Sarcoplasmic Reticulum Vesicles Research Articles

Subconductance Gating and Voltage Sensitivity of Sarcoplasmic Reticulum K+ Channels: A Modeling Approach

Sarcoplasmic reticulum (SR) K+ channels are voltage-regulated channels that are thought to be actively gating when the membrane potential across the SR is close to zero as is expected physiologically. A characteristic of SR K+ channels is that they gate to subconductance open states but the relevance of the subconductance events and their contribution to the overall current flowing through the channels at physiological membrane potentials is not known. We have investigated the relationship between subconductance and full conductance openings and developed kinetic models to describe the voltage sensitivity of channel gating. Because there may be two subtypes of SR K+ channels (TRIC-A and TRIC-B) present in most tissues, to conduct our study on a homogeneous population of SR K+ channels, we incorporated SR vesicles derived from Tric-a knockout mice into artificial membranes to examine the remaining SR K+ channel (TRIC-B) function. The channels displayed very low open probability (Po) at negative potentials (≤0 mV) and opened predominantly to subconductance open states. Positive holding potentials primarily increased the frequency of subconductance state openings and thereby increased the number of subsequent transitions into the full open state, although a slowing of transitions back to the sublevels was also important. We investigated whether the subconductance gating could arise as an artifact of incomplete resolution of rapid transitions between full open and closed states; however, we were not able to produce a model that could fit the data as well as one that included multiple distinct current amplitudes. Our results suggest that the apparent subconductance openings will provide most of the K+ flux when the SR membrane potential is close to zero. The relative contribution played by openings to the full open state would increase if negative charge developed within the SR thus increasing the capacity of the channel to compensate for ionic imbalances.

FRET-Based RyR-Selective Detection Reveals No Significant Competition between CaM and S100A1 Binding to RyRs

Using fluorescence resonance energy transfer (FRET), we directly tested the hypothesis that S100A1 competes with calmodulin (CaM) for binding to intact, functional ryanodine receptors type I (RyR1) and II (RyR2) from skeletal and cardiac muscle, respectively. This hypothesis is largely based on competition assays using isolated sarcoplasmic reticulum (SR) membranes from skeletal muscle and evidence that S100A1 binds to peptides corresponding to one of the proposed CaM binding domains on RyR1. However, competition between S100A1 and CaM for RyR binding has not been directly detected. We targeted a donor-labeled FKBP12.6 (D-FKBP) to the cytoplasmic domain of RyR1 or RyR2 in SR vesicles isolated from pig skeletal or cardiac muscle. FRET was detected as a decrease of D-FKBP fluorescence in the presence of 100nM acceptor-labeled CaM (A-CaM) and used to index CaM binding to RyR. Upon pre-incubating SR with [S100A1] ranging from 0.01 to >100 μM, we found partial inhibition of FRET, with μM KI, for both skeletal and cardiac SR. By comparison, FRET was completely inhibited by unlabeled WT-CaM (KI ∼100nM), indicating that A-CaM and WT-CaM bind RyR with similar affinities and at the same site. Similar results were obtained using co-sedimentation assays conducted under similar experimental conditions, to detect competition between S100A1 and CaM binding to SR membranes. Taken together, these results indicate that CaM-RyR binding may not be significantly competed by S100A1 under normal physiologic conditions. Furthermore, structural analysis of FRET data suggests that S100A1 allosterically interacts with RyR-CaM binding. Initial results from a complementary FRET approach, using acceptor-labeled S100A1, further support the conclusion that S100A1 does not significantly compete with CaM-RyR binding in skeletal or cardiac muscle.

Measurement and characterization of superoxide generation from xanthine dehydrogenase: a redox-regulated pathway of radical generation in ischemic tissues.

