EMD 57033 partially reverses ventilator-induced diaphragm muscle fibre calcium desensitisation

In critically ill patients, ventilator-induced diaphragm muscle fibre dysfunction (VIDD) contributes to weaning problems, increasing hospitalisation time and related costs. VIDD pathophysiology remains partially unknown, especially the characterisation of the contractile dysfunction. In the present study, it was hypothesised that Ca²⁺ activation is affected during VIDD. Ca²⁺ sensitivity of contraction was therefore evaluated at the single skinned diaphragm muscle fibre level in piglets randomised into sham operation or 5-day mechanical ventilation. Ca²⁺ sensitivities of force and stiffness in fibres were significantly impaired in all mechanically ventilated piglets compared with sham-operated controls, suggesting a less efficient Ca²⁺ activation of cells, i.e. a lower relative number of strongly attached cross-bridges for each sub-maximal concentration of Ca²⁺. In an attempt to test whether this negative effect of VIDD is reversible, single muscle fibres were exposed to the EMD 57033 Ca²⁺ sensitiser. EMD 57033 (30 µM) improved the Ca²⁺ sensitivity of force and stiffness in fibres from animals that were mechanically ventilated for 5 days as well as in sham-operated piglets. Thus, EMD 57033 partly restored the Ca²⁺ activation of cells, reducing VIDD. This finding offers a strong basis for evaluating the effect of Ca²⁺ sensitisers on diaphragm function in vivo.     

高气道压力机械通气致大鼠膈肌损伤的细胞凋亡机制

目的在前期试验中我们发现,给予大鼠40 cmH_2O气道压力控制通气20min对膈肌收缩功能有损伤。本研究进一步观察高气道压力控制通气下大鼠膈肌细胞超微结构的变化,并探讨膈肌细胞凋亡在呼吸机相关性膈肌损伤中的可能机制。方法将30只SD大鼠随机分为3组。给予40 cmH_2O气道压力控制通气20min组(HAP20min),5min组(HAP5min)、及对照组。HAP20min及HAP5min组设置50次,分的呼吸频率完全抑制了膈肌的电活动。机械通气后取膈肌组织经固定、常规超薄切片制备,进行超微结构观察。结果20min40cmH_2O气道压力控制通气可导致膈肌细胞发生凋亡的形态学改变,包括:核固缩,核膜模糊并有溶解。核内异染色质呈致密的块状,异染色质边聚呈环状,胞质内线粒体肿胀,部分嵴溶解。而HAP5min仅有细胞核的轻度改变。结论20min的高气道压力控制通气会造成大鼠膈肌肌纤维细胞凋亡,这可能是膈肌收缩功能下降的原因之一。     

Malignant hyperthermia and excitation–contraction coupling

Malignant hyperthermia (MH) is a state of elevated skeletal muscle metabolism that may occur during general anaesthesia in genetically pre‐disposed individuals. Malignant hyperthermia results from altered control of sarcoplasmic reticulum (SR) Ca²⁺ release. Mutations have been identified in MH‐susceptible (MHS) individuals in two key proteins of excitation–contraction (EC) coupling, the Ca²⁺ release channel of the SR, ryanodine receptor type 1 (RyR1) and the α1‐subunit of the dihydropyridine receptor (DHPR, L‐type Ca²⁺ channel). During EC coupling, the DHPR senses the plasma membrane depolarization and transmits the information to the ryanodine receptor (RyR). As a consequence, Ca²⁺ is released from the terminal cisternae of the SR. One of the human MH‐mutations of RyR1 (Arg614Cys) is also found at the homologous location in the RyR of swine (Arg615Cys). This animal model permits the investigation of physiological consequences of the homozygously expressed mutant release channel. Of particular interest is the question of whether voltage‐controlled release of Ca²⁺ is altered by MH‐mutations in the absence of MH‐triggering substances. This question has recently been addressed in this laboratory by studying Ca²⁺ release under voltage clamp conditions in both isolated human skeletal muscle fibres and porcine myotubes.     

Physiology and pathophysiology of excitation–contraction coupling: the functional role of ryanodine receptor

Calcium (Ca²⁺) release from intracellular stores plays a key role in the regulation of skeletal muscle contraction. The type 1 ryanodine receptors (RyR1) is the major Ca²⁺ release channel on the sarcoplasmic reticulum (SR) of myocytes in skeletal muscle and is required for excitation–contraction (E–C) coupling. This article explores the role of RyR1 in skeletal muscle physiology and pathophysiology.     

