Activation Of Ryanodine Receptors Research Articles

Role of Nociceptor CaMKII in Transition from Acute to Chronic Pain (Hyperalgesic Priming) in Male and Female Rats

We have previously shown that activation of protein kinase Cε (PKCε) in male rats induces a chronic, long-lasting change in nociceptors such that a subsequent exposure to proinflammatory mediators produces markedly prolonged mechanical hyperalgesia. This neuroplastic change, hyperalgesic priming, is dependent on activation of cytoplasmic polyadenylation element-binding protein (CPEB), downstream of PKCε, and consequent translation of mRNAs in the peripheral terminal of the nociceptor. Since α calmodulin-dependent protein kinase II (αCaMKII), a molecule implicated in neuroplasticity, is a target of CPEB and can also affect CPEB function, we investigated its role in the transition from acute to chronic pain. Priming induced by direct activation of PKCε can be prevented by inhibition of αCaMKII. In addition, direct activation of αCaMKII induces priming, which was not prevented by pretreatment with PKCε antisense, suggesting that αCaMKII is downstream of PKCε in the induction of priming. Activation of ryanodine receptors (RyRs), which can lead to activation of αCaMKII, also induced priming, in a calcium- and αCaMKII-dependent manner. Similarly, inhibition of the RyR and a calcium buffer prevented induction of priming by PKCε. Unlike activation of PKCε, ryanodine and αCaMKII induced priming in female as well as male rats. Our results demonstrate a contribution of αCaMKII to induction of hyperalgesic priming, a phenomenon implicated in the transition from acute to chronic pain.

Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists

We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K(+) (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca(2+), resulting from ryanodine receptor (RyR) activation via Ca(2+)-induced Ca(2+) release, triggered by Ca(2+) influx through L-type Ca(2+) (CaV) channels. Carbachol inhibited tBK current by reducing Ca(2+) influx and Ca(2+) release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca(2+) was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd(2+) (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca(2+) release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca(2+) release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on urethral tone.

Structure–activity relationship of non-coplanar polychlorinated biphenyls toward skeletal muscle ryanodine receptors in rainbow trout (Oncorhynchus mykiss)

Research addressing the health impacts of polychlorinated biphenyls (PCBs) has primarily focused on the effects of coplanar, or dioxin-like (DL), congeners, which is especially true for research assessing impacts in fish species. Ortho substituted non-coplanar, termed non-dioxin-like (NDL), PCBs have received less attention. In mammals, NDL PCBs enhance the activity of ryanodine receptors (RyR), calcium release channels necessary for engaging excitation–contraction (EC) coupling in striated muscle. We utilized in vitro receptor binding analysis to determine whether NDL PCB congeners detected in aquatic environments alter the activity of RyR isoform 1 (RyR1) found in the skeletal muscle of rainbow trout. Congeners 52, 95, 136, and149 were the most efficacious leading to an increase in receptor activity that was approximately 250% greater than that found under solvent control conditions. Other environmentally relevant congeners, namely PCB 153, 151 and 101, which all contain two or more chlorines in the ortho-position, enhanced receptor activity by greater than 160% of baseline. The mono-ortho congeners or the non-ortho PCB 77 had negligible impact on the RyR1. When combined, in binary or environmentally relevant mixtures, congeners shown to enhance receptor activity appeared to display additivity and when the active PCB 95 was present with the non-active congener PCB 77 the impact on receptor activity was reduced from 250% to 230%. The important role of the RyR and the demonstrated additive nature of NDL congeners toward altering channel function calls for further investigation into the ecological implications of altered RyR function in fish with high PCB burdens.

