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Hydrogen sulfide (H2S) is gaining interest as a mammalian signalling molecule with wide ranging effects. S-sulfhydration is one mechanism that is emerging as a key post translational modification through which H2S acts. Ion channels and neuronal receptors are key target proteins for S-sulfhydration and this can influence a range of neuronal functions. Voltage-gated K+ channels, including Kv2.1, are fundamental components of neuronal excitability. Here, we show that both recombinant and native rat Kv2.1 channels are inhibited by the H2S donors, NaHS and GYY4137. Biochemical investigations revealed that NaHS treatment leads to S-sulfhydration of the full length wild type Kv2.1 protein which was absent (as was functional regulation by H2S) in the C73A mutant form of the channel. Functional experiments utilising primary rat hippocampal neurons indicated that NaHS augments action potential firing and thereby increases neuronal excitability. These studies highlight an important role for H2S in shaping cellular excitability through S-sulfhydration of Kv2.1 at C73 within the central nervous system.

The receptor tyrosine kinase inhibitor imatinib mesylate has improved patient cancer survival rates but has been linked to long-term cardiotoxicity. This study investigated the effects of imatinib on cell viability, apoptosis, autophagy and necroptosis in human cardiac progenitor cells in vitro. After 24 hours, imatinib significantly reduced cell viability (75.9±2.7% vs._100.0±0.0%, n=5, p0.05) increase in viability after imatinib and wortmannin co-treatment. Imatinib-induced necroptosis was associated with an 8.5±2.5-fold increase in activation of mixed lineage kinase domain-like pseudokinase. Imatinib-induced toxicity was rescued by RIP1 inhibition relative to the control; 88.6±3.0% vs. 100.0±0.0% (n=4, p>0.05). In summary, imatinib applied to human cardiac progenitor cells depolarizes mitochondria and induces cell death through necroptosis, which can be recovered by inhibition of RIP1, with an additional partial role for autophagy in the cell death pathway. These data provide two possible targets for co-therapies to address imatinib-induced long-term cardiotoxicity.

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals.

Graphical abstract, Highlights • Inhibition of Kv7 channels induce Ca2+ signals through L- and T-type VGCCs in rat and human vascular smooth muscle cells. • Kv7.5 is functionally the most important subunit to mediate these effects. • Phospholipase C mediated Kv7 inhibition is likely to contribute to Ca2+ oscillations induced in VSMCs by vasopressin. • Kv7 activator, retigabine, strongly suppresses [Ca2+]i oscillations induced by vasopressin or direct Kv7 channel inhibition., Voltage-gated Kv7 (or KCNQ) channels control activity of excitable cells, including vascular smooth muscle cells (VSMCs), by setting their resting membrane potential and controlling other excitability parameters. Excitation-contraction coupling in muscle cells is mediated by Ca2+ but until now, the exact role of Kv7 channels in cytosolic Ca2+ dynamics in VSMCs has not been fully elucidated. We utilised microfluorimetry to investigate the impact of Kv7 channel activity on intracellular Ca2+ levels and electrical activity of rat A7r5 VSMCs and primary human internal mammary artery (IMA) SMCs. Both, direct (XE991) and G protein coupled receptor mediated (vasopressin, AVP) Kv7 channel inhibition induced robust Ca2+ oscillations, which were significantly reduced in the presence of Kv7 channel activator, retigabine, L-type Ca2+ channel inhibitor, nifedipine, or T-type Ca2+ channel inhibitor, NNC 55-0396, in A7r5 cells. Membrane potential measured using FluoVolt exhibited a slow depolarisation followed by a burst of sharp spikes in response to XE991; spikes were temporally correlated with Ca2+ oscillations. Phospholipase C inhibitor (edelfosine) reduced AVP-induced, but not XE991-induced Ca2+ oscillations. AVP and XE991 induced a large increase of [Ca2+]i in human IMA, which was also attenuated with retigabine, nifedipine and NNC 55-0396. RT-PCR, immunohistochemistry and electrophysiology suggested that Kv7.5 was the predominant Kv7 subunit in both rat and human arterial SMCs; CACNA1C (Cav1.2; L-type) and CACNA1 G (Cav3.1; T-type) were the most abundant voltage-gated Ca2+ channel gene transcripts in both types of VSMCs. This study establishes Kv7 channels as key regulators of Ca2+ signalling in VSMCs with Kv7.5 playing a dominant role.

Increased dissolved carbon monoxide decreases $I_{Ca,L} I_{KI}$ and $I_{Kr}$ and increases late $I_{Na}$ currents in rat and guinea pig patch-clamped isolated ventricular myocytes. Action potentials are prolonged. These effects are reproduced by scaling the currents in the Gattoni et al., 2016 (rat) and Luo and Rudy, 1994 (guinea-pig) cell models. Using the same scaling of currents in the O‘Hara-Rudy (2011) models the endo-, mid-myo- and epi-cardial APD90 is prolonged. CO abolishes alternans in endo-, and induces alternans in mid-myo -cardial cell models at cycle lengths < 280ms. In the homogenous human ventricular tissue models these CO effects decrease epi-, endocardial conduction velocities from 0.4 to 0.32m/s, and increase the widths of the vulnerable windows by +9%, +8%. In the ventricular wall model (a third each of endo-, mid-myo- and epicardial) CO increased transmural propagation times from 44 to 55 ms and maximal difference in propagating APD from 68 to 73 ms. The computed effects of CO on human ventricular tissue are pro-arrhythmogenic.

Background. Missense variants in the ryanodine receptor 1 gene (RYR1) are associated with malignant hyperthermia but only a minority of these have met the criteria for use in predictive DNA diagnosis. We examined the utility of a simplified method of segregation analysis and a functional assay for determining the pathogenicity of recurrent RYR1 variants associated with malignant hyperthermia. Methods We identified previously uncharacterised RYR1 variants found in four or more malignant hyperthermia families and conducted simplified segregation analyses. An efficient cloning and mutagenesis strategy was used to express ryanodine receptor protein containing one of six RYR1 variants in HEK293 cells. Caffeine-induced calcium release, measured using a fluorescent calcium indicator, was compared in cells expressing each variant to that in cells expressing wild type ryanodine receptor protein. Results We identified 43 malignant hyperthermia families carrying one of the six RYR1 variants. There was segregation of genotype with the malignant hyperthermia susceptibility phenotype in families carrying the p.E3104K and p.D3986E variants, but the number of informative meioses limited the statistical significance of the associations. HEK293 functional assays demonstrated an increased sensitivity of RyR1 channels containing the p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I compared with wild type, but cells expressing p.D3986E had a similar caffeine sensitivity to cells expressing wild type RyR1. Conclusions Segregation analysis is of limited value in assessing pathogenicity of RYR1 variants in malignant hyperthermia. Functional analyses in HEK293 cells provided evidence to support the use of p.R2336H, p.R2355W, p.E3104K, p.G3990V and p.V4849I for diagnostic purposes but not p.D3986E.

