Redox modulation of diaphragm contractility: Interaction between DHPR and RyR channels

Previous reports indicate that reactive oxygen species (ROS) may modulate contractility in skeletal muscle. Although Ca(2+)-sensitivity of the contractile apparatus appears to be a primary site of regulation, dihydropyridine receptor (DHPR or L-type Ca(2+) channels) and calcium efflux in isolated sarcoplasmic reticulum (SR) vesicles appear to be redox sensitive as well. However, DHPR as a target is poorly understood in intact muscles at body temperature, particularly in the diaphragm, a muscle more dependent on external Ca(2+) than locomotor muscles. Previously, we reported that oxidant challenge via xanthine oxidase (XO) alters the K(+) contractures in diaphragm fiber bundles, suggestive of a role of L-type Ca(2+) channels. Contractility of isolated rat diaphragm fiber bundles revealed a biphasic response to ROS challenge that was dose and time dependent. Potentiation of twitch and low-frequency diaphragm fiber bundle contractility with 0.02 U?ml(-1) XO was reversible or partially preventable with washout, dithiothreitol, and the SOD/catalase mimetic EUK-134. The RyR antagonist ruthenium red inhibited xanthine oxidase-induced potentiation, while the RyR agonist caffeine elevated diaphragm twitch and low-frequency tension in a non-additive manner by 55% when introduced simultaneously with ROS challenge. The DHPR antagonist nitrendipine (15 μM) inhibited elevation in low-frequency diaphragm tension produced by ROS challenge. Caffeine threshold tension curves were shifted to the left with 0.02 U?ml(-1) XO, but this effect was partially reversed with 15 μM nitrendipine. These results are consistent with the hypothesis that DHPR redox state and RyR function are modulated in an interactive manner, affecting contractility in intact diaphragm fiber bundles.     

Muscle metabolic, SR Ca(2+) -cycling responses to prolonged cycling, with and without glucose supplementation.

This study investigated the effects of prolonged exercise, with and without glucose supplementation, on metabolism and sarcoplasmic reticulum (SR) Ca(2+)-handling properties in working vastus lateralis muscle. Fifteen untrained volunteers [peak O(2) consumption (Vo(2peak)) = 3.45 +/- 0.17 l/min; mean +/- SE] cycled at approximately 60% Vo(2peak) on two occasions, during which they were provided with either an artificially sweetened placebo beverage (NG) or a 6% glucose (G) beverage (~1.00 g carbohydrate/kg body mass). Beverage supplementation started at 30 min of exercise and continued every 15 min thereafter. SR Ca(2+) handling, metabolic, and substrate responses were assessed in tissue extracted from the vastus lateralis at rest, after 30 min and 90 min of exercise, and at fatigue in both conditions. Plasma glucose during G was 15-23% higher (P < 0.05) than those observed during NG following 60 min of exercise until fatigue. Cycle time to fatigue was increased (P < 0.05) by approximately 19% during G (137 +/- 7 min) compared with NG (115 +/- 6 min). Prolonged exercise reduced (P < 0.05) maximal Ca(2+)-ATPase activity (-18.4%), SR Ca(2+) uptake (-27%), and both Phase 1 (-22.2%) and Phase 2 (-34.2%) Ca(2+)-release rates during NG. The exercise-induced reductions in SR Ca(2+)-cycling properties were not altered during G. The metabolic responses to exercise were all unaltered by glucose supplementation, since no differences in respiratory exchange ratios, carbohydrate and lipid oxidation rates, and muscle metabolite and glycogen contents were observed between NG and G. These results indicate that the maintenance of blood glucose homeostasis by glucose supplementation is without effect in modifying the muscle metabolic, endogenous glycogen, or SR Ca(2+)-handling responses.     

Effects of progressive exercise and hypoxia on human muscle sarcoplasmic reticulum function.

