Mitochondrial Calcium Regulation Research Articles

Transcriptome analysis and gene expression profiles of early apoptosis-related genes in Streptococcus pyogenes-infected epithelial cells.

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and GAS infection of epithelial cells has been suggested to induce apoptosis. We previously reported that the induction of apoptosis is strongly associated with the protein F1-mediated invasion. We present here the gene expression profiles of the human epithelial HEp-2 cells during GAS-induced apoptosis, using serial gene analysis of expression (SAGE) analysis and macroarray analysis of apoptosis-related genes. Serial gene analysis of expression revealed the downregulation of voltage-dependent anion channels 1 and 2 genes and the upregulation of the cytochrome c oxidase and calcium binding protein genes (calpactin, calgizzarin and programmed cell death 6). Macroarray analysis and quantitative RT-PCR analysis also revealed that the genes for IL-1beta, IL-12 p35, IL12 p40, and GM-CSF are also markedly induced by GAS invasion. Furthermore, caspase-1, -9, and -14 genes are significantly upregulated during GAS invasion. These observations indicated that apoptosis associated with GAS invasion is mainly induced by mitochondrial dysfunction and calcium regulation as well as by stress, and that these transcriptional controls may regulate the cellular response to GAS invasion.

Mechanism of estrogen-mediated neuroprotection: regulation of mitochondrial calcium and Bcl-2 expression.

Estrogens are neuroprotective against glutamate excitotoxicity caused by an excessive rise in intracellular calcium ([Ca(2+)](i)). In this study, we demonstrate that 17 beta-estradiol (E(2)) treatment of hippocampal neurons attenuated the excitotoxic glutamate-induced rise in bulk-free [Ca(2+)](i) despite potentiating the influx of Ca(2+) induced by glutamate. E(2)-induced attenuation of bulk-free [Ca(2+)](i) depends on mitochondrial sequestration of Ca(2+), which is blocked in the presence of the combination of rotenone and oligomycin or in the presence of antimycin, which collapse the mitochondrial membrane potential, thereby preventing mitochondrial Ca(2+) transport. Release of mitochondrial Ca(2+) by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) after excitotoxic glutamate treatment resulted in a greater [Ca(2+)](i) in E(2)-treated cells, indicating an E(2)-induced increase in the mitochondrial calcium ([Ca(2+)](m)) load. The increased [Ca(2+)](m) load was accompanied by increased expression of Bcl-2, which can promote mitochondrial Ca(2+) load tolerance. These findings provide a mechanism of E(2)-induced neuronal survival by attenuation of excitotoxic glutamate [Ca(2+)](i) rise via increased mitochondrial sequestration of cytosolic Ca(2+) coupled with an increase in Bcl-2 expression to sustain mitochondrial Ca(2+) load tolerance and function.

Mitochondrial calcium response in human transformed lymphoblastoid cells

Human lymphoblastoid cell line (LCL) transformed by Epstein-Barr Virus (EBV) is a unique cellular model for the study of human diseases. Although pathophysiological significance of mitochondrial calcium regulation is drawing attention, it is not known whether or not mitochondria in LCLs play a role in intracellular calcium signaling. In this study, role of mitochondria of the lymphoblastoid cell line in calcium signaling was examined. Intra-mitochondrial calcium concentration ([Ca 2+]m) was successfully measured using dihydro-Rhod-2, revealed by the decrease of fluorescence after application of carbonyl cyanide m-chlorophenylhydrazone (CCCP) and intracellular localization patterns imaged by florescent microscope. Platelet activating factor (PAF) concentration-dependently increased cytosolic calcium concentration ([Ca 2+]i), while no increase of [Ca 2+]m was observed. In contrast, 10 μM thapsigargin increased [Ca 2+]i as well as [Ca 2+]m. LCLs may be used for the study of possible pathophysiological role of mitochondrial calcium regulation in human diseases.

Calcium signaling by HBx protein in hepatitis B virus DNA replication.

Hepatitis B virus (HBV) infects more than 300 million people and is a leading cause of liver cancer and disease. The HBV HBx protein is essential for infection; HBx activation of Src is important for HBV DNA replication. In our study, HBx activated cytosolic calcium-dependent proline-rich tyrosine kinase-2 (Pyk2), a Src kinase activator. HBx activation of HBV DNA replication was blocked by inhibiting Pyk2 or calcium signaling mediated by mitochondrial calcium channels, which suggests that HBx targets mitochondrial calcium regulation. Reagents that increased cytosolic calcium substituted for HBx protein in HBV DNA replication. Thus, alteration of cytosolic calcium was a fundamental requirement for HBV replication and was mediated by HBx protein.

