Activation Of Ryanodine Receptors Research Articles

The inhibitory glutathione transferase M2-2 binding site is located in divergent region 3 of the cardiac ryanodine receptor

The muscle-specific glutathione transferase GSTM2-2 modulates the activity of ryanodine receptor (RyR) calcium release channels: it inhibits the activity of cardiac RyR (RyR2) channels with high affinity and activates skeletal RyR (RyR1) channels with low affinity. The C terminal domain of GSTM2-2 (GSTM2C) alone physically binds to RyR2 and inhibits its activity, but it does not bind to RyR1. We have now used yeast two-hybrid analysis, chemical cross-linking, intrinsic tryptophan fluorescence and Ca2+ release studies to determine that the binding site for GSTM2C is in divergent region 3 (D3) of RyR2. The D3 region encompasses residues 1855–1890 in RyR2. Specific mutagenesis shows the binding primarily involves electrostatic interactions with residues K1875, K1886, R1887 and K1889, all residues that are present in RyR2, but not in RyR1. The significant sequence differences between the D3 regions of RyR2 and RyR1 explain why GSTM2-2 specifically inhibits RyR2. This specific inhibition of RyR2 could modulate Ca cycling and be useful for the treatment of heart failure. RyR2 inhibition during diastole may improve filling of the SR with Ca2+ and improve contractility.

Presynaptic receptors regulating the time course of neurotransmitter release from vertebrate nerve endings

A number of different types of presynaptic receptors was revealed in central and peripheral chemical synapses activated both by main mediator and co-mediators released simultaneously. Physiological significance and mechanisms of functioning of these receptors are not clear yet. They are assumed to provide negative or positive feedback decreasing or increasing the number of neurotransmitter quanta released in response to nerve impulse and thus regulating synaptic transmission. At the same time, there is one more way of secretion process modulation associated with the changes of timing of transmitter release. This mechanism was shown to contribute to the efficiency of synaptic transmission. The role of presynaptic receptors in regulation of the kinetics of quanta release is one of the interesting questions of modern neurophysiology. This paper overviews the results obtained by the authors that demonstrate the contribution of presynaptic receptors of different types into the regulation of temporal parameters of quantal secretion at the vertebrates neuromuscular junction. It was shown that activation of the cholinergic nicotinic receptors leads to a decrease of the amplitude of postsynaptic response not only due to reduction of the quantity of released quanta but also due to increased the level of asynchronous release. On the contrary, the facilitating effect of catecholamines on the neuromuscular synapse is the result of activation of presynaptic β1-adrenoreceptors which leads to greater synchronization of release process and, consequently, to the increase of the amplitude of the postsynaptic response. Presynaptic purine receptors, involved in the modulation the intensity of secretion, are also capable of alteration of the time course of secretion. Activation of ryanodine receptors results in the increase of the number of quanta released with prolonged latencies leading to appearance of the phase of delayed asynchronous neurotransmitter release.

Decoding Neurodevelopment: Findings on Environmental Exposures and Synaptic Plasticity

What makes one person different from the next? In large part it’s that our individual brains are wired a bit differently; each neuron in each person’s brain has a different set of synapses connecting to a different set of neurons. When we’re born, most of the major functional subregions of the brain and their interconnections are in place, but various experiences and environmental exposures affect which synaptic connections become stronger and which become weaker. Different areas of the brain have this synaptic plasticity—or ability to grow stronger or weaker connections—at different time periods, and the timing of these “critical periods” is tightly regulated. Thermogram showing hormone receptors in the hippocampus of an infant. Exposures to certain neurotoxic agents during critical periods of synaptic plasticity may set the stage for developmental disorders. © Explorer/Photo Researchers, Inc. The field of neurobiology is humming with new findings on the intricate workings that orchestrate this process, too many to describe in one story. But of special interest are key discoveries by investigators at the National Institute of Environmental Health Sciences (NIEHS) about some of the basic mechanisms involved in synaptic plasticity, and work by other investigators that explores the hypothesis that environmental toxicants that disrupt synaptic plasticity at critical periods play a role in disorders that have roots in early brain development, such as autism spectrum disorders (ASDs), attention deficit/hyperactivity disorder, and schizophrenia.1,2

