Sarcoplasmic Reticulum Channels Research Articles

Voltage-induced calcium release in Caenorhabditis elegans body muscles

Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In voltage-induced calcium release (VICR), CaV1 voltage sensing domains are directly coupled to ryanodine receptors (RYRs), an SR calcium channel. In calcium-induced calcium release (CICR), calcium ions flowing through activated CaV1 channels bind and activate RYR channels. VICR is thought to occur exclusively in vertebrate skeletal muscle while CICR occurs in all other muscles (including all invertebrate muscles). Here, we use calcium-activated SLO-2 potassium channels to analyze CaV1-SR coupling in Caenorhabditis elegans body muscles. SLO-2 channels were activated by both VICR and external calcium. VICR-mediated SLO-2 activation requires two SR calcium channels (RYRs and IP3 Receptors), JPH-1/Junctophilin, a PDZ (PSD95, Dlg1, ZO-1 domain) binding domain (PBD) at EGL-19/CaV1's carboxy-terminus, and SHN-1/Shank (a scaffolding protein that binds EGL-19's PBD). Thus, VICR occurs in invertebrate muscles.

Abstract 14182: A Missense Mutation in Ryanodine Receptor 2 Leads to Structural Remodeling of the Heart and Cardiac Arrhythmia in a Rabbit Model

Ryanodine receptor 2 (RyR2) is the Ca 2+ release channel of sarcoplasmic reticulum that provides the majority of Ca 2+ necessary for contractions of the heart. RyR2 mutations are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmogenic disorder with normal heart structure. There are reports suggesting that RyR2 mutations can also lead to structural heart disease, notably arrhythmogenic right ventricular cardiomyopathy (ARVC); however, additional research is required to support this association. We generated a rabbit model carrying the missense mutation RyR2-V2475F, linked to CPVT in humans. Heterozygous (V2475F +/- ) rabbits show a CPVT-like syndrome and display arrhythmia under pharmacological stress. Homozygotes (V2475F +/+ ) show a stronger phenotype, with 37% of the animals (13/35) experiencing sudden death at 22.5±3.8 weeks old during regular housing. Remarkably, gross examination of V2475F +/+ hearts showed significant structural remodeling, characterized by fibrous infiltrations and thinning of the right ventricle with or without left ventricle involvement. This phenotype is reminiscent of ARVC. Lead II ECG obtained in conscious rabbits by telemetry showed that V2475F +/+ animals have significant bradycardia (144.6±11.1 bpm) with prolonged QT interval (QTc: 180.4±8.2 ms, n=6) compared to WT (HR: 224.9±4.2 bpm, p=0.04; QTc: 157.4±5.3 ms, p=0.02, n=5). After subcutaneous injection of 0.5 mg/kg of isoproterenol, WT rabbits showed the expected positive chronotropic response (HR, 367±10 bpm, n=3) while V2475F +/+ animals developed bouts of ventricular tachycardia. Mild emotional stress applied by transporting the animal around the laboratory, also induced ventricular arrhythmia in 100% (5/5) V2475F +/+ rabbits, while WT littermates were unaffected. RyR2-V2475F expressed in a rabbit model triggers remarkably different phenotypes based on the zygosity of the animals, ranging from a purely arrhythmogenic syndrome (CPVT) in V2475F +/- animals to severe structural disease compatible with ARVC in V2475F +/+ rabbits. The RyR2-V2475F rabbit model is therefore an excellent model to dissect the intersection of arrhythmic and structural phenotypes due to RyR2 dysfunction.

Periodic Stretching of Cultured Myotubes Enhances Myofibril Assembly.

