC coupling. We define SOCE to occur on the same timescale as EC coupling and determine the temporal coherence of SOCE activation to SR Ca²⁺ release. We derive a delay of 0.3 ms reflecting diffusive Ca²⁺-equilibration at the luminal ryanodine receptor 1 (RyR1) channel mouth upon SR Ca²⁺-release. Numerical simulations of Ca²⁺-calsequestrin binding estimates a characteristic diffusion length and confines an upper limit for the spatial distance between STIM1 and RyR1. Experimental evidence for a 4- fold change in t-system Ca²⁺-permeability upon prolonged electrical stimulation in conjunction with numerical simulations of Ca²⁺-STIM1 binding suggests a Ca²⁺ dissociation constant of STIM1 below 0.35 mM. Our results show that phasic SOCE is intimately linked with RyR opening and closing, with only μs delays, because [Ca²⁺] in the terminal cisternae is just above the threshold for Ca²⁺ dissociation from STIM1 under physiological resting conditions.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

The gating of the sheep skeletal sarcoplasmic reticulum Ca2+-release channel is regulated by luminal Ca2+

The effects of changes in luminal [Ca²⁺] have been investigated in sheep skeletal sarcoplasmic reticulum (SR) Ca²⁺-release channels after activation of the channels by different ligands from the cytosolic side of the channel. Native heavy SR membrane vesicles were incorporated into planar phospholipid bilayers under voltage-clamp conditions. Experiments were carried out in symmetrical 250 mm Cs⁺. Lifetime analysis indicates that channels activated solely by cytosolic Ca²⁺ exhibit at least two open and five closed states. The open events are very brief and are close to the minimum resolvable duration. When channels are activated solely by cytosolic Ca²⁺, luminal Ca²⁺ does not appear to exert any regulatory effect. The P ₀ and duration of the open and closed lifetimes are unchanged. However, if channels are activated by ATP alone or by ATP plus cytosolic Ca²⁺, increases in luminal [Ca²⁺] produce marked increases in P ₀ and in the duration of the open lifetimes. Our results demonstrate that maximum activation of the skeletal SR Ca²⁺-release channel by ATP cannot be obtained in the absence of millimolar luminal [Ca²⁺].We are grateful to the British Heart Foundation for financial support.     

Kinetics on Demand Is a Simple Mathematical Solution that Fits Recorded Caffeine-Induced Luminal SR Ca2+ Changes in Smooth Muscle Cells

The process of Ca²⁺ release from sarcoplasmic reticulum (SR) comprises 4 phases in smooth muscle cells. Phase 1 is characterized by a large increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) with a minimal reduction of the free luminal SR [Ca²⁺] ([Ca²⁺]_FSR). Importantly, active SR Ca²⁺ ATPases (SERCA pumps) are necessary for phase 1 to occur. This situation cannot be explained by the standard kinetics that involves a fixed amount of luminal Ca²⁺ binding sites. A new mathematical model was developed that assumes an increasing SR Ca²⁺ buffering capacity in response to an increase of the luminal SR [Ca²⁺] that is called Kinetics-on-Demand (KonD) model. This approach can explain both phase 1 and the refractory period associated with a recovered [Ca²⁺]_FSR. Additionally, our data suggest that active SERCA pumps are a requisite for KonD to be functional; otherwise luminal SR Ca²⁺ binding proteins switch to standard kinetics. The importance of KonD Ca²⁺ binding properties is twofold: a more efficient Ca²⁺ release process and that [Ca²⁺]_FSR and Ca²⁺-bound to SR proteins ([Ca²⁺]_BSR) can be regulated separately allowing for Ca²⁺ release to occur (provided by Ca²⁺-bound to luminal Ca²⁺ binding proteins) without an initial reduction of the [Ca²⁺]_FSR.     

Quantitative measurement of Ca²(+) in the sarcoplasmic reticulum lumen of mammalian skeletal muscle

Skeletal muscle stores Ca²⁺ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca²⁺ in the SR ([Ca²⁺]_SR) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca²⁺]_SR in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca²⁺]_{SR, free} is 390 μM. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca²⁺]_{SR, free} to ∼8 μM, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca²⁺ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca²⁺ and for future studies of the role of SR Ca²⁺ in healthy and diseased mammalian muscle.     