Role of Stored Calcium in the Regulation of Neurotransmitter Quantum Size

Release of stored calcium ions during activation of ryanodine receptors with ryanodine or caffeine elevates the mean amplitude of spontaneous miniature end-plate potentials. Blockade of these receptors with selective antagonists abolishes this effect. Preliminary loading of the motor nerve terminals with intracellular calcium buffer EGTA-AM, but not with BAPTA-AM, also completely prevented the ryanodine-induced increment of miniature end-plate potential amplitude probably induced by the release of stored calcium. Vesamicol, a selective blocker of acetylcholine transport into vesicles, prevented the ryanodine-induced increment of the mean amplitude of miniature end-plate potentials. This increment was 2-fold more pronounced after preliminary blockade of protein kinase C with chelerythrine and was completely abolished by blockade of protein kinase A with H-89.

Differences in locomotor performance between individuals: importance of parvalbumin, calcium handling and metabolism

Locomotor performance is linked to fitness and health of animals and is expected to be under strong selection. However, interindividual variation in locomotor performance is pronounced in many species. It was our aim to investigate the relative importance of energy metabolism and calcium handling in determining sprint and sustained locomotion in the zebrafish (Danio rerio). Sprint and sustained performance (U(crit)) varied independently from each other. Using in vivo electroporation, we found that increased parvalbumin protein concentration improved both sprint and sustained locomotion. This is the first demonstration that parvalbumin plays a role in determining whole-animal performance. High sprint performance fish had greater mRNA concentrations of the metabolic regulators PPARδ and PGC1β compared with fish with poor sprint performance. High sustained performance fish, in contrast, had greater concentrations of PGC-1α and PGC-1β. The increased expression of these metabolic regulators indicates an enhancement of the metabolic machinery in high performance animals. Sprint performance is also enhanced by creatine kinase activity, which may be associated with increased PPARδ mRNA concentration. Ryanodine receptor (RyR) and sarcoplasmic reticulum Ca(2+)-ATPase 1 (SERCA1) mRNA concentrations were significantly increased in high sustained performance fish, while parvalbumin 2, dihydropyridine (DHPR) receptor and SERCA2 mRNA levels were increased in fish with high sprint velocities. Sustained performance was more sensitive to experimentally induced decreases in RyR and DHPR activity than sprint performance. We provide mechanistic explanations of why locomotor performance differs between individuals, which is important for understanding ecological and sporting success, disease and the evolutionary processes underlying selection.

Ca2+ Heterogeneity Within a Ca2+ Spark

The Ca2+ spark is the elemental Ca2+ release event in excitation-contraction coupling. The synchronized summation of thousands (10,000 to 20,000 per cell) of these local Ca2+ release events will give rise to the normal Ca2+ transient that underlies contraction. Ca2+ sparks represent a widely found Ca2+ release process among different types of tissue (muscles, neurons and even non-excitable cells) and species (rat, mouse, guinea-pig, rabbit, dog, cat, human) although the details are tissue and species specific. Ca2+ sparks are the local events showing the increase of Ca2+ in the cytosol following activation of ryanodine receptors (RyR) at the junctional sarcoplasmic reticulum (jSR). This increase of [Ca2+] in the cytosol is matched by a decrease of Ca2+ within the jSR (Ca2+ blink). Ca2+ blinks were detected by using the low affinity Ca2+ indicator fluo-5N loaded into the SR. Ca2+ blinks are even more localized events (FWHM=1 μM versus 2.2 μM for sparks) than Ca2+ sparks, reflecting the inner structure of the SR. Simultaneous visualization of Ca2+ sparks and Ca2+ blinks has allowed the detection of a new sub-population of Ca2+ release events that are smaller than Ca2+ sparks and these have been called “quarky Ca2+ release events (QCR)” (Brochet et al., 2011) because they are intermediate in size between Ca2+ release by a single RyR channel and the entire cluster of RyR in the jSR. QCR events have also been hypothesized to be commingled with the dynamics of Ca2+ release during a spark. Here, we visualize heterogeneity of Ca2+ release within a spark. The importance of this observation and its relationship with the organization of RyR at the jSR will be discussed. These results provide important new understanding of cardiac Ca2+ signaling.