Interference with Calcium-Dependent Mitochondrial Bioenergetics in Cardiac Myocytes Isolated from Doxorubicin-Treated Rats

In previous studies we showed that doxorubicin (DOX) interferes with mitochondrial calcium homeostasis and that cardiac mitochondria from DOX-treated rats express a dose-dependent, cumulative, and irreversible decrease in calcium-loading capacity. Associated with this is an increased sensitivity to calcium-induced cell killing of cardiac myocytes from DOX-treated rats. Because mitochondria play an important role in cytosolic calcium regulation, we questioned whether cardiac myocytes isolated from DOX-treated rats express an exaggerated response to interventions that increase cytosolic calcium. Adult male Sprague–Dawley rats received six weekly (sc) injections of either 2 mg/kg of doxorubicin or an equivalent volume of saline and were killed the following week. Cardiac myocytes were isolated and exposed in vitro to 25 μM of A23187, 20 mM of caffeine, or 500 μM of ouabain. A23187 and ouabain caused a dose-dependent increase in intracellular calcium, as measured fluorometrically with Fura-2AM. In the case of ouabain, the increase in cytosolic calcium was greater for myocytes from DOX-treated rats than for cells from control rats. With caffeine, however, the increase in intracellular calcium was not evident unless the mitochondria were depolarized. A23187 also caused depolarization of mitochondria, the extent of which was greater for cardiac myocytes from DOX-treated rats. All three agents caused depletion of cardiac myocyte ATP and cell killing, both of which were more profound in cells from DOX-treated rats than in controls. We suggest that by interfering with mitochondrial calcium regulation, long-term treatment with DOX renders myocytes susceptible to agents that increase cytosolic calcium, presumably by increasing the calcium-dependent disruption of mitochondrial function, leading to depletion of ATP and eventually cell death. This interference with mitochondrial calcium regulation may underlie the pathogenesis of DOX-induced cardiomyopathy.

Modulation of mitochondrial Ca 2+ in ECV304 endothelial cells by agents which elevate CAMP

In the human umbilical vein endothelial cell-derived cell line, ECV304, we have previously shown that the elevation of [Ca 2+] m in response to agonist stimulation is dependent on Ca 2+ influx, i.e. an ATP-induced sustained increase in [Ca 2+] c results in a slow-onset, sustained elevation in [Ca 2+] m [Lawrie A.M., Rizzuto R., Pozzan T., Simpson A.W.M. A role for calcium influx in the regulation of mitochondrial calcium in endothelial cells. J Biol Chem 1996; 271: 10753–10759]. In this study, we have investigated the effect of raising CAMP on ATP-evoked elevations in both [Ca 2+] m and [Ca 2+] c by: (i) activating adenylate cyclase with the forskolin analogue — forskolin 6-[3′-(N,N-dimethylamino-propionyl)]-HCI (1 μM) (FA); (ii) addition of membrane permeable dibutyryl-CAMP (100 μM) (dbcAMP); and (iii) a combination of FA plus inhibition of CAMP phosphodiesterase using RO 20–1724 (17.5 μM) (RO);. We have found that protocols aimed at elevating CAMP significantly reduce the ATP-evoked (1–10 μM) rise in [Ca 2+] m ( n = 14); however, the [Ca 2+] c response to ATP was not affected ( n = 33). This new evidence shows that a second messenger system, other than Ca 2+ itself, may influence [Ca 2+]m changes in response to agonist stimulation.

Muscular degeneration in Duchenne's dystrophy may be caused by a mitochondrial defect

Duchenne's dystrophy (DMD), a recessive chromosome X-related disease, is the most common and severe form of myopathy. The different theories (vascular, neurogenic, membranous, calcic and auto-immune) formulated to account for this disease have not been swept away by the discovery of the DMD gene and the deficient protein, dystrophin, since the exact cellular role played by the latter is still unknown. Our work on skeletal muscle has demonstrated a mitochondrial deficiency of the calcium-specific protein, calmitine, in degenerating muscle of myopathic persons and animals. Considering its great affinity for calcium, this protein specific to skeletal muscle could be essential to mitochondrial calcium regulation and thus to the functioning of the entire muscle cell. Its deficiency in Duchenne's and Becker type muscular dystrophy could be due to a mitochondrial genome alteration solely accountable for muscular degeneration. This hypothesis challenges the supposedly essential but still undefined role that researchers have attributed to dystrophin.

Disruption of Mitochondrial Calcium Homeostasis Following Chronic Doxorubicin Administration

Doxorubicin (Adriamycin) is an anthracycline antibiotic with broad antineoplastic activity. However, the clinical success is limited by the incidence of cumulative cardiomyopathy. In vitro, doxorubicin elicits a cyclosporine A-sensitive release of calcium from cardiac mitochondria. It has been suggested that this leads to mitochondrial calcium cycling and depolarization of membrane potential, which may account for the inhibition of mitochondrial respiration and cytotoxicity observed with the drug. Implication of a similar mechanism in the manifestation of clinical doxorubicin toxicity requires evidence for a disruption of mitochondrial calcium homeostasis following chronic in vivo administration. Cardiac mitochondria isolated from doxorubicin-treated rats (2 mg/kg/week, s.c. × 13 weeks) had a lower RCR but no change in ADP/O compared to controls and exhibited an enhanced cyclosporine A-sensitive release of mitochondrial calcium. Associated with this was a calcium-induced depolarization of membrane potential, which was inhibited by either cyclosporine A or ruthenium red suggesting the induction of mitochondrial calcium cycling following chronic doxorubicin treatment. The persistence of these effects on mitochondrial calcium regulation 4-7 days after the last drug treatment is consistent with the cumulative cardiotoxicity associated with doxorubicin therapy. Cardiac mitochondria isolated from rats treated with iminodaunorubicin, a noncardiotoxic analog of doxorubicin, showed no differences from control suggesting that this disruption of mitochondrial calcium homeostasis in vivo may be an important determinant of the cardiomyopathy observed clinically with doxorubicin.

