Characterization of the Effect of the Mitochondrial Protein Hint2 on?Intracellular Ca2+ dynamics

Hint2, one of the five members of the superfamily of the histidine triad AMP-lysine hydrolase proteins, is expressed in mitochondria of various cell types. In human adrenocarcinoma cells, Hint2 modulates Ca²⁺ handling by mitochondria. As Hint2 is highly expressed in hepatocytes, we investigated if this protein affects Ca²⁺ dynamics in this cell type. We found that in hepatocytes isolated from Hint2^−/− mice, the frequency of Ca²⁺ oscillations induced by 1 μM noradrenaline was 150% higher than in the wild-type. Using spectrophotometry, we analyzed the rates of Ca²⁺ pumping in suspensions of mitochondria prepared from hepatocytes of either wild-type or Hint2^−/− mice; we found that Hint2 accelerates Ca²⁺ pumping into mitochondria. We then resorted to computational modeling to elucidate the possible molecular target of Hint2 that could explain both observations. On the basis of a detailed model for mitochondrial metabolism proposed in another study, we identified the respiratory chain as the most probable target of Hint2. We then used the model to predict that the absence of Hint2 leads to a premature opening of the mitochondrial permeability transition pore in response to repetitive additions of Ca²⁺ in suspensions of mitochondria. This prediction was then confirmed experimentally.     

Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b-/- mouse model of Wilson's disease

Cellular copper overload as found in Wilson's disease may disturb mitochondrial function and integrity. Atp7b^−/− mice accumulate copper in the liver and serve as an animal model for this inherited disease. The molecular mechanism of copper toxicity in hepatocytes is poorly understood. Total mitochondrial lipids from liver of wild-type mice were subjected to oxidative stress by the Cu²⁺/H₂O₂/ascorbate system. Phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were detected as cardiolipin fragmentation products by thin-layer chromatography combined with MALDI-TOF mass spectrometry in oxidized samples, but not in unperturbed ones. The formation of PA and PHA in copper-treated model membrane correlated well with the decrease of cardiolipin. Mitochondrial lipids from Atp7b^−/− mice of different age were analyzed for the presence of PA. While 32-weeks old wild-type (control) and Atp7b^−/− mice did not show any PA, there was a steady increase in the amount of this lipid in Atp7b^−/− mice in contrast to control with increasing age. Hepatocytes from elder Atp7b^−/−mice contained morphologically changed mitochondria unlike cells from wild-type animals of the same age. We concluded that free-radical fragmentation of cardiolipin with the formation of PA is a likely mechanism that damages mitochondria under conditions of oxidative stress due to copper overload. Our findings are relevant for better understanding of molecular mechanisms for liver damage found in Wilson's disease.     

Glucagon Regulation of Oxidative Phosphorylation Requires an Increase in Matrix Adenine Nucleotide Content through Ca2+ Activation of the Mitochondrial ATP-Mg/Pi Carrier SCaMC-3

It has been known for a long time that mitochondria isolated from hepatocytes treated with glucagon or Ca²⁺-mobilizing agents such as phenylephrine show an increase in their adenine nucleotide (AdN) content, respiratory activity, and calcium retention capacity (CRC). Here, we have studied the role of SCaMC-3/slc25a23, the mitochondrial ATP-Mg/P_i carrier present in adult mouse liver, in the control of mitochondrial AdN levels and respiration in response to Ca²⁺ signals as a candidate target of glucagon actions. With the use of SCaMC-3 knock-out (KO) mice, we have found that the carrier is responsible for the accumulation of AdNs in liver mitochondria in a strictly Ca²⁺-dependent way with an S_0.5 for Ca²⁺ activation of 3.3 ± 0.9 μm. Accumulation of matrix AdNs allows a SCaMC-3-dependent increase in CRC. In addition, SCaMC-3-dependent accumulation of AdNs is required to acquire a fully active state 3 respiration in AdN-depleted liver mitochondria, although further accumulation of AdNs is not followed by increases in respiration. Moreover, glucagon addition to isolated hepatocytes increases oligomycin-sensitive oxygen consumption and maximal respiratory rates in cells derived from wild type, but not SCaMC-3-KO mice and glucagon administration in vivo results in an increase in AdN content, state 3 respiration and CRC in liver mitochondria in wild type but not in SCaMC-3-KO mice. These results show that SCaMC-3 is required for the increase in oxidative phosphorylation observed in liver mitochondria in response to glucagon and Ca²⁺-mobilizing agents, possibly by allowing a Ca²⁺-dependent accumulation of mitochondrial AdNs and matrix Ca²⁺, events permissive for other glucagon actions.     

