Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca²⁺. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer''s and Parkinson diseases. One key regulator that underlies cell survival and Ca²⁺ homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca²⁺ dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca²⁺ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca²⁺ through the InsP3 receptor (InsP3R). The Ca²⁺ efflux in IRE1α-deficient cells correlates with dissociation of the Ca²⁺-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca²⁺ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca²⁺ dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca²⁺ influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca²⁺ homeostasis and cell survival during ER stress and reveal a previously unknown Ca²⁺-mediated cell death signaling between the IRE1α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.     

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

Glycogen synthase kinase-3β (GSK3β) is a multifunctional kinase whose inhibition is known to limit myocardial ischemia–reperfusion injury. However, the mechanism mediating this beneficial effect still remains unclear. Mitochondria and sarco/endoplasmic reticulum (SR/ER) are key players in cell death signaling. Their involvement in myocardial ischemia–reperfusion injury has gained recognition recently, but the underlying mechanisms are not yet well understood. We questioned here whether GSK3β might have a role in the Ca²⁺ transfer from SR/ER to mitochondria at reperfusion. We showed that a fraction of GSK3β protein is localized to the SR/ER and mitochondria-associated ER membranes (MAMs) in the heart, and that GSK3β specifically interacted with the inositol 1,4,5-trisphosphate receptors (IP₃Rs) Ca²⁺ channeling complex in MAMs. We demonstrated that both pharmacological and genetic inhibition of GSK3β decreased protein interaction of IP₃R with the Ca²⁺ channeling complex, impaired SR/ER Ca²⁺ release and reduced the histamine-stimulated Ca²⁺ exchange between SR/ER and mitochondria in cardiomyocytes. During hypoxia reoxygenation, cell death is associated with an increase of GSK3β activity and IP₃R phosphorylation, which leads to enhanced transfer of Ca²⁺ from SR/ER to mitochondria. Inhibition of GSK3β at reperfusion reduced both IP₃R phosphorylation and SR/ER Ca²⁺ release, which consequently diminished both cytosolic and mitochondrial Ca²⁺ concentrations, as well as sensitivity to apoptosis. We conclude that inhibition of GSK3β at reperfusion diminishes Ca²⁺ leak from IP₃R at MAMs in the heart, which limits both cytosolic and mitochondrial Ca²⁺ overload and subsequent cell death.Glycogen synthase kinase-3 (GSK3) was originally identified as a phosphorylating kinase for glycogen synthase.^{1, 2} It has two isoforms, α and β, that possess strong homology in their kinase domains with, however, distinct functions.³ GSK3 is constitutively active but it can be inhibited by phosphorylation on serine 21 (Ser21) for GSK3α and Ser9 for GSK3β.⁴ In the heart, GSK3β has several important roles in cardiac hypertrophy⁵ and ischemia–reperfusion (IR) injury.⁶ Accumulating evidence indicates that phospho-Ser9-GSK3β-mediated cytoprotection is achieved by an increased threshold for permeability transition pore (PTP) opening.^{6, 7, 8, 9} The mechanism by which GSK3β delays PTP opening still remains unclear. It has been reported that GSK3β could interact with ANT at the inner mitochondrial membrane in the heart⁹ and/or to phosphorylate voltage-dependent anion channel (VDAC) and cyclophilin D (CypD) in cancer cells.^{10, 11} GSK3β also has other proposed mechanisms of action, including a poorly characterized role in calcium (Ca²⁺) homeostasis regulation¹² and protein–protein interactions,⁹ as well as functions in different subcellular fractions such as the nucleus, cytosol and mitochondria.¹³Reperfusion is the most powerful intervention to salvage ischemic myocardium. However, it can also paradoxically lead to cardiomyocyte injury and death.¹⁴ One of the main actors of this lethal reperfusion injury is cellular Ca²⁺ overload,¹⁵ which results in part from excessive sarco/endoplasmic reticulum (SR/ER) Ca²⁺ release and Ca²⁺ influx through the plasma membrane (e.g. through L-type Ca²⁺channel and NCX (sodium-calcium exchanger)).¹⁶ Although ryanodine receptors (RyRs) are the major cardiac SR/ER Ca²⁺-release channels involved in excitation–contraction coupling (ECC)¹⁷ and ischemia–reperfusion (IR) injury,¹⁸ recent studies reported an increasing role for inositol 1,4,5-trisphosphate receptors (IP₃Rs) Ca²⁺-release channels in the modulation of ECC and cell death.^{19, 20} Ca²⁺-handling proteins of ER and mitochondria are highly concentrated at mitochondria-associated ER membranes (MAMs), providing a direct and proper mitochondrial Ca²⁺ signaling, including VDAC, Grp75 and IP₃R1.^{20, 21, 22}Here, we provide evidence that, following IR, a fraction of cellular GSK3β is localized at the SR/ER and MAMs. At the MAMs interface, GSK3β can specifically interact and regulate the protein composition of the IP₃R Ca²⁺ channeling complex and modulate Ca²⁺ transfer between SR/ER and mitochondria. These findings support a novel mechanism of action of GSK3β in cell death process during reperfusion injury.     