Regulation of cardiac mitochondrial calcium by average extramitochondrial calcium

A system to perifuse isolated rat heart mitochondria was designed to study the relationship between mitochondrial matrix free Ca2+ and extramitochondrial free Ca2+ under conditions in which the latter concentration could oscillate over a range typical of that expected in vivo. We tested the hypothesis that the level of intramitochondrial Ca2+ responds to the average extramitochondrial Ca2+ in the heart. Mitochondria were immobilized within an optical chamber for measurement of endogenous NAD(P)H and fura 2 fluorescence. NAD(P)H increased significantly on provision of substrates and decreased reversibly in the presence of ADP, indicating maintenance intact coupled respiration by this preparation. Matrix free Ca2+ was measured using fura 2-loaded mitochondria and, in parallel experiments, media free Ca2+ was measured with fura 2 in the absence of mitochondria. Oscillation of extramitochondrial Ca2+ from < 0.1 microM to approximately 2 microM at frequencies of 0.5, 1.0, and 1.25 cycles/s produced steady-state levels of matrix Ca2+ that were independent of frequency but proportional to the average media free Ca2+ concentration. Matrix Ca2+ increased to a steady state on an increase in the extramitochondrial average Ca2+ concentration with a half-time (t1/2) of approximately 2 min at 22 degrees C. Oscillation of mitochondrial Ca2+ was not observed under any conditions tested. The data are taken to indicate that in vivo, the concentration of mitochondrial matrix free Ca2+ is a steady state that is proportional to the average extramitochondrial Ca2+ concentration and that changes in the latter represent a mechanism of signal transduction from the cytosol to the mitochondrial matrix.

Changes in mitochondrial calmitine and calcium in rat denervated skeletal muscle after injection of a myotoxic drug, chlorpromazine

1. Calmitine and mitochondrial calcium were studied after injection of chlorpromazine into control and denervated gastrocnemius muscle in rat. 2. Calmitine decreased under the effect of chlorpromazine and then increased again. Regenerative capacity was more marked for denervated than control muscle. Calcium increased and then returned to its normal level in control muscle while remaining elevated in denervated muscle. 3. Thus, it would appear that calmitine synthesis can occur in the absence of innervation and that denervation, which probably causes disturbances in mitochondrial calcium regulation systems, may prevent total regeneration of muscle after an injury.

Mitochondrial free calcium regulation in hypoxia and reoxygenation: relation to cellular injury.

The role of mitochondria in myocardial ischemic and hypoxic injury is discussed. Increases in mitochondrial Ca content and ionized Ca2+ concentration are observed during and after ischemic and hypoxic exposure and have traditionally been considered to impair mitochondrial function. New data are discussed in which it is shown that increases in mitochondrial [Ca2+] do not necessarily reflect irreversible myocyte injury. Further, it is shown that irreversible cellular injury may occur in hypoxic myocytes in association with increases in mitochondrial [Ca2+] that would ordinarily be considered to fall within a physiologic range. The significance of these observations is considered in context with observations relating to the assessment of post-hypoxic mitochondrial function.

Effect of the sarcoplasmic reticulum on regulation of cytosolic and mitochondrial free calcium in anoxic cardiac myocytes

Effect of the sarcoplasmic reticulum on regulation of cytosolic and mitochondrial free calcium in anoxic cardiac myocytes

Relationships between skeletal muscle mitochondrial calcium regulation and porcine constitution

ZusammenfassungBeziehungen zwischen der mitochondrialen Kalziumregulation im Skelettmuskel und der Konstitution beim SchweinAn einem Tiermaterial von 97 männlichen Schlachtschweinen der Rassen DE, DL und PI wurde die Bedeutung der mitochondrialen Kalziumregulation für die Konstitution des Schweines untersucht. Durch die Verwendung für drei Rassen wurde eine möglichst breite konstitutionelle Basis abgedeckt. Eine Kinetik der mitochondrialen Kalziumregulation wurde mittels Kalziumselektiver Elektroden abgeleitet. Es ergab sich eine deutliche Variabilität des mitochondrialen Kalziumregulationsvermögens. Mittels multivariater Analyseverfahren wurde die mitochondriale Kalziumregulation in 6 Hauptkomponenten zerlegt. Eine Gruppierung anhand der Ausprägung dieser Komponenten ergab drei Reaktionstypen, die sich sowohl im Hinblick auf ihr mitochondriales Kalziumregulationsvermögen, als auch auf ihre Konstitution signifikant unterschieden.