Endoplasmic Reticulum-Plasma Membrane Contact Sites.

The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

Ca2+ dynamics in interstitial cells: foundational mechanisms for the motor patterns in the gastrointestinal tract.

The gastrointestinal (GI) tract displays multiple motor patterns that move nutrients and wastes through the body. Smooth muscle cells (SMCs) provide the forces necessary for GI motility, but interstitial cells, electrically coupled to SMCs, tune SMC excitability, transduce inputs from enteric motor neurons, and generate pacemaker activity that underlies major motor patterns, such as peristalsis and segmentation. The interstitial cells regulating SMCs are interstitial cells of Cajal (ICC) and PDGF receptor (PDGFR)α+ cells. Together these cells form the SIP syncytium. ICC and PDGFRα+ cells express signature Ca2+-dependent conductances: ICC express Ca2+-activated Cl- channels, encoded by Ano1, that generate inward current, and PDGFRα+ cells express Ca2+-activated K+ channels, encoded by Kcnn3, that generate outward current. The open probabilities of interstitial cell conductances are controlled by Ca2+ release from the endoplasmic reticulum. The resulting Ca2+ transients occur spontaneously in a stochastic manner. Ca2+ transients in ICC induce spontaneous transient inward currents and spontaneous transient depolarizations (STDs). Neurotransmission increases or decreases Ca2+ transients, and the resulting depolarizing or hyperpolarizing responses conduct to other cells in the SIP syncytium. In pacemaker ICC, STDs activate voltage-dependent Ca2+ influx, which initiates a cluster of Ca2+ transients and sustains activation of ANO1 channels and depolarization during slow waves. Regulation of GI motility has traditionally been described as neurogenic and myogenic. Recent advances in understanding Ca2+ handling mechanisms in interstitial cells and how these mechanisms influence motor patterns of the GI tract suggest that the term "myogenic" should be replaced by the term "SIPgenic," as this review discusses.

Piperine enhances contractile force in slow- and fast-twitch muscle.

Piperine has been shown to bind to myosin and shift the distribution of conformational states of myosin molecules from the super-relaxed state to the disordered relaxed state. However, little is known about the implications for muscle force production and potential underlying mechanisms. Muscle contractility experiments were performed using isolated muscles and single fibres from rats and mice. The dose-response effect of piperine on muscle force was assessed at several stimulation frequencies. The potentiation of muscle force was also tested in muscles fatigued by eccentric contractions. Potential mechanisms of force potentiation were assessed by measuring Ca2+ levels during stimulation in enzymatically dissociated muscle fibres, while myofibrillar Ca2+ sensitivity was assessed in chemically skinned muscle fibres. Piperine caused a dose-dependent increase in low-frequency force with no effect on high-frequency force in both slow- and fast-twitch muscle, with similar relative increases in twitch force, rate of force development and relaxation rate. The potentiating effect of piperine on low-frequency force was reversible, and piperine partially recovered low-frequency force in fatigued muscle. Piperine had no effect on myoplasmic free [Ca2+] levels in mouse muscle fibres, whereas piperine substantially augmented the force response to submaximal levels of [Ca2+] in rat MyHCII fibres and MyHCI fibres along with a minor increase in maximum Ca2+-activated force. Piperine enhances low-frequency force production in both fast- and slow-twitch muscle. The effects are reversible and can counteract muscle fatigue. The primary underlying mechanism appears to be an increase in Ca2+ sensitivity. KEY POINTS: Piperine is a plant alkaloid derived from black pepper. It is known to bind to skeletal muscle myosin and enhance resting ATP turnover but its effects on contractility are not well known. We showed for the first time a piperine-induced force potentiation that was pronounced during low-frequency electrical stimulation of isolated muscles. The effect of piperine was observed in both slow and fast muscle types, was reversible, and could counteract the force decrements observed after fatiguing muscle contractions. Piperine treatment caused an increase in myofibrillar Ca2+ sensitivity in chemically skinned muscle fibres, while we observed no effect on intracellular Ca2+ concentrations during electrical stimulation in enzymatically dissociated muscle fibres.

