In the present study, both Se-deficient chicken muscle models and selenoprotein W (SelW) gene knockdown myoblast and embryo models were used to study the mechanism. The results showed that Se deficiency-induced typical muscular injuries accompanied with Ca2+ leak and oxidative stress (P < 0.05) injured the ultrastructure of the sarcoplasmic reticulum (SR) and mitochondria; decreased the levels of the Ca2+ channels, SERCA, SLC8A, CACNA1S, ORAI1, STIM1, TRPC1, and TRPC3 (P < 0.05); and increased the levels of Ca2+ channel PMCA (P < 0.05). Similarly, SelW knockdown also induced Ca2+ leak from the SR and cytoplasm; increased mitochondrial Ca2+ levels and oxidative stress; injured SR and mitochondrial ultrastructure; decreased levels of SLC8A, CACNA1S, ORA1, TRPC1, and TRPC3; and caused abnormal activities of Ca2+ channels in response to inhibitors in myoblasts and chicken embryos. Thus, both Se deficiency and SelW knockdown induced Ca2+ leak, oxidative stress, and Ca2+ channel reduction. In addition, Ca2+ levels and the expression of the Ca2+ channels, RyR1, SERCA, CACNA1S, TRPC1, and TRPC3 were recovered to normal levels by N-acetyl-L-cysteine (NAC) treatment compared with SelW knockdown cells. Thus, with regard to the decreased Ca2+ channels, SelW knockdown closely correlated Se deficiency with Ca2+ leak in muscles. The redox regulation role of SelW is crucial in Se deficiency-induced Ca2+ leak in muscles.Selenium (Se) deficiency induces Ca Howe2+ plays important roles in the contraction, signal transduction, and enzyme active site in skeletal muscles [2+ is regulated by several types of Ca2+ channels, including intracellular Ca2+-release channel: ryanodine receptor channel  and Ca2+ pump channel (SERCA); extracellular Ca2+-entry channels: L-type voltage-dependent Ca2+ channel dihydropyridine receptors (DHPR or CACNA1S), transient receptor potential channels , Ca2+-release-activated Ca2+ current channels (CRAC); and extracellular Ca2+-entry balancing channels: Na+/Ca2+ exchanger (NCX), plasma membrane Ca2+-ATPases (PMCA) and others [2+ channels are closely related to the biological function of skeletal muscles and diseases of the muscles. Early observations demonstrated that the sarcoplasmic reticulum (SR) of Se deficiency muscle is defective in Ca2+ sequestration, resulting in extensive calcification of the muscle tissues [2+ disruption or Ca2+ channel inactivation in different types of cells was also correlated with disordered selenoproteins  [2+ signal disruption. However, the effect of Se deficiency on Ca2+ homeostasis has been less studied, and the mechanism of Ca2+ disruption in Se deficiency muscles still remains unclear. Although Se deficiency diseases models have been established in mice, pig, cow, lamb, and chicken [2+ leak and calcification, in Se deficiency diseases can be replicated using currently available practical diets has yet to be determined.Ca muscles . In skeld others . The actRyR1, RyR and Ca2+or SelM) –12, the RyR1, RyR and Ca2+ chicken , 14, whe2+ levels in the SR and calcification in muscles [2+ signals and Se deficiency-related muscle damage. However, to the extent of our knowledge, there is still no direct report on the role of SelW in Ca2+ regulation or the link between SelW and Ca2+ disorder in Se deficiency muscles. Thus, we conducted the present study to determine 1) whether and how Se deficiency influences Ca2+ signals in chicken muscles and 2) whether SelW plays a role in the regulation of Ca2+ homeostasis in vitro and/or in vivo. The present study provides insights into the effects of Se and SelW on Ca2+ signaling and the physiological role of SelW in muscular dystrophy.Selenoprotein W (SelW) is the first selenoprotein linked to Se deficiency-related muscular disorders . SelW is muscles . Therefo2+ leak and muscle calcification were always accompanied with muscle injuries in lamb [2+ levels in the muscles using ICP-MS technology . In addition, the distribution of Ca2+ in skeletal muscles by SRμ-XRF demonstrated lower Ca2+ levels in muscle sections  and increased Ca2+ levels in mitochondria . These data also support the hypothesis that SelW-related muscular disease is defective of Ca2+ levels in SR [In the present study, we silenced the expression of SelW by siRNA for 48 h and used different Caegatives , and them Figure  and SR  and alleviated the Ca2+ release from SR . These results demonstrated that NAC alleviated the effect of SelW deficiency (P < 0.05) in the cytoplasm and mitochondria. However, the Ca2+ signal in SR is still lower than the control (P < 0.05). Thus, SelW may partially influence the Ca2+ signal by regulating the oxidative stress.Following SelW deficiency, we treated cells with NAC, a global antioxidant. In our previous study, we showed that NAC treatment decreased ROS levels and apoptosis, following the SelW deficiency and the Heatments . Therefo+ Figure  and decr+ Figure  to normaR Figure  . Therefore, the SelW knockdown embryo models were established by Cas9 technology. Then, we screened the SelW knockdown models to check the levels of Ca2+ and other biomarkers. The results . The in vitro experiment further verified the role of SelW in the regulation of Ca2+ and that SelW deficiency was closely related to Ca2+ leak in muscles.To further identify the Cas Figure  demonstrs Figure  was valis Figure  showed t2+ channels: including RyR1, RyR3, SERCA1s, TRPC1, TRPC3, CACNA1S, CRAC , NCX (SLC8A1), and PMCA1  and the increase of PMCA (P < 0.05), but it did not influence RyR1 or RyR3 (P > 0.05). All extracellular Ca2+-entry channels were decreased, while intracellular Ca2+-release channels were not influenced. Lower levels of extracellular Ca2+-entry channels decrease the Ca2+ transporting activities, but higher PMCA, lower SERCA, and unchanged RyR1 and RyR3 keep Ca2+ leak from SR. Thus, taking into account the lower levels of total muscle Ca2+ levels, it is reasonable to hypothesize that Se deficiency induced Ca2+ leak in muscles by influencing Ca2+ channels.In the present study, we detected 10 Ca1 Figure . Se defiP < 0.05) but did not influence SLC8A1 or PMCA Ca2+ channels (P > 0.05). Thus, the effect of SelW deficiency on Ca2+ channels  was the same as Se deficiency in muscles.In chicken myoblasts, SelW deficiency Figure  decrease2+ channels, we also treated myoblasts with the specific Ca2+ channel inhibitors  and activator (caffeine for RyR1). After the cell fluorescence was equilibrated and stabilized, we treated the cells with different inhibitors and activators separately and observed the cellular response. The results  than control and were even reduced to a lower level than normal levels. Therefore, the sensitivity of SERCA to thapsigargin is increased in siRNA SelW. Ni treatment induced a higher and more rapid response in control cells with shorter duration than siRNA cells. Ni treatment maintained a higher Ca2+ level in siRNA cells. In siRNA cells, the NCX (SLC8A1) channels showed a sustained response to Ni. Cd treatment decreased the Ca2+ levels in siRNA cells, but did not influence Ca2+ levels in control cells. SelW deficiency increased the sensitivity of cells to Cd treatment. Thus, excepting the inhibitory effect of SelW deficiency on the expression of Ca2+ channels, SelW deficiency also influenced the activities of RyR1, SERCA, CACNA1S, and SLC8A1 in myoblasts.To identify the effect of SelW on Cas Figure  demonstr2+ channels were also influenced by SelW deficiency. NAC treatment increased RyR1, CACNA1S, TRPC1, and TRPC3 to a normal or higher level  and alleviated the effect of SelW deficiency on RyR3, SERCA, and STIM1 (P < 0.05). Therefore, the effect of SelW on Ca2+ channels was partially dependent on the redox-regulation role.In addition, after the treatment of NAC, the Cal Figure  , but other Ca2+ channels were not influenced (P > 0.05). It was observed that SelW influenced less Ca2+ channels than Se deficiency, but the effect of SelW knockdown on Ca2+ channels  was similar. In the in vivo experiment, SelW primarily decreased the levels of extracellular Ca2+-entry channels, similar to Se deficiency.Similar to the Se deficiency muscles, we also detected 10 Cas Figure  showed t2O2 and MDA levels. The results . However, in embryo muscle , not GPx and SOD (P > 0.05). The H2O2 and MDA levels were increased (P < 0.05). In our previous study, we also detected the ROS concentration, MDA levels, and antioxidative enzymes in myoblasts, which showed oxidative stress in SelW-silenced myoblasts [To examine the redox state in chicken and embryo, we detected the activities of antioxidative enzymes GPx, SOD, and CAT and measured Hs Figure  showed te Figure , SelW knyoblasts , 17. The2+ leak and oxidative stress [In the present study, we detected the ultrastructure of chicken muscles, myoblast, and embryo muscles. The results Figure  demonstre stress . Myoblas2+ and mitochondrial Ca2+. In the present study, we measured ΔΨm using two different technologies. Both results , while the NAC treatment alleviated the ΔΨm (P < 0.05). Similar to previous studies, which demonstrated that excessive Ca2+ accumulation in mitochondria induced mitochondrial dysfunction and injury, ΔΨm was also influenced by the treatment of NAC [Mitochondrial membrane potential (ΔΨm) is always influenced by substantial amounts of cytosolic Cas Figure  showed tt of NAC , 19. The2+ leak and calcification in skeletal muscles was reported in Se deficiency-related muscular dystrophy [2+ disorder in Se deficiency muscle injuries. However, fewer reports demonstrated the effect of Se deficiency on Ca2+ signal in animals, and the mechanism by which pathological changes occur. In chicken, we successfully replicated Se deficiency-related muscle disease and observed the typical pathological and molecular changes, such as oxidative stress, apoptosis, disordered selenoproteins [2+ leak was reported only in lamb [2+ disruption was involved in the muscle disease induced by Se deficiency. To the extent of our knowledge, this is the first report to show the effect of Se deficiency on Ca2+ signal in chicken muscles.Caystrophy , which iproteins , SR, and in lamb . The pre2+ signal in muscles is tightly regulated by several types of Ca2+ channels and other molecules [2+ channels will influence the Ca2+ signal, and induce Ca2+ channelopathies [2+ leak or disruption with TRPCs and NCX [2+ signal in chicken muscles is available. In the present study, we selected 10 Ca2+ channels and hoped to screen out one or some Ca2+ channels related to Se deficiency muscle injuries. The results showed that Se deficiency decreased the expression of SLC8A, CACNA1S, ORAI1, STIM1, SERCA, TRPC1, and TRPC3; increased the expression of PMCA; and did not influence RyR1 and RyR3 levels. The reduced extracellular Ca2+-entry channels and SERCA may well explain the Ca2+ leak in muscles; however, Ca2+ leak may also influence the levels of some Ca2+ channels [2+ channels and Ca2+ leak in muscles is a complex. Excepting the close relationship of TRPCs, STIM1, and SLC8A channels with Ca2+ leak, the most important Ca2+ channels involved in the regulation of SR Ca2+ levels are RyRs and SERCA [2+ channels to be related to Se treatment [2+ channels TRPC1, TRPC3, STIM1, SLC8A, SERCA, PMCA1, and CACNA1S are involved in Se deficiency-induced Ca2+ leak. Compared with previous studies, we reported two new Ca2+ channels, PMCA1 and CACNA1S that were related to Se deficiency and Ca2+ leak but found that RyRs were not involved in the Se deficiency-induced Ca2+ leak.Caolecules . The disopathies , 21–25.  