Mitochondrial localization of NCXs: Balancing calcium and energy homeostasis

It is well established that mitochondria are the main source of ATP production within cells. However, mitochondria have other remarkable functions, serving as important modulators of cellular Ca²⁺ signaling, and it is now generally recognized that control over Ca²⁺ homeostasis is intrinsically interwoven with mitochondrial abilities to adjust and tune ATP production. In this review, we describe the mechanisms that mitochondria use to balance Ca²⁺ homeostasis maintenance and cell energy metabolism. In recent years, the knowledge on the molecular machinery mediating Ca²⁺ influx/efflux has been improved and, albeit still open to further investigations, several lines of evidence converge on the hypothesis that plasma membrane Na⁺/Ca²⁺ exchanger (NCX) isoforms are also expressed at the mitochondrial level, where they contribute to the Ca²⁺ and Na⁺ homeostasis maintenance. In particular, the connection between mitochondrial NCX activity and metabolic substrates utilization is further discussed here. We also briefly focus on the alterations of both mitochondrial Ca²⁺ handling and cellular bioenergetics in neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease.     

Proteasome inhibitor lactacystin disturbs the intracellular calcium homeostasis of dopamine neurons in ventral mesencephalic cultures

Ubiquitin proteasome system (UPS) impairment has been implicated in the pathology of Parkinson's disease, but the mechanisms underlying the UPS impairment-induced dopamine (DA) neuron degeneration remain obscure. To test whether calcium homeostasis disturbance is involved in the DA neuronal injury resulting from UPS impairment, we treated the primary ventral mesencephalic (VM) cultures with the proteasome inhibitor lactacystin, and observed its effects on the expression of the gene Homer 1a that is related to calcium homeostasis, and the intracellular free calcium ([Ca²⁺]_i) levels as well as the DA neuron survival. We also investigated a possible role of the L-type voltage dependent calcium channels (L-VDCC) in these events. We found that the lactacystin exposure induced the Homer 1a expression, lowered the [Ca²⁺]_i levels, reduced the depolarization-induced calcium entry and DA release in the VM cultures, and caused a significant DA neuron loss. Activation of L-VDCC by potassium chloride or its agonists alleviated the effects of lactacystin on the [Ca²⁺]_i levels and promoted DA neuron survival, whereas L-VDCC antagonists blocked the depolarization-mediated neuroprotective effect, and at high concentrations the L-VDCC antagonists aggravated the lactacystin-induced DA neuronal injury. These results indicate that calcium homeostasis disturbance may be a novel pathological mechanism leading to DA neuronal injury under conditions of proteasome inhibition.     

Rosiglitazone transiently disturbs calcium homeostasis in monocytic cells

The PPARγ agonist Rosiglitazone exerts anti-hyperglycaemic effects by regulating the long-term expression of genes involved in metabolism, differentiation and inflammation. In the present study, Rosiglitazone treatment rapidly inhibited (5-30 min) the ER Ca²⁺ ATPase SERCA2b in monocytic cells (IC₅₀ = 1.88 μM; p < 0.05), thereby disrupting short-term Ca²⁺ homeostasis (resting [Ca²⁺]_cyto = 121.2 ± 2.9% basal within 1 h; p < 0.05). However, extended Rosiglitazone treatment (72 h) induced dose-dependent SERCA2b up-regulation, and restored calcium homeostasis, in monocytic cells (SERCA2b mRNA: 138.7 ± 5.7% basal (1 μM)/215.0 ± 30.9% basal (10 μM); resting [Ca²⁺]_cyto = 97.3 ± 8.3% basal (10 μM)). As unfavourable cardiovascular outcomes, possibly related to disrupted cellular Ca²⁺ homeostasis, have been linked to Rosiglitazone, this effect may be of clinical interest. In contrast, in PPRE-luciferase reporter-gene assays, Rosiglitazone induced non-dose-dependent PPARγ-dependent effects (1 μM: 152.5 ± 4.9% basal; 10 μM: 136.1 ± 5.1% basal (p < 0.05 for 1 μM vs. 10 μM)). Thus, we conclude that Rosiglitazone can exert PPARγ-independent non-genomic effects, such as the SERCA2b inhibition seen here, but that long-term Rosiglitazone treatment did not perturb resting [Ca]_cyto in this study.     