Cytosolic and mitochondrial Ca2+ concentrations in primary hepatocytes change with ageing and in consequence of an mtDNA mutation

Mitochondrial Ca²⁺ flux is crucial for the regulation of cell metabolism. Ca²⁺ entry to the mitochondrial matrix is mediated by VDAC1 and MCU with its regulatory molecules. We investigated hepatocytes isolated from conplastic C57BL/6NTac-mt^NODLtJ mice (mtNOD) that differ from C57BL/6NTac mice (controls) by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase, resulting in functional and morphological mitochondrial adaptations. Mice of both strains up to 12 months old were compared using mitochondrial GEM-GECO1 and cytosolic CAR-GECO1 expression to gain knowledge of age-dependent alterations of Ca²⁺ concentrations. In controls we observed a significant increase in glucose-induced cytosolic Ca²⁺ concentration with ageing, but only a minor elevation in mitochondrial Ca²⁺ concentration. Conversely, glucose-induced mitochondrial Ca²⁺ concentration significantly declined with ageing in mtNOD mice, paralleled by a slight decrease in cytosolic Ca²⁺ concentration. This was consistent with a significant reduction of the MICU1 to MCU expression ratio and a decline in MCUR1. Our results can best be explained in terms of the adaptation of Ca²⁺ concentrations to the mitochondrial network structure. In the fragmented mitochondrial network of ageing controls there is a need for high cytosolic Ca²⁺ influx, because only some of the isolated mitochondria are in direct contact with the endoplasmic reticulum. This is not important in the hyper-fused elongated mitochondrial network found in ageing mtNOD mice which facilitates rapid Ca²⁺ distribution over a large mitochondrial area.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

Ca²⁺ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca²⁺-dependent enzymes. However, excessive mitochondrial Ca²⁺ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca²⁺ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca²⁺ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca²⁺ levels ([Ca²⁺]_c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca²⁺ content (Ca^2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca²⁺]_c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca²⁺ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca²⁺m that was not further increased during prolonged incubations, despite a sustained [Ca²⁺]_c elevation. By contrast, thapsigargin induced a net Ca²⁺ accumulation in mitochondria that continued for up to 30 min and reached Ca^2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca²⁺ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca²⁺ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca²⁺ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca²⁺ through artificial means, enabling mitochondria to function as a Ca²⁺ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)     

Transport of Ca2+ and Ca2+-dependent permeability transition in heart mitochondria in the early stages of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive skeletal muscle disease that is associated with severe cardiac complications in the late stages. Significant mitochondrial dysfunction is reportedly responsible for the development of cardiomyopathy with age. At the same time, adaptive changes in mitochondrial metabolism in cardiomyocytes were identified in the early stages of DMD. In this work, we evaluate the functioning of calcium transport systems (MCU and NCLX), and MPT pore in the heart mitochondria of young dystrophin-deficient mice. As compared to wild-type animals, heart mitochondria of mdx mice have been found to be more efficient both in respect to Ca²⁺ uniport and Na⁺-dependent Ca²⁺ efflux. The data obtained indicate that the increased rate of Ca²⁺ uptake by heart mitochondria of mdx mice may be due to an increase in the ratio of MCU and MCUb subunits. In turn, an increase in the rate of Ca²⁺ efflux from organelles in DMD may be the result of a significant increase in the level of NCLX. Moreover, the heart mitochondria of mdx mice were more resistant to MPT pore opening, which may be due to an increase in the microviscosity of mitochondrial membranes of DMD mice. At the same time, the level of putative MPT pore proteins did not change. The paper discusses the effect of rearrangements of the mitochondrial proteome involved in the transport and accumulation of calcium on the adaptation of this organ to DMD.     