Guanine Nucleotides in the Meiotic Maturation of Starfish Oocytes: Regulation of the Actin Cytoskeleton and of Ca2+ Signaling

Background

Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca²⁺ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca²⁺ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation.

Methodology/Principal Findings

By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca²⁺ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca²⁺; iii) GDPβS has little effect on the amplitude of the Ca²⁺ peak, but significantly expedites the initial Ca²⁺ waves induced by InsP₃ photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca²⁺ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca²⁺ signaling.

Conclusions/Significance

Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca²⁺ release.     

BAX inhibitor-1 is a Ca2+ channel critically important for immune cell function and survival

The endoplasmic reticulum (ER) serves as the major intracellular Ca²⁺ store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca²⁺ leak channel also implicated in the response against protein misfolding, thereby connecting the Ca²⁺ store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca²⁺ levels. When put into culture, purified knockout T cells and even more so B cells die spontaneously. This is preceded by increased activity of the mitochondrial initiator caspase-9 and correlated with a significant surge in mitochondrial Ca²⁺ levels, suggesting an exhausted mitochondrial Ca²⁺ buffer capacity as the underlying cause for cell death in vitro. In vivo, T-cell-dependent experimental autoimmune encephalomyelitis and B-cell-dependent antibody production are attenuated, corroborating the ex vivo results. These results suggest that BI-1 has a major role in the functioning of the adaptive immune system by regulating intracellular Ca²⁺ homeostasis in lymphocytes.The endoplasmic reticulum (ER) serves as the major intracellular calcium (Ca²⁺) store, the release of which controls a vast array of cellular functions from short-term responses such as contraction and secretion to long-term regulation of cell growth and proliferation.¹ Dysregulated release of ER Ca²⁺, in contrast, initiates programmed cell death by several mechanisms including mitochondrial Ca²⁺ overload, depolarization, ATP loss and cytochrome c release.² Besides this, the ER also has a key role in the synthesis, folding and sorting of proteins destined for the secretory pathway. The deleterious consequences of an increase in unfolded proteins is called ER stress and can be antagonized by the unfolded protein response (UPR), a mechanism that coordinates a simultaneous increase in the ER folding capacity and a decrease in folding load. In the case of insufficient adaptation to ER stress, cells undergo apoptosis.³BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is an evolutionarily conserved protein that bridges both the Ca²⁺ homeostasis and UPR functions of the ER.⁴ BI-1 was first identified in a screen for human proteins capable of inhibiting BAX-mediated cell death in yeast.⁵ In mammalian cells, BI-1''s antiapoptotic function is most pronounced in paradigms of ER stress⁶ and involves changes in the amount of Ca²⁺ that can be released from intracellular stores.^{6, 7} BI-1 is a highly hydrophobic protein that forms a Ca²⁺ pore responsible for its Ca²⁺ leak properties⁸ and is the founding member of a family of six proteins with similar properties.⁹ The increase in the ER Ca²⁺ leak mediated by BI-1 is blocked at a more acidic pH¹⁰ – a function recently corroborated by a structural analysis of a bacterial homolog of BI-1.¹¹Despite its evolutionarily conserved role in important functions such as ER stress and Ca²⁺ regulation, bi-1−/− mice were reported to have no phenotypic abnormalities but increased infarct volumes in a stroke model, and increased sensitivity to tunicamycin-induced kidney toxicity.⁶ Moreover, livers from BI-1-deficient mice regenerate faster than those from wild-type (WT) mice and this correlates with increased nuclear translocation of nuclear factor of activated T cells (NFATs)¹², a Ca²⁺-dependent process. BI-1 knockout (KO) mice also express more of the spliced form of X-box-binding protein-1 (sXBP-1) in their liver and kidney,¹³ which is generated by the endoribonuclease activity of inositol requiring enzyme 1 (IRE1), and is considered an indicator of increased UPR activity. This was later reproduced and attributed to an inhibitory function of BI-1 on IRE1α mediated via a direct interaction of the two proteins.¹⁴In our study, we found that bi-1−/− mice are more obese and suffer from leukopenia. T and B cells from these mice show significant changes in cellular Ca²⁺ homeostasis and dynamics, and are more prone to spontaneous death in culture but, surprisingly, demonstrate no signs of ongoing ER stress within the homeostatic system of the living animal. These changes lead to an attenuated functioning of the adaptive immune system in vivo. Our results suggest that a major role of BI-1 in vivo involves its effects on the intracellular Ca²⁺ homeostasis in lymphocytes in line with its function as an ER Ca²⁺ leak channel.     

