Sarcoplasmic/endoplasmic Reticulum Calcium ATPase Research Articles

Ca2+ Depletion in the ER Causes Store-Operated Ca2+ Entry via the TRPC6 Channel in Mouse Brown Adipocytes.

beta3-adrenergic activation causes Ca2+ release from the mitochondria and subsequent Ca2+ release from the endoplasmic reticulum (ER), evoking store-operated Ca2+ entry (SOCE) due to Ca2+ depletion from the ER in mouse brown adipocytes. In this study, we investigated how Ca2+ depletion from the ER elicits SOCE in mouse brown adipocytes using fluorometry of intracellular Ca2+ concentration ([Ca2+]i). The administration of cyclopiazonic acid (CPA), a reversible sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump blocker in the ER, caused an increase in [Ca2+]i. Moreover, CPA induced SOCE was suppressed by the administration of a Ca2+ free Krebs solution and the transient receptor potential canonical 6 (TRPC6) selective blockers 2-APB, ML-9 and GsMTx-4 but not Pico145, which blocks TRPC1/4/5. Administration of TRPC6 channel agonist 1-oleoyl-2-acetyl-sn-glycerol (OAG) and flufenamic acid elicited Ca2+ entry. Moreover, our RT-PCR analyses detected mRNAs for TRPC6 in brown adipose tissues. In addition, western blot analyses showed the expression of the TRPC6 protein. Thus, TRPC6 is one of the Ca2+ pathways involved in SOCE. These modes of Ca2+ entry provide the basis for heat production via activation of Ca2+-dependent dehydrogenase and the expression of uncoupling protein 1 (UCP1). Enhancing thermogenic metabolism in brown adipocytes may serve as broad therapeutic utility to reduce obesity and metabolic syndrome.

Development of Chemical Tools Based on GSK-7975A to Study Store-Operated Calcium Entry in Cells

Many physiological functions, such as cell differentiation, proliferation, muscle contraction, neurotransmission and fertilisation, are regulated by changes of Ca2+ levels. The major Ca2+ store in cells is the endoplasmic reticulum (ER). Certain cellular processes induce ER store depletion, e.g. by activating IP3 receptors, that in turn induces a store refilling process known as store-operated calcium entry (SOCE). This refilling process entails protein-protein interactions between Ca2+ sensing stromal interaction molecules (STIM) in the ER membrane and Orai proteins in the plasma membrane. Fully assembled STIM/Orai complexes then form highly selective Ca2+ channels called Ca2+ release activated Ca2+ Channels (CRAC) through which Ca2+ ions flow into the cytosol and subsequently are pumped into the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Abnormal SOCE has been associated with numerous human diseases and cancers, and therefore key players STIM and Orai have attracted significant therapeutic interest. Several potent experimental and clinical candidate compounds have been developed and have helped to study SOCE in various cell types. We have synthesized multiple novel small-molecule probes based on the known SOCE inhibitor GSK-7975A. Here we present GSK-7975A derivatives, which feature photo-caging, photo-crosslinking, biotin and clickable moieties, and also contain deuterium labels. Evaluation of these GSK-7975A probes using a fluorometric imaging plate reader (FLIPR)-Tetra-based Ca2+ imaging assay showed that most synthetic modifications did not have a detrimental impact on the SOCE inhibitory activity. The photo-caged GSK-7975A was also used in patch-clamp electrophysiology experiments. In summary, we have developed a number of active, GSK-7975A-based molecular probes that have interesting properties and therefore are useful experimental tools to study SOCE in various cells and settings.

The Contractile Response to Oxytocin in Non-pregnant Rat Uteri Is Modified After the First Pregnancy