Nelfinavir Inhibits Intra-Mitochondrial Calcium Influx and Protects Brain against Hypoxic-Ischemic Injury in Neonatal Mice

Nelfinavir (NLF), an antiretroviral agent, preserves mitochondrial membranes integrity and protects mature brain against ischemic injury in rodents. Our study demonstrates that in neonatal mice NLF significantly limits mitochondrial calcium influx, the event associated with protection of the brain against hypoxic-ischemic insult (HI). Compared to the vehicle-treated mice, cerebral mitochondria from NLF-treated mice exhibited a significantly greater tolerance to the Ca²⁺-induced membrane permeabilization, greater ADP-phosphorylating activity and reduced cytochrome C release during reperfusion. Pre-treatment with NLF or Ruthenium red (RuR) significantly improved viability of murine hippocampal HT-22 cells, reduced Ca²⁺ content and preserved membrane potential (Ψm) in mitochondria following oxygen-glucose deprivation (OGD). Following histamine-stimulated Ca²⁺ release from endoplasmic reticulum, in contrast to the vehicle-treated cells, the cells treated with NLF or RuR also demonstrated reduced Ca²⁺ content in their mitochondria, the event associated with preserved Ψm. Because RuR inhibits mitochondrial Ca²⁺ uniporter, we tested whether the NLF acts via the mechanism similar to the RuR. However, in contrast to the RuR, in the experiment with direct interaction of these agents with mitochondria isolated from naïve mice, the NLF did not alter mitochondrial Ca²⁺ influx, and did not prevent Ca²⁺ induced collapse of the Ψm. These data strongly argues against interaction of NLF and mitochondrial Ca²⁺ uniporter. Although the exact mechanism remains unclear, our study is the first to show that NLF inhibits intramitochondrial Ca²⁺ flux and protects developing brain against HI-reperfusion injury. This novel action of NLF has important clinical implication, because it targets a fundamental mechanism of post-ischemic cell death: intramitochondrial Ca²⁺ overload → mitochondrial membrane permeabilization → secondary energy failure.     

cAMP and CaInvolvement in the Mitochondrial Response of Cultured Fetal Rat Hepatocytes to Adrenaline

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca²⁺concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca²⁺concentrations, suggesting that the effects of cAMP may be mediated by free Ca²⁺concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca²⁺act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both α- and β-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.     

Duality of effect of La3+ on mitochondrial permeability transition pore depending on the concentration

In order to explore the role of mitochondria in proliferation promotion and/or apoptosis induction of lanthanum, the mutual influences between La³⁺ and Ca²⁺ on mitochondrial permeability transition pore (PTP) opening were investigated with isolated mitochondria from rat liver. The experimental results revealed that La³⁺ influence the state of mitochondria in a concentration-dependent biphasic manner. La³⁺ in nanomolar concentrations, acting as a Ca²⁺ analog, entered mitochondrial matrix via the RuR sensitive Ca²⁺ channel and elevated ROS level, leading to opening of PTP indicated by mitochondrial swelling, reduction of ΔΨ_m and cytochrome c release. Inhibition of PTP with 10 μM CsA attenuated the effects of La³⁺. However, micromolar concentrations La³⁺ acted mainly as a Ca²⁺ antagonist, inhibiting PTP opening induced by Ca²⁺. We postulated that this action of La³⁺ on mitochondria through interaction with Ca²⁺ might be involved in the proliferation-promoting and apoptosis induction by La³⁺.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

Studies elucidating the mechanisms of calcium induced mitochondrial depolarization in individual isolated brain mitochondria