This study examined the effects of progressive exercise to fatigue in normoxia (N) on muscle sarcoplasmic reticulum (SR) Ca(2+) cycling and whether alterations in SR Ca(2+) cycling are related to the blunted peak mechanical power output (PO(peak)) and peak oxygen consumption (Vo(2 peak)) observed during progressive exercise in hypoxia (H). Nine untrained men (20.7 +/- 0.42 yr) performed progressive cycle exercise to fatigue on two occasions, namely during N (inspired oxygen fraction = 0.21) and during H (inspired oxygen fraction = 0.14). Tissue extracted from the vastus lateralis before exercise and at power output corresponding to 50 and 70% of Vo(2 peak) (as determined during N) and at fatigue was used to investigate changes in homogenate SR Ca(2+)-cycling properties. Exercise in H compared with N resulted in a 19 and 21% lower (P < 0.05) PO(peak) and Vo(2 peak), respectively. During progressive exercise in N, Ca(2+)-ATPase kinetics, as determined by maximal activity, the Hill coefficient, and the Ca(2+) concentration at one-half maximal activity were not altered. However, reductions with exercise in N were noted in Ca(2+) uptake (before exercise = 357 +/- 29 micromol x min(-1) x g protein(-1); at fatigue = 306 +/- 26 micromol x min(-1) x g protein(-1); P < 0.05) when measured at free Ca(2+) concentration of 2 microM and in phase 2 Ca(2+) release (before exercise = 716 +/- 33 micromol x min(-1) x g protein(-1); at fatigue = 500 +/- 53 micromol x min(-1) x g protein(-1); P < 0.05) when measured in vitro in whole muscle homogenates. No differences were noted between N and H conditions at comparable power output or at fatigue. It is concluded that, although structural changes in SR Ca(2+)-cycling proteins may explain fatigue during progressive exercise in N, they cannot explain the lower PO(peak) and Vo(2 peak) observed during H.     

不同频率电刺激膈神经导致隔肌肌浆网Ca^2＋—ATPase和Ca^2＋释放—摄取动力学改变

研究不同频率慢性电刺激（CES）后兔膈肌肌浆网（SR）Ca^2 -ATPase活性以及SRC^2 摄取－释放动力学对不同频率CES的活应性变化,建立不同频率CES组,用定磷法测定SR Ca^2 -ATPaes活性,用Fura-2荧光法测定SR Ca^2 摄取－释放动力学,与对照组比较,慢性低频电刺激10Hz和20Hz组的SR Ca^2 －ATPase活性明显降低（P＜0．01）,Ca^2 释放－摄动力学也显著降低（P＜0．01）,慢性高频电刺激50Hz和100Hz组的SRCa^2 -ATPase活性则显著升高（P＜0．01）,Ca^2 释放－摄取动力学亦明显升高（P＜0．01）,实验提示,ECS后不同频率CES导致膈肌SRCa^2 -ATPase,Ca^2 摄取－释放动力学产生不同的适应性变化,对不同功能状态的膈应用不同频谱的慢性电刺激可能具有重要的临床意义。     

Interactions between intracellular calcium and phosphate in intact mouse muscle during fatigue

Fatigue was studied in intact tibialis anterior muscle of anesthetized mice. The distal tendon was detached and connected to a force transducer while blood flow continued normally. The muscle was stimulated with electrodes applied directly to the muscle surface and fatigued by repeated (1 per 4 s), brief (0.4 s), maximal (100-Hz stimulation frequency) tetani. Force declined monotonically to 49 ± 5% of the initial value with a half time of 36 ± 5 s and recovered to 86 ± 4% after 4 min. Intracellular phosphate concentration ([P(i)]) was measured by (31)P-NMR on perchloric acid extracts of muscles. [P(i)] increased during fatigue from 7.6 ± 1.7 to 16.0 ± 1.6 mmol/kg muscle wet wt and returned to control during recovery. Intracellular Ca(2+) was measured with cameleons whose plasmids had been transfected in the muscle 2 wk before the experiment. Yellow cameleon 2 was used to measure myoplasmic Ca(2+), and D1ER was used to measure sarcoplasmic reticulum (SR) Ca(2+). The myoplasmic Ca(2+) during tetani declined steadily during the period of fatigue and showed complete recovery over 4 min. The SR Ca(2+) also declined monotonically during fatigue and showed a partial recovery with rest. These results show that the initial phase of force decline is accompanied by a rise in [P(i)] and a reduction in the tetanic myoplasmic Ca(2+). We suggest that both changes contribute to the fatigue. A likely cause of the decline in tetanic myoplasmic Ca(2+) is precipitation of CaP(i) in the SR.     

