Elevation Of Cytoplasmic Ca2 Research Articles

Polybrene induces neural degeneration by bidirectional Ca2+ influx-dependent mitochondrial and ER\u2013mitochondrial dynamics

Hexadimethrine bromide (Polybrene) was once used clinically as a heparin neutralizer and has recently found use as a promoter in virus-mediated gene therapy trials and gene transfer in research. However, the potential for tissue-specific toxicity of polybrene at low doses has been ignored so far. Here, we found that after intracerebroventricular (ICV) polybrene injection, mice showed disability of movement accompanied neural death and gliosis in brain, and in human neurons, polybrene induces concentration-dependent neuritic beading and fragmentation. Mechanistically, polybrene induces a rapid voltage-dependent calcium channel (VDCC)-mediated influx of extracellular Ca2+. The elevated cytoplasmic Ca2+ activates DRP1, which leads to mitochondrial fragmentation and metabolic dysfunction. At the same time, Ca2+ influx induces endoplasmic reticulum (ER) fragmentation and tightened associations between ER and mitochondria, which makes mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected neuronal toxicity of polybrene, wherein Ca2+ influx serves as a regulator for both mitochondrial dynamics and ER–mitochondrial remodeling.

Compromised astrocyte function and survival negatively impact neurons in infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCLs) are the most common cause of childhood dementia and are invariably fatal. Early localized glial activation occurs in these disorders, and accurately predicts where neuronal loss is most pronounced. Recent evidence suggests that glial dysfunction may contribute to neuron loss, and we have now explored this possibility in infantile NCL (INCL, CLN1 disease). We grew primary cultures of astrocytes, microglia, and neurons derived from Ppt1 deficient mice (Ppt1−/−) and assessed their properties compared to wildtype (WT) cultures, before co-culturing them in different combinations (astrocytes with microglia, astrocytes or microglia with neurons, all three cell types together). These studies revealed that both Ppt1−/− astrocytes and microglia exhibit a more activated phenotype under basal unstimulated conditions, as well as alterations to their protein expression profile following pharmacological stimulation. Ppt1- /− astrocytes also displayed abnormal calcium signalling and an elevated cytoplasmic Ca2+ level, and a profound defect in their survival. Ppt1−/− neurons displayed decreased neurite outgrowth, altered complexity, a reduction in cell body size, and impaired neuron survival with prolonged time in culture. In co-cultures, the presence of both astrocytes and microglia from Ppt1−/− mice further impaired the morphology of both wild type and Ppt1−/− neurons. This negative influence was more pronounced for Ppt1−/− microglia, which appeared to trigger increased Ppt1−/− neuronal death. In contrast, wild type glial cells, especially astrocytes, ameliorated some of the morphological defects observed in Ppt1−/− neurons. These findings suggest that both Ppt1−/− microglia and astrocytes are dysfunctional and may contribute to the neurodegeneration observed in CLN1 disease. However, the dysfunctional phenotypes of Ppt1−/− glia are different from those present in CLN3 disease, suggesting that the pathogenic role of glia may differ between NCLs.

InsP3R-SEC5 interaction on phagosomes modulates innate immunity to Candida albicans by promoting cytosolic Ca2+ elevation and TBK1 activity

BackgroundCandida albicans (C. albicans) invasion triggers antifungal innate immunity, and the elevation of cytoplasmic Ca2+ levels via the inositol 1,4,5-trisphosphate receptor (InsP3R) plays a critical role in this process. However, the molecular pathways linking the InsP3R-mediated increase in Ca2+ and immune responses remain elusive.ResultsIn the present study, we find that during C. albicans phagocytosis in macrophages, exocyst complex component 2 (SEC5) promotes InsP3R channel activity by binding to its C-terminal α-helix (H1), increasing cytosolic Ca2+ concentrations ([Ca2+]c). Immunofluorescence reveals enriched InsP3R-SEC5 complex formation on phagosomes, while disruption of the InsP3R-SEC5 interaction by recombinant H1 peptides attenuates the InsP3R-mediated Ca2+ elevation, leading to impaired phagocytosis. Furthermore, we show that C. albicans infection promotes the recruitment of Tank-binding kinase 1 (TBK1) by the InsP3R-SEC5 interacting complex, leading to the activation of TBK1. Subsequently, activated TBK1 phosphorylates interferon regulatory factor 3 (IRF-3) and mediates type I interferon responses, suggesting that the InsP3R-SEC5 interaction may regulate antifungal innate immune responses not only by elevating cytoplasmic Ca2+ but also by activating the TBK1-IRF-3 pathway.ConclusionsOur data have revealed an important role of the InsP3R-SEC5 interaction in innate immune responses against C. albicans.