Role of Ryanodine Receptors in the Effects of Hydrogen Sulfide on Transmitter Release from the Frog Motor Nerve Ending

We studied the role of ryanodine receptors in the effects of hydrogen sulfide on transmitter release from frog motor nerve ending. Sodium hydrosulfide (300 μM), a donor of hydrogen sulfide, reversibly increased the frequency of miniature endplate current without changes in its amplitude-time parameters. These effects were associated with reversible increase in endplate current amplitude, which was abolished by activation of ryanodine receptors of intracellular Ca(2+)stores with caffeine (3 mM) and ryanodine (0.5 μM). Under conditions of ryanodine receptors blockade with ryanodine (10 μM), sodium hydrosulfide had no effect on induced transmitter release, but its effects remained unchanged during ryanodine receptors blockade with dantrolene (25 μM). We concluded that an enhanced acetylcholine release induced by hydrogen sulfide is related to an increase of intracellular Ca(2+)concentration due to activation of ryanodine receptors for intracellular Ca(2+)-pool.

Long term hypoxia impairs ryanodine receptor function and regulation by cyclic nucleotides in immature and mature pulmonary arterial myocytes

Calcium sparks are due to ryanodine receptor (RyR) activation and are intimate to local and global Ca2+ signals in pulmonary arterial (PA) myocytes. One possibility is that impaired RyR activity contributes to altered PA reactivity following long‐term hypoxia (LTH) in developing arteries. Cyclic nucleotides (CNs), including cAMP and cGMP, can augment localized Ca2+ sparks and global Ca2+ waves due to RyR activation. Yet, the influence of LTH on CN regulation of RyRs of PA myocytes is unknown. These studies tested the hypothesis that cAMP and cGMP increase Ca2+ sparks and waves in PA myocytes and that LTH moderates this effect. Spark and wave activity was evaluated by visual analysis of line‐scan confocal recordings of PA myocytes from term‐fetal, ~ 10 day old, or adult sheep that lived at low (720 m) or at high altitude (3,200 m) for >;100 days. Similar percentages of term‐fetal, newborn, and adult myocytes had sparks and waves before CN treatment. LTH blunted spark activity and impaired CN reduction of sparks in fetus and adult. However, CNs inhibited sparks of LTH but not normoxic newborns. Ca2+ wave activity and regulatory influences by CNs were diminished by LTH in all age groups. The data suggest the effects of LTH on CN dependent regulation of RyRs may contribute to PA dysfunction and are highly dependent on the developmental age examined. (NSF MRI923559, NIH R03HD69746, P01HD31226, R01HD3807, LLU basic sciences fellowship)

Alterations in Intracellular Ca2+ Release via CD38‐Dependent Pathways in Pulmonary Arterial Smooth Muscle of Rat by Chronic Hypoxia

CD38 is a multifunctional enzyme for synthesis of cyclic ADP‐ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), which activate ryanodine receptors of sarcoplasmic reticulum and NAADP‐sensitive two‐pore channels (TPCs) of lysosomal stores, respectively, leading to intracellular Ca2+ release. It is thought to play important roles in regulation of intracellular Ca2+ concentration ([Ca2+]i) and vascular functions. Ca2+ homeostasis in pulmonary arteries (PAs) is altered during the development of pulmonary hypertension by chronic hypoxia (CH). However, it still remains unclear whether those CD38–dependent pathways are affected. Here, we examined the changes in the expression of CD38 and TPCs in endothelium denuded PA, and angiotensin II (AngII)‐induced Ca2+ release (AICR) in PA smooth muscle cells (PASMCs). The expressions of CD38 and TPCs in PAs of CH rats were significantly increased. In addition, AICR was found to be mediated in part via the CD38‐dependent pathways. AICR in PASMCs with hypoxic exposure was significantly enhanced compared to control and was reduced by various types of inhibitors for the CD38‐dependent pathway. In summary, our results show that CH exposure leads to changes in expression of CD38 and TPC1/2 in PA smooth muscle as well as increased their associated responses in PASMCs, which may contribute to the alterations in Ca2+ homeostasis. (Supported by R01‐HL075134)

Quercetin as a fluorescent probe for the ryanodine receptor activity in Jurkat cells

