Nanoscale coupling of junctophilin-2 and ryanodine receptors regulates vascular smooth muscle cell contractility.

Junctophilin proteins maintain close contacts between the endoplasmic/sarcoplasmic reticulum (ER/SR) and the plasma membrane in many types of cells, as typified by junctophilin-2 (JPH2), which is necessary for the formation of the cardiac dyad. Here, we report that JPH2 is the most abundant junctophilin isotype in native smooth muscle cells (SMCs) isolated from cerebral arteries and that acute knockdown diminishes the area of sites of interaction between the SR and plasma membrane. Superresolution microscopy revealed nanometer-scale colocalization of JPH2 clusters with type 2 ryanodine receptor (RyR2) clusters near the cell surface. Knockdown of JPH2 had no effect on the frequency, amplitude, or kinetics of spontaneous Ca2+ sparks generated by transient release of Ca2+ from the SR through RyR2s, but it did nearly abolish Ca2+ spark-activated, large-conductance, Ca2+-activated K+ (BK) channel currents. We also found that JPH2 knockdown was associated with hypercontractility of intact cerebral arteries. We conclude that JPH2 maintains functional coupling between RyR2s and BK channels and is critically important for cerebral arterial function.

Potential contribution of ryanodine receptor 2 upregulation to cGMP/PKG signaling-induced cone degeneration in cyclic nucleotide-gated channel deficiency.

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca2+ influx in rod and cone photoreceptors. Mutations in cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. Mice lacking functional cone CNG channel show endoplasmic reticulum (ER) stress-associated cone degeneration. The elevated cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) signaling and upregulation of the ER Ca2+ channel ryanodine receptor 2 (RyR2) have been implicated in cone degeneration. This work investigates the potential contribution of RyR2 to cGMP/PKG signaling-induced ER stress and cone degeneration. We demonstrated that the expression and activity of RyR2 were highly regulated by cGMP/PKG signaling. Depletion of cGMP by deleting retinal guanylate cyclase 1 or inhibition of PKG using chemical inhibitors suppressed the upregulation of RyR2 in CNG channel deficiency. Depletion of cGMP or deletion of Ryr2 equivalently inhibited unfolded protein response/ER stress, activation of the CCAAT-enhancer-binding protein homologous protein, and activation of the cyclic adenosine monophosphate response element-binding protein, leading to early-onset cone protection. In addition, treatment with cGMP significantly enhanced Ryr2 expression in cultured photoreceptor-derived Weri-Rb1 cells. Findings from this work demonstrate the regulation of cGMP/PKG signaling on RyR2 in the retina and support the role of RyR2 upregulation in cGMP/PKG signaling-induced ER stress and photoreceptor degeneration.

Impact of NR5A2 and RYR2 3'UTR polymorphisms on the risk of breast cancer in a Chinese Han population.

OBJECTIVES: The NR5A2 and RYR2 genes are important players in steroid metabolism and play an important role in cancer research. In this research, we want to evaluate the effect of NR5A2 and RYR2 polymorphisms on breast cancer (BC). METHODS: Four single nucleotide polymorphisms on NR5A2 and RYR2 were selected to genotype by Agena MassARRAY in 379 BC patients and 407 healthy controls. Using the PLINK software to calculate the Odds ratio (OR) and 95% confidence intervals (CIs) via the logistic regression analysis to evaluate the risk for BC. RESULTS: We found that NR5A2 rs2246209 significantly decreased the risk of BC with the AA genotype (OR 0.58, 95%CI 0.34-0.99, p = 0.049), and recessive model (OR 0.59, 95%CI 0.35-0.99, p = 0.046); rs12594 in the RYR2 gene significantly decreased the risk of BC in the GG genotype (OR 0.44, 95%CI 0.22-0.88, p = 0.020), and recessive model (OR 0.43, 95%CI 0.21-0.85, p = 0.016). Further stratification analysis showed that NR5A2 rs2246209 was related to a lower incidence of BC affected by age, lymph nodes metastasis, and tumor stage; RYR2 rs12594 was related to a decreased BC risk restricted by age, estrogen receptor (ER), progesterone receptor (PR), menopausal status, tumor size, and tumor stage. Rs12594 in the RyR2 gene remained significant on the genetic susceptibility of PR-positive BC after Bonferroni correction (p < 0.0125). CONCLUSIONS: This study provides an evidence that NR5A2 rs2246209 and RYR2 rs12594 decreased the risk of breast cancer.