Constitutive lysosome exocytosis releases ATP and engages P2Y receptors in human monocytes

Elucidating mechanisms by which Ca(2+) signals are generated by monocytes is important for understanding monocyte function in health and disease. We have investigated mechanisms underlying Ca(2+) signals generated following disruption of lysosomes by exposure to the cathepsin C substrate glycyl-L-phenylalanine-β-napthylamide (GPN). Exposure to 0.2 mM GPN resulted in robust increases in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence of extracellular Ca(2+). The response was antagonised by thapsigargin and evoked capacitative Ca(2+) entry. Dantrolene-sensitive Ca(2+) responses were observed at higher concentrations of GPN (0.4 mM) but not at 0.2 mM. Strikingly, GPN-evoked Ca(2+) responses and β-hexosaminidase secretion were inhibited by the ATPase/ADPase apyrase. Simultaneous measurement of [Ca(2+)](i) and extracellular ATP revealed a concomitant secretion of ATP during GPN-evoked Ca(2+) signalling. Furthermore, the ability of GPN to raise [Ca(2+)](i) was inhibited by P2Y receptor antagonists or by inhibiting vesicular exocytosis with N-ethylmaleimide (NEM). NEM treatment was associated with an inability of GPN to trigger ATP secretion, a drop in baseline [Ca(2+)](i) and reduction in extracellular ATP concentration. Antagonism of purinergic signalling also caused a reduction in baseline [Ca(2+)](i). In summary, these data suggest that P2Y receptor activation contributes significantly to GPN-evoked Ca(2+) signalling, and that constitutive secretion of lysosomal ATP is a major determinant of Ca(2+) homeostasis in monocytes. Lysosomal Ca(2+) stores can communicate with ER Ca(2+) stores either directly through activation of ryanodine receptors, or indirectly through release of ATP and engagement of P2Y receptors.

Can the Sodium-Calcium Exchanger Initiate or Supress Calcium Sparks in Cardiac Myocytes?

Calcium (Ca) sparks in cardiac myocyte are formed by positive feedback (Ca induced Ca release). At the resting membrane potential (Vm ∼-80 mV), the sodium-calcium exchanger (NCX) removes Ca from the cell (forward mode). If Ca released from the sarcoplasmic reticulum (SR) is quickly removed via forward mode NCX before the positive feedback process starts, the Ca release becomes non-spark Ca leak. However, at higher Vm, Ca enters through the NCX (reverse mode) and Ca entry through reverse mode NCX may activate ryanodine receptors (RyRs) and initiate Ca sparks. These are also influenced by the cleft/non-cleft distribution of NCX, which is still unknown. In this study, using physiologically detailed mathematical model of the subcellular Ca cycling, we investigate how the magnitude of NCX current and the distribution of NCXs alter Ca spark formation. We find that at the resting membrane potential if all NCXs are localized to the cleft, Ca sparks are significantly reduced (at [Ca]SR=700 μM, there is a 40% reduction vs. the case where all NCXs locate outside of the cleft). During excitation-contraction coupling, most Ca sparks are induced by L-type Ca current (ICaL) and only a small fraction of Ca sparks are due to NCX current at Vm = 0mV. If Vm is higher (+40 mV), since ICaL becomes smaller and NCX becomes larger, more sparks are induced by NCX. But the absolute number of Ca sparks is limited. These results also strongly depend on the distribution of NCXs. If many NCXs locate close to RyRs, NCX currents initiate Ca sparks much more efficiently. This underscores the criticality of NCX localization regarding functional impact on SR Ca release.

