Sarcoplasmic-endoplasmic Reticulum Ca Research Articles

Atrial cardiomyocytes contribute to the inflammatory status associated with atrial fibrillation in right heart disease.

Right heart disease (RHD), characterized by right ventricular (RV) and atrial (RA) hypertrophy, and cardiomyocytes' (CM) dysfunctions have been described to be associated with the incidence of atrial fibrillation (AF). Right heart disease and AF have in common, an inflammatory status, but the mechanisms relating RHD, inflammation, and AF remain unclear. We hypothesized that right heart disease generates electrophysiological and morphological remodelling affecting the CM, leading to atrial inflammation and increased AF susceptibility. Pulmonary artery banding (PAB) was surgically performed (except for sham) on male Wistar rats (225-275 g) to provoke an RHD. Twenty-one days (D21) post-surgery, all rats underwent echocardiography and electrophysiological studies (EPS). Optical mapping was performed in situ, on Langendorff-perfused hearts. The contractility of freshly isolated CM was evaluated and recorded during 1 Hz pacing in vitro. Histological analyses were performed on formalin-fixed RA to assess myocardial fibrosis, connexin-43 levels, and CM morphology. Right atrial levels of selected genes and proteins were obtained by qPCR and Western blot, respectively. Pulmonary artery banding induced severe RHD identified by RV and RA hypertrophy. Pulmonary artery banding rats were significantly more susceptible to AF than sham. Compared to sham RA CM from PAB rats were significantly elongated and hypercontractile. Right atrial CM from PAB animals showed significant augmentation of mRNA and protein levels of pro-inflammatory interleukin (IL)-6 and IL1β. Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) and junctophilin-2 were decreased in RA CM from PAB compared to sham rats. Right heart disease-induced arrhythmogenicity may occur due to dysfunctional SERCA2a and inflammatory signalling generated from injured RA CM, which leads to an increased risk of AF.

Comparative Efficacy of Levosimendan, Ramipril, and Sacubitril/ Valsartan in Isoproterenol-induced Experimental Heart Failure: A Hemodynamic and Molecular Approach.

Cardiac ischemia-related myocardial damage has been considered a major reason for heart failure. We aimed to investigate the role of levosimendan (LEVO) in comparison to ramipril and sacubitril/valsartan (Sac/Val) in preventing damage associated with isoproterenol (ISO) induced myocardial infarction. Myocardial infarction was induced by injecting subcutaneous isoproterenol (5 mg/kg once for 7 consecutive days) to establish an experimental heart failure model. Simultaneously, LEVO (1 mg/kg/day), ramipril (3mg/kg/day) and Sac/Val (68 mg/kg/day) suspension were administered orally for four weeks. We observed a significant correlation between ISO-induced ischemia with cardiac remodeling and alterations in myocardial architecture. LEVO, ramipril, and Sac/Val significantly prevented lipid peroxidation and damaged antioxidant enzymes like superoxide dismutase, catalase, glutathione and thioredoxin reductase. We also observed their ameliorative effects in myocardium's cardiac hypertrophy, evidenced by reduced heart weight to body weight ratio and transforming growth factor β related collagen deposition. LEVO, ramipril, and Sac/Val also maintained cardiac biomarkers like lactate dehydrogenase, creatine kinase-MB, brain natriuretic peptide and cardiac Troponin-I, indicating reduced myocardial damage that was further demonstrated by histopathological examination. Decreased sarcoplasmic endoplasmic reticulum Ca2+ATPase2a and sodium-calcium exchanger-1 protein depletion after LEVO, ramipril, and Sac/Val administration indicated improved Ca2+ homeostasis during myocardial contractility. Our findings suggest that LEVO has comparable effects to ramipril, and Sac/Val in preventing myocardial damage via balancing oxidant-antioxidant system, decreased collagen deposition, reduced myocardial stress as well as improved Ca2+ homeostasis during myocardial contractility.