Calcium Signaling in Mammalian Eggs at Fertilization

The innovation and development of live-cell fluorescence imaging methods have revealed the dynamic aspects of intracellular Ca2+ in a wide variety of cells. The fertilized egg, the very first cell to be a new individual, has long been under extensive investigations utilizing Ca2+ imaging since its early days, and spatiotemporal Ca2+ dynamics and underlying mechanisms of Ca2+ mobilization, as well as physiological roles of Ca2+ at fertilization, have become more or less evident in various animal species. In this article, we illustrate characteristic patterns of Ca2+ dynamics in mammalian gametes and molecular basis for Ca2+ release from intracellular stores leading to the elevation in cytoplasmic Ca2+ concentration, and describe the identity and properties of sperm-borne egg-activating factor in relation to the induction of Ca2+ waves and Ca2+ oscillations, referring to its potential use in artificial egg activation as infertility treatment. In addition, a possible Ca2+ influx-driven mechanism for slow and long-lasting Ca2+ oscillations characteristic of mammalian eggs is proposed, based on the recent experimental findings and mathematical modeling. Cumulative knowledge about the roles of Ca2+ in the egg activation leading to early embryogenesis is summarized, to emphasize the diversity of functions that Ca2+ can perform in a single type of cell.

Red blood cells with elevated cytoplasmic Ca(2+) are primarily taken up by splenic marginal zone macrophages and CD207+ dendritic cells.

The normal red blood cell (RBC) life span may be significantly reduced when RBCs are stored under blood bank conditions, resulting in a reduced 24-hour survival after transfusion. The damage of stored RBCs is probably multifactorial as stored RBCs share features of both senescence and suicidal RBC death (eryptosis). Since an increased intracellular Ca(2+) concentration ([Ca(2+) ]i ) is one key feature of eryptosis, we here investigated if stored human RBCs had increased [Ca(2+) ]i and the mechanisms behind uptake of such RBCs in a murine model. The intracellular Ca(2+) content of RBCs was determined using the Ca(2+) probe Fluo-3 and flow cytometry. In vivo uptake of Ca(2+) ionophore-treated murine RBCs (Ca(2+) -RBCs) was investigated in recipient mice, using flow cytometry and immunohistochemical analysis. A small fraction of human RBCs accumulated [Ca(2+) ]i during storage for up to 42 days under blood bank conditions. In a murine model, where fresh or Ca(2+) -RBCs were transfused, Ca(2+) -RBCs were mainly trapped by MARCO+ splenic marginal zone macrophages and CD11c+ CD207+ dendritic cells (DCs) within 1 hour after transfusion. In marked contrast, freshly transfused RBCs aging normally in circulation were cleared much slower and preferentially by F4/80+ red pulp macrophages. CD47 on the Ca(2+) -RBCs did not affect their clearance by splenic phagocytic cells. A small fraction of RBCs accumulate [Ca(2+) ]i during storage, and in a murine model such RBCs are recognized by splenic macrophages and DCs in ways similar to what has been reported for nucleated apoptotic cells.

STIM1 activation is regulated by a 14 amino acid sequence adjacent to the CRAC activation domain.