Frog α- and β-Ryanodine Receptors Provide Distinct Intracellular Ca2+ Signals in a Myogenic Cell Line

Background

In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, α-RyR and β-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/Principal Findings

In this study, we expressed either or both α-RyR and β-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that α-RyR and β-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca²⁺ imaging studies, each isoform exhibited caffeine-induced Ca²⁺ transients, an indicative of Ca²⁺-induced Ca²⁺ release (CICR). However, the fashion of Ca²⁺ release events was fundamentally different: α-RyR mediated graded and sustained Ca²⁺ release observed uniformly throughout the cytoplasm, whereas β-RyR supported all-or-none type regenerative Ca²⁺ oscillations and waves. α-RyR but not β-RyR exhibited Ca²⁺ transients triggered by membrane depolarization with high [K⁺]_o that were nifedipine-sensitive, indicating that only α-RyR mediates depolarization-induced Ca²⁺ release. Myotubes co-expressing α-RyR and β-RyR demonstrated high [K⁺]_o-induced Ca²⁺ transients which were indistinguishable from those with myotubes expressing α-RyR alone. Furthermore, procaine did not affect the peak height of high [K⁺]_o-induced Ca²⁺ transients, suggesting minor amplification of Ca²⁺ release by β-RyR via CICR in 1B5 myotubes.

Conclusions/Significance

These findings suggest that α-RyR and β-RyR provide distinct intracellular Ca²⁺ signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.     

Filtering of calcium transients by the endoplasmic reticulum in pancreatic beta-cells

Calcium handling in pancreatic β-cells is important for intracellular signaling, the control of electrical activity, and insulin secretion. The endoplasmic reticulum (ER) is a key organelle involved in the storage and release of intracellular Ca²⁺. Using mathematical modeling, we analyze the filtering properties of the ER and clarify the dual role that it plays as both a Ca²⁺ source and a Ca²⁺ sink. We demonstrate that recent time-dependent data on the free Ca²⁺ concentration in pancreatic islets and β-cell clusters can be explained with a model that uses a passive ER that takes up Ca²⁺ when the cell is depolarized and the cytosolic Ca²⁺ concentration is elevated, and releases Ca²⁺ when the cell is repolarized and the cytosolic Ca²⁺ is at a lower concentration. We find that Ca²⁺-induced Ca²⁺ release is not necessary to explain the data, and indeed the model is inconsistent with the data if Ca²⁺-induced Ca²⁺ release is a dominating factor. Finally, we show that a three-compartment model that includes a subspace compartment between the ER and the plasma membrane provides the best agreement with the experimental Ca²⁺ data.     

A Highlights from MBoC Selection: Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress–induced cell death during the unfolded protein response

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress–induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop^−/− cells are partially resistant to ER stress–induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK''s down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress–induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a “two-hit” model of ER stress–induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.     

Heat-Shock-Induced Changes in Intracellular Ca2+ Level in Tobacco Seedlings in Relation to Thermotolerance

Exposure of plants to elevated temperatures results in a complex set of changes in gene expression that induce thermotolerance and improve cellular survival to subsequent stress. Pretreatment of young tobacco (Nicotiana plumbaginifolia) seedlings with Ca²⁺ or ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid enhanced or diminished subsequent thermotolerance, respectively, compared with untreated seedlings, suggesting a possible involvement of cytosolic Ca²⁺ in heat-shock (HS) signal transduction. Using tobacco seedlings transformed with the Ca²⁺-sensitive, luminescent protein aequorin, we observed that HS temperatures induced prolonged but transient increases in cytoplasmic but not chloroplastic Ca²⁺. A single HS initiated a refractory period in which additional HS signals failed to increase cytosolic Ca²⁺. However, throughout this refractory period, seedlings responded to mechanical stimulation or cold shock with cytosolic Ca²⁺ increases similar to untreated controls. These observations suggest that there may be specific pools of cytosolic Ca²⁺ mobilized by heat treatments or that the refractory period results from a temporary block in HS perception or transduction. Use of inhibitors suggests that HS mobilizes cytosolic Ca²⁺ from both intracellular and extracellular sources.     

