Increase In Mitochondrial Ca2 Research Articles

TNFα decreases mitochondrial movement in human airway smooth muscle.

In airway smooth muscle (ASM) cells, excitation-contraction coupling is accomplished via a cascade of events that connect an elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) with cross-bridge attachment and ATP-consuming mechanical work. Excitation-energy coupling is mediated by linkage of the elevation of [Ca2+]cyt to an increase in mitochondrial Ca2+ concentration, which in turn stimulates ATP production. Proximity of mitochondria to the sarcoplasmic reticulum (SR) and plasma membrane is thought to be an important mechanism to facilitate mitochondrial Ca2+ uptake. In this regard, mitochondrial movement in ASM cells may be key in establishing proximity. Mitochondria also move where ATP or Ca2+ buffering is needed. Mitochondrial movement is mediated through interactions with the Miro-Milton molecular complex, which couples mitochondria to kinesin motors at microtubules. We examined mitochondrial movement in human ASM cells and hypothesized that, at basal [Ca2+]cyt levels, mitochondrial movement is necessary to establish proximity of mitochondria to the SR and that, during the transient increase in [Ca2+]cyt induced by agonist stimulation, mitochondrial movement is reduced, thereby promoting transient mitochondrial Ca2+ uptake. We further hypothesized that airway inflammation disrupts basal mitochondrial movement via a reduction in Miro and Milton expression, thereby disrupting the ability of mitochondria to establish proximity to the SR and, thus, reducing transient mitochondrial Ca2+ uptake during agonist activation. The reduced proximity of mitochondria to the SR may affect establishment of transient "hot spots" of higher [Ca2+]cyt at the sites of SR Ca2+ release that are necessary for mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter.

The effect of chronic alcohol consumption on mitochondrial calcium handling in hepatocytes.

The damage to liver mitochondria is universally observed in both humans and animal models after excessive alcohol consumption. Acute alcohol treatment has been shown to stimulate calcium (Ca2+) release from internal stores in hepatocytes. The resultant increase in cytosolic Ca2+ is expected to be accumulated by neighboring mitochondria, which could potentially lead to mitochondrial Ca2+ overload and injury. Our data indicate that total and free mitochondrial matrix Ca2+ levels are, indeed, elevated in hepatocytes isolated from alcohol-fed rats compared with their pair-fed control littermates. In permeabilized hepatocytes, the rates of mitochondrial Ca2+ uptake were substantially increased after chronic alcohol feeding, whereas those of mitochondrial Ca2+ efflux were decreased. The changes in mitochondrial Ca2+ handling could be explained by an up-regulation of the mitochondrial Ca2+ uniporter and loss of a cyclosporin A-sensitive Ca2+ transport pathway. In intact cells, hormone-induced increases in mitochondrial Ca2+ declined at slower rates leading to more prolonged elevations of matrix Ca2+ in the alcohol-fed group compared with controls. Moreover, treatment with submaximal concentrations of Ca2+-mobilizing hormones markedly increased the levels of mitochondrial reactive oxygen species (ROS) in hepatocytes from alcohol-fed rats, but did not affect ROS levels in controls. The changes in mitochondrial Ca2+ handling are expected to buffer and attenuate cytosolic Ca2+ increases induced by acute alcohol exposure or hormone stimulation. However, these alterations in mitochondrial Ca2+ handling may also lead to Ca2+ overload during cytosolic Ca2+ increases, which may stimulate the production of mitochondrial ROS, and thus contribute to alcohol-induced liver injury.

FOXO3a regulates BNIP3 and modulates mitochondrial calcium, dynamics, and function in cardiac stress.

