Inositol trisphosphate enhances Ca2+ oscillations but not Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] in excitation-contraction coupling in cardiac muscle is still unclear, although many laboratories are beginning to assume a critical role for this putative second messenger. Earlier studies from this laboratory [Nosek et al. (1986) Am J Physiol 250:C807] found that Ins(1,4,5)P ₃ enhanced spontaneous Ca²⁺ release and the caffeine sensitivity of Ca²⁺ release from myocardial sarcoplasmic reticulum (SR) and proposed an increase in the Ca²⁺ sensitivity of the release as a possible mechanism. In order to clarify the phyisological relevance of these actions of Ins(1,4,5)P ₃ and specifically to test the effect of Ins(1,4,5)P ₃ on the Ca²⁺ sensitivity of Ca²⁺ release, we compared the effects of Ins(1,4,5)P ₃ on Ca²⁺ oscillations and on Ca²⁺-induced Ca²⁺ release (CICR) from the SR in saponin-skinned rat papillary muscle. We found that: (a) 30 M Ins(1,4,5)P ₃ enhanced the Ca²⁺ oscillations (measured by tension oscillations) from the rat cardiac SR, consistent with the previous report on guinea pig tissue; (b) both GTP and GTP[S] enhanced Ca²⁺ oscillations. The effect was not additive to that of Ins(1,4,5)P ₃ indicating that two different Ca²⁺-release pools do not exist in cardiac SR; (c) 30 M Ins(1,4,5)P ₃ had no effect on the Ca²⁺ sensitivity of CICR; (d) Ins(1,4,5)P ₃ (up to 30 M) had no effect on SR Ca²⁺ loading. The studies were performed in the presence of Cd²⁺ or 2,3-bisphosphoglycerate, agents that inhibit Ins(1,4,5)P ₃ hydrolysis. These results suggest that: (a) two different mechanisms underlie Ca²⁺ oscillations and CICR, Ins(1,4,5)P ₃ influencing Ca²⁺ oscillations but not CICR; (b) Ins(1,4,5)P ₃ does not increase the Ca²⁺ sensitivity of Ca²⁺ release from the SR; (c) cardiac muscle is different from smooth muscle where Ca²⁺ release from the SR is dependent upon GTP; (d) the physiological role of Ins(1,4,5)P ₃ in excitation-contraction coupling in cardiac muscle is minimal. In contrast, Ins(1,4,5)P ₃ may play a pathological role in cardiac arrhythmogenesis by enhancing spontaneous Ca²⁺ ocsillations.     

Mechanisms of Ca2+ handling in zebrafish ventricular myocytes

The zebrafish serves as a promising transgenic animal model that can be used to study cardiac Ca²⁺ regulation. However, mechanisms of sarcoplasmic reticulum (SR) Ca²⁺ handling in the zebrafish heart have not been systematically explored. We found that in zebrafish ventricular myocytes, the action potential-induced Ca²⁺ transient is mainly (80 %) mediated by Ca²⁺ influx via L-type Ca²⁺ channels (LTCC) and only 20 % by Ca²⁺ released from the SR. This small contribution of the SR to the Ca²⁺ transient was not the result of depleted SR Ca²⁺ load. We found that the ryanodine receptor (RyR) expression level in zebrafish myocytes was ～72 % lower compared to rabbit myocytes. In permeabilized myocytes, increasing cytosolic [Ca²⁺] from 100 to 350 nM did not trigger SR Ca²⁺ release. However, an application of a low dose of caffeine activated Ca²⁺ sparks. These results show that the zebrafish cardiac RyR has low sensitivity to the mechanism of Ca²⁺-induced Ca²⁺ release. Activation of protein kinase A by forskolin increased phosphorylation of the RyR in zebrafish myocardium. In half of the studied cells, an increased Ca²⁺ transient by forskolin was entirely mediated by augmentation of LTCC current. In the remaining myocytes, the forskolin action was associated with an increase of both LTCC and SR Ca²⁺ release. These results indicate that the mechanism of excitation–contraction coupling in zebrafish myocytes differs from the mammalian one mainly because of the small contribution of SR Ca²⁺ release to the Ca²⁺ transient. This difference is due to a low sensitivity of RyRs to cytosolic [Ca²⁺].     