Because channels of intracellular organelles are not directly accessible to the patch-clamp technique, the activity (open probability) of intracellular ion channels in intact cells has so far eluded direct examination. Here, we present strong evidence that the ratio F380/F440 of the quercetin-specific cellular fluorescence emitted at 540nm upon excitation at 380/440nm reflects the open probability of an endoplasmic reticulum Ca(2+) release channel, the ryanodine receptor (RyR), in both intact and permeabilized Jurkat cells. The time course of the Ca(2+) release signal induced by high levels of quercetin in intact cells and that of F380/F440 were strongly correlated. The RyR specific inhibitor, ryanodine, the RyR type 3 and 1 but not type 2 specific inhibitor, dantrolene, as well as the non-specific RyR inhibitor, ruthenium red, depressed consistently the quercetin-induced Ca(2+) transient. Confocal microscopy confirmed that the dual fluorescent signal emitted by quercetin colocalizes with the endoplasmic reticulum, not the mitochondria. A novel regulatory mechanism was identified whereby RyR activity under physiological conditions is partially suppressed (hindered channel), whereas the channel becomes nearly fully activated after exposure to millimolar concentrations of bulk cytosolic Ca(2+) and subsequent chelation of Ca(2+) (rectified channel). Upon rectification, the dependence of F380/F440 on the cytosolic Ca(2+) concentration was remarkably similar to that of the open probability of the RyR type 3, not 1 or 2, reported from bilayer experiments. So, quercetin appears to be a semi-specific fluorescent probe for the activity of ryanodine receptors, which in our Jurkat (clone E6.1) cell preparations probably reports the type 3 RyR activity.

Dual Effects of Isoflurane on Proliferation, Differentiation, and Survival in Human Neuroprogenitor Cells

Previous studies have demonstrated that isoflurane can provide both neuroprotection and neurotoxicity in various tissue culture models and in rodent developing brains. The cellular and molecular mechanisms mediating these dual effects are not clear, but the exposure level and duration of isoflurane appear to be determinant factors. Using the ReNcell CX (Millipore, Billerica, MA) human neural progenitor cell line, the authors investigated the impact of prolonged exposure to varying isoflurane concentrations on cell survival and neurogenesis. In addition, the authors assessed the impact of short isoflurane preconditioning on elevation of cytosolic Ca concentration and cytotoxic effects mediated by prolonged isoflurane exposures and the contribution of inositol-1,4,5-trisphosphate or ryanodine receptor activation to these processes. Short exposures to low isoflurane concentrations promote proliferation and differentiation of ReNcell CX cells, with no cell damage. However, prolonged exposures to high isoflurane concentrations induced significant ReNcell CX cell damage and inhibited cell proliferation. These prolonged exposures suppressed neuronal cell fate and promoted glial cell fate. Preconditioning of ReNcell CX cultures with short exposures to low concentrations of isoflurane ameliorated the effects of prolonged exposures to isoflurane. Pretreatment of ReNcell cultures with inositol-1,4,5-trisphosphate or ryanodine receptor antagonists mostly prevented isoflurane-mediated effects on survival, proliferation, and differentiation. Finally, isoflurane-preconditioned cultures showed significantly less isoflurane-evoked changes in calcium concentration. The commonly used general anesthetic isoflurane exerts dual effects on neuronal stem cell survival, proliferation, and differentiation, which may be attributed to differential regulation of calcium release through activation of endoplasmic reticulum localized inositol-1,4,5-trisphosphate and/or ryanodine receptors.

Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice

Muscle strength declines with age in part due to a decline of Ca(2+) release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Ca(v)1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Ca(v)1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Ca(v)1.1 and the sarcoplasmic reticulum Ca(2+) storage protein calsequestrin (CASQ1). Here we show that JP45 and CASQ1 strengthen skeletal muscle contraction by modulating Ca(v)1.1 channel activity. Using muscle fibres from JP45 and CASQ1 double knockout mice, we demonstrate that Ca(2+) transients evoked by tetanic stimulation are the result of massive Ca(2+) influx due to enhanced Ca(v)1.1 channel activity, which restores muscle strength in JP45/CASQ1 double knockout mice. We envision that JP45 and CASQ1 may be candidate targets for the development of new therapeutic strategies against decay of skeletal muscle strength caused by a decrease in sarcoplasmic reticulum Ca(2+) content.