Increased mitochondrial nanotunneling activity, induced by calcium imbalance, affects intermitochondrial matrix exchanges.

Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca2+ imbalance by depressing RyR2 channel activity during excitation-contraction coupling, resulting in random bursts of Ca2+ release probably due to Ca2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2A4860G+/- cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower.

Congenital myopathy results from misregulation of a muscle Ca2+ channel by mutant Stac3.

Skeletal muscle contractions are initiated by an increase in Ca2+ released during excitation-contraction (EC) coupling, and defects in EC coupling are associated with human myopathies. EC coupling requires communication between voltage-sensing dihydropyridine receptors (DHPRs) in transverse tubule membrane and Ca2+ release channel ryanodine receptor 1 (RyR1) in the sarcoplasmic reticulum (SR). Stac3 protein (SH3 and cysteine-rich domain 3) is an essential component of the EC coupling apparatus and a mutation in human STAC3 causes the debilitating Native American myopathy (NAM), but the nature of how Stac3 acts on the DHPR and/or RyR1 is unknown. Using electron microscopy, electrophysiology, and dynamic imaging of zebrafish muscle fibers, we find significantly reduced DHPR levels, functionality, and stability in stac3 mutants. Furthermore, stac3NAM myofibers exhibited increased caffeine-induced Ca2+ release across a wide range of concentrations in the absence of altered caffeine sensitivity as well as increased Ca2+ in internal stores, which is consistent with increased SR luminal Ca2+ These findings define critical roles for Stac3 in EC coupling and human disease.

3D dSTORM imaging reveals novel detail of ryanodine receptor localization in rat cardiac myocytes.

KEY POINTS: Using 3D direct stochastic optical reconstruction microscopy (dSTORM), we developed novel approaches to quantitatively describe the nanoscale, 3D organization of ryanodine receptors (RyRs) in cardiomyocytes. Complex arrangements of RyR clusters were observed in 3D space, both at the cell surface and within the cell interior, with allocation to dyadic and non-dyadic pools. 3D imaging importantly allowed discernment of clusters overlapping in the z-axis, for which detection was obscured by conventional 2D imaging techniques. Thus, RyR clusters were found to be significantly smaller than previous 2D estimates. Ca2+ release units (CRUs), i.e. functional groupings of neighbouring RyR clusters, were similarly observed to be smaller than earlier reports. Internal CRUs contained more RyRs in more clusters than CRUs on the cell surface, and yielded longer duration Ca2+ sparks. ABSTRACT: Cardiomyocyte contraction is dependent on Ca2+ release from ryanodine receptors (RyRs). However, the precise localization of RyRs remains unknown, due to shortcomings of imaging techniques which are diffraction limited or restricted to 2D. We aimed to determine the 3D nanoscale organization of RyRs in rat cardiomyocytes by employing direct stochastic optical reconstruction microscopy (dSTORM) with phase ramp technology. Initial observations at the cell surface showed an undulating organization of RyR clusters, resulting in their frequent overlap in the z-axis and obscured detection by 2D techniques. Non-overlapping clusters were imaged to create a calibration curve for estimating RyR number based on recorded fluorescence blinks. Employing this method at the cell surface and interior revealed smaller RyR clusters than 2D estimates, as erroneous merging of axially aligned RyRs was circumvented. Functional groupings of RyR clusters (Ca2+ release units, CRUs), contained an average of 18 and 23 RyRs at the surface and interior, respectively, although half of all CRUs contained only a single 'rogue' RyR. Internal CRUs were more tightly packed along z-lines than surface CRUs, contained larger and more numerous RyR clusters, and constituted ∼75% of the roughly 1 million RyRs present in an average cardiomyocyte. This complex internal 3D geometry was underscored by correlative imaging of RyRs and t-tubules, which enabled quantification of dyadic and non-dyadic RyR populations. Mirroring differences in CRU size and complexity, Ca2+ sparks originating from internal CRUs were of longer duration than those at the surface. These data provide novel, nanoscale insight into RyR organization and function across cardiomyocytes.