During pregnancy, the uterus undergoes several modifications under the influence of hormonal and mechanical stimuli. We hypothesize that while most of these modifications are reverted during involution, some of the physiological properties of the uterus are permanently altered. To investigate this hypothesis, we conducted motility experiments to evaluate the contractility response of uterine tissue samples from non-pregnant virgin and proven breeder female rats to oxytocin (10−10 to 10−5 M). We found that the virgin tissue contracts more robustly than proven breeder tissue in the absence of oxytocin, yet with oxytocin, proven breeder samples displayed a significantly higher increase in activity. These results could depend on a more elevated expression of oxytocin receptor and/or on an alteration in the intracellular pathways affected by the activation of the oxytocin receptors. Here, we explored the impact of some structures involved in the management of intracellular calcium on the dose response to oxytocin recorded from virgin and proven breeder uterine strips. Specifically, we replicated the dose response experiments in low extracellular calcium (10 μM), in the presence of the intracellular calcium channel blocker ruthenium red (10 μM), and in the presence of the sarcoplasmic-endoplasmic reticulum calcium ATP-ase pump inhibitor, cyclopiazonic acid (10 μM). The results of these experiments suggest that also the expression of proteins that control intracellular calcium availability is affected by the experience of pregnancy. Molecular biology experiments will give us more detail on the magnitude of these expression changes.

Endoplasmic reticulum stress regulates autophagic response that is involved in Saikosaponin a-induced liver cell damage

Saikosaponin a (Ssa) is an active ingredient of the Chinese herbal plant Radix Bupleuri (RB) and has severe hepatotoxicity. However, biomolecular mechanisms involved in Ssa-induced hepatotoxicity are not yet entirely clear. Previous studies reported that Ssd (an isomer of Ssa) as a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitor can induce autophagy in apoptotic defective cells, leading to autophagy-dependent cell death. Therefore, we speculate that endoplasmic reticulum (ER) stress and autophagy may also play an important role in Ssa-induced hepatocyte death. This study aimed to explore the connection between ER stress and autophagy and Ssa-induced hepatotoxicity. Experiments in vitro showed that the cell viability of L-02 cells in the Ssa treatment group decreased, the level of autophagy marker LC3-II/LC3-I and Beclin1 increased, the level of p62 decreased, the colocalization of autophagosome and lysosome increased, and the cell viability was significantly increased after the application of autophagy inhibitors 3-MA. In addition, SSa can induce ER stress in L-02 cells in vitro. Further studies demonstrated that SSa activated the PERK/eIF2α/ATF4/CHOP pathway, IRE1-TRAF2 pathway, ATF6 pathway, and AMPK/mTOR pathway associated with ER stress. Application of ER stress inhibitors 4-PBA can significantly down-regulate the level of autophagy and improve cell viability. Results of in vivo experiments showed that treatment with 150 and 300 mg/kg Ssa significantly elevated the liver/body weight ratio and caused histological injury in mice liver. Furthermore, Ssa treatment induced significantly downregulated p62 expression but upregulated LC3-II, CHOP, and GRP78 expression in mice livers. Taken together, our results showed that SSa can activate endoplasmic reticulum stress, promote toxic autophagy, and then induce cell death. We revealed an alternative mechanism involving autophagy and ERs, by which Ssa induced hepatotoxicity.

Research Note: Disturbance of intracellular calcium signal in salpingitis simulation of laying hens

This study investigated whether there is disturbance of calcium signal in the simulated salpingitis of laying hens. A total of 90 Roman Pink layers (81 wk; 1.916 ± 0.17 kg) were divided into 3 groups (Control treated with PBS, 1.85 mg lipopolysaccharide (LPS)/layer as LPS group, 1.85 mg LPS/layer as LPS+organic chemical reagent (OCR) group) with 6 replicates of 5 layers. Compared with the Control, the mRNA expression of calcium/calmodulin dependent protein kinase IV (CaMK IV), sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and plasma membrane calcium-transporting ATPase (PMCA) were not only decreased (P < 0.05) in magnum of laying hens from LPS and LPS+OCR groups, but also in isthmus and uterus of hens from LPS+OCR group. Moreover, the mRNA expression of calcium sensing receptor (CaSR) and Orai1 in uterus from LPS+OCR group were higher (P < 0.05) than that from Control. The relative fluorescence intensity of Ca2+ in uterus from LPS and LPS+OCR groups were significantly higher than that from Control (P < 0.05). In conclusion, it existed that the linkage of simulated salpingitis treated with LPS+OCR and altered intracellular calcium signals in layers, which provided a new insight for alleviating salpingitis and uterine dysfunction of laying hens.