Recruitment of Muscle Genes as an Effect of Brown Adipose Tissue Ablation in Cold-Acclimated Brandt’s Voles (Lasiopodomys brandtii)

Skeletal muscle-based nonshivering thermogenesis (NST) plays an important role in the regulation and maintenance of body temperature in birds and large mammals, which do not contain brown adipose tissue (BAT). However, the relative contribution of muscle-based NST to thermoregulation is not clearly elucidated in wild small mammals, which have evolved an obligate thermogenic organ of BAT. In this study, we investigated whether muscle would become an important site of NST when BAT function is conditionally minimized in Brandt's voles (Lasiopodomys brandtii). We surgically removed interscapular BAT (iBAT, which constitutes 52%~56% of total BAT) and exposed the voles to prolonged cold (4 °C) for 28 days. The iBAT-ablated voles were able to maintain the same levels of NST and body temperature (~37.9 °C) during the entire period of cold acclimation as sham voles. The expression of uncoupling protein 1 (UCP1) and its transcriptional regulators at both protein and mRNA levels in the iBAT of cold-acclimated voles was higher than that in the warm group. However, no difference was observed in the protein or mRNA levels of these thermogenesis-related markers except for PGC-1α in other sites of BAT (including infrascapular region, neck, and axilla) between warm and cold groups either in sham or iBAT-ablated voles. The iBAT-ablated voles showed higher UCP1 expression in white adipose tissue (WAT) than sham voles during cold acclimation. The expression of sarcolipin (SLN) and sarcoplasmic endoplasmic reticulum Ca2+-dependent adenosine triphosphatase (SERCA) in skeletal muscles was higher in cold than in warm, but no alteration in phospholamban (PLB) and phosphorylated-PLB (P-PLB) was observed. Additionally, there was increased in iBAT-ablated voles compared to that in the sham group in cold. Moreover, these iBAT-ablated voles underwent extensive remodeling of mitochondria and genes of key components related with mitochondrial metabolism. These data collectively indicate that recruitment of skeletal muscle-based thermogenesis may compensate for BAT impairment and suggest a functional interaction between the two forms of thermogenic processes of iBAT and skeletal muscle in wild small mammals for coping cold stress.

Evidence for Bell-Shaped Dose-Response Emetic Effects of Temsirolimus and Analogs: The Broad-Spectrum Antiemetic Efficacy of a Large Dose of Temsirolimus Against Diverse Emetogens in the Least Shrew (Cryptotis parva).

Temsirolimus is a prodrug form of sirolimus (rapamycin). With its analogs (everolimus, ridaforolimus, and rapamycin), it forms a group of anticancer agents that block the activity of one of the two mammalian targets of rapamycin (mTOR) complexes, mTORC1. We investigated the emetic potential of varying doses (0, 0.5, 1, 2.5, 5, 10, 20, and 40 mg/kg, i.p.) of temsirolimus in the least shrew. Temsirolimus caused a bell-shaped and dose-dependent increase in both the mean vomit frequency and the number of shrews vomiting with maximal efficacy at 10 mg/kg (p < 0.05 and p < 0.02, respectively). Its larger doses (20 or 40 mg/kg) had no significant emetic effect. We also evaluated the emetic potential of its analogs (5, 10, and 20 mg/kg, i.p.), all of which exhibited a similar emetic profile. Our observational studies indicated that temsirolimus can reduce the shrew motor activity at 40 mg/kg, and subsequently, we examined the motor effects of its lower doses. At 10 and 20 mg/kg, it did not affect the spontaneous locomotor activity (distance moved) but attenuated the mean rearing frequency in a U-shaped manner at 10 mg/kg (p < 0.05). We then determined the broad-spectrum antiemetic potential of a 20 mg/kg (i.p.) dose of temsirolimus against diverse emetogens, including selective and nonselective agonists of 1) dopaminergic D2/3 receptors (apomorphine and quinpirole); 2) serotonergic 5-HT3 receptors [5-HT (serotonin) and 2-methyl-5-HT]; 3) cholinergic M1 receptors (pilocarpine and McN-A-343); 4) substance P neurokinin NK1 receptors (GR73632); 5) the L-type calcium (Ca2+) channel (LTCC) (FPL64176); 6) the sarcoplasmic endoplasmic reticulum Ca2+ ATPase inhibitor, thapsigargin; 7) the CB1 receptor inverse agonist/antagonist, SR141716A; and 8) the chemotherapeutic cisplatin. Temsirolimus prevented vomiting evoked by the aforementioned emetogens with varying degrees. The mechanisms underlying the pro- and antiemetic effects of temsirolimus evaluated by immunochemistry for c-fos expression demonstrated a c-fos induction in the AP and NTS, but not DMNX with the 10 mg/kg emetic dose of temsirolimus, whereas its larger antiemetic dose (20 mg/kg) had no significant effect. Our study is the first to provide preclinical evidence demonstrating the promising antiemetic potential of high doses of temsirolimus and possibly its analogs in least shrews.