The enzyme xanthine oxidoreductase (XOR) is an important source of oxygen free radicals and related postischemic injury. Xanthine dehydrogenase (XDH), the major form of XOR in tissues, can be converted to xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis. The conversion of XDH to XO has been assumed to be required for radical generation and tissue injury. It is also possible that XDH could generate significant quantities of superoxide, •O2–, for cellular signaling or injury; however, this possibility and its potential ramifications have not been previously considered. To unambiguously determine if XDH can be a significant source of •O2–, experiments were performed to measure and characterize •O2– generation using XDH from chicken liver that is locked in the dehydrogenase conformation. Electron paramagnetic resonance spin trapping experiments with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide demonstrated that XDH in the presence of xanthine produces significant amounts of •O2–. NAD+ and NADH inhibited the generation of •O2– from XDH in a dose-dependent manner, with NAD+ exhibiting stronger inhibition than NADH at low physiological concentrations. Decreased amounts of NAD+ and NADH, which occur during and following tissue ischemia, enhanced the generation of •O2– from XDH in the presence of xanthine. It was observed that XDH-mediated oxygen radical generation markedly depressed Ca2+-ATPase activity of isolated sarcoplasmic reticulum vesicles from cardiac muscle, and this was modulated by NAD+ and NADH. Thus, XDH can be an important redox-regulated source of •O2– generation in ischemic tissue, and conversion to XO is not required to activate radical formation and subsequent tissue injury.

A cell-penetrating phospholamban-specific RNA aptamer enhances Ca2 + transients and contractile function in cardiomyocytes

The sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a)–phospholamban (PLN) system of sarcoplasmic reticulum plays a pivotal role in regulation of intracellular Ca2+ cycling in ventricular cardiomyocytes. Given that Ca2+ cycling is impaired in heart failure, proteins that contribute to this process are potential targets for the treatment of this condition. We have now isolated PLN-specific aptamers with a phosphorothioate-modified backbone from a library of RNA molecules containing a randomized 40-nucleotide sequence by application of the systematic evolution of ligands by exponential enrichment (SELEX) protocol with a fusion protein containing the cytoplasmic region of human PLN. One of these aptamers was shortened to a 30-nucleotide oligomer (RNA-Apt30) without loss of function. RNA-Apt30 showed a high affinity for the cytoplasmic region of PLN (Kd=11nM), but it did not bind to the phosphorylated form of PLN or to a phosphomimetic mutant. It also increased SERCA2a activity in isolated cardiac SR vesicles with an EC50 of 18nM by relieving PLN-mediated inhibition. Conjugation of RNA-Apt30 to a cell-penetrating peptide allowed its delivery into adult rat cardiomyocytes, in which it enhanced both Ca2+ transients and contractile function. These effects of the aptamer were also apparent in the presence of the β-adrenergic receptor antagonist propranolol. This cell-penetrating PLN aptamer may thus provide a basis for the development of new therapeutic agents for heart failure without the need for gene transfer or a change in endogenous protein expression.

Adverse effects of doxorubicin and its metabolic product on cardiac RyR2 and SERCA2A.

The use of anthracycline chemotherapeutic drugs is restricted owing to potentially fatal cardiotoxic side effects. It has been hypothesized that anthracycline metabolites have a primary role in this cardiac dysfunction; however, information on the molecular interactions of these compounds in the heart is scarce. Here we provide novel evidence that doxorubicin and its metabolite, doxorubicinol, bind to the cardiac ryanodine receptor (RyR2) and to the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA2A) and deleteriously alter their activity. Both drugs (0.01 μM-2.5 μM) activated single RyR2 channels, and this was reversed by drug washout. Both drugs caused a secondary inhibition of RyR2 activity that was not reversed by drug washout. Preincubation with the reducing agent dithiothreitol (DTT, 1 mM) prevented drug-induced inhibition of channel activity. Doxorubicin and doxorubicinol reduced the abundance of thiol groups on RyR2, further indicating that oxidation reactions may be involved in the actions of the compounds. Ca(2+) uptake into sarcoplasmic reticulum vesicles by SERCA2A was inhibited by doxorubicinol, but not doxorubicin. Unexpectedly, in the presence of DTT, doxorubicinol enhanced the rate of Ca(2+) uptake by SERCA2A. Together the evidence provided here shows that doxorubicin and doxorubicinol interact with RyR2 and SERCA2A in similar ways, but that the metabolite acts with greater efficacy than the parent compound. Both compounds modify RyR2 and SERCA2A activity by binding to the proteins and also act via thiol oxidation to disrupt SR Ca(2+) handling. These actions would have severe consequences on cardiomyocyte function and contribute to clinical symptoms of acute anthracycline cardiotoxicity.

Amide-type local anesthetics action on the sarcoplasmic reticulum Ca-ATPase from fast-twitch skeletal muscle.