Calpain-3 is autolyzed and hence activated in human skeletal muscle 24 h following a single bout of eccentric exercise.

The function and normal regulation of calpain-3, a muscle-specific Ca(2+)-dependent protease, is uncertain, although its absence leads to limb-girdle muscular dystrophy type 2A. This study examined the effect of eccentric exercise on calpain-3 autolytic activation, because such exercise is known to damage sarcomeric structures and to trigger adaptive changes that help prevent such damage on subsequent exercise. Six healthy human subjects performed a 30-min bout of one-legged, eccentric, knee extensor exercise. Torque measurements, vastus lateralis muscle biopsies, and venous blood samples were taken before and up to 7 days following the exercise. Peak isometric muscle torque was depressed immediately and at 3 h postexercise and recovered by 24 h, and serum creatine kinase concentration peaked at 24 h postexercise. The amount of autolyzed calpain-3 was unchanged immediately and 3 h after exercise, but increased markedly (from approximately 16% to approximately 35% of total) 24 h after the exercise, and returned to preexercise levels within 7 days. In contrast, the eccentric exercise produced little autolytic activation of the ubiquitous Ca(2+)-activated protease, mu-calpain. Eccentric exercise is the first physiological circumstance shown to result in calpain-3 activation in vivo.     

Changes in skeletal muscle SR Ca2+ pump in congestive heart failure due to myocardial infarction are prevented by angiotensin II blockade

In order to understand the mechanisms of exercise intolerance and muscle fatigue, which are commonly observed in congestive heart failure, we studied sarcoplasmic reticulum (SR) Ca(2+)-transport in the hind-leg skeletal muscle of rats subjected to myocardial infarction (MI). Sham-operated animals were used for comparison. On one hand, the maximal velocities (Vmax) for both SR Ca(2+)-uptake and Ca(2+)-stimulated ATPase activities in skeletal muscle of rats at 8 weeks of MI were higher than those of controls. On the other hand, the Vmax values for both SR Ca(2+)-uptake and Ca(2+)-stimulated ATPase activities were decreased significantly at 16 weeks of MI when compared with controls. These alterations in Ca(2+)-transport activities were not associated with any change in the affinity (1/Ka) of the SR Ca(2+)-pump for Ca2+. Furthermore, the stimulation of SR Ca(2+)-stimulated ATPase activity by cyclic AMP-dependent protein kinase was not altered at 8 or 16 weeks of MI when compared with the respective control values. Treatment of 3-week infarcted animals with angiotensin-converting enzyme (ACE) inhibitors such as captopril, imidapril, and enalapril or an angiotensin receptor (AT1R) antagonist, losartan, for a period of 13 weeks not only attenuated changes in left ventricular function but also prevented defects in SR Ca(2+)-pump in skeletal muscle. These results indicate that the skeletal muscle SR Ca(2+)-transport is altered in a biphasic manner in heart failure due to MI. It is suggested that the initial increase in SR Ca(2+)-pump activity in skeletal muscle may be compensatory whereas the depression at late stages of MI may play a role in exercise intolerance and muscle fatigue in congestive heart failure. Furthermore, the improvements in the skeletal muscle SR Ca(2+)-transport by ACE inhibitors may be due to the decreased activity of renin-angiotensin system in congestive heart failure.     