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca(2+) -sensitive K(+) channels. More recent studies have revealed the ability of CO to inhibit T-type Ca(2+) channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Background: CO poisoning causes cardiac arrhythmias, in part via modulation of the cardiac Na+ channel, Nav1.5. Results: CO inhibition of peak recombinant Nav1.5 current occurs via nitric oxide formation and is also dependent on channel redox state. Conclusion: CO inhibits peak recombinant Nav1.5 current via a mechanism distinct from activation of the late Na+ current. Significance: CO may induce Brugada-like arrhythmias via inhibition of peak Na+ current., Sublethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias, and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na+ channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na+ current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognized CO-sensitive intracellular signaling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of NO formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to DTT immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, l-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor Nω-nitro-l-arginine methyl ester hydrochloride, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na+ current (which can lead to Brugada syndrome-like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation, and is dependent on channel redox state.

In adult rat ventricular myocytes (ARVMs), local Ca2+-release from the Golgi apparatus (GA) was stimulated by a low level of isoproterenol (10 nM) in combination with (1) phosphodiesterase-3 inhibitor (milrinone, 30 µM), (2) phosphodiesterase-4 inhibitor (rolipram, 30 µM) or (3) Epac activator 8-CPT (20 µM); while isoproterenol alone (10 nM) was ineffective. Experiments were done to investigate whether conditions that promote GA Ca2+ release (GCR) influence receptor trafficking. In all cells under control conditions, the VEGFR-1 receptor exhibited a punctate intracellular distribution, which co-localized with the GA resident protein GM130. A minority of cells (29.1 ± 9.3%, n=5) also exhibited a striated VEGFR-1 pattern, which co-localized with the distribution of Nav1.1 located within the sarcolemma/t-tubules. Incubation of ARVMs with isoproterenol alone, which had no effect on Golgi Ca2+ release, did not affect the cellular distribution of VEGFR-1 (p>0.05, n=5). Isoproterenol in combination with milrinone, rolipram or 8-CPT markedly increased the percentage of cells with a striated VEGFR-1 distribution to 67.2 ± 6.4 % (n=4, p

The primary reason for cessation of statin therapy is skeletal myopathy but the underlying mechanism and apparent absence of detrimental effects of statins on cardiac muscle are not fully understood. We have reported that statin treatment in vivo causes a profound leak of Ca2+ from the sarcoplasmic reticulum (SR) of intact skeletal myocytes, a common myopathic mechanism. Here we compare the effect of simvastatin on Ca2+ sparks in rat skeletal and cardiac myocytes. Simvastatin treatment in vivo (40 mg/kg/day for 4 weeks) markedly increased Ca2+ spark frequency (280%) and duration (140%) in fluo4-loaded flexor digitorum brevis (FDB)fibres (P

International audience; Rationale: Clinical reports describe life-threatening cardiac arrhyth-mias after environmental exposure to carbon monoxide (CO) or accidental CO poisoning. Numerous case studies describe disruption of repolarization and prolongation of the QT interval, yet the mechanisms underlying CO-induced arrhythmias are unknown. Objectives: To understand the cellular basis of CO-induced arrhyth-mias and to indentify an effective therapeutic approach. Methods: Patch-clamp electrophysiology and confocal Ca 21 and nitric oxide (NO) imaging in isolated ventricular myocytes was performed together with protein S-nitrosylation to investigate the effects of CO at the cellular and molecular levels, whereas telemetry was used to investigate effects of CO on electrocardiogram recordings in vivo. Measurements and Main Results: CO increased the sustained (late) component of the inward Na 1 current, resulting in prolongation of the action potential and the associated intracellular Ca 21 transient. In more than 50% of myocytes these changes progressed to early after-depolarization–like arrhythmias. CO elevated NO levels in myocytes and caused S-nitrosylation of the Na1 channel, Nav1.5. All proarrhythmic effects of CO were abolished by the NO synthase inhibitor L-NAME, and reversed by ranolazine, an inhibitor of the late Na1 current. Ranolazine also corrected QT variability and arrhyth- mias induced by CO in vivo, as monitored by telemetry. Conclusions: Our data indicate that the proarrhythmic effects of CO arise from activation of NO synthase, leading to NO-mediated nitro- sylation of NaV1.5 and to induction of the late Na1 current. We also show that the antianginal drug ranolazine can abolish CO-induced early after-depolarizations, highlighting a novel approach to the treatment of CO-induced arrhythmias.

International audience; E. Cardiac arrhythmia mechanisms in rats with heart failure induced by pulmonary hypertension.Pulmonary hypertension provokes right heart failure and arrhythmias. Better understanding of the mechanisms underlying these arrhythmias is needed to facilitate new therapeutic approaches for the hypertensive, failing right ventri-cle (RV). The aim of our study was to identify the mechanisms generating arrhythmias in a model of RV failure induced by pulmonary hypertension. Rats were injected with monocrotaline to induce either RV hypertrophy or failure or with saline (control). ECGs were measured in conscious, unrestrained animals by telemetry. In isolated hearts, electrical activity was measured by optical mapping and myofiber orientation by diffusion tensor-MRI. Sarcoplasmic reticular Ca 2ϩ handling was studied in single myocytes. Compared with control animals, the T-wave of the ECG was prolonged and in three of seven heart failure animals, prominent T-wave alternans occurred. Discordant action potential (AP) alternans occurred in isolated failing hearts and Ca 2ϩ transient alternans in failing myocytes. In failing hearts, AP duration and dispersion were increased; conduction velocity and AP restitution were steeper. The latter was intrinsic to failing single myocytes. Failing hearts had greater fiber angle disarray; this correlated with AP duration. Failing myocytes had reduced sarco(en-do)plasmic reticular Ca 2ϩ-ATPase activity, increased sarcoplasmic reticular Ca 2ϩ-release fraction, and increased Ca 2ϩ spark leak. In hypertrophied hearts and myocytes, dysfunctional adaptation had begun, but alternans did not develop. We conclude that increased electrical and structural heterogeneity and dysfunctional sarcoplasmic reticular Ca 2ϩ handling increased the probability of alternans, a proarrhythmic predictor of sudden cardiac death. These mechanisms are potential therapeutic targets for the correction of arrhythmias in hypertensive, failing RVs. electrocardiography; monocrotaline; calcium signaling; voltage-sensitive dye imaging; alternans ALTHOUGH RIGHT VENTRICULAR (RV) failure most often develops as a consequence of left ventricular (LV) failure, RV failure occurs in many, increasingly common, diseases associated with dysfunction of the pulmonary circulation (6, 18, 58). This includes pulmonary hypertension, where RV failure is the major cause of death in patients (6). Pulmonary hypertension is associated with RV electrical remodeling (23, 26, 27) and a higher risk of arrhythmias (17). Sudden death occurs in 30-40% of sufferers (10, 63), and lethal arrhythmias are thought to be one cause, in addition to other mechanisms, such as pulmonary embolism and pulmonary artery dissection. Interestingly, the RV is not currently a target for therapeutic intervention in this disease (19). There are several established mechanisms by which arrhyth-mias can arise. Increased electrical heterogeneity and steeper electrical restitution (1, 32, 53) increase the probability of alternans and arrhythmias. This is because heterogeneity increases the dispersion of refractoriness and steeper restitution decreases the stability of action potential (AP) duration (APD) and diastolic interval adaptation to a change in heart rate, also increasing the dispersion of refractoriness. This makes conduction block and reentry more likely (9, 35, 49, 60). Alternans describe the events where parameters such as APD or intracel-lular Ca 2ϩ concentration ([Ca 2ϩ ] i) transient amplitude alternate in size on a beat-to-beat basis. They are established predictors of sudden cardiac death, and discordant alternans, where the parameter in question is out of phase in different regions of the ventricles, are most likely to generate reentrant arrhythmias (9, 35, 49, 60). Defective Ca 2ϩ handling also leads to the generation of arrhythmias. Increased sarcoplasmic reticulum (SR) Ca 2ϩ leak, SR release fraction, SR Ca 2ϩ content, and slowed SR Ca 2ϩ reuptake are thought to contribute to the generation of Ca 2ϩ transient alternans and thus to APD and T-wave alternans (via modulation of Ca 2ϩ-dependent currents) by facilitating beat-to-beat fluctuations in SR Ca 2ϩ release (38, 49). This is because increased SR Ca 2ϩ content favors SR Ca 2ϩ leak and places activated ryanodine receptors in a refractory state for the next beat, leading to fluctuations in SR Ca 2ϩ release (48). Additionally, elevated SR Ca 2ϩ content and release fraction in the presence of a slowed SR Ca 2ϩ reuptake lead to intra-SR fluctuations in Ca 2ϩ content (13). Structural changes, such as an alteration in connexin expression or increased fibrosis, which disrupts the normal myofiber organization, can slow conduction, create electrical heteroge-neity (31), and contribute to dysfunctional electrical activity (16). Given the number of potential arrhythmic mechanisms, the specific occurrence and relative importance of these mechanisms need to be characterized for any given pathology, if that pathology is to be fully understood and treated.