The effects of mechanical stress on cultured muscle cells were examined with particular interest in myofibril assembly by using a cell-stretching system. We observed that formation and maintenance of cross-striated myofibrils in chick muscle cell cultures was suppressed in the media containing higher concentration of KCl, tetrodotoxin, or ML-9 (an inhibitor of myosin light chain kinase), but periodic stretching of myotubes for several days enabled formation of striated myofibrils just as in standard muscle cultures. However, ryanodine (a blocker of the Ca2 + channel in sarcoplasmic reticulum) and BDM (an inhibitor of myosin-actin interaction) suppressed the stretch-induced myofibrillogenesis. We further found that stretching of myotubes causes quick and transient elevation of the intracellular Ca2 + concentration and this elevation is disturbed by inhibition of Ca2 + channels of sarcoplasmic reticulum and suppression of Ca2 + influx from culture medium. These observations indicate that periodic stretching induces elevation of intracellular Ca2 + concentration and that this elevation may be due to release of Ca2 + from sarcoplasmic reticulum and Ca2 + influx from outside of the cells. The increased Ca2 + may activate actin-myosin interaction by interacting with troponin that is located along actin filaments and/or inducing phosphorylation of myosin light chains and thereby promote myofibril assembly.

Abstract 12877: Magnesium Deficiency Causes Reversible Metabolic Diastolic and Systolic Cardiomyopathies

Introduction: Low circulating magnesium (Mg) level is associated with increased cardiovascular mortality, and conversely, dietary Mg intake is associated with a decreased risk of developing heart failure. Hypothesis: We investigated whether Mg deficiency alone could cause cardiomyopathy. Methods: C57BL/6J mice were fed with a low-Mg diet (low-Mg, 15-30 mg/kg Mg) or a normal diet (nl-Mg, 600 mg/kg Mg) for 6 weeks. To test reversibility, half of the low-Mg mice were fed then with normal diet for another 6 weeks (low→nl-Mg group). Results: Mg deficiency increased mortality, especially in female mice. After 6 weeks of low-Mg diet, surviving mice showed significantly decreased serum Mg (0.9±0.1 vs. 2.8±0.1 mg/dL for nl-Mg) and a reciprocal increase in serum Ca, K, and Na. Low-Mg mice exhibited a decreased cardiac ejection fraction (EF%, 39.8±1.9% vs. 52.0±1.7% of nl-Mg) and impaired relaxation (E/e’: 21.1±1.1 vs. 15.4±0.4 of nl-Mg) in echocardiography. At the cellular level, ATP, Ca transient amplitude, sarcoplasmic reticulum (SR) Ca load, resting sarcomere length, and sarcomere shortening were all decreased significantly in low-Mg hearts and cardiomyocytes. These changes were accompanied by evidence of mitochondrial dysfunction with significantly increased mitochondrial ROS production and mitochondrial membrane depolarization. Mg repletion normalized electrolytes, contraction, relaxation, and cellular changes. The SR Ca pump (SERCA) was decreased, the SR Ca channel RyR2 oxidized, and cardiac myosin binding protein C S-glutathionylated in low-Mg mouse hearts. These changes were normalized with Mg repletion. In vivo , mitoTEMPO treatment during low Mg diet improved the cardiac relaxation and increased cellular ATP levels without improving contraction. Conclusions: Mg deficiency caused a reversible diastolic and systolic cardiomyopathy associated with mitochondrial dysfunction. This cardiomyopathy may explain the relationship of hypomagnesemia and worsening heart failure. Mg intake could reverse these changes, reinforcing the known correlation of increased Mg intake and reduced heart failure symptoms and mortality. In deficiency states, Mg supplementation may represent a novel treatment for systolic and diastolic heart failure.

Enhanced activity of multiple TRIC-B channels: an endoplasmic reticulum/sarcoplasmic reticulum mechanism to boost counterion currents.