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.     

Effects of fatigue and training on sarcoplasmic reticulum Ca(2+) regulation in human skeletal muscle.

Little is known about fatigue and training effects on sarcoplasmic reticulum (SR) function in human muscle, and we therefore investigated this in eight untrained controls (UT), eight endurance-trained (ET), and eight resistance-trained athletes (RT). Muscle biopsies (vastus lateralis) taken at rest and after 50 maximal quadriceps contractions (180 degrees/s, 0.5 Hz) were analyzed for fiber composition, metabolites and maximal SR Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase activity. Fatigue reduced (P < 0.05) Ca(2+) release (42.1 +/- 3.8%, 43.4 +/- 3.9%, 31.3 +/- 6.1%), Ca(2+) uptake (43.0 +/- 5.2%, 34.1 +/- 4.6%, 28.4 +/- 2.8%), and Ca(2+)-ATPase activity (38.6 +/- 4.2%, 48.5 +/- 5.7%, 29.6 +/- 5.0%), in UT, RT, and ET, respectively. These decreases were correlated with fatigability and with type II fiber proportion (P < 0.05). Resting SR measures were correlated with type II proportion (r > or = 0.51, P < 0.05). ET had lower resting Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase (P < 0.05) than UT and RT (P < 0.05), probably because of their lower type II proportion; only minor effects were found in RT. Thus SR function is markedly depressed with fatigue in controls and in athletes, is dependent on fiber type, and appears to be minimally affected by chronic training status.     

Physical coupling supports the local Ca2+ transfer between sarcoplasmic reticulum subdomains and the mitochondria in heart muscle

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.     

Relaxation of diaphragm muscle.

Relaxation is the process by which, after contraction, the muscle actively returns to its initial conditions of length and load. In rhythmically active muscles such as diaphragm, relaxation is of physiological importance because diaphragm must return to a relatively constant resting position at the end of each contraction-relaxation cycle. Rapid and complete relaxation of the diaphragm is likely to play an important role in adaptation to changes in respiratory load and breathing frequency. Regulation of diaphragm relaxation at the molecular and cellular levels involves Ca(2+) removal from the myofilaments, active Ca(2+) pumping by the sarcoplasmic reticulum (SR), and decrease in the number of working cross bridges. The relative contribution of these mechanisms mainly depends on sarcomere length, muscle tension, and the intrinsic contractile function. Increased capacity of SR to take up Ca(2+) can arise from increased density of active SR pumping sites or in slow-twitch fibers from phosphorylation of phospholamban, whereas impaired coupling between ATP hydrolysis and Ca(2+) transport into the SR or intracellular acidosis reduces SR Ca(2+) pump activity. In experimental conditions of decreased contractile performance, slowed, enhanced, or unchanged relaxation rates have been reported in vitro. In vivo, a slowing in the rate of decline of the respiratory pressure is generally considered an early reliable index of respiratory muscle fatigue. Impaired relaxation rate may, in turn, favor mismatch between blood flow and metabolic demand, especially at high breathing frequencies.     

Phospholamban knockout increases CaM kinase II activity and intracellular Ca2+ wave activity and alters contractile responses of murine gastric antrum

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA), and this inhibition is relieved by Ca(2+) calmodulin-dependent protein kinase II (CaM kinase II) phosphorylation. We previously reported significant differences in contractility, SR Ca(2+) release, and CaM kinase II activity in gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase II. In this study, we used PLB-knockout (PLB-KO) mice to directly examine the effect of PLB absence on contractility, CaM kinase II activity, and intracellular Ca(2+) waves in gastric antrum smooth muscles. The frequencies and amplitudes of spontaneous phasic contractions were elevated in antrum smooth muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic contractions in antrum smooth muscles from both control and PLB-KO mice. Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of antrum smooth muscle strips from PLB-KO mice, but the effects were less pronounced compared with control strips. The CaM kinase II inhibitor KN-93 was less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. Similarly, the intracellular Ca(2+) wave frequency was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings suggest that PLB is an important modulator of gastric antrum smooth muscle contractility by modulation of SR Ca(2+) release and CaM kinase II activity.     

Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

Local Ca(2+) transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca(2+) release to activate mitochondrial Ca(2+) uptake and to evoke a matrix [Ca(2+)] ([Ca(2+)](m)) rise. [Ca(2+)](m) exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca(2+) release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca(2+) sensitivity of both the Ca(2+) release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca(2+) accumulation in various apoptotic paradigms, methods are available for buffering of [Ca(2+)], for dissipation of the driving force of the mitochondrial Ca(2+) uptake and for inhibition of the mitochondrial Ca(2+) transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca(2+) handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca(2+) uptake on cytosolic [Ca(2+)] and [Ca(2+)](m) in intact cultured cells.     

Separation of active and inactive (nonphosphorylating) Ca(2+)-ATPase in sarcoplasmic reticulum subfractions from low-frequency-stimulated rabbit muscle.

Chronic low-frequency stimulation elicits in rabbit fast-twitch muscle a partial inactivation of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase and Ca(2+)-uptake activities. Inactive Ca(2+)-ATPase was enriched in a light microsomal fraction by sucrose density gradient centrifugation after calcium oxalate loading in the presence of ATP. This fraction showed a reduced specific activity and phosphoprotein formation of the Ca(2+)-transport ATPase. These results suggest that the inactivation of the Ca(2+)-ATPase as induced by increased contractile activity, is confined to a specific SR vesicle population.     

Impaired calcium release during fatigue.

Impaired calcium release from the sarcoplasmic reticulum (SR) has been identified as a contributor to fatigue in isolated skeletal muscle fibers. The functional importance of this phenomenon can be quantified by the use of agents, such as caffeine, which can increase SR Ca(2+) release during fatigue. A number of possible mechanisms for impaired calcium release have been proposed. These include reduction in the amplitude of the action potential, potentially caused by extracellular K(+) accumulation, which may reduce voltage sensor activation but is counteracted by a number of mechanisms in intact animals. Reduced effectiveness of SR Ca(2+) channel opening is caused by the fall in intracellular ATP and the rise in Mg(2+) concentrations that occur during fatigue. Reduced Ca(2+) available for release within the SR can occur if inorganic phosphate enters the SR and precipitates with Ca(2+). Further progress requires the development of methods that can identify impaired SR Ca(2+) release in intact, blood-perfused muscles and that can distinguish between the various mechanisms proposed.     

Sarcoplasmic reticulum Ca(2+) release and muscle fatigue.

Efforts to examine the relevant mechanisms involved in skeletal muscle fatigue are focusing on Ca(2+) handling within the active muscle cell. It has been demonstrated time and again that reductions in sarcoplasmic reticulum (SR) Ca(2+) release resulting from increased or intense muscle contraction will compromise tension development. This review seeks to accomplish two related goals: 1) to provide an up-to-date molecular understanding of the Ca(2+)-release process, with considerable attention devoted to the SR Ca(2+) channel, including its associated proteins and their regulation by endogenous compounds; and 2) to examine several putative mechanisms by which cellular alterations resulting from intense and/or prolonged contractile activity will modify SR Ca(2+) release. The mechanisms that are likely candidates to explain the reductions in SR Ca(2+) channel function following contractile activity include elevated Ca(2+) concentrations, alterations in metabolic homeostasis within the "microcompartmentalized" triadic space, and modification by reactive oxygen species.     