Spontaneous action potentials in retinal horizontal cells of goldfish (Carassius auratus) are dependent upon L-type Ca2+ channels and ryanodine receptors.

Horizontal cells (HCs) are interneurons of the outer retina that undergo graded changes in membrane potential during the light response and provide feedback to photoreceptors. We characterized spontaneous Ca2+-based action potentials (APs) in isolated goldfish (Carassius auratus) HCs with electrophysiological and intracellular imaging techniques. Transient changes in intracellular Ca2+ concentration ([Ca2+]i) were observed with fura-2 and were abolished by removal of extracellular Ca2+ or by inhibition of Ca2+ channels by 50 µM Cd2+ or 100 µM nifedipine. Inhibition of Ca2+ release from stores with 20 µM ryanodine or 50 µM dantrolene abolished Ca2+ transients and increased baseline [Ca2+]i. This increased baseline was prevented by blocking L-type Ca2+ channels with nifedipine, suggesting that Ca2+-induced Ca2+ release from stores may be needed to inactivate membrane Ca2+ channels. Caffeine (3 mM) increased the frequency of Ca2+ transients, and the store-operated channel antagonist 2-aminoethyldiphenylborinate (100 µM) counteracted this effect. APs were detected with voltage-sensitive dye imaging (FluoVolt) and current-clamp electrophysiology. In current-clamp recordings, regenerative APs were abolished by removal of extracellular Ca2+ or in the presence of 5 mM Co2+ or 100 µM nifedipine, and APs were amplified with 15 mM Ba2+. Collectively, our data suggest that during APs Ca2+ enters through L-type Ca2+ channels and that Ca2+ stores (gated by ryanodine receptors) contribute to the rise in [Ca2+]i. This work may lead to further understanding of the possible role APs have in vision, such as transitioning from light to darkness or modulating feedback from HCs to photoreceptors.NEW & NOTEWORTHY Horizontal cells (HCs) are interneurons of the outer retina that provide inhibitory feedback onto photoreceptors. HCs respond to light via graded changes in membrane potential. We characterized spontaneous action potentials in HCs from goldfish and linked action potential generation to a rise in intracellular Ca2+ via plasma membrane channels and ryanodine receptors. Action potentials may play a role in vision, such as transitioning from light to darkness, or in modulating feedback from HCs to photoreceptors.

A computational model of large conductance voltage and calcium activated potassium channels: implications for calcium dynamics and electrophysiology in detrusor smooth muscle cells.

The large conductance voltage and calcium activated potassium (BK) channels play a crucial role in regulating the excitability of detrusor smooth muscle, which lines the wall of the urinary bladder. These channels have been widely characterized in terms of their molecular structure, pharmacology and electrophysiology. They control the repolarising and hyperpolarising phases of the action potential, thereby regulating the firing frequency and contraction profiles of the smooth muscle. Several groups have reported varied profiles of BK currents and I-V curves under similar experimental conditions. However, no single computational model has been able to reconcile these apparent discrepancies. In view of the channels' physiological importance, it is imperative to understand their mechanistic underpinnings so that a realistic model can be created. This paper presents a computational model of the BK channel, based on the Hodgkin-Huxley formalism, constructed by utilising three activation processes - membrane potential, calcium inflow from voltage-gated calcium channels on the membrane and calcium released from the ryanodine receptors present on the sarcoplasmic reticulum. In our model, we attribute the discrepant profiles to the underlying cytosolic calcium received by the channel during its activation. The model enables us to make heuristic predictions regarding the nature of the sub-membrane calcium dynamics underlying the BK channel's activation. We have employed the model to reproduce various physiological characteristics of the channel and found the simulated responses to be in accordance with the experimental findings. Additionally, we have used the model to investigate the role of this channel in electrophysiological signals, such as the action potential and spontaneous transient hyperpolarisations. Furthermore, the clinical effects of BK channel openers, mallotoxin and NS19504, were simulated for the detrusor smooth muscle cells. Our findings support the proposed application of these drugs for amelioration of the condition of overactive bladder. We thus propose a physiologically realistic BK channel model which can be integrated with other biophysical mechanisms such as ion channels, pumps and exchangers to further elucidate its micro-domain interaction with the intracellular calcium environment.

