Role Of Mitochondrial Calcium Uptake Research Articles

Monitoring mitochondrial calcium in cardiomyocytes during coverslip hypoxia using a fluorescent lifetime indicator

An increase in mitochondrial calcium via the mitochondrial calcium uniporter (MCU) has been implicated in initiating cell death in the heart during ischemia-reperfusion (I/R) injury. Measurement of calcium during I/R has been challenging due to the pH sensitivity of indicators coupled with the fall in pH during I/R. The development of a pH-insensitive indicator, mitochondrial localized Turquoise Calcium Fluorescence Lifetime Sensor (mito-TqFLITS), allows for quantifying mitochondrial calcium during I/R via fluorescent lifetime imaging. Mitochondrial calcium was monitored using mito-TqFLITS in neonatal mouse ventricular myocytes (NMVM) isolated from germline MCU-KO mice and MCUfl/fl treated with CRE-recombinase to acutely knockout MCU. To simulate ischemia, a coverslip was placed on a monolayer of NMVMs to prevent access to oxygen and nutrients. Reperfusion was induced by removing the coverslip. Mitochondrial calcium increases threefold during coverslip hypoxia in MCU-WT. There is a significant increase in mitochondrial calcium during coverslip hypoxia in germline MCU-KO, but it is significantly lower than in MCU-WT. We also found that compared to WT, acute MCU-KO resulted in no difference in mitochondrial calcium during coverslip hypoxia and reoxygenation. To determine the role of mitochondrial calcium uptake via MCU in initiating cell death, we used propidium iodide to measure cell death. We found a significant increase in cell death in both the germline MCU-KO and acute MCU-KO, but this was similar to their respective WTs. These data demonstrate the utility of mito-TqFLITS to monitor mitochondrial calcium during simulated I/R and further show that germline loss of MCU attenuates the rise in mitochondrial calcium during ischemia but does not reduce cell death.

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

The debate continues – What is the role of MCU and mitochondrial calcium uptake in the heart?

Since the identification of the mitochondrial calcium uniporter (MCU) in 2011, several studies utilizing genetic models have attempted to decipher the role of mitochondrial calcium uptake in cardiac physiology. Confounding results in various mutant mouse models have led to an ongoing debate regarding the function of MCU in the heart. In this review, we evaluate and discuss the totality of evidence for mitochondrial calcium uptake in the cardiac stress response and highlight recent reports that implicate MCU in the control of homeostatic cardiac metabolism and function. This review concludes with a discussion of current gaps in knowledge and remaining experiments to define how MCU contributes to contractile function, cell death, metabolic regulation, and heart failure progression.

Mitochondrial Calcium Uptake Is Instrumental to Alternative Macrophage Polarization and Phagocytic Activity.

Macrophages are highly plastic and dynamic cells that exert much of their function through phagocytosis. Phagocytosis depends on a coordinated, finely tuned, and compartmentalized regulation of calcium concentrations. We examined the role of mitochondrial calcium uptake and mitochondrial calcium uniporter (MCU) in macrophage polarization and function. In primary cultures of human monocyte-derived macrophages, calcium uptake in mitochondria was instrumental for alternative (M2) macrophage polarization. Mitochondrial calcium uniporter inhibition with KB-R7943 or MCU knockdown, which prevented mitochondrial calcium uptake, reduced M2 polarization, while not affecting classical (M1) polarization. Challenging macrophages with E. coli fragments induced spikes of mitochondrial calcium concentrations, which were prevented by MCU inhibition or silencing. In addition, mitochondria remodelled in M2 macrophages during phagocytosis, especially close to sites of E. coli internalization. Remarkably, inhibition or knockdown of MCU significantly reduced the phagocytic capacity of M2 macrophages. KB-R7943, which also inhibits the membrane sodium/calcium exchanger and Complex I, reduced mitochondria energization and cellular ATP levels, but such effects were not observed with MCU silencing. Therefore, phagocytosis inhibition by MCU knockdown depended on the impaired mitochondrial calcium buffering rather than changes in mitochondrial and cellular energy status. These data uncover a new role for MCU in alternative macrophage polarization and phagocytic activity.

Increased mitochondrial calcium uniporter in adipocytes underlies mitochondrial alterations associated with insulin resistance.