Altered intracellular Ca<Superscript>2+</Superscript> regulation in chronic rat heart failure

Altered intracellular Ca²⁺ handling by the sarcoplasmic reticulum (SR) plays a crucial role in the pathogenesis of heart failure (HF). Despite extensive effort, the underlying causes of abnormal SR Ca²⁺ handling in HF have not been clarified. To determine whether the diastolic SR Ca²⁺ leak along with reduced Ca²⁺ reuptake is required for decreased contractility, we investigated the cytosolic Ca²⁺ transients and SR Ca²⁺ content and assessed the expression of ryanodine receptor (RyR2), FK506 binding protein (FKBP12.6), SR-Ca²⁺ ATPase (SERCA2a), and L-type Ca²⁺ channel (LTCC) using an SD-rat model of chronic HF. We found that the cytosolic Ca²⁺ transients were markedly reduced in amplitude in HF myocytes (ΔF/F ₀ = 12.3 ± 0.8) compared with control myocytes (ΔF/F ₀ = 17.7 ± 1.2, P < 0.01), changes paralleled by a significant reduction in the SR Ca²⁺ content (HF: ΔF/F ₀ = 12.4 ± 1.1, control: ΔF/F ₀ = 32.4 ± 1.9, P < 0.01). Moreover, we demonstrated that the expression of FKBP12.6 associated with RyR2, SERCA2a, and LTCC was significantly reduced in rat HF. These results provide evidence for phosphorylation-induced detachment of FKBP12.6 from RyRs and down-regulation of SERCA2a and LTCC in HF. We conclude that diastolic SR Ca²⁺ leak (due to dissociation of FKBP12.6 from RyR2) along with reduced SR Ca²⁺ uptake (due to down-regulation of SERCA2a) and defective E-C coupling (due to down-regulation of LTCC) could contribute to HF.     

Prolonged exercise potentiates sarcoplasmic reticulum Ca<Superscript>2+</Superscript> uptake in rat diaphragm

The effects of a single bout of prolonged treadmill exercise [mean=81 (13) min] on sarcoplasmic reticulum (SR) Ca²⁺ release, uptake and ATPase activity were determined in the costal region of rat diaphragm (D) and red gastrocnemius (RG). Glycogen depletion measurements made immediately following exercise suggested that treadmill running substantially recruited the fibers throughout both muscles. SR Ca²⁺ ATPase activity, measured in isolated SR vesicles, decreased in the RG by 33% but remained unchanged in D in response to the exercise bout. This effect in RG was matched by a 37% decline in Ca²⁺ uptake and a 28% depression in Ca²⁺ release when measured in muscle homogenates. Conversely, Ca²⁺ uptake increased between 157% and 263% in the D in the absence of any change in Ca²⁺ release. These data show that the attenuation of SR function that has been consistently observed in limb muscle over the last several decades is absent in diaphragm despite the fact that its fibers appear to experience sufficient activity to deplete their glycogen. In fact, the large increase in Ca²⁺ uptake in D shows that prolonged activity actually potentiates the ability of SR vesicles to sequester Ca²⁺ in the absence of any increase in energy cost. Thus, it appears necessary to re-evaluate the role of exercise in regulating Ca²⁺ sequestration by the SR as different muscles may respond in ways that are dictated by their function. Electronic Publication     

Trifluoperazine: a rynodine receptor agonist

Trifluoperazine (TFP), a phenothiazine, is a commonly used antipsychotic drug whose therapeutic effects are attributed to its central anti-adrenergic and anti-dopaminergic actions. However, TFP is also a calmodulin (CaM) antagonist and alters the Ca²⁺ binding properties of calsequestrin (CSQ). The CaM and CSQ proteins are known modulators of sarcoplasmic reticulum (SR) Ca²⁺ release in ventricular myocytes. We explored TFP actions on cardiac SR Ca²⁺ release in cells and single type-2 ryanodine receptor (RyR2) channel activity in bilayers. In intact and permeabilized ventricular myocytes, TFP produced an initial activation of RyR2-mediated SR Ca²⁺ release and over time depleted SR Ca²⁺ content. At the single channel level, TFP or nortryptiline (NRT; a tricyclic antidepressant also known to modify CSQ Ca²⁺ binding) increased the open probability (Po) of CSQ-free channels with an EC₅₀ of 5.2 μM or 8.9 μM (respectively). This Po increase was due to elevated open event frequency at low drug concentrations while longer mean open events sustained Po at higher drug concentrations. Activation of RyR2 by TFP occurred in the presence or absence of CaM. TFP may also inhibit SR Ca uptake as well as increase RyR2 opening. Our results suggest TFP and NRT can alter RyR2 function by interacting with the channel protein directly, independent of its actions on CSQ or CaM. This direct action may contribute to the clinical adverse cardiac side effects associated with these drugs.     

Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

Ryanodine receptor 1 (RyR1) is well-known to be expressed in systemic and pulmonary vascular smooth muscle cells (SMCs); however, its functional roles remain largely unknown. In the present study, we attempted to determine the potential importance of RyR1 in membrane depolarization-, neurotransmitter-, and hypoxia-induced Ca²⁺ release and contraction in pulmonary artery SMCs (PASMCs) using RyR1 homozygous and heterozygous gene deletion (RyR1^−/− and RyR1^+/−) mice. Our results indicate that spontaneous local Ca²⁺ release and caffeine-induced global Ca²⁺ release are significantly reduced in embryonic RyR1^−/− and adult RyR^+/− cells. An increase in [Ca²⁺]_i following membrane depolarization with high K⁺ is markedly attenuated in RyR1^−/− and RyR1^+/− PASMCs in normal Ca²⁺ or Ca²⁺-free extracellular solution. Similarly, muscle contraction evoked by membrane depolarization is reduced in RyR1^+/− pulmonary arteries in the presence or absence of extracellular Ca²⁺. Neurotransmitter receptor agonists and inositol 1,4,5-triphosphate elicit a much smaller increase in [Ca²⁺]_i in both RyR1^−/− and RyR1^+/− cells. We have also found that neurotransmitter-evoked muscle contraction is significantly inhibited in RyR1^+/− pulmonary arteries. Hypoxia-induced increase in [Ca²⁺]_i and contraction are largely blocked in RyR1^−/− and/or RyR1^+/− PASMCs. Collectively, our findings provide genetic evidence for the functional importance of RyR1 in spontaneous local Ca²⁺ release, and membrane depolarization-, neurotransmitter-, as well as hypoxia-induced global Ca²⁺ release and attendant contraction in PASMCs.     

Ryanodine modification of RyR1 retrogradely affects L-type Ca2+ channel gating in skeletal muscle

In skeletal muscle, there is bidirectional signalling between the L-type Ca²⁺ channel (1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine-sensitive Ca²⁺ release channel (RyR1) of the sarcoplasmic reticulum (SR). In the case of “orthograde signalling” (i.e., excitation-contraction coupling), the conformation of RyR1 is controlled by depolarization-induced conformational changes of the DHPR resulting in Ca²⁺ release from the SR. “Retrograde coupling” is manifested as enhanced L-type current. The nature of this retrograde signal, and its dependence on RyR1 conformation, are poorly understood. Here, we have examined L-type currents in normal myotubes after an exposure to ryanodine (200 μM, 1 h at 37°C) sufficient to lock RyR1 in a non-conducting, inactivated, conformational state. This treatment caused an increase in L-type current at less depolarized test potentials in comparison to myotubes similarly exposed to vehicle as a result of a ~5 mV hyperpolarizing shift in the voltage-dependence of activation. Charge movements of ryanodine-treated myotubes were also shifted to more hyperpolarizing potentials (~13 mV) relative to vehicle-treated myotubes. Enhancement of the L-type current by ryanodine was absent in dyspedic (RyR1 null) myotubes, indicating that ryanodine does not act directly on the DHPR. Our findings indicate that in retrograde signaling, the functional state of RyR1 influences conformational changes of the DHPR involved in activation of L-type current. This raises the possibility that physiological regulators of the conformational state of RyR1 (e.g., Ca²⁺, CaM, CaMK, redox potential) may also affect DHPR gating.     