Effect of calmodulin on Ca2+-induced Ca2+ release of skeletal muscle from mutant mice expressing either ryanodine receptor type 1 or type 3

 We analyzed the effects of calmodulin (CaM) on Ca²⁺-induced Ca²⁺ release (CICR) in mouse skeletal muscle cells expressing only ryanodine receptor type 1 (RyR-1) or type 3 (RyR-3) following targeted disruption of one of the RyR genes. Under Mg²⁺-free conditions, CaM potentiated CICR via RyR-3 at low Ca²⁺ concentrations (pCa≥6) but inhibited CICR at high Ca²⁺ concentrations (pCa≤5). On the other hand, CaM potentiated CICR via RyR-1 between pCa 7 and pCa 5. Greater concentrations of CaM were required for potentiation of CICR at pCa 6 than for the inhibition at pCa 5 in the RyR-3-expressing cells. Similarly, higher concentrations of CaM were required for the potentiation of CICR via RyR-1 at pCa 6 than potentiation at pCa 5. In the presence of Mg²⁺ and β,γ-methyleneadenosine 5′-trisphosphate (AMPOPCP), the same differential effects of CaM on the CICR via the different subtypes of RyR were observed. These data suggest that multiple CaM-binding sites are involved in the differential effects on RyR-1 and RyR-3. These effects of CaM are important for the evaluation of the physiological roles of RyRs. Received: 5 May 1998 / Received after revision: 14 August 1998 / Accepted: 3 September 1998     

Ca(2+)-dependent negative control mechanism for Ca(2+)-induced Ca2+ release in crayfish muscle.

The mechanism of termination of Ca(2+)-induced Ca2+ release (CICR) from the sarcoplasmic reticulum has been investigated in voltage clamped cut crayfish muscle fibres loaded with rhod-2. During depolarizing steps evoking calcium current (ICa), Ca2+ release was first activated. Then the release rapidly (tau approximately 6 ms) declined, as evidenced by the rate of change of the intracellular fluorescence signal representing a Ca2+ transient. The rapid termination of release was not accounted for by inactivation of the trigger ICa or depletion of Ca2+ from the SR, since the rate at which release declined was constant under conditions where the rate of ICa inactivation and the amount of Ca2+ released varied widely. Pre-elevations of [Ca2+]i with prepulses or photolysis of caged Ca2+ caused depression of Ca2+ release during a subsequent test pulse. When the rate of ICa onset was varied by applying voltage ramps with different slopes, currents with fast onset elicited larger Ca2+ release than calcium currents with slower onset, even though the amplitude of the currents was the same. These results suggest that a Ca(2+)-dependent negative control mechanism exists which mediates the termination of CICR independently of the duration of the trigger ICa and before significant depletion of Ca2+ in the SR occurs.     

Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum

Prolonged exercise decreased the rate of Ca⁺ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 M AgNO₃. [³H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca²⁺ released by Ag⁺ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca²⁺ release in the presence of ruthenium red, a known inhibitor of the Ca²⁺ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca²⁺ release from SR by directly modifying the Ca²⁺ release channel. Current address: Department of Physics, Portland State University, Portland, OR 97207, USA     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

 The effects of the sarcoplasmic reticulum (SR) Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca²⁺ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca²⁺] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca²⁺] of 100 nM, introduction of 20 μM CPA induced a slow release of Ca²⁺ from the SR. The following one to two caffeine-induced Ca²⁺ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca²⁺ transients decreased progressively and were barely detectable 6–7 min after introduction of CPA. However, increasing the bathing [Ca²⁺] or increasing the Ca²⁺ loading period resulted in a partial recovery of the caffeine-induced Ca²⁺ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 μM CPA. The slow Ca²⁺ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca²⁺ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca²⁺ transient reflects a balance between efflux via the SR Ca²⁺ channel and reuptake by the Ca pump. Ca²⁺ release upon addition of CPA may result from inhibition of SR Ca²⁺ uptake, which reveals a tonic Ca²⁺ efflux that is independent of the Ca²⁺ release channels. Received: 26 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

Effects of ATP and related compounds on the Ca-induced Ca release mechanism of theXenopus SR