Drug Discovery Using Evolutionary Similarities in Chemical Binding to Inhibit Patient-Derived Hepatocellular Carcinoma.

Drug resistance causes therapeutic failure in refractory cancer. Cancer drug resistance stems from various factors, such as patient heterogeneity and genetic alterations in somatic cancer cells, including those from identical tissues. Generally, resistance is intrinsic for cancers; however, cancer resistance becomes common owing to an increased drug treatment. Unfortunately, overcoming this issue is not yet possible. The present study aimed to evaluate a clinical approach using candidate compounds 19 and 23, which are sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) inhibitors, discovered using the evolutionary chemical binding similarity method. mRNA sequencing indicated SERCA as the dominant marker of patient-derived anti-cancer drug-resistant hepatocellular carcinoma (HCC), but not of patient-derived anti-cancer drug-sensitive HCC. Candidate compounds 19 and 23 led to significant tumor shrinkage in a tumor xenograft model of anti-cancer drug-resistant patient-derived HCC cells. Our results might be clinically significant for the development of novel combinatorial strategies that selectively and efficiently target highly malignant cells such as drug-resistant and cancer stem-like cells.

Structural Analysis of the Interaction of Pyrazole and Benzimidazole Core Compounds with PfATP4

P‐type cation ATPase in Plasmodium falciparum(PfATP4) serves as one of the important emerging drug targets for the treatment of the malaria. PfATP4 maintains sodium homeostasis in the parasite and its inhibition causes the death of the parasite through a mechanism that mimics premature schizogony. Over the past few years, we and others have identified ~20 different chemotypes of molecules that can disrupt sodium homeostasis via PfATP4. We have developed two series of potent PfATP4 inhibitors with a pyrazole and/or benzimidazole core and tested them on wild type and lab resistant strains of P. falciparum. However, due to lack of PfATP4 crystal structure, we have limited understanding of the structure‐activity relationship among the analogs of these molecules and the mechanism of inhibition of PfATP4 which has hindered the rational optimization of the discovered inhibitors. In this study, we modeled the E1 and E2 states of PfATP4 using homology modeling with the corresponding state templates from the mammalian Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA). Models of PfATP4 were refined using molecular dynamics simulations and ~200 pyrazole and benzimidazole compounds were docked to the binding site using GOLD molecular docking software. Results from the docking study suggests that the compounds preferred mode of binding is to the proton bound E2 state of PfATP4. The docking scores significantly correlated with IC50 values with a correlation co‐efficient &gt; 0.7. Further MD simulations of the docked complexes suggested residues GLN172, GLN351, SER175 and ASN207 play a key role in ligand binding which may have a role in the development of resistance to PfATP4 inhibitors which needs further experimental validation.

Calcium handling genes are regulated by promoter DNA methylation in colorectal cancer cells

Calcium signaling regulates various cellular processes, including proliferation and cell death. DNA methylation of gene promoters is an epigenetic modification that facilitates transcriptional suppression. Disruption of calcium homeostasis and DNA methylation in cancer are each linked to tumor development and progression. However, the possible connection between these two processes has not been thoroughly studied. Therefore, we measured the expression of six gene families involved in calcium regulation (ATP2A, ITPR, ORAI, RyR, STIM, and TRPC) in a colorectal cancer cell model, HCT116, with either genetic (Double Knock-out/DKO) or pharmacological (5-aza-2′-deoxycytidine/DAC) inhibition of DNA methyltransferases. Fourteen of the 20 examined calcium handling genes were expressed at higher levels in DKO cells as compared to HCT116. Expression of five genes was increased in HCT116 cells treated with DAC, three matching DKO. Due to a unique expression pattern of the three ATP2A genes in our model, encoding the Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA) pumps, we chose to evaluate the methylation status of these genes, protein expression, and potential associated physiological effects, using the SERCA inhibitor thapsigarin (TG). We observed an expected pattern of promoter methylation coinciding with reduced expression and vice versa. This differential mRNA expression was associated with altered SERCA3 protein expression and cytosolic calcium levels with TG exposure. As a result, DKO cells displayed less TG-induced cytotoxicity, as compared to HCT116 cells. Overall, it is likely that at least several calcium regulatory genes are transcriptionally regulated by DNA methylation, and this may play a role in tumorigenesis through altering apoptosis in cancer.