Regulation of the gating of the sheep cardiac sarcoplasmic reticulum ca2+-release channel by luminal Ca2+

We investigated the effects of changes in luminal [Ca²⁺] on the gating of native andpurified sheep cardiac sarcoplasmic reticulum (SR) Ca²⁺-release channels reconstituted intoplanar phospholipid bilayers. The open probability (P _o )of channels activated solely by cytosolic Ca²⁺ was greater at positive than negative holding potentials. Channels activatedsolely by 10 m cytosolic Ca²⁺ exhibited no change in steady-stateP _o or in the relationship betweenP _o and voltage when the luminal[Ca²⁺] was increased from nanomolar to millimolar concentrations. In the absence of activating concentrationsof cytosolic Ca²⁺, the channel can be activated by the phosphodiesterase inhibitor sulmazole (AR-L 115BS). However, cytosolicCa²⁺-independent activation of the channel by sulmazole requires luminal Ca²⁺. In the presence ofsulmazole, at picomolar luminal [Ca²⁺] the channel remains completely closed. Increasing the luminal [Ca²⁺]to millimolar levels markedly increases the P _o via an increase in theduration of open events. The P _o and duration of the sulmazole-activated, luminalCa²⁺-dependent channel openings are voltage dependent. In the presence of micromolar luminal Ca²⁺, theP _o and duration of sulmazole-activated openings are greater atnegative voltages. However, at millimolar luminal [Ca²⁺], long openings are also observed at positive voltages and theP _o appears to be similar at positive and negative voltages. Our findings indicate thatthe regulation of channel gating by luminal Ca²⁺ depends on the mechanism of channel activation.We would like to thank Dr Allan Lindsay for the preparation of the purified SR Ca²⁺-release channels. This work was supported by the British Heart Foundation.     

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart

Sarcoplasmic reticulum (SR) Ca²⁺ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca²⁺ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca²⁺ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (V_m) and free intra-SR [Ca²⁺] ([Ca²⁺]_SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca²⁺ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca²⁺] ([Ca²⁺]_i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca²⁺]_SR is in the millimolar range. In addition to revealing SR Ca²⁺ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of V_m, allowing for investigations into the bidirectional relationship between V_m and SR Ca²⁺ and the role of SR Ca²⁺ in arrhythmogenic phenomena.     

Variable luminal sarcoplasmic reticulum Ca buffer capacity in smooth muscle cells

Sarcoplasmic reticulum contains the internal Ca²⁺ store in smooth muscle cells and its lumen appears to be a continuum that lacks diffusion barriers. Accordingly, the free luminal Ca²⁺ level is the same all throughout the SR; however, whether the Ca²⁺ buffer capacity is the same in all the SR is unknown. We have estimated indirectly the luminal Ca²⁺ buffer capacity of the SR by comparing the reduction in SR Ca²⁺ levels with the corresponding increase in [Ca²⁺]_i during activation of either IP₃Rs with carbachol or RyRs with caffeine, in smooth muscle cells from guinea pig urinary bladder. We have determined that carbachol-sensitive SR has a 2.4 times larger Ca²⁺ buffer capacity than caffeine-sensitive SR. Rapid inhibition of SERCA pumps with thapsigargin revealed that this pump activity accounts for 80% and 60% of the Ca²⁺ buffer capacities of carbachol- and caffeine-sensitive SR, respectively. Moreover, the Ca²⁺ buffer capacity of carbachol-sensitive SR was similar to caffeine-sensitive SR when SERCA pumps were inhibited. Similar rates of Ca²⁺ replenishments suggest similar levels of SERCA pump activities for either carbachol- or caffeine-sensitive SR. Paired pulses of caffeine, in conditions of low Ca²⁺ influx, indicate the relevance of luminal SR Ca²⁺ buffer capacity in the [Ca²⁺]_i response. To further study the importance of luminal SR Ca²⁺ buffer capacity in the release process we used low levels of heparin to partially inhibit IP₃Rs. This condition revealed carbachol-induced transient increase of luminal SR Ca²⁺ levels provided that SERCA pumps were active. It thus appears that SERCA pump activity keeps the luminal SR Ca²⁺-binding proteins in the high-capacity, low-affinity conformation, particularly for IP₃R-mediated Ca²⁺ release.     