Among other mitochondrial functions, energy production and Ca²⁺ uptake are crucial for maintaining neuronal viability. Both of these functions are critically dependent on mitochondrial membrane potential (ΔΨ_m). Mitochondrial Ca²⁺ overload causing a dissipation of ΔΨ_m is a key component of several neuronal pathologies. However, the mechanism of Ca²⁺-induced depolarization in neuronal mitochondria remains unclear. Typically, ΔΨ_m has been evaluated as a single overall estimate from all mitochondria present in a given cell or tissue. However, recent data showed that the population of mitochondria isolated from tissues is not homogeneous, and averaged parameters from the whole population do not necessarily reflect the processes taking place in a single organelle. This review summarizes our recent studies of Ca²⁺-induced depolarization in individual mitochondria isolated from rat forebrain and immobilized to coverslips. Fluorescence imaging techniques and potentiometric fluorescent dyes were effectively used to study ΔΨ_m changes. The data have shown that Ca²⁺ triggers ΔΨ_m oscillations in brain mitochondria followed by a complete depolarization. Further investigation of this phenomenon led us to suggest that Ca²⁺-induced ΔΨ_m oscillations can represent an intermediate unstable state that may lead to irreversible mitochondrial dysfunction. Therefore, further study of this phenomenon would help to understand what causes the irreversible damage of mitochondria during cytosolic/mitochondrial Ca²⁺ overload. Here we discuss the effects of different modulators of the mitochondrial permeability transition pore on Ca²⁺-induced depolarization in brain mitochondria and in liver mitochondria, where the mechanism of Ca²⁺-depolarization is better understood. A comparison of these effects in brain and liver mitochondria led us to conclude that Ca²⁺ can induce reversible “low conductance” permeability transition in brain mitochondria, the phenomenon which requires a transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore. The article is published in the original.     

Dysfunction of mitochondria Ca uptake in cystic fibrosis airway epithelial cells

In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca²⁺ mobilization is abnormally increased. Because the uptake of Ca²⁺ by mitochondria during Ca²⁺ influx or Ca²⁺ release from ER stores may be crucial for maintaining a normal Ca²⁺ homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (ΔΨ_mit) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2–AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca²⁺. The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the ΔΨ_mit of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca²⁺ uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca²⁺ uptake.     

Calcium Movement in Cardiac Mitochondria

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca²⁺]_i) in heart. These buffers can remove up to one-third of the Ca²⁺ that enters the cytosol during the [Ca²⁺]_i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca²⁺ movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca²⁺ signals (i.e., Ca²⁺ sparks and [Ca²⁺]_i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨ_m), the role of the mitochondria in buffering cytosolic Ca²⁺ signals was investigated. We show here that rapid loss of ΔΨ_m leads to no significant changes in cytosolic Ca²⁺ signals. Second, we make direct measurements of mitochondrial [Ca²⁺] ([Ca²⁺]_m) using a mitochondrially targeted Ca²⁺ probe (MityCam) and these data suggest that [Ca²⁺]_m is near the [Ca²⁺]_i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca²⁺ signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca²⁺ cycling suggests that mitochondrial Ca²⁺ uptake would need to be at least ∼100-fold greater than the current estimates of Ca²⁺ influx for mitochondria to influence measurably cytosolic [Ca²⁺] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca²⁺ uptake does not significantly alter cytosolic Ca²⁺ signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca²⁺]_i under physiological conditions in heart.     

CaMKII‐dependent ryanodine receptor phosphorylation mediates sepsis‐induced cardiomyocyte apoptosis

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis‐induced apoptosis. Wild‐type (WT) CASP mice hearts showed an increase in apoptosis respect to WT‐Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3‐I) were protected against sepsis‐induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca²⁺ release, prevented apoptosis in WT‐CASP. To examine whether CaMKII‐dependent RyR2 phosphorylation mediates diastolic Ca²⁺ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation‐dependent enhancement in diastolic SR Ca²⁺ release leading to mitochondrial Ca²⁺ overload, mitochondrial Ca²⁺ retention capacity was reduced in mitochondria isolated from WT‐CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene‐treated mice. We conclude that in sepsis, CaMKII‐dependent RyR2 phosphorylation results in diastolic Ca²⁺ release from SR which leads to mitochondrial Ca²⁺ overload and apoptosis.     