Increasing SERCA function promotes initiation of calcium sparks and breakup of calcium waves.

Increasing sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump activity enhances sarcoplasmic reticulum calcium (Ca) load, which increases both ryanodine receptor opening and driving force of Ca release flux. Both of these effects promote Ca spark formation and wave propagation. However, increasing SERCA activity also accelerates local cytosolic Ca decay as the wave front travels to the next cluster, which limits wave propagation. As a result, increasing SERCA pump activity has a biphasic effect on the propensity of arrhythmogenic Ca waves, but a monotonic effect to increase Ca spark frequency and amplitude. Waves of sarcoplasmic reticulum (SR) calcium (Ca) release can cause arrhythmogenic afterdepolarizations in cardiac myocytes. Ca waves propagate when Ca sparks at one Ca release unit (CRU) recruit new Ca sparks in neighbouring CRUs. Under normal conditions, Ca sparks are too small to recruit neighbouring Ca sparks where Ca sensitivity is also low. However, under pathological conditions such as a Ca overload or ryanodine receptor (RyR) sensitization, Ca sparks can be larger and propagate more readily as macro-sparks or full Ca waves. Increasing SERCA pump activity promotes SR Ca load, which promotes RyR opening and increases driving force of the Ca release flux from SR to cytosol, promoting Ca waves. However, high sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) activity can also decrease local cytosolic [Ca] as it approaches the next CRU, thereby reducing wave appearance and propagation. In this study, we use a physiologically detailed model of subcellular Ca cycling and experiments in phospholamban-knockout mice, to show how Ca waves are initiated and propagate and how different conditions contribute to the generation and propagation of Ca waves. We show that reducing diffusive coupling between Ca sparks by increasing SERCA activity prevents Ca waves by reducing [Ca] at the next CRU, as do Ca buffers, low intra-SR Ca diffusion and distance between CRUs. Increasing SR Ca uptake rate has a biphasic effect on Ca wave propagation; initially it enhances Ca spark probability and amplitude and CRU coupling, thereby promoting arrhythmogenic Ca wave propagation, but at higher levels SR Ca uptake can abort those arrhythmogenic Ca waves.

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.

Synthesis and Pharmacological Characterization of 2-Aminoethyl Diphenylborinate (2-APB) Derivatives for Inhibition of Store-Operated Calcium Entry (SOCE) in MDA-MB-231 Breast Cancer Cells.

Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca2+ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP3) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca2+ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca2+ channels that facilitate a measurable Ca2+ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.

Modulation of SERCA in Patients with Persistent Atrial Fibrillation Treated by Epicardial Thoracoscopic Ablation: The CAMAF Study.

Objectives: To evaluate atrial fibrillation (AF) recurrence and Sarcoplasmic Endoplasmic Reticulum Calcium ATPase (SERCA) levels in patients treated by epicardial thoracoscopic ablation for persistent AF. Background: Reduced levels of SERCA have been reported in the peripheral blood cells of patients with AF. We hypothesize that SERCA levels can predict the response to epicardial ablation. Methods: We designed a prospective, multicenter, observational study to recruit, from October 2014 to June 2016, patients with persistent AF receiving an epicardial thoracoscopic pulmonary vein isolation. Results: We enrolled 27 patients. Responders (n = 15) did not present AF recurrence after epicardial ablation at one-year follow-up; these patients displayed a marked remodeling of the left atrium, with a significant reduction of inflammatory cytokines, B type natriuretic peptide (BNP), and increased levels of SERCA compared to baseline and to nonresponders (p < 0.05). Furthermore, mean AF duration (Heart rate (HR) 1.235 (1.037–1.471), p < 0.05), Left atrium volume (LAV) (HR 1.755 (1.126–2.738), p < 0.05), BNP (HR 1.945 (1.895–1.999), p < 0.05), and SERCA (HR 1.763 (1.167–2.663), p < 0.05) were predictive of AF recurrence. Conclusions: Our data indicate for the first time that baseline values of SERCA in patients with persistent AF might be predictive of failure to epicardial ablative approach. Intriguingly, epicardial ablation was associated with increased levels of SERCA in responders. Therefore, SERCA might be an innovative therapeutic target to improve the response to epicardial ablative treatments.