Dual ablation of the RyR2-Ser2808 and RyR2-Ser2814 sites increases propensity for pro-arrhythmic spontaneous Ca2+ releases.

During exercise or stress, the sympathetic system stimulates cardiac contractility via ß-adrenergic receptor (ß-AR) activation, resulting in phosphorylation of the cardiac ryanodine receptor (RyR2). Three RyR2 phosphorylation sites have taken prominence in excitation-contraction coupling: S2808 and S2030 are described as protein kinase A specific and S2814 as a Ca2+/calmodulin kinase type-2-specific site. To examine the contribution of these phosphosites to Ca2+ signalling, we generated double knock-in (DKI) mice in which Ser2808 and Ser2814 phosphorylation sites have both been replaced by alanine (RyR2-S2808A/S2814A). These mice did not exhibit an overt phenotype. Heart morphology and haemodynamic parameters were not altered. However, they had a higher susceptibility to arrhythmias. We performed confocal Ca2+ imaging and electrophysiology experiments. Isoprenaline was used to stimulate ß-ARs. Measurements of Ca2+ waves and latencies in myocytes revealed an increased propensity for spontaneous Ca2+ releases in DKI myocytes, both in control conditions and during ß-AR stimulation. In DKI cells, waves were initiated from a lower threshold concentration of Ca2+ inside the sarcoplasmic reticulum, suggesting higher Ca2+ sensitivity of the RyRs. The refractoriness of Ca2+ spark triggering depends on the Ca2+ sensitivity of the RyR2. We found that RyR2-S2808A/S2814A channels were more Ca2+ sensitive in control conditions. Isoprenaline further shortened RyR refractoriness in DKI cardiomyocytes. Together, our results suggest that ablation of both the RyR2-Ser2808 and RyR2-S2814 sites increases the propensity for pro-arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. Given that the DKI cells present a full response to isoprenaline, the data suggest that phosphorylation of Ser2030 might be sufficient for ß-AR-mediated sensitization of RyRs. KEY POINTS: Phosphorylation of cardiac sarcoplasmic reticulum Ca2+-release channels (ryanodine receptors, RyRs) is involved in the regulation of cardiac function. Ablation of both the RyR2-Ser2808 and RyR2-Ser2814 sites increases the propensity for pro-arrhythmic spontaneous Ca2+ releases, as previously suggested for hyperphosphorylated RyRs. The intra-sarcoplasmic reticulum Ca2+ threshold for spontaneous Ca2+ wave generation is lower in RyR2-double-knock-in cells. The RyR2 from double-knock-in cells exhibits increased Ca2+ sensitivity. Phosphorylation of Ser2808 and Ser2814 might be important for basal activity of the channel. Phosphorylation of Ser2030 might be sufficient for a ß-adrenergic response.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1: A new calcium-sensitive protein functionally activated by endoplasmic reticulum calcium release and calmodulin binding in astrocytes.