Myotoxic effects related to intracellular Ca(2+) disturbances have been reported for local anesthetics. Such effects might derive from Ca-ATPase dysfunction. The aim of this work was to describe the effect of lidocaine and bupivacaine on the sarcoplasmic reticulum (SR) Ca-ATPase from fast-twitch skeletal muscle and to identify the affected steps of the enzyme's cycle. SR sealed vesicles were isolated from rabbit fast-twitch muscles by ultracentrifugation. The effect of the anesthetics on Ca-ATPase activity was assessed with a colorimetric method and Ca(2+) binding, uptake, phosphorylation of the enzyme by ATP, Ca(2+) dissociation kinetics and phosphoenzyme formation and decomposition levels were tested with radioisotopic methods. Lidocaine and bupivacaine inhibited Ca-ATPase activity with half-maximal inhibitory concentrations (Ki) of 25.3 and 31.4mM, respectively, and the steady-state Ca(2+) transport ability with Ki values of 33.6 and 46.5mM, decreasing the maximal transport rate without modification of the Ca(2+) or ATP affinity for the enzyme. This is consistent with an absence of competition for the transport and catalytic sites. The anesthetics did not inhibit Ca(2+) binding but inhibited the phosphorylation partial reactions. Ca(2+) dissociation kinetics was not affected, but the phosphoenzyme levels were decreased, and the decomposition rate of the phosphoenzyme became faster in the presence of the anesthetics. It is concluded that lidocaine and bupivacaine at concentrations available in pharmaceutical formulations for clinical medical and dental uses inhibit the SR Ca-ATPase through inhibition of key phosphorylation steps of the enzymatic cycle.

The potassium channel opener NS1619 modulates calcium homeostasis in muscle cells by inhibiting SERCA

NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one) is widely used as a large-conductance Ca2+-activated K+ (BKCa) channel opener. It was previously reported that activation of BKCa channels by NS1619 could protect the cardiac muscle against ischaemia and reperfusion injury. This study reports the effects of NS1619 on intracellular Ca2+ homeostasis in H9C2 and C2C12 cells as well as its molecular mechanism of action. The effects of NS1619 on Ca2+ homeostasis in C2C12 and H9C2 cells were assessed using the Fura-2 fluorescence method. Ca2+ uptake by sarcoplasmic reticulum (SR) vesicles isolated from rat skeletal muscles and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity were measured. The effect of NS1619 on the isometric force of papillary muscle contraction in the guinea pig heart was also examined. H9C2 and C2C12 cells treated with NS1619 released Ca2+ from internal stores in a concentration-dependent manner. Ca2+ accumulation by the SR vesicles was inhibited by NS1619 treatment. NS1619 also decreased the activity of SERCA derived from rat skeletal muscle. The calcium release from cell internal stores and inhibition of SERCA by NS1619 are pH dependent. Finally, NS1619 had a profound effect on the isometric force of papillary muscle contraction in the guinea pig heart. These results indicate that NS1619 is a potent modulator of the intracellular Ca2+ concentration in H9C2 and C1C12 cells due to its interaction with SRs. The primary target of NS1619 is SERCA, which is located in SR vesicles. The effect of NS1619-mediated SERCA inhibition on cytoprotective processes should be considered.

A Novel Artificial MicroRNA Expressing AAV Vector for Phospholamban Silencing in Cardiomyocytes Improves Ca2+ Uptake into the Sarcoplasmic Reticulum

In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.

β1a490–508, a 19-Residue Peptide from C-Terminal Tail of Cav1.1 β1a Subunit, Potentiates Voltage-Dependent Calcium Release in Adult Skeletal Muscle Fibers

The α1 and β1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca2+ release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the β1a subunit and this effect can be mimicked by a peptide (β1a490–524) corresponding to the 35-residue C-terminal tail of the β1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the β1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (β1a490–508) is sufficient to reproduce activating effects of β1a490–524. Here we examined the effects of β1a490–508 on Ca2+ release and Ca2+ currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. β1a490–508 (25 nM) increased the peak Ca2+ release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of β1a490–508 in fibers. β1a490–508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of β1a490–508 peptide was used as negative control and produced negligible effects on Ca2+ release flux and RyR1 activity. Our results show that the β1a490–508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.