The Role of Curcumin as An Antimalarial Agent

Malaria remains a global health burden, with 219 million cases worldwide in 2017, resulting in 435,000 deaths. The main obstacle in malaria treatment is resistance to antimalarial drugs, rendering patients at risk of complications. Consequently, traditional herbs have been widely developed and studied as adjuvant therapies for malaria. Curcumin, an active compound of turmeric has been known to possess antimalarial effects against various Plasmodium species. It has a beneficial impact on both parasite removal and endothelial protection and may promote the immune response against Plasmodium via increasing the reactive oxygen species production, activating PPARγ/Nrf2 and upregulating CD36 expression in monocytes or macrophages to phagocytose parasite-infected erythrocytes. It can also downregulate the proinflammatory cytokine response and the expression of various adhesion molecules in endothelial cells and may bind to the heterodimer interface of the α/β-tubulin, causing the depolarisation of microtubules in the mitosis phase during cell division. Curcumin also has a role as an antiplasmodial agent via targeting PfATP6, the parasite orthologue of mammalian SERCA (sarcoplasmic-endoplasmic reticulum Ca2+-ATPase). Furthermore, it inhibits the formation of β-hematin and glycogen synthase kinase-3β (GSK3β), a protein kinase that mediates an anti-inflammatory response through NF-ƘB activation. Moreover, it is a histone acetyltransferase (HAT) inhibitor that is involved in parasite chromatin modifications. In summary, curcumin has a potency to be used as an adjuvant therapy for malaria.

Sarcoplasmic reticulum calcium mishandling: central tenet in heart failure?

Excitation-contraction coupling links excitation of the sarcolemmal surface membrane to mechanical contraction. In the heart this link is established via a Ca2+-induced Ca2+ release process, which, following sarcolemmal depolarisation, prompts Ca2+ release from the sarcoplasmic reticulum (SR)though the ryanodine receptor (RyR2). This substantially raises the cytoplasmic Ca2+ concentration to trigger systole. In diastole, Ca2+ is removed from the cytoplasm, primarily via the sarcoplasmic-endoplasmic reticulum Ca2+-dependent ATPase (SERCA) pump on the SRmembrane, returning Ca2+ to the SR store. Ca2+ movement across the SR is thus fundamental to the systole/diastole cycle and plays an essential role in maintaining cardiac contractile function. Altered SR Ca2+ homeostasis (due to disrupted Ca2+ release, storage, and reuptake pathways) is a central tenet of heart failure and contributes to depressed contractility, impaired relaxation, and propensity to arrhythmia. This review will focus on the molecular mechanisms that underlie asynchronous Ca2+ cycling around the SR in the failing heart. Further, this review will illustrate that the combined effects of expression changes and disruptions to RyR2 and SERCA2a regulatory pathways are critical to the pathogenesis of heart failure.

Hydrogen sulfide dilates the isolated retinal artery mainly via the activation of myosin phosphatase

AimsHydrogen sulfide (H2S) is shown in ocular tissues and suggested to involve in the regulation of retinal circulation. However, the mechanism of H2S-induced relaxation on retinal artery is not clarified yet. Herein, we aimed to evaluate the role of several calcium (Ca2+) signaling and Ca2+ sensitization mechanisms in the relaxing effect of H2S donor, NaHS, on retinal arteries. Materials and methodsRelaxing effects of NaHS (10−5–3 × 10−3M) were determined on precontracted retinal arteries in Ca2+ free medium as well as in the presence of the inhibitors of Ca2+ signaling and Ca2+ sensitization mechanisms. Additively, Ca2+ sensitivity of the contractile apparatus were evaluated by CaCl2-induced contractions in the presence of NaHS (3 × 10−3M). Functional experiments were furtherly assessed by protein and/or mRNA expressions, as appropriate. Key findingsThe relaxations to NaHS were preserved in Ca2+ free medium while NaHS pretreatment decreased the responsiveness to CaCl2. The inhibitors of plasmalemmal Ca2+-ATPase, sarcoplasmic-endoplasmic reticulum Ca2+-ATPase, Na+-Ca2+ ion-exchanger and myosin light chain kinase (MLCK) unchanged the relaxations to NaHS. Likewise, Ca2+ sensitization mechanisms including, rho kinase, protein kinase C and tyrosine kinase were unlikely to mediate the relaxation to NaHS in retinal artery. Whereas, a marked reduction was determined in NaHS-induced relaxations in the presence of MLCP inhibitor, calyculin A. Supportively, NaHS pretreatment significantly reduced phosphorylation of MYPT1-subunit of MLCP. SignificanceThe relaxing effect of NaHS in retinal artery is likely to be related to the activation of MLCP and partly, to decrement in Ca2+ sensitivity of contractile apparatus.