The forkhead box O3a (FOXO3a) transcription factor has been shown to regulate glucose metabolism, muscle atrophy, and cell death in postmitotic cells. Its role in regulation of mitochondrial and myocardial function is not well studied. Based on previous work, we hypothesized that FOXO3a, through BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), modulates mitochondrial morphology and function in heart failure (HF). We modulated the FOXO3a-BNIP3 pathway in normal and phenylephrine (PE)-stressed adult cardiomyocytes (ACM) in vitro and developed a cardiotropic adeno-associated virus serotype 9 encoding dominant-negative FOXO3a (AAV9.dn-FX3a) for gene delivery in a rat model of HF with preserved ejection fraction (HFpEF). We found that FOXO3a upregulates BNIP3 expression in normal and PE-stressed ACM, with subsequent increases in mitochondrial Ca2+, leading to decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis. Whereas dn-FX3a attenuated the increase in BNIP3 expression and its consequences in PE-stressed ACM, AAV9.dn-FX3a delivery in an experimental model of HFpEF decreased BNIP3 expression, reversed adverse left ventricular remodeling, and improved left ventricular systolic and, particularly, diastolic function, with improvements in mitochondrial structure and function. Moreover, AAV9.dn-FX3a restored phospholamban phosphorylation at S16 and enhanced dynamin-related protein 1 phosphorylation at S637. Furthermore, FOXO3a upregulates maladaptive genes involved in mitochondrial apoptosis, autophagy, and cardiac atrophy. We conclude that FOXO3a activation in cardiac stress is maladaptive, in that it modulates Ca2+ cycling, Ca2+ homeostasis, and mitochondrial dynamics and function. Our results suggest an important role of FOXO3a in HF, making it an attractive potential therapeutic target.

Functional roles of MICU1 and MICU2 in mitochondrial Ca2 + uptake

MICU1 and MICU2 are the main regulators of the mitochondrial Ca2+-uniporter (MCU), but their precise functional role is still under debate. We show here that MICU2 behaves as a pure inhibitor of MCU at low cytosolic [Ca2+] ([Ca2+]c), though its effects decrease as [Ca2+]c is increased and disappear above 7μM. Regarding MICU1, studying its effects is more difficult because knockdown of MICU1 leads also to loss of MICU2. However, while knockdown of MICU2 induces only a persistent increase in mitochondrial Ca2+ uptake, knockdown of MICU1 also induces a peculiar use-dependent transient activation of MCU that cannot be attributed to the parallel loss of MICU2. Therefore, MICU1 is endowed with a specific inhibitory effect on MCU at low [Ca2+]c, separate and kinetically different from that of MICU2. On the other hand, we and others have shown previously that MICU1 activates MCU at [Ca2+]c above 2.5μM. Thus, MICU1 has a double role in MCU regulation, inhibitory at low [Ca2+]c and activatory at high [Ca2+]c.

Mitochondrial Dysfunction and Ca2+ Overload Contributes to Hesperidin Induced Paraptosis in Hepatoblastoma Cells, HepG2

Paraptosis is a programmed cell death which is morphologically and biochemically different from apoptosis. In this study, we have investigated the role of Ca(2+) in hesperidin-induced paraptotic cell death in HepG2 cells. Increase in mitochondrial Ca(2+) level was observed in hesperidin treated HepG2 cells but not in normal liver cancer cells. Inhibition of inositol-1,4,5-triphosphate receptor (IP3 R) and ryanodine receptor also block the mitochondrial Ca(2+) accumulation suggesting that the release of Ca(2+) from the endoplasmic reticulum (ER) may probably lead to the increase in mitochondrial Ca(2+) level. Pretreatment with ruthenium red (RuRed), a Ca(2+) uniporter inhibitor inhibited the hesperidin-induced mitochondrial Ca(2+) overload, swelling of mitochondria, and cell death in HepG2 cells. It has also been demonstrated that mitochondrial Ca(2+) influxes act upstream of ROS and mitochondrial superoxide production. The increased ROS production further leads to mitochondrial membrane loss in hesperidin treated HepG2 cells. Taken together our results show that IP3 R and ryanodine receptor mediated release of Ca(2+) from the ER and its subsequent influx through the uniporter into mitochondria contributes to hesperidin-induced paraptosis in HepG2 cells.