Oligomerization of the Ca2+ sensor, STIM1, in the endoplasmic reticulum (ER) membrane, caused by depletion of ER Ca2+ stores, results in STIM1 coupling to the plasma membrane Ca2+ channel protein, Orai1, to activate Ca2+ influx in a process known as store-operated Ca2+ entry. We use fluorimetry-based fluorescence resonance energy transfer (FRET) to monitor changes in STIM1 oligomerization in COS7 cells transfected with STIM1 constructs containing selected truncations, deletions, and point mutations, and labeled with donor and acceptor fluorescent proteins at either the luminal (N-terminal) or the cytoplasmic (C-terminal) ends. Our results with sequential truncations of STIM1 from the C-terminus support previous evidence that the CRAC activation domain (CAD/SOAR, human sequence 342–448) is an oligomer-promoting segment of STIM1, and they show that truncation just after CAD/SOAR (1–448) causes significantly elevated basal cytoplasmic Ca2+ and spontaneous STIM1 clustering. We find that a 14 amino acid sequence just C-terminal of CAD/SOAR (449–462) prevents spontaneous clustering and activation of STIM1 in COS7 cells. In response to store depletion, C-terminally labeled STIM1 without CAD/SOAR clusters together with CAD/SOAR-containing STIM1 constructs. However, these donor-acceptor pairs do not undergo a stimulated increase in FRET, exhibiting instead a decrease in FRET consistent with a stimulated conformational extension in full length STIM1. We find that the 14 amino acid sequence plays a regulatory role in this process. Overall, our FRET results provide evidence in live cells that Ca2+ store depletion stimulates a conformational extension in the cytoplasmic segment of STIM1 that accompanies its oligomerization.

Panaxydol, a component of Panax ginseng, induces apoptosis in cancer cells through EGFR activation and ER stress and inhibits tumor growth in mouse models.

We reported previously that panaxydol, a component of Panax ginseng roots, induced mitochondria-mediated apoptosis preferentially in transformed cells. This study demonstrates that EGFR activation and the resulting ER stress mediate panaxydol-induced apoptosis, and that panaxydol suppresses in vivo tumor growth in syngeneic and xenogeneic mouse tumor models. In addition, we elucidated that CaMKII and TGF-β-activated kinase (TAK1) participate in p38/JNK activation by elevated cytoplasmic Ca(2+) concentration ([Ca(2+)]c). In MCF-7 cells, EGFR was activated immediately after exposure to panaxydol, and this activation was necessary for induction of apoptosis, suggesting that panaxydol might be a promising anticancer candidate, especially for EGFR-addicted cancer. Activation of PLCγ followed EGFR activation, resulting in Ca(2+) release from the endoplasmic reticulum (ER) via inositol triphosphate and ryanodine receptors. ER Ca(2+) release triggered mitochondrial Ca(2+) uptake indirectly through oxidative stress and ensuing ER stress. Elevated [Ca(2+)]c triggered sequential activation of calmodulin/CaMKII, TAK1 and p38/JNK. As shown previously, p38 and JNK activate NADPH oxidase. Here, it was shown that the resulting oxidative stress triggered ER stress. Among the three signaling branches of the unfolded protein response, protein kinase R-like ER kinase (PERK), but not inositol-requiring enzyme 1 or activating transcription factor 6, played a role in transmitting the apoptosis signal. PERK induced C/EBP homologous protein (CHOP), and CHOP elevated Bim expression, initiating mitochondrial Ca(2+) uptake and apoptosis. In summary, we identified roles of EGFR, the CAMKII-TAK1-p38/JNK pathway, and ER stress in panaxydol-induced apoptosis and demonstrated the in vivo anticancer effect of panaxydol.

Research advances on potential neurotoxicity of quantum dots.

With rapid development of nanotechnology, quantum dots (QDs) as advanced nanotechnology products have been widely used in biological and biomedical studies, including neuroscience, due to their superior optical properties. In recent years, there has been intense concern regarding the toxicity of QDs with a growing number of studies. However, the knowledge of neurotoxic consequences of QDs applied in living organisms is lagging behind their development, while a potential risk of neurotoxicity arises if mass production of QDs leads to increased exposure and distribution in the nervous system. Owing to the quantum size effect of QDs, they are capable of crossing the blood-brain barrier or moving along neural pathways and entering the brain. Nevertheless, the interactions of QDs with cells and tissues in the central nervous system are not well understood. This review highlighted research advances on the neurotoxicity of QDs in the central nervous system, including oxidative stress injury, elevated cytoplasmic Ca(2+) levels and autophagy to damage in vitro neural cells, and impairments of synaptic transmission and plasticity as well as brain functions in tested animals, with the hope of throwing light on future research directions of QD neurotoxicity, which is a demanding topic that requires further exploration.