Ultra-low doses of the transient receptor potential vanilloid 1 agonist, resiniferatoxin, prevents vomiting evoked by diverse emetogens in the least shrew (Cryptotis parva).

Published studies have shown that the transient receptor potential vanilloid 1 (TRPV1) receptor agonist, resiniferatoxin (RTX), has pro and antiemetic effects. RTX can suppress vomiting evoked by a variety of nonselective emetogens such as copper sulfate and cisplatin in several vomit-competent species. In the least shrew, we have already demonstrated that combinations of ultra-low doses of RTX and low doses of the cannabinoid CB1/2 receptor agonist delta-9-tetrahydrocannabinol (Δ-THC) produce additive antiemetic effects against cisplatin-evoked vomiting. In the current study, we investigated the broad-spectrum antiemetic potential of very low nonemetic doses of RTX against a diverse group of specific emetogens including selective and nonselective agonists of serotonergic 5-hydroxytrptamine (5-HT3) receptor (5-HT and 2-Me-5-HT), dopaminergic D2 receptor (apomorphine and quinpirole), cholinergic M1 receptor (pilocarpine and McN-A-343), as well as the selective substance P neurokinin NK1 receptor agonist GR73632, the selective L-Type calcium channel agonist FPL64176, and the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA) inhibitor thapsigargin. When administered subcutaneously, ultra-low (0.01 µg/kg) to low (5.0 µg/kg) doses of RTX suppressed vomiting induced by the aforementioned emetogens in a dose-dependent fashion with 50% inhibitory dose values ranging from 0.01 to 1.26 µg/kg. This study is the first to demonstrate that low nanomolar nonemetic doses of RTX have the capacity to completely abolish vomiting caused by diverse receptor specific emetogens in the least shrew model of emesis.

P4466Empagliflozin improves heart function after myocardial infarction in the rat

Abstract Background Selective Sodium-glucose cotransporter 2 (SGLT2) inhibition with Empagliflozin reduced progression of left ventricular dysfunction and improved tolerance of physical exercise in heart failure by normoglycemic rats. Here, we hypothesized that Empagliflozin prevents cardiac dysfunction after myocardial infarction (MI). Purpose This study aimed to investigate whether Empagliflozin protects the heart in the early phase after experimental MI in normoglycemic rats. Methods MI was induced in Wistar rats via permanent ligation of the left coronary artery. Treatment with Empagliflozin (1 mg/kg/daily per os) was started after MI and continued for 7 days. Sham operated and vehicle treated animals served as controls (n=8). Hemodynamic parameters were measured via transthoracic echocardiography and intracardiac Samba catheter. Glucose concentration was determined in serum and urine. Protein expression of Na+/H+ exchanger isoform-1 (NHE-1), sodium bicarbonate co-transporter (NBC), Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), transforming growth factor beta 1 (TGF-beta1), Smad2 in the left ventricle were also studied. Additionally, NHE-1 regulation was investigated in cardiomyocyte cell line H9c2. Results Systolic heart function was improved in Empagliflozin treated MI animals compared to vehicle as demonstrated by Global Longitudinal Strain (GLS) (20,9% vs. 16,6%; p&lt;0.05). E/A ratio was decreased by tendency and blood pressure was not affected. Ejection fraction (p&lt;0.05), fractional shortening (p&lt;0.05), stroke volume (p&lt;0.01) were increased in Empagliflozin treated control rats as compared with the sham group. Moreover, application of Empagliflozin (1mg/kg, i.v. bolus) to healthy rats in 30 min increased maximal pressure in the left ventricle as compared with vehicle (110,5±15,3 mmHg vs 79,1±11,9 mmHg; p&lt;0.05). Parallel, dP/dtmax was increased while dP/dtmin was decreased by tendency. Empagliflozin treatment did not affect glucose concentration in serum and urine. Treatment of cardiac H9c2 cells with Empagliflozin (1μM) down-regulated NHE-1 by 27%. Conclusion SGLT2 inhibitor Empagliflozin improved systolic function in the early phase post MI independently from glucose regulation. The cardioprotective mechanisms of SGLT2 inhibitors may involve cardiac NHE-1 exchanger inhibition.