Single channel characterization of the mitochondrial ryanodine receptor in heart mitoplasts

Heart mitochondria utilize multiple Ca(2+) transport mechanisms. Among them, the mitochondrial ryanodine receptor provides a fast Ca(2+) uptake pathway across the inner membrane to control "excitation and metabolism coupling." In the present study, we identified a novel ryanodine-sensitive channel in the native inner membrane of heart mitochondria and characterized its pharmacological and biophysical properties by directly patch clamping mitoplasts. Four distinct channel conductances of ～100, ～225, ～700, and ～1,000 picosiemens (pS) in symmetrical 150 mm CsCl were observed. The 225 pS cation-selective channel exhibited multiple subconductance states and was blocked by high concentrations of ryanodine and ruthenium red, known inhibitors of ryanodine receptors. Ryanodine exhibited a concentration-dependent modulation of this channel, with low concentrations stabilizing a subconductance state and high concentrations abolishing activity. The 100, 700, and 1,000 pS conductances exhibited different channel characteristics and were not inhibited by ryanodine. Taken together, these findings identified a novel 225 pS channel as the native mitochondrial ryanodine receptor channel activity in heart mitoplasts with biophysical and pharmacological properties that distinguish it from previously identified mitochondrial ion channels.     

Visible light excited ratiometric-GECIs for long-term in-cellulo monitoring of calcium signals

Genetically Encoded Calcium Indicators (GECIs) are powerful molecular tools for monitoring calcium (Ca²⁺) signaling in the cytosol and organellar compartments. However, currently available ratiometric indicators that allow measurements of resting Ca²⁺ levels have limitations in long-term Ca²⁺ imaging. They either are ultraviolet (UV)-excited ones with strong photo-toxicity, or have poor performance. To overcome this hurdle, we developed a set of visible light excited ratiometric-GECIs (VR-GECIs) based on existing mono-colored GECIs. With performance comparable to their corresponding mono-color prototypes, this set of VR-GECIs enables long-term measurements of intra-cellular or intra-organellar Ca²⁺ signals. Using these VR-GECIs together with a newly developed off-line analysis tool, we achieved long-term measurements of Ca²⁺ homeostasis of moving or dividing cells. Our tools may find broad applications in decoding Ca²⁺-modulated physiological or pathological processes.     

Mitochondrial nitric oxide inhibits ATP synthesis. Effect of free calcium in rat heart

Summary.  Nitric oxide is a small potentially toxic molecule and a diatomic free radical. We report the interaction of L-arginine, oxygen and calcium with the synthesis of nitric oxide in heart mitochondria. Nitric oxide synthesis is increased in broken rat heart mitochondria compared with intact and permeabilized mitochondria. Intact mitochondria subjected to hypoxia-reoxygenation conditions accumulated nitric oxide that inhibits oxygen consumption and ATP synthesis. ATPase activity is not affected during this augment of nitric oxide. Physiological free calcium concentrations protected mitochondria from the damage caused by the accumulation of nitric oxide. Higher concentrations of the divalent cation increase the damage exerted by nitric oxide. Received April 15, 2002 Accepted June 17, 2002 Published online November 14, 2002 Acknowledgements This work was supported in part by Mexican Grants from CONACYT (to A.S.M. during its sabbatical) and CIC-UMSNH (2.5). Authors' address: Alfredo Saavedra-Molina, Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-3. C.U., Morelia, Mich. 58030, México, Fax: 52-443-326-5788, E-mail: saavedra@zeus.umich.mx     

Intracellular calcium and the relationship to contractility in an avian model of heart failure