Calcium-dependent signaling mechanisms and soleus fiber remodeling under gravitational unloading

The decrease in postural muscle fiber size, diminishing of their contractile properties, slow-to-fast shift in myosin heavy chain expression pattern are known to be the main consequences of gravitational unloading. The Ca²⁺ role in these processes has been studied for about 20 years. Ingalls et al. [J Appl Physiol 87(1):382–390, 1999] found the resting Ca²⁺ level increase in soleus fibers of hindlimb unloaded mice. Results obtained in our laboratory showed that systemic or local application of nifedipine (L-type Ca²⁺ channels’ blocker) prevents Ca²⁺ accumulation in fibers. Thus, activation of dihydropyridine calcium channels can be supposed to promote resting Ca²⁺ loading under disuse. So, calcium-dependent signaling pathways may play an important role in the development of some key events observed under unloading. Since 90th the increased activities of Ca²⁺-dependent proteases (calpains) were considered as the crucial effect of hypogravity-induced muscle atrophy, which was proved later. We observed maintenance of titin and nebulin relative content in soleus muscle under unloading combined with Ca²⁺ chelators administration. Nifedipine administration was shown to considerably restrict the slow-to-fast transition of myosin heavy chains (MHC) under unloading (at the RNA level and at the protein level as well). To clarify the role of calcineurin/NFAT signaling system in MHC pattern transition under unloading, we blocked this pathway by cyclosporine A application. Hereby, we demonstrated that calcineurin/NFAT pathway possesses a stabilizing function counteracting the myosin phenotype transformation under gravitational unloading.     

Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca²⁺ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca²⁺ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca²⁺ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 M) significantly increased the Ca²⁺ sensitivity of the contractile apparatus (decreasing Ca²⁺ required for the half-maximal response from 1.56±0.04 M to 1.46±0.05 M) but had no effect on the maximal Ca²⁺-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca²⁺ release or Ca²⁺-induced Ca²⁺ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca²⁺ release by ruthenium red in skinned fibers suggests that the SR Ca²⁺ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.     

A Mutation in the CASQ1 Gene Causes a Vacuolar Myopathy with Accumulation of Sarcoplasmic Reticulum Protein Aggregates

A missense mutation in the calsequestrin‐1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high‐affinity Ca²⁺‐binding sites of CASQ1 and alters the kinetics of Ca²⁺ release in muscle fibers. Expression of the mutated CASQ1 protein in COS‐7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron‐dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca²⁺ release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.     

Anisotropic mechanosensitive pathways in the diaphragm and their implications in muscular dystrophies

The diaphragm is the “respiratory pump;” the muscle that generates pressure to allow ventilation. Diaphragm muscles play a vital function and thus are subjected to continuous mechanical loading. One of its peculiarities is the ability to generate distinct mechanical and biochemical responses depending on the direction through which the mechanical forces applied to it. Contractile forces originated from its contractile components are transmitted to other structural components of its muscle fibers and the surrounding connective tissue. The anisotropic mechanical properties of the diaphragm are translated into biochemical signals that are directionally mechanosensitive by mechanisms that appear to be unique to this muscle. Here, we reviewed the current state of knowledge on the biochemical pathways regulated by mechanical signals emphasizing their anisotropic behavior in the normal diaphragm and analyzed how they are affected in muscular dystrophies.     

Toward the roles of store-operated Ca2+ entry in skeletal muscle

Store-operated Ca²⁺ entry (SOCE) has been found to be a rapidly activated robust mechanism in skeletal muscle fibres. It is conducted across the junctional membranes by stromal interacting molecule 1 (STIM1) and Orai1, which are housed in the sarcoplasmic reticulum (SR) and tubular (t-) system, respectively. These molecules that conduct SOCE appear evenly distributed throughout the SR and t-system of skeletal muscle, allowing for rapid and local control in response to depletions of Ca²⁺ from SR. The significant depletion of SR Ca²⁺ required to reach the activation threshold for SOCE could only be achieved during prolonged bouts of excitation–contraction coupling (EC coupling) in a healthy skeletal muscle fibre, meaning that this mechanism is not responsible for refilling the SR with Ca²⁺ during periods of fibre quiescence. While Ca²⁺ in SR remains below the activation threshold for SOCE, a low-amplitude persistent Ca²⁺ influx is provided to the junctional cleft. This article reviews the properties of SOCE in skeletal muscle and the proposed molecular mechanism, assesses its potential physiological roles during EC coupling, namely refilling the SR with Ca²⁺ and simple balancing of Ca²⁺ within the cell, and also proposes the possibility of SOCE as a potential regulator of t-system and SR membrane protein function.     