and NCX ; SERCA,  and NCX , 28; STI and NCX ; and PMC and NCX . Howeverchannels , 31. Thereatment , 33 and reatment , 35. The2+ disruption is the selenoproteins. Selenoproteins, such as SelN, SelM, SelT, SelK, SelW, and Txnrd, have either direct or indirect relation with Ca2+ signal and/or Ca2+ channels regulation [2+ regulation. Among these, SelW has a close relationship with the biological function and injuries of muscles [2+ levels in cytoplasm and SR, but accumulation in mitochondria. Similar to the myoblast, SelW knockdown in embryo muscles also induced Ca2+ leak. In addition, SelW deficiency also induced typical SR and mitochondrial injuries, which were closely related to Ca2+ leak, oxidative stress, and disordered Ca2+ channels [in vivo and in vitro experiments indicated that Ca2+ leak and the related muscles injuries occur subsequent to SelW deficiency, which supported the idea that SelW deficiency is actively involved in the mechanism of Se deficiency-induced Ca2+ leak in muscles. However, by detecting the response of Ca2+ channels to SelW deficiency, we found that SelW deficiency also influenced the Ca2+ channels. In myoblasts, SelW deficiency reduced RyR1, RyR3, SERCA, CACNA1S, TRPC1, TRPC3, STIM1 and ORAI1 Ca2+ channels. In embryos, SelW deficiency reduced SLC8A, CACNA1S, ORA1, TRPC1, and TRPC3 Ca2+ channels. The effect of SelW deficiency on CACNA1S, ORA1, TRPC1, and TRPC3 was similar to the treatment of Se deficiency. Therefore, SelW may play roles in the Se deficiency-induced Ca2+ leak by regulating the expressions of some typical Ca2+ channels.Another possible link between Se deficiency and Cagulation –12, 32. gulation , only Tx muscles , 36, 37.channels , 38. Bot2+ signal and the Ca2+ channels remains an unsolved question. In the present study, we treated myoblasts with different Ca2+ channel inhibitors and an activator to check the response of cells. The effect of ryanodine, Ni, Cd, and thapsigargin on cells was different. SelW deficiency reduced the response of RyR1 to ryanodine, increased the sensitivity of SERCA to thapsigargin, and CACNA1S to Cd, or sustained and delayed the effect of Ni on NCX. However, caffeine does not induce any change in response in the control or siRNA cells. Therefore, the expression levels were influenced by SelW, as were the activities of some Ca2+ channels. As SelW is the typical antioxidative selenoprotein in myoblast, the redox regulation role may link SelW closely with these Ca2+ channels. In addition, oxidative stress was detected in Se deficiency chicken muscles and SelW knockdown embryo muscles and myoblasts [2+ channels were regulated by SelW in a redox-dependent manner. As in the Ca2+ channels, the Ca2+ levels in cytoplasm and mitochondria and the ROS levels [2+ leak was alleviated by NAC treatment. In addition, the SR and mitochondria injuries induced by SelW deficiency were also alleviated by NAC treatment. Therefore, the regulation of Ca2+ by SelW was partially dependent on the redox regulation function of SelW. This agrees with previous studies showing that redox activities of some Ca2+ channels, such as SERCA, RyR1, and TRPC1 [2+ leak.How Se and SelW deficiency influenced the Cayoblasts , 17. Thend TRPC1 , 35, cound TRPC1 , 39. The2+ leak functions with its redox regulation. One possible explanation in this context is that the SelW deficiency may decrease the antioxidative ability of myoblast [2+ channels such as SERCA and TRPCs, and leads to Ca2+ leak. In contrast, Ca2+ leak induced by SelW deficiency increased the accumulation of Ca2+ in mitochondria, and then this increased mitochondrial Ca2+ led to the production of reactive oxygen species [2+ leak and oxidative stress.An important unanswered question that emerges from our experimentation is how SelW related Camyoblast  and musc species , 34 and 2+ leak can be successfully replicated by solo feeding broiler chicken with a Se-deficient diet. In this process, Se deficiency primarily decreased the extracellular Ca2+-entry channels and SERCA, and influenced the Ca2+ signals. In addition, SelW deficiency induced the Ca2+ leak in both in vitro myoblast and in vivo embryo muscles, and influenced the Ca2+ channels in embryo muscles, which indicates that SelW is involved in the process of Ca2+ leak induced by Se deficiency. In the present study, we screened Ca2+ channels: TRPC1, TRPC3, STIM1, ORA1, SLC8A, SERCA, PMCA1, and CACNA1S in chicken muscles and SLC8A, CACNA1S, ORA1, TRPC1, and TRPC3 in myoblasts and embryos that were involved in the Ca2+ leak. By treating the myoblasts with different Ca2+ channel inhibitors and NAC, we also found that redox-regulation role of SERCA, CACNA1S, TRPC1, and TRPC3 by SelW was closely related to Ca2+ leak. We concluded that SelW plays an important role in the Se deficiency-induced muscles Ca2+ leak.In summary, the typical pathological changes of skeletal muscles CaAll procedures used in this study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University . A total4 cells/cm2 and were allowed to proliferate for 24 h in 5% CO2 at 37°C. Later, the differentiation of myoblasts was induced by replacing the proliferation medium with differentiation medium.Primary cultures of chicken embryo-driven myoblasts were prepared as described . BrieflyAfter the chicken myoblasts were plated in six-well plates at 70-80% confluence, the cells were transfected with 3 μL of 20 μM siRNAs and 3 μL of Lipofectamine RNAiMAX Reagent (Invitrogen) in 2 mL of Opti-MEM. After transfection for approximately 48 h, the cells were harvested for analysis. In the N-acetyl-L-cysteine (NAC) group, the cells were co-incubated with 2.5 mM NAC for 6 h after transfection and then harvested for analysis.CRISPR/Cas9 plasmid for SelW was constructed by Inovogen Tech. Co., and the gRNA sequences for SelW are shown in Table Total RNA was isolated  from musPrimer Premier Software  was used to design specific primers based on known chicken sequences Table .Protein extracts from the muscles, myoblasts, and embryos were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. Separated proteins were then transferred to nitrocellulose membranes using a tank transfer for 2 h at 200 mA in Tris-glycine buffer containing 20% methanol. The membranes were blocked with 5% skim milk for 2 h, and then incubated over night with diluted primary antibodies against SERCA  and SelW (made by our lab) followed by a horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit IgG . To verify equivalent loading of samples, the membrane was incubated with monoclonal β-actin antibody , followed by an HRP-conjugated goat anti-mouse IgG (1:3000). The signal was detected using an enhanced chemiluminescence system .Histological analysis was performed according to our previous study . After n2+ in the chicken muscles, inductively coupled plasma mass spectrometry ICP-MS  was used. The instrumental parameters of the equipment used are summarized in Table To determine the levels of Ca2+ concentrations were determined in by an acid digest of samples according to the method of Uluozlu et al. [3 (65%) and 2 mL H2O2 (30%) in a microwave digestion system and diluted to 10 mL with deionized water. A blank digest was carried out in the same way. All sample solutions were clear. Digestion conditions for the microwave system were applied as 1800 W for 3 min at 100°C, 1800 W for 10 min at 150°C, and 1800 W for 45 min at 180°C. All digested samples were filled with ultra-pure water to the final volume before analysis by ICP-MS.The Cau et al. . One gra2+ localization in muscle tissues were surveyed with synchrotron radiation micro X-ray fluorescence (SRμ-XRF). Muscle tissue specimens were rapidly fixed in 10% neutral-buffered formalin solution for at least 24 h. Fixed specimens were dehydrated through a graded series of ethanol, cleared in xylene, embedded in paraffin, and then cut into 5 mm-thick sections. The slices were fixed onto a 1 mm-thick glass slide, and then analyzed by SRμ-XRF according to the method of Li [Caod of Li .2+ fluorescence indicators: 5 μM Fluo3 acetoxymethylester (AM)  for the cytoplasm, 10 μM Rhod2 (Invitrogen) for the mitochondria [2, 0.5 MgCl2, 10 Hepes, and 5.6 glucose, pH = 7.4) for 10 min until the cell fluorescence equilibrated. Images were acquired using excitation wavelengths of 488 nm for Fluo3, Fluo5N, and Mito-Tracker Green and 563-587 nm for Rhod2 and ER-Tracker Red. The signals were collected at 505-530 nm for Fluo3, Fluo5N and Mito Tracker Green and at 590 nm for Rhod2 and ER-Tracker Red. After the cell fluorescence was equilibrated and stabilized, Ni, ryanodine, Cd, caffeine, and thapsigargin were added to the cells, and the fluorescence was collected and imaged with a confocal laser scanning microscope, (FluoviewTM FV 1000) using a 40× oil lens, and analyzed using the Olympus Fluoview Ver. 2.0a Viewer software. The fluorescence intensity levels are presented relative to baseline and shown as F/F0, where F0 is the initial fluorescence as described previously [The cells were cultured in 35 mm glass-bottomed dishes and loaded for 30-60 min (37°C) with three different Cachondria , and 5 μchondria –50. Rhodchondria . To remochondria . To estichondria . In addichondria . Cells wg to collect supernatants. Ca2+ levels were determined using the assay kits (Nanjing Jiancheng Bioengineering Institute). Protein levels were determined using the protein assay kit (Nanjing Jiancheng Bioengineering Institute). The biochemical assays were performed according to the manufacturer's instructions with a UV-visible spectrophotometer .Embryo muscle samples were homogenized on ice in physiological saline and centrifuged at 700 × g to collect supernatants for the biochemical assays. GPx, SOD, and CAT activities; MDA concentration; H2O2 levels; and protein concentrations were determined according to the kit's instructions  and were recorded with Multimode Plate Readers .Chicken and embryo muscle samples were homogenized on ice in physiological saline and centrifuged at 700× The mitochondrial membrane potential was monitored using 5, 5′, 6, 6′- tetra-chloro-1, 1′, 3, 3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), a lipophilic cationic fluorescence dye  , 55. Celvia microscopy. The microphotographs were taken with a transmission electron microscope (TEM).The technique adopted to observe ultrastructural changes was similar to that of our previous study : the colt-test or one-way ANOVA. The data are expressed as the means ± standard deviation. Differences were considered to be significant at P < 0.05.The data analysis was performed using SPSS statistical software for Windows . Differences between different groups were assessed using Student's