Control of Diastolic Activity of the RyR2 Channel by Luminal Calcium and ATP

The mechanism of activation of the cardiac ryanodine receptor (RyR2) by luminal Ca2+ in the presence of ATP was examined in planar lipid bilayers. The dose response of the RyR2 channel to ATP was characterized at a range of cytosolic (100-400 nM) and luminal (0.01 - 53 mM) Ca2+ concentrations. Luminal Ca2+ markedly increased the maximal open probability in the presence of ATP (POmax) and markedly decreased EC50 for ATP (EC50ATP). Cytosolic Ca2+, without substantially activating the RyR2 channel in the absence of ATP, greatly amplified the effects of 1 mM [Ca2+]L on POmax and EC50ATP. An allosteric model of RyR2 interaction with ATP showed that the increase in RyR2 open probability by luminal Ca2+ in the presence of ATP is induced by a decrease of the ATP dissociation constant (KATP) at low [Ca2+]L, by an increase in the positive allosteric effect of ATP on channel opening (decrease of fATP) at intermediate [Ca2+]L, and by an increase in the stability of the ATP-free RyR2 open state (decrease of KO0) at the highest [Ca2+]L. Increasing [Ca2+]C did not affect KATP but led to a parallel decrease of KO0 and fATP. These results suggest that the allosteric effect of ATP might be mediated by the energy of the ATP-free open state, i.e., indirectly by the effect of the occupancy of the channel by Ca2+ at the cytosolic and luminal sites on the stability of the open state. The increase of RyR2 open probability at diastolic levels of cytosolic Ca2+ by elevated luminal Ca2+ may play a role in the calcium overload induced Ca2+ release.Supported by APVV-0721-10, APVV-0628-10, VEGA2/0118/09 and VEGA 2/0190/10

Inhibition of Akt Attenuates Isoproterenol-Induced RyR-Dependent Diastolic Ca2+ Release in Rabbit Ventricular Myocytes

Cardiac ryanodine receptor (RyR)-dependent diastolic SR Ca2+ release is increased by beta-adrenergic receptor (ß-AR) stimulation. Increased diastolic RyR activity can lead to arrhythmogenic spontaneous SR Ca2+ release. Our studies have shown that calmodulin-dependent protein kinase II (CaMKII) activation by nitric oxide (NO) implicates nitric oxide synthase (NOS) in the pathway. Here we investigate the role Akt may play in activating NOS, thus increasing diastolic SR Ca2+ release during ß-AR stimulation in isolated rabbit ventricular myocytes. Western blot analysis showed a dose-dependent increase in phosphorylated Akt in response to the β-AR agonist isoproterenol (ISO). Cytosolic Ca2+ was measured using fluo-4 loaded intact myocytes. SR Ca2+ was varied by field-stimulated to steady state at different frequencies. Tetracaine (1mM) was used to rapidly and reversibly block the RyR. The tetracaine-dependent shift of Ca2+ from the cytosol to the SR is proportional to diastolic Ca2+ release. Primary myocytes were treated with 250nM ISO with and without Akt inhibitor-X (AIX; 5μM; 30min pre-incubation and continuous perfusion). ISO-induced increase in the diastolic SR Ca2+ release was abolished by the treatment of myocytes with AIX. When data were selected such that SR[Ca2+] was matched in each group (ISO: 155.22±5.1μM; ISO+AIX: 153.92±3.8μM), myocytes treated with ISO had significantly higher tetracaine-dependent increase in diastolic SR[Ca2+] (12.43±3.8μM) vs. those treated with ISO and AIX (1±2.2μM) (P=0.01, t-test). The results suggest that the ISO-dependent increase in diastolic SR Ca2+ release is dependent upon Akt. To further test this hypothesis myocytes were cultured for 24 hours with adenovirus to express a dominant-negative Akt construct (Akt-dn). Preliminary evidence shows reduced diastolic SR[Ca2+] release for a given SR[Ca2+] in Akt-dn myocytes. This evidence indicates that Akt activation may be an important upstream effector involved in the β-AR-induced diastolic SR Ca2+ release.