Early Alterations of Hippocampal Neuronal Firing Induced by Abeta42.

We studied the effect of Amyloid ß 1-42 oligomers (Abeta42) on Ca2+ dependent excitability profile of hippocampal neurons. Abeta42 is one of the Amyloid beta peptides produced by the proteolytic processing of the amyloid precursor protein and participates in the initiating event triggering the progressive dismantling of synapses and neuronal circuits. Our experiments on cultured hippocampal network reveal that Abeta42 increases intracellular Ca2+ concentration by 46% and inhibits firing discharge by 19%. More precisely, Abeta42 differently regulates ryanodine (RyRs), NMDA receptors (NMDARs), and voltage gated calcium channels (VGCCs) by increasing Ca2+ release through RyRs and inhibiting Ca2+ influx through NMDARs and VGCCs. The overall increased intracellular Ca2+ concentration causes stimulation of K+ current carried by big conductance Ca2+ activated potassium (BK) channels and hippocampal network firing inhibition. We conclude that Abeta42 alters neuronal function by means of at least 4 main targets: RyRs, NMDARs, VGCCs, and BK channels. The development of selective modulators of these channels may in turn be useful for developing effective therapies that could enhance the quality of life of AD patients during the early onset of the pathology.

Venous Vasomotion.

Veins exhibit spontaneous contractile activity, a phenomenon generally termed vasomotion. This is mediated by spontaneous rhythmical contractions of mural cells (i.e. smooth muscle cells (SMCs) or pericytes) in the wall of the vessel. Vasomotion occurs through interconnected oscillators within and between mural cells, entraining their cycles. Pharmacological studies indicate that a key oscillator underlying vasomotion is the rhythmical calcium ion (Ca2+) release-refill cycle of Ca2+ stores. This occurs through opening of inositol 1,4,5-trisphosphate receptor (IP3R)- and/or ryanodine receptor (RyR)-operated Ca2+ release channels in the sarcoplasmic/endoplasmic (SR/ER) reticulum and refilling by the SR/ER reticulum Ca2+ATPase (SERCA). Released Ca2+ from stores near the plasma membrane diffuse through the cytosol to open Ca2+-activated chloride (Cl-) channels, this generating inward current through an efflux of Cl-. The resultant depolarisation leads to the opening of voltage-dependent Ca2+ channels and possibly increased production of IP3, which through Ca2+-induced Ca2+ release (CICR) of IP3Rs and/or RyRs and IP3R-mediated Ca2+ release provide a means by which store oscillators entrain their activity. Intercellular entrainment normally involves current flow through gap junctions that interconnect mural cells and in many cases this is aided by additional connectivity through the endothelium. Once entrainment has occurred the substantial Ca2+ entry that results from the near-synchronous depolarisations leads to rhythmical contractions of the mural cells, this often leading to vessel constriction. The basis for venous/venular vasomotion has yet to be fully delineated but could improve both venous drainage and capillary/venular absorption of blood plasma-associated fluids.

Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation.

Ventilator-induced diaphragmatic dysfunction (VIDD) refers to the diaphragm muscle weakness that occurs following prolonged controlled mechanical ventilation (MV). The presence of VIDD impedes recovery from respiratory failure. However, the pathophysiological mechanisms accounting for VIDD are still not fully understood. Here, we show in human subjects and a mouse model of VIDD that MV is associated with rapid remodeling of the sarcoplasmic reticulum (SR) Ca(2+) release channel/ryanodine receptor (RyR1) in the diaphragm. The RyR1 macromolecular complex was oxidized, S-nitrosylated, Ser-2844 phosphorylated, and depleted of the stabilizing subunit calstabin1, following MV. These posttranslational modifications of RyR1 were mediated by both oxidative stress mediated by MV and stimulation of adrenergic signaling resulting from the anesthesia. We demonstrate in the murine model that such abnormal resting SR Ca(2+) leak resulted in reduced contractile function and muscle fiber atrophy for longer duration of MV. Treatment with ß-adrenergic antagonists or with S107, a small molecule drug that stabilizes the RyR1-calstabin1 interaction, prevented VIDD. Diaphragmatic dysfunction is common in MV patients and is a major cause of failure to wean patients from ventilator support. This study provides the first evidence to our knowledge of RyR1 alterations as a proximal mechanism underlying VIDD (i.e., loss of function, muscle atrophy) and identifies RyR1 as a potential target for therapeutic intervention.

