ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic in Casq2 -/- Mice.

Intracellular Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in RyR2 single channel and [3H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single channels and RyR2-mediated spontaneous Ca2+ release in Casq2 -/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 minutes and half-life of 45 minutes after intraperitoneal administration of 3 mg/kg in mice. In vivo, ent-B1 significantly reduced catecholamine-induced ventricular arrhythmias in Casq2 -/- mice in a dose-dependent manner. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. SIGNIFICANCE STATEMENT: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

ent -Verticilide B1 inhibits type 2 ryanodine receptor channels and is antiarrhythmic in Casq2-/- mice.

Ca 2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca 2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent- (+)-verticilide ( ent -1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product ( nat -(-)-verticilide). Here, we examined its 18-membered ring-size oligomer ( ent -verticilide B1; " ent -B1") in single RyR2 channel assays, [ 3 H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent -B1 inhibited RyR2 single-channels and [ 3 H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca 2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent -B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent -B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent -B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity - ent -B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. Significance statement: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

Dantrolene inhibition of ryanodine channels (RyR2) in artificial lipid bilayers depends on FKBP12.6.

Dantrolene is a neutral hydantoin that is clinically used as a skeletal muscle relaxant to prevent overactivation of the skeletal muscle calcium release channel (RyR1) in response to volatile anesthetics. Dantrolene has aroused considerable recent interest as a lead compound for stabilizing calcium release due to overactive cardiac calcium release channels (RyR2) in heart failure. Previously, we found that dantrolene produces up to a 45% inhibition RyR2 with an IC50 of 160 nM, and that this inhibition requires the physiological association between RyR2 and CaM. In this study, we tested the hypothesis that dantrolene inhibition of RyR2 in the presence of CaM is modulated by RyR2 phosphorylation at S2808 and S2814. Phosphorylation was altered by incubations with either exogenous phosphatase (PP1) or kinases; PKA to phosphorylate S2808 or endogenous CaMKII to phosphorylate S2814. We found that PKA caused selective dissociation of FKBP12.6 from the RyR2 complex and a loss of dantrolene inhibition. Rapamycin-induced FKBP12.6 dissociation from RyR2 also resulted in the loss of dantrolene inhibition. Subsequent incubations of RyR2 with exogenous FKBP12.6 reinstated dantrolene inhibition. These findings indicate that the inhibitory action of dantrolene on RyR2 depends on RyR2 association with FKBP12.6 in addition to CaM as previously found.

A bivalent remipede toxin promotes calcium release via ryanodine receptor activation.

Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis, a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

RYR2 Channel Inhibition Is the Principal Mechanism of Flecainide Action in CPVT.

RATIONALE: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT. OBJECTIVE: To determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. METHODS AND RESULTS: We synthesized N-methylated flecainide analogues (QX-flecainide and N-methyl flecainide) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes-lacking intact sarcolemma and devoid of sodium channel contribution-flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N-methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas N-methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. CONCLUSIONS: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

Mammalian TRP ion channels are insensitive to membrane stretch.

TRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading and detection of cerebrospinal fluid flow. However, in many instances it is unclear whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six mammalian subfamilies. We found that these TRP channels were insensitive to short membrane stretches in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that, when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and, thus, are more likely to be activated by cytoplasmic tethers or downstream components and to act as amplifiers of cellular mechanosensory signaling cascades.

Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia.

BACKGROUND: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. METHODS: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patch-clamp recordings in isolated cardiomyocytes. RESULTS: SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. CONCLUSIONS: SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.

Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814.

Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a Kd = 121 ±â€¯14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca2+-spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.

Regulation of the RyR channel gating by Ca2+ and Mg2.

Ryanodine receptors (RyRs) are the Ca2+ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca2+ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca2+. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca2+ affinities of the order of 1 µM and 1 mM. Magnesium competes with Ca2+ at these sites to inhibit RyRs and this plays an important role in modulating their Ca2+-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca2+ and Mg2+ and how these mechanisms control Ca2+ release in excitation-contraction coupling and cardiac pacemaking.