SERCA Activity Controls the Systolic Calcium Increase in the Nucleus of Cardiac Myocytes.

In cardiomyocytes, nuclear calcium is involved in regulation of transcription and, thus, remodeling. The cellular mechanisms regulating nuclear calcium, however, remain elusive. Therefore, the aim of this study was to identify and characterize the factors that regulate nuclear calcium in cardiomyocytes. We focused on the roles of (1) the cytoplasmic calcium transient (CaT), (2) the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and (3) intracellular calcium stores for nuclear calcium handling. Experiments were performed on rat ventricular myocytes loaded with Fluo-4/AM. Subcellularly resolved CaTs were visualized using confocal microscopy. The cytoplasmic CaT was varied by reducing extracellular calcium (from 1.5 to 0.3 mM) or by exposure to isoprenaline (ISO, 10 nM). SERCA was blocked by thapsigargin (5 μM). There was a strict linear dependence of the nucleoplasmic CaT on the cytoplasmic CaT over a wide range of calcium concentrations. Increasing SERCA activity impaired, whereas decreasing SERCA activity augmented the systolic calcium increase in the nucleus. Perinuclear calcium store load, on the other hand, did not change with either 0.3 mM calcium or ISO and was not a decisive factor for the nucleoplasmic CaT. The results indicate, that the nucleoplasmic CaT is determined largely by the cytoplasmic CaT via diffusion of calcium through nuclear pores. They identify perinuclear SERCA activity, which limits the systolic calcium increase in the nucleus, as a novel regulator of the nuclear CaT in cardiac myocytes.

Computational modeling of amylin-induced calcium dysregulation in rat ventricular cardiomyocytes

Hyperamylinemia is a condition that accompanies obesity and precedes type II diabetes, and it is characterized by above-normal blood levels of amylin, the pancreas-derived peptide. Human amylin oligomerizes easily and can deposit in the pancreas [1], brain [2], and heart [3], where they have been associated with calcium dysregulation. In the heart, accumulating evidence suggests that human amylin oligomers form moderately cation-selective [4,5] channels that embed in the cell sarcolemma (SL). The oligomers increase membrane conductance in a concentration-dependent manner [5], which is correlated with elevated cytosolic Ca2+. These findings motivate our core hypothesis that non-selective inward Ca2+ conduction afforded by human amylin oligomers increase cytosolic and sarcoplasmic reticulum (SR) Ca2+ load, which thereby magnifies intracellular Ca2+ transients. Questions remain however regarding the mechanism of amylin-induced Ca2+ dysregulation, including whether enhanced SL Ca2+ influx is sufficient to elevate cytosolic Ca2+ load [6], and if so, how might amplified Ca2+ transients perturb Ca2+-dependent cardiac pathways. To investigate these questions, we modified a computational model of cardiomyocytes Ca2+ signaling to reflect experimentally-measured changes in SL membrane permeation and decreased sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) function stemming from acute and transgenic human amylin peptide exposure. With this model, we confirmed the hypothesis that increasing SL permeation alone was sufficient to enhance Ca2+ transient amplitudes. Our model indicated that amplified cytosolic transients are driven by increased Ca2+ loading of the SR and that greater fractional release may contribute to the Ca2+-dependent activation of calmodulin, which could prime the activation of myocyte remodeling pathways. Importantly, elevated Ca2+ in the SR and dyadic space collectively drive greater fractional SR Ca2+ release for human amylin expressing rats (HIP) and acute amylin-exposed rats (+Amylin) mice, which contributes to the inotropic rise in cytosolic Ca2+ transients. These findings suggest that increased membrane permeation induced by oligomeratization of amylin peptide in cell sarcolemma contributes to Ca2+ dysregulation in pre-diabetes.