Atherosclerosis affects calcium signalling in endothelial cells from apolipoprotein E knockout mice before plaque formation

Little is known about how hypercholesterolaemia affects Ca²⁺ signalling in the vasculature of ApoE^−/− mice, a model of atherosclerosis. Our objectives were therefore to determine (i) if hypercholesterolaemia alters Ca²⁺ signalling in aortic endothelial cells before overt atherosclerotic lesions occur, (ii) how Ca²⁺ signals are affected in older plaque-containing mice, and (iii) whether Ca²⁺ signalling changes were translated into contractility differences. Using confocal microscopy we found agonist-specific Ca²⁺ changes in endothelial cells. ATP responses were unchanged in ApoE^−/− cells and methyl-β-cyclodextrin, which lowers cholesterol, was without effect. In contrast, Ca²⁺ signals to carbachol were significantly increased in ApoE^−/− cells, an effect methyl-β-cyclodextrin reversed. Ca²⁺ signals were more oscillatory and store-operated Ca²⁺ entry decreased as mice aged and plaques formed. Despite clearly increased Ca²⁺ signals, aortic rings pre-contracted with phenylephrine had impaired relaxation to carbachol. This functional deficit increased with age, was not related to ROS generation, and could be partially rescued by methyl-β-cyclodextrin. In conclusion, carbachol-induced calcium signalling and handling are significantly altered in endothelial cells of ApoE^−/− mice before plaque development. We speculate that reduction in store-operated Ca²⁺ entry may result in less efficient activation of eNOS and thus explain the reduced relaxatory response to CCh, despite the enhanced Ca²⁺ response.     

Ca2+ handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease

We investigated Ca²⁺ handling in isolated brain synaptic and non‐synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non‐synaptic mitochondria from 2‐ and 12‐month‐old YAC128 mice had larger Ca²⁺ uptake capacity than mitochondria from YAC18 and wild‐type FVB/NJ mice. Synaptic mitochondria from 12‐month‐old YAC128 mice had further augmented Ca²⁺ capacity compared with mitochondria from 2‐month‐old YAC128 mice and age‐matched YAC18 and FVB/NJ mice. This increase in Ca²⁺ uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12‐month‐old YAC128 mice. We speculate that this may happen because of mHtt‐mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca²⁺‐induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca²⁺ followed by recovery to near resting levels. Following recovery of cytosolic Ca²⁺, mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca²⁺, suggesting similar Ca²⁺ release and, consequently, Ca²⁺ loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca²⁺ handling in brain mitochondria of YAC128 mice.

     

Bnip3-mediated mitochondrial autophagy is independent of the mitochondrial permeability transition pore

Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many cells. In this study, we have investigated the mechanism by which Bnip3 induces autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive autophagy in adult cardiac myocytes. Fluorescent microscopy studies and ultrastructural analysis revealed selective degradation of mitochondria by autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased levels of intracellular Ca²⁺ have been reported by others to induce autophagy, but Bnip3-induced autophagy was not abolished by antioxidant treatment or the Ca²⁺ chelator BAPTA-AM. We also investigated the role of the mitochondrial permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the mPTP has previously been implicated in the induction of autophagy and selective removal of damaged mitochondria by autophagosomes, mitochondria sequestered by autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability transition, and treatment with the mPTP inhibitor cyclosporine A did not inhibit mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is an essential component of the mPTP and Bnip3 induced autophagy to the same extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. These results support a model where Bnip3 induces selective removal of the mitochondria in cardiac myocytes, and that Bnip3 triggers induction of autophagy independent of Ca²⁺, ROS generation, and mPTP opening.     

Diversity of mitochondrial Ca signaling in rat neonatal cardiomyocytes: Evidence from a genetically directed Ca probe,mitycam-E31Q