An alkaline follicular fluid fraction induces capacitation and limited release of oviduct epithelium-bound stallion sperm.

Induction of hyperactivated motility is considered essential for triggering the release of oviduct-bound mammalian spermatozoa in preparation for fertilization. In this study, oviduct-bound stallion spermatozoa were exposed for 2 h to: i) pre-ovulatory and ii) post-ovulatory oviductal fluid; iii) 100% and iv) 10% follicular fluid (FF); v) cumulus cells, vi) mature equine oocytes, vii) capacitating and viii) non-capacitating medium. None of these triggered sperm release or hyperactivated motility. Interestingly, native FF was detrimental to sperm viability, an effect that was negated by heat inactivation, charcoal treatment and 30 kDa filtration alone or in combination. Moreover, sperm suspensions exposed to treated FF at pH 7.9 but not pH 7.4 showed Ca(2+)-dependent hypermotility. Fluo-4 AM staining of sperm showed elevated cytoplasmic Ca(2+) in hyperactivated stallion spermatozoa exposed to treated FF at pH 7.9 compared to a modest response in defined capacitating conditions at pH 7.9 and no response in treated FF at pH 7.4. Moreover, 1 h incubation in alkaline, treated FF induced protein tyrosine phosphorylation in 20% of spermatozoa. None of the conditions tested induced widespread release of sperm pre-bound to oviduct epithelium. However, the hyperactivating conditions did induce release of 70-120 spermatozoa per oviduct explant, of which 48% showed protein tyrosine phosphorylation and all were acrosome-intact, but capable of acrosomal exocytosis in response to calcium ionophore. We conclude that, in the presence of elevated pH and extracellular Ca(2+), a heat-resistant, hydrophilic, <30 kDa component of FF can trigger protein tyrosine phosphorylation, elevated cytoplasmic Ca(2+) and hyperactivated motility in stallion sperm, but infrequent release of sperm pre-bound to oviduct epithelium.

Store-operated calcium entry contributes to abnormal Ca2+ signalling in dystrophic mdx mouse myoblasts

Sarcolemma damage and activation of various calcium channels are implicated in altered Ca2+ homeostasis in muscle fibres of both Duchenne muscular dystrophy (DMD) sufferers and in the mdx mouse model of DMD. Previously we have demonstrated that also in mdx myoblasts extracellular nucleotides trigger elevated cytoplasmic Ca2+ concentrations due to alterations of both ionotropic and metabotropic purinergic receptors. Here we extend these findings to show that the mdx mutation is associated with enhanced store-operated calcium entry (SOCE). Substantially increased rate of SOCE in mdx myoblasts in comparison to that in control cells correlated with significantly elevated STIM1 protein levels. These results reveal that mutation in the dystrophin-encoding Dmd gene may significantly impact cellular calcium response to metabotropic stimulation involving depletion of the intracellular calcium stores followed by activation of the store-operated calcium entry, as early as in undifferentiated myoblasts. These data are in agreement with the increasing number of reports showing that the dystrophic pathology resulting from dystrophin mutations may be developmentally regulated. Moreover, our results showing that aberrant responses to extracellular stimuli may contribute to DMD pathogenesis suggest that treatments inhibiting such responses might alter progression of this lethal disease.