Key points There are two subtypes of trimeric intracellular cation (TRIC) channels but their distinct single‐channel properties and physiological regulation have not been characterized. We examined the differences in function between native skeletal muscle sarcoplasmic reticulum (SR) K+‐channels from wild‐type (WT) mice (where TRIC‐A is the principal subtype) and from Tric‐a knockout (KO) mice that only express TRIC‐B.We find that lone SR K+‐channels from Tric‐a KO mice have a lower open probability and gate more frequently in subconducting states than channels from WT mice but, unlike channels from WT mice, multiple channels gate with high open probability with a more than six‐fold increase in activity when four channels are present in the bilayer.No evidence was found for a direct gating interaction between ryanodine receptor and SR K+‐channels in Tric‐a KO SR, suggesting that TRIC‐B–TRIC‐B interactions are highly specific and may be important for meeting counterion requirements during excitation–contraction coupling in tissues where TRIC‐A is sparse or absent. The trimeric intracellular cation channels, TRIC‐A and TRIC‐B, represent two subtypes of sarcoplasmic reticulum (SR) K+‐channel but their individual functional roles are unknown. We therefore compared the biophysical properties of SR K+‐channels derived from the skeletal muscle of wild‐type (WT) or Tric‐a knockout (KO) mice. Because TRIC‐A is the major TRIC‐subtype in skeletal muscle, WT SR will predominantly contain TRIC‐A channels, whereas Tric‐a KO SR will only contain TRIC‐B channels. When lone SR K+‐channels were incorporated into bilayers, the open probability (Po) of channels from Tric‐a KO mice was markedly lower than that of channels from WT mice; gating was characterized by shorter opening bursts and more frequent brief subconductance openings. However, unlike channels from WT mice, the Po of SR K+‐channels from Tric‐a KO mice increased as increasing channel numbers were present in the bilayer, driving the channels into long sojourns in the fully open state. When co‐incorporated into bilayers, ryanodine receptor channels did not directly affect the gating of SR K+‐channels, nor did the presence or absence of SR K+‐channels influence ryanodine receptor activity. We suggest that because of high expression levels in striated muscle, TRIC‐A produces most of the counterion flux required during excitation‐contraction coupling. TRIC‐B, in contrast, is sparsely expressed in most cells and, although lone TRIC‐B channels exhibit low Po, the high Po levels reached by multiple TRIC‐B channels may provide a compensatory mechanism to rapidly restore K+ gradients and charge differences across the SR of tissues containing few TRIC‐A channels.

Abnormal calcium signalling and the caffeine–halothane contracture test

BackgroundThe variable clinical presentation of malignant hyperthermia (MH), a disorder of calcium signalling, hinders its diagnosis and management. Diagnosis relies on the caffeine–halothane contracture test, measuring contraction forces upon exposure of muscle to caffeine or halothane (FC and FH, respectively). Patients with above-threshold FC or FH are diagnosed as MH susceptible. Many patients test positive to halothane only (termed ‘HH’). Our objective was to determine the characteristics of these HH patients, including their clinical symptoms and features of cytosolic Ca2+ signalling related to excitation–contraction coupling in myotubes. MethodsAfter institutional ethics committee approval, recruited patients undergoing contracture testing at Toronto's MH centre were assigned to three groups: HH, doubly positive (HS), and negative patients (HN). A clinical index was assembled from musculoskeletal symptoms and signs. An analogous calcium index summarised four measures in cultured myotubes: resting [Ca2+]cytosol, frequency of spontaneous cytosolic Ca2+ events, Ca2+ waves, and cell-wide Ca2+ spikes after electrical stimulation. ResultsThe highest values of both indexes were found in the HH group; the differences in calcium index between HH and the other groups were statistically significant. The principal component analysis confirmed the unique cell-level features of the HH group, and identified elevated resting [Ca2+]cytosol and spontaneous event frequency as the defining HH characteristics. ConclusionsThese findings suggest that HH pathogenesis stems from excess Ca2+ leak through sarcoplasmic reticulum channels. This identifies HH as a separate diagnostic group and opens their condition to treatment based on understanding of pathophysiological mechanisms.