Intracellular calcium influences an array of pathways and affects cellular processes. With the rapidly progressing research investigating the molecular identity and the physiological roles of the mitochondrial calcium uniporter (MCU) complex, we now have the tools to understand the functions of mitochondrial Ca2+ in the regulation of pathophysiological processes. Herein, we describe the role of key MCU complex components in insulin resistance in mouse and human adipose tissue. Adipose tissue gene expression was analyzed from several models of obese and diabetic rodents and in 72 patients with obesity as well as in vitro insulin-resistant adipocytes. Genetic manipulation of MCU activity in 3T3-L1 adipocytes allowed the investigation of the role of mitochondrial calcium uptake. In insulin-resistant adipocytes, mitochondrial calcium uptake increased and several MCU components were upregulated. Similar results were observed in mouse and human visceral adipose tissue (VAT) during the progression of obesity and diabetes. Intriguingly, subcutaneous adipose tissue (SAT) was spared from overt MCU fluctuations. Furthermore, MCU expression returned to physiological levels in VAT of patients after weight loss by bariatric surgery. Genetic manipulation of mitochondrial calcium uptake in 3T3-L1 adipocytes demonstrated that changes in mitochondrial calcium concentration ([Ca2+]mt) can affect mitochondrial metabolism, including oxidative enzyme activity, mitochondrial respiration, membrane potential, and reactive oxygen species formation. Finally, our data suggest a strong relationship between [Ca2+]mt and the release of IL-6 and TNFα in adipocytes. Altered mitochondrial calcium flux in fat cells may play a role in obesity and diabetes and may be associated with the differential metabolic profiles of VAT and SAT.

Characterization of Mitochondrial Calcium Uptake in Skeletal Muscle

Mitochondrial Ca2+ uptake provides fundamental input to the control of oxidative metabolism and contributes to cell survival regulating signaling mechanisms. Early studies of mitochondria isolated from various tissues have demonstrated that the Ca2+ uptake is driven by the membrane potential (ΔΨm), is mediated by an electrogenic uniport, which is completely inhibited by ruthenium red (RuRed) and was attributed to the “Ca2+ uniporter” (mtCU). mtCU has been recently identified as a protein complex constituted by the pore-forming MCU, its dominant-negative form, MCUb, a scaffold, EMRE, and the Ca2+-sensitive regulators, MICU1, MICU2 and MICU3 which determine both the threshold and cooperative activation of the mtCU by Ca2+. Although mtCU is ubiquitously present in the whole body, mitochondria isolated from several tissues displayed a range of Ca2+ current activities through the inner membrane: very low in heart, moderate in liver and kidney, and high in skeletal muscle (SM). This suggests an important role of mitochondrial calcium uptake specifically in SM function. Indeed, calcium homeostasis is crucial for both excitation-contraction (EC) coupling and relaxation. However, mtCU composition and Ca2+ transport activity in SM remains to be characterized. The goal of this project was to fill this void. We evaluated the abundance of each mtCU forming protein and found that both MCU and MICU1 showed a high expression level in SM compared to other tissues. Furthermore, SM mitochondria took up Ca2+ at surprisingly low concentrations and showed a high maximal activity (assessed by both fluorometric measurement of mitochondrial clearance of added Ca2+ and 45Ca2+ sequestration), supporting the hypothesis of a relevant contribution of mitochondria to clearance of sarcoplasmic Ca2+ in SM.

Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals

Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.

Calcium-mediated activation of pyruvate dehydrogenase in severely injured postischemic myocardium.

Indirect evidence suggests that activity of pyruvate dehydrogenase (PDH) influences recovery of the myocardium after transient ischemia. The present study examined the relationship between postischemic injury and activity of PDH and the role of mitochondrial calcium uptake for observed changes in PDH activity. Isovolumically beating isolated rat hearts perfused with erythrocyte-enriched buffer containing glucose, palmitate, and insulin were submitted to either 20 or 35 min of no-flow ischemia. After 20 min of no-flow ischemia, hearts exhibited complete recovery of developed left ventricular pressure (DLVP). The proportion of myocardial PDH in the active state was modestly increased to 38% (compared with 13% in control hearts) without a change in glucose oxidation. In contrast, in hearts subjected to 35 min of no-flow ischemia (which exhibited poor recovery of DLVP), there was marked stimulation of glucose oxidation (+460%; P < 0.01) and pronounced increase in the active fraction of PDH to 72% (P < 0.01). Glycolytic flux was not significantly altered. Ruthenium red (6 microM) completely abolished the activation of PDH and the increase in glucose oxidation. The results indicate that variable stimulation of glucose oxidation during reperfusion is related to different degrees of activation of PDH, which depends on the severity of the ischemic injury. Activation of PDH seems to be mediated by myocardial calcium uptake.

Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis.

Perturbed cellular calcium homeostasis has been implicated in both apoptosis and necrosis, but the role of altered mitochondrial calcium handling in the cell death process is unclear. The temporal ordering of changes in cytoplasmic ([Ca2+]C) and intramitochondrial ([Ca2+]M) calcium levels in relation to mitochondrial reactive oxygen species (ROS) accumulation and membrane depolarization (MD) was examined in cultured neural cells exposed to either an apoptotic (staurosporine; STS) or a necrotic (the toxic aldehyde 4-hydroxynonenal; HNE) insult. STS and HNE each induced an early increase of [Ca2+]C followed by delayed increase of [Ca2+]M. Overexpression of Bcl-2 blocked the elevation of [Ca2+]M and the MD in cells exposed to STS but not in cells exposed to HNE. The cytoplasmic calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red prevented both apoptosis and necrosis. STS and HNE each induced mitochondrial ROS accumulation and MD, which followed the increase of [Ca2+]M. Cyclosporin A prevented both apoptosis and necrosis, indicating critical roles for MD in both forms of cell death. Caspase activation occurred only in cells undergoing apoptosis and preceded increased [Ca2+]M. Collectively, these findings suggest that mitochondrial calcium overload is a critical event in both apoptotic and necrotic cell death.

Postischaemic reperfusion injury in the isolated rat heart: effect of ruthenium red

The aim was to investigate the effect of attenuating mitochondrial calcium uptake with ruthenium red on myocardial function and the resultant necrosis following prolonged ischaemia and reperfusion in isolated rat hearts. Mitochondrial dysfunction, secondary to increased calcium uptake, has been implicated as an important mediator of reperfusion injury in the heart. To examine the role of mitochondrial calcium uptake in mediating ischaemic and reperfusion injury, isolated rat hearts were perfused with ruthenium red (n = 6), a polysaccharide dye which inhibits calcium uptake by mitochondria, and were compared to control perfused hearts (n = 7). After stabilisation, hearts were subjected to 60 min no flow ischaemia, immediately followed by 40 min reperfusion. Hearts were used from male Wistar rats weighing 300-350 g. Cardiac high energy phosphates (ATP, phosphocreatine, inorganic phosphate) and pH were continuously monitored during ischaemia and reperfusion using phosphorus magnetic resonance spectroscopy. Contractility (dP/dT), coronary flow, creatine kinase release, and the time to the onset of ischaemic contracture were also measured. No differences in metabolic abnormalities or time to peak contraction during ischaemia were found between groups, suggesting that ruthenium red does not alter the metabolic consequences of ischaemia. However, upon reperfusion, the following differences in the ruthenium red perfused hearts were observed when compared to control hearts (p less than 0.05): ATP and phosphocreatine recovery were more complete, myocardial contractility was greater, coronary flow was greater, and myocyte necrosis was attenuated. Combined with the known inhibitory effect of ruthenium red on mitochondrial calcium uptake, these data suggest that an important component of myocardial injury following ischaemia and reperfusion in the isolated rat heart is the result of mitochondrial calcium accumulation.

Intestinal mitochondrial calcium uptake during adaptation to dietary calcium restriction.

In order to evaluate the role of mitochondrial calcium uptake in intestinal calcium absorption, the effect of adaptation to dietary calcium deficiency on the in vitro uptake of calcium by isolated duodenal mitochondria was studied. Experiments were performed utilizing 28-day-old cockerels which had received a diet adequate in vitamin D3 and phosphate and containing either 0.08% or 1.20% calcium. Mitochondrial 45Ca uptake from chicks deprived of dietary calcium was not significantly different from controls. These results suggest that increased calcium uptake by intestinal mitochondria is not crucial for adaptation to a low calcium diet.

A Possible regulatory role of mitochondrial calcium uptake in uterine contractions.