Leaky RyR2 channels unleash a brainstem spreading depolarization mechanism of sudden cardiac death.

Cardiorespiratory failure is the most common cause of sudden unexplained death in epilepsy (SUDEP). Genetic autopsies have detected "leaky" gain-of-function mutations in the ryanodine receptor-2 (RyR2) gene in both SUDEP and sudden cardiac death cases linked to catecholaminergic polymorphic ventricular tachycardia that feature lethal cardiac arrhythmias without structural abnormality. Here we find that a human leaky RyR2 mutation, R176Q (RQ), alters neurotransmitter release probability in mice and significantly lowers the threshold for spreading depolarization (SD) in dorsal medulla, leading to cardiorespiratory collapse. Rare episodes of sinus bradycardia, spontaneous seizure, and sudden death were detected in RQ/+ mutant mice in vivo; however, when provoked, cortical seizures frequently led to apneas, brainstem SD, cardiorespiratory failure, and death. In vitro studies revealed that the RQ mutation selectively strengthened excitatory, but not inhibitory, synapses and facilitated SD in both the neocortex as well as brainstem dorsal medulla autonomic microcircuits. These data link defects in neuronal intracellular calcium homeostasis to the vulnerability of central autonomic brainstem pathways to hypoxic stress and implicate brainstem SD as a previously unrecognized site and mechanism contributing to premature death in individuals with leaky RYR2 mutations.

Dietary Nitrate Enhances the Contractile Properties of Human Skeletal Muscle.

Dietary nitrate, a source of nitric oxide (NO), improves the contractile properties of human muscle. We present the hypothesis that this is due to nitrosylation of the ryanodine receptor and increased NO signaling via the soluble guanyl cyclase-cyclic guanosine monophosphate-protein kinase G pathway, which together increase the free intracellular Ca concentration along with the Ca sensitivity of the myofilaments themselves.

Stac adaptor proteins regulate trafficking and function of muscle and neuronal L-type Ca2+ channels.

Excitation-contraction (EC) coupling in skeletal muscle depends upon trafficking of CaV1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca(2+) channel), to plasma membrane regions at which the DHPRs interact with type 1 ryanodine receptors (RyR1) in the sarcoplasmic reticulum. A distinctive feature of this trafficking is that CaV1.1 expresses poorly or not at all in mammalian cells that are not of muscle origin (e.g., tsA201 cells), in which all of the other nine CaV isoforms have been successfully expressed. Here, we tested whether plasma membrane trafficking of CaV1.1 in tsA201 cells is promoted by the adapter protein Stac3, because recent work has shown that genetic deletion of Stac3 in skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and CaV1.1 traffic together to the tsA201 plasma membrane, whereas CaV1.1 is retained intracellularly when Stac3 is absent. Moreover, L-type Ca(2+) channel function in tsA201 cells coexpressing Stac3 and CaV1.1 is quantitatively similar to that in myotubes, despite the absence of RyR1. Although Stac3 is not required for surface expression of CaV1.2, the principle subunit of the cardiac/brain L-type Ca(2+) channel, Stac3 does bind to CaV1.2 and, as a result, greatly slows the rate of current inactivation, with Stac2 acting similarly. Overall, these results indicate that Stac3 is an essential chaperone of CaV1.1 in skeletal muscle and that in the brain, Stac2 and Stac3 may significantly modulate CaV1.2 function.

Cardiac sarcoplasmic reticulum calcium leak: basis and roles in cardiac dysfunction.