Stage-Dependent Benefits and Risks of Pimobendan in Genetic Dilated Cardiomyopathy Mice with Progressive Heart Failure

The Ca2+ sensitizer pimobendan is a unique inotropic agent that, compared with traditional inotropes, improves cardiac contractility with less oxygen consumption and potentially fewer adverse effects on myocardial remodelling and arrhythmia. However, clinical trials report contradictory effects of pimobendan in heart failure (HF) patients. We provide mechanistic experimental evidence of the efficacy of pimobendan using a novel mouse model of progressive HF.A knock-in mouse model of human genetic dilated cardiomyopathy, which shows a clear transition from compensatory to end-stage HF at a fixed time during growth, was used to evaluate the efficacy of pimobendan and explore the underlying molecular and cellular mechanisms.Pimobendan prevented myocardial remodelling in compensated HF and significantly extended the life span in both compensated and end-stage HF, but dose-dependently increased the sudden death in end-stage HF. In cardiomyocytes isolated from end-stage HF mice, pimobendan induced triggered activity probably due to early or delayed afterdepolarizations via markedly up-regulated electrogenic sodium/calcium exchanger 1 activation. The L-type Ca2+ channel blocker verapamil decreased the incidence of triggered activity, suggesting that this was from overly elevated cytoplasmic Ca2+ through increased Ca2+ entry by phosphodiesterase 3 inhibition under diminished sarcoplasmic reticulum Ca2+ reuptake and increased Ca2+ leakage from sarcoplasmic reticulum in end-stage HF.Pimobendan is beneficial irrespective of HF stage, but increases sudden cardiac death in end-stage HF with extensive remodelling ofCa2+ handling. Reduction of cytoplasmic Ca2+ elevated by phosphodiesterase 3 inhibition may decrease the risk of sudden cardiac death.

Heat stress induces apoptosis through a Ca²⁺-mediated mitochondrial apoptotic pathway in human umbilical vein endothelial cells.

BackgroundHeat stress can be acutely cytotoxic, and heat stress-induced apoptosis is a prominent pathological feature of heat-related illnesses, although the precise mechanisms by which heat stress triggers apoptosis are poorly defined.MethodsThe percentages of viability and cell death were assessed by WST-1 and LDH release assays. Apoptosis was assayed by DNA fragmentation and caspase activity. Expression of cleaved PARP, Apaf-1, phospho-PERK, Phospho-eIF2a, ATF4, XBP-1s, ATF6, GRP78, phospho-IP3R, RYR and SERCA was estimated by Western blot. The effect of calcium overload was determined using flow cytometric analysis with the fluorescent probe Fluo-3/AM. The generation of ROS (O2 −, H2O2, NO) was labeled by confocal laser scanning microscopy images of fluorescently and flow cytometry.ResultsIn this study, we found that heat stress in HUVEC cells activated initiators of three major unfolded protein response (UPR) signaling transduction pathways: PERK-eIF2a-ATF4, IRE1-XBP-1S and ATF6 to protect against ER stress, although activation declined over time following cessation of heat stress. Furthermore, we show that intense heat stress may induce apoptosis in HUVEC cells through the calcium-mediated mitochondrial apoptotic pathway, as indicated by elevation of cytoplasmic Ca2+, expression of Apaf-1, activation of caspase-9 and caspase-3, PARP cleavage, and ultimately nucleosomal DNA fragmentation; Reactive oxygen species (ROS) appear to act upstream in this process. In addition, we provide evidence that IP3R upregulation may promote influx of Ca2+ into the cytoplasm after heat stress.ConclusionOur findings describe a novel mechanism for heat stress-induced apoptosis in HUVEC cells: following elevation of cytoplasm Ca2+, activation of the mitochondrial apoptotic pathway via the IP3R upregulation, with ROS acting as an upstream regulator of the process.