In malignant hyperthermia (MH), mutations in RyR1 underlie direct activation of the channel by volatile anesthetics, leading to muscle contracture and a life-threatening increase in core body temperature. The aim of the present study was to establish whether the associated depletion of sarcoplasmic reticulum (SR) Ca(2+) triggers sarcolemmal Ca(2+) influx via store-operated Ca(2+) entry (SOCE). Samples of vastus medialis muscle were obtained from patients undergoing assessment for MH susceptibility using the in vitro contracture test. Single fibers were mechanically skinned, and confocal microscopy was used to detect changes in [Ca(2+)] either within the resealed t-system ([Ca(2+)](t-sys)) or within the cytosol. In normal fibers, halothane (0.5 mM) failed to initiate SR Ca(2+) release or Ca(2+)(t-sys) depletion. However, in MH-susceptible (MHS) fibers, halothane induced both SR Ca(2+) release and Ca(2+)(t-sys) depletion, consistent with SOCE. In some MHS fibers, halothane-induced SR Ca(2+) release took the form of a propagated wave, which was temporally coupled to a wave of Ca(2+)(t-sys) depletion. SOCE was potently inhibited by "extracellular" application of a STIM1 antibody trapped within the t-system but not when the antibody was denatured by heating. In conclusion, (i) in human MHS muscle, SR Ca(2+) depletion induced by a level of volatile anesthetic within the clinical range is sufficient to induce SOCE, which is tightly coupled to SR Ca(2+) release; (ii) sarcolemmal STIM1 has an important role in regulating SOCE; and (iii) sustained SOCE from an effectively infinite extracellular Ca(2+) pool may contribute to the maintained rise in cytosolic [Ca(2+)] that underlies MH.

Sepsis is associated with ventricular dysfunction and increased incidence of atrial and ventricular arrhythmia however the underlying pro-arrhythmic mechanisms are unknown. Serum levels of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) are elevated during sepsis and affect Ca2+ regulation. We investigated whether pro-inflammatory cytokines disrupt cellular Ca2+ cycling leading to reduced contractility, but also increase the probability of pro-arrhythmic spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). Isolated rat ventricular myocytes were exposed to TNF-alpha (0.05 ng ml(-1)) and IL-1beta (2 ng ml(-1)) for 3 hr and then loaded with fura-2 or fluo-3 to record the intracellular Ca2+ concentration ([Ca2+](i)). Cytokine treatment decreased the amplitude of the spatially averaged Ca2+ transient and the associated contraction, induced asynchronous Ca2+ release during electrical stimulation, increased the frequency of localized Ca2+ release events, decreased the SR Ca2+ content and increased the frequency of spontaneous Ca2+ waves at any given cytoplasmic Ca2+. These data suggest that TNF-alpha and IL-1beta increase the SR Ca2+ leak from the SR, which contributes to the depressed Ca2+ transient and contractility. Increased susceptibility to spontaneous SR Ca2+ release may contribute to arrhythmias in sepsis as the resulting Ca2+ extrusion via NCX is electrogenic, leading to cell depolarisation.

In this study we present 3 families with malignant hyperthermia (MH), all of Indian subcontinent descent. One individual from each of these families was fully sequenced for RYR1 and presented with the non-synonymous change c.11315G>A/p.R3772Q. When present in the homozygous state c.11315*A is associated with myopathic symptoms.

Changes in skeletal muscle volume induce localized sarcoplasmic reticulum (SR) Ca(2+) release (LCR) events, which are sustained for many minutes, suggesting a possible signaling role in plasticity or pathology. However, the mechanism by which cell volume influences SR Ca(2+) release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrode's solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (E(m)), and intracellular Ca(2+) ([Ca(2+)](i)) were determined. To allow comparison with previous studies, solutions were made hyperosmotic by the addition of sugars or divalent cations, or they were made hypoosmotic by reducing [NaCl](o). All hyperosmotic solutions induced a sustained decrease in cell volume, which was accompanied by membrane depolarization (by 14-18 mV; n = 40) and SR Ca(2+) release. However, sugar solutions caused a global increase in [Ca(2+)](i), whereas solutions made hyperosmotic by the addition of divalent cations only induced LCR. Decreasing osmolarity induced an increase in cell volume and a negative shift in E(m) (by 15.04 +/- 1.85 mV; n = 8), whereas [Ca(2+)](i) was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and E(m) was associated with LCR. Both global and localized SR Ca(2+) release were abolished by the dihydropyridine receptor inhibitor nifedipine by sustained depolarization of the sarcolemmal or by the addition of the ryanodine receptor 1 inhibitor tetracaine. Inhibitors of the Na-K-2Cl (NKCC) cotransporter markedly inhibited the depolarization associated with hyperosmotic shrinkage and the associated SR Ca(2+) release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca(2+) release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in E(m). The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions.