SRP-35, a newly identified protein of the skeletal muscle sarcoplasmic reticulum, is a retinol dehydrogenase

In the present study we provide evidence that SRP-35, a protein we identified in rabbit skeletal muscle sarcoplasmic reticulum, is an all-trans-retinol dehydrogenase. Analysis of the primary structure and tryptic digestion revealed that its N-terminus encompasses a short hydrophobic sequence bound to the sarcoplasmic reticulum membrane, whereas its C-terminal catalytic domain faces the myoplasm. SRP-35 is also expressed in liver and adipocytes, where it appears in the post-microsomal supernatant; however, in skeletal muscle, SRP-35 is enriched in the longitudinal sarcoplasmic reticulum. Sequence comparison predicts that SRP-35 is a short-chain dehydrogenase/reductase belonging to the DHRS7C [dehydrogenase/reductase (short-chain dehydrogenase/reductase family) member 7C] subfamily. Retinol is the substrate of SRP-35, since its transient overexpression leads to an increased production of all-trans-retinaldehyde. Transfection of C2C12 myotubes with a fusion protein encoding SRP-35-EYFP (enhanced yellow fluorescent protein) causes a decrease of the maximal Ca²⁺ released via RyR (ryanodine receptor) activation induced by KCl or 4-chloro-m-chresol. The latter result could be mimicked by the addition of retinoic acid to the C2C12 cell tissue culture medium, a treatment which caused a significant reduction of RyR1 expression. We propose that in skeletal muscle SRP-35 is involved in the generation of all-trans-retinaldehyde and may play an important role in the generation of intracellular signals linking Ca2+ release (i.e. muscle activity) to metabolism.

Regulation of sarcoplasmic reticulum Ca²⁺ leak by cytosolic Ca²⁺ in rabbit ventricular myocytes.

Sarcoplasmic reticulum (SR) Ca(2+) leak determines SR Ca(2+) content and, therefore, the amplitude of global Ca(2+) transients in ventricular myocytes. However, it remains unresolved to what extent Ca(2+) leak can be modulated by cytosolic [Ca(2+)] ([Ca(2+)](i)). Here, we studied the effects of [Ca(2+)](i) on SR Ca(2+) leak in permeabilized rabbit ventricular myocytes. Using confocal microscopy we monitored SR Ca(2+) leak as the change in [Ca(2+)](SR) (with Fluo-5N) after complete SERCA inhibition with thapsigargin (10 μm). Increasing [Ca(2+)](i) from 150 to 250 nM significantly increased SR Ca(2+) leak over the entire range of [Ca(2+)](SR). This increase was associated with an augmentation of both Ca(2+) spark- and non-spark-mediated Ca(2+) leak. Further increasing [Ca(2+)](i) to 350 nM led to rapid [Ca](2+)](SR) depletion due to the occurrence of Ca(2+) waves. The augmentation of SR Ca(2+) leak by high [Ca(2+)](i) was insensitive to inhibition of Ca(2+)-calmodulin-dependent protein kinase II. In contrast, lowering [Ca(2+)](i) to 50 nM markedly decreased SR Ca(2+) leak rate and nearly abolished Ca(2+) sparks. When the ryanodine receptor (RyR) was completely inhibited with ruthenium red (50 μM), changes in [Ca(2+)](i) between 50 and 350 nM did not produce any significant effect on SR Ca(2+) leak, indicating that [Ca(2+)](i) alters SR Ca(2+) leak solely by regulating RyR activity. In summary, [Ca(2+)](i) in the range of 50-350 nM has a significant effect on SR Ca(2+) leak rate mainly via direct regulation of RyR activity. As RyR activity depends highly on [Ca(2+)](i) and [Ca(2+)](SR), SR Ca(2+) leak remains relatively constant during the declining phase of the Ca(2+) transient when [Ca(2+)](SR) and [Ca(2+)](i) change in opposite directions.