The effects of ATP and related compounds on the Ca²⁺ release mechanism of the sarcoplasmic reticulum (SR) was studied by using skinned skeletal muscle fibers ofXenopus laevis. ATP evoked marked Ca²⁺ release at very low level of Mg²⁺. , -Methylene analogue of ATP was almost as effective as ATP, which suggests Ca²⁺ release evoked by ATP is elicited without ATP hydrolysis. ADP and AMP also evoked Ca²⁺ release from the SR, but the effect of them became gradually weaker than that of ATP as the number of phosphates decreased. CTP, UTP and ITP were less potent than ATP. Adenosine also evoked more effective Ca²⁺ release than inosine. The compounds with adenine base, therefore, seem to elicit more potent Ca²⁺ release than those which have the same number of phosphates but do not consist of adenine base. AMP and Ca²⁺ ion evoked Ca²⁺ release synergistically, and the Ca²⁺ release responses evoked by ATP and related compounds showed the same pharmacological characteristics as Ca-induced Ca release. So, these Ca²⁺ release responses are construed as the manifestation of the same mechanism as Ca-induced Ca release. Effective concentration range of ATP and the effect of pyrophosphate on Ca²⁺ release evoked by ATP suggest that neither the high affinity ATP catalytic site of (Ca²⁺+Mg²⁺) ATPase of the SR nor the low affinity ATP binding site, reported by Dupont (1977), is implicated in the enhancement of these Ca²⁺ release responses from the SR.     

Free radical-dependent Ca2+ signaling: role of Ca2+-induced Ca2+ release

Previously we have shown that Fe3+/ascorbate-induced Ca2+ release from scallop sarcoplasmic reticulum (SR) is due to Ca2+-channel gating by free radicals. This study is aimed at demonstrating that Ca2+-induced Ca2+ release (CICR) plays a role in this kind of Ca2+ release. Scallop SR vesicles were incubated with fluo-3 and exposed to Fe3+/ascorbate. Fluorimetric recordings showed massive Ca2+ release, with maximum rate and 50% release occurring at 30 min after exposure. Conversely, the use of the probe for reactive oxygen species dihydrorhodamine or the assay of malondialdehyde allowed oxyradical production to be traced for approximately 5 min only. Hence, although Ca2+ release started just after exposure to Fe3+/ascorbate, most release occurred after free radical exhaustion. Ruthenium red addition after Fe3+/ascorbate slowed down the Ca2+ release, whereas cyclic adenosine 5'-diphosphoribose addition accelerated it, indicating that the free radical-induced Ca2+ release from SR vesicles triggers a mechanism of CICR that dramatically increases the initial effect.     

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres

 The effect of intracellular Cl^– on Ca²⁺ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg²⁺] and [ATP] and sarcoplasmic reticulum (SR) Ca²⁺ loading. Both in rat and toad fibres, the presence of 20 mM Cl^–in the myoplasm increased Ca²⁺ leakage from the SR at pCa (i.e. –log₁₀ [Ca²⁺]) 6.7, but not at pCa 8. Ca²⁺ uptake was not significantly affected by the presence of Cl^–. This Ca²⁺-dependent effect of Cl^– on Ca²⁺ leakage was most likely due to a direct action on the ryanodine receptor/Ca²⁺ release channel, and could influence channel sensitivity and the resting [Ca²⁺] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl^– to the myoplasm caused a 40–50% reduction in Ca²⁺ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl^– induces a potential across the SR (lumen negative) which might reduce Ca²⁺ release via several different mechanisms. Received: 20 October 1997 / Received after revision: 1 December 1997 / Accepted: 2 December 1997     

Propagation of excitation-contraction coupling into ventricular myocytes

This paper examines the [Ca²⁺]_i transient in isolated rat heart cells using a laser scanning confocal microscope and the calcium indicator fluo-3. We find that the depolarization-evoked [Ca²⁺]_i transient is activated synchronously near the surface and in the middle of the heart cell with similar kinetics of activation. The time of rise of the transient did not depend on whether the sarcoplasmic reticulum (SR) Ca-release was abolished (by thapsigargin and ryanodine). The synchrony of activation and the similarity of levels of [Ca²⁺]_i at the peripheral and deeper myoplasm (regardless of the availability of SR Ca-release) shows that sarcolemmal Ca channels and SR Ca-release channels are distributed throughout the rat heart cell and that the propagation of the action potential into the interior of the cell is rapid. In addition, the activation of calcium release from the SR by CICR is rapid (2 ms) when compared to the time-course of calcium influx via the sarcolemmal Ca channel.     