Smooth Muscle Nitric Oxide Responsiveness and Clinical Maturation of Hemodialysis Arteriovenous Fistulae

The arteriovenous fistula is the preferred type of hemodialysis vascular access for patients with end-stage renal disease, but a high proportion of newly created fistulas fail to mature for use. Stenosis caused by neointimal hyperplasia often is present in fistulas with maturation failure, suggesting that local mechanisms controlling vascular smooth muscle cell (SMC) migration and proliferation are important contributors to maturation failure. SMCs cultured from explants of vein tissue obtained at the time of fistula creation from 19 patients with end-stage renal disease were studied to determine whether smooth muscle responsiveness to nitric oxide is associated with fistula maturation outcomes. Nitric oxide-induced inhibition of smooth muscle cell migration, but not proliferation, was greater in cells from patients with subsequent fistula maturation success than from patients with subsequent fistula maturation failure (mean inhibition percentage, 17 versus 5.7, respectively; P=0.035). Impaired nitric oxide responsiveness was associated with oxidation of the calcium regulatory protein, sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), and was reversed by overexpressing SERCA (1.8-fold increase in inhibition, P=0.0128) or down-regulating Nox4-based NADPH oxidase (2.3-fold increase in inhibition; P=0.005). Our data suggest that the nitric oxide responsiveness of SMC migration is associated with fistula maturation success and raises the possibility that therapeutic restoration of nitric oxide responsiveness through manipulation of local mediators may prevent fistulamaturation failure.

Content decline of SERCA inhibitors saikosaponin a and d attenuates cardiotoxicity and hepatotoxicity of vinegar-baked Radix bupleuri

Improper usage of unprocessed Radix bupleuri root (chaihu) may cause cardiotoxicity and liver injury. Baking herb with vinegar is believed to attenuate the adverse responses. However, the chemical and molecular basis involved remained unclear. To this end, we investigated the in vitro toxicity of saikosaponin a, c, d, and their hydrolysates saikosaponin b1 and b2. Results showed that SSa and SSd possessed higher affinity with sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) by molecular docking, and exhibited stronger toxic responses on cardiomyocytes and hepatocytes than the other three saikosaponins in equivalent concentrations. Further, SSa and SSd induced LC3 puncta formation in U2OS-mCherry-EGFP-LC3 cells. Blockage of autophagy by 3-methyladenine did not abrogate the cytotoxicities induced by SSa and SSd. In parallel, none of SSc, SSb1, or SSb2 caused cell injury. Our study reveals how changes in chemical ingredients are connected to the toxicity of Chaihu during vinegar baking process and also provides a guidance for structure optimization to reduce drug induced toxicity.

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

3',5'-cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced β-adrenergic receptor-cAMP signalling to SERCA.

αB-Crystallin R120G variant causes cardiac arrhythmias and alterations in the expression of Ca(2+) -handling proteins and endoplasmic reticulum stress in mice.

Mutations of αB-crystallin (CryαB), a small heat shock protein abundantly expressed in cardiac and skeletal muscles, are known to cause desmin-related myopathies. The CryαB R120G allele has been linked to a familial desminopathy and, in transgenic mice, causes a sudden death at about 28weeks of age. To investigate the mechanisms of the sudden cardiac arrest of CryαB R120G transgenic mice, we prepared protein samples from left ventricular tissues of two different age groups (10 and 28weeks) and examined Ca(2+) -handling proteins. Expression of sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) 2, phospholamban, ryanodine receptor 2 and calsequestrin 2 was significantly decreased in 28- versus 10-week-old CryαB R120G transgenic mice. In addition, low heart rate variability, including heart rate, total power and low frequency, was observed and continuous electrocardiogram monitoring revealed cardiac arrhythmias, such as ventricular tachycardia, atrioventricular block and atrial flutter, in 28-week-old CryαB R120G transgenic mice. In contrast, expression of endoplasmic reticulum (ER) degradation enhancing α-mannosidase-like protein, inositol requirement 1 and X-box binding protein 1 were increased significantly in 28- versus 10-week-old CryαBR120G transgenic mice, suggesting that the CryαBR120G transgenic mice exhibit increased ER stress compared with wild-type mice. Together, the data suggest that the CryαB R120G dominant variant induces ER stress and impairs Ca(2+) regulation, leading to ageing-related cardiac dysfunction, arrhythmias and decreased autonomic tone with shortened lifespan.