In skeletal muscle, Mg 2+ exerts a dual inhibitory effect on RyR1, by competing with Ca 2+ at the activation site and binding to a low affinity Ca 2+ /Mg 2+ inhibitory site. Pharmacological activators of RyR1 must overcome the inhibitory action of Mg 2+ before Ca 2+ efflux can occur. In normal muscle, where the free [Mg 2+ ] i is ∼1 mM, even prolonged exposure to millimolar levels of volatile anesthetics does not initiate SR Ca 2+ release. However, when the cytosolic [Mg 2+ ] is reduced below the physiological range, low levels of volatile anesthetic within the clinically relevant range (⩽1 mM) can initiate SR Ca 2+ release, in the form of a propagating Ca 2+ wave. In human muscle fibers from malignant hyperthermia susceptible patients, such Ca 2+ waves occur when 1 mM halothane is applied at physiological [Mg 2+ ] i . There is increasing evidence to suggest that defective Mg 2+ regulation of RyR1 confers susceptibility to malignant hyperthermia. At the molecular level, interactions between critical RyR1 subdomains may explain the clustering of RyR1 mutations and associated effects on Mg 2+ regulation.

Ca(2+) release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. We found that the Golgi apparatus was the source of prolonged Ca(2+) release events that originated near the nuclei of primary cardiomyocytes. Golgi Ca(2+) release was unaffected by depletion of sarcoplasmic reticulum Ca(2+), and disruption of the Golgi apparatus abolished Golgi Ca(2+) release without affecting sarcoplasmic reticulum function, suggesting functional and spatial independence of Golgi and sarcoplasmic reticulum Ca(2+) stores. β1-Adrenoceptor stimulation triggers the production of the second messenger cAMP, which activates the Epac family of Rap guanine nucleotide exchange factors and the kinase PKA (protein kinase A). Phosphodiesterases (PDEs), including those in the PDE3 and PDE4 families, degrade cAMP. Activation of β1-adrenoceptors stimulated Golgi Ca(2+) release, an effect that required activation of Epac, PKA, and the kinase CaMKII. Inhibition of PDE3s or PDE4s potentiated β1-adrenergic-induced Golgi Ca(2+) release, which is consistent with compartmentalization of cAMP signaling near the Golgi apparatus. Interventions that stimulated Golgi Ca(2+) release appeared to increase the trafficking of vascular endothelial growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane of cardiomyocytes. In cardiomyocytes from rats with heart failure, decreases in the abundance of PDE3s and PDE4s were associated with increased Golgi Ca(2+) release events. These data suggest that the Golgi apparatus is a focal point for β1-adrenergic-stimulated Ca(2+) signaling and that the Golgi Ca(2+) store functions independently from the sarcoplasmic reticulum and the global Ca(2+) transients that trigger contraction in cardiomyocytes.

In adult rat ventricular myocytes, local Ca2+-release from the Golgi apparatus (GA) was stimulated by phosphodiesterase-3 (milrinone, 30 μM) or −4 (rolipram, 30 μM) inhibition, while treatment with a high level of isoproterenol alone (1 μM) had comparatively little effect. PDE 3 or 4 inhibition also increased the frequency of SR Ca2+ sparks. With rolipram, this occurred without a significant increase in SR Ca2+ content suggesting that the increase in spark frequency was primarily mediated by a direct effect on RyR2. The selective Epac activator 8-CPT (30 µM) also stimulated GA Ca2+ release events and increased Ca2+ spark frequency without increasing the SR Ca2+ content. A pull-down assay was used to measure the level of active (GTP-bound) Rap1, providing an index of Epac activation. Basal Rap1 activation was consistently detectable in ventricular myocytes and was significantly (p

The need to understand the mechanism of statin-induced myopathy is increasing as the cardiovascular risk threshold for statin prescription is reduced across the globe. Here we determine for the first time the effect of chronic statin treatment in vivo (simvastatin 40 mg/kg/day by oral gavage over a 4 week period) on Ca2+ homeostasis in intact (non-permeabilised) type II rat skeletal muscle fibres, and investigate a role for mitochondria and NO. In fluo-4 loaded FDB fibres, sarcoplasmic reticulum (SR) Ca2+ leak (indexed in the presence of the SERCA inhibitor cyclopiazonic acid) was significantly higher (P

Background In normal resting muscle, cytosolic Mg 2+ exerts a potent inhibitory influence on the sarcoplasmic reticulum (SR) Ca 2+ release channel (ryanodine receptor, RyR1). Impaired Mg 2+ -regulation of RyR1 has been proposed as a causal factor in malignant hyperthermia (MH). The aim of this study was to compare the effects of cytosolic Mg 2+ on SR Ca 2+ release induced by halothane or sevoflurane in normal (MHN) and MH susceptible (MHS) human skeletal muscle fibres. Methods Samples of vastus medialis muscle were obtained from patients under investigation for MH susceptibility. Single fibres were mechanically skinned and perfused with solutions mimicking the intracellular milieu. Changes in [Ca 2+ ] i were detected using fura-2 fluorescence after application of equimolar halothane or sevoflurane. Results In MHN fibres, concentrations of sevoflurane or halothane as high as 10 mM typically failed to induce SR Ca 2+ release at physiological free [Mg 2+ ] (1 mM). However, when [Mg 2+ ] was decreased to 0.4 mM, SR Ca 2+ release occurred in 51% (16/33) and 6% (2/33) of MHN fibres after the addition of 1 mM halothane or 1 mM sevoflurane, respectively. Further decreases in [Mg 2+ ] increased the proportion of responsive fibres. In the presence of 0.1 mM [Mg 2+ ], Ca 2+ release occurred in all fibres (33/33) after the introduction of 1 mM halothane or 1 mM sevoflurane. In MHS fibres, 1 mM halothane or 1 mM sevoflurane-induced Ca 2+ release in 54% (7/13) or 15% (2/13) of fibres, respectively, at 1 mM Mg 2+ . A decrease in [Mg 2+ ] to 0.2 mM Mg 2+ was sufficient to render 100% of MHS fibres (13/13) responsive to 1 mM halothane or 1 mM sevoflurane. Conclusions In both MHS and MHN fibres (i) halothane is a more potent activator of SR Ca 2+ release than sevoflurane and (ii) as with halothane, the efficacy of sevoflurane-induced SR Ca 2+ release exhibits a marked dependence on cytosolic [Mg 2+ ]. The marked potentiation of SR Ca 2+ release after a moderate reduction in cytosolic [Mg 2+ ] suggests that conditions which cause hypomagnesaemia will increase the probability and possibly severity of an MH event. Conversely, maintenance of a normal or slightly increased cytosolic [Mg 2+ ] may reduce the probability of MH.