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

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

o-Phthalaldehyde activates the Ca(2+) release mechanism from skeletal muscle sarcoplasmic reticulum.

o-Phthalaldehyde (OPA) is a bifunctional reagent that forms an isoindole derivative by reacting with cysteine and lysine residues separated by approximately 0.3 nm. OPA inhibits sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity at low micromolar concentrations and induces Ca(2+) release from actively loaded SR vesicles by activating the ryanodine receptor from fast twitch skeletal muscle. Both ryanodine binding and single-channel activity show a biphasic concentration dependence. At low OPA concentrations (<100 microM), ryanodine binding and single channel activity are stimulated, while at higher concentrations, a time-dependent sequential activation and inhibition of receptor binding is observed. Activation is characterized by a Ca(2+)-independent increase in maximal receptor occupancy. Data are presented to support a model in which Ca(2+) channel and ryanodine binding activity are enhanced due to an intramolecular cross-linking of nearby lysine and nonhyperreactive cysteine residues. OPA complexation with endogenous lysine residue(s) is critical for receptor activation.     

Myocardial calcium cycling defect in furazolidone cardiomyopathy.

We have previously demonstrated that in furazolidone-induced congestive heart failure in turkeys the specific Ca(2+)-ATPase activity of myocardial sarcoplasmic reticulum (SR) is 60% increased in compensation for a 50% depression in net Ca(2+)-sequestration activity. This study tested the hypothesis that SR Ca(2+)-uptake and Ca(2+)-ATPase activities were uncoupled in this cardiomyopathy because of increased Ca(2+)-release channel activity. A novel microassay was used to monitor Ca2+ transport by myocardial homogenates using the fluorescent Ca2+ dye indo 1 to indicate extravesicular ionized Ca2+. The method is applied to cyropreserved biopsy specimens of myocardium and requires only 50 mg tissue. Both SR Ca(2+)-pump and SR Ca(2+)-channel activity were estimated using the channel-inhibitor ruthenium red (RR) and the mitochondrial inhibitor sodium azide. The specificity of the RR inhibition was confirmed using ryanodine. Cardiomyopathy was induced in 2-week-old turkey poults by the addition of 0.07% furazolidone to their feed for 4 weeks. Compared with controls, myocardial maximal Ca(2+)-channel activity relative to maximal Ca(2+)-pump activity was 22% greater and duration of Ca(2+)-channel activity was 100% increased. However, the heart failure birds had 43 and 53% decreases in absolute maximal Ca(2+)-pumping and Ca(2+)-channel activities, respectively. The abnormal Ca(2+)-channel activity resulted in 200% greater time before initiation of net Ca2+ sequestration and 700% greater final myocardial Ca2+ concentrations. For all birds, the Ca(2+)-accumulating activity was highly correlated with Ca(2+)-release activity (all p less than 0.05). These data indicate that in this animal model of congestive heart failure there is defective SR Ca(2+)-channel function resulting in abnormal Ca2+ homeostasis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Eugenol-induced contractions of saponin-skinned fibers are inhibited by heparin or by a ryanodine receptor blocker

The effects of eugenol on the sarcoplasmic reticulum (SR) and contractile apparatus of chemically skinned skeletal muscle fibers of the frog Rana catesbeiana were investigated. In saponin-skinned fibers, eugenol (5 mmol/L) induced muscle contractions, probably by releasing Ca(2+) from the SR. The Ca(2+)-induced Ca(2+) release blocker ruthenium red (10 micromol/L) inhibited both caffeine- and eugenol-induced muscle contractions. Ryanodine (200 micromol/L), a specific ryanodine receptor/Ca(2+) release channel blocker, promoted complete inhibition of the contractions induced by caffeine, but only partially blocked the contractions induced by eugenol. Heparin (2.5 mg/mL), an inositol 1,4,5-trisphosphate (InsP3) receptor blocker, strongly inhibited the contractions induced by eugenol but had only a small effect on the caffeine-induced contractions. Eugenol neither altered the Ca(2+) sensitivity nor the maximal force in Triton X-100 skinned muscle fibers. These data suggest that muscle contraction induced by eugenol involves at least 2 mechanisms of Ca(2+) release from the SR: one related to the activation of the ryanodine receptors and another through a heparin-sensitive pathway.