Synchronized SR calcium (Ca) release is critical to normal cardiac myocyte excitation-contraction coupling, and ideally this release shuts off completely between heartbeats. However, other SR Ca release events are referred to collectively as SR Ca leak (which includes Ca sparks and waves as well as smaller events not detectable as Ca sparks). Much, but not all, of the SR Ca leak occurs via ryanodine receptors and can be exacerbated in pathological states such as heart failure. The extent of SR Ca leak is important because it can (a) reduce SR Ca available for release, causing systolic dysfunction; (b) elevate diastolic [Ca]i, contributing to diastolic dysfunction; (c) cause triggered arrhythmias; and (d) be energetically costly because of extra ATP used to repump Ca. This review addresses quantitative aspects and manifestations of SR Ca leak and its measurement, and how leak is modulated by Ca, associated proteins, and posttranslational modifications in health and disease.

The Anthracycline Metabolite Doxorubicinol Abolishes RyR2 Sensitivity to Physiological Changes in Luminal Ca2+ through an Interaction with Calsequestrin.

The chemotherapeutic anthracycline metabolite doxorubicinol (doxOL) has been shown to interact with and disrupt the function of the cardiac ryanodine receptor Ca2+ release channel (RyR2) in the sarcoplasmic reticulum (SR) membrane and the SR Ca2+ binding protein calsequestrin 2 (CSQ2). Normal increases in RyR2 activity in response to increasing diastolic SR [Ca2+] are influenced by CSQ2 and are disrupted in arrhythmic conditions. Therefore, we explored the action of doxOL on RyR2's response to changes in luminal [Ca2+] seen during diastole. DoxOL abolished the increase in RyR2 activity when luminal Ca2+ was increased from 0.1 to 1.5 mM. This was not due to RyR2 oxidation, but depended entirely on the presence of CSQ2 in the RyR2 complex. DoxOL binding to CSQ2 reduced both the Ca2+ binding capacity of CSQ2 (by 48%-58%) and its aggregation, and lowered CSQ2 association with the RyR2 complex by 67%-77%. Each of these effects on CSQ2, and the lost RyR2 response to changes in luminal [Ca2+], was duplicated by exposing native RyR2 channels to subphysiologic (≤1.0 µM) luminal [Ca2+]. We suggest that doxOL and low luminal Ca2+ both disrupt the CSQ2 polymer, and that the association of the monomeric protein with the RyR2 complex shifts the increase in RyR2 activity with increasing luminal [Ca2+] away from the physiologic [Ca2+] range. Subsequently, these changes may render the channel insensitive to changes of luminal Ca2+ that occur through the cardiac cycle. The altered interactions between CSQ2, triadin, and/or junctin and RyR2 may produce an arrhythmogenic substrate in anthracycline-induced cardiotoxicity.

Coupling of excitation to Ca2+ release is modulated by dysferlin.

KEY POINTS: Dysferlin, the protein missing in limb girdle muscular dystrophy 2B and Miyoshi myopathy, concentrates in transverse tubules of skeletal muscle, where it stabilizes voltage-induced Ca2+ transients against loss after osmotic shock injury (OSI). Local expression of dysferlin in dysferlin-null myofibres increases transient amplitude to control levels and protects them from loss after OSI. Inhibitors of ryanodine receptors (RyR1) and L-type Ca2+ channels protect voltage-induced Ca2+ transients from loss; thus both proteins play a role in injury in dysferlin's absence. Effects of Ca2+ -free medium and S107, which inhibits SR Ca2+ leak, suggest the SR as the primary source of Ca2+ responsible for the loss of the Ca2+ transient upon injury. Ca2+ waves were induced by OSI and suppressed by exogenous dysferlin. We conclude that dysferlin prevents injury-induced SR Ca2+ leak. ABSTRACT: Dysferlin concentrates in the transverse tubules of skeletal muscle and stabilizes Ca2+ transients when muscle fibres are subjected to osmotic shock injury (OSI). We show here that voltage-induced Ca2+ transients elicited in dysferlin-null A/J myofibres were smaller than control A/WySnJ fibres. Regional expression of Venus-dysferlin chimeras in A/J fibres restored the full amplitude of the Ca2+ transients and protected against OSI. We also show that drugs that target ryanodine receptors (RyR1: dantrolene, tetracaine, S107) and L-type Ca2+ channels (LTCCs: nifedipine, verapamil, diltiazem) prevented the decrease in Ca2+ transients in A/J fibres following OSI. Diltiazem specifically increased transients by ∼20% in uninjured A/J fibres, restoring them to control values. The fact that both RyR1s and LTCCs were involved in OSI-induced damage suggests that damage is mediated by increased Ca2+ leak from the sarcoplasmic reticulum (SR) through the RyR1. Congruent with this, injured A/J fibres produced Ca2+ sparks and Ca2+ waves. S107 (a stabilizer of RyR1-FK506 binding protein coupling that reduces Ca2+ leak) or local expression of Venus-dysferlin prevented OSI-induced Ca2+ waves. Our data suggest that dysferlin modulates SR Ca2+ release in skeletal muscle, and that in its absence OSI causes increased RyR1-mediated Ca2+ leak from the SR into the cytoplasm.