In vitro experiments have shown that the ryanodine receptor-2 (RyR2) central domain peptide DPc10 (Gly(2460)-Pro(2495)) mimics channel dysfunction associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) by acting competitively to reduce stabilizing interactions between the N-terminal and central domains. In the present study, DPc10 was used as a tool to establish an adult cell model of the disease and to analyse the underlying mechanisms.Rat ventricular myocytes were permeabilized with saponin and perfused with solutions approximating the intracellular milieu containing fluo-3. Sarcoplasmic reticulum (SR) Ca(2+) release was detected using confocal microscopy. DPc10 (10 or 50 microM) was compared with 0.2 mM caffeine, which is known to activate RyR2 and to facilitate Ca(2+)-induced Ca(2+) release (CICR).Introduction of DPc10 induced a transient increase in spark frequency and a sustained rise in resting [Ca(2+)]. Under conditions causing initial Ca(2+) overload of the SR, DPc10 reduced the frequency and amplitude of spontaneous, propagated Ca(2+) release (SPCR). Following equilibration with 10microM DPc10, the cytosolic [Ca(2+)] threshold for SPCR was markedly reduced and the proportion of spontaneously active cells increased. Caffeine induced a similar, transient increase in spark frequency and a reduction in the [Ca(2+)] threshold for SPCR. However, unlike DPc10, caffeine increased SPCR frequency and had no sustained effect on resting [Ca(2+)]. These results suggest that the net effect of DPc10 (and CPVT mutations) on RyR2 function in situ is not only to increase the sensitivity to CICR as caffeine does, but also to potentiate Ca(2+) leakage from the SR. As SPCR can trigger delayed after-depolarisations, the decrease in [Ca(2+)] threshold may contribute to arrhythmias in CPVT patients during exercise or stress.

Objective : Previous work suggests that modification of sarcoplasmic reticulum (SR) function may contribute to the cardioprotective effect of halothane during ischaemia and reperfusion. The aim of this study was to investigate the effects of halothane on spontaneous Ca2+ release from the sarcoplasmic reticulum (Ca2+ sparks and waves). Methods : Rat atrial myocytes were permeabilized with saponin and perfused with solutions approximating to the intracellular milieu and containing fluo-3. SR Ca2+ release was detected using confocal microscopy. Results : In the presence of 5 mM ATP, halothane (0.25–2 mM) had no significant effect on the amplitude or frequency of spontaneous Ca2+ waves. However, in the presence of 0.05 mM ATP, halothane (0.25–2 mM) induced a concentration-dependent decrease in the amplitude and an increase in the frequency of spontaneous Ca2+ waves, e.g., 1 mM halothane decreased the amplitude by 34.7 ± 3.5% ( n =9) and increased the frequency by 67 ± 19.9% ( n =7). In the presence of 5 mM ATP, 1 mM halothane had no significant effect on the amplitude or frequency of Ca2+ sparks. When [ATP] was reduced to 0.05 mM, Ca2+ spark frequency decreased by 67.9 ± 14% and the amplitude increased by 27.5 ± 4.9% ( n =13). Subsequent introduction of halothane (0.5–1 mM) induced a transient burst of Ca2+ sparks, consistent with ryanodine receptor (RyR) activation. Further experiments showed that the decrease in Ca2+ spark frequency following ATP depletion was associated with a progressive increase in the SR Ca2+ content over 1–2 min. This rise in SR Ca2+ content did not occur when 1 mM halothane was present during ATP depletion. Conclusions : These data suggest that the sensitivity of the RyR to activation by halothane increases at low [ATP]. In metabolically impaired cells, halothane would be expected to lessen any rise in SR Ca2+ content and to reduce the amplitude of spontaneous Ca2+ release. These effects of halothane are considered in relation to the events that occur during ischaemia and reperfusion.

Background Recent work suggests that impaired Mg(2+) regulation of the ryanodine receptor is a common feature of both pig and human malignant hyperthermia. Therefore, the influence of [Mg(2+)] on halothane-induced Ca(2+) release from the sarcoplasmic reticulum was studied in malignant hyperthermia-susceptible (MHS) or -nonsusceptible (MHN) muscle. Methods Vastus medialis fibers were mechanically skinned and perfused with solutions containing physiologic (1 mm) or reduced concentrations of free [Mg(2+)]. Sarcoplasmic reticulum Ca(2+) release was detected using fura-2 or fluo-3. Results In MHN fibers, 1 mm halothane consistently did not induce sarcoplasmic reticulum Ca(2+) release in the presence of 1 mm Mg(2+). It was necessary to increase the halothane concentration to 20 mm or greater before Ca release occurred. However, when [Mg(2+)] was reduced below 1 mm, halothane became an increasingly effective stimulus for Ca(2+) release; e.g., at 0.4 mm Mg(2+), 58% of MHN fibers responded to halothane. In MHS fibers, 1 mm halothane induced Ca(2+) release in 57% of MHS fibers at 1 mm Mg(2+). Reducing [Mg(2+)] increased the proportion of MHS fibers that responded to 1 mm halothane. Further experiments revealed differences in the characteristics of halothane-induced Ca(2+) release in MHS and MHN fibers: In MHN fibers, at 1 mm Mg(2+), halothane induced a diffuse increase in [Ca(2+)], which began at the periphery of the fiber and spread slowly inward. In MHS fibers, halothane induced a localized C(2+)a release, which then propagated along the fiber. However, propagated Ca(2+) release was observed in MHN fibers when halothane was applied at an Mg(2+) concentration of 0.4 mm or less. Conclusions When Mg(2+) inhibition of the ryanodine receptor is reduced, the halothane sensitivity of MHN fibers and the characteristics of the Ca release process approach that of the MHS phenotype. In MHS fibers, reduced Mg(2+) inhibition of the ryanodine receptor would be expected to have a major influence on halothane sensitivity. The Mg dependence of the halothane response in MHN and MHS may have important clinical implications in circumstances where intracellular [Mg(2+)] deviates from normal physiologic concentrations.

Regulation of the ryanodine receptor (RYR) by Mg(2+) and SR luminal Ca(2+) was studied in mechanically skinned malignant hyperthermia susceptible (MHS) and non-susceptible (MHN) fibres from human vastus medialis. Preparations were perfused with solutions mimicking the intracellular milieu and changes in [Ca(2+)] were detected using fura-2 fluorescence. At 1 mM cytosolic Mg(2+), MHS fibres had a higher sensitivity to caffeine (2-40 mM) than MHN fibres. The inhibitory effect of Mg(2+) on caffeine-induced Ca(2+) release was studied by increasing [Mg(2+)] of the solution containing 40 mM caffeine. Increasing [Mg(2+)] from 1 to 3 mM reduced the amplitude of the caffeine-induced Ca(2+) transient by 77 +/- 7.4 % (n = 8) in MHN fibres. However, the caffeine-induced Ca(2+) transient decreased by only 24 +/- 8.1 % (n = 9) in MHS fibres. In MHN fibres, reducing the Ca(2+) loading period from 4 to 1 min (at 1 mM Mg(2+)) decreased the fraction of the total sarcoplasmic reticulum (SR) Ca(2+) content released in response to 40 mM caffeine by 90.4 +/- 6.2 % (n = 6). However, in MHS fibres the response was reduced by only 31.2 +/- 17.4 % (n = 6) under similar conditions. These results suggest that human malignant hyperthermia (MH) is associated with reduced inhibition of the RYR by (i) cytosolic Mg(2+) and (ii) SR Ca(2+) depletion. Both of these effects may contribute to increased sensitivity of the RYR to caffeine and volatile anaesthetics.