Beta amyloid peptide plaques fail to alter evoked neuronal calcium signals in APP/PS1 Alzheimer's disease mice

Alzheimer's disease (AD) is a multifactorial disorder of unknown etiology. Mechanistically, beta amyloid peptides (Aβ) and elevated Ca2+ have been implicated as proximal and likely interactive features of the disease process. We tested the hypothesis that proximity to Aβ plaque might exacerbate activity-dependent neuronal Ca2+ signaling in hippocampal pyramidal neurons from APPSWE/PS1M146V mice. Using combined approaches of whole cell patch clamp recording and 2-photon imaging of neuronal Ca2+ signals with thioflavin-S plaque labeling in hippocampal slices, we found no correlation between thioflavin-S labeled Aβ plaque proximity and Ca2+ responses triggered by ryanodine receptor (RyR) activation or action potentials in either dendrites or somata of AD mice, regardless of age. Baseline and RyR-stimulated spontaneous excitatory postsynaptic potentials also showed little difference in relation to Aβ plaque proximity. Consistent with previous studies, RyR-evoked Ca2+ release in APPSWE/PS1M146V mice was greater than in nontransgenic controls. Within the soma, RyR-evoked Ca2+ release was elevated in older APPSWE/PS1M146V mice compared with younger APPSWE/PS1M146V mice, but was still independent of plaque proximity. The results indicate that early Ca2+ signaling disruptions can become yet more severe with age through mechanisms independent of Aβ plaques, suggesting that alternative pathogenic mechanisms might contribute to AD-associated dysfunction.

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.

Type 1 Inositol (1,4,5)-Trisphosphate Receptor Activates Ryanodine Receptor 1 to Mediate Calcium Spark Signaling in Adult Mammalian Skeletal Muscle

Functional coupling between inositol (1,4,5)-trisphosphate receptor (IP(3)R) and ryanodine receptor (RyR) represents a critical component of intracellular Ca(2+) signaling in many excitable cells; however, the role of this mechanism in skeletal muscle remains elusive. In skeletal muscle, RyR-mediated Ca(2+) sparks are suppressed in resting conditions, whereas application of transient osmotic stress can trigger activation of Ca(2+) sparks that are restricted to the periphery of the fiber. Here we show that onset of these spatially confined Ca(2+) sparks involves interaction between activation of IP(3)R and RyR near the sarcolemmal membrane. Pharmacological prevention of IP(3) production or inhibition of IP(3)R channel activity abolishes stress-induced Ca(2+) sparks in skeletal muscle. Although genetic ablation of the type 2 IP(3)R does not appear to affect Ca(2+) sparks in skeletal muscle, specific silencing of the type 1 IP(3)R leads to ablation of stress-induced Ca(2+) sparks. Our data indicate that membrane-delimited signaling involving cross-talk between IP(3)R1 and RyR1 contributes to Ca(2+) spark activation in skeletal muscle.

Benzodiazepines and Benzothiazepines as Modulators of the Sarcoplasmic Reticulum Calcium ATP-ase and Ryanodine Receptors in Striated Muscle