Endothelial cells cultured under physiologic O2 conditions (5% O2) exhibit a reduced cytosolic Ca2+ response to stimulation. The mechanism for reduced plateau [Ca2+]i upon stimulation was due to increased sarco/endoplasmic reticulum Ca2+ ATPase (SERCA)-mediated reuptake rather than changes in Ca2+ influx capacity. Agonist-stimulated phosphorylation of the SERCA regulatory protein phospholamban was increased in cells cultured under 5% O2. Elevation of cytosolic and mitochondrial [Ca2+] and cell death after prolonged ionomycin treatment, as a model of Ca2+ overload, were lower when cells were cultured long-term under 5% compared with 18% O2. This protection was abolished by cotreatment with the SERCA inhibitor cyclopiazonic acid. Taken together, these results demonstrate that culturing cells under hyperoxic conditions reduces their ability to efficiently regulate [Ca2+]i, resulting in greater sensitivity to cytotoxic stimuli.—Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. Reduced SERCA activity underlies dysregulation of Ca2+ homeostasis under atmospheric O2 levels.Unregulated increases in cellular Ca In the n HUVECs  and chonn HUVECs , whereasn HUVECs . Reactivvein ECs  and C2C1vein ECs  under hytectable . In this2 levels was achieved by transferring cells from a standard CO2 incubator (∼18% O2) to an atmosphere-regulated workstation set to 5% O2 and 5% CO2 . Cells were adapted to 5% O2 from passage 2 for a minimum of 5 d, and subculture was performed at 5% O2 with all media and buffers pre-equilibrated to this O2 level.Umbilical cords were obtained from St. Thomas’ Hospital, and venous ECs were isolated using a collagenase solution. HUVECs were cultured in M199 + 20% fetal calf serum and used at passage 3. Culture under physiologic (5%) O2+ was performed as described previously -2,5-diphenyltetrazolium bromide (MTT) assay or annexin V fluorescence. Briefly, cells were incubated with 0.5 mg/ml MTT in M199 for 4 h after treatment. The resulting formazan crystals were dissolved in DMSO, and absorption was measured at 562 nm using a plate reader . Results are expressed relative to vehicle  treatment. To assess annexin V staining, HUVECs were stained using a CFM640-conjugated annexin V label  with Hoechst 33342 (Sigma-Aldrich) used to counterstain nuclei. Images were obtained using an Orca-03G camera  and quantified using ImageJ software .2+]i are representative of results obtained from at least three different donors. In the figures, data in bar graphs report the means ± sem responses from at least three different donors, where the mean from three to four replicate wells represents a single value for each donor (the quoted n is the number of donors). Significance was assessed using a paired Student’s t test or ANOVA where appropriate. A value of P < 0.05 was considered significant.The raw traces of [Ca2 significantly lowers plateau [Ca2+]i in human ECs  stimulated with histamine, ATP, or ionomycin  was assessed using the Mn2+ quench assay, also showing no alteration in Ca2+ influx capacity under 5% O2. Similar findings were observed in HUVECs challenged with ATP .We have previously shown that culture at 5% Oonomycin . Represeadd-back B, C was 2+ influx appeared unaltered in HUVECs cultured at 5% O2, the lower plateau [Ca2+]i most likely results from enhanced Ca2+ efflux from the cytosol. Four Ca2+ efflux pathways exist in ECs: plasma membrane ATPase (PMCA), SERCA, NCX, and mitochondrial Ca2+ uniporter (MCU). To investigate the relative contribution of SERCA and MCU, the pharmacological agents cyclopiazonic acid (CPA) and the protonophore carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) were used. Treatment with 10 µM CPA   and histamine (10 µM) to empty internal Ca2+ stores. With SERCA inhibited, the subsequent decline in [Ca2+]i must represent extrusion across the plasma membrane via either PMCA or NCX. Because the latter is not thought to contribute to Ca2+ homeostasis in the absence of altered Na+ regulation   and after cotreatment with ionomycin and CPA . Cotreatment with ionomycin and CPA significantly increased [Ca2+]i under both O2 levels and to a similar level (ii), and subsequent addition of FCCP had no further effect, probably reflecting either the limits of detection for Fura-2 or a complete breakdown in Ca2+ homeostasis. A correlation was observed between the plateau [Ca2+]i after stimulation with ionomycin and the amount of FCCP-releasable Ca2+  B, and feµM, 1 h) C. To cor and CPA D in HUVEa2+ load , was sig load  robustly alters cellular phenotype, which may account for artifactual findings. For example, many primary human cell types show pronounced senescence or apoptosis during routine cell culture, which can be attributed to hyperoxia and the overproduction of reactive oxygen species associated with elevated O2 levels , suggesting the mitochondria may play a subtle role in ER Ca2+ homeostasis under nonphysiologic conditions. Enhanced ER capacity may buffer elevations in mitochondrial Ca2+, although increased Ca2+ storage in the ER has also been shown to reduce cell viability under pathologic conditions (The fact that histamine-stimulated peak i. This likely reflects the fact that most of these processes are dependent on Ca2+ influx and not on net [Ca2+] (2 (2+]i. Rather, Ca2+ clearance efficiency can become an important factor in maintaining cellular function during episodes of prolonged [Ca2+]i elevation (2+ can ultimately determine cell survival. We have demonstrated that increased SERCA activity can protect HUVECs against ionomycin-induced Ca2+ overload at 5% O2 (2+ ER homeostasis (2+ extrusion mechanisms (2+ reuptake may protect cells against acute Ca2+ overload. Prolonged culture under hyperoxic conditions detrimentally affects Ca2+ homeostasis, ultimately sensitizing cells toward damaging stimuli. We therefore propose that culture under physiologic O2 levels may provide a more suitable in vitro model to investigate treatments aimed at alleviating dysregulation of Ca2+ handling.As we recently demonstrated , key Ca2t [Ca2+] . MoreoveCa2+] (2  may complevation . Under tat 5% O2 . Increaseostasis  or Ca2+ chanisms . Our reshttp://www.fasebj.org to obtain this information.This article includes supplemental data. Please visit Click here for additional data file.


Background: Prolongation of the QT interval of the electrocardiogram (ECG), underlain by prolongation of the action potential duration (APD) at the cellular level, is linked to increased vulnerability to cardiac arrhythmia. Pharmacological management of arrhythmia associated with QT prolongation is typically achieved through attempting to restore APD to control ranges, reversing the enhanced vulnerability to Ca2+-dependent afterdepolarisations (arrhythmia triggers) and increased transmural dispersion of repolarisation (arrhythmia substrate) associated with APD prolongation. However, such pharmacological modulation has been demonstrated to have limited effectiveness. Understanding the integrative functional impact of pharmacological modulation requires simultaneous investigation of both the trigger and substrate.Methods: We implemented a multi-scale (cell and tissue) in silico approach using a model of the human ventricular action potential, integrated with a model of stochastic 3D spatiotemporal Ca2+ dynamics, and parameter modification to mimic prolonged QT conditions. We used these models to examine the efficacy of the hERG activator MC-II-157c in restoring APD to control ranges, examined its effects on arrhythmia triggers and substrates, and the interaction of these arrhythmia triggers and substrates.Results: QT prolongation conditions promoted the development of spontaneous release events underlying afterdepolarisations during rapid pacing. MC-II-157c applied to prolonged QT conditions shortened the APD, inhibited the development of afterdepolarisations and reduced the probability of afterdepolarisations manifesting as triggered activity in single cells. In tissue, QT prolongation resulted in an increased transmural dispersion of repolarisation, which manifested as an increased vulnerable window for uni-directional conduction block. In some cases, MC-II-157c further increased the vulnerable window through its effects on INa. The combination of stochastic release event modulation and transmural dispersion of repolarisation modulation by MC-II-157c resulted in an integrative behavior wherein the arrhythmia trigger is reduced but the arrhythmia substrate is increased, leading to variable and non-linear overall vulnerability to arrhythmia.Conclusion: The relative balance of reduced trigger and increased substrate underlies a multi-dimensional role of MC-II-157c in modulation of cardiac arrhythmia vulnerability associated with prolonged QT interval. Conditions in which the QT interval of the electrocardiogram (ECG) is prolonged, such as heart failure Hart,  and inhe2+ handling , causing spontaneous SR Ca2+ release events and delayed-afterdepolarisations through activation of the forward-mode Na+-Ca2+ exchange current (INaCa), which in turn can result in triggered activity if the delayed afterdepolarisation is of sufficient magnitude  arise because APD prolongation is rarely homogenous in and between the different regions of the ventricles  , thus reducing the APD  IKr   triggers and substrates interact to induce arrhythmias .The potentially pro-arrhythmic effects of hERG activators, such as NS1643 has prompted the search for novel hERG activators that do not display these effects. One recently-identified compound is MC-II-157c, an NS1643 analog. MC-II-157c activates Computational models provide powerful research tools to understand the intricacies of arrhythmia trigger and substrate interaction, as they allow us to modify parameters under precisely controlled conditions and quantify the resultant tissue-level arrhythmic behavior, and can predict how these arrhythmias will manifest in a clinical setting . We therefore used a computational approach to quantify the interaction of pharmacologically-modified arrhythmia trigger and substrate, using the novel hERG activator MC-II-157c as an example.2+ loading and subsequent spontaneous SR Ca2+ release events; (iii) the probability of spontaneous SR Ca2+ release manifesting as triggered action potentials in single cell and ectopic activity in tissue; and (iv) the vulnerability to the initiation of re-entrant like conduction patterns.We wanted to quantify the modified triggers and substrate that result from MC-II-157c ion channel actions. To this end, we use detailed single cell and tissue level models to study the effect of QT prolongation and its modulation by MC-II-157c on: (i) APD heterogeneity in isolated cells; (ii) SR Cain silico approach to study the interactions between arrhythmia trigger and substrate in conditions associated with prolonged QT intervals, and their modulation by the hERG activator MC-II-157c.We implemented a multi-scale 2+ release events, a spatial model of intracellular Ca2+ handling which explicitly accounts for stochastic state transitions and spatial coupling is required. We therefore implemented an efficient, idealized reduction of a previously developed and validated general model of spatio-temporal Ca2+ handling with realistic structure , subspace ([Ca2+]SS) and bulk-cytosolic space ([Ca2+]i), and the network and junctional SR . The bulk cytosol, subspace and network SR are diffusively coupled to neighboring CRUs; the dyadic cleft space and junctional SR are not spatially coupled to neighbors. The fundamental model equations describing this system are:In order to simulate triggered and ectopic activity underlain by spontaneous Cawhere transfer between compartments is given by:vx is the volume of compartment x, βx is an instantaneous buffering term, and φx is the reaction