The sarcoplasmic reticulum Ca2+ store arrangement in vascular smooth muscle

In vascular smooth muscle cells, Ca²⁺ release via IP₃ receptors (IP₃R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) Ca²⁺ store contributes significantly to the regulation of cellular events such as gene regulation, growth and contraction. Ca²⁺ release from various regions of a structurally compartmentalized SR, it is proposed, may selectively activate different cellular functions. Multiple SR compartments with various receptor arrangements are proposed also to exist at different stages of smooth muscle development and in proliferative vascular diseases such as atherosclerosis. The conclusions on SR organization have been derived largely from the outcome of functional studies. This study addresses whether the SR Ca²⁺ store is a single continuous interconnected network or multiple separate Ca²⁺ pools in single vascular myocytes. To do this, the consequences of depletion of the SR in small restricted regions on the Ca²⁺ available throughout the store was examined using localized photolysis of caged-IP₃ and focal application of ryanodine in guinea-pig voltage-clamped single portal vein myocytes. From one small site on the cell, the entire SR could be depleted via either RyR or IP₃R. The entire SR could also be refilled from one small site on the cell. The results suggest a single luminally continuous SR exists. However, the opening of IP₃R and RyR was regulated by the Ca²⁺ concentration within the SR (luminal [Ca²⁺]). As the luminal [Ca²⁺] declines, the opening of the receptors decline and stop, and there may appear to be stores with either only RyR or only IP₃R. The SR Ca²⁺ store is a single luminally continuous entity which contains both IP₃R and RyR and within which Ca²⁺ is accessed freely by each receptor. While the SR is a single continuous entity, regulation of IP₃R and RyR by luminal [Ca²⁺] explains the appearance of multiple stores in some functional studies.     

Enhanced ryanodine receptor-mediated calcium leak determines reduced sarcoplasmic reticulum calcium content in chronic canine heart failure

In this study, we investigated the role of elevated sarcoplasmic reticulum (SR) Ca²⁺ leak through ryanodine receptors (RyR2s) in heart failure (HF)-related abnormalities of intracellular Ca²⁺ handling, using a canine model of chronic HF. The cytosolic Ca²⁺ transients were reduced in amplitude and slowed in duration in HF myocytes compared with control, changes paralleled by a dramatic reduction in the total SR Ca²⁺ content. Direct measurements of [Ca²⁺]_SR in both intact and permeabilized cardiac myocytes demonstrated that SR luminal [Ca²⁺] is markedly lowered in HF, suggesting that alterations in Ca²⁺ transport rather than fractional SR volume reduction accounts for the diminished Ca²⁺ release capacity of SR in HF. SR Ca²⁺ ATPase (SERCA2)-mediated SR Ca²⁺ uptake rate was not significantly altered, and Na⁺/Ca²⁺ exchange activity was accelerated in HF myocytes. At the same time, SR Ca²⁺ leak, measured directly as a loss of [Ca²⁺]_SR after inhibition of SERCA2 by thapsigargin, was markedly enhanced in HF myocytes. Moreover, the reduced [Ca²⁺]_SR in HF myocytes could be nearly completely restored by the RyR2 channel blocker ruthenium red. The effects of HF on cytosolic and SR luminal Ca²⁺ signals could be reasonably well mimicked by the RyR2 channel agonist caffeine. Taken together, these results suggest that RyR2-mediated SR Ca²⁺ leak is a major factor in the abnormal intracellular Ca²⁺ handling that critically contributes to the reduced SR Ca²⁺ content of failing cardiomyocytes.     

Cellular Hypertrophy and Increased Susceptibility to Spontaneous Calcium-Release of Rat Left Atrial Myocytes Due to Elevated Afterload