Mitochondrial dysfunctions during progression of dystrophic cardiomyopathy

Duchenne muscular dystrophy (DMD) is a progressive muscle disease with severe cardiac complications. It is believed that cellular oxidative stress and augmented Ca2+ signaling drives the development of cardiac pathology. Some mitochondrial and metabolic dysfunctions have also been reported. Here we investigate cellular mechanisms responsible for impaired mitochondrial metabolism in dystrophic cardiomyopathy at early stages of the disease. We employed electrophysiological and imaging techniques to study mitochondrial structure and function in cardiomyocytes from mdx mice, an animal model of DMD. Here we show that mitochondrial matrix was progressively oxidized in myocytes isolated from mdx mice. Moreover, an abrupt increase in workload resulted in significantly more pronounced oxidation of mitochondria in dystrophic cells. Electron micrographs revealed a gradually increased number of damaged mitochondria in mdx myocytes. Degradation in mitochondrial structure was correlated with progressive increase in mitochondrial Ca2+ sequestration and mitochondrial depolarization, despite a substantial and persistent elevation in resting cytosolic sodium levels. Treatment of mdx cells with cyclosporine A, an inhibitor of mitochondrial permeability transition pore (mPTP), shifted both resting and workload-dependent mitochondrial redox state to the levels recorded in control myocytes. It also significantly reduced workload dependent depolarization of mitochondrial membrane in dystrophic cardiomyocytes. Overall, our studies highlight age dependent deterioration of mitochondrial function in dystrophic cardiomyocytes, which seems to be associated with excessive opening of mPTP due to oxidative stress and cellular Ca2+ overload.

Apoptosis caused by excessive mitochondrial albumin uptake in renal cells is initiated by increased mitochondrial calcium concentration

Recent advances in the development of organelle-targeted low affinity Ca2+ sensitive dyes have lead to new understanding of the role of Ca2+ in subcellular compartments. Here we report that the onset of the mitochondrial apoptotic pathway is preceded by an increase in mitochondrial Ca2+. Excessive Ca2+ uptake in renal cells causes apoptosis and is a major contributor to the progression of chronic kidney disease. Primary rat proximal tubule cells (RPTC) were incubated with 10mg/ml lipid-free albumin and monitored with regard to mitochondrial Ca2+ concentration ([Ca2+]mt), mitochondrial membrane potential (Δψm) and mitochondrial accumulation of the apoptotic factor Bax. To monitor changes in [Ca2+]mt we expressed RCaMP1h, a mitochondria targeted genetic Ca2+ indicators in RPTC. [Ca2+]mt started to increase significantly after 5 min of albumin incubation and continued to increase during the observation period. At 30 min it was 20 % higher than during control conditions. The increase in [Ca2+]mt was followed by a significant Δψm depolarization after 45 min, and mitochondrial accumulation of the apoptotic factor Bax, which became significant after 30 min. Low concentration of ouabain (10 nM) protected from depolarization and Bax accumulation by activating a Na+, K+-ATPase/IP3R signalling pathway. Thapsigargin, an inhibitor of the SERCA pump causes apoptosis in all cells but its mechanism of action has been elusive. Here we found that exposure of RPTC to cyclopiazonic acid, an analogue of thapsigargin, caused an increase in mitochondrial Ca2+ that paralleled the increase in cytosolic Ca2+. Conclusion Selective sustained increase of mitochondrial Ca2+ concentration may be a major factor responsible for the onset of the intrinsic apoptotic pathway.

Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

The seleno-organic compound ebselen impairs mitochondrial physiology and induces cell death in AR42J cells