We have reported that CGP-37157, a benzothiazepine (BZT) derivative of clonazepam utilized as a blocker of the mitochondrial Na+/Ca2+ exchanger, also activates ryanodine receptors (RyRs) and inhibits the sarcoplasmic reticulum (SR) Ca2+-stimulated ATPase (SERCA). We extended the studies to other BZT (e.g., clonazepan, diltiazem) as well as to benzodiazepines (BZD; e.g. diazepan, lorazepan). We aimed to determine if these drug classes have as a common trait the ability to modulate RyRs and/or SERCA. The effects of BZD and BZT on RyRs activity were tested in SR microsomes with a Ca2+ leak assay as well as after reconstituting RyRs into lipid bilayers. The agents tested had variable potency to increase RyR-mediated Ca2+ leak from skeletal SR microsomes. As an example, while diazepam significantly increased RyR activity, most of the others (clonazepam, lorazepam) had minor effects at high doses. In contrast, diltiazem produced moderate inhibition. Planar bilayer studies confirmed the leak observations with both cardiac and skeletal RyR. In the presence of ruthenium red, most agents decreased the rate of loading which indicates inhibitory effects on SERCA activity. The effects of BZT and BZD on loading correlated with a decrease in ATPase activity of SERCA-enriched skeletal SR fractions. In summary, most BZT and BZD utilized in therapeutics or as pharmacological tools have an inhibitory action on SERCA. In contrast, they show a variety of effects on RyRs ranging from inhibition to activation. Hence, the pharmacological action of BZT and BZD on cellular Ca2+ homeostasis reported in the literature of cardiac and skeletal muscle as well as other non-muscle systems may require taking into consideration the contributions of all drug-sensitive intracellular Ca2+ transporters. (Supported by NIH-GM078665 and AHA-MWA 12180038).

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 leads to the impairment of muscle function. The 1,4-benzothiazepine derivative JTV519, and 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 that SR vesicles enriched in RyR1 bound a large number of S107 molecules with micromolar affinity. The effects of S107 and FKBP12 on RyR1 were examined under conditions that altered the redox state of RyR1. In SR vesicles, S107 increased FKBP12 binding to RyR1 in presence of reduced glutathione and the NO-donor NOC12. S107 was without effect in the presence of oxidized glutathione. Addition of 0.15 μM FKBP12 to SR vesicles prevented FKBP12 dissociation, whereas in the presence of oxidized glutathione and NOC12 FKBP12 dissociation was observed in skeletal muscle homogenates that contained 0.43 μM myoplasmic FKBP12, and this dissociation was attenuated by S107. In single channel measurements, pretreatment of FKBP12-depleted RyR1s with S107 in the absence and presence of NOC12 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.

Intracellular Na+ Overload of Cardiomyocytes Causes ROS Induced CaMKII Activation, Leading to RYR Dysfunction and Diastolic Ca2+ Mishandling

Introduction: Intracellular sodium (Na+i) overload of cardiomyocytes is associated with abnormal calcium (Ca2+i) homeostasis and cell dysfunction. We hypothesized that the impaired Ca2+ handling is triggered by reactive oxygen species (ROS) -activated Ca2+/Calmodulin-dependent protein kinase II (CaMKII) which in turn on modulation of ryanodine receptor (RyR) activity, leading to Ca2+ leak from the sarcoplasmic reticulum (SR).Methods: We used confocal fluorescent imaging of intact and membrane-permeabilized rabbit ventricular cardiomyocytes to study Na+i overload, Ca2+i transients, and ROS production. Na+i overload in intact cardiomyocytes was induced by anemone toxin-II (ATX-II), a late Na+ current (INaL) enhancer. CaMKII oxidation and phosphorylation were measured using Western Blot analysis.Results: Rabbit ventricular myocytes treated with ATX-II (5 nM) exhibited Na+i overload, aberrant Ca2+i transients, and enhanced ROS production, accompanied by CaMKII oxidation and phosphorylation. CaMKII activation was reduced by the INaL inhibitor ranolazine (10 μM), by antioxidants (5 mM DDT or 10 μM Q10co), or by the CaMKII inhibitor KN93 (10 μM). These findings indicate a pathogenic cascade of events from Na+i overload to ROS production and to CaMKII activation. To determine how ROS and CaMKII contribute to the Ca2+i mishandling caused by Na+i overload, we studied spontaneous Ca2+ waves in membrane-permeabilized myocytes. Superfusion with high Na+ solutions elevated diastolic Ca2+i and accelerated Ca2+ waves frequency. These effects indicated Ca2+ leak through RyRs. These Ca2+ changes were dependent from ROS and CaMKII inhibition.Conclusions: The data show that Na+i overload causes Ca2+i mishandling through increased ROS production and CaMKII activation. Na+i overload enhances ROS production, which activates CaMKII, leading to the phosphorylation of RyRs, resulting in SR Ca2+ leak and increase in diastolic Ca2+.