Systolic [Ca2+ ]i regulates diastolic levels in rat ventricular myocytes.

KEY POINTS: For the heart to function as a pump, intracellular calcium concentration ([Ca2+ ]i ) must increase during systole to activate contraction and then fall, during diastole, to allow the myofilaments to relax and the heart to refill with blood. The present study investigates the control of diastolic [Ca2+ ]i in rat ventricular myocytes. We show that diastolic [Ca2+ ]i is increased by manoeuvres that decrease sarcoplasmic reticulum function. This is accompanied by a decrease of systolic [Ca2+ ]i such that the time-averaged [Ca2+ ]i remains constant. We report that diastolic [Ca2+ ]i is controlled by the balance between Ca2+ entry and Ca2+ efflux during systole. The results of the present study identify a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+ ]i . ABSTRACT: The intracellular Ca concentration ([Ca2+ ]i ) must be sufficently low in diastole so that the ventricle is relaxed and can refill with blood. Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+ ]i , in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane, are unclear. We investigated the effects on diastolic [Ca2+ ]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo-3 in voltage clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+ ]i . This increase of [Ca2+ ]i was larger when SR function was impaired either by making the ryanodine receptor leaky (with caffeine or ryanodine) or by decreasing sarco/endoplasmic reticulum Ca-ATPase activity with thapsigargin. The increase of diastolic [Ca2+ ]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient, such that there was no change of time-averaged [Ca2+ ]i . Time-averaged [Ca2+ ]i was increased by ß-adrenergic stimulation with isoprenaline and increased in a saturating manner with increased stimulation frequency; average [Ca2+ ]i was a linear function of Ca entry per unit time. Diastolic and time-averaged [Ca2+ ]i were decreased by decreasing the L-type Ca current (with 50 µm cadmium chloride). We conclude that diastolic [Ca2+ ]i is controlled by the balance between Ca entry and efflux during systole. Furthermore, manoeuvres that decrease the amplitude of the Ca transient (without decreasing Ca influx) will therefore increase diastolic [Ca2+ ]i . This identifies a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+ ]i .

Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC-A.

KEY POINTS: The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric-a gene knockout (KO) alters the single-channel function of skeletal RyR (RyR1). We find that RyR1 from Tric-a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+ , ATP, caffeine and luminal Ca2+ . In the presence of Mg2+ , ATP cannot effectively activate RyR1 from Tric-a KO mice. Additionally, RyR1 from Tric-a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of ß-adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+ -release. The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+ -release observed in skeletal muscle from Tric-a KO mice, further highlighting the importance of TRIC-A for normal physiological regulation of SR Ca2+ -release in skeletal muscle. ABSTRACT: The type A trimeric intracellular cation channel (TRIC-A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric-a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric-a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage-clamp conditions. We find that RyR1 channels from Tric-a KO mice respond normally to cytosolic Ca2+ , ATP, adenine, caffeine and to luminal Ca2+ . However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+ , ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC-A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC-A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC-A.

Intracellular calcium release channels: an update.

Ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3 Rs) are calcium (Ca2+ ) release channels on the endo/sarcoplasmic reticulum (ER/SR). Here we summarize the latest advances in the field, describing the recently discovered mechanistic roles of intracellular Ca2+ release channels in the regulation of mitochondrial fitness and endothelial function, providing novel therapeutic options for the treatment of heart failure, hypertension, and diabetes mellitus.