The Efficacy of Microcurrent Therapy on Eccentric Contraction-Induced Muscle Damage in Rat Fast-Twitch Skeletal Muscle

Microcurrent (MC) therapy, in which a very small electric current is applied to the body, has widely been used to promote tissue healing and relieve symptoms. The aim of this study was to examine the effect of MC treatment on eccentric contraction (ECC)-induced muscle damage in rat fast-twitch skeletal muscles. Tibialis anterior muscles underwent 200 repeated ECCs in situ and were then stimulated (25 μA, 0.3 Hz) for 20 min (MC treatment). MC treatment was performed immediately after ECC and during a recovery period of 3 days (a total of 4 times). Three days after ECC, the muscles were excised and used for measure of force output and for biochemical analyses. In MC-treated muscles, tetanic forces at 20 Hz and 100 Hz were partially and fully restored, respectively, whereas in non-treated muscles, both forces remained depressed. Biochemical analyses revealed that MC treatment partially or completely inhibited ECC-induced reductions: in 1) the Ca2+-release function of sarcoplasmic reticulum (SR), 2) proteolysis of ryanodine receptor, a Ca2+ release channel of SR, and 3) myosin ATPase activity. On the other hand, MC treatment was unable to lessen increases in the activity of calpain, a cytosolic, Ca2+-activated neutral protease. These results indicate that MC treatment results in beneficial effects, such as restoration of muscle performance following ECC, although the precise mechanisms are still unknown at this time.

Calcium release-dependent inactivation precedes formation of the tubular system in developing rat cardiac myocytes.

Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.

Functional Role of TRPC1 Channels in Neonatal Cardiomyocytes

Transient receptor potential cation (TRPC) 1 channels are thought to play a role in store-operated and receptor-operated calcium entry in various mammalian cell types. However, our understanding of the functional role of these channels in cardiomyocytes remains to be ill-defined. Here, we test the hypothesis that the channels are involved in calcium leak from the sarcoplasmic reticulum (SR) and modulate the cytosolic calcium concentration in cardiomyocytes. Our studies were performed on neonatal rodent ventricular myocytes. After cell isolations, cells were plated on glass slides. Two days after isolation cells were infected with adenoviral vectors of TRPC1 tagged with eGFP. Measurements were performed 4-5 days after infection. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular density of native TRPC1 and TRPC1 expressed via adenoviral vectors. TRPC1 was not associated with the sarcolemma, but the SR. We measured the rest decay and caffeine induced peak calcium release using rapid scanning confocal microscopy on infected cells loaded with the calcium sensitive dye Rhod-3. We found an increased SR calcium content in the presence of the TRPC channel blocker SKF-96365. SR calcium content exhibited a decreasing relationship with TRPC1 expression. In a computational model, activated SR TRPC1 channels increased the systolic and diastolic cytosolic calcium concentration with only minor effects on action potential and SR calcium content. Our studies indicate that TRPC1 channels are not involved in sarcolemmal electrophysiology of rodent ventricular myocytes, but localized in the SR. The studies support our hypothesis that the channels play a role as SR calcium leak channels. The findings could guide us to an understanding of TRPC channels as physiological modulators of intracellular calcium and contractility in cardiac myocytes.

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle

Calsequestrin, the only known protein with cyclical storage and supply of calcium as main role, is proposed to have other functions, which remain unproven. Voluntary movement and the heart beat require this calcium flow to be massive and fast. How does calsequestrin do it? To bind large amounts of calcium in vitro, calsequestrin must polymerize and then depolymerize to release it. Does this rule apply inside the sarcoplasmic reticulum (SR) of a working cell? We answered using fluorescently tagged calsequestrin expressed in muscles of mice. By FRAP and imaging we monitored mobility of calsequestrin as [Ca2+] in the SR--measured with a calsequestrin-fused biosensor--was lowered. We found that calsequestrin is polymerized within the SR at rest and that it depolymerized as [Ca2+] went down: fully when calcium depletion was maximal (a condition achieved with an SR calcium channel opening drug) and partially when depletion was limited (a condition imposed by fatiguing stimulation, long-lasting depolarization, or low drug concentrations). With fluorescence and electron microscopic imaging we demonstrated massive movements of calsequestrin accompanied by drastic morphological SR changes in fully depleted cells. When cells were partially depleted no remodeling was found. The present results support the proposed role of calsequestrin in termination of calcium release by conformationally inducing closure of SR channels. A channel closing switch operated by calsequestrin depolymerization will limit depletion, thereby preventing full disassembly of the polymeric calsequestrin network and catastrophic structural changes in the SR.