Dual role of junctin in the regulation of ryanodine receptors and calcium release in cardiac ventricular myocytes

Junctin, a 26 kDa intra-sarcoplasmic reticulum (SR) protein, forms a quaternary complex with triadin, calsequestrin and the ryanodine receptor (RyR) at the junctional SR membrane. The physiological role for junctin in the luminal regulation of RyR Ca(2+) release remains unresolved, but it appears to be essential for proper cardiac function since ablation of junctin results in increased ventricular automaticity. Given that the junctin levels are severely reduced in human failing hearts, we performed an in-depth study of the mechanisms affecting intracellular Ca(2+) homeostasis in junctin-deficient cardiomyocytes. In concurrence with sparks, JCN-KO cardiomyocytes display increased Ca(2+) transient amplitude, resulting from increased SR [Ca(2+)] ([Ca(2+)](SR)). Junctin ablation appears to affect how RyRs 'sense' SR Ca(2+) load, resulting in decreased diastolic SR Ca(2+) leak despite an elevated [Ca(2+)](SR). Surprisingly, the β-adrenergic enhancement of [Ca(2+)](SR) reverses the decrease in RyR activity and leads to spontaneous Ca(2+) release, evidenced by the development of spontaneous aftercontractions. Single channel recordings of RyRs from WT and JCN-KO cardiac SR indicate that the absence of junctin produces a dual effect on the normally linear response of RyRs to luminal [Ca(2+)]: at low luminal [Ca(2+)] (<1 mmol l(-1)), junctin-devoid RyR channels are less responsive to luminal [Ca(2+)]; conversely, high luminal [Ca(2+)] turns them hypersensitive to this form of channel modulation. Thus, junctin produces complex effects on Ca(2+) sparks, transients, and leak, but the luminal [Ca(2+)]-dependent dual response of junctin-devoid RyRs demonstrates that junctin normally acts as an activator of RyR channels at low luminal [Ca(2+)], and as an inhibitor at high luminal [Ca(2+)]. Because the crossover occurs at a [Ca(2+)](SR) that is close to that present in resting cells, it is possible that the activator-inhibitor role of junctin may be exerted under periods of prevalent parasympathetic and sympathetic activity, respectively.

Abstract P076: Essential Role of Neuronal Nitric Oxide Synthase in Ca 2+ /Calmodulin-Dependent Protein Kinase II Activation in Cardiac Myocytes During β-Adrenergic Stimulation

RATIONALE: Stimulation of the beta-adrenergic (beta-AR) pathway leads to positive inotropy, and is the major regulator of heart function. In addition to the traditional PKA pathway, activation of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and neuronal nitric oxide synthase (NOS1) signaling also play important roles in the positive inotropy by modulating ryanodine receptor (RyR) activity. OBJECTIVE: The upstream activators of CaMKII during beta-AR stimulation are not well defined. The purpose of this study is to investigate if there is any cross-talk between the CaMKII and NOS1 signaling pathways. METHODS AND RESULTS: Myocytes were isolated from wildtype (WT, C57Bl/6) and NOS1 −/− mice. Ca 2+ transients (Fluo-4) and cell shortening (edge detection) were simultaneously measured. RyR activity was measured using the SR Ca 2+ leak/load relationship. CaMKII was acutely inhibited by KN93. In WT myocytes, KN93 decreased beta-AR stimulated contraction (Ca2+ transients (Fluo-4) and cell shortening). In NOS1 −/− myocytes, beta-AR stimulated contraction was blunted compared to WT, and KN93 had no further effect on contraction. Furthermore, beta-AR stimulated RyR activity was blunted in NOS1 −/− compared to WT myocytes. As with contraction, KN93 decreased beta-AR stimulated RyR activity in WT myocytes, but had no effect in NOS1 −/− myocytes. CONCLUSION: These data suggest that NOS1 is required for CaMKII-mediated RyR activation which contributes to positive inotropy during beta-AR stimulation. Further study of this pathway is warranted since CAMKII expression and activity are increased in cardiac hypertrophy and heart failure. A better understanding of the NOS1/CaMKII pathway during beta-AR stimulation has beneficial therapeutic potential for heart diseases.