The role of P-type IIA and P-type IIB Ca2+-ATPases in plant development and growth.

As sessile organisms, plants have evolved mechanisms to adapt to variable and rapidly fluctuating environmental conditions. Calcium (Ca2+) in plant cells is a versatile intracellular second messenger that is essential for stimulating short- and long-term responses to environmental stresses through changes in its concentration in the cytosol ([Ca2+]cyt). Increases in [Ca2+]cyt direct the strength and length of these stimuli. In order to terminate them, the cells must then remove the cytosolic Ca2+ against a concentration gradient, either taking it away from the cell or storing it in organelles such as the endoplasmic reticulum (ER) and/or vacuoles. Here, we review current knowledge about the biological roles of plant P-type Ca2+-ATPases as potential actors in the regulation of this cytosolic Ca2+ efflux, with a focus the IIA ER-type Ca2+-ATPases (ECAs) and the IIB autoinhibited Ca2+-ATPases (ACAs). While ECAs are analogous proteins to animal sarcoplasmic-endoplasmic reticulum Ca2+-ATPases (SERCAs), ACAs are equivalent to animal plasma membrane-type ATPases (PMCAs). We examine their expression patterns in cells exhibiting polar growth and consider their appearance during the evolution of the plant lineage. Full details of the functions and coordination of ECAs and ACAs during plant growth and development have not yet been elucidated. Our current understanding of the regulation of fluctuations in Ca2+ gradients in the cytoplasm and organelles during growth is in its infancy, but recent technological advances in Ca2+ imaging are expected to shed light on this subject.

Possible Role of the Ca2+/Mn2+ P-Type ATPase Pmr1p on Artemisinin Toxicity through an Induction of Intracellular Oxidative Stress.

Artemisinins are widely used to treat Plasmodium infections due to their high clinical efficacy; however, the antimalarial mechanism of artemisinin remains unresolved. Mutations in P. falciparum ATPase6 (PfATP6), a sarcoplasmic endoplasmic reticulum Ca2+-transporting ATPase, are associated with increased tolerance to artemisinin. We utilized Saccharomyces cerevisiae as a model to examine the involvement of Pmr1p, a functional homolog of PfATP6, on the toxicity of artemisinin. Our analysis demonstrated that cells lacking Pmr1p are less susceptible to growth inhibition from artemisinin and its derivatives. No association between sensitivity to artemisinin and altered trafficking of the drug efflux pump Pdr5p, calcium homeostasis, or protein glycosylation was found in pmr1∆ yeast. Basal ROS levels are elevated in pmr1∆ yeast and artemisinin exposure does not enhance ROS accumulation. This is in contrast to WT cells that exhibit a significant increase in ROS production following treatment with artemisinin. Yeast deleted for PMR1 are known to accumulate excess manganese ions that can function as ROS-scavenging molecules, but no correlation between manganese content and artemisinin resistance was observed. We propose that loss of function mutations in Pmr1p in yeast cells and PfATP6 in P. falciparum are protective against artemisinin toxicity due to reduced intracellular oxidative damage.