Ebselen is a seleno-organic compound that causes cell death in several cancer cell types. The mechanisms underlying its deleterious effects have not been fully elucidated. In this study, the effects of ebselen (1μM–40μM) on AR42J tumor cells have been examined. Cell viability was studied using AlamarBlue® test. Cell cycle phase determination was carried out by flow cytometry. Changes in intracellular free Ca2+ concentration were followed by fluorimetry analysis of fura-2-loaded cells. Distribution of mitochondria, mitochondrial Ca2+ concentration and mitochondrial membrane potential were monitored by confocal microscopy of cells loaded with Mitotracker Green™ FM, rhod-2 or TMRM respectively. Caspase-3 activity was calculated following the luorogenic substrate ACDEVD-AMC signal with a spectrofluorimeter. Results show that cell viability decreased in the presence of ebselen. An increase in the number of cells in the S-phase of the cell cycle was observed. Ebselen induced a concentration-dependent mobilization of Ca2+ from agonist- and thapsigargin-sensitive Ca2+ pools. Ebselen induced also a transient increase in mitochondrial Ca2+ concentration, a progressive decrease of the mitochondrial membrane potential and a disruption of the mitochondrial network. Finally, a concentration-dependent increase in caspase-3 activity was detected. We conclude that ebselen exerts deleterious actions on the cells that involve the impairment of mitochondrial physiology and the activation of caspase-3-mediated apoptotic pathway.

Cardiac troponin I phosphorylation and the force–length relationship

Cardiac troponin I phosphorylation and the force–length relationship

The role of mitochondrial permeability transition pore in regulating the shedding of the platelet GPIbα ectodomain

Agonists induce platelet activation or apoptosis with concomitant shedding of the platelet receptor glycoprotein Ibα (GPIbα) ectodomain in a disintegrin and metalloproteinase 17 (ADAM17)-dependent mechanism. Mitochondrial permeability transition pore (MPTP) plays a pivotal role in platelet activation or apoptosis. However, its impact on ADAM17-mediated GPIbα ectodomain shedding remains unclear. Here, we aimed to test the hypothesis that MPTP regulates ADAM17-dependent GPIbα ectodomain shedding. We showed that calcium ionophore A23187- or thrombin plus collagen-induced GPIbα ectodomain shedding was partially inhibited by a MPTP inhibitor, and a MPTP potentiator promoted A23187-induced GPIbα cleavage. Furthermore, A23187-induced elevation of mitochondrial Ca2+ levels was blocked by the mitochondrial calcium uniporter (MCU) inhibitor Ru360 or treatment with the membrane-permeable Ca2+ chelator BAPTA-AM. We also demonstrated that an increase in mitochondrial Ca2+ levels triggered MPTP opening, as revealed by the examination of mitochondrial inner transmembrane potential depolarization. In addition, A23187 induced-mitochondrial reactive oxygen species (ROS) generation was blocked by the MPTP inhibitor or by treatment with the mitochondria-targeted ROS scavenger. Lastly, A23187-induced GPIbα shedding was partially blocked by inhibitors of either ROS or calpain, and was completely inhibited when platelets were exposed to both inhibitors. Therefore, these observations indicate that MPTP opening triggered mitochondrial ROS production, which plays an important role in regulating ADAM17-mediated shedding of the GPIbα ectodomain in A23187-treated platelets.

Selective Inhibition by Ethanol of Mitochondrial Calcium Influx Mediated by Uncoupling Protein-2 in Relation to N-Methyl-D-Aspartate Cytotoxicity in Cultured Neurons

BackgroundWe have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca2+ levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca2+ levels in cultured murine neocortical neurons.Methodology/Principal FindingsIn neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca2+ levels determined by Rhod-2 at concentrations of 0.1 to 100 µM. Further addition of 250 mM ethanol significantly inhibited the increase by glutamate and NMDA in Rhod-2 fluorescence, while similarly potent inhibition of the NMDA-induced increase was seen after exposure to ethanol at 50 to 250 mM in cultured neurons. Lentiviral overexpression of UCP2 significantly accelerated the increase by NMDA in Rhod-2 fluorescence in neurons, without affecting Fluo-3 fluorescence for intracellular Ca2+ levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca2+ levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca2+ levels irrespective of UCP2 overexpression. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by ethanol in neurons exposed to glutamate. In HEK293 cells with NMDAR containing GluN2B subunit, more efficient inhibition was similarly induced by ethanol at 50 and 250 mM on the NMDA-induced increase in mitochondrial Ca2+ levels than in those with GluN2A subunit. Decreased protein levels of GluN2B, but not GluN2A, subunit were seen in immunoprecipitates with UCP2 from neurons with brief exposure to ethanol at concentrations over 50 mM.Conclusions/SignificanceEthanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca2+ incorporation and cell death after NMDAR activation in neurons.

Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals

The recent identification of the mitochondrial Ca2+ uniporter gene (Mcu/Ccdc109a) has enabled us to address its role, and that of mitochondrial Ca2+ uptake, in neuronal excitotoxicity. Here we show that exogenously expressed Mcu is mitochondrially localized and increases mitochondrial Ca2+ levels following NMDA receptor activation, leading to increased mitochondrial membrane depolarization and excitotoxic cell death. Knockdown of endogenous Mcu expression reduces NMDA-induced increases in mitochondrial Ca2+, resulting in lower levels of mitochondrial depolarization and resistance to excitotoxicity. Mcu is subject to dynamic regulation as part of an activity-dependent adaptive mechanism that limits mitochondrial Ca2+ overload when cytoplasmic Ca2+ levels are high. Specifically, synaptic activity transcriptionally represses Mcu, via a mechanism involving the nuclear Ca2+ and CaM kinase-mediated induction of Npas4, resulting in the inhibition of NMDA receptor-induced mitochondrial Ca2+ uptake and preventing excitotoxic death. This establishes Mcu and the pathways regulating its expression as important determinants of excitotoxicity, which may represent therapeutic targets for excitotoxic disorders.

Wolfram Syndrome protein, Miner1, regulates sulphydryl redox status, the unfolded protein response, and Ca 2+ homeostasis

Miner1 is a redox-active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1−/− mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca2+ stores, a dramatic increase in mitochondrial Ca2+ load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD+/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti-oxidant N-acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease.

Role of VDAC in coronary endothelial cell dysfunction in type 1 diabetic mice

Endothelial dysfunction is a common feature of diabetic vascular complications. Rarefaction of capillaries in the heart, as a result of coronary endothelial dysfunction, represents one of the most critical mechanisms involved in cardiac ischemia. The voltage‐dependent anion channel (VDAC) plays an important role in the regulation of mitochondrial metabolic function and mitochondria‐mediated apoptosis. The objective of this study is to examine the role of VDAC in coronary endothelial dysfunction in diabetes. Endothelial cells (ECs) were more apoptotic in the left ventricle of diabetic mice and hyperglycemia induced significant increase in mitochondrial Ca2+ concentration and VDAC protein levels in coronary ECs. The expression of VDAC‐shRNA in diabetic MCECs not only decreased the mitochondrial Ca2+ concentration, but also attenuated mitochondrial superoxide anion (O2−) production and the activity of the mitochondrial permeability transition pore opening (an indirect indicator of cell apoptosis) toward control levels. These data suggest that the increased VDAC protein expression in diabetic MCECs leads to the activation of pro‐apoptotic cascade and normalizing VDAC protein level may help to decrease endothelial cell apoptosis, increase capillary density in the heart, and subsequently decrease the incidence of cardiac ischemia in diabetes.This research was supported by grant HL115578.

L-type Ca2+ channel contributes to alterations in mitochondrial calcium handling in the mdx ventricular myocyte