BACKGROUND: MLC1 is a membrane protein highly expressed in brain perivascular astrocytes and whose mutations account for the rare leukodystrophy (LD) megalencephalic leukoencephalopathy with subcortical cysts disease (MLC). MLC is characterized by macrocephaly, brain edema and cysts, myelin vacuolation and astrocyte swelling which cause cognitive and motor dysfunctions and epilepsy. In cultured astrocytes, lack of functional MLC1 disturbs cell volume regulation by affecting anion channel (VRAC) currents and the consequent regulatory volume decrease (RVD) occurring in response to osmotic changes. Moreover, MLC1 represses intracellular signaling molecules (EGFR, ERK1/2, NF-kB) inducing astrocyte activation and swelling following brain insults. Nevertheless, to date, MLC1 proper function and MLC molecular pathogenesis are still elusive. We recently reported that in astrocytes MLC1 phosphorylation by the Ca2+/Calmodulin-dependent kinase II (CaMKII) in response to intracellular Ca2+ release potentiates MLC1 activation of VRAC. These results highlighted the importance of Ca2+ signaling in the regulation of MLC1 functions, prompting us to further investigate the relationships between intracellular Ca2+ and MLC1 properties. METHODS: We used U251 astrocytoma cells stably expressing wild-type (WT) or mutated MLC1, primary mouse astrocytes and mouse brain tissue, and applied biochemistry, molecular biology, video imaging and electrophysiology techniques. RESULTS: We revealed that WT but not mutant MLC1 oligomerization and trafficking to the astrocyte plasma membrane is favored by Ca2+ release from endoplasmic reticulum (ER) but not by capacitive Ca2+ entry in response to ER depletion. We also clarified the molecular events underlining MLC1 response to cytoplasmic Ca2+ increase, demonstrating that, following Ca2+ release, MLC1 binds the Ca2+ effector protein calmodulin (CaM) at the carboxyl terminal where a CaM binding sequence was identified. Using a CaM inhibitor and generating U251 cells expressing MLC1 with CaM binding site mutations, we found that CaM regulates MLC1 assembly, trafficking and function, being RVD and MLC-linked signaling molecules abnormally regulated in these latter cells. CONCLUSION: Overall, we qualified MLC1 as a Ca2+ sensitive protein involved in the control of volume changes in response to ER Ca2+ release and astrocyte activation. These findings provide new insights for the comprehension of the molecular mechanisms responsible for the myelin degeneration occurring in MLC and other LD where astrocytes have a primary role in the pathological process.

Rapamycin Controls Lymphoproliferation and Reverses T-Cell Responses in a Patient with a Novel STIM1 Loss-of-Function Deletion.

PURPOSE: Deficiency of stromal interaction molecule 1 (STIM1) results in combined immunodeficiency accompanied by extra-immunological findings like enamel defects and myopathy. We here studied a patient with a STIM1 loss-of-function mutation who presented with severe lymphoproliferation. We sought to explore the efficacy of the mTOR inhibitor rapamycin in controlling disease manifestations and reversing aberrant T-cell subsets and functions, which has never been used previously in this disorder. METHODS: Clinical findings of the patient were collected over time. We performed immunological evaluations before and after initiation of rapamycin treatment, including detailed lymphocyte subset analyses, alterations in frequencies of circulating T follicular helper (cTFH) and regulatory T (Treg) cells and their subtypes as well as T cell activation and proliferation capacities. RESULTS: A novel homozygous exon 2 deletion in STIM1 was detected in a 3-year-old girl with severe lymphoproliferation, recurrent infections, myopathy, iris hypoplasia, and enamel hypoplasia. Lymphoproliferation was associated with severe T-cell infiltrates. The deletion resulted in a complete loss of protein expression, associated with a lack of store-operated calcium entry response, defective T-cell activation, proliferation, and cytokine production. Interestingly, patient blood contained fewer cTFH and increased circulating follicular regulatory (cTFR) cells. Abnormal skewing towards TH2-like responses in certain T-cell subpopulations like cTFH, non-cTFH memory T-helper, and Treg cells was associated with increased eosinophil numbers and serum IgE levels. Treatment with rapamycin controlled lymphoproliferation, improved T-cell activation and proliferation capacities, reversed T-cell responses, and repressed high IgE levels and eosinophilia. CONCLUSIONS: This study enhances our understanding of STIM1 deficiency by uncovering additional abnormal T-cell responses, and reveals for the first time the potential therapeutic utility of rapamycin for this disorder.

RyR2 C-terminal truncating variants identified in patients with arrhythmic phenotypes exert a dominant negative effect through formation of wildtype-truncation heteromers.