Maintenance of Calcium Homeostasis in the Endoplasmic Reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca²⁺) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca²⁺ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca²⁺-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca²⁺ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca²⁺ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca²⁺ pool in untreated cells when extracellular Ca²⁺ is low. However, low extracellular Ca²⁺ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca²⁺ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca²⁺ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.     

Strontium-Induced Repetitive Calcium Spikes in a Unicellular Green Alga

The divalent cation Sr²⁺ induced repetitive transient spikes of the cytosolic Ca²⁺ activity [Ca²⁺]_cy and parallel repetitive transient hyperpolarizations of the plasma membrane in the unicellular green alga Eremosphaera viridis. [Ca²⁺]_cy measurements, membrane potential measurements, and cation analysis of the cells were used to elucidate the mechanism of Sr²⁺-induced [Ca²⁺]_cy oscillations. Sr²⁺ was effectively and rapidly compartmentalized within the cell, probably into the vacuole. The [Ca²⁺]_cy oscillations cause membrane potential oscillations, and not the reverse. The endoplasmic reticulum (ER) Ca²⁺-ATPase blockers 2,5-di-tert-butylhydroquinone and cyclopiazonic acid inhibited Sr²⁺-induced repetitive [Ca²⁺]_cy spikes, whereas the compartmentalization of Sr²⁺ was not influenced. A repetitive Ca²⁺ release and Ca²⁺ re-uptake by the ER probably generated repetitive [Ca²⁺]_cy spikes in E. viridis in the presence of Sr²⁺. The inhibitory effect of ruthenium red and ryanodine indicated that the Sr²⁺-induced Ca²⁺ release from the ER was mediated by a ryanodine/cyclic ADP-ribose type of Ca²⁺ channel. The blockage of Sr²⁺-induced repetitive [Ca²⁺]_cy spikes by La³⁺ or Gd³⁺ indicated the necessity of a certain influx of divalent cations for sustained [Ca²⁺]_cy oscillations. Based on these data we present a mathematical model that describes the baseline spiking [Ca²⁺]_cy oscillations in E. viridis.     

Cardiomyocyte Contractile Dysfunction in the APPswe/PS1dE9 Mouse Model of Alzheimer's Disease

Objectives

Ample clinical and experimental evidence indicated that patients with Alzheimer''s disease display a high incidence of cardiovascular events. This study was designed to examine myocardial histology, cardiomyocyte shortening, intracellular Ca²⁺ homeostasis and regulatory proteins, electrocardiogram, adrenergic response, endoplasmic reticulum (ER) stress and protein carbonyl formation in C57 wild-type (WT) mice and an APPswe/PS1dE9 transgenic (APP/PS1) model for Alzheimer''s disease.

Methods

Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR), maximal velocity of shortening and relengthening (±dL/dt), intracellular Ca²⁺ transient rise and decay.

Results

Little histological changes were observed in APP/PS1 myocardium. Cardiomyocytes from APP/PS1 but not APP or PS1 single mutation mice exhibited depressed PS, reduced±dL/dt, normal TPS and TR compared with WT mice_. Rise in intracellular Ca²⁺ was lower accompanied by unchanged resting/peak intracellular Ca²⁺ levels and intracellular Ca²⁺ decay in APP/PS1 mice. Cardiomyocytes from APP/PS1 mice exhibited a steeper decline in PS at high frequencies. The responsiveness to adrenergic agonists was dampened although β₁-adrenergic receptor expression was unchanged in APP/PS1 hearts. Expression of the Ca²⁺ regulatory protein phospholamban and protein carbonyl formation were downregulated and elevated, respectively, associated with unchanged SERCA2a, Na⁺-Ca²⁺ exchanger and ER stress markers in APP/PS1 hearts. Our further study revealed that antioxidant N-acetylcysteine attenuated the contractile dysfunction in APP/PS1 mice.

Conclusions

Our results depicted overt cardiomyocyte mechanical dysfunction in the APP/PS1 Alzheimer''s disease model, possibly due to oxidative stress.     

Interaction between Age and Obesity on Cardiomyocyte Contractile Function: Role of Leptin and Stress Signaling

Objectives

This study was designed to evaluate the interaction between aging and obesity on cardiac contractile and intracellular Ca²⁺ properties.