The L-type Ca(2+) channel is the main route for calcium entry into cardiac myocytes, and it is essential for contraction. Alterations in whole cell L-type Ca(2+) channel current and Ca(2+) homeostasis have been implicated in the development of cardiomyopathies. Cytoskeletal proteins can influence whole cell L-type Ca(2+) current and mitochondrial function. Duchenne muscular dystrophy is a fatal X-linked disease that leads to progressive muscle weakness due to the absence of cytoskeletal protein dystrophin. This includes dilated cardiomyopathy, but the mechanisms are not well understood. We sought to identify the effect of alterations in whole cell L-type Ca(2+) channel current on mitochondrial function in the murine model of Duchenne muscular dystrophy (mdx). Activation of the L-type Ca(2+) channel with the dihydropyridine agonist BayK(-) caused a significantly larger increase in cytosolic Ca(2+) in mdx vs. wild-type (wt) ventricular myocytes. Consistent with elevated cytosolic Ca(2+), resting mitochondrial Ca(2+), NADH, and mitochondrial superoxide were significantly greater in mdx vs. wt myocytes. Activation of the channel with BayK(-) caused a further increase in mitochondrial Ca(2+), NADH, and superoxide in mdx myocytes. The ratios of the increases were similar to the ratios recorded in wt myocytes. In mitochondria isolated from 8-wk-old mdx hearts, respiration and mitochondrial electron transport chain complex activity were similar to mitochondria isolated from wt hearts. We conclude that mitochondria function at a higher level of resting calcium in the intact mdx myocyte and activation of the L-type Ca(2+) channel contributes to alterations in calcium handling by the mitochondria. This perturbation may contribute to the development of cardiomyopathy.

Mechanisms involved in postconditioning protection of cardiomyocytes against acute reperfusion injury

Experimental and clinical studies demonstrated that postconditioning confers protection against myocardial ischemia/reperfusion injury. However the underlying cellular mechanisms responsible for the beneficial effect of postconditioning are still poorly understood. The aim of the present study was to examine the role of cytosolic and mitochondrial Ca2+-handling. For this purpose adult rat cardiomyocytes were subjected to simulated in vitro ischemia (glucose-free hypoxia at pH6.4) followed by simulated reperfusion with a normoxic buffer (pH7.4; 2.5mmol/L glucose). Postconditioning, i.e., 2 repetitive cycles of normoxic (5s) and hypoxic (2.5min) superfusion, was applied during the first 5min of reoxygenation. Mitochondrial membrane potential (ΔΨm), cytosolic and mitochondrial Ca2+ concentrations, cytosolic pH and necrosis were analysed applying JC-1, fura-2, fura-2/manganese, BCECF and propidium iodide, respectively. Mitochondrial permeability transition pore (MPTP) opening was detected by calcein release. Hypoxic treatment led to a reduction of ΔΨm, an increase in cytosolic and mitochondrial Ca2+ concentration, and acidification of cardiomyocytes. During the first minutes of reoxygenation, ΔΨm transiently recovered, but irreversibly collapsed after 7min of reoxygenation, which was accompanied by MPTP opening. Simultaneously, mitochondrial Ca2+ increased during reperfusion and cardiomyocytes developed spontaneous cytosolic Ca2+ oscillations and severe contracture followed by necrosis after 25min of reoxygenation. In postconditioned cells, the collapse in ΔΨm as well as the leak of calcein, the increase in mitochondrial Ca2+, cytosolic Ca2+ oscillations, contracture and necrosis were significantly reduced. Furthermore postconditioning delayed cardiomyocyte pH recovery. Postconditioning by hypoxia/reoxygenation was as protective as treatment with cyclosporine A. Combining cyclosporine A and postconditioning had no additive effect. The data of the present study demonstrate that postconditioning by hypoxia/reoxygenation prevents reperfusion injury by limiting mitochondrial Ca2+ load and thus opening of the MPTP in isolated cardiomyocytes. These effects seem to be supported by postconditioning-induced delay in pH recovery and suppression of Ca2+ oscillations. This article is part of a Special Issue entitled “Calcium Signaling in Heart”.

GTPase of the immune-associated nucleotide-binding protein 5 (GIMAP5) regulates calcium influx in T-lymphocytes by promoting mitochondrial calcium accumulation