EGFR tyrosine kinase inhibition decreases cardiac remodeling and SERCA2a/NCX1 depletion in streptozotocin induced cardiomyopathy in C57/BL6 mice

AimEpidermal growth factor receptor (EGFR) related reactive oxygen species (ROS) generation results in myocardial damage. We aimed to investigate the role of gefitinib (EGFR-tyrosine kinase inhibitor) in diabetic cardiomyopathy (DbCM). MethodDbCM was induced by injecting streptozotocin (55 mg/kg for 5 consecutive days) to C57/BL6 mice intraperitoneally. Diabetic C57/BL6 mice (fasting blood glucose level ≥ 250 mg/dl) were allocated into four study groups and treated with two doses of gefitinib (30 mg/kg and 350 mg/kg per day) as well as ramipril (3 mg/kg/day) for four weeks. Key findingsWe observed a significant correlation between persistent hyperglycaemia with cardiac remodeling and alterations in myocardial architecture. Gefitinib significantly prevented lipid peroxidation (MDA), damage of antioxidant enzymes like superoxide dismutase (SOD), Catalase, glutathione (GSH) and thioredoxin reductase (TrxR). Gefitinib also prevented hypertrophy of myocardium evidenced by reduced heart weight to body weight ratio and TGF‑β related collagen deposition. Gefitinib maintained cardiac biomarkers like lactate dehydrogenase (LDH), Creatine Kinase‑MB, brain natriuretic peptide (BNP) and cardiac Troponin‑I (cTroponinI) indicating reduced myocardial damage. Decreased sarcoplasmic endoplasmic reticulum Ca2 + ATPase2a (SERCA2a) and sodium‑calcium exchanger-1 (NCX1) protein depletion after gefitinib administration indicated improved Ca2+ homeostasis during myocardial contractility. Histopathology and transmission electron microscopy clearly showed almost normal myofibrils and mitochondrial arrangements in gefitinib treated mice. SignificanceOur findings suggest that gefitinib protects myocardial damage in DbCM via balancing oxidant-antioxidant system, decreased collagen deposition as well as improved CKMB, BNP, cTroponinI and SERCA2a/NCX-1. Thereby, it indicated that gefitinib may be a potential therapeutic drug for treating DbCM.

Restoring the impaired cardiac calcium homeostasis and cardiac function in iron overload rats by the combined deferiprone and N-acetyl cysteine

Intracellular calcium [Ca2+]i dysregulation plays an important role in the pathophysiology of iron overload cardiomyopathy. Although either iron chelators or antioxidants provide cardioprotection, a comparison of the efficacy of deferoxamine (DFO), deferiprone (DFP), deferasirox (DFX), N-acetyl cysteine (NAC) or a combination of DFP plus NAC on cardiac [Ca2+]i homeostasis in chronic iron overload has never been investigated. Male Wistar rats were fed with either a normal diet or a high iron (HFe) diet for 4 months. At 2 months, HFe rats were divided into 6 groups and treated with either a vehicle, DFO (25 mg/kg/day), DFP (75 mg/kg/day), DFX (20 mg/kg/day), NAC (100 mg/kg/day), or combined DFP plus NAC. At 4 months, the number of cardiac T-type calcium channels was increased, whereas cardiac sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA) was decreased, leading to cardiac iron overload and impaired cardiac [Ca2+]i homeostasis. All pharmacological interventions restored SERCA levels. Although DFO, DFP, DFX or NAC alone shared similar efficacy in improving cardiac [Ca2+]i homeostasis, only DFP + NAC restored cardiac [Ca2+]i homeostasis, leading to restoring left ventricular function in the HFe-fed rats. Thus, the combined DFP + NAC was more effective than any monotherapy in restoring cardiac [Ca2+]i homeostasis, leading to restored myocardial contractility in iron-overloaded rats.

Localization and in-Vivo Characterization of Thapsia garganica CYP76AE2 Indicates a Role in Thapsigargin Biosynthesis.

The Mediterranean plant Thapsia garganica (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase calcium pump in mammals and is of industrial importance as the active moiety of the anticancer drug mipsagargin, currently in clinical trials. Knowledge of thapsigargin in planta storage and biosynthesis has been limited. Here, we present the putative second step in thapsigargin biosynthesis, by showing that the cytochrome P450 TgCYP76AE2, transiently expressed in Nicotiana benthamiana, converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts. This emphasizes the involvement of these cells in the biosynthesis of thapsigargin. This study paves the way for further studies of thapsigargin biosynthesis.

Eicosapentaenoic acid triggers Ca2+ release and Ca2+ influx in mouse cerebral cortex endothelial bEND.3 cells.