Atrial remodeling due to elevated arterial pressure predisposes the heart to atrial fibrillation (AF). Although abnormal sarcoplasmic reticulum (SR) function has been associated with AF, there is little information on the effects of elevated afterload on atrial Ca²⁺-handling. We investigated the effects of ascending aortic banding (AoB) on Ca²⁺-handling in rat isolated atrial myocytes in comparison to age-matched sham-operated animals (Sham). Myocytes were either labelled for ryanodine receptor (RyR) or loaded with fluo-3-AM and imaged by confocal microscopy. AoB myocytes were hypertrophied in comparison to Sham controls (P<0.0001). RyR labeling was localized to the z-lines and to the cell edge. There were no differences between AoB and Sham in the intensity or pattern of RyR-staining. In both AoB and Sham, electrical stimulation evoked robust SR Ca²⁺-release at the cell edge whereas Ca²⁺ transients at the cell center were much smaller. Western blotting showed a decreased L-type Ca channel expression but no significant changes in RyR or RyR phosphorylation or in expression of Na⁺/Ca²⁺ exchanger, SR Ca²⁺ ATPase or phospholamban. Mathematical modeling indicated that [Ca²⁺]_i transients at the cell center were accounted for by simple centripetal diffusion of Ca²⁺ released at the cell edge. In contrast, caffeine (10 mM) induced Ca²⁺ release was uniform across the cell. The caffeine-induced transient was smaller in AoB than in Sham, suggesting a reduced SR Ca²⁺-load in hypertrophied cells. There were no significant differences between AoB and Sham cells in the rate of Ca²⁺ extrusion during recovery of electrically-stimulated or caffeine-induced transients. The incidence and frequency of spontaneous Ca²⁺-transients following rapid-pacing (4 Hz) was greater in AoB than in Sham myocytes. In conclusion, elevated afterload causes cellular hypertrophy and remodeling of atrial SR Ca²⁺-release.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca2+ Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

Voltage-Activated Elementary Calcium Release Events in Isolated Mouse Skeletal Muscle Fibers

The elementary Ca²⁺-release events underlying voltage-activated myoplasmic Ca²⁺ transients in mammalian muscle remain elusive. Here, we looked for such events in confocal line-scan (x,t) images of fluo-3 fluorescence taken from isolated adult mouse skeletal muscle fibers held under voltage-clamp conditions. In response to step depolarizations, spatially segregated fluorescence signals could be detected that were riding on a global increase in fluorescence. These discrete signals were separated using digital filtering in the spatial domain; mean values for their spatial half-width and amplitude were 1.99 ± 0.09 μm and 0.16 ± 0.005 ΔF/F ₀ (n = 151), respectively. Under control conditions, the duration of the events was limited by the pulse duration. In contrast, in the presence of maurocalcine, a scorpion toxin suspected to disrupt the process of repolarization-induced ryanodine receptor (RyR) closure, events uninterrupted by the end of the pulse were readily detected. Overall results establish these voltage-activated low-amplitude local Ca²⁺ signals as inherent components of the physiological Ca²⁺-release process of mammalian muscle and suggest that they result from the opening of either one RyR or a coherently operating group of RyRs, under the control of the plasma membrane polarization.     

Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca2+ release during the quasi-steady level of release in twitch fibers from frog skeletal muscle

Experiments were performed to characterize the properties of the intrinsic Ca²⁺ buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca_T]_SR and [Ca²⁺]_SR) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca²⁺ indicator). Results indicate SR Ca²⁺ buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca²⁺. Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca²⁺]_SR and [Ca_T]_SR are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca²⁺ permeability of the SR, namely d[Ca_T]_SR/dt ÷ [Ca²⁺]_SR (denoted release permeability), in experiments in which only [Ca_T]_SR or [Ca²⁺]_SR is measured. In response to a voltage-clamp step to −20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ∼50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca²⁺ release of 2.3 SR Ca²⁺ release channels neighboring each channel activated by its associated voltage sensor. Release permeability at −60 mV increases as [Ca_T]_SR decreases from its resting physiological level to ∼0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca²⁺]_SR inhibits release when [Ca_T]_SR declines to a low level.     

N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium

Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca²⁺]_i) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca²⁺]_i changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca²⁺]_i mobilization using several extraction methods. The spatial distribution pattern of [Ca²⁺]_i among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 μM and 50 μM AHL triggered opposing osteoblast fates. At 50 μM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 μM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca²⁺]_i levels in osteoblasts treated with 50 μM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 μM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 μM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca²⁺]_i changes. Thus, AHL acts as a double-edged sword for osteoblast function.     