The action of perchlorate on malignant-hyperthermia-susceptible muscle

 To better understand the altered skeletal muscle excitation-contraction (E-C) coupling that occurs in malignant hyperthermia, we have examined the potentiating actions of perchlorate in intact muscle fiber bundles, isolated sarcoplasmic reticulum (SR) vesicles, and the purified ryanodine receptor/Ca²⁺ release channel (RyR) isolated from malignant-hyperthermia-susceptible (MHS) and normal porcine muscle. The concentration of perchlorate that half-maximally potentiated twitch tension (2.5–3.5 mM) was not significantly different for MHS and normal muscles. The effect of perchlorate on fractional twitch force was significantly greater for normal than for MHS muscle, although the absolute twitch potentiation was similar for both muscle types. The K-contracture threshold of MHS muscle bundles is significantly lower than that of normal bundles; perchlorate shifted the K-contraction activation curves of both MHS and normal muscle bundles to lower K⁺ concentrations. Perchlorate both increased ryanodine binding to MHS and normal SR vesicles and increased single-channel open probability of the purified MHS and normal RyR. In both cases, the percentage increase was greater for normal than for MHS preparations; however, the absolute increase in activity was not different for MHS and normal RyR indicating that there is no difference in the perchlorate sensitivity of MHS and normal SR Ca²⁺ release channels. Thus, the greater absolute responses of the MHS Ca²⁺ release channel in the presence of perchlorate is likely to be due to the greater basal activity of the MHS release channel and does not reflect an underlying defect in the site of action of perchlorate on the MHS skeletal muscle Ca²⁺ release channel. Received: 17 April 1997 / Accepted: 9 July 1997     

Influence of LVAD function on mechanical unloading and electromechanical delay: a simulation study

This study hypothesized that a left ventricular assist device (LVAD) shortens the electromechanical delay (EMD) by mechanical unloading. The goal of this study is to examine, by computational modeling, the influence of LVAD on EMD for four heart failure (HF) cases ranging from mild HF to severe HF. We constructed an integrated model of an LVAD-implanted cardiovascular system, then we altered the Ca²⁺ transient magnitude, with scaling factors 1, 0.9, 0.8, and 0.7 representing HF1, HF2, HF3, and HF4, respectively, in order of increasing HF severity. The four HF conditions are classified into two groups. Group one is the four HF conditions without LVAD, and group two is the conditions treated with continuous LVAD pump. The single-cell mechanical responses showed that EMD was prolonged with the higher load. The findings indicated that in group one, the HF-induced Ca2 + transient remodeling prolonged the mechanical activation time (MAT) and decreased the contractile tension, which reduced the left ventricle (LV) pressure, and increased the end-diastolic strain. In group two, LVAD shortened MAT of the ventricles. Furthermore, LVAD reduced the contractile tension, and end-diastolic strain, but increased the aortic pressure. The computational study demonstrated that LVAD shortens EMD by mechanical unloading of the ventricle.     

Impact of IL-10 on diaphragmatic cytokine expression and contractility during Pseudomonas Infection

Severe weakness of the respiratory muscles, with attendant respiratory failure and death, has been documented in sepsis. In this study, we show that during murine pulmonary infection with Pseudomonas aeruginosa, multiple proinflammatory genes are up-regulated not only within the lungs, but also within the diaphragm. Significant induction of TNF-alpha, IL-1alpha, IL-1beta, IL-6, and IL-18 gene expression occurred within the diaphragm in a bacterial dose-dependent manner. We determined whether the anti-inflammatory cytokine IL-10 could blunt proinflammatory gene expression within the diaphragm under these conditions. The IL-10 receptor was found to be expressed by the diaphragm in vivo as well as in primary diaphragmatic muscle cell cultures. Transduction of myoblasts with an adenoviral vector (Ad-IL-10) induced strong IL-10 expression, and intramuscular injection of the same vector in vivo produced significant increases in IL-10 serum levels. Ad-IL-10 treatment of mice infected with P. aeruginosa significantly inhibited the induction of proinflammatory cytokines within the diaphragm, but not in the infected lungs. Ad-IL-10 treatment also led to greatly improved diaphragmatic force production in infected mice. These results suggest that pulmonary infection triggers proinflammatory gene expression by the diaphragm along with diaphragmatic weakness. Shifting the balance between pro- and anti-inflammatory mediators in favor of the latter by IL-10 gene delivery was able to restore normal diaphragmatic force-generating capacity under these conditions, suggesting a possible avenue for therapeutic intervention.     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca²⁺-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca²⁺-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca²⁺. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca²⁺-activated K⁺ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca²⁺ influx was triggered through the Na⁺/Ca²⁺ exchanger and the other Ca²⁺ entry pathways were blocked. Thapsigargin, another specific SR Ca²⁺-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca²⁺-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca²⁺ influx presumably via nifedipine-sensitive Ca²⁺ channels. This study suggests the important role of SR Ca²⁺-ATPase in the modulation of the muscle tone and the function of SR as a “buffer barrier” to Ca²⁺ entry in the cat gastric fundus smooth muscle. Received: 10 August 1995/Received after revision: 9 November 1995/Accepted: 10 November 1995     