Eicosapentaenoic acid (EPA), an omega-3 fatty acid abundant in fish oil, protects endothelial cells (EC) from lipotoxicity and triggers EC NO release. The latter is related to an elevation of cytosolic Ca2+. Although EPA has been shown to cause human EC cytosolic Ca2+ elevation, the mechanism is unclear. Microfluorimetric imaging was used here to measure free cytosolic Ca2+ concentration. EPA was shown to cause intracellular Ca2+ release in mouse cerebral cortex endothelial bEND.3 cells; interestingly, the EPA-sensitive intracellular Ca2+ pool(s) appeared to encompass and was larger than the Ca2+ pool mobilized by sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition by cyclopiazonic acid. EPA also opened a Ca2+ influx pathway pharmacologically distinct from store-operated Ca2+ influx. Surprisingly, EPA-triggered Ca2+ influx was Ni2+-insensitive; and EPA did not trigger Mn2+ influx. Further, EPA-triggered Ca2+ influx did not involve Na+-Ca2+ exchangers. Thus, our results suggest EPA triggered unusual mechanisms of Ca2+ release and Ca2+ influx in EC.

Resveratrol protects against lipopolysaccharide-induced cardiac dysfunction by enhancing SERCA2a activity through promoting the phospholamban oligomerization.

The bacterial endotoxin lipopolysaccharide (LPS) is a main culprit responsible for cardiac dysfunction in sepsis. This study examined whether resveratrol could protect against LPS-induced cardiac dysfunction by improving the sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity. Echocardiographic parameters, cardiomyocyte contractile and Ca2+ transient properties, markers for cardiac inflammation, cell death, and oxidative stress, SERCA2a activity, and the ratios of phospholamban (PLB) monomer to oligomer were measured. Cardiac function was decreased >50% after LPS challenge (6 mg/kg for 6 h), which was improved by resveratrol. There was neither difference in plasma tumor necrosis factor-α and troponin I levels nor in infiltration of CD45+ cells in cardiac tissue between resveratrol-treated and untreated groups. In cardiomyocytes, LPS significantly decreased contractile amplitude, elongated relengthening time, diminished Ca2+ transient, reduced SERCA2a activity, and increased superoxide generation. These pathological alterations were attenuated by resveratrol treatment. Immunoblot analysis showed that LPS-treated mice had increased levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and the monomer form of PLB, along with decreases in the levels of SERCA2a, the oligomer form of PLB and nuclear factor erythroid 2-related factor (Nrf-2). Resveratrol treatment upregulated SERCA2a, the oligomer form of PLB, and Nrf-2 expression and function, and downregulated MDA, 4-HNE, and the monomer form of PLB. Our data suggest that the activity of SERCA2a in endotoxemia is inhibited, possibly due to increases in the monomer form of PLB. Resveratrol protects the heart from LPS-induced injuries at least in part through promoting the oligomerization of PLB that leads to enhanced SERCA2a activity.

Methylxanthine-evoked perturbation of spontaneous and evoked activities in isolated newborn rat hippocampal networks

Treatment of apnea of prematurity with methylxanthines like caffeine, aminophylline or theophylline can evoke hippocampal seizures. However, it is unknown at which interstitial brain concentrations methylxanthines promote such neonatal seizures or interfere with physiological ‘early network oscillations’ (ENOs) that are considered as pivotal for maturation of hippocampal neural networks. We studied theophylline and caffeine effects on ENOs in CA3 neurons (CA3-ENOs) and CA3 electrical stimulation-evoked monosynaptic CA1 field potentials (CA1-FPs) in sliced and intact hippocampi, respectively, from 8 to 10-days-old rats. Submillimolar doses of theophylline and caffeine, blocking adenosine receptors and phosphodiesterase-4 (PDE4), did not affect CA3-ENOs, ENO-associated cytosolic Ca2+ transients or CA1-FPs nor did they provoke seizure-like discharges. Low millimolar doses of theophylline (⩾1mM) or caffeine (⩾5mM), blocking GABAA and glycine receptors plus sarcoplasmic-endoplasmic reticulum Ca2+ ATPase (SERCA)-type Ca2+ ATPases, evoked seizure-like discharges with no indication of cytosolic Ca2+ dysregulation. Inhibiting PDE4 with rolipram or glycine receptors with strychnine had no effect on CA3-ENOs and did not occlude seizure-like events as tested with theophylline. GABAA receptor blockade induced seizure-like discharges and occluded theophylline-evoked seizure-like discharges in the slices, but not in the intact hippocampi. In summary, submillimolar methylxanthine concentrations do not acutely affect spontaneous CA3-ENOs or electrically evoked synaptic activities and low millimolar doses are needed to evoke seizure-like discharges in isolated developing hippocampal neural networks. We conclude that mechanisms of methylxanthine-related seizure-like discharges do not involve SERCA inhibition-related neuronal Ca2+ dysregulation, PDE4 blockade or adenosine and glycine receptor inhibition, whereas GABAA receptor blockade may contribute partially.