Detubulation of rat ventricular myocytes has been used to investigate the role of the t-tubules in Ca 2+ cycling during excitation-contraction coupling in rat ventricular myocytes. Ca 2+ was monitored using fluo-3 and confocal microscopy. In control myocytes, electrical stimulation caused a spatially uniform increase in intracellular [Ca 2+ ] across the cell width. After detubulation, [Ca 2+ ] rose initially at the cell periphery and then propagated into the center of the cell. Application of caffeine to control myocytes resulted in a rapid and uniform increase of intracellular [Ca 2+ ]; the distribution and amplitude of this increase was the same in detubulated myocytes, although its decline was slower. On application of caffeine to control cells, there was a large, rapid, and transient rise in extracellular [Ca 2+ ] as Ca 2+ was extruded from the cell; this rise was significantly smaller in detubulated cells, and the remaining increase was blocked by the sarcolemmal Ca 2+ ATPase inhibitor carboxyeosin. The treatment used to produce detubulation had no significant effect on Ca 2+ efflux in atrial cells, which lack t-tubules. Detubulation of ventricular myocytes also resulted in loss of Na + -Ca 2+ exchange current, although the density of the fast Na + current was unaltered. It is concluded that Na + -Ca 2+ exchange function, and hence Ca 2+ efflux by this mechanism, is concentrated in the t-tubules, and that the concentration of Ca 2+ flux pathways in the t-tubules is important in producing a uniform increase in intracellular Ca 2+ on stimulation.

The effects of phosphocreatine (PCr) on sarcoplasmic reticulum (SR) Ca(2+) regulation were investigated in saponin-permeabilized rat ventricular myocytes. Cells were perfused continuously with weakly Ca(2+)-buffered solutions approximating to the intracellular milieu. Ca(2+) release from the SR was detected using Fura-2 or Fluo-3. Withdrawal of PCr reduced the frequency of spontaneous Ca(2+) release by 12.8 +/- 3.4 % (n = 9) and the amplitude of the spontaneous Ca(2+) transient by 17.4 +/- 3.1 % (n = 9). Stepwise reductions in [PCr] progressively increased the time for the spontaneous Ca(2+) transient to rise from 25 to 100 % of the maximum value (TP75) and to fall by 75 % of the peak level (DT75). Following complete PCr withdrawal, the TP75 and the DT75 were 147.1 +/- 13.2 and 174.8 +/- 23.2 % of the control values, respectively. Experiments involving confocal microscopy showed that PCr withdrawal decreased the propagation velocity of spontaneous Ca(2+) waves. PCr withdrawal also reduced the frequency and amplitude, but increased the duration of spontaneous Ca(2+) sparks. Rapid application of 20 mM caffeine was used to assess the SR Ca(2+) content at the point of spontaneous Ca(2+) release. In the absence of PCr, the amplitude of the caffeine-induced Ca(2+) transient was 18.4 +/- 2.7 % (n = 9) lower than in the presence of 10 mM PCr. This suggests that PCr withdrawal reduces the maximum SR Ca(2+) content that can be sustained before spontaneous Ca(2+) release occurs. These results suggest that local ADP buffering by PCr is essential for normal Ca(2+) regulation by the SR. Prolongation of the descending phase of the spontaneous Ca(2+) transient is consistent with a reduction in the efficiency of the SR Ca(2+) pump due to ADP accumulation. The fact that spontaneous Ca(2+) release occurs at a lower SR Ca(2+) content in the absence of PCr suggests that the Ca(2+) release mechanism may also be affected. These effects may be of relevance in circumstances where PCr depletion and Ca(2+) overload occur, such as myocardial ischaemia or anoxia.

Abstract— Confocal imaging was used to study the influence of cytosolic ATP on the properties of spontaneous Ca 2+ sparks in permeabilized ventricular myocytes. Cells were perfused with mock intracellular solutions containing fluo 3. Reducing [ATP] to 2+ sparks. In the presence of 20 μmol/L ATP, the amplitude increased by 48.7±10.9%, and the frequency decreased by 77.07±3.8%, relative to control responses obtained at 5 mmol/L ATP. After exposure to a solution containing zero ATP, the frequency of Ca 2+ sparks decreased progressively and approached zero within 90 seconds. As ATP washed out of the cell, the sarcoplasmic reticulum (SR) Ca 2+ content increased, until reaching a maximum after 3 minutes. Subsequent introduction of adenylyl imidodiphosphate precipitated a burst of large-amplitude Ca 2+ sparks. This was accompanied by a rapid decrease in SR Ca 2+ content to 80% to 90% of the steady-state value obtained in the presence of 5 mmol/L ATP. Thereafter, the SR Ca 2+ content declined much more slowly over 5 to 10 minutes. The effects of ATP withdrawal on Ca 2+ sparks may reflect reduced occupancy of the adenine nucleotide site on the SR Ca 2+ channel. These effects may contribute to previously reported changes in SR function during myocardial ischemia and reperfusion, in which ATP depletion and Ca 2+ overload occur.

1. The effects of creatine phosphate (CP) and inorganic phosphate (Pi) on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in mechanically skinned muscle fibres from rat extensor digitorum longus (EDL) muscles. Changes in [Ca2+] were detected using fura-2 fluorescence, during continuous perfusion or when the solution surrounding the preparation was restricted to approximately 6 microl by stopping perfusion. 2. In solutions with 5 mM ATP and 10 mM CP, stopping the flow for 2-3 min had no effect on [Ca2+] within the bath. This suggests that SR Ca2+ uptake is balanced by an efflux under these conditions. 3. In solutions with CP, the introduction of Pi induced a small transient rise in [Ca2+], due to Ca2+ loss from the SR. Following equilibration with solutions containing Pi (> or = 5 mM), a maintained decrease in [Ca2+] occurred when the flow was stopped. This is consistent with calcium phosphate (Ca-Pi) precipitation within the SR, resulting in maintained Ca2+ uptake. 4. In the absence of CP, the [Ca2+] within the bath increased progressively when the flow was stopped. This rise in [Ca2+] was inhibited by an alternative ATP regenerating system comprising phosphoenolpyruvate (PEP) and pyruvate kinase (PK). Therefore, the loss of Ca2+ from the SR may result from local ADP accumulation and the consequent reversal of the SR Ca2+ pump. 5. In the absence of CP, the initial Ca2+ release associated with the introduction of Pi increased markedly. Following prolonged equilibration with solutions containing Pi, a rise in [Ca2+] occurred within the bath when the flow was stopped. Maintained Ca2+ uptake associated with Ca-Pi precipitation was not apparent at any level of Pi tested (1-60 mM), when CP was absent. 6. These results suggest that withdrawal of CP is associated with activation of a SR Ca2+ efflux pathway. This may involve reversal of the SR Ca2+ pump, due to local ADP accumulation. In the absence of CP, the dominant influence of Pi appears to involve further Ca2+ efflux via the SR Ca2+ pump. The possible relevance of these effects to skeletal muscle fatigue is considered.