Stimulation of NOX2 in isolated hearts reversibly sensitizes RyR2 channels to activation by cytoplasmic calcium

The response of ryanodine receptor (RyR) channels to cytoplasmic free calcium concentration ([Ca2+]) is redox sensitive. Here, we report the effects of a mild oxidative stress on cardiac RyR (RyR2) channels in Langendorff perfused rat hearts. Single RyR2 channels from control ventricles displayed the same three responses to Ca2+ reported in other mammalian tissues, characterized by low, moderate, or high maximal activation. A single episode of 5min of global ischemia, followed by 1min of reperfusion, enhanced 2.3-fold the activity of NOX2 compared to controls and changed the frequency distribution of the different responses of RyR2 channels to calcium, favoring the more active ones: high activity response increased and low activity response decreased with respect to controls. This change was fully prevented by perfusion with apocynin or VAS 2870 before ischemia and totally reversed by the extension of the reperfusion period to 15min. In vitro activation of NOX2 in control SR vesicles mimicked the effect of the ischemia/reperfusion episode on the frequencies of emergence of single RyR2 channel responses to [Ca2+] and increased 2.2-fold the rate of calcium release in Ca2+-loaded SR vesicles. In vitro changes were reversed at the single channel level by DTT and in isolated SR vesicles by glutaredoxin. Our results indicate that in whole hearts a mild oxidative stress enhances the response of cardiac RyR2 channels to calcium via NOX2 activation, probably by S-glutathionylation of RyR2 protein. This change is transitory and fully reversible, suggesting a possible role of redox modification in the physiological response of cardiac RyR2 to cellular calcium influx.

Effects of endogenous cannabinoid anandamide on excitation–contraction coupling in rat ventricular myocytes

A role for anandamide (N-arachidonoyl ethanolamide; AEA), a major endocannabinoid, in the cardiovascular system in various pathological conditions has been reported in earlier reports. In the present study, the effects of AEA on contractility, Ca2+ signaling, and action potential (AP) characteristics were investigated in rat ventricular myocytes. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the fluorescent indicator fura-2 AM. AEA (1μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically evoked Ca2+ transients following caffeine application were not altered. Biochemical studies in sarcoplasmic reticulum (SR) vesicles from rat ventricles indicated that AEA affected Ca2+-uptake and Ca2+-ATPase activity in a biphasic manner. [3H]-ryanodine binding and passive Ca2+ release from SR vesicles were not altered by 10μM AEA. Whole-cell patch-clamp technique was employed to investigate the effect of AEA on the characteristics of APs. AEA (1μM) significantly decreased the duration of AP. The effect of AEA on myocyte shortening and AP characteristics was not altered in the presence of pertussis toxin (PTX, 2μg/ml for 4h), AM251 and SR141716 (cannabinoid type 1 receptor antagonists; 0.3μM) or AM630 and SR 144528 (cannabinoid type 2 receptor antagonists; 0.3μM). The results suggest that AEA depresses ventricular myocyte contractility by decreasing the action potential duration (APD) in a manner independent of CB1 and CB2 receptors.

Mechanisms of Anthracycline-Induced Dysfunction of Calcium Handling Proteins in the Heart

Anthracyclines are powerful chemotherapy agents, whose use is limited due to the onset of potentially fatal cardiotoxicity which includes arrhythmogenesis and heart failure. Several proteins important in intracellular Ca2+ signalling have been identified as drug binding targets, including the cardiac ryanodine receptor Ca2+ release channel (RyR2), the Ca2+ binding protein calsequestrin (CSQ2) and the Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA2a). The effects of the drug metabolites have been poorly characterized but are believed to be important in the devastating cardiac effects of these drugs.The functional effect of doxorubicin and its metabolite, doxorubicinol on RyR2 was assessed by adding clinically relevant drug concentrations to single RyR2 channels in lipid bilayers. Both drugs caused biphasic modulation of RyR2 activity where there was an early increase in channel activity followed by a later, inhibitory phase. RyR2 channel activation, but not inhibition, could be reversed by drug washout, typical of a ligand binding effect. Conversely, the irreversible nature of the inhibitory effect suggested a non-ligand binding effect. Treatment with doxorubicin/doxorubicinol reduced the number of thiols on RyR2, indicative of drug-induced thiol oxidation. Additionally, doxorubicinol abolished the response of RyR2 to changes in luminal Ca2+. Further experiments revealed that the loss of luminal Ca2+ sensing was due to an interaction between doxOL and CSQ2. Finally, we found that doxorubicinol inhibits SERCA2a Ca2+ uptake into SR vesicles and that this was prevented by pretreatment with DTT. These results provide novel insight into the cellular mechanisms of anthracyclines. We suggest that by targeting multiple Ca2+ handling proteins in cardiac muscle, anthracyclines severely disturb cardiomyocyte Ca2+ homeostasis and that these effects have an important role in the onset of anthracycline-mediated arrhythmia and heart failure.