Abstract 13785: Left Ventricular Contractile Entropy in 99mTc-Sestamibi SPECT is a Novel Prognostic Predictor in Patients with Non-ischemic Dilated Cardiomyopathy

Background: It has been reported that mechanical dispersion of myocardial contraction is increased in failing myocardium. However little is known about the association between contractile entropy evaluated by myocardial scintigraphy and prognosis in patients with non-ischemic dilated cardiomyopathy (NIDCM). Purpose: We aimed to investigate the prognostic value of contractile entropy in patients with NIDCM. Methods: Forty-seven patients (38 male, 55.1 years) with NIDCM were performed gated 99mTc-sestamibi myocardial perfusion SPECT (GMPS) and endomyocardial biopsy. Entropy was automatically calculated as a result of contractile phase analysis for each myocardial sampling point from GMPS, and it reflects a dispersion of global mechanical contraction. All patients were allocated into two groups based on the median of entropy; HE-group: entropy≥0.61 and LE-group: entropy&lt;0.61. All patients were followed up at the mean of 2.8 years. Results: The mean QRS duration, left ventricular ejection fraction (LVEF) and plasma brain natriuretic peptide (BNP) levels were 114 msec, 35% and 225 pg/mL, respectively. Although there were no significant differences in QRS duration and plasma BNP levels between the two groups, LVEF was lower in the HE-group than in the LE-group (31.1% vs 39.8%, p=0.002). In Kaplan-Meier survival analysis, cardiac event rate was significantly higher in the HE-group (Figure). Cox proportional hazard analysis revealed that the HE-group was a significant determinant of cardiac events (Hazard Ratio: 7.66; 95%CI: 0.070-2.532; p=0.033). The mRNA expression level of sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA2a) in biopsy specimens was significantly lower in the LE-group (p=0.015). Conclusion: Contractile entropy, reflecting an impairment of global left ventricular contraction, might be useful to predict a poor prognosis in patients with NIDCM.

Left Ventricular Contractile Entropy in 99mTc-Sestamibi SPECT is a Novel Prognostic Predictor in Patients with Non-ischemic Dilated Cardiomyopathy

Background: It has been reported that mechanical dispersion of myocardial contraction is increased in failing myocardium. However little is known about the association between contractile entropy evaluated by myocardial scintigraphy and prognosis in patients with non-ischemic dilated cardiomyopathy (NIDCM). Purpose: We aimed to investigate the prognostic value of contractile entropy in patients with NIDCM. Methods: Forty-seven patients (38 male, 55.1 years) with NIDCM were performed gated 99mTc-sestamibi myocardial perfusion SPECT (GMPS) and endomyocardial biopsy. Entropy was automatically calculated as a result of contractile phase analysis for each myocardial sampling point from GMPS, and it reflects a dispersion of global mechanical contraction. All patients were allocated into two groups based on the median of entropy; HE-group: entropy≥0.61 and LE-group: entropy Results: The mean QRS duration, left ventricular ejection fraction (LVEF) and plasma brain natriuretic peptide (BNP) levels were 114 msec, 35% and 225 pg/mL, respectively. Although there were no significant differences in QRS duration and plasma BNP levels between the two groups, LVEF was lower in the HE-group than in the LE-group (31.1% vs 39.8%, p=0.002). In Kaplan-Meier survival analysis, cardiac event rate was significantly higher in the HE-group (Figure). Cox proportional hazard analysis revealed that the HE-group was a significant determinant of cardiac events (Hazard Ratio: 7.66; 95%CI: 0.070-2.532; p=0.033). The mRNA expression level of sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA2a) in biopsy specimens was significantly lower in the LE-group (p=0.015). Conclusion: Contractile entropy, reflecting an impairment of global left ventricular contraction, might be useful to predict a poor prognosis in patients with NIDCM.