The effects of cytosolic ATP on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in saponin-permeabilised rat ventricular myocytes. [Ca2+] within the cells was monitored using Fura-2 or Fluo-3 fluorescence. Spontaneous cyclic Ca2+ release from the SR was induced by increasing the bathing [Ca2+] to 200–300 nM, in solutions weakly Ca2+ buffered with 0.05 mm EGTA. Alternatively, Ca2+-induced Ca2+ release (CICR) was triggered by a rapid increase in [Ca2+] induced by flash photolysis of Nitr-5 (0.08 mm), replacing EGTA in the solution. Stepwise reductions in [ATP] were associated with corresponding decreases in the frequency and increases in the amplitude of spontaneous Ca2+ transients. A decrease from 5 mm to 0.1 mm ATP, reduced the release frequency by 48.6 ± 7 % (n = 7) and almost doubled the amplitude of the Ca2+ transient. Marked prolongation of the spontaneous Ca2+ transient occurred when [ATP] was further reduced to 10 μM, consistent with inhibition of the SR Ca2+ pump. These effects of ATP were compared with other interventions that inhibit Ca2+ uptake or reduce the sensitivity of the SR Ca2+ release mechanism. Inhibition of the SR Ca2+ pump with cyclopiazonic acid (CPA) markedly reduced the spontaneous Ca2+ release frequency, without changing the amplitude. The descending phase of the Ca2+ transient was prolonged in the presence of CPA, while the rising phase was unaffected. In contrast, desensitisation of the SR Ca2+ release mechanism with tetracaine decreased the frequency of spontaneous release, but markedly increased the amplitude. CICR triggered by flash photolysis of Nitr-5 appeared to be more sensitive to cytosolic [ATP] than spontaneous release and was generally delayed by a decrease to 2.5 mm ATP. In the presence of 0.1-0.2 mm ATP, release often failed completely or was not consistently triggered. Some preparations exhibited Ca2+ release ‘alternans’, whereby every alternate trigger induced a response. These results suggest that the increase in spontaneous Ca2+ release amplitude and the decrease in frequency that occurs as [ATP] is reduced from 1 mm to 100 μM, is mainly due to desensitisation of the SR Ca2+ release mechanism, which allows the SR Ca2+ content to reach a higher level before release occurs. At very low [ATP], a reduction in the SR Ca2+ uptake rate may also contribute to the decrease in release frequency. CICR triggered by photolysis of Nitr-5 appeared to be more sensitive to cytosolic [ATP]. The possible underlying mechanisms and the relevance of these results to myocardial ischaemia or hypoxia is considered. In the first few minutes of anoxia or global ischaemia, inhibition of aerobic metabolism is accompanied by a pronounced decrease in contractile force (reviewed by Allen & Orchard, 1987). During this phase, the average intracellular concentration of ATP ([ATP]i) remains relatively constant as ADP is re-phosphorylated by creatine phosphate (CrP), catalysed by creatine kinase. The [CrP]i decreases rapidly from 15–20 mm to undetectable levels and the intracellular inorganic phosphate concentration ([Pi]i) increases from about 2 mm to 20 mm. After depletion of CrP, ATP synthesis continues via anaerobic glycolysis, resulting in lactic acid production and a decrease in intracellular pH (pHi). With more prolonged periods of global ischaemia, anaerobic glycolysis ceases and a further increase in [Pi]i occurs due to the net hydrolysis of ATP. Previous studies have shown that these characteristic changes in pH, [Pi] and [CrP] have pronounced effects on SR Ca2+ regulation and the myofilament force production (Orchard & Kentish, 1990; Smith & Steele, 1992; Steele et al. 1995). However, comparatively little is known about the effects of ATP depletion on SR Ca2+ regulation and in particular the CICR mechanism. In isolated SR vesicles, ATP increases the maximum Ca2+-activated efflux rate and the apparent Ca2+ sensitivity of the release mechanism (Meissner & Henderson, 1987). Experiments on isolated SR Ca2+ channels have also shown that, in the presence of activating levels of Ca2+, millimolar levels of ATP and other adenine nucleotides activate the channel (Rousseau et al. 1986; McGarry & Williams, 1994). However, in recent studies on isolated channels under simulated ischaemic conditions, the dominant effect of ATP was a reduction in the degree of channel inactivation as [Ca2+] increased above 30 μM (Xu et al. 1996). At lower [Ca2+] channels appeared to be relatively insensitive to ATP: reducing the [ATP] from 5 to 1 mm had little effect on the opening probability (Po), while complete removal of ATP resulted in a 20 % reduction in Po. Based on single channel studies alone, it is difficult to assess what effect changes in cytosolic [ATP] may have on the sensitivity of the CICR mechanism in intact cardiac cells under more physiological conditions. In the present study, we have investigated the effects of cytosolic ATP on SR Ca2+ regulation in permeabilised rat ventricular myocytes. In this preparation, the effects of ATP on the Ca2+ uptake and release process can be studied under conditions in which the SR remains in situ. The [Ca2+] within the cell was monitored using Fura-2 or Fluo-3 fluorescence. Stepwise reductions in [ATP] were associated with corresponding decreases in the frequency and increases in the amplitude of spontaneous CICR transients. A decrease from 5 to 0.1 mm ATP, reduced the CICR frequency and almost doubled the amplitude of the Ca2+ transient. Marked prolongation of the spontaneous CICR transient occurred when [ATP] was further reduced to 10 μM, consistent with inhibition of the SR Ca2+ pump. These effects on spontaneous Ca2+ release were compared with CICR triggered by a rapid increase in [Ca2+] induced by flash photolysis of the caged calcium chelator Nitr-5. Release triggered by flash photolysis at regular time intervals appeared more sensitive to the cytosolic [ATP] and was generally delayed by a decrease to 2.5 mm. In the presence of 0.1–0.2 mm ATP, some preparations exhibited release ‘alternans’, whereby every alternate trigger induced a response. The mechanism underlying these effects of cytosolic ATP on CICR and the possible relevance to ischaemia or anoxia are considered.

The effects of Pion sarcoplasmic reticulum (SR) Ca2+regulation were studied in mechanically skinned rat skeletal muscle fibers. Brief application of caffeine was used to assess the SR Ca2+content, and changes in concentration of Ca2+([Ca2+]) within the cytosol were detected with fura 2 fluorescence. Introduction of Pi(1–40 mM) induced a concentration-dependent Ca2+efflux from the SR. In solutions lacking creatine phosphate (CP), the amplitude of the Pi-induced Ca2+transient approximately doubled. A similar potentiation of Pi-induced Ca2+release occurred after inhibition of creatine kinase (CK) with 2,4-dinitrofluorobenzene. In the presence of ruthenium red or ryanodine, caffeine-induced Ca2+release was almost abolished, whereas Pi-induced Ca2+release was unaffected. However, introduction of the SR Ca2+ATPase inhibitor cyclopiazonic acid effectively abolished Pi-induced Ca2+release. These data suggest that Piinduces Ca2+release from the SR by reversal of the SR Ca2+pump but not via the SR Ca2+channel under these conditions. If this occurs in intact skeletal muscle during fatigue, activation of a Ca2+efflux pathway by Pimay contribute to the reported decrease in net Ca2+uptake and increase in resting [Ca2+].