Nerve-induced responses of mouse vaginal smooth muscle.

Neural and agonist-induced contractions of proximal (i.e. upper half adjacent to the cervix) and distal mouse vaginal smooth muscle strips were investigated. We hypothesised that nerve-mediated vaginal contractions arise through activity of cholinergic nerves. Nerve activation by bursts of electrical field stimulation (EFS) caused a primary transient contraction often accompanied by a secondary transient contraction, both larger in proximal than distal tissues (i.e. primary: 7-fold larger; secondary: 3-fold larger). Our hypothesis was supported as we found that cholinergic nerves mediated the primary transient contraction in both proximal and distal vaginal strips, as EFS responses were enhanced by neostigmine an anticholinesterase, massively inhibited by the competitive muscarinic receptor antagonist atropine and not affected by the non-selective α-adrenergic receptor antagonist phentolamine. Primary transient contractions were halved in amplitude by the L-type Ca2+ channel blocker nifedipine and markedly inhibited by the sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibitor cyclopiazonic acid (CPA). Resultant secondary transient contractions were abolished by nifedipine. Notably, the selective α1-adrenergic receptor agonist phenylephrine caused tonic contracture in distal but not proximal strips. Low-frequency EFS often initiated recurrent transient contractions similar to those elicited by CCh. Immunohistochemical studies demonstrated innervation of the smooth muscle. Findings of enhanced proximal cholinergic nerve-induced transient contractions, evidence that maintained nerve stimulation could cause recurrent contractions and the finding of distal phenylephrine-mediated tonic contraction have implications on insemination.

Cardiac Calcium Release Channel (Ryanodine Receptor 2) Regulation by Halogenated Anesthetics.

BACKGROUND: Halogenated anesthetics activate cardiac ryanodine receptor 2-mediated sarcoplasmic reticulum Ca release, leading to sarcoplasmic reticulum Ca depletion, reduced cardiac function, and providing cell protection against ischemia-reperfusion injury. Anesthetic activation of ryanodine receptor 2 is poorly defined, leaving aspects of the protective mechanism uncertain. METHODS: Ryanodine receptor 2 from the sheep heart was incorporated into artificial lipid bilayers, and their gating properties were measured in response to five halogenated anesthetics. RESULTS: Each anesthetic rapidly and reversibly activated ryanodine receptor 2, but only from the cytoplasmic side. Relative activation levels were as follows: halothane (approximately 4-fold; n = 8), desflurane and enflurane (approximately 3-fold,n = 9), and isoflurane and sevoflurane (approximately 1.5-fold, n = 7, 10). Half-activating concentrations (Ka) were in the range 1.3 to 2.1 mM (1.4 to 2.6 minimum alveolar concentration [MAC]) with the exception of isoflurane (5.3 mM, 6.6 minimum alveolar concentration). Dantrolene (10 µM with 100 nM calmodulin) inhibited ryanodine receptor 2 by 40% but did not alter the Ka for halothane activation. Halothane potentiated luminal and cytoplasmic Ca activation of ryanodine receptor 2 but had no effect on Mg inhibition. Halothane activated ryanodine receptor 2 in the absence and presence (2 mM) of adenosine triphosphate (ATP). Adenosine, a competitive antagonist to ATP activation of ryanodine receptor 2, did not antagonize halothane activation in the absence of ATP. CONCLUSIONS: At clinical concentrations (1 MAC), halothane desflurane and enflurane activated ryanodine receptor 2, whereas isoflurane and sevoflurane were ineffective. Dantrolene inhibition of ryanodine receptor 2 substantially negated the activating effects of anesthetics. Halothane acted independently of the adenine nucleotide-binding site on ryanodine receptor 2. The previously observed adenosine antagonism of halothane activation of sarcoplasmic reticulum Ca release was due to competition between adenosine and ATP, rather than between halothane and ATP.

The emerging role of calmodulin regulation of RyR2 in controlling heart rhythm, the progression of heart failure and the antiarrhythmic action of dantrolene.