It is generally known that mitochondria isolated from a great variety of tissues have the capacity to take up calcium ions efficiently against a concentration gradient (for reviews, see Lehninger et al., 1967; Carafoli, 1973). The transport process receives the necessary energy either directly from coupled electron transport in the respiratory chain, in which case calcium uptake is abolished by respiratory inhibitors but not by oligomycin, or from hydrolysis of adenosine triphosphate (ATP) by the mitochondrial ATPase, in which case transport is unaffected by respiratory chain inhibitors but is inhibited by oligomycin. These relationships are summarized in Fig. 1. The exact nature of the mitochondrial “energy pressure” in equilibrium with the redox reactions of the respiratory chain as well as with the ATP/ADP-Pi system is not known with certainty (for review, see, e.g., Slater, 1971). It has been proposed that mitochondrial calcium transport is catalyzed by a specific carrier situated in the mitochondrial inner membrane. (Lehninger and Carafoli, 1970; Carafoli, 1973). However, the metabolic significance of mitochondrial calcium transport has not been unequivocally established, although it seems evident that it must be involved in the control of the cytoplasmic calcium concentration, a parameter which, in turn, very delicately controls a variety of intracellular activities (see e.g., Rasmussen, 1970).KeywordsCalcium UptakeUterine ContractionCalcium TransportCalcium AccumulationMitochondrial CalciumThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Phosphorylase, calcium, and cyclic AMP in smooth-muscle contraction.

ARTICLESPhosphorylase, calcium, and cyclic AMP in smooth-muscle contractionJ DiamondJ DiamondPublished Online:01 Oct 1973https://doi.org/10.1152/ajplegacy.1973.225.4.930MoreSectionsPDF (2 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformation Cited ByBibliography16 October 2014Glycogen Metabolism in Smooth MuscleGlycogen Metabolism in Smooth MuscleThe direct excitatory effects of cimetidine on the smooth muscle of guinea pig ileumAgents and Actions, Vol. 11, No. 3Effects of Strontium as Substitute for Extracellular Calcium on Contraction and Relaxation of Vascular Smooth MusclePapaverine-Induced Inhibition of Electrical and Mechanical Activity and Calcium Movements of Rat Ileal Smooth Muscle1 June 1980 | Journal of Experimental Biology, Vol. 86, No. 1EFFECTS OF HYDRALAZINE AND VERAPAMIL ON PHOSPHORYLASE ACTIVITY AND GUANOSINE CYCLIC 3′,5′-MONOPHOSPHATE LEVELS IN GUINEA-PIG TAENIA COLI19 July 2012 | British Journal of Pharmacology, Vol. 68, No. 2Cytochemical demonstration of adenylate and guanylate cyclases in vascular smooth muscle of circle of WillisJournal of Neurosurgery, Vol. 49, No. 2Mechanisms of contractile response of cerebral artery to externally-applied fresh bloodJournal of Neurosurgery, Vol. 43, No. 1The role of cyclic nucleotides in central synaptic function21 June 2005The Role of Cyclic Nucleotides in Central Synaptic FunctionEnzymes of glycogen metabolism in developing embryos of a teleostWilhelm Roux's Archives of Developmental Biology, Vol. 177, No. 2Calcium regulation in the uterusPharmacology & Therapeutics. Part B: General and Systematic Pharmacology, Vol. 1, No. 4Response of single nephron glomerular filtration rate to distal nephron microperfusionKidney International, Vol. 6, No. 4Alteration of Contractile Function and Calcium Ion Movements in Vascular Smooth Muscle by Gentamicin and Other Aminoglycoside AntibioticsAntimicrobial Agents and Chemotherapy, Vol. 5, No. 6A Possible Regulatory Role of Mitochondrial Calcium Uptake in Uterine Contractions More from this issue > Volume 225Issue 4October 1973Pages 930-937 Copyright & PermissionsCopyright © 1973 by American Physiological Societyhttps://doi.org/10.1152/ajplegacy.1973.225.4.930PubMed4355181History Published online 1 October 1973 Published in print 1 October 1973 Metrics

The role of mitochondrial calcium uptake in contraction and relaxation of the human myometrium.

Abstract Ca2+ binding by the mitochondrial and microsomal fractions of human myometrium was studied in the presence of ATP. Mitochondria in contrast to microsomes were able to lower the Ca2+ concentration to