HNO Enhances SERCA2a Activity and Cardiomyocyte Function by Promoting Redox-Dependent Phospholamban Oligomerization

Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca(2+) uptake and myofilament Ca(2+) sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) in a redox-dependent manner, improving Ca(2+) handling in isolated myocytes/hearts. Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln(-/-)) mice. Compared to WT, pln(-/-) myocytes displayed enhanced resting sarcomere shortening, peak Ca(2+) transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca(2+) transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln(-/-) cells/hearts. HNO enhanced SR Ca(2+) uptake in WT but not pln(-/-) SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca(2+)-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca(2+) handling in failing hearts.

Electrophoretic mobility of sarcoplasmic reticulum vesicles — Analytical model includes amino acid residues of A + P + N domain of Ca2 +-ATPase and charged lipids

This work is an experimental and theoretical study of electrostatic and hydrodynamic properties of the surface of sarcoplasmic reticulum (SR) membrane using particle electrophoresis. The essential structural components of SR membrane include a lipid matrix and a dense layer of Ca2+-ATPases embedded in the matrix. The Ca2+-ATPase layer both drives and impedes vesicle mobility. To analyze the experimental mobility data, obtained at pH4.0, 4.7, 5.0, 6.0, 7.5, and 9.0 in 0.1M monovalent (1:1) electrolyte, an analytical solution for the vesicle mobility and electroosmotic flow velocity distribution was obtained by solving the Poisson–Boltzmann and the Navier–Stokes–Brinkman equations. The electrophoretic mobility model includes two sets of charges that represent: (a) charged lipids of the lipid matrix of the vesicle core, and (b) charged amino acid residues of APN domains of Ca2+-ATPases. APN domains are assumed to form a charged plane displaced from the surface of lipid matrix. The charged plane is embedded in a frictional layer that represents the surface layer of calcium pumps. Electrophoretic mobility is driven by the charged APN domain and by lipid matrix while the surface layer provides hydrodynamic friction. The charge of APN domain is determined by ionized amino acid residues obtained from the amino acid composition of SERCA1a Ca2+-ATPase. Agreement between the measured and the predicted mobility is evaluated by the weighted sum of mobility deviation squared. This model reproduces the experimental dependence of mobility on pH and predicts that APN domains are located in the upper half of the SR vesicle surface layer.

Oxygen-coupled Redox Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+ Release Channel (RyR1): SITES AND NATURE OF OXIDATIVE MODIFICATION

In mammalian skeletal muscle, Ca(2+) release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca(2+)-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO2) through O2-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O2) versus low pO2 (1% O2), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO2-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO2 only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO2-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO2-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca(2+) channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in "hot spot" regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO2-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.

Effect of Cyclic Hydroxamic Acids Derived from Glycine and D,L-Alanine on Activity of Ca2+, Mg2+-ATPase Hydrolases of Sarcoplasmic Reticulum and Cyclic Guanosine Monophosphate Phosphodiesterase

The effects of cyclic hydroxamic acids (CHAs) derived from glycine and D,L-alanine on the enzymatic activity of Ca2+,Mg2+-ATPase from sarcoplasmic reticulum (Ca2+,Mg2+-ATPase SR) and cyclic guanosine monophosphate phosphodiesterase (PDEcGMP) were investigated. CHAs I (C5H10N2O2), II (C6H12N2O2), III (C8H15N3O2), IV (C9H17N3O2), V (C11H21N3O2), and VI (C12H23N3O2) were modulators of Ca2+,Mg2+-ATPase SR enzyme activity. Compounds I-VI decoupled to various extents the hydrolytic and transport functions of Ca2+,Mg2+-ATPase SR, disrupting the ratio of intra- and extracellular Ca2+ ions. This affected the adhesion of metastatic cells to capillary endothelium. Compounds IV and VI had the highest metastasis inhibition indices (MII%) for B-16 melanoma of 33 and 81%, respectively. This correlated with a decreased Ca2+ transmembrane transfer coefficient into SR vesicles of 0.75 for IV and 0.5 for VI compared with a [Ca2+]/[ATP] ratio in the control of 1.4. CHAs I-VI did not affect the functioning of PDEcGMP. The results enabled potential antimetastatic drugs in the CHA series to be predicted.