Cardiac output and rhythm depend on the release and the take-up of calcium from the sarcoplasmic reticulum (SR). Excessive diastolic calcium leak from the SR due to dysfunctional calcium release channels (RyR2) contributes to the formation of delayed after-depolarizations, which underlie the fatal arrhythmias that occur in heart failure and inherited syndromes. Calmodulin (CaM) is a calcium-binding protein that regulates target proteins and acts as a calcium sensor. CaM is comprised of two calcium-binding EF-hand domains and a flexible linker. CaM is an accessory protein that partially inhibits RyR2 channel activity. CaM is critical for normal cardiac function, and altered CaM binding and efficacy may contribute to defects in SR calcium release. The present paper reviews CaM binding to RyR2 and how it regulates RyR2 channel activity. It then goes on to review how mutations in the CaM amino acid sequence give rise to inherited syndromes such as Catecholaminergic Polymorphic Ventricular Tachychardia (CPVT) and long QT syndrome (LQTS). In addition, the role of reduced CaM binding to RyR2 that results from RyR2 phosphorylation or from oxidation of either RyR2 or CaM contributes to the progression of heart failure is reviewed. Finally, this manuscript reviews recent evidence that CaM binding to RyR2 is required for the inhibitory action of a pharmaceutical agent (dantrolene) on RyR2. Dantrolene is a clinically used muscle relaxant that has recently been found to exert antiarrhythmic effects against SR Ca2+ overload arrhythmias.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene.

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca(2+)] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca(2+) release in the absence of CaM, but reduced Ca(2+) wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.

Single mechanically-gated cation channel currents can trigger action potentials in neocortical and hippocampal pyramidal neurons.

The mammalian brain is a mechanosensitive organ that responds to different mechanical forces ranging from intrinsic forces implicated in brain morphogenesis to extrinsic forces that can cause concussion and traumatic brain injury. However, little is known of the mechanosensors that transduce these forces. In this study we use cell-attached patch recording to measure single mechanically-gated (MG) channel currents and their affects on spike activity in identified neurons in neonatal mouse brain slices. We demonstrate that both neocortical and hippocampal pyramidal neurons express stretch-activated MG cation channels that are activated by suctions of ~25mm Hg, have a single channel conductance for inward current of 50-70pS and show weak selectivity for alkali metal cations (i.e., Na(+)

Polarized and persistent Ca²âº plumes define loci for formation of wall ingrowth papillae in transfer cells.

Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca(2+) by co-operative functioning of plasma membrane Ca(2+)-permeable channels and Ca(2+)-ATPases. Viewed paradermally, and proximal to the cytosol-plasma membrane interface, the Ca(2+) signal was organized into discrete patches that aligned spatially with clusters of Ca(2+)-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca(2+) were consistent with inward-directed plumes of elevated [Ca(2+)]cyt. Plume formation depended upon an alternating distribution of Ca(2+)-permeable channels and Ca(2+)-ATPase clusters. On further inward diffusion, the Ca(2+) plumes coalesced into a uniform Ca(2+) signal. Blocking or dispersing the Ca(2+) plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca(2+) plumes define the loci at which wall ingrowth papillae are deposited.

Differences in the regulation of RyR2 from human, sheep, and rat by Ca²âº and Mg²âº in the cytoplasm and in the lumen of the sarcoplasmic reticulum.

Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca(2+) and Mg(2+) plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca(2+)- and Mg(2+)-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca(2+) activation (K(a) = 4 µM) and inhibition by cytoplasmic Mg(2+) (K(i) = 10 µM at 100 nM Ca(2+)) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca(2+), RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg(2+) inhibition than those from sheep and rat. The K(a) values for luminal Ca(2+) activation were similar in the three species (35 µM for human, 12 µM for sheep, and 10 µM for rat). From the relationship between open probability and luminal [Ca(2+)], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ~10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg(2+) as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca(2+) and Mg(2+) only occurred when cytoplasmic [Ca(2+)] was <3 µM. The activation response of RyR2 to luminal and cytoplasmic Ca(2+) was strongly dependent on the Mg(2+) concentration. Addition of physiological levels (1 mM) of Mg(2+) raised the K(a) for cytoplasmic Ca(2+) to 30 µM (human and sheep) or 90 µM (rat) and raised the K(a) for luminal Ca(2+) to ~1 mM in all species. This is the first report of the regulation by Ca(2+) and Mg(2+) of native RyR2 receptor activity from healthy human hearts.