Mechanism of Ca++ Release from the Sarcoplasmic Reticulum: A Computer Model

The proposed model describes myocyte calcium (Ca⁺⁺) cycling, emphasizing the kinetics of sarcoplasmic reticulum (SR) Ca⁺⁺ release channels. The suggested SR channel regulating mechanism includes two types of Ca⁺⁺ binding sites: (1) low affinity sites with high binding rates, regulating the opening of Ca⁺⁺ channels and (2) high affinity sites with low binding rates, which regulate their closing. The amount of Ca⁺⁺ released from the SR and the peak value of Ca⁺⁺ ion concentration [Ca⁺⁺] in the cytoplasm were found to depend on the rate of the increase of [Ca⁺⁺], similar to Ca⁺⁺ induced Ca⁺⁺ release experiments. The model describes spontaneous release of Ca⁺⁺ from overloaded SR. The dependence of the control mechanism on the activating and inactivating sites is substantiated by simulations of ryanodine intervention, providing results similar to experimental results. Simulations under conditions of isolated SR vesicles produced Ca⁺⁺ release results similar to measured data. Consequently, it is suggested that the recovery of Ca⁺⁺ release channels represents the rate limiting factor in the process of mechanical restitution. © 1998 Biomedical Engineering Society. PAC98: 8722Fy, 8710+e     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 1996     

Inhibitors of Bcl-2 protein family deplete ER Ca2+ stores in pancreatic acinar cells

Physiological stimulation of pancreatic acinar cells by cholecystokinin and acetylcholine activate a spatial-temporal pattern of cytosolic [Ca⁺²] changes that are regulated by a coordinated response of inositol 1,4,5-trisphosphate receptors (IP₃Rs), ryanodine receptors (RyRs) and calcium-induced calcium release (CICR). For the present study, we designed experiments to determine the potential role of Bcl-2 proteins in these patterns of cytosolic [Ca⁺²] responses. We used small molecule inhibitors that disrupt the interactions between prosurvival Bcl-2 proteins (i.e. Bcl-2 and Bcl-xl) and proapoptotic Bcl-2 proteins (i.e. Bax) and fluorescence microfluorimetry techniques to measure both cytosolic [Ca⁺²] and endoplasmic reticulum [Ca⁺²]. We found that the inhibitors of Bcl-2 protein interactions caused a slow and complete release of intracellular agonist-sensitive stores of calcium. The release was attenuated by inhibitors of IP₃Rs and RyRs and substantially reduced by strong [Ca²⁺] buffering. Inhibition of IP₃Rs and RyRs also dramatically reduced activation of apoptosis by BH3I-2′. CICR induced by different doses of BH3I-2′ in Bcl-2 overexpressing cells was markedly decreased compared with control. The results suggest that Bcl-2 proteins regulate calcium release from the intracellular stores and suggest that the spatial-temporal patterns of agonist-stimulated cytosolic [Ca⁺²] changes are regulated by differential cellular distribution of interacting pairs of prosurvival and proapoptotic Bcl-2 proteins.     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Activation of Ca(2+)-dependent protein kinase II during repeated contractions in single muscle fibres from mouse is dependent on the frequency of sarcoplasmic reticulum Ca(2+) release