Non-contact High-Frequency Ultrasound Microbeam Stimulation for Studying Mechanotransduction in Human Umbilical Vein Endothelial Cells

We describe how contactless high-frequency ultrasound microbeam stimulation (HFUMS) is capable of eliciting cytoplasmic calcium (Ca2+) elevation in human umbilical vein endothelial cells. The cellular mechanotransduction process, which includes cell sensing and adaptation to the mechanical micro-environment, has been studied extensively in recent years. A variety of tools for mechanical stimulation have been developed to produce cellular responses. We developed a novel tool, a highly focused ultrasound microbeam, for non-contact cell stimulation at a microscale. This tool, at 200 MHz, was applied to human umbilical vein endothelial cells to investigate its potential to elicit an elevation in cytoplasmic Ca2+ levels. It was found that the response was dose dependent, and moreover, extracellular Ca2+ and cytoplasmic Ca2+ stores were involved in the Ca2+ elevation. These results suggest that high-frequency ultrasound microbeam stimulation is potentially a novel non-contact tool for studying cellular mechanotransduction if the acoustic pressures at such high frequencies can be quantified.

Reticulon 4 Is Necessary for Endoplasmic Reticulum Tubulation, STIM1-Orai1 Coupling, and Store-operated Calcium Entry

Despite recent advances in understanding store-operated calcium entry (SOCE) regulation, the fundamental question of how ER morphology affects this process remains unanswered. Here we show that the loss of RTN4, is sufficient to alter ER morphology and severely compromise SOCE. Mechanistically, we show this to be the result of defective STIM1-Orai1 coupling because of loss of ER tubulation and redistribution of STIM1 to ER sheets. As a functional consequence, RTN4-depleted cells fail to sustain elevated cytoplasmic Ca(2+) levels via SOCE and therefor are less susceptible to Ca(2+) overload induced apoptosis. Thus, for the first time, our results show a direct correlation between ER morphology and SOCE and highlight the importance of RTN4 in cellular Ca(2+) homeostasis.

Oligomeric Aβ-induced synaptic dysfunction in Alzheimer's disease.

Alzheimer’s disease (AD) is a devastating disease characterized by synaptic and neuronal loss in the elderly. Compelling evidence suggests that soluble amyloid-β peptide (Aβ) oligomers induce synaptic loss in AD. Aβ-induced synaptic dysfunction is dependent on overstimulation of N-methyl-D-aspartate receptors (NMDARs) resulting in aberrant activation of redox-mediated events as well as elevation of cytoplasmic Ca2+, which in turn triggers downstream pathways involving phospho-tau (p-tau), caspases, Cdk5/dynamin-related protein 1 (Drp1), calcineurin/PP2B, PP2A, Gsk-3β, Fyn, cofilin, and CaMKII and causes endocytosis of AMPA receptors (AMPARs) as well as NMDARs. Dysfunction in these pathways leads to mitochondrial dysfunction, bioenergetic compromise and consequent synaptic dysfunction and loss, impaired long-term potentiation (LTP), and cognitive decline. Evidence also suggests that Aβ may, at least in part, mediate these events by causing an aberrant rise in extrasynaptic glutamate levels by inhibiting glutamate uptake or triggering glutamate release from glial cells. Consequent extrasynaptic NMDAR (eNMDAR) overstimulation then results in synaptic dysfunction via the aforementioned pathways. Consistent with this model of Aβ-induced synaptic loss, Aβ synaptic toxicity can be partially ameliorated by the NMDAR antagonists (such as memantine and NitroMemantine). PSD-95, an important scaffolding protein that regulates synaptic distribution and activity of both NMDA and AMPA receptors, is also functionally disrupted by Aβ. PSD-95 dysregulation is likely an important intermediate step in the pathological cascade of events caused by Aβ. In summary, Aβ-induced synaptic dysfunction is a complicated process involving multiple pathways, components and biological events, and their underlying mechanisms, albeit as yet incompletely understood, may offer hope for new therapeutic avenues.