I_Ca-gated Ca²⁺ release (CICR) from the cardiac SR is the main mechanism mediating the rise of cytosolic Ca²⁺, but the extent to which mitochondria contribute to the overall Ca²⁺ signaling remains controversial. To examine the possible role of mitochondria in Ca²⁺ signaling, we developed a low affinity mitochondrial Ca²⁺ probe, mitycam-E31Q (300–500 MOI, 48–72 h) and used it in conjunction with Fura-2AM to obtain simultaneous TIRF images of mitochondrial and cytosolic Ca²⁺ in cultured neonatal rat cardiomyocytes. Mitycam-E31Q staining of adult feline cardiomyocytes showed the typical mitochondrial longitudinal fluorescent bandings similar to that of TMRE staining, while neonatal rat cardiomyocytes had a disorganized tubular or punctuate appearance. Caffeine puffs produced rapid increases in cytosolic Ca²⁺ while simultaneously measured global mitycam-E31Q signals decreased more slowly (increased mitochondrial Ca²⁺) before decaying to baseline levels. Similar, but oscillating mitycam-E31Q signals were seen in spontaneously pacing cells. Withdrawal of Na⁺ increased global cytosolic and mitochondrial Ca²⁺ signals in one population of mitochondria, but unexpectedly decreased it (release of Ca²⁺) in another mitochondrial population. Such mitochondrial Ca²⁺ release signals were seen not only during long lasting Na⁺ withdrawal, but also when Ca²⁺ loaded cells were exposed to caffeine-puffs, and during spontaneous rhythmic beating. Thus, mitochondrial Ca²⁺ transients appear to activate with a delay following the cytosolic rise of Ca²⁺ and show diversity in subpopulations of mitochondria that could contribute to the plasticity of mitochondrial Ca²⁺ signaling.     

Measurements of mitochondrial calcium in vivo

Mitochondria play a pivotal role in intracellular Ca²⁺ signalling by taking up and releasing the ion upon specific conditions. In order to do so, mitochondria depend on a number of factors, such as the mitochondrial membrane potential and spatio-temporal constraints. Whereas most of the basic principles underlying mitochondrial Ca²⁺ handling have been successfully deciphered over the last 50 years using assays based on in vitro preparations of mitochondria or cultured cells, we have only just started to understand the actual physiological relevance of these processes in the whole animal. Recent advancements in imaging and genetically encoded sensor technologies have allowed us to visualise mitochondrial Ca²⁺ transients in live mice. These studies used either two-photon microscopy or bioluminescence imaging of cameleon or aequorin-GFP Ca²⁺ sensors, respectively. Both methods revealed a consistent picture of Ca²⁺ uptake into mitochondria under physiological conditions even during very short-lasting elevations of cytosolic Ca²⁺ levels. The big future challenge is to understand the functional impact of such Ca²⁺ signals on the physiology of the observed tissue as well as of the whole organism. To that end, the development of multiparametric in vivo approaches will be mandatory.     

Goldilocks calcium concentrations and the regulation of oxidative phosphorylation: Too much,too little,or just right

Calcium (Ca²⁺) is a key regulator in diverse intracellular signaling pathways and has long been implicated in metabolic control and mitochondrial function. Mitochondria can actively take up large amounts of Ca²⁺, thereby acting as important intracellular Ca²⁺ buffers and affecting cytosolic Ca²⁺ transients. Excessive mitochondrial matrix Ca²⁺ is known to be deleterious due to opening of the mitochondrial permeability transition pore (mPTP) and consequent membrane potential dissipation, leading to mitochondrial swelling, rupture, and cell death. Moderate Ca²⁺ within the organelle, on the other hand, can directly or indirectly activate mitochondrial matrix enzymes, possibly impacting on ATP production. Here, we aimed to determine in a quantitative manner if extra- or intramitochondrial Ca²⁺ modulates oxidative phosphorylation in mouse liver mitochondria and intact hepatocyte cell lines. To do so, we monitored the effects of more modest versus supraphysiological increases in cytosolic and mitochondrial Ca²⁺ on oxygen consumption rates. Isolated mitochondria present increased respiratory control ratios (a measure of oxidative phosphorylation efficiency) when incubated with low (2.4 ± 0.6 μM) and medium (22.0 ± 2.4 μM) Ca²⁺ concentrations in the presence of complex I–linked substrates pyruvate plus malate and α-ketoglutarate, respectively, but not complex II–linked succinate. In intact cells, both low and high cytosolic Ca²⁺ led to decreased respiratory rates, while ideal rates were present under physiological conditions. High Ca²⁺ decreased mitochondrial respiration in a substrate-dependent manner, mediated by mPTP. Overall, our results uncover a Goldilocks effect of Ca²⁺ on liver mitochondria, with specific “just right” concentrations that activate oxidative phosphorylation.