Gain-of-function missense variants in the cardiac ryanodine receptor (RyR2) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), whereas RyR2 loss-of-function missense variants cause Ca2+ release deficiency syndrome (CRDS). Recently, truncating variants in RyR2 have also been associated with ventricular arrhythmias (VAs) and sudden cardiac death. However, there are limited insights into the potential clinical relevance and in vitro functional impact of RyR2 truncating variants. We performed genetic screening of patients presenting with syncope, VAs, or unexplained sudden death and in vitro characterization of the expression and function of RyR2 truncating variants in HEK293 cells. We identified two previously unknown RyR2 truncating variants (Y4591Ter and R4663Ter) and one splice site variant predicted to result in a frameshift and premature termination (N4717 + 15Ter). These 3 new RyR2 truncating variants and a recently reported RyR2 truncating variant, R4790Ter, were generated and functionally characterized in vitro. Immunoprecipitation and immunoblotting analyses showed that all 4 RyR2 truncating variants formed heteromers with the RyR2-wildtype (WT) protein. Each of these C-terminal RyR2 truncations was non-functional and suppressed [3H]ryanodine binding to RyR2-WT and RyR2-WT mediated store overload induced spontaneous Ca2+ release activity in HEK293 cells. The expression of these RyR2 truncating variants in HEK293 cells was markedly reduced compared with that of the full-length RyR2 WT protein. Our data indicate that C-terminal RyR2 truncating variants are non-functional and can exert a dominant negative impact on the function of the RyR2 WT protein through formation of heteromeric WT/truncation complex.

Reduced O-GlcNAcylation diminishes cardiomyocyte Ca2+ dependent facilitation and frequency dependent acceleration of relaxation.

Ca2+ dependent facilitation (CDF) and frequency dependent acceleration of relaxation (FDAR) are regulatory mechanisms that potentiate cardiomyocyte Ca2+ channel function and increase the rate of Ca2+ sequestration following a Ca2+-release event, respectively, when depolarization frequency increases. CDF and FDAR likely evolved to maintain EC coupling at increased heart rates. Ca2+/calmodulin-dependent kinase II (CaMKII) was shown to be indispensable to both; however, the mechanisms remain to be completely elucidated. CaMKII activity can be modulated by post-translational modifications but if and how these modifications impact CDF and FDAR is unknown. Intracellular O-linked glycosylation (O-GlcNAcylation) is a post-translational modification that acts as a signaling molecule and metabolic sensor. In hyperglycemic conditions, CaMKII was shown to be O-GlcNAcylated resulting in pathologic activity. Here we sought to investigate whether O-GlcNAcylation impacts CDF and FDAR through modulation of CaMKII activity in a pseudo-physiologic setting. Using voltage-clamp and Ca2+ photometry we show that cardiomyocyte CDF and FDAR are significantly diminished in conditions of reduced O-GlcNAcylation. Immunoblot showed that CaMKIIδ and calmodulin expression are increased but the autophosphorylation of CaMKIIδ and the muscle cell-specific CaMKIIß isoform are reduced by 75% or more when O-GlcNAcylation is inhibited. We also show that the enzyme responsible for O-GlcNAcylation (OGT) can likely be localized in the dyad space and/or at the cardiac sarcoplasmic reticulum and is precipitated by calmodulin in a Ca2+ dependent manner. These findings will have important implications for our understanding of how CaMKII and OGT interact to impact cardiomyocyte EC coupling in normal physiologic settings as well as in disease states where CaMKII and OGT may be aberrantly regulated.

Ca2+ release via InsP3Rs enhances RyR recruitment during Ca2+ transients by increasing dyadic [Ca2+] in cardiomyocytes.