Remodeling of Atrial Ca Handling in Canine Model of Chronic Heart Failure Points to Non-Triggered Mechanisms of Atrial Fibrillation

Contractile dysfunction of atria and atrial arrhythmias are common comorbidities in heart failure (HF). Although abnormal Ca handling has been recognized to underlie both reduced contractility and increased propensity to arrhythmias in failing left ventricle, its role in HF-related atrial pathogenesis is not well established. We studied Ca handling in control and HF myocytes isolated from left atrial appendage using canine model of chronic heart failure. In this model, chronic HF was associated with reduced left atrial contractility, increased inducibility of atrial fibrillation, and shortened atrial action potential. Amplitude of Ca transients and cellular shortening was significantly reduced in field-stimulated myocytes from HF compared to control group. Diminished Ca transients recorded in voltage-clamped HF myocytes were associated with reduced amplitude of the L-type Ca currents and decreased gain of excitation-contraction coupling. HF myocytes displayed spatial segregation of Ca signaling manifested in impaired propagation of Ca transient from surface to the center. Frequency of Ca sparks in permeabilzed myocytes was higher in HF, while sarcoplasmic reticulum (SR) Ca content was similar in both control and HF groups. Despite of the increased functional activity of ryanodine receptors, rate of occurrence of diastolic Ca waves and corresponding delayed after depolarizations recorded in paced intact myocytes in a presence of beta-adrenergic receptor agonist isoproterenol was significantly lower in HF than in controls. We conclude that reduced atrial contractility observed in canine model of chronic HF can be attributed, at least in part, to the decreased efficacy of Ca current to trigger SR Ca release. Furthermore, our cellular data do not support Ca-dependent triggered activity in left atrial appendage as a mechanism for atrial arrhythmia in chronic HF.

A Cytoplasmic Interaction between Junctin and RyRs with Major Consequences for RyR1 and RyR2 Activity In Vitro

Junctin is an intrinsic sarcoplasmic reticulum (SR) membrane protein in cardiac and skeletal muscle. The bulk of the protein is within the lumen of the SR where it binds to ryanodine receptor (RyR) calcium release channels and regulates Ca2+ release from the SR. We have previously reported that junctin added to the luminal solution increases RyR1 activity in lipid bilayers [1].The contributions of the luminal and cytoplasmic domains of junctin to RyR activation have not previously been explored, although it is assumed that activation depends on interactions between their luminal domains. To explore this further, we expressed junctin's C-terminal domain (Cjun), synthesised a cytoplasmic N-terminal domain peptide (Njun) and isolated full length junctin (FLjun) from skeletal muscle. Consistent with previous findings [1,2], FLjun in luminal solutions activated purified RyR1 and RyR2. We predicted that luminal Cjun would similarly activate RyRs if the proteins interact only via their luminal domains. Unexpectedly, (a) luminal Cjun strongly inhibited RyR1 and RyR2 and (b) cytoplasmic Njun activated channels to a greater degree than luminal FLjun and also bound to cytoplasmic fragments of RyR1 [3]. Neither luminal Njun, nor scrambled Njun in cytoplasmic solution influenced channel activity. To explore cytoplasmic Njun and luminal Cjun effects further, we added both domains sequentially to channels. Excess activation by Njun was reduced to FLjun levels by adding Cjun, while significant inhibition by Cjun added first was reduced by Njun, it remained significantly different from the activation by FLjun. Therefore cytoplasmic interactions between junctin and RyRs determines the overall effect of junctin on channel activity.1. Wei et al. (2009) Int J Biochem Cell Biol41;2214.2. Gyorke et al. (2004) Biophys J 86:2121.3. Mirza et al. (2013) Biophysics abstract.