term. Stochastic dynamics are modeled for the RyRs and LTCCs, which are part of the dyadic cleft space reaction term. All parameters and reaction terms are given in the where INa, IKr, IKs, IK1, Ito, INaK) were included, without the further details of phosphorylation included in the original study; Ca2+ currents  are described by the Ca2+ handling model. These simplifications were implemented to improve computational efficiency and for integration with the general stochastic Ca2+ handling model described above. The model provides specific formulations to describe the heterogeneity in ionic currents of the transmural cell types  found in the left ventricular free wall of the human heart  human ventricular cell model . Default model action potentials and cytosolic Ca2+ transients, for the three transmural cell types of the simplified ORd model with stochastic Ca2+ handling, are shown in Figure 2+ transient properties similar to the original cell model and within the range of experimental data presented in the original study . The model is therefore considered suitable for the mechanistic study undertaken.This integrated stochastic framework captures spontaneous Ca+ current (IKs) maximal conductance by 50%, similar to LQTS1, which we term “prolonged QT variant a” (PQTa) in the remainder of the manuscript; (ii) downregulation of IKr maximal conductance by 50% (PQTb), similar to LQTS2; and (iii) upregulation of ICa,L maximal conductance by 50% (PQTc), similar to LQT8  in one of three ways: (i) downregulation of the slow delayed rectifier KIKr were modeled by modifying the IKr formulation according to experimental data . Effects of MC-II-157c on blocking INa  of 40 ms  for use in tissue-level simulations, to capture spontaneous Ca2+ release events at significantly reduced computational cost and with complete controllability. The non-spatial model consists of a single CRU  with additional analytical functions which describe the RyR waveform associated with whole-cell spontaneous release events, derived from analysis of the fully detailed spatial cell model, similar to the approach used in Campos et al. :where initiation time and the duration. The stochastic nature of spontaneous Ca2+ release is captured by randomly selecting these parameters from functions describing their physiological distributions. The distributions describing initiation time of whole-cell spontaneous release events in the spatial cell model are typically skewed  and the ti at the cumulative probability of 0.25, ti_sep, completely control the resulting distribution. The duration, D, of the RyR waveform can be determined from distributions in an analogous manner:where the gradient parameters of the two sigmoidal functions . We fix ti_sep in simulations such that only the width of the initiation time distribution  and the median of the duration distribution are varied: ti distribution widths of 350, 550, and 1,000 ms . Parallelisation was implemented with OpenMP. Cell APD was measured from the time the membrane potential crossed −80 mV during the upstroke of the action potential, to when the membrane potential crossed back over −80 mV during the repolarisation phase.Models were coded in C/C++ and run on a Linux desktop machine, or using the University of Leeds ARC2 High Performance Computing facilities. Equations for isolated cell models were solved using a forward Euler method with a time step of ΔIKr) has the largest effect on prolonging APD , due to the larger IKr conductance in human cells relative to IKs conductance, and its primary role in AP repolarisation. All three PQT conditions increase the transmural difference in APD (the difference between the longest and shortest APDs in the three cell types), from 88 ms in WT to 97, 279 and 122 ms with PQTa, PQTb, and PQTc, respectively.The single cell model was paced at a cycle length of 1,000 ms for 100 beats under control (WT), PQT and PQT + MC-II-157c conditions to evaluate the efficacy of MC-II-157c on reversing PQT induced APD prolongation Figure . Figure IKs) due to the minimal effect this condition has on initially prolonging APD, but the largest decrease was seen with PQTb in midmyocardial cells, where APD was reduced from 649 to 540 ms, a reduction of 17% . QT-prolongation was observed for all three remodeling types Figure , with PQIKr, is illustrated in Figure The mechanism by which MC-II-157c shortens APD, despire reducing the maximal conductance of 2+ release events . The maximal flux rate of intracellular uptake was increased, simulating the effect of sympathetic stimulation, in order to promote SR loading. An increase by a factor of two was chosen as this loaded the SR-Ca2+ in WT and the PQTc condition in isolated epicardial cells illustrates the effect on SR-Ca2+ loading under rapid pacing and highlights that APD prolongation associated with PQT can promote loading  does not promote SR loading compared to WT (peak SR Ca2+ load of 1.07 mM in WT and 1.06 mM in PQTa), but the PQTb (downregulation of IKr) and PQTc  conditions do , with PQTc having the largest effect due to the increase in the transmembrane Ca2+ current in this condition. MC-II-157c reverses SR Ca2+ loading in PQTb, reducing peak SR Ca2+ load at periodic steady-state to 1.05 mM, which is below WT levels. However, the drug has only a very small effect in PQTc (down to 1.108 mM) due to the minimal effect MC-II-157c has on APD in this condition, and the subsequently small change in the time course of ICa,L.The time series of SR-Cag Figure . The pea2+ can manifest as significant differences in the vulnerability to spontaneous Ca2+ release due to the non-linear threshold dependence on SR Ca2+ concentration . Example distributions are shown in Figure 2+ as a result of MC-II-157c significantly reduces the probability of spontaneous release  as well as widening the distribution . Due to the choice of loading parameters giving the WT close to threshold, no spontaneous release occurs for either PQTa or PQTb under the application of MC-II-157c as in these conditions threshold SR Ca2+ is not reached.These relatively small changes in SR-Ca Figures . The proN = 1,000 per condition) for four different RyR waveform duration distributions .The effect of MC-II-157c on the probability of DADs manifesting as full triggered action potentials was investigated using the simplified, non-spatial model such that spontaneous release waveforms could be directly controlled. The initiation time was set to 1,000 ms and behavior of the cell models for WT, PQT and PQT + MC-II-157c was compared over multiple simulations . In all cases, the IKr modification reduces the probability of triggered activity , but the role of INa is less clear; blocking INa is important, but the degree of INa block has different effects depending on the condition. The mechanism of the drug's action is shown in Figure IKr acts during the DAD to keep the cell's membrane potential below the threshold for triggered activity; reduced INa also pays a role  in the drug's mechanism of action as it reduces excitability of the cell, again reducing the ease with which the cell's membrane potential can reach threshold.Examples of 100 simulations for WT and PQTb, with and without MC-II-157c, are shown in Figure ti and duration median distributions; Epicardial cell model) wherein the tissue model was paced to steady state and then left quiescent for two simulation seconds within which time the spontaneous release occurs.The vulnerability to the development of ectopic activity  was assessed in a 2D homogeneous sheet to provide a medium for the synchronization of independent stochastic release events. Ten simulations were performed for each condition (WT and PQT variants ± MC-II-157c for the combinations of 2+ transients), and conversely inhibited by wide distributions of initiation time  and long RyR waveforms . For example, no ectopic activity was observed for any condition with ti distribution widths of 1,000 ms or duration medians of 300 ms or longer, whereas a ti distribution width of 350 ms combined with median durations of 150 or 200 ms resulted in ectopic activity occurring in 100% of simulations . Similarly in-line with single-cell results, MC-II-157c can inhibit ectopic activity , as well as with only the MC-II-157c IKr modification. These VWs are quantified by length (over which they occur in the 1D strand) and area . In both WT and PQTa conditions, the drug increases both the length and the area of the VW . Again, the influence of INa is different depending on the condition: including the effects of INa  increases the length of tissue over which the VW occurs in all conditions , but reduces the overall area of the VW .Examples of propagation applied during the repolarisation phase in 1D tissue simulations are shown in Figure INa) and repolarising (IKr) membrane ionic currents targeted by the drug affects arrhythmia triggers and substrates, our findings also provide general insight into the role of ion-currents in controlling triggers and substrate at multiple scales which, along with insight from other in silico studies [see  Although MC-II-157c acts on membrane ionic currents carrying K+ and Na+, the drug has indirect effects on intracellular Ca2+ handling, particularly SR Ca2+ loading and related spontaneous SR Ca2+ release events and subsequent DADs, through modulation of the AP; (iii) Increased IKr (through its repolarising effects) and reduced INa (by decreasing cell excitability) act to reduce the probability of a DAD reaching threshold and developing into triggered activity at both cellular and tissue scales; and (iv) Despite MC-II-157c reducing triggered activity at the cell level, the drug can increase both the spatial region of tissue over which a VW for unidirectional conduction block occurs, as well as the temporal width of the VW at all points along the tissue, and in doing so increases the total spatiotemporal size of the VW. These results highlight the complex considerations which underlie overall vulnerability to arrhythmia at multiple scales.Our key findings are that: (i) Despite the hERG activator MC-II-157c reducing the maximal conductance of 2+ loading, reduces the occurrences of spontaneous SR Ca2+ release events, reduces DAD occurrences, and reduces the probability of a DAD developing into a triggered action potential. At the tissue level, the combination of these factors leads to a significantly reduced vulnerability to the development of ectopic beats; MC-II-157c reduces the probability of an ectopic beat development at given spontaneous release function distributions  as well as inhibiting SR Ca2+ loading and thus reducing synchronization . In relation to the development of Ca2+ induced triggers at single cell and tissue levels, therefore, our simulation results suggest that the drug has efficacy as an anti-arrhythmic.At the isolated cell level, MC-II-157c reduces SR Caand the substrates that underlie arrhythmia initiation: there is a delicate balance between increased/decreased trigger and increased/decreased substrate  that determines whether any given trigger stimulus  will result in unidirectional propagation and, potentially, initiation of a re-entrant arrhythmia. This is one potential reason that single cell studies showing efficacious effects of putative anti-arrhythmic drugs may not translate to the