Circulation Research “In This Issue” Anthology

Circulation Research “In This Issue” Anthology

Arrhythmogenic mechanisms in ryanodine receptor channelopathies.

Ryanodine receptors (RyRs) are the calcium release channels of sarcoplasmic reticulum (SR) that provide the majority of cal-cium ions (Ca2+) necessary to induce contraction of cardiac and skeletal muscle cells. In their intracellular environment, RyR channels are regulated by a variety of cytosolic and luminal factors so that their output signal (Ca2+) induces finely-graded cell contraction without igniting cellular processes that may lead to aberrant electrical activity (ventricular arrhythmias) or cellular remodeling. The importance of RyR dysfunction has been recently highlighted with the demonstration that point mutations in RYR2, the gene encoding for the cardiac isoform of the RyR (RyR2), are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia in individuals with an apparently normal heart. Here we summarize the state of the field in regards to the main arrhythmogenic mechanisms triggered by RyR2 channels harboring mutations linked to CPVT. Most CPVT mutations characterized to date endow RyR2 channels with a gain of function, resulting in hyperactive channels that release Ca2+ spontaneously, especially during diastole. The spontaneous Ca2+ release is extruded by the electrogenic Na+/Ca2+ exchanger, which depolarizes the external membrane (delayed afterdepolarization or DAD) and may trigger untimely action potentials. However, a rare set of CPVT mutations yield RyR2 channels that are intrinsically hypo-active and hypo-responsive to stimuli, and it is unclear whether these channels release Ca2+ spontaneously during diastole. We discuss novel cellular mechanisms that appear more suitable to explain ventricular arrhythmias due to RyR2 loss-of-function mutations.

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes.

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca2+]i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca2+-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20µM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10µM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10µM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca2+]i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.

Calcium influx and release mechanism(s) in histamine-induced myometrial contraction in buffaloes

The present study was undertaken to characterize the presence of histamine H1R using molecular biology tools and unravel the influx and release mechanism(s) involved in calcium signalling cascades in histamine-induced myometrial contraction in buffaloes. The presence of H1R mRNA transcript and immunoreactive membrane protein in buffalo myometrium was confirmed by RT-PCR and Western blot analysis. Further, histamine produced concentration-dependent (1nM–10μM) contraction in buffalo myometrium with a potency of 7.13±0.11. When myometrial strips were pre-incubated either with Ca2+ free solution or with nifedipine, a L-type Ca2+ channel blocker, dose response curve (DRC) of histamine was significantly (P<0.05) shifted towards right with decline in maximal contraction (Emax). Reduction in Emax of histamine in the presence of nifedipine (55.75±3.10%) was significantly (P<0.001) greater than that in the presence of ruthenium red (93.61±3.43%), a blocker of IP3-gated and RyR-sensitive Ca2+ channels. Moreover, histamine produced only 26.87±1.99% of the maximum contraction in the presence of both nifedipine and CPA (blocker of sarco-endoplasmic reticulum Ca2+-ATPase). Interestingly, following concurrent exposure to U-73122 (a PL-C inhibitor) and nifedipine, the DRC of histamine was significantly (P<0.05) shifted towards left with increase in maximal contraction (126.30±3.36%). Our findings in buffalo uterus thus suggest that influx of extracellular calcium plays a major role in histamine-induced myometrial contraction, while release of intracellular calcium through calcium-release channels of sarcoplasmic reticulum has a minor role. A possible involvement of non-selective cation channels in histamine-induced myometrial contraction cannot be ruled out, and therefore requires further investigations.