Calcium uptake and release modulated by counter-ion conductances in the sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum (SR) plays the central role in regulating the free myoplasmic Ca²⁺ level for the contractile activation of skeletal muscle. The initial stages of the voltage-controlled Ca²⁺ release mechanism are known in molecular detail. However, there is still very little known about the later stages of Ca²⁺ uptake and total Ca²⁺ turnover in the contraction–relaxation cycle under normal physiological conditions or under conditions influenced by fatigue or disease. Ca²⁺ uptake and release are both accompanied by ‘counter-ion’ movements across the SR membrane which prevent or reduce the generation of SR membrane potentials and balance for electroneutrality in the SR lumen. The SR membrane is permeable for the cations K⁺, Na⁺, H⁺ and Mg²⁺ and the anion Cl^-. Using electron-probe X-ray microanalysis, it has been shown that during tetanic stimulation the Ca²⁺ release was mainly balanced by uptake of K⁺ and Mg²⁺, leaving a charge deficit that was assumed to be neutralized via H⁺ ion or organic counter-ion movement. The low time resolution of electron-probe X-ray microanalysis leaves the possibility of other transient concentration changes in the SR, e.g. for Cl^- ions. Possible physiological roles of the SR counter-ion conductances can be tested using skinned muscle fibre preparations with intact sarcoplasmic reticulum and removed or chemically permeabilized outer sarcolemma. In skinned fibres, the SR K⁺ conductance can be effectively reduced with SR K⁺ channel blockers such as 4-aminopyridine, tetraethylammonium and decamethonium. Interestingly, these blockers increase Ca²⁺ loading as well as Ca²⁺ release, whereas other less specific blockers, such as 1.10-bis-quanidino-n-decane, seem to reduce Ca²⁺ release, possibly also via blocking Ca²⁺ release channels. Thus, it seems very important also to test the effects of counter-currents carried by K⁺, Mg²⁺, H⁺ or Cl^- ions on intact and voltage-clamped single-fibre preparations.     

Stromal interaction molecule 1 (STIM1) regulates sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a (SERCA1a) in skeletal muscle

Stromal interaction molecule 1 (STIM1) mediates Ca²⁺ movements from the extracellular space to the cytosol through a store-operated Ca²⁺ entry (SOCE) mechanism in various cells including skeletal muscle cells. In the present study, to reveal the unidentified functional role of the STIM1 C terminus from 449 to 671 amino acids in skeletal muscle, binding assays and quadrupole time-of-flight mass spectrometry were used to identify proteins binding in this region along with proteins that mediate skeletal muscle contraction and relaxation. STIM1 binds to sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 1a (SERCA1a) via this region (called STIM1-SBR). The binding was confirmed in endogenous full-length STIM1 in rabbit skeletal muscle and mouse primary skeletal myotubes via co-immunoprecipitation assay and immunocytochemistry. STIM1 knockdown in mouse primary skeletal myotubes decreased Ca²⁺ uptake from the cytosol to the sarcoplasmic reticulum (SR) through SERCA1a only at micromolar cytosolic Ca²⁺ concentrations, suggesting that STIM1 could be required for the full activity of SERCA1a possibly during the relaxation of skeletal muscle. Various Ca²⁺ imaging experiments using myotubes expressing STIM1-SBR suggest that STIM1 is involved in intracellular Ca²⁺ distributions between the SR and the cytosol via regulating SERCA1a activity without affecting SOCE. Therefore, in skeletal muscle, STIM1 could play an important role in regulating Ca²⁺ movements between the SR and the cytosol.