Excitation-contraction coupling (ECC) relies on temporally synchronized sarcoplasmic reticulum (SR) Ca2+ release via ryanodine receptors (RyRs) at dyadic membrane compartments. Neurohormones, such as endothelin-1 (ET-1), that act via Gαq-associated G protein-coupled receptors (GPCRs) modulate Ca2+ dynamics during ECC and induce SR Ca2+ release events involving Ca2+ release via inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs). How the relatively modest Ca2+ release via InsP3Rs elicits this action is not resolved. Here, we investigated whether the actions of InsP3Rs on Ca2+ handling during ECC were mediated by a direct influence on dyadic Ca2+ levels and whether this mechanism contributes to the effects of ET-1. Using a dyad-targeted genetically encoded Ca2+ reporter, we found that InsP3R activation augmented dyadic Ca2+ fluxes during Ca2+ transients and increased Ca2+ sparks. RyRs were required for these effects. These data provide the first direct demonstration of GPCR and InsP3 effects on dyadic Ca2+, and support the notion that Ca2+ release via InsP3Rs influences Ca2+ transients during ECC by facilitating the activation and recruitment of proximal RyRs. We propose that this mechanism contributes to neurohormonal modulation of cardiac function. This article has an associated First Person interview with the first author of the paper.

Dual effects of the small-conductance Ca2+-activated K+ current on human atrial electrophysiology and Ca2+-driven arrhythmogenesis: an in silico study.

By sensing changes in intracellular Ca2+, small-conductance Ca2+-activated K+ (SK) channels dynamically regulate the dynamics of the cardiac action potential (AP) on a beat-to-beat basis. Given their predominance in atria versus ventricles, SK channels are considered a promising atrial-selective pharmacological target against atrial fibrillation (AF), the most common cardiac arrhythmia. However, the precise contribution of SK current (ISK) to atrial arrhythmogenesis is poorly understood, and may potentially involve different mechanisms that depend on species, heart rates, and degree of AF-induced atrial remodeling. Both reduced and enhanced ISK have been linked to AF. Similarly, both SK channel up- and downregulation have been reported in chronic AF (cAF) versus normal sinus rhythm (nSR) patient samples. Here, we use our multiscale modeling framework to obtain mechanistic insights into the contribution of ISK in human atrial cardiomyocyte electrophysiology. We simulate several protocols to quantify how ISK modulation affects the regulation of AP duration (APD), Ca2+ transient, refractoriness, and occurrence of alternans and delayed afterdepolarizations (DADs). Our simulations show that ISK activation shortens the APD and atrial effective refractory period, limits Ca2+ cycling, and slightly increases the propensity for alternans in both nSR and cAF conditions. We also show that increasing ISK counteracts DAD development by enhancing the repolarization force that opposes the Ca2+-dependent depolarization. Taken together, our results suggest that increasing ISK in human atrial cardiomyocytes could promote reentry while protecting against triggered activity. Depending on the leading arrhythmogenic mechanism, ISK inhibition may thus be a beneficial or detrimental anti-AF strategy.NEW & NOTEWORTHY Using our established framework for human atrial myocyte simulations, we investigated the role of the small-conductance Ca2+-activated K+ current (ISK) in the regulation of cell function and the development of Ca2+-driven arrhythmias. We found that ISK inhibition, a promising atrial-selective pharmacological strategy against atrial fibrillation, counteracts the reentry-promoting abbreviation of atrial refractoriness, but renders human atrial myocytes more vulnerable to delayed afterdepolarizations, thus potentially increasing the propensity for ectopic (triggered) activity.

Sexual dimorphism in bidirectional SR-mitochondria crosstalk in ventricular cardiomyocytes.

Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with ß-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.

The roles of transmembrane family proteins in the regulation of store-operated Ca2+ entry.

Store-operated Ca2+ entry (SOCE) is a major pathway for calcium signaling, which regulates almost every biological process, involving cell proliferation, differentiation, movement and death. Stromal interaction molecule (STIM) and ORAI calcium release-activated calcium modulator (ORAI) are the two major proteins involved in SOCE. With the deepening of studies, more and more proteins are found to be able to regulate SOCE, among which the transmembrane (TMEM) family proteins are worth paying more attention. In addition, the ORAI proteins belong to the TMEM family themselves. As the name suggests, TMEM family is a type of proteins that spans biological membranes including plasma membrane and membrane of organelles. TMEM proteins are in a large family with more than 300 proteins that have been already identified, while the functional knowledge about the proteins is preliminary. In this review, we mainly summarized the TMEM proteins that are involved in SOCE, to better describe a picture of the interaction between STIM and ORAI proteins during SOCE and its downstream signaling pathways, as well as to provide an idea for the study of the TMEM family proteins.