clinic.However, even though arrhythmic triggered activity is reduced with the drug, the spatiotemporal area of the VW increased, and so the anti-arrhythmic effects of the reduced probability of an arrhythmia trigger occurring is opposed by the pro-arrhythmic effects of an increased arrhythmia substrate. This highlights the importance of examining the effects of pharmacological compounds on both the triggers IKr and INa, both of which modify transmural dispersion of repolarisation. Activation of IKr causes heterogeneous changes to APD at the tissue level, the same mechanism as in our previous studies examining the effects of NS1643  reduces the probability of DADs developing into triggered activity , with more block reducing this probability; that is to say, the greater the INa block, the more anti-arrhythmic (in terms of reducing triggered activity) the effects. However, in the PQTa condition, while 40% block of INa reduced the probability of triggered activity occurring, increasing block of the current to 80% slightly increased the probability of triggered activity; in this condition, a small amount of INa block has anti-arrhythmic effects, but increasing this small level of block is pro-arrhythmic. In the PQTc condition, INa block of any magnitude increased the probability of triggered activity , with the probability of triggered activity occurring increasing as INa block is increased. Similarly varied results were found for the 300 ms Ca2+ release duration distribution  increases this length, potentially due to an increase in transmural dispersion of repolarisation with INa block; analyses of these results alone would conclude that INa block is pro-arrhythmic. However, despite the spatial width of the VW increasing, block of INa results in the total spatiotemporal area of the VW decreasing , i.e., an anti-arrhythmic result, likely due to the reduced excitability that results from INa block reducing the likelihood that any triggered activity would propagate.One further note of caution with regards to INa block per se is an effective antiarrhythmic strategy  electrophysiological consequences in a manner that is not intuitive. The mechanisms underlying these varying effects of INa block on arrhythmogenesis, particularly on DAD initiation and their development into triggered activity, remain to be elucidated.Our tissue-level findings therefore indicate that 2+ release events under certain conditions), one interesting finding did emerge in relation to arrhythmia triggers that can result from abnormal intracellular Ca2+ handling: Modification of membrane ion channels carrying ions other than Ca2+ (K+ and Na+ in this case) can have beneficial effects in terms of restoring abnormal intracellular Ca2+ handling, through their actions in shortening APD. This was shown in Figure 2+ loading (and resultant spontaneous SR Ca2+ release events) seen under PQT conditions was reversed by upregulating IKr and downregulating INa, which in turn shortened APD. This reduces the duration over which ICa,L is activated, reduces the amount of Ca2+ crossing the cell membrane and entering the cell via that current, and thus reduces SR Ca2+ load. Furthermore, the combined action of both of these current modifications reduced the probability of DADs manifesting as triggered action potentials in single cell as well as triggered action potentials manifesting as fully propagating ectopic beats at the tissue scale. Targeting cell membrane ion channels carrying ions other than Ca2+ in order to restore abnormal intracellular Ca2+ handling  will alter the delicate homeostasis of an already-compromised system, yielding negative results  which were not considered in the present study. However, the aim of this study was to investigate the multi-scale interaction between trigger and substrate, and the use of the simplified spontaneous release functions in single-cell and tissue simulations allowed this to be analyzed in a general manner and across a large range of conditions, independent of spontaneous release mechanism. In future, combining detailed single cell studies of the mechanisms of spontaneous release in disease conditions with tissue simulations of the same conditions would provide further mechanistic insight; a dynamic simplified spontaneous release model would furthermore allow the study of long-term interactions between trigger and substrate in tissue e.g., during re-entry.In this study, a general model of intracellular Ca2+ release) is primarily suitable for interpretation of general trends only, rather than as a quantitative analysis of the efficacy of MC-II-157c in modulating Ca2+ release dependent triggers; it is encouraging to note that our results are consistent with those of a previous study , but it is important to note that there were significant differences between the simulation and experimental data  in a similar manner to the way in which the kinetics of IKr are modified. Thus, further experimental characterisation of the drug's effects on both IKr and INa are required.Furthermore, recent work, (e.g., Li et al., The relative balance of reduced trigger and increased substrate underlies a multi-dimensional role of MC-II-157c in modulation of arrhythmia vulnerability associated with prolonged QT interval. Our results highlight that studies examining the efficacy of putative anti-arrhythmic drugs need to assess the effects of the drug on both the triggers and the substrates involved in arrhythmogenesis, i.e., such studies should adopt a multiscale approach to examine both cell- and tissue-level effects.All authors conceived and designed the study; MC, EP, and AB carried out simulations, and acquired and analyzed data; all authors interpreted the data; MC and AB prepared the first draft of the manuscript text; MC and EP prepared the figures; all authors edited the manuscript; all authors approved the final version of the manuscript; all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer KF and handling Editor declared their shared affiliation.


Interactions between (ER)-located Ca2+ sensors  and plasma membrane-located Ca2+ channel-forming protein (Orai1) underlie SOCE and are well described in non-excitable cells. In neurons, however, SOCE appears to be more complex because of the importance of Ca2+ influx via voltage-gated or ionotropic receptor-operated Ca2+ channels. We found that the SOCE inhibitors ML-9 and SKF96365 reduced α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced [Ca2+]i amplitude by 80% and 53%, respectively. To assess the possible involvement of AMPA receptors (AMPARs) in SOCE, we used their specific inhibitors. As estimated by Fura-2 acetoxymethyl (AM) single-cell Ca2+ measurements in the presence of CNQX or NBQX, thapsigargin (TG)-induced Ca2+ influx decreased 2.2 or 3.7 times, respectively. These results suggest that under experimental conditions of SOCE when Ca2+ stores are depleted, Ca2+ can enter neurons also through AMPARs. Using specific antibodies against STIM proteins or GluA1/GluA2 AMPAR subunits, co-immunoprecipitation assays indicated that when Ca2+ levels are low in the neuronal ER, a physical association occurs between endogenous STIM proteins and endogenous AMPAR receptors. Altogether, our data suggest that STIM proteins in neurons can control AMPA-induced Ca2+ entry as a part of the mechanism of SOCE.The process of store-operated calcium entry (SOCE) leads to refilling the endoplasmic reticulum (ER) with calcium ions (Ca Interactions between STIMs and Orais lead to the formation of complexes that are visible as puncta  channels  pump to refill ER stores and can be used for signaling  and ligand-gated Ca2+-channels (LGCCs), such as ionotropic glutamate receptors . For the co-immunoprecipitation assays and Western blot, neurons were seeded on poly-D-lysine-precoated BioCoat plastic Petri dishes (BD Biosciences Discovery Labware) at a density of 7 × 106 cells/plate. Neurons were grown in Neurobasal medium (Invitrogen) supplemented with 2% B27 (Invitrogen), 0.5 mM glutamine (Sigma), 12.5 μM glutamate (Sigma), and a penicillin (100 U/ml)/streptomycin (100 mg/ml) mixture (Gibco). Cultures were maintained at 37°C in a humidified 5% CO2/95% air atmosphere. Every 3–4 days, half of the conditioned medium was removed and replaced by fresh growth medium. The experiments were performed on 15-day-old cultures  or 16- to 17-day-old cultures (Ca2+ measurements).Cortical neuronal cultures were prepared from embryonic day 19 (E19) Wistar rat brains. Pregnant female Wistar rats were provided by the Animal House of the Mossakowski Medical Research Centre, Polish Academy of Sciences . Animal care was in accordance with the European Communities Council Directive (86/609/EEC). The experimental procedures were approved by the Local Commission for the Ethics of Animal Experimentation no. 1 in Warsaw. Brains were removed from rat embryos and collected in cold Hanks solution supplemented with 15 mM HEPES buffer and penicillin/streptomycin. The cortices were isolated, rinsed three times in cold Hanks solution, and treated with trypsin for 35 min. The tissue was then rinsed in warm Hanks solution, washed three times, and dissociated by pipetting. For CaTo make the glial cultures, a cortical cell suspension was plated on poly-D-lysine-precoated BioCoat plastic Petri dishes (BD Biosciences Discovery Labware) and grown in Dulbecco’s Modified Eagle Medium (DMEM) that contained 10% fetal bovine serum and a penicillin (100 U/ml)/streptomycin (100 mg/ml) mixture (Gibco). The cells were grown to confluence (after ~1 week) and were replated to avoid non-glial contamination. The medium was replaced once every 3–4 days. For Western blot, the cells were cultured for 15 days prior to the experiments.2 atmosphere.HeLa cells were grown in DMEM that contained 10% fetal bovine serum and a penicillin (100 U/ml)/streptomycin (100 mg/ml) mixture (Gibco) at 37°C in a 5% CO2+ levels in cortical neurons were recorded using the ratiometric Ca2+ indicator dye Fura-2 acetoxymethyl ester (Fura-2 AM). Cells were grown on eight-well chamber slides and loaded with 2 μM Fura-2 AM for 30 min at 37°C in Hanks Balanced Salt Solution (HBSS) that contained 145 mM NaCl, 5 mM KCl, 0.75 mM Na2HPO4, 10 mM glucose, 10 mM HEPES (pH 7.4) and 1 mM MgCl2 supplemented with 2 mM CaCl2 at 37°C (high Ca2+ medium) and then rinsed and left undisturbed for 30 min at 37°C to allow for de-esterification. Measurements of intracellular Ca2+ levels were performed every 1 s at 37°C using an Olympus Scan∧R&Cell∧R imaging system that consisted of an IX81 microscope , 10× 0.40 NA UPlanS Apo objective , and Hamamatsu EM-CCD C9100-02 camera . Changes in intracellular Ca2+ concentration ([Ca2+]i) in individual neuronal cell bodies are expressed as the F340/F380 ratio after subtracting background fluorescence. This ratio represents the emission intensities at 510 nm obtained after excitation at 340 and 380 nm. The low Ca2+ medium (Ca2+-free solution) contained 0.5 mM ethylene glycol tetraacetic acid (EGTA) in the standard buffer. At the end of the experiments, 50 mM KCl in the presence of 2 mM CaCl2 was added to assess which cells were neurons , scraped and centrifuged at 1000× g for 5 min at 4°C. The pellet was suspended in lysate buffer, pH 7.5, that contained 50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% NP-40, and 1 mM