A Chloride Channel Blocker Prevents Inorganic Phosphate Accumulation and Its Effects in the Sarcoplasmic Reticulum of Frog Permeabilized Skeletal Muscle Fibers

In skeletal muscle intense activity is accompanied by an increase in intracellular inorganic phosphate (Pi) due to breakdown of ATP and phoshocreatine. It is widely accepted that Pi enters the sarcoplasmic reticulum (SR) where it precipitates as Ca2+ salt, thus contributing to the impairment of Ca2+ release in muscle fatigue (Fryer et al, J Physiol 1995; Launikonis et al., Biophys J 2005). We studied the effects of increasing Pi concentrations on elementary Ca2+ release events (sparks) in permeabilized fibers. Spark frequency F/F0 increased with [Pi] up to 7-10 mM/l and decreased monotonically reaching 0.1 at [Pi] = 55 mM. These changes were highly correlated with the level of [Ca2+]SR, monitored with Fluo5N or Mag-Fluo4. SR anion channels, probably chloride channels (Laver et al.,J Physiol2001), provide the principal entry path of Pi to the SR. The chloride channel blocker 9-anthracenecarboxylic acid (9AC, at 400 uM) increased spark frequency by about 15% and shifted the Pi effects to slightly higher concentrations. Because 9AC blocks the channel from the SR-luminal side another approach was taken to enhance its action. Fibers were incubated in the presence of 5 mM Mg2+ and 2 mM [9AC] for 30 minutes. There after 9AC was reduced to 400 uM and Mg2+ to 0.4 mM/l. After this protocol, Pi concentrations up to 55 mM/l had no effect on frequency or spark morphology. Correspondingly, [Ca2+]SR-monitoring signals were not affected. These results indicate that chloride channels constitute the main entrance of Pi to the SR, confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR and provide approaches to study these mechanisms quantitatively.Funded by CSIC and NIH.

Malignant Hyperthermia: Clinical and Molecular Aspects

Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disorder that affects genetically predisposed individuals. It manifests in susceptible individuals in response to exposure to Inhalant anesthetics, depolarizing muscle relaxants or extreme physical activity in hot environments. During exposure to these triggering agents, there is a rapid and sustained increase of myoplasmic calcium (Ca(2+)) concentration induced by hyperactivation of ryanodine receptor of skeletal muscle (RyR1), causing a profound change in Ca(2+) homeostasis, featuring a hypermetabolic state. RyR1, Ca(2+) release channels of sarcoplasmic reticulum, is the primary locus for MH susceptibility. Several mutations in the gene encoding the protein RyR1 have been identified; however, other genes may be involved. Actually, the standard method for diagnosing MH susceptibility is the muscle contracture test for exposure to halothane-caffeine (CHCT) and the only treatment is the use of dantrolene. However, with advances in molecular genetics, a full understanding of the disease etiology may be provided, favoring the development of an accurate diagnosis, less invasive, with DNA test, and also will provide the development of new therapeutic strategies for treatment of MH. Thus, this brief review aims to integrate molecular and clinical aspects of MH, gathering input for a better understanding of this channelopathy.

Role of Triadin in the Organization of Reticulum Membrane at the Muscle Triad

The terminal cisternae represent one of the functional domains of the skeletal muscle sarcoplasmic reticulum (SR). They are closely apposed to plasma membrane invaginations, the T-tubules, with which they form structures called triads. In triads, the physical interaction between the T-tubule-anchored voltage-sensing channel DHPR and the SR calcium channel RyR1 is essential because it allows the depolarization-induced calcium release that triggers muscle contraction. This interaction between DHPR and RyR1 is based on the peculiar membrane structures of both T-tubules and SR terminal cisternae. However, little is known about the molecular mechanisms governing the formation of SR terminal cisternae. We have previously shown that ablation of triadins, a family of SR transmembrane proteins that interact with RyR1, induced skeletal muscle weakness in knockout mice as well as a modification of the shape of triads. Here we explore the intrinsic molecular properties of the longest triadin isoform Trisk 95. We show that when ectopically expressed, Trisk 95 can modulate reticulum membrane morphology. The membrane deformations induced by Trisk 95 are accompanied by modifications of the microtubule network organization. We show that multimerization of Trisk 95 by disulfide bridges, together with interaction with microtubules, are responsible for the ability of Trisk 95 to structure reticulum membrane. When domains responsible for these molecular properties are deleted, anchoring of Trisk 95 to the triads in muscle cells is strongly decreased, suggesting that oligomers of Trisk 95 and microtubules contribute to the organization of the SR terminal cisternae in a triad.