Structure and Function of Plant and Mammalian TPC Channels.

Two-pore channels (TPCs) belong to the family of voltage-gated tetrameric cation channels and are ubiquitously expressed in organelles of animals and plants. These channels are believed to be evolutionary intermediates between homotetrameric voltage-gated potassium/sodium channels and the four-domain, single subunit, voltage-gated sodium/calcium channels. Each TPC subunit contains 12 transmembrane segments that can be divided into two homologous copies of an S1-S6 Shaker-like 6-TM domain. A functional TPC channel assembles as a dimer - the equivalent of a voltage-gated tetrameric cation channel. The plant TPC channel is localized in the vacuolar membrane and is also called the SV channel for generating the slow vacuolar (SV) current observed long before its molecular identification. Three subfamilies of mammalian TPC channels have been defined - TPC1, 2, and 3 - with the first two being ubiquitously expressed in animals and TPC3 being expressed in some animals but not in humans. Mammalian TPC1 and TPC2 are localized to the endolysosomal membrane and their functions are associated with various physiological processes. TPC3 is localized in the plasma membrane and its physiological function is not well defined.

Calcium Signaling Consequences of RyR2-S4938F Mutation Expressed in Human iPSC-Derived Cardiomyocytes.

Type-2 ryanodine receptor (RyR2) is the major Ca2+ release channel of the cardiac sarcoplasmic reticulum (SR) that controls the rhythm and strength of the heartbeat, but its malfunction may generate severe arrhythmia leading to sudden cardiac death or heart failure. S4938F-RyR2 mutation in the carboxyl-terminal was expressed in human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) using CRISPR/Cas9 gene-editing technique. Ca2+ signaling and electrophysiological properties of beating cardiomyocytes carrying the mutation were studied using total internal reflection fluorescence microscopy (TIRF) and patch clamp technique. In mutant cells, L-type Ca2+ currents (ICa), measured either by depolarizations to zero mV or repolarizations from +100 mV to -50 mV, and their activated Ca2+ transients were significantly smaller, despite their larger caffeine-triggered Ca2+ release signals compared to wild type (WT) cells, suggesting ICa-induced Ca2+ release (CICR) was compromised. The larger SR Ca2+ content of S4938F-RyR2 cells may underlie the higher frequency of spontaneously occurring Ca2+ sparks and Ca2+ transients and their arrhythmogenic phenotype.

Anti-Inflammatory Effects of Alphitolic Acid Isolated from Agrimonia coreana Nakai Extracts Are Mediated via the Inhibition of ICRAC Activity in T Cells.

Agrimonia pilosa Ledeb., an important medicinal herb in traditional East Asian medicine, is primarily used to treat abdominal pain, dysentery, and hemostasis. There are ten other reported species of Agrimonia plants, including Agrimonia coreana Nakai-a naturally growing species in South Korea-and Agrimonia eupatoria Linn. Although recent studies have isolated numerous active constituents and investigated their effects, the medicinal utility of this herb is not yet fully explored. Through patch-clamp recording, a previous study reported that Agrimonia plant extracts inhibit the function of Ca2+ release-activated Ca2+ channels (CRACs). Herein, we aimed to identify and isolate the main compounds in A. coreana responsible for CRAC inhibition while assessing the anti-inflammatory effects mediated by this inhibition. We demonstrated for the first time that alphitolic acid isolated from A. coreana has a dose-dependent inhibitory effect on CRAC activity and, thus, an inhibitory effect on intracellular calcium increase. Furthermore, analysis of human CD4+ T cell proliferation via the carboxyfluorescein diacetate succinimidyl ester method revealed that alphitolic acid inhibited T cell proliferation in a concentration-dependent manner. Our findings provide a theoretical basis for the potential therapeutic use of alphitolic acid in the treatment of inflammatory diseases.