Aim: To investigate the importance and contribution of calmodulin‐dependent protein kinase II (CaMKII) activity on sarcoplasmic reticulum (SR) Ca²⁺‐release in response to different work intensities in single, intact muscle fibres. Methods: CaMKII activity was blocked in single muscle fibres using either the inhibitory peptide AC3‐I or the pharmacological inhibitor KN‐93. The effect on tetanic force production and [Ca²⁺]_i was determined during work of different intensities. The activity of CaMKII was assessed by mathematical modelling. Results: Using a standard protocol to induce fatigue (50× 70 Hz, 350 ms duration, every 2 s) the number of stimuli needed to induce fatigue was decreased from 47 ± 3 contractions in control to 33 ± 3 with AC3‐I. KN‐93 was a more potent inhibitor, decreasing the number of contractions needed to induce fatigue to 15 ± 3. Tetanic [Ca²⁺]_i was 100 ± 11%, 97 ± 11% and 67 ± 11% at the end of stimulation in control, AC3‐I and KN‐93 respectively. A similar inhibition was obtained using a high intensity protocol (20× 70 Hz, 200 ms duration, every 300 ms). However, using a long interval protocol (25× 70 Hz, 350 ms duration, every 5 s) no change was observed in either tetanic [Ca²⁺]_i or force when inhibiting CaMKII. A mathematical model used to investigate the activation pattern of CaMKII suggests that there is a threshold of active CaMKII that has to be surpassed in order for CaMKII to affect SR Ca²⁺ release. Conclusion: Our results show that CaMKII is crucial for maintaining proper SR Ca²⁺ release and that this is regulated in a work intensity manner.     

Sarcoplasmic reticulum Ca2+ loading in rabbits 8 and 15 weeks after coronary artery ligation

 Calcium uptake by cardiac sarcoplasmic reticulum (SR) is reported to be reduced in heart failure in the human and in a number of animal models. However, the majority of studies have examined end-stage heart failure in the human and few animal studies have taken account of the duration and severity of left ventricular dysfunction. In this study we have compared SR Ca²⁺ loading in a haemodynamically assessed, coronary artery ligation model of heart failure at 8 and 15 weeks after ligation. Trabeculae were isolated from the right ventricle and mounted for isometric tension measurement. They were treated with saponin to permeabilize the sarcolemma but retain SR function and bathed in a mock intracellular solution including adenosine triphosphate (ATP) and buffered Ca²⁺. Caffeine was used to release Ca²⁺ from the SR. The amplitude of the caffeine-induced contracture was used as a quantitative gauge of the Ca²⁺ content of the SR. Eight weeks after ligation, trabeculae demonstrated enhanced SR Ca²⁺ uptake as manifest by larger caffeine-induced contractures (e.g. 200 nM [Ca²⁺], 120 s loading – 38.2±9.2 versus 67.3±10.1% of maximum Ca²⁺-activated force, F _{Ca, max}, P=0.03). At 15 weeks, trabeculae from ligated hearts were not significantly different from controls with SR Ca²⁺ loading returning to control levels (e.g. 200 nM [Ca²⁺], 120 s loading – 47.3±9.6 versus 30.2±12.8% F _{Ca, max}, P=0.12). These data suggest that SR Ca²⁺ loading may increase in the early stages of heart failure and fall back to normal with an increasing duration of left ventricular dysfunction. Increased incidence of spontaneous Ca²⁺ release observed from the SR at 8 weeks and not at 15 weeks may represent an arrhythmogenic mechanism specific to the early phase of heart failure. Received: 21 January 1998 / Received after revision and accepted: 3 April 1998     

Influence of caffeine,Ca2+, and Mg2+ on ryanodine depression of the tension transient in skinned myocardial fibers of the rabbit

Ryanodine, a blocker for Ca²⁺-release channels of the sarcoplasmic reticulum (SR Ca²⁺-release channels), induces depression of myocardial contraction in isolated intact muscle, which is consistent with depression of the caffeine-induced tension transient in skinned muscle fibers. In isolated SR, ryanodine binds to a specific receptor with high affinity, and this binding is enhanced by caffeine and increasing Ca²⁺ and decreased by increasing Mg²⁺. The aim of this study was to test the hypothesis that depression of myocardial contraction is mediated by changes in ryanodine-receptor binding properties. Accordingly, factors (caffeine, Ca²⁺, and Mg²⁺) affecting ryanodine-receptor binding properties in the isolated SR membrane were studied in skinned myocardial fibers from adult rabbits. The depression of the caffeine-induced tension transient by ryanodine (ryanodine depression) influenced by these three factors was measured. In a dose-dependent manner, increasing caffeine or Ca²⁺ concentrations enhanced the ryanodine depression. The concentrations for 50% ryanodine depression (IC₅₀) approximated 7mM for caffeine, and pCa 5.25 for Ca²⁺. When 1 M ryanodine and 25 mM caffeine were combined, ryanodine depression was independent of Ca²⁺ at low Ca²⁺ concentrations (20%–30% at pCa>8 and 7.5) and was a direct function of Ca²⁺ at higher concentrations (pCa 7.5–6.0 with IC₅₀ approx. pCa 6.75). In contrast, increasing Mg²⁺ reduced the ryanodine depression with IC₅₀ approximately equal to pMg 3.3. In conclusion, the caffeineor Ca²⁺-enhanced, and Mg²⁺-reduced ryanodine depression observed in this study is consistent with known ryanodinereceptor binding properties.     