Diesterified Nitrone Rescues Nitroso-Redox Levels and Increases Myocyte Contraction Via Increased SR Ca2+ Handling

Nitric oxide (NO) and superoxide (O2 −) are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O2 .− must exist at defined levels. Unfortunately, the NO and O2 .− levels are altered in many cardiomyopathies (heart failure, ischemia, hypertrophy, etc.) leading to contractile dysfunction and adverse remodeling. Hence, rescuing the nitroso-redox levels is a potential therapeutic strategy. Nitrone spin traps have been shown to scavenge O2 .− while releasing NO as a reaction byproduct; and we synthesized a novel, cell permeable nitrone, 2–2–3,4-dihydro-2H-pyrrole 1-oxide (EMEPO). We hypothesized that EMEPO would improve contractile function in myocytes with altered nitroso-redox levels. Ventricular myocytes were isolated from wildtype (C57Bl/6) and NOS1 knockout (NOS1−/−) mice, a known model of NO/O2 .− imbalance, and incubated with EMEPO. EMEPO significantly reduced O2 .− (lucigenin-enhanced chemiluminescence) and elevated NO (DAF-FM diacetate) levels in NOS1−/− myocytes. Furthermore, EMEPO increased NOS1−/− myocyte basal contraction (Ca2+ transients, Fluo-4AM; shortening, video-edge detection), the force-frequency response and the contractile response to β-adrenergic stimulation. EMEPO had no effect in wildtype myocytes. EMEPO also increased ryanodine receptor activity (sarcoplasmic reticulum Ca2+ leak/load relationship) and phospholamban Serine16 phosphorylation (Western blot). We also repeated our functional experiments in a canine post-myocardial infarction model and observed similar results to those seen in NOS1−/− myocytes. In conclusion, EMEPO improved contractile function in myocytes experiencing an imbalance of their nitroso-redox levels. The concurrent restoration of NO and O2 .− levels may have therapeutic potential in the treatment of various cardiomyopathies.

Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy

Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak. Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.

In Cardiomyocytes, Binding of Unzipping Peptide Activates Ryanodine Receptor 2 and Reciprocally Inhibits Calmodulin Binding

One hypothesis for elevated Ca(2+) leak through cardiac ryanodine receptors (ryanodine receptor 2 [RyR2]) in heart failure is interdomain unzipping that can enhance aberrant channel activation. A peptide (domain peptide corresponding to RyR2 residues 2460-2495 [DPc10]) corresponding to RyR2 central domain residues 2460-2495 recapitulates this arrhythmogenic RyR2 leakiness by unzipping N-terminal and central domains. Calmodulin (CaM) and FK506-binding protein (FKBP12.6) bind to RyR2 and stabilize the closed channel. Little is known about DPc10 binding to the RyR2 and how that may interact with binding (and effects) of CaM and FKBP12.6 to RyR2. To measure, directly in cardiac myocytes, the kinetics and binding affinity of DPc10 to RyR2 and how that affects RyR2 interaction with FKBP12.6 and CaM. We used permeabilized rat ventricular myocytes and fluorescently labeled DPc10, FKBP12.6, and CaM. DPc10 access to its binding site is extremely slow in resting RyR2 but is accelerated by promoting RyR opening or unzipping (by unlabeled DPc10). RyR2-bound CaM (but not FKBP12.6) drastically slowed DPc10 binding. Conversely, DPc10 binding significantly reduced CaM (but not FKBP12.6) binding to the RyR2. Fluorescence resonance energy transfer measurements indicate that DPc10-binding and CaM-binding sites are separate and allow triangulation of the structural DPc10 binding locus on RyR2 vs FKBP12.6-binding and CaM-binding sites. DPc10-RyR2 binding is sterically limited by the resting zipped RyR2 state. CaM binding to RyR2 stabilizes this zipped state, whereas RyR2 activation or prebound DPc10 enhances DPc10 access. DPc10-binding and CaM-binding sites are distinct but are allosterically interacting RyR2 sites. Neither DPc10 nor FKBP12.6 influences RyR2 binding of the other.