phenylmethylsulfonyl fluoride supplemented with complete Ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail (Roche) and lysed with an insulin syringe (18×). After incubation for 2 h on ice and centrifugation at 15,000× g  for 20 min at 4°C, cleared lysates were pre-incubated with 30 μl of washed protein A-Sepharose (Roche) for 3 h at 4°C. Sepharose resin and bound components were recovered by centrifugation at 15,000× g for 20 min at 4°C. Precleared lysates were subsequently incubated overnight at 4°C on a rocking platform with 30 μl of A-Sepharose that was pre-incubated earlier for 3 h with 3 μg of antibody . As a negative control when indicated, lysates were omitted or were incubated with anti-Flag (Sigma) or anti-IgG antibody (Sigma). Precipitated samples were then washed three times with repeated centrifugation, eluted in 50 μl of 2× Laemmli Buffer. Protein extracts were resolved by 6% SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a Protran nitrocellulose membrane (Whatman), and blocked for 2 h at room temperature in TBST  plus 5% dry non-fat milk. Nitrocellulose sheets were then incubated in blocking solution with primary antibodies against STIM1 , STIM2 , GluA1  and GluA2  at 4°C overnight. The appropriate horseradish peroxidase-conjugated secondary antibody IgG (Sigma) was added at a dilution of 1:10,000 for 1 h. The peroxidase was detected with a Chemiluminescent Substrate Reagent Kit .On Western blot containing glial cells extracts, the optical density of the bands was estimated using a GS-800 Calibrated Densitometer and Quantity One software (Bio-Rad). GAPDH was run to normalize the protein loading.p < 0.05. Statistical significance was assessed using the nonparametric Mann-Whitney U test for comparisons between the mean values of unpaired groups. All of the experiments were performed at least in triplicate.The statistical analysis was performed using Prism 5.02 software . All of the data are expressed as mean ± standard error of the mean (SEM), and differences were considered significant at 2+]i responses are affected. Rat cortical neurons were loaded with the Fura-2 AM Ca2+ indicator in 2 mM CaCl2-containing medium, and the Ca2+ signal was recorded. The cells were then treated with 2 mM Ca2+ medium supplemented with 100 μM AMPA in the absence or presence of 100 μM ML-9. After the addition of AMPA, an increase in [Ca2+]i changes was observed in both neuronal cultures  receptor antagonist DAP-5 in the presence of NM  by the AMPA/kainate receptor antagonist CNQX  blockers were applied in the presence of ML-9. Figure 2+]i responses were reduced by ML-9 in the presence of NM and DAP-5 by 75%, thus confirming its inhibitory effects. To further examine the impact of SOCE on AMPA-induced [Ca2+]i response we used another SOCE inhibitor—SKF96365. In the presence of SKF96365 the AMPA-induced changes in [Ca2+]i were decreased by 53%  and either NBQX or CNQX. After 5 min, a medium with 2 mM CaCl2 was added to induce SOCE in the presence of 30 μM NBQX or 30 μM CNQX. To distinguish neurons from other cells that were present in the culture, KCl was added at the end of the experiments, and the Ca2+ signal was recorded (data not shown). Only cells that responded with fast and transient increases in Ca2+ were considered neurons  and the TG-treatment increased the level of both proteins in the membrane fractions  contains GluA2 subunit of AMPAR, whereas non-pyramidal cells (mainly GABAergic inhibitory interneurons) preferentially express GluA1-specific mRNA  inhibitor that is commonly used to inhibit SOCE  does not affect SOCE or STIM1 redistribution (Smyth et al.,  Figures  is proba Figures , which c2+ entry is linked to InsP3-dependent Ca2+ store depletion induced by activation of metabotropic glutamate receptors (Berridge, 2+ influx pathway activated by glutamate was found. This influx pathway, attributable to AMPA/kainite receptors, is sensitive to NBQX but insensitive to SOCE blocker La3+ (Pizzo et al., It is well known that a sustained Ca2+ stores in glia, in which TG-induced SOCE should lead to greater Ca2+ influx than in neurons. Similar observations were described previously (Arakawa et al., 2+ entry may be attributable to differences in SOCE channels or their mechanism of activation between glial and neuronal cells. We found that NBQX decreased glial SOCE by 46%, whereas it decreased neuronal SOCE by 73%. This likely reflects the amount and type of AMPARs in both types of cells (Figure 2+-permeable AMPARs with Q/R-unedited GluR2 subunits than neurons (Whitney et al., 2+ influx in rat cortical astrocytes was also shown to be sensitive to LaCl3, which inhibits SOCE (Pizzo et al., In the present study, we found that SOCE in neurons was approximately two-times lower than in glial cells. This seems to reflect the larger size of Cas Figure . Althougs Figure , the glis Figure . The exps Figure . In corts Figure . However2+ homeostasis that are observed in several neurological disorders, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, chronic epilepsy, amyotrophic lateral sclerosis, painful nerve injury and cerebral ischemia (Leissring et al., 2+ store depletion, and slower oligomerization rate of STIM2 compared with STIM1 (Stathopulos et al., 2+ signals by recruiting STIM1 and Orai1 to the postsynaptic density (Korkotian et al., V1.2 (Park et al., V3.1 (Nguyen et al., 2+ influx, which may be involved in the neuronal dysfunction that is observed in schizophrenia, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and global ischemia (Akbarian et al., 2+]i responses in neurons are a part of the SOCE process, suggesting a possible relationship between SOCE, STIM proteins, and AMPARs in these diseases.Recently, SOCE in neurons has become the subject of intense research with regard to disturbances in Ca2+ signal. Together with the studies by Park et al. (2+ channels present in the plasma membrane.In conclusion, the present data demonstrate that SOCE is functional in neurons as an AMPA-dependent Cak et al.  and Garck et al. , our resJG-B conceived, designed and performed experiments, analyzed the data, and wrote the manuscript. MS performed the experiments, analyzed the data, and wrote the manuscript. JK designed the experiments, analyzed the data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


By themselves, NOMs increased lipid peroxidation, Ca2++Mg2+-ATPase, and/or carbonic anhydrase activities (significant in seven of 15 NOM-endpoint combinations), whereas metals by themselves did not affect the first two endpoints, but Cu and Pb increased carbonic anhydrase activities. In combination, the effects of NOMs predominated, with the metal exerting no additional effect in 33 out of 45 combinations. While NOM effects varied amongst different isolates, there was no clear pattern with respect to optical or chemical properties. When NOMs were treated as a single source by data averaging, NOM had no effect on Ca2++Mg2+-ATPase activity but markedly stimulated carbonic anhydrase activity and lipid peroxidation, and there were no additional effects of any metal. Our results indicate that marine NOMs may have direct effects on this model marine organism, as well as protective effects against metal toxicity, and the quality of marine NOMs may be an important factor in these actions.Metals are present in aquatic environments as a result of natural and anthropogenic inputs, and may induce toxicity to organisms. One of the main factors that influence this toxicity in fresh water is natural organic matter (NOM) but all NOMs are not the same in this regard. In sea water, possible protection by marine NOMs is not well understood. Thus, our study isolated marine NOMs by solid-phase extraction from five different sites and characterized them by excitation-emission fluorescence analysis—one inshore (terrigenous origin), two offshore (autochthonous origin), and two intermediate in composition (indicative of a mixed origin). The physiological effects of these five NOMS alone (at 8 mg/L), of three metals alone , and of each metal in combination with each NOM, were evaluated in 48-h exposures of mussel larvae. Endpoints were whole body Ca Nevertheless, these may not be the only mechanisms of toxicity, and there is emerging evidence that metals may also induce oxidative stress , and that all NOMs are not the same in this regard . TraditiIn sea water, there is now evidence that NOM strongly protects against Cu toxicity, but the particular source or chemical nature of the NOM may or may not have a large influence . On the Mytilus galloprovincialis. These organisms are cultured commercially on the west coast of Vancouver Island, British Columbia, close to the sites where most of the NOMs were collected. The various NOMs were characterized by excitation-emission fluorescence  that at least some of the NOMs would exert positive effects on the organism; (ii) that metals alone would exert negative effects, which would differ according to the metal; (iii) that at least some of the NOMs would be protective, ameliorating the negative effects of the metals; and (iv) that these effects would vary both amongst the different NOMs, and among the different metals.The objectives of the present study were to test the influence of a range of true seawater NOMs actually collected from the marine environment on physiological indices of toxicity, in the presence or absence of sublethal concentrations of Cu, Pb, or Zn, using developing larvae of the blue mussel, rescence . The sit®, Brazil) and acidified to pH 2 with HCl. The acidified water was passed through PPL cartridges  to remove highly polar species. The salts retained in the PPL cartridges were removed using acidified ultrapure water (pH 2). At the end of the process, the NOM elution was performed using methanol that was then removed by lyophilization, and the concentrated NOM solutions were prepared with ultrapure water. Activation of the PPL cartridges, removal of salts, and rinsing, as well as NOM elution, lyophilization, and storage were performed as described by ® filter; Millipore, Billerica, MA, USA), diluted appropriately, and the dissolved organic carbon (DOC) was measured .Test solutions were similarly assayed for DOC, without dilution. The five NOMs from different sources were stored in the dark at 4 °C.NOM extraction followed the solid-phase method described by a priori assumption that there were three components  with 1 cm path-length quartz cuvettes. The fluorescence spectra were measured at excitation wavelengths from 200 to 450 nm using 10 nm increments, and emission wavelengths in the range of 250–650 nm in 1 nm increments. Each matrix contains fluorescence information for specific excitation/emission wavelength pairs. The excitation-emission matrices EEMs were plotted as contours to identify the fluorescing components. Parallel Factor Analysis (PARAFAC) was then used to identify and quantify the component peaks in the EEMs, as implemented in the PLS Toolbox  running on a Matlab™ platform. PARAFAC resolves this information into component spectra and relative component concentrations . PARAFACM. galloprovincialis were obtained by courtesy of Northwest Aquaculture Farm located on Effingham Inlet in Barkley Sound on the west coast of Vancouver Island. At Bamfield Marine Sciences Centre (BMSC), they were held in 10 L tanks at 10–15 °C in aerated seawater (30 ppt). Males and females were induced to spawn through the heat shock method, according to Adults of the mussel M. galloprovincialis were exposed to isolated NOMs from each of the five different sources or combined treatments of NOMs and metals  starting at about 1 h post-fertilization. For all the tests, the embryos were transferred into seawater exposure solutions  at a density of 10 individuals/mL, 15 °C for 48 h. These parameters were based on the acute toxicity test protocol with embryos of saltwater bivalve molluscs , and also exposed to single metals in Bamfield sea water without addition of NOMs. At the end of 48 h exposure, the samples were filtered using an 8-µm polycarbonate filter  under gentle vacuum, and the D-veliger larvae were collected in 1.5-mL vials and stored at −80 °C for later analyses.Embryos of d metals . All tremolluscs . The isorossolus , measurerossolus , but prerossolus , so the 2+ + Mg2+-ATPase and carbonic anhydrase activities (see below). Ca2+ + Mg2+-ATPase measurements were performed using an assay originally described by +, but one with Mg2+ present but no Ca2+, and the other with both Ca2+ and Mg2+ present. The first is intended to yield Mg2+ ATPase activity, and the difference between the two is intended to yield Ca2+ATPase activity. In our hands, the two incubation media generally yielded very similar activities such that Ca2+ATPase activity could not be reliably distinguished. This suggests that the enzyme in blue mussel larvae is rather unspecific in its substrate specificity, being activated in the presence of either divalent cation. Therefore, we have reported only the results obtained in the medium with both cations present  as “Ca2++Mg2+-ATPase activity”. Pi concentration was determined using a commercial reagent kit . Protein concentration in the homogenate of larvae was determined using the method of Samples were homogenized in a buffer solution  for measurement of both CaM. galloprovincialis was assayed as described by 2-saturated solution by the enzyme with subsequent release of H+ and consequent reduction of the pH. The larvae were homogenized in the same buffer solution used for Ca2++Mg2+-ATPase measurements, with the protein concentration again determined by the method of 2HPO4) and an aliquot of a CO2 saturated solution was added. The pH was measured every 5 s up to 30 s. The enzyme activity was calculated based on the slope of the linear regression for the pH change over time and the protein content in the cell homogenate. The specific enzyme activity was expressed as an arbitrary unit of carbonic anhydrase (slope of the linear regression)/mg protein/h.Carbonic anhydrase activity in the larvae of the The extent of lipid peroxidation was determined by the malondialdehyde (MDA) reaction produced during the lipoperoxidation process with thiobarbituric acid, according to n = 3). Each sample (n) represents a pool of ∼2,500 larvae of M. galloprovincialis. Isolated effects of NOM exposures  in larvae were calculated by one-way analysis of variance (ANOVA) followed by Tukey’s test. ANOVA assumptions  were checked and the data transformed as necessary to satisfy these assumptions. For simplicity, as there are so many possible comparisons, the differences among NOMs have not been marked. Thus, the statistical differences were indicated only for comparisons between specific NOMs and the absolute control condition (Bamfield sea water with no added NOM) using an asterisk (*). The effect of metal addition in each treatment  was evaluated by Student’s two tailed t-tests, comparing the presence and absence of metal in the exposure within the same source of NOM. Significant differences have been marked with # signs. The significance level adopted was 95% (α = 0.05).Data are expressed as means ± standard error  in comparison to the effects, or lack of effects, of the NOM alone. With respect to significant effects of metals in the combined exposures # in –4, in fo2++Mg2+-ATPase activities remained similar to their respective NOM controls, except in the case of copper which decreased Ca2++Mg2+-ATPase activity in the presence of Bamfield NOM (The two “mixed” NOMs (Port and Bamfield) showed the greatest number of enzymatic alterations after metal addition. Carbonic anhydrase activity was increased significantly by the addition of copper, lead and zinc in the presence of Port and Bamfield, with the single exception of lead with Bamfield  and 4B. ield NOM .Addition of metals in the presence of autochthonous NOMs (Offshore-CA and Offshore-BR) resulted in no additional effects at all with Offshore-BR but some differences with Offshore-CA. When copper was added with Offshore-CA, carbonic anhydrase activity declined, and when zinc was added with Offshore-CA, both enzymatic activities fell  and 4B.In the presence of the terrigenous NOM (Pachena), metals exerted no stimulatory effects. However, lipid peroxidation decreased with zinc exposure  but rema2++Mg2+ ATPase activity as positive effects (four of the seven changes) and stimulation of lipid peroxidation as a negative effect (three of the seven changes). Another unexpected result was that our second hypothesis that metals alone would exert negative effects which would differ according to the metal, was largely not supported. Indeed metals alone had no influence on Ca2++Mg2+ ATPase activity or lipid peroxidation, whereas both copper and lead stimulated carbonic anhydrase activity. Therefore in the absence of negative effects of metals alone, our third hypothesis, that some of the NOMs would be protective, ameliorating the negative effects of the metals, became problematical. However, it is noteworthy that the most common pattern was that the metal exerted no additional effect  in comparison to the effect, or lack of effect, of the NOM alone. In four of the combined exposure endpoints, the effect of the added metal was less than that of the NOM alone, whereas, in eight cases, the added metal caused additional stimulation. Clearly direct effects of the NOMs were predominant in most cases. Our final hypothesis, that effects would vary both amongst the different NOMs and among the different metals, was strongly supported for the NOMs, but less strongly for the different metals, as discussed subsequently. Finally, it is important to emphasize that the current conclusions relate only to developmental status at the end of the first 48 h of mussel life (development to the D-larva stage), and that different effects of NOMs and metals may well occur in the intervening developmental events prior to 48 h, and after 48 h as veliger development proceeds subsequently.The marked effects of NOMs alone on physiological endpoints confirmed our first hypothesis that at least some of the NOMs would exert positive effects on the organisms. Indeed, significant changes were seen in seven of 15 NOM-endpoint combinations, though not all were necessarily beneficial. In this regard, we interpret stimulation of carbonic anhydrase activity and CaExtraction of NOMs from five different regions resulted in three chemically distinct groups. Offshore-CA and Offshore-BR NOMs had very similar chemical characteristics typical of autochthonous origin, with large tyrosine-like and tryptophan-like components. Pachena, which is close to the outflow of a brown-water river draining evergreen forest land, had a typical terrigenous signature high in humic substances. Bamfield and Port NOMs, which would be subject to both land runoff and open ocean influences, were classified as of “mixed” terrigenous and autochthonous. PARAFAC demonstrated Pachena to be the most terrigenous with >80% humic substance contributions to total fluorescence and FI identified the off shore samples as most authochthonous (FI greater than 1.5) with Bamfield and Pachena having intermediate properties of the end-member sources. The uptake of dissolved organic matter by marine organisms has been known since the classic work of The NOM exposures, by themselves, clearly demonstrated pronounced physiological effects which varied amongst the different NOMs. However, there was no clear pattern with respect to physico-chemical properties or origins of the various NOMS. Both autochthonous, optically light NOMs (Offshore-CA and Offshore-BR) and terrigenous, optically dark NOM (Pachena) resulted in significant oxidative stress as indicated by increased lipid peroxidation, whereas the intermediate NOMs (Port and Bamfield) did not affect this endpoint. Previously, oxidative stress has also been reported in freshwater amphipods exposed to NOMs from different sources, as evidenced by decreased peroxidase and increased catalase activities . The firM. galloprovincialis also increased Ca2++Mg2+-ATPase (Offshore-BR) or carbonic anhydrase activities (Offshore-CA and Pachena). These enzymatic alterations could be related to oxidative stress stimulating tyrosine phosphorylation which has been described as a “key-process” in the transduction and regulation of enzyme activity in diverse cell types  were not seen; indeed both copper and lead stimulated carbonic anhydrase activity. There are several possible explanations. Firstly, these effects have been described previously mainly in freshwater organisms (see Introduction), and the very different physiologies of seawater organisms, as well as the very different physical chemistry of sea water, may prevent their occurrence. Secondly, there is considerable evidence that the effects caused by metals are both time-dependent and concentration-dependent  caused additional stimulation of carbonic anhydrase activity (except for Bamfield and lead) and lipid peroxidation . This could be interpreted as either a lack of protection, or an exacerbation of toxic effect, reflecting the intermediate or mixed nature of the chemical structures.2+ + Mg2+-ATPase and carbonic anhydrase activities were seen when Zn was added to Offshore-CA. Offshore-CA NOM had a lesser amount of humic substance-like components but more of the proteinaceous materials (tryptophan-like and tyrosine-like). As shown in some studies, these types of compounds affect directly the capacity to bind metals , our study reinforces the importance of the quality of NOMs both in their direct effects and their interactions with metal effects in a model marine organisms. All marine NOMs are not alike. The latter has previously been shown in several other seawater studies . HoweverCurrently, computational models such as the Biotic Ligand Model  that pre2++Mg2+-ATPase activity, the combined NOM treatment, alone or in combination with various metals, had no significant effect  concentrations of the NOMs from different sources are Pachena = 7.4 mg DOC/L, Bamfield = 6.3 mg DOC/L, Port = 7.9 mg DOC/L, Off-CA = 6.0 mg DOC/L, Off-BR= 9.1 mg DOC/L.Click here for additional data file.10.7717/peerj.3141/supp-2Table S1N = 3) using approximately 2500 embryos each.Treatments were performed in triplicate .Asterisks (*) indicate only significant differences between metals and the absolute control condition (Bamfield sea water with no added NOM). (B) Represent p-values from comparisons between the absolute control condition (Bamfield sea water with no added NOM). Bold type indicates statistical differences. The significance level adopted was 95% ( Click here for additional data file.