Impact of mitochondria on local calcium release in murine sinoatrial nodal cells.

Roles of mitochondria in sinoatrial nodal cells (SANCs) have not been fully clarified. We have previously demonstrated that mitochondrial Ca2+ efflux through the Na+-Ca2+ exchanger, NCXm, modulates sarcoplasmic reticulum (SR) Ca2+ content and automaticity of HL-1 cardiomyocytes. In this study, we extended this line of investigation to clarify the spatial and functional association between mitochondria and local calcium release (LCR) from the SR in murine SANCs. High-speed two dimensional (2D) and confocal line-scan imaging of SANCs revealed that LCRs in the early phase of the Ca2+ transient cycle length (CL) appeared with a higher probability near mitochondria. Although LCR increased toward the late phase of CL, no significant difference was noted in the occurrence of late LCRs near and distant from mitochondria. LCRs, especially in the late phase of CL, induced temporal and spatial heterogeneity of the Ca2+ transient amplitude. Attenuating mitochondrial Ca2+ efflux using an NCXm inhibitor, CGP-37157 (1 µM), reduced the amplitude, duration and size of LCR. It also attenuated early LCR occurrence, and simultaneously prolonged LCR period and CL. Additionally, CGP-37157 reduced caffeine-induced Ca2+ transient. Therefore, the inhibitory effect on LCR was attributable to the reduction of the SR Ca2+ content through NCXm inhibition. No obvious off-target effects of 1 µM CGP-37157 were found on T- and L-type voltage-gated Ca2+ currents and hyperpolarization-activated inward current. Taken together, these results suggest that mitochondria are involved in LCR generation by modulating the SR Ca2+ content through NCXm-mediated Ca2+ efflux in murine SANCs.

Inositol 1,4,5-trisphosphate receptor - reactive oxygen signaling domain regulates excitation-contraction coupling in atrial myocytes.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is up-regulated in patients with atrial fibrillation (AF) and InsP3-induced Ca2+ release (IICR) is linked to pro-arrhythmic spontaneous Ca2+ release events. Nevertheless, knowledge of the physiological relevance and regulation of InsP3Rs in atrial muscle is still limited. We hypothesize that InsP3R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP3R agonist affinity and thereby Ca2+ release. To quantitate the contribution of IICR to atrial excitation-contraction coupling (ECC) atrial myocytes (AMs) were isolated from wild type and NOX2 deficient (Nox2-/-) mice and changes in the cytoplasmic Ca2+ concentration ([Ca2+]i; fluo-4/AM, indo-1) or ROS (2',7'-dichlorofluorescein, DCF) were monitored by fluorescence microscopy. Superfusion of AMs with Angiotensin II (AngII: 1 µmol/L) significantly increased diastolic [Ca2+]i (F/F0, Ctrl: 1.00 ± 0.01, AngII: 1.20 ± 0.03; n = 7; p < 0.05), the field stimulation induced Ca2+ transient (CaT) amplitude (ΔF/F0, Ctrl: 2.00 ± 0.17, AngII: 2.39 ± 0.22, n = 7; p < 0.05), and let to the occurrence of spontaneous increases in [Ca2+]i. These changes in [Ca2+]i were suppressed by the InsP3R blocker 2-aminoethoxydiphenyl-borate (2-APB; 1 µmol/L). Concomitantly, AngII induced an increase in ROS production that was sensitive to the NOX2 specific inhibitor gp91ds-tat (1 µmol/L). In NOX2-/- AMs, AngII failed to increase diastolic [Ca2+]i, CaT amplitude, and the frequency of spontaneous Ca2+ increases. Furthermore, the enhancement of CaTs by exposure to membrane permeant InsP3 was abolished by NOX inhibition with apocynin (1 µM). AngII induced IICR in Nox2-/- AMs could be restored by addition of exogenous ROS (tert-butyl hydroperoxide, tBHP: 5 µmol/L). In saponin permeabilized AMs InsP3 (5 µmol/L) induced Ca2+ sparks that increased in frequency in the presence of ROS (InsP3: 9.65 ± 1.44 sparks*s-1*(100µm)-1; InsP3 + tBHP: 10.77 ± 1.5 sparks*s-1*(100µm)-1; n = 5; p < 0.05). The combined effect of InsP3 + tBHP was entirely suppressed by 2-APB and Xestospongine C (XeC). Changes in IICR due to InsP3R glutathionylation induced by diamide could be reversed by the reducing agent dithiothreitol (DTT: 1 mmol/L) and prevented by pretreatment with 2-APB, supporting that the ROS-dependent post-translational modification of the InsP3R plays a role in the regulation of ECC. Our data demonstrate that in AMs the InsP3R is under dual control of agonist induced InsP3 and ROS formation and suggest that InsP3 and NOX2-derived ROS co-regulate atrial IICR and ECC in a defined InsP3R/NOX2 signaling domain.