CGMP reduces the sarcoplasmic reticulum Ca2+ loading in airway smooth muscle cells: a putative mechanism in the regulation of Ca2+ by cGMP

Ca(2+) and cGMP have opposite roles in many physiological processes likely due to a complex negative feedback regulation between them. Examples of opposite functions induced by Ca(2+) and cGMP are smooth muscle contraction and relaxation, respectively. A main Ca(2+) storage involved in contraction is sarcoplasmic reticulum (SR); nevertheless, the role of cGMP in the regulation of SR-Ca(2+) has not been completely understood. To evaluate this role, intracellular Ca(2+) concentration ([Ca(2+)]i) was determinated by a ratiometric method in isolated myocytes from bovine trachea incubated with Fura-2/AM. The release of Ca(2+) from SR induced by caffeine was transient, whereas caffeine withdrawal was followed by a [Ca(2+)]i undershoot. Caffeine-induced Ca(2+) transient peak and [Ca(2+)]i undershoot after caffeine were reproducible in the same cell. Dibutyryl cGMP (db-cGMP) blocked the [Ca(2+)]i undershoot and reduced the subsequent caffeine peak (SR-Ca(2+) loading). Both, the opening of SR channels with ryanodine (10 μM) and the blockade of SR-Ca(2+) ATPase with cyclopiazonic acid inhibited the [Ca(2+)]i undershoot as well as the SR-Ca(2+) loading. The addition of db-cGMP to ryanodine (10 μM) incubated cells partially restored the SR-Ca(2+) loading. Cyclic GMP enhanced [Ca(2+)]i undershoot induced by the blockade of ryanodine channels with 50 μM ryanodine. In conclusion, the reduction of SR-Ca(2+) content in airway smooth muscle induced by cGMP can be explained by the combination of SR-Ca(2+) loading and the simultaneous release of SR-Ca(2+). The reduction of SR-Ca(2+) content induced by cGMP might be a putative mechanism limiting releasable Ca(2+) in response to a particular stimulus.

Arrhythmogenic Ion-Channel Remodeling in Heart Failure and Upstream Approach

Class I antiarrhythmic drugs with Na+ channel blocking action cannot be readily used for the treatment of various arrhythmias associated with heart failure because the antiarrhthmic drugs may worsen cardiac function. In failing hearts ion-channel remodeling, such as downregulation of K channels and connexin 43, upregulation of Na-Ca exchanger and HCN channels, and instability of Ca release channels in sarcoplasmic reticulum, can be observed. These electrophysiological abnormalities can produce severe ventricular tachyarrhythmias through reentrant and automatic mechanisms. Neurohumoral factors such as renin-angiotensin system, endothelin system and sympathetic nervous system play an important role in the establishment of the ion-channel remodeling. Upstream approach with blockade of the neurohumoral factors may prevent lethal ventricular arrhythmias. In congestive heart failure atrial tissues are expanded and susceptibility to atrial fibrillation is increased. Control of neurohumoral factors may be also beneficial for the prevention of atrial fibrillation. Recently it has been suggested that inflammatory mechanisms may be also involved in not only the deterioration of heart failure but also induction of atrial fibrillation and ventricular tachyarrhythmias. Drugs with anti-inflammatory action, including the antiarrhythmic drug amiodarone, may be effective for the treatment of arrhythmias associated with heart failure. Current perspective of upstream approach to preventing atrial and ventricular arrhythmias associated with heart failure will be discussed.

On the origin of rhythmic calcium transients in the ICC-MP of the mouse small intestine

Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP(3)R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP(3) and IP(3)-induced calcium release from the SR. Immunohistochemistry proved the expression of IP(3)R type I (IP(3)R-I), but not type II (IP(3)R-II) and type III (IP(3)R-III) in ICC-MP, indicating the involvement of the IP(3)R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICC-MP. In summary, ongoing IP(3) synthesis and IP(3)-induced calcium release from the SR, via the IP(3)R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker frequency, which is likely directly linked to kinetics of the IP(3)-activated SR calcium channel and IP(3) metabolism.