Pharmacological approaches that slow lymphatic flow as a snakebite first aid.

BACKGROUND: This study examines the use of topical pharmacological agents as a snakebite first aid where slowing venom reaching the circulation prevents systemic toxicity. It is based on the fact that toxin molecules in most snake venoms are large molecules and generally first enter and traverse the lymphatic system before accessing the circulation. It follows on from a previous study where it was shown that topical application of a nitric oxide donor slowed lymph flow to a similar extent in humans and rats as well as increased the time to respiratory arrest for subcutaneous injection of an elapid venom (Pseudonaja textilis, Ptx; Eastern brown snake) into the hind feet of anaesthetized rats. METHODOLOGY/PRINCIPAL FINDINGS: The effects of topical application of the L-type Ca(2+) channel antagonist nifedipine and the local anesthetic lignocaine in inhibiting lymph flow and protecting against envenomation was examined in an anaesthetized rat model. The agents significantly increased dye-measured lymph transit times by 500% and 390% compared to controls and increased the time to respiratory arrest to foot injection of a lethal dose of Ptx venom by 60% and 40% respectively. The study also examined the effect of Ptx venom dose over the lethal range of 0.4 to 1.5 mg/kg finding a negative linear relationship between increase in venom dose and time to respiratory arrest. CONCLUSIONS/SIGNIFICANCE: The findings suggest that a range of agents that inhibit lymphatic flow could potentially be used as an adjunct treatment to pressure bandaging with immobilization (PBI) in snakebite first aid. This is important given that PBI (a snakebite first aid recommended by the Australian National Health and Medical research Council) is often incorrectly applied. The use of a local anesthetic would have the added advantage of reducing pain.

ß-Adrenergic stimulation increases RyR2 activity via intracellular Ca2+ and Mg2+ regulation.

Here we investigate how ß-adrenergic stimulation of the heart alters regulation of ryanodine receptors (RyRs) by intracellular Ca(2+) and Mg(2+) and the role of these changes in SR Ca(2+) release. RyRs were isolated from rat hearts, perfused in a Langendorff apparatus for 5 min and subject to 1 min perfusion with 1 µM isoproterenol or without (control) and snap frozen in liquid N2 to capture their phosphorylation state. Western Blots show that RyR2 phosphorylation was increased by isoproterenol, confirming that RyR2 were subject to normal ß-adrenergic signaling. Under basal conditions, S2808 and S2814 had phosphorylation levels of 69% and 15%, respectively. These levels were increased to 83% and 60%, respectively, after 60 s of ß-adrenergic stimulation consistent with other reports that ß-adrenergic stimulation of the heart can phosphorylate RyRs at specific residues including S2808 and S2814 causing an increase in RyR activity. At cytoplasmic [Ca(2+)] <1 µM, ß-adrenergic stimulation increased luminal Ca(2+) activation of single RyR channels, decreased luminal Mg(2+) inhibition and decreased inhibition of RyRs by mM cytoplasmic Mg(2+). At cytoplasmic [Ca(2+)] >1 µM, ß-adrenergic stimulation only decreased cytoplasmic Mg(2+) and Ca(2+) inhibition of RyRs. The Ka and maximum levels of cytoplasmic Ca(2+) activation site were not affected by ß-adrenergic stimulation. Our RyR2 gating model was fitted to the single channel data. It predicted that in diastole, ß-adrenergic stimulation is mediated by 1) increasing the activating potency of Ca(2+) binding to the luminal Ca(2+) site and decreasing its affinity for luminal Mg(2+) and 2) decreasing affinity of the low-affinity Ca(2+)/Mg(2+) cytoplasmic inhibition site. However in systole, ß-adrenergic stimulation is mediated mainly by the latter.

Extraction of sub-microscopic Ca fluxes from blurred and noisy fluorescent indicator images with a detailed model fitting approach.

The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This 'forward method' allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.