The sarcoplasmic–endoplasmic reticulum Ca2+-ATPase (SERCA) is the likely molecular target for the acute toxicity of the brominated flame retardant hexabromocyclododecane (HBCD)

Hexabromocyclododecane (HBCD) is a widely utilised brominated flame retardant (BFR). It has been shown to bio-accumulate within organisms, including man, and possibly cause neurological disorders. The acute neurotoxicity of HBCD, and six other unrelated BFRs, were assessed in SH-SY5Y human neuroblastoma cells by 24h viability assays and HBCD proved to be the most lethal (LC50, 3μM). In addition, the effects of these BFRs were also assessed for their potency at inhibiting the sarcoplasmic–endoplasmic reticulum Ca2+ ATPase (SERCA) derived from the SH-SY5Y cells and again HBCD was the most potent (IC50, 2.7μM). The data for the other BFRs tested showed a direct correlation (coefficient 0.94) between the potencies of inducing cell death and inhibiting the Ca2+ ATPase, indicating that SERCA is likely to be the molecular target for acute toxicity. Mechanistic studies of HBCD on the Ca2+ ATPase suggest that it affects ATP binding, phosphorylation as well as the E2 to E1 transition step.

Insulin Secretion and Ca2+ Dynamics in β-Cells Are Regulated by PERK (EIF2AK3) in Concert with Calcineurin

Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (EIF2AK3) is essential for normal development and function of the insulin-secreting β-cell. Although genetic ablation of PERK in β-cells results in permanent neonatal diabetes in humans and mice, the underlying mechanisms remain unclear. Here, we used a newly developed and highly specific inhibitor of PERK to determine the immediate effects of acute ablation of PERK activity. We found that inhibition of PERK in human and rodent β-cells causes a rapid inhibition of secretagogue-stimulated subcellular Ca(2+) signaling and insulin secretion. These dysfunctions stem from alterations in store-operated Ca(2+) entry and sarcoplasmic endoplasmic reticulum Ca(2+)-ATPase activity. We also found that PERK regulates calcineurin, and pharmacological inhibition of calcineurin results in similar defects on stimulus-secretion coupling. Our findings suggest that interplay between calcineurin and PERK regulates β-cell Ca(2+) signaling and insulin secretion, and that loss of this interaction may have profound implications in insulin secretion defects associated with diabetes.

Effects of GLP‐1 receptor agonist on Ca2+ handling in coronary smooth muscle cells from metabolic syndrome Ossabaw swine with coronary artery disease

Chronic effects of glucagon‐like peptide (GLP‐1) receptor agonists were studied in Ossabaw swine with metabolic syndrome (MetS). MetS was induced by hypercaloric atherogenic diet for 6 months. Subcutaneous injections were given twice daily for 6 months after induction of MetS. Consistent with clinical effects, food consumption and weight gain were attenuated. MetS increases coronary artery disease (CAD) and coronary smooth muscle (CSM) Ca2+ dysfunction. CSM cells were isolated enzymatically from the coronary arteries and Ca2+ was digitally imaged with fluorescent fura‐2. In normal physiological salt solution we released sarcoplasmic reticulum (SR) stores with caffeine. Treated cells showed a larger Ca2+ transient, ΔF360/380=0.26±0.02, compared to placebo cells, ΔF360/380=0.16±0.01 (p&lt;0.001). In lean CSM, acute exposure to exenatide markedly increased activity of the sarcoplasmic‐endoplasmic reticulum Ca2+ ATPase (SERCA). This activity was confirmed by cyclopizaonic acid block. The increased SERCA activity may explain increased SR Ca2+ store in chronic MetS CSM. Acute exposure of placebo MetS CSM to exenatide has shown no effect on SERCA activity (n=43). Evidence for myocardial resistance to GLP‐1 in MetS is consistent with the lack of acute response. Our interpretation is that chronic exposure attenuates the decline in CSM Ca2+ handling associated with more severe CAD and dedifferentiation of CSM.(Support: NIH HL062552, TL1 TR000162, Amylin Pharmaceuticals Inc.)