Lysosomal alkalinization, lipid oxidation, and reduced phagosome clearance triggered by activation of the P2X7 receptor

Lysosomal enzymes function optimally at low pH; as accumulation of waste material contributes to cell aging and disease, dysregulation of lysosomal pH may represent an early step in several pathologies. Here, we demonstrate that stimulation of the P2X7 receptor (P2X7R) for ATP alkalinizes lysosomes in cultured human retinal pigmented epithelial (RPE) cells and impairs lysosomal function. P2X7R stimulation did not kill RPE cells but alkalinized lysosomes by 0.3 U. Receptor stimulation also elevated cytoplasmic Ca(2+); Ca(2+) influx was necessary but not sufficient for lysosomal alkalinization. P2X7R stimulation decreased access to the active site of cathepsin D. Interestingly, lysosomal alkalinization was accompanied by a rise in lipid oxidation that was prevented by P2X7R antagonism. Likewise, the autofluorescence of phagocytosed photoreceptor outer segments increased by lysosomal alkalinization was restored 73% by a P2X7R antagonist. Together, this suggests that endogenous autostimulation of the P2X7R may oxidize lipids and impede clearance. The P2X7R was expressed on apical and basolateral membranes of mouse RPE; mRNA expression of P2X7R and extracellular ATP marker NTPDase1 was raised in RPE tissue from the ABCA4(-/-) mouse model of Stargardt's retinal degeneration. In summary, P2X7R stimulation raises lysosomal pH and impedes lysosomal function, suggesting a possible role for overstimulation in diseases of accumulation.

Effects of CaMKII-Mediated Phosphorylation of Ryanodine Receptor Type 2 on Islet Calcium Handling, Insulin Secretion, and Glucose Tolerance

Altered insulin secretion contributes to the pathogenesis of type 2 diabetes. This alteration is correlated with altered intracellular Ca2+-handling in pancreatic β cells. Insulin secretion is triggered by elevation in cytoplasmic Ca2+ concentration ([Ca2+]cyt) of β cells. This elevation in [Ca2+]cyt leads to activation of Ca2+/calmodulin-dependent protein kinase II (CAMKII), which, in turn, controls multiple aspects of insulin secretion. CaMKII is known to phosphorylate ryanodine receptor 2 (RyR2), an intracellular Ca2+-release channel implicated in Ca2+-dependent steps of insulin secretion. Our data show that RyR2 is CaMKII phosphorylated in a pancreatic β-cell line in a glucose-sensitive manner. However, it is not clear whether any change in CaMKII-mediated phosphorylation underlies abnormal RyR2 function in β cells and whether such a change contributes to alterations in insulin secretion. Therefore, knock-in mice with a mutation in RyR2 that mimics its constitutive CaMKII phosphorylation, RyR2-S2814D, were studied. This mutation led to a gain-of-function defect in RyR2 indicated by increased basal RyR2-mediated Ca2+ leak in islets of these mice. This chronic in vivo defect in RyR2 resulted in basal hyperinsulinemia. In addition, S2814D mice also developed glucose intolerance, impaired glucose-stimulated insulin secretion and lowered [Ca2+]cyt transients, which are hallmarks of pre-diabetes. The glucose-sensitive Ca2+ pool in islets from S2814D mice was also reduced. These observations were supported by immunohistochemical analyses of islets in diabetic human and mouse pancreata that revealed significantly enhanced CaMKII phosphorylation of RyR2 in type 2 diabetes. Together, these studies implicate that the chronic gain-of-function defect in RyR2 due to CaMKII hyperphosphorylation is a novel mechanism that contributes to pathogenesis of type 2 diabetes.

Abstract 58: Modeling Pathogenesis in Familial Hypertrophic Cardiomyopathy Using Patient-Specific Induced Pluripotent Stem Cells