Global contractile heart failure was induced in turkey poults by furazolidone feeding (700 ppm). Abnormal calcium regulation appears to be a key factor in the pathophysiology of heart failure, but the cellular mechanisms contributing to changes in calcium fluxes have not been clearly defined. Isolated ventricular myocytes from non-failing and failing hearts were therefore used to determine whether the whole heart and ventricular muscle contractile dysfunctions were realized at the single cell level. Whole cell current- and voltage-clamp techniques were used to evaluate action potential configurations and L-type calcium currents, respectively. Intracellular calcium transients were evaluated in isolated myocytes with fura-2 and in isolated left ventricular muscles using aequorin. Action potential durations were prolonged in failing myocytes, which correspond to slowed cytosolic calcium clearing. Calcium current-voltage relationships were normal in failing myocytes; preliminary evidence suggests that depressed transient outward potassium currents contribute to prolonged action potential durations. The number of calcium channels (as measured by radioligand binding) were also similar in non-failing and failing hearts. Isolated ventricular muscles from failing hearts had enhanced inotropic responses, in a dose-dependent fashion, to a calcium channel agonist (Bay K 8644). These data suggest that changes in intracellular calcium mobilization kinetics and longer calcium-myofilament interaction may be able to compensate for contractile failure. We conclude that the relationship between calcium current density and sarcoplasmic reticulum calcium release is a dynamic process that may be altered in the setting of heart failure at higher contraction rates. Accepted: 1 March 2000     

Ultrastructural study of calcium distribution in cardiac muscle cells

Summary To investigate calcium distribution in cardiac muscle cells, two methods, one using oxalate and another using lead acetate, were used concomitantly to determine the subcellular localization of calcium. Particular attention was paid to the specificity of the methods employed. Chemical and physical analyses of the electron-opaque deposits of the reaction end-products was performed by chelation with EGTA or X-ray and electron diffraction. Results obtained show that the distribution of the calcium deposits in the cardiac muscle cells is more complex than that described for striated muscle fibers. The implications of these findings are discussed and an original viewpoint on the calcium distribution and movement in cardiac muscle cells is presented.     

蓖麻毒素A链的基因克隆

作为导向药物蓖麻毒素A(Ricin-A)链在大肠杆菌中表达时不含糖基侧链,在体内半衰期长,可提高共作为导向药物的疗效。我们根据Ricin基因核苷酸序列,设计Ricin-A的上、下游引物,通过PCR(多聚酶链式反应)方法,扩增出Ricin-A链基因。与pUC_(19)载体连接,转化到JM103大肠杆菌中,得到重组克隆。对其进行几种酶切鉴定,证明酶切位点正确,又经序列分析,读出与文献发表的Ricin-A序列只有两个碱基不同,但无氨基酸残基的改变。有关Ricin-A的表达工作正在进行中。     

The effects of ricin on the sympathetic vascular neuroeffector system of the rabbit

Ricin is a toxic lectin that inhibits protein synthesis. Because ricin decreases arterial pressure and causes cardiovascular collapse, its effects on the vascular neuroeffector system were investigated. Rabbits were given either of two doses of ricin, and then norepinephrine (NE) release from aorta to transmural stimulation, NE uptake into aorta, NE content of aorta, monoamine oxidase activity, and catechol-O-methyl transferase activity in aorta were determined 18 hours, 4 days or 7 days later. Norepinephrine uptake and enzyme activities in the aorta were not altered by ricin administration. Norepinephrine release and content of aorta were increased at most time periods following ricin administration, significantly so for NE content at 4 days and for release at 18 hours following the lower dose of ricin. We conclude that the mechanisms involved in the release of NE from sympathetic nerves in the vasculature are not impaired by ricin administration, but rather show changes that indicate increased compensatory activity.     

逆向转运过程中ARFRP1与蓖麻毒素相互作用的研究

利用MTT法检测了ADP糖基化因子相关蛋白1(ADP-ribosylation factor-related protein 1,ARFRP1)在蓖麻毒素逆向转运中的作用。研究发现,转染ARFRP1和ARFRP1(Q97L)后,蓖麻毒素对细胞的毒性增强,而转染ARFRP1(T31N)后,毒性有所降低;用brefeldin A阻断高尔基体上的蛋白运输后,转染ARFRP1和ARFRP1(Q97L)后的细胞依然对蓖麻毒素敏感;低温培养细胞,过量表达ARFRP1的细胞抵抗毒素的能力与其他细胞相比变化明显。结果说明ARFRP1能够促进蓖麻毒素在细胞内的逆向转运。     

The ultrastructure of the sinuatrial ring bundle and of the caudal extension of the sinus node in the right atrium of the rabbit heart