Prolonged loss of force and power following fatiguing contractions in rat soleus muscles. Is low-frequency fatigue an issue during dynamic contractions?

Low-frequency fatigue (LFF) is defined by a relatively larger deficit in isometric force elicited by low-frequency electrical stimulation compared with high-frequency stimulation. However, the effects of LFF on power during dynamic contractions elicited at low and high frequencies have not been thoroughly characterized. In the current study, rat soleus muscles underwent fatiguing either concentric, eccentric, or isometric contractions. Before and 1 h after the fatiguing contractions, a series of brief isometric and dynamic contractions elicited at 20 and 80 Hz stimulation to establish force-velocity relationships. Maximal force (Fmax), velocity (Vmax), and power (Pmax) were assessed for each frequency. Sarcoplasmic reticulum (SR) Ca2+ release and reuptake rates were assessed pre- and postfatigue. Prolonged fatigue was observed as a loss of Fmax and Pmax in muscles fatigued by concentric or eccentric, but not by isometric contractions. When quantified as a decrease in the ratio between 20 Hz and 80 Hz contractile output, LFF was more pronounced for isometric force than for power (-21% vs. -16% for concentrically fatigued muscles, P = 0.003; 29 vs. 13% for eccentrically fatigued muscles, P < 0.001). No changes in SR Ca2+ release or reuptake rates were observed. We conclude that LFF is less pronounced when expressed in terms of power deficits than when expressed in terms of force deficits, and that LFF, therefore, likely affects performance to a lesser degree during fast concentric contractions than during static or slow contractions.

RyR2 disease mutations at the C-terminal domain intersubunit interface alter closed-state stability and channel activation.

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca2+- and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca2+-independent basal activity and Ca2+-dependent activation of [3H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations.

A gain-of-function mutation in the ITPR1 gating domain causes male infertility in mice.

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an intracellular Ca2+ release channel critical for numerous cellular processes. Despite its ubiquitous physiological significance, ITPR1 mutations have thus far been linked to primarily movement disorders. Surprisingly, most disease-associated ITPR1 mutations generate a loss of function. This leaves our understanding of ITPR1-associated pathology oddly one-sided, as little is known about the pathological consequences of ITPR1 gain of function (GOF). To this end, we generated an ITPR1 gating domain mutation (D2594K) that substantially enhanced the inositol trisphosphate (IP3 )-sensitivity of ITPR1, and a mouse model expressing this ITPR1-D2594K+/- GOF mutation. We found that heterozygous ITPR1-D2594K+/- mutant mice exhibited male infertility, azoospermia, and acrosome loss. Furthermore, we functionally characterized a human ITPR1 variant V494I identified in the UK Biobank database as potentially associated with disorders of the testis. We found that the ITPR1-V494I variant significantly enhanced IP3 -induced Ca2+ release in HEK293 cells. Thus, ITPR1 hyperactivity may increase the risk of testicular dysfunction.