Background: Hypertrophic cardiomyopathy (HCM) is a prevalent familial cardiac disorder linked to development of heart failure, arrhythmia, and sudden cardiac death. Molecular genetic studies have demonstrated HCM is caused by mutations in genes encoding for the cardiac sarcomere. However, the pathways by which sarcomeric mutations result in myocyte hypertrophy and contractile abnormalities are not well understood. Methods: We aimed to elucidate the molecular mechanisms underlying the development of HCM through the generation of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from dermal fibroblasts of a 10 member family, five of whom carry a hereditary HCM missense mutation (Arg663His) in the MYH7 gene. Results: As compared to control iPSC-CMs derived from healthy family members, HCM iPSC-CMs exhibited enlarged cell size, increased atrial natriuretic factor (ANF) expression, nuclear translocation of nuclear factor of activated T-cells (NFAT), and aggravated contractile dysfunction in response to stimulation by β-adrenergic agonists. Interestingly, both video analysis of beating cells and whole cell patch clamping revealed arrhythmia in a significant portion of diseased iPSC-CMs at the single cell level. Ca 2+ imaging demonstrated elevated cytoplasmic Ca 2+ content and irregular transients in HCM iPSC-CMs prior to the onset of cellular hypertrophy, suggesting the HCM phenotype is triggered by dysfunction in Ca 2+ cycling. Treatment of irregular Ca 2+ homeostasis by the Ca 2+ channel blocker verapamil prevented development of cellular hypertrophy and arrhythmia. Conclusions: We hypothesize the cellular abnormalities observed in HCM iPSC-CMs are caused by deficiencies in Ca 2+ regulation. We anticipate our findings will elucidate the mechanisms underlying HCM development and identify novel targets for treatment of the disease.

Regulation of the methylation status of G protein-coupled receptor kinase 1 (rhodopsin kinase)

Rhodopsin kinase (GRK1) is a member of G protein-coupled receptor kinase family and a key enzyme in the quenching of photolysed rhodopsin activity and desensitisation of the rod photoreceptor neurons. Like some other rod proteins involved in phototransduction, GRK1 is posttranslationally modified at the C terminus by isoprenylation (farnesylation), endoproteolysis and α-carboxymethylation. In this study, we examined the potential mechanisms of regulation of GRK1 methylation status, which have remained unexplored so far. We found that considerable fraction of GRK1 is endogenously methylated. In isolated rod outer segments, its methylation is inhibited and demethylation stimulated by low-affinity nucleotide binding. This effect is not specific for ATP and was observed in the presence of a non-hydrolysable ATP analogue AMP-PNP, GTP and other nucleotides, and thus may involve a site distinct from the active site of the kinase. GRK1 demethylation is inhibited in the presence of Ca2+ by recoverin. This inhibition requires recoverin myristoylation and the presence of the membranes, and may be due to changes in GRK1 availability for processing enzymes upon its redistribution to the membranes induced by recoverin/Ca2+. We hypothesise that increased GRK1 methylation in dark-adapted rods due to elevated cytoplasmic Ca2+ levels would further increase its association with the membranes and recoverin, providing a positive feedback to efficiently suppress spurious phosphorylation of non-activated rhodopsin molecules and thus maximise senstivity of the photoreceptor. This study provides the first evidence for dynamic regulation of GRK1 α-carboxymethylation, which might play a role in the regulation of light sensitivity and adaptation in the rod photoreceptors.

Ca2+ signaling and gene transcription in glucose-stimulated insulinoma cells

Glucose stimulation induces expression of the transcription factors Egr-1 and c-Jun as well as phosphorylation of CREB and c-Jun in insulinoma cells. The activator protein-1 (AP-1) binding sites within the c-Jun promoter, the serum response elements (SREs) within the Egr-1 promoter, and cyclic AMP response elements (CREs) function as glucose responsive element. Glucose-induced transcriptional regulation was attenuated by inhibition of L-type Ca2+ channels, or chelating cytoplasmic Ca2+. It has been proposed that a rise in nuclear Ca2+ is required for CREB-mediated transcription of CRE-regulated genes, while elevated cytoplasmic Ca2+ levels trigger an upregulation of SRE-containing genes. Here, we show that a rise in cytoplasmic Ca2+ is required for AP-1, CRE, and SRE mediated transcription in glucose-stimulated insulinoma cells. Buffering Ca2+ in the nucleus or the endoplasmic reticulum had no inhibitory effect upon transcription. However, overexpression of the mitochondrial protein Fis-1 or inhibition of the GTPase Drp-1 impaired glucose-stimulated gene transcription in insulinoma cells, suggesting that the mitochondria play an important role in regulating Ca2+ mediated gene transcription. The extracellular signal-regulated protein kinase functions as an essential link connecting glucose stimulation, the rise in cytoplasmic Ca2+, and enhanced transcription in insulinoma cells.