Summary The cells of the sinuatrial ring bundle are smaller than the ordinary myocardial cells; they have a regular outline and a large content of myofibrils exhibiting distinct H-bands and M-lines. Rudimentary T-tubules are found. The cells are connected by frequent nexus junctions, desmosomes and regions of interfibrillar contact, both on the well developed intercalated discs and at the periphery of the cells.The cells in the cauda of the sinus node, which extends alongside the crista terminalis together with the right branch of the sinuatrial ring bundle, are irregularly outlined and have a highly variable diameter (1–10 m). They occur in clusters of closely packed cells. The content of myofibrils is sparse and the fibrils exhibit no M-lines and only weak H-bands. No T-tubules are found. The cells are not connected by intercalated discs and no nexus junctions are found.Both tissues contain unmyelinated nerves and nerve fibres. Varicosities with mitochondria and vesicles are found in close apposition to the muscle cells.     

Ricin A chain reaches the endoplasmic reticulum after endocytosis

Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.     

Mitochondrial calcium as a key regulator of mitochondrial ATP production in mammalian cells

Mitochondrial Ca²⁺ transport was initially considered important only in buffering of cytosolic Ca²⁺ by acting as a “sink” under conditions of Ca²⁺ overload. The main regulator of ATP production was considered to be the relative concentrations of high energy phosphates. However, work by Denton and McCormack in the 1970s and 1980s showed that free intramitochondrial Ca²⁺ ([Ca²⁺]_m) activated dehydrogenase enzymes in mitochondria, leading to increased NADH and hence ATP production. This leads them to propose a scheme, subsequently termed a “parallel activation model” whereby increases in energy demand, such as hormonal stimulation or increased workload in muscle, produced an increase in cytosolic [Ca²⁺] that was relayed by the mitochondrial Ca²⁺ transporters into the matrix to give an increase in [Ca²⁺]_m. This then stimulated energy production to meet the increased energy demand. With the development of methods for measuring [Ca²⁺]_m in living cells that proved [Ca²⁺]_m changed over a dynamic physiological range rather than simply soaking up excess cytosolic [Ca²⁺], this model has now gained widespread acceptance. However, work by ourselves and others using targeted probes to measure changes in both [Ca²⁺] and [ATP] in different cell compartments has revealed variations in the interrelationships between these two in different tissues, suggesting that metabolic regulation by Ca²⁺ is finely tuned to the demands and function of the individual organ.     

Impaired mitochondrial bioenergetics determines glutamate-induced delayed calcium deregulation in neurons

Background

Accumulation of glutamate in ischaemic CNS is thought to amplify neuronal death during a stroke. Exposure of neurons to toxic glutamate concentrations causes an initial transient increase in [Ca²⁺]_c followed by a delayed increase commonly termed delayed [Ca²⁺]_c deregulation (DCD).

Methods

We have used fluorescence imaging techniques to explore differences in glutamate-induced DCD in rat hippocampal neurons after different periods of time in culture (days in vitro; DIV).

Results

The amplitude of both the initial [Ca²⁺]_c signal and the number of cells showing DCD in response to glutamate increased with the duration of culture. The capacity of mitochondria to accumulate calcium in permeabilised neurons decreased with time in culture, although mitochondrial membrane potential at rest did not change. The rate of ATP consumption, measured as an increase in [Mg²⁺]_c following inhibition of ATP synthesis, was lower in ‘young’ neurons. The sensitivity of ‘young’ neurons to glutamate-induced DCD approximated to that of ‘old’ neurons when mitochondrial function was impaired using either FCCP or oligomycin. Further, following such treatment, cells showed a DCD-like response to increased [Ca²⁺]_c induced by KCl induced depolarisation which was never otherwise seen.

General significance

Thus, changes in cellular bioenergetics dictate the onset of DCD in response to glutamate.     