Istaroxime stimulates SERCA2a and accelerates calcium cycling in heart failure by relieving phospholamban inhibition

Calcium handling is known to be deranged in heart failure. Interventions aimed at improving cell Ca(2) (+) cycling may represent a promising approach to heart failure therapy. Istaroxime is a new luso-inotropic compound that stimulates cardiac contractility and relaxation in healthy and failing animal models and in patients with acute heart failure (AHF) syndrome. Istaroxime is a Na-K ATPase inhibitor with the unique property of increasing sarcoplasmic reticulum (SR) SERCA2a activity as shown in heart microsomes from humans and guinea pigs. The present study addressed the molecular mechanism by which istaroxime increases SERCA2a activity. To study the effect of istaroxime on SERCA2a-phospholamban (PLB) complex, we applied different methodologies in native dog healthy and failing heart preparations and heterologous canine SERCA2a/PLB co-expressed in Spodoptera frugiperda (Sf21) insect cells. We showed that istaroxime enhances SERCA2a activity, Ca(2) (+) uptake and the Ca(2) (+) -dependent charge movements into dog healthy and failing cardiac SR vesicles. Although not directly demonstrated, the most probable explanation of these activities is the displacement of PLB from SERCA2a.E2 conformation, independently from cAMP/PKA. We propose that this displacement may favour the SERCA2a conformational transition from E2 to E1, thus resulting in the acceleration of Ca(2) (+) cycling. Istaroxime represents the first example of a small molecule that exerts a luso-inotropic effect in the failing human heart through the stimulation of SERCA2a ATPase activity and the enhancement of Ca(2) (+) uptake into the SR by relieving the PLB inhibitory effect on SERCA2a in a cAMP/PKA independent way.

Mechanism of calsequestrin regulation of single cardiac ryanodine receptor in normal and pathological conditions

Release of Ca2+ from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca2+ regulation of SR Ca2+ release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca2+ regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca2+ regulates ryanodine receptor (RyR)2-mediated SR Ca2+ release through mechanisms localized inside the SR; one of these involves luminal Ca2+ interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function.CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca2+] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca2+ activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca2+ activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca2+ sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice.

Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine

The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca2+ release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca2+ uptake completion prevented this response. Maurocalcine enhanced equilibrium [3H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent Ki for Mg2+ inhibition of [3H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca2+] from 10μM to 0.1μM or at cytoplasmic [Mg2+]≥30μM. At 0.1μM [Ca2+], only channels that displayed poor activation by Ca2+ were readily activated by 5nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.

Stabilization of the Skeletal Muscle Ryanodine Receptor Ion Channel-FKBP12 Complex by the 1,4-Benzothiazepine Derivative S107

Activation of the skeletal muscle ryanodine receptor (RyR1) complex results in the rapid release of Ca2+ from the sarcoplasmic reticulum and muscle contraction. Dissociation of the small FK506 binding protein 12 subunit (FKBP12) increases RyR1 activity and impairs muscle function. The 1,4-benzothiazepine derivative JTV519, and the more specific derivative S107 (2,3,4,5,-tetrahydro-7-methoxy-4-methyl-1,4-benzothiazepine), are thought to improve skeletal muscle function by stabilizing the RyR1-FKBP12 complex. Here, we report a high degree of nonspecific and specific low affinity [3H]S107 binding to SR vesicles. SR vesicles enriched in RyR1 bound ∼48 [3H]S107 per RyR1 tetramer with EC50 ∼52 µM and Hillslope ∼2. The effects of S107 and FKBP12 on RyR1 were examined under conditions that altered the redox state of RyR1. S107 increased FKBP12 binding to RyR1 in SR vesicles in the presence of reduced glutathione and the NO-donor NOC12, with no effect in the presence of oxidized glutathione. Addition of 0.15 µM FKBP12 to SR vesicles prevented FKBP12 dissociation; however, in the presence of oxidized glutathione and NOC12, FKBP12 dissociation was observed in skeletal muscle homogenates that contained 0.43 µM myoplasmic FKBP12 and was attenuated by S107. In single channel measurements with FKBP12-depleted RyR1s, in the absence and presence of NOC12, S107 augmented the FKBP12-mediated decrease in channel activity. The data suggest that S107 can reverse the harmful effects of redox active species on SR Ca2+ release in skeletal muscle by binding to RyR1 low affinity sites.