The GK rat is a widely used nonobese, nonhypertensive genetic model of T2DM which is characterized by insulin resistance, elevated blood glucose, alterations in blood lipid profile, and cardiac dysfunction.The association between diabetes mellitus (DM) and high mortality linked to cardiovascular disease (CVD) is a major concern worldwide. Clinical and preclinical studies have demonstrated a variety of diastolic and systolic dysfunctions in patients with type 2 diabetes mellitus (T2DM) with the severity of abnormalities depending on the patients' age and duration of diabetes. The cellular basis of hemodynamic dysfunction in a type 2 diabetic heart is still not well understood. The aim of this review is to evaluate our current understanding of contractile dysfunction and disturbances of Ca Diabetes mellitus (DM) is a metabolic disease characterized by abnormal glucose homeostasis and defects in insulin metabolism. Cardiovascular disease (CVD) is the leading cause of death in the diabetic population. However, the molecular mechanisms underlying diabetic cardiomyopathy remain unclear.in vivo and in isolated pancreata, raised glycosylated hemoglobin, hepatic and peripheral insulin resistance, altered heart and body weight, and a variety of late complications, including cardiomyopathy, nephropathy, and neuropathy [Animal models are increasingly being used to elucidate the mechanisms underlying diabetic cardiomyopathy in both type 1 and type 2 diabetes. One of the most widely used animal models of type 2 diabetes mellitus (T2DM) is the Goto-Kakizaki (GK) rat. The GK rat is a polygenic nonobese model of T2DM. This model is generated by selective inbreeding of mildly glucose-intolerant Wistar rats over many generations [uropathy –11. In curopathy .β-cell mass and reduced insulin secretion [β-cell proliferation and reduction in β-cell mass), reduced insulin sensitivity in the liver, and moderate insulin resistance in peripheral tissues [Three genetic loci are responsible for coding and transferring diabetic pathology to the fetus, and these include genes that are responsible for a reduction in ecretion . During  tissues , 13.β-cell dysfunction. In fact, the pancreatic islets of adult GK rats show decreased β-cell number and insulin content as compared to their age-matched control animals [Persistent hyperglycemia over time provokes pancreatic islet inflammation, oxidative stress, fibrosis, and finally  animals .β-cell mass and reduced β-cell function are key characteristics found in this animal model [β-cell compensation in the pathogenesis of T2DM.GK rats have been considered as one of the best nonobese type 2 diabetic animal models. GK rats exhibit valuable characteristics that are more or less common and functionally present in human diabetic patients. This animal model is considered appropriate to examine various pathologic mechanisms of T2DM , 14. As al model . Therefoal model  in orderAn earlier study has shown that GK islet fibrosis is accompanied by marked inflammation which is a characteristic that has been reported in islets of type 2 diabetic patients . Other cAs in humans, GK rats also develop renal lesions, structural changes in peripheral nerves, and retinal damage . For exaβ-cell to sufficiently compensate, which leads to hyperglycemia [T2DM is characterized by insulin resistance and the inability of the glycemia . In addiglycemia , 22. Manglycemia . The genglycemia , 23.ob/ob mice, Leprdb/db mice, and Zucker diabetic fatty rats are the most commonly used models of monogenic obesity. They have a disrupted leptin signaling pathway, leading to hyperphagia and obesity [Y mice, New Zealand obese (NZO) mice, TallHo/Jng mice, and Otsuka Long Evans Tokushima Fat (OLETF) rat. Obesity can also be induced by feeding the rodent a high-fat diet (diet-induced models). The weight gain in these animals is associated with insulin resistance and abnormal glucose metabolism [Lep obesity . Polygen obesity . These itabolism , 13, 23.β-cell mass and/or β-cell function [β-cell mass [β-cell mass. Blood glucose is elevated only after the first 3-4 weeks of animal's age, and blood glucose rises significantly after a glucose challenge [In contrast to the animal models mentioned above, the GK rat is a non-obese animal model of T2DM. It is characterized by reduced function . The GK ell mass  and/or fell mass . Adult Ghallenge , 27. Thehallenge , 12. Othhallenge .Blood insulin, glucose, and lipid profiles in the GK rats compared to controls are summarized in Tables Body weight and heart weight measures in GK rats compared to controls are summarized in Tables β1 (TGF-β1) in the ventricular tissue [β1 reproduces most of the hallmarks seen in structural remodeling. Specifically, TGF-β1 induces expression levels of extracellular matrix (ECM) constituents by cardiac fibroblasts , self-amplifies its own expression in both cardiac myocytes and fibroblast [β1 and phosphorylation of protein kinase B (PKB)/Akt and its downstream effectors mediate the hypertrophic effects of TGF-β1 in the prediabetic GK left ventricle [+/H+ exchanger plays a role in the molecular mechanisms involved in cardiac hypertrophy. It is likely that the activation of the Akt pathway mediates the hypertrophic effect of myocardial Na+/H+ exchanger in the GK rat model of T2DM [Earlier studies have reported that chronic mild hyperglycemia produces molecular and structural correlates of hypertrophic myopathy in GK rats . Severalr tissue . TGF-β1 broblast , 54, andbroblast , 56. D'Sentricle . The hypentricle . Earlier of T2DM . Interes of T2DM , 32, 57.In vivo hemodynamic function and related measures in GK rats compared to controls are summarized in in vivo in GK and control hearts  via activation of the ryanodine receptor (RyR), by the process termed calcium-induced calcium release (CICR). The transient rise in intracellular Ca2+ (Ca2+ transient) results in the binding of Ca2+ to troponin C which initiates and regulates the process of cardiac muscle cell contraction. During the process of relaxation, Ca2+ is pumped back into the SR via the SR Ca2+-ATPase (SERCA2) and extruded from the cell, primarily via the Na+/Ca2+ exchanger (NCX) [During the process of excitation-contraction coupling (ECC), the arrival of an action potential causes depolarization of the cardiac myocyte plasma membrane. This depolarization opens voltage-gated L-type Caer (NCX) , 61. Chaer (NCX) , 63. Theer (NCX) .2+ in myocytes from GK rats compared to controls are shown in 2+ is unaltered [2+ transient is unaltered [2+ transient is unaltered [2+ transient is unaltered [2+ current are unaltered [Characteristics of intracellular Canaltered , 47, 48 naltered ; TPK Ca2naltered , 48, 50 naltered ; and thenaltered , 41, 48,naltered  in myocy2+ in cardiac cells is maintained by Ca2+ influx  and efflux  [2+ release (via the ryanodine receptors) and uptake by both SR (through SERCA2) and mitochondria, it is possible that the observed differences in these results may be attributed to differential changes in Ca2+ transport activities in these organelles. Furthermore, the observed alterations in intracellular Ca2+ may also be due to differences in the stage and severity of diabetes [Since intracellular Cahe cell) , as welldiabetes , 66.2+ uptake and release mechanisms would impair cardiac cell function. Several studies have reported changes in cardiac SR Ca2+ transport during the development of chronic diabetes [2+ uptake activity by SR was associated with a decrease in SERCA2a activity [2+ uptake activity was markedly decreased in the diabetic rat heart [2+ content and ryanodine binding sites in diabetic hearts, indicating that the SR functions of storage and release of Ca2+ were depressed [2+ uptake activity in chronic diabetes may contribute to the occurrence of intracellular Ca2+ overload [It is well known that alterations in SR Cadiabetes –71. For activity . Furtherat heart . Yu et aepressed . It shouoverload .2+ current and Ca2+ transients were simultaneously measured in endocardial (ENDO) and epicardial (EPI) myocytes from the left ventricle of GK rats [2+ current, over a wide range of test potentials, was unaltered in ENDO and EPI myocytes from the left ventricle of GK rat. However, the amplitude of the Ca2+ transients was reduced and by similar extents, in ENDO and EPI myocytes from the GK rat heart. The THALF decay of the Ca2+ transients was reduced in EPI and ENDO myocytes from GK rats compared to controls. Interestingly, while a reduction in the amplitude of L-type Ca+ current has been reported in earlier studies on a diabetic heart [2+ transients. This is because many reports show no change in L-type Ca2+ current despite the reduction in both contractions and Ca2+ transients [2+ transients and the consequent contractile dysfunction may be due to depletion of SR Ca2+, which may result from RYR-dependent Ca2+ leak, an increased Ca2+ extrusion through NCX, or a reduced function of SERCA [2+ transport in myocytes from the GK rat. Sheikh et al. [2+ transient peaks as compared to controls. This finding supports the idea that altered activity of sarcolemmal NCX during Ca2+ efflux contributes to the decrease in Ca2+ transient-observed GK myocytes. Previous experiments in ventricular myocytes from the streptozotocin-induced diabetic rats have reported reduced caffeine-evoked Ca2+ transients [2+ uptake [2+ channel (ryanodine receptor) activity [2+ content, Ca2+ uptake, and Ca2+ release mechanisms in ventricular myocytes from the streptozotocin-induced diabetic rat.In our recently published data, L-type Ca GK rats . Consist GK rats , the ampic heart , 76, it ansients , 77–79. of SERCA , 80. Furh et al.  demonstransients –91, SERC+ uptake , 92–94 aactivity , 95 sugg2+, which serves as a protective mechanism during cardiac dysfunction and intracellular Ca2+ overload. Therefore, altered mitochondrial uptake of Ca2+ during diabetes may contribute to the reported decreased Ca2+ transients. Although the mitochondria contribute to Ca2+ signaling, their exact role in diabetic cardiomyopathy remains to be investigated.Under pathological conditions, such as chronic diabetes, the mitochondria are able to accumulate large amounts of Ca2+ handling [Recent investigations, using animal models, suggest that mitochondrial dysfunction may also play a critical role in the pathogenesis of diabetic cardiomyopathy , 71. Pothandling , 108.2+ sinks in the cell by acting as a local buffering system, removing Ca2+ and modulating cytosolic Ca2+concentrations [2+ concentration, mitochondria dynamically interact with the cytosol and intracellular Ca2+ handling machineries to shape the cellular Ca2+ signaling network [2+ between the mitochondria and the cytosol and that mitochondrial Ca2+ uptake increases mitochondrial ATP production [2+ overload in cardiomyocytes under different pathological conditions. For example, during the development of cardiac dysfunction and intracellular Ca2+ overload in chronic diabetes, mitochondria are believed to continue accumulating Ca2+, thereby serving as a protective mechanism [2+ concentration exceeds its buffering capacity, irreversible swelling occurs leading to mitochondrial dysfunction. As a result, energy production as well as energy stores are depleted. Collectively, these defects may contribute to the development of cardiac dysfunction in diabetic cardiomyopathy [It should be noted that the main function of the mitochondria in the heart is to produce energy in the form of ATP, which is required for cardiac contractile activity. However, mitochondria are known to serve as Catrations , 109. In network . Recent oduction . Therefoechanism , 71. How2+ handling has been demonstrated in animal models of both type 1 and type 2 diabetes. For example, in streptozotocin- (STZ-) induced diabetic rats, hyperglycemia was associated with lower rates of mitochondrial Ca2+ uptake [2+. In STZ-induced diabetic rats, Oliveira et al. observed that Ca2+ uptake was similar in control versus diabetic hearts; however, mitochondria in diabetic hearts were unable to retain the accumulated Ca2+. This effect was not observed in the presence of cyclosporin, an MPTP inhibitor [2+ release upon electrical stimulation, slowed intracellular Ca2+ decay rate, and impaired mitochondrial Ca2+ handling were observed [2+ levels and a reduction in the rate of Ca2+ decay in isolated cardiomyocytes from db/db animals, suggesting impaired mitochondrial Ca2+ uptake [2+ handling is impaired in diabetic myocardium, resulting in compromised energy metabolism and thus reduced contractility.Evidence of deficits in mitochondrial Ca+ uptake . This renhibitor . In typeobserved , 112. Si+ uptake . Taken tin vivo and in the isolated perfused heart. Hyperglycemia is usually associated with alterations in heart rate, blood pressure, blood pumping capability, and/or coronary blood flow. Contractile function, in terms of amplitude and kinetics of shortening, is frequently disturbed in the GK type 2 diabetic heart. Several mechanisms may contribute to cardiac dysfunction including mitochondrial dysfunction, myocardial fibrosis, hypertrophy, and apoptosis. Many studies show no change in L-type Ca2+ current despite the reduction in both contractions and Ca2+ transient. Instead, reduction of Ca2+ transients and the consequent contractile dysfunction may be attributed to both depletion of SR Ca2+, which may result from RyR-dependent Ca2+ leak, an increased Ca2+ extrusion through NCX, or a reduced function of SERCA (2+ signaling will provide opportunities for the development of new treatments to improve heart function in T2DM patients.Although diabetic cardiomyopathy is a frequent and important complication of DM, its physiological bases are still not completely understood. The GK type 2 diabetic heart displays a variety of abnormal hemodynamic characteristics of SERCA . Underst