Dual probe fluorescence monitoring of intracellular free calcium during ischemia in mouse heart by using continuous compensation for pH dependence of the dissociation constant of Fura-2, and the interference of myoglobin

Mitochondrial damage is the main source of cellular injury upon ischemia-reperfusion, and calcium loading has been implicated in this phenomenon. The use of optical probes for calcium monitoring of the intact heart is hampered by internal filter effects of intracellular hemoproteins, endogenous fluorescence, and their sensitivity to pH. We describe here a method for measurement of intracellular free calcium in isolated myoglobin-deficient perfused mouse hearts under conditions of large intracellular pH fluctuations by simultaneous fluorescence monitoring of the calcium-probe Fura-2 and the pH probe BCECF through dual wavelength excitation of both probes. In myoglobin-containing mouse heart endogenous chromophores interfere with Fura-2 fluorometry. It is shown that a paradoxical decrease in Fura-2 fluorescence occurs during ischemia in isolated mouse hearts. Simultaneous recording of BCECF fluorescence (calibrated against pH measurement with phosphorus NMR) and data reduction based on continual recalculation of the apparent dissociation constant of the calcium-probe complex revealed that a marked increase in intracellular free calcium occurs, and that the Fura-2 fluorescence decrease was caused by an increase in dissociation constant due to intracellular acidification. Intracellular free calcium rose almost linearly during a 20-min period of ischemia and returned to basal values rapidly upon the commencement of perfusion.     

蓖麻毒素与其单克隆抗体相互作用动力学研究

表面等离子体激元共振(SPR)是一种可微量、实时、动态地监测生物分子相互作用的生物传感技术。蓖麻毒素为核糖体失活蛋白,具有很强的细胞毒性作用。通过SPR技术研究了两种抗蓖麻毒素的单克隆抗体C5、D12与蓖麻毒素相互作用的动力学,计算出两者的亲和常数分别为2.49×108mol-1·L和7.9×108mol-1·L,并对两种抗体的抗原表位进行了分析。     

Calcium and magnesium levels in isolated cardiac mitochondria from mice injected with isoproterenol

Summary Calcium (Ca) and Magnesium (Mg) are determined by atomic absorption flame spectrometry in isolated cardiac mitochondria from mice receiving subcutaneous injections of DL-isoproterenol HC1 (ISO), and in mitochondria of untreated controls. In the controls, mitochondria were isolated in the presence or absence of ruthenium red. On the absence of ruthenium red in the isolation medium, mitochondrial Ca levels increase by about 300%, while levels of Mg remain unchanged. Focal myocardial necrosis following a single ISO-injection is shown by electron microscopy. Ca and Mg levels are largely unaffected by a single dose of ISO until 24 h after the injection. A slight increase in Ca occurs in the 48 h samples. When multiple injections of ISO are given every 12th hour for 48 h, 72 h and 96 h, respectively, endogenous Ca and Mg increase significantly. It is suggested that this increase might be associated with ISO-induced cardiac hypertrophy rather than with the pharmacological effects of ISO per se.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

Early calcium handling imbalance in pressure overload-induced heart failure with nearly normal left ventricular ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a common clinical syndrome associated with high morbidity and mortality. Therapeutic options are limited due to a lack of knowledge of the pathology and its evolution. We investigated the cellular phenotype and Ca²⁺ handling in hearts recapitulating HFpEF criteria. HFpEF was induced in a portion of male Wistar rats four weeks after abdominal aortic banding. These animals had nearly normal ejection fraction and presented elevated blood pressure, lung congestion, concentric hypertrophy, increased LV mass, wall stiffness, impaired active relaxation and passive filling of the left ventricle, enlarged left atrium, and cardiomyocyte hypertrophy. Left ventricular cell contraction was stronger and the Ca²⁺ transient larger. Ca²⁺ cycling was modified with a RyR2 mediated Ca²⁺ leak from the sarcoplasmic reticulum and impaired Ca²⁺ extrusion through the Sodium/Calcium exchanger (NCX), which promoted an increase in diastolic Ca²⁺. The Sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA2a) and NCX protein levels were unchanged. The phospholamban (PLN) to SERCA2a ratio was augmented in favor of an inhibitory effect on the SERCA2a activity. Conversely, PLN phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLN-Thr17), which promotes SERCA2A activity, was increased as well, suggesting an adaptive compensation of Ca²⁺ cycling. Altogether our findings show that cardiac remodeling in hearts with a HFpEF status differs from that known for heart failure with reduced ejection fraction. These data also underscore the interdependence between systolic and diastolic “adaptations” of Ca²⁺ cycling with complex compensative interactions between Ca²⁺ handling partner and regulatory proteins.