Mature T-lymphocytes undergo spontaneous apoptosis in the biobreeding diabetes-prone strain of rats due to the loss of the functional GIMAP5 (GTPase of the immune-associated nucleotide-binding protein 5) protein. The mechanisms underlying the pro-survival function of GIMAP5in T-cells have not yet been elucidated. We have previously shown that GIMAP5 deficiency in T-cells impairs Ca2+ entry via plasma membrane channels following exposure to thapsigargin or stimulation of the T-cell antigen receptor. In the present study we report that this reduced Ca2+ influx in GIMAP5-deficient T-cells is associated with the inability of their mitochondria to sequester Ca2+ following capacitative entry, which is required for sustained Ca2+ influx via the plasma membrane channels. Consistent with a role for GIMAP5in regulating mitochondrial Ca2+, overexpression of GIMAP5in HEK (human embryonic kidney)-293 cells resulted in increased Ca2+ accumulation within the mitochondria. Disruption of microtubules, but not the actin cytoskeleton, abrogated mitochondrial Ca2+ sequestration in primary rat T-cells, whereas both microtubules and actin cytoskeleton were needed for the GIMAP5-mediated increase in mitochondrial Ca2+ in HEK-293 cells. Moreover, GIMAP5 showed partial colocalization with tubulin in HEK-293 cells. On the basis of these findings, we propose that the pro-survival function of GIMAP5 in T-lymphocytes may be linked to its requirement to facilitate microtubule-dependent mitochondrial buffering of Ca2+ following capacitative entry.

The cocoa flavanol (−)‐epicatechin exerts its cardioprotective effects by protecting myocardial bioenergetics

Targeting the mitochondria during ischemia reperfusion (IR) may confer cardioprotection and lead to improved clinical outcomes. This study tested the potential of the cocoa flavanol (−)‐epicatechin (EPI) to exert cardioprotection during IR via modulation of mitochondrial function. Ischemia was induced in rats via a 45 min occlusion. Reperfusion was allowed for 1h, 48h, or 3 wks. EPI (10 mg/kg) was administered IV 15 min prior to reperfusion for the single dose group and again 12h later for the double dose group. Controls received water. A single dose of EPI significantly reduced infarct size by 27% and 28% at 48h and 3wk, respectively, compared to controls. Double EPI treatment further decreased infarct size at 48h (80% reduction), which was sustained at 3 wk (52% reduction). In order to assess if this cardioprotection was mediated by protection of mitochondrial function, mitochondria were isolated from the left ventricle of sham, IR, and IR+EPI animals 1h after ischemia. IR animals had a significant decrease in mitochondrial O2 consumption, significant increase in mitochondrial Ca2+ levels, and decreased ATP and NADH pools. Epicatechin protected against these changes and had levels similar to sham animals. Taken together, the results suggest that EPI appears to enhance myocardial bioenergetics to confer cardioprotection. This study addresses an important topic related to the identification of mechanisms of action of an inexpensive, yet apparently effective dietary supplement for the potential treatment of cardiovascular disease. This work was supported by NIH 1R24DK092154 and Cardero Therapeutics.

Miner1, mutated in Wolfram Syndrome, is an endoplasmic reticulum protein that regulates cellular redox status and Ca2+ homeostasis

Miner1 (Cisd2) is a redox‐active 2Fe2S cluster protein linked to the development of Wolfram Syndrome, an untreatable disease associated with diabetes, blindness, and deafness. Using mouse embryonic fibroblasts derived from Miner1 deficient mice, we demonstrate that loss of Miner1 leads to endoplasmic reticular (ER) stress and the induction of the unfolded protein response. Miner1 has a profound effect on Ca2+ homeostasis, leading to a depletion of ER Ca2+ stores and an increase in mitochondrial Ca2+ load. These findings were associated with an increased rate of ATP utilization in intact cells, yet a slower growth rate, suggesting futile energy cycling. Furthermore, we observed ultrastructural alterations in the mitochondria lacking Miner1, and an increase in maximal O2 consumption without a change in the level of mitochondrial uncoupling. The cellular pyridine nucleotide and sulfhydryl redox status were shifted to a more oxidized state. Treatment with the antioxidant N‐acetylcysteine reversed most of the abnormalities in the Miner1 deficient cells. Thus, we establish Miner1 as a protein that regulates the redox status of the cell, and propose that Wolfram Syndrome is fundamentally a disease resulting from oxidative stress. Supported by the American Diabetes Assoc 1‐08‐RA‐139, and NIH 4R42DK081298.