Transport of Ca2+ from Sarcoplasmic Reticulum to Mitochondria in Rat Ventricular Myocytes

Studies with electron microscopy have shown that sarcoplasmic reticulum (SR) andmitochondria locate close to each other in cardiac muscle cells. We investigated the hypothesis thatthis proximity results in a transient exposure of mitochondrial Ca²⁺ uniporter (CaUP) to highconcentrations of Ca²⁺ following Ca²⁺ release from the SR and thus an influx of Ca²⁺into mitochondria. Single ventricular myocytes of rat were skinned by exposing them to aphysiological solution containing saponin (0.2 mg/ml). Cytosolic Ca²⁺ concentration ([Ca²⁺]_c)and mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) were measured with fura-2 and rhod2,respectively. Application of caffeine (10 mM) induced a concomitant increase in[Ca²⁺]_c and [Ca²⁺]_m.Ruthenium red, at concentrations that block CaUP but not SR release, diminished thecaffeine-induced increase in [Ca²⁺]_m but not[Ca²⁺]_c. In the presence of 1 mM BAPTA, a Ca²⁺ chelator,the caffeine-induced increase in [Ca²⁺]_m was reduced substantially less than [Ca²⁺]_c. Moreover,inhibition of SR Ca²⁺ pump with two different concentrations of thapsigargin caused anincrease in [Ca²⁺]_m, which was related to the rate of [Ca²⁺]_c increase. Finally, electronmicroscopy showed that sites of junctions between SR and T tubules from which Ca²⁺ is released,or Ca²⁺ release units, CRUs, are preferentially located in close proximity to mitochondria.The distance between individual SR Ca²⁺ release channels (feet or ryanodine receptors) isvery short, ranging between approximately 37 and 270 nm. These results are consistent withthe idea that there is a preferential coupling of Ca²⁺ transport from SR to mitochondria incardiac muscle cells, because of their structural proximity.     

Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

Model of Sarcomeric Ca2+ Movements,Including ATP Ca2+ Binding and Diffusion,during Activation of Frog Skeletal Muscle

Cannell and Allen (1984. Biophys. J. 45:913–925) introduced the use of a multi-compartment model to estimate the time course of spread of calcium ions (Ca²⁺) within a half sarcomere of a frog skeletal muscle fiber activated by an action potential. Under the assumption that the sites of sarcoplasmic reticulum (SR) Ca²⁺ release are located radially around each myofibril at the Z line, their model calculated the spread of released Ca²⁺ both along and into the half sarcomere. During diffusion, Ca²⁺ was assumed to react with metal-binding sites on parvalbumin (a diffusible Ca²⁺- and Mg²⁺-binding protein) as well as with fixed sites on troponin. We have developed a similar model, but with several modifications that reflect current knowledge of the myoplasmic environment and SR Ca²⁺ release. We use a myoplasmic diffusion constant for free Ca²⁺ that is twofold smaller and an SR Ca²⁺ release function in response to an action potential that is threefold briefer than used previously. Additionally, our model includes the effects of Ca²⁺ and Mg²⁺ binding by adenosine 5′-triphosphate (ATP) and the diffusion of Ca²⁺-bound ATP (CaATP). Under the assumption that the total myoplasmic concentration of ATP is 8 mM and that the amplitude of SR Ca²⁺ release is sufficient to drive the peak change in free [Ca²⁺] (Δ[Ca²⁺]) to 18 μM (the approximate spatially averaged value that is observed experimentally), our model calculates that (a) the spatially averaged peak increase in [CaATP] is 64 μM; (b) the peak saturation of troponin with Ca²⁺ is high along the entire thin filament; and (c) the half-width of Δ[Ca²⁺] is consistent with that observed experimentally. Without ATP, the calculated half-width of spatially averaged Δ[Ca²⁺] is abnormally brief, and troponin saturation away from the release sites is markedly reduced. We conclude that Ca²⁺ binding by ATP and diffusion of CaATP make important contributions to the determination of the amplitude and the time course of Δ[Ca²⁺].     

Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

Release of Ca²⁺ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca²⁺ regulation of SR Ca²⁺ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca²⁺ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca²⁺ regulates ryanodine receptor (RyR)2-mediated SR Ca²⁺ release through mechanisms localized inside the SR; one of these involves luminal Ca²⁺ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function.CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca²⁺] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca²⁺ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca²⁺ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca²⁺ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.