MicroRNA-1 and -133 Increase Arrhythmogenesis in Heart Failure by Dissociating Phosphatase Activity from RyR2 Complex

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca2+ release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca2+ waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca2+/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca2+ waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca2+ waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca2+ cycling, and increased propensity to arrhythmogenesis.

Lessons from calsequestrin-1 ablation in vivo: much more than a Ca2+ buffer after all

Calsequestrin type-1 (CASQ1), the main sarcoplasmic reticulum (SR) Ca(2+) binding protein, plays a dual role in skeletal fibers: a) it provides a large pool of rapidly-releasable Ca(2+) during excitation-contraction (EC) coupling; and b) it modulates the activity of ryanodine receptors (RYRs), the SR Ca(2+) release channels. We have generated a mouse lacking CASQ1 in order to further characterize the role of CASQ1 in skeletal muscle. Contrary to initial expectations, CASQ1 ablation is compatible with normal motor activity, in spite of moderate muscle atrophy. However, CASQ1 deficiency results in profound remodeling of the EC coupling apparatus: shrinkage of junctional SR lumen; proliferation of SR/transverse-tubule contacts; and increased density of RYRs. While force development during a twitch is preserved, it is nevertheless characterized by a prolonged time course, likely reflecting impaired Ca(2+) re-uptake by the SR. Finally, lack of CASQ1 also results in increased rate of SR Ca(2+) depletion and inability of muscle to sustain tension during a prolonged tetani. All modifications are more pronounced (or only found) in fast-twitch extensor digitorum longus muscle compared to slow-twitch soleus muscle, likely because the latter expresses higher amounts of calsequestrin type-2 (CASQ2). Surprisingly, male CASQ1-null mice also exhibit a marked increased rate of spontaneous mortality suggestive of a stress-induced phenotype. Consistent with this idea, CASQ1-null mice exhibit an increased susceptibility to undergo a hypermetabolic syndrome characterized by whole body contractures, rhabdomyolysis, hyperthermia and sudden death in response to halothane- and heat-exposure, a phenotype remarkably similar to human malignant hyperthermia and environmental heat-stroke. The latter findings validate the CASQ1 gene as a candidate for linkage analysis in human muscle disorders.

How Does Stochastic Ryanodine Receptor-Mediated Ca Leak Fail to Initiate a Ca Spark?

Spontaneous calcium (Ca) sparks are initiated by single ryanodine receptor (RyR) opening. Once one RyR channel opens, it elevates local [Ca] in the cleft space ([Ca]Cleft), which opens other RyR channels in the same Ca release unit (CaRU) via Ca-induced Ca-release. Experiments by Zima et al. (J. Physiol. 588:4743–4757, 2010) demonstrate that spontaneous Ca sparks occur only when intrasarcoplasmic-reticulum (SR) [Ca] ([Ca]SR) is above a threshold level, but that RyR-mediated SR Ca leak exists without Ca sparks well below this threshold [Ca]SR. We examine here how single RyR opening at lower [Ca]SR can fail to recruit Ca sparks at a CaRU, while still contributing to SR Ca leak. We assess this using a physiologically detailed mathematical model of junctional SR Ca release in which RyR gating is regulated by [Ca]SR and [Ca]Cleft. We find that several factors contribute to the failure of Ca sparks as [Ca]SR declines: 1), lower [Ca]SR reduces driving force and thus limits local [Ca]Cleft achieved and the rate of rise during RyR opening; 2), low [Ca]SR limits RyR open time (τO), which further reduces local [Ca]Cleft attained; 3), low τO and fast [Ca]Cleft dissipation after RyR closure shorten the opportunity for neighboring RyR activation; 4), at low [Ca]SR, the RyR exhibits reduced [Ca]Cleft sensitivity. We conclude that all of these factors conspire to reduce the probability of Ca sparks as [Ca]SR declines, despite continued RyR-mediated Ca leak. In addition, these same factors explain the much lower efficacy of L-type Ca channel opening to trigger local SR Ca release at low [Ca]SR during excitation-contraction coupling. Conversely, all of these factors are fundamentally important for increasing the propensity for pro-arrhythmic Ca sparks and waves in cardiac myocytes at high [Ca]SR.