Methods

Cardiomyocytes from young (4-mo) and aging (12- and 18-mo) male lean and the leptin deficient ob/ob obese mice were treated with leptin (0.5, 1.0 and 50 nM) for 4 hrs in vitro. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obesity models at young and older age were used for comparison. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), intracellular Ca²⁺ levels and decay. O₂ ⁻ levels were measured by dihydroethidium fluorescence.

Results

Our results revealed reduced survival in ob/ob mice. Aging and obesity reduced PS, ± dL/dt, intracellular Ca²⁺ rise, prolonged TR₉₀ and intracellular Ca²⁺ decay, enhanced O₂ ⁻ production and p ^47phox expression without an additive effect of the two, with the exception of intracellular Ca²⁺ rise. Western blot analysis exhibited reduced Ob-R expression and STAT-3 phosphorylation in both young and aging ob/ob mice, which was restored by leptin. Aging and obesity reduced phosphorylation of Akt, eNOS and p38 while promoting pJNK and pIκB. Low levels of leptin reconciled contractile, intracellular Ca²⁺ and cell signaling defects as well as O₂ ⁻ production and p ^47phox upregulation in young but not aging ob/ob mice. High level of leptin (50 nM) compromised contractile and intracellular Ca²⁺ response as well as O₂ ⁻ production and stress signaling in all groups. High fat diet-induced and db/db obesity displayed somewhat comparable aging-induced mechanical but not leptin response.

Conclusions

Taken together, our data suggest that aging and obesity compromise cardiac contractile function possibly via phosphorylation of Akt, eNOS and stress signaling-associated O₂ ⁻ release.     

Calcium Flux between the Endoplasmic Reticulum and Mitochondrion Contributes to Poliovirus-Induced Apoptosis

We show that poliovirus (PV) infection induces an increase in cytosolic calcium (Ca²⁺) concentration in neuroblastoma IMR5 cells, at least partly through Ca²⁺ release from the endoplasmic reticulum lumen via the inositol 1,4,5-triphosphate receptor (IP₃R) and ryanodine receptor (RyR) channels. This leads to Ca²⁺ accumulation in mitochondria through the mitochondrial Ca²⁺ uniporter and the voltage-dependent anion channel (VDAC). This increase in mitochondrial Ca²⁺ concentration in PV-infected cells leads to mitochondrial dysfunction and apoptosis.Poliovirus (PV), the prototype member of the Picornaviridae family, is the etiological agent of paralytic poliomyelitis (26, 27). This acute human disease of the central nervous system results from the destruction of motor neurons associated with PV replication. In PV-infected mice, motor neurons die through apoptosis (16). However, the mechanisms involved are poorly understood (5).Apoptosis is an active cell death process triggered by various stimuli, including viral infections (18). This process leads to DNA fragmentation and is triggered by two main pathways (22): (i) the extrinsic pathway, mediated by the activation of cell surface death receptors such as Fas/CD95, and (ii) the intrinsic pathway, characterized notably by mitochondrial membrane permeabilization (MMP). In many models, this process implies a loss of mitochondrial transmembrane potential (Δψ_m) and the release of proapoptotic molecules, including cytochrome c, from the mitochondrial intermembrane space into the cytosol. The apoptotic program initiated by PV infection has been shown to involve mitochondrial dysfunction in several cell lines (2-4, 17).The intrinsic pathway also can originate from the endoplasmic reticulum (ER) (30). The ER participates in protein synthesis and folding, cellular responses to stress, and intracellular calcium (Ca²⁺) homeostasis. Nevertheless, under stress conditions, it may induce apoptosis via several different mechanisms, one of which involves ER cross-talk with mitochondria, mediated by Ca²⁺ release from ER stores through the inositol 1,4,5-triphosphate receptor (IP₃R) and ryanodine receptor (RyR) channels (7, 12, 15). Several recent studies have identified Ca²⁺ signaling as a key cellular target for viral infection (for a review, see reference 8). Upon PV infection, cells display an increase in cytosolic Ca²⁺ concentration (20). Phospholipase C also is activated, leading to an increase in IP₃ concentration in PV-infected cells (19), potentially accounting for the observed increase in cytosolic Ca²⁺ concentration. However, the role of Ca²⁺ efflux from the ER in PV-induced apoptosis has yet to be studied.Here, we postulated that an increase in cytosolic Ca²⁺ following PV infection can have an impact on cell fate and investigated the cellular response in terms of mitochondrial function and apoptosis in neuroblastoma IMR5 cells.