Histamine-evoked Ca2+ oscillations in HeLa cells are sensitive to methylxanthines but insensitive to ryanodine

The relative contribution of inositol-trisphosphate(InsP ₃)-sensitive and InsP ₃-insensitive Ca²⁺ stores to the agonist-evoked oscillatory release of Ca²⁺ in HeLa cells was investigated using fura-2 cytosolic Ca²⁺ measurements and whole-cell recordings of Ca²⁺-activated K⁺ currents [K(Ca²⁺)]. The experimental approach chosen consisted in studying the effects on Ca²⁺ oscillations of a variety of pharmacological agents such as ryanodine, ruthenium red, caffeine and theophylline, which are known to affect the Ca²⁺ channels responsible for Ca²⁺-induced Ca²⁺ release (CICR) in excitable cells. The results obtained essentially indicate (a) that neither ryanodine nor ruthenium red affects the generation of periodic K(Ca²⁺) current pulses in whole-cell experiments, and (b) that histamine-induced Ca²⁺ oscillations are inhibited by caffeine and theophylline in a dose-dependent manner. However, these methylxanthines were unable, at concentrations ranging from 0.1 mM to 10 mM, either to mobilize Ca²⁺ from internal stores or to block the initial Ca²⁺ rise evoked by histamine. In addition, both methylxanthines showed at high concentrations (10–20 mM) a moderate inhibitory action on the production of InsP ₃ induced by histamine. This effect was not essential to the action of caffeine on the oscillatory release of Ca²⁺, since an inhibition by caffeine of InsP ₃-induced Ca²⁺ oscillations was still observed in whole-cell experiments where the InsP ₃ concentration was kept constant. The results also show (c) that the application of either caffeine or theophylline during histamine stimulation leads systematically to an increased Ca²⁺ sequestration in InsP ₃-sensitive Ca²⁺ pools, the effect observed with theophylline being stronger than that resulting from the application of caffeine, and finally (d) that the action of caffeine and theophylline is not related to an increase in cAMP concentration since neither forskolin (10–50 M) nor 8-Br-cAMP (1 mM) caused an inhibition of the InsP ₃-induced Ca²⁺ oscillations. It is concluded on the basis of these results that the agonist-evoked Ca²⁺ oscillations in HeLa cells do not involve directly or indirectly a ryanodine-sensitive Ca²⁺-release channel with CICR properties, but rather arise from a control by Ca²⁺ of the InsP ₃ Ca²⁺-release process.     

Functional significance of Ca2+ in long-lasting fatigue of skeletal muscle

Repeated activation of skeletal muscle causes fatigue, which involves a reduced ability to produce force and slowed contraction regarding both the speed of shortening and relaxation. One important component in skeletal muscle fatigue is a reduced sarcoplasmic reticulum (SR) Ca²⁺ release. In the present review we will describe different types of fatigue-induced inhibition of SR Ca²⁺ release. We will focus on a type of long-lasting failure of SR Ca²⁺ release which is called low-frequency fatigue, because this type of fatigue may be involved in the muscle dysfunction and chronic pain experienced by computer workers. Paradoxically it appears that the Ca²⁺ released from the SR, which is required for contraction, may actually be responsible for the failure of SR Ca²⁺ release during low-frequency fatigue. We will also discuss the relationship between gross morphological changes in muscle fibres and long-lasting failure of SR Ca²⁺ release. Finally, a model linking muscle cell dysfunction and muscle pain is proposed. Accepted: 6 June 2000