Subcellular Heterogeneity of Ryanodine Receptor Properties in Ventricular Myocytes with Low T-Tubule Density

RationaleIn ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).ObjectiveTo characterize RyR functional properties in relation to TT proximity, at baseline and after MI.MethodsMyocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.ResultsIn CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI.ConclusionTT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves.

Variation in expression of calcium-handling proteins is associated with inter-individual differences in mechanical performance of rat (Rattus norvegicus) skeletal muscle

An important constraint on locomotor performance is the trade-off between sprint and endurance performance. One intuitive explanation for this trade-off is that an individual muscle cannot excel at generating both maximal force/power and high fatigue resistance. The underlying reasons for this muscle trade-off are poorly defined. The aim of this study was to test the hypothesis that inter-individual variation in muscle mechanics is associated with inter-individual differences in metabolic capacities and expression of calcium-handling proteins. Lateral gastrocnemius muscles were isolated from 20 rats (Rattus norvegicus) and analysed to determine metabolic capacity, sarco/endoplasmic reticulum calcium ATPase (SERCA)1 protein concentration, total SERCA activity, and mRNA concentrations of SERCA1, SERCA2, troponin I and ryanodine receptors. Isometric studies of lateral gastrocnemius muscles at 30°C showed that muscles with higher sprint performance had lower fatigue resistance. More rapid muscle contraction was correlated with higher lactate dehydrogenase activity and increased expression of ryanodine receptor 1. More rapid muscle relaxation was correlated with increased expression of troponin I type 2 (fast) isoform and decreased expression of SERCA2 (slow) isoform. Treating muscles with dantrolene confirmed that ryanodine receptor activity is important in determining tetanus force and muscle contraction rates, but has no effect on fatigue resistance. Thapsigargin treatment revealed that SERCA activity determines fatigue resistance but does not affect maximal muscle force or contraction rates. We conclude that the opposing roles of SERCA activity and expression of ryanodine receptors in determining fatigue resistance and force production, respectively, at least partly explain differences in sprint and endurance performance in isolated rat gastrocnemius muscle.

Trisk 32 regulates IP3 receptors in rat skeletal myoblasts

To date, four isoforms of triadins have been identified in rat skeletal muscle. While the function of the 95-kDa isoform in excitation-contraction coupling has been studied in detail, the role of the 32-kDa isoform (Trisk 32) remains elusive. Here, Trisk 32 overexpression was carried out by stable transfection in L6.G8 myoblasts. Co-localization of Trisk 32 and IP(3) receptors (IP(3)R) was demonstrated by immunocytochemistry, and their association was shown by co-immunoprecipitation. Functional effects of Trisk 32 on IP(3)-mediated Ca(2+) release were assessed by measuring changes in [Ca(2+)](i) following the stimulation by bradykinin or vasopressin. The amplitude of the Ca(2+) transients evoked by 20 μM bradykinin was significantly higher in Trisk 32-overexpressing (p < 0.01; 426 ± 84 nM, n = 27) as compared to control cells (76 ± 12 nM, n = 23). The difference remained significant (p < 0.02; 217 ± 41 nM, n = 21, and 97 ± 29 nM, n = 31, respectively) in the absence of extracellular Ca(2+). Similar observations were made when 0.1 μM vasopressin was used to initiate Ca(2+) release. Possible involvement of the ryanodine receptors (RyR) in these processes was excluded, after functional and biochemical experiments. Furthermore, Trisk 32 overexpression had no effect on store-operated Ca(2+) entry, despite a decrease in the expression of STIM1. These results suggest that neither the increased activity of RyR, nor the amplification of SOCE, is responsible for the differences observed in bradykinin- or vasopressin-evoked Ca(2+) transients; rather, they were due to the enhanced activity of IP(3)R. Thus, Trisk 32 not only co-localizes with, but directly contributes to, the regulation of Ca(2+) release via IP(3)R.