1. The effect of creatine phosphate (PCr) on sarcoplasmic reticulum (SR) Ca2+ regulation was studied in mechanically skinned skeletal muscle fibres from rat extensor digitorium longus (EDL). Preparations were perfused with solutions mimicking the intracellular milieu and the [Ca2+] within the muscle was monitored continuously using fura-2. 2. Brief application of 40 mM caffeine caused a transient increase in [Ca2+] due to SR Ca2+ release, and an associated tension response. Withdrawal of PCr resulted in (i) a slow transient release of Ca2+ from the SR (ii) a marked prolongation of the descending phase of the caffeine-induced fluorescence ratio transient and (iii) a decrease in the Ca2+ transient amplitude to 69.2 +/- 2.7 % (n = 16) of control responses. 3. Prolongation of the caffeine-induced Ca2+ transient also occurred following application of the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). This suggests that (i) the descending phase of the caffeine-induced Ca2+ transient is dependent on the rate of Ca2+ uptake by the SR and (ii) prolongation associated with PCr withdrawal may also reflect a decrease in the net Ca2+ uptake rate. 4. The effects of PCr withdrawal were mimicked by addition of the creatine kinase (CK) inhibitor 2,4-dinitro-1-fluorobenzene (DNFB). Hence, reducing the [PCr] may influence SR Ca2+ regulation by limiting local ATP regeneration by endogenous CK. After treatment with DNFB, PCr withdrawal had no effect on the Ca2+ transient, confirming that PCr does not have an additional direct effect on the SR. 5. The Ca2+ efflux associated with PCr withdrawal was insensitive to ryanodine or Ruthenium Red, but was effectively abolished by pretreatment with the SR Ca2+ pump inhibitor cyclopiazonic acid (CPA). This suggests that the Ca2+ efflux associated with PCr withdrawal is independent of the SR Ca2+ channel, but may involve reversal or inhibition of the Ca2+ ATPase. 6. These data suggest that Ca2+ regulation by the SR is strongly dependent on the supply of ATP via endogenous CK. Depletion of PCr may contribute to impaired SR Ca2+ regulation known to occur in intact skeletal muscle under conditions of fatigue.

1. The effect of caffeine and adenine nucleotides on the sarcoplasmic reticulum (SR) Ca2+ release mechanism was investigated in permeabilized frog skeletal muscle fibres. Caffeine was rapidly applied and the resulting release of Ca2+ from the SR detected using fura-2 fluorescence. Decreasing the [ATP] from 5 to 0.1 mM reduced the caffeine-induced Ca2+ transient by 89 +/- 1.4% (mean +/- s.e.m., n = 16), while SR Ca2+ uptake was unaffected. 2. The dependence of caffeine-induced Ca2+ release on cytosolic [ATP] was used to study the relative ability of other structurally related compounds to substitute for, or compete with, ATP at the adenine nucleotide binding site. It was found that AMP, ADP and the non-hydrolysable analogue adenylyl imidodiphosphate (AMP-PNP) partially substituted for ATP, although none was as potent in facilitating the Ca2+-releasing action of caffeine. 3. Adenosine reversibly inhibited caffeine-induced Ca2+ release, without affecting SR Ca2+ uptake. Five millimolar adenosine markedly reduced the amplitude of the caffeine-induced Ca2+ transient by 64 +/- 4% (mean +/- s.e.m., n = 11). The degree of inhibition was dependent upon the cytosolic [ATP], suggesting that adenosine may act as a competitive antagonist at the adenine nucleotide binding site. 4. These data show that (i) the sensitivity of the in situ SR Ca2+ channel to caffeine activation is strongly dependent upon the cytosolic [ATP], (ii) the number of phosphates attached to the 5' carbon of the ribose ring influences the efficacy of the ligand, and (iii) removal of a single phosphate group transforms AMP from a partial agonist, to adenosine, which acts as a competitive antagonist under these conditions.

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

Previous studies have used analysis of Ca(2+) sparks extensively to investigate both normal and pathological Ca(2+) regulation in cardiac myocytes. The great majority of these studies used line-scan confocal imaging. In part, this is because the development of open-source software for automatic detection of Ca(2+) sparks in line-scan images has greatly simplified data analysis. A disadvantage of line-scan imaging is that data are collected from a single row of pixels, representing only a small fraction of the cell, and in many instances x-y confocal imaging is preferable. However, the limited availability of software for Ca(2+) spark analysis in two-dimensional x-y image stacks presents an obstacle to its wider application. This study describes the development and characterization of software to enable automatic detection and analysis of Ca(2+) sparks within x-y image stacks, implemented as a plugin within the open-source image analysis platform ImageJ. The program includes methods to enable precise identification of cells within confocal fluorescence images, compensation for changes in background fluorescence, and options that allow exclusion of events based on spatial characteristics.

We have investigated the effect of the selective ‘exchange protein directly activated by cAMP' (Epac) activator 8-pCPT-2'-O-Me-cAMP (8-CPT) on the viability of adult rat ventricular myocytes (ARVMs), in the presence or absence or isoproterenol (ISO). ARVMs were exposed to 8-CPT, ISO or ISO + 8-CPT for 24 hours under cell culture conditions. In the presence of 8-CPT alone, cell viability decreased by 6.6 ± 2.6% (mean ± s.e.m, n=14) although this effect was not significant (p>0.05). ISO (0.1 or 1 μM) decreased cell viability by 40.9 ± 2.7 and 51.4 ± 3.25 % respectively (n=14, p

Statins are widely prescribed for the treatment of hypercholesterolemia. Although effective in lowering cholesterol levels, statin use has been associated with muscle weakness, myopathies and, in rare cases, rhabdomyolysis. The mechanisms underlying toxicity have not been elucidated, however, cerivastatin, a compound removed from the market due to reports of rhabdomyolysis, has been shown to trigger Ca2+ release from isolated skeletal sarcoplasmic reticulum (SR) vesicles (Inoue et al., 2003). To investigate whether statins can directly activate RyR channels, we incorporated single sheep cardiac RyR2 or mouse skeletal RyR1 channels into planar phospholipid bilayers under voltage-clamp conditions and added simvastatin, a commonly prescribed statin, to the cytosolic channel side. In the presence of 10 μM cytosolic [Ca2+], 10 μM simvastatin (carboxylate form), significantly increased RyR1 open probability (Po) from 0.020±0.013 to 0.269±0.068 (SEM, n=13, p

To The Editor: We read with interest the recent article by Liu et al, in Circulation Research , on the mechanism underlying the antiarrhythmic effects of flecainide in catecholaminergic polymorphic ventricular tachycardia (CPVT).1 They conclude that Na+ channel block but not inhibition of the cardiac ryanodine receptor (RyR2) has a key role in the antiarrhythmic effects of flecainide in CPVT, because they found flecainide suppresses triggered activity without a reduction of Ca2+ waves in RyR2R4496C+/− mice. However, we have previously reported that flecainide directly inhibits RYR2 and thereby prevents CPVT.2 Evidence that flecainide reduces the frequency of triggered beats to a greater extent than that of spontaneous Ca2+ waves in cardiac calsequestrin Casq2 null (Casq2−/−) mice, another model of CPVT, led us to propose a dual mode of flecainide action in CPVT: suppression of …