Uptake of safranine by cardiac mitochondria. Competition with calcium ions and dependence on anions.
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ABSTRACT:
SUBMITTER: Harris EJ
PROVIDER: S-EPMC1162370 | biostudies-other | 1980 Nov
REPOSITORIES: biostudies-other
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Project description:Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca(2+)]i) in heart. These buffers can remove up to one-third of the Ca(2+) that enters the cytosol during the [Ca(2+)]i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca(2+) movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca(2+) signals (i.e., Ca(2+) sparks and [Ca(2+)]i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (??m), the role of the mitochondria in buffering cytosolic Ca(2+) signals was investigated. We show here that rapid loss of ??m leads to no significant changes in cytosolic Ca(2+) signals. Second, we make direct measurements of mitochondrial [Ca(2+)] ([Ca(2+)]m) using a mitochondrially targeted Ca(2+) probe (MityCam) and these data suggest that [Ca(2+)]m is near the [Ca(2+)]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 Ca(2+) signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca(2+) cycling suggests that mitochondrial Ca(2+) uptake would need to be at least ?100-fold greater than the current estimates of Ca(2+) influx for mitochondria to influence measurably cytosolic [Ca(2+)] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca(2+) uptake does not significantly alter cytosolic Ca(2+) signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca(2+)]i under physiological conditions in heart.
| S-EPMC4167535 | biostudies-literature
Project description:Efflux of Ca2+ from previously Ca2+-loaded heart mitochondria was measured after inhibiting respiratory activity. The efflux was increased by p-chloromercuribenzoate, methylmercuric chloride, Cu2+, Fe2+, 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (uncoupler). 1,1,1-trifluoro-3-(2-thienylacetone and indomethacin; after such increase it could be diminished by dithiothreitol. The induced loss of the Ca2+ was accompanied by a loss of endogenous adenine nucleotide. Methylmercuric chloride was particularly effective, since it was active at ratios of about 1 nmol/mg of mitochondrial protein. The non-respiring mitochondria were found to regenerate bound thiol groups after their original complement had reacted with thiol-blocking reagent. This regeneration was diminished by the Ca2+-efflux stimulatig agents that were not themselves thiol-blocking reagents, such as thyroxine, uncoupler, trifluorothienylacetone and indomethacin. The external exposure of thiol groups was also diminished by thyroxine, uncoupler and trifluorothienylacetone. The results support the proposal made previously that the membrane is maintained in a state of low permeability by adenine nucleotide and Mg2+ being bound to thiol-dependent sites.
| S-EPMC1161707 | biostudies-other
Project description:Elevated calcium and reactive oxygen species (ROS) are responsible for the bulk of cell death occurring in a variety of clinical settings that include acute coronary events, cerebrovascular accidents, and acute kidney injury. It is commonly believed that calcium and ROS participate in a viscous cycle during these events. However, the precise feedback mechanisms are unknown. We quantitatively demonstrate in this study that, on the contrary, calcium does not stimulate free radical production but suppresses it. Isolated mitochondria from guinea pig hearts were energized with a variety of substrates and exposed to calcium concentrations designed to induce moderate calcium overload conditions associated with ischemia/reperfusion injury but do not elicit the well-known mitochondrial permeability transition phenomenon. Metabolic function and free radical emission were simultaneously quantified using high-resolution respirometry and fluorimetry. Membrane potential, high amplitude swelling, and calcium dynamics were also quantified in parallel. Our results reveal that calcium overload does not lead to excessive ROS emission but does decrease ADP stimulated respiration rates for NADH-dependent pathways. Moreover, we developed an empirical model of mitochondrial free radical homeostasis to identify the processes that are different for each substrate and calcium condition. In summary, we show that in healthy guinea pig mitochondria, calcium uptake and free radical generation do not contribute to a viscous cycle and that the relationship between net free radical production and oxygen concentration is hyperbolic. Altogether, these results lay out an important foundation necessary to quantitatively determine the role of calcium in IR injury and ROS production.
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Project description:Under high Ca(2+) load conditions, Ca(2+) concentrations in the extra-mitochondrial and mitochondrial compartments do not display reciprocal dynamics. This is due to a paradoxical increase in the mitochondrial Ca(2+) buffering power as the Ca(2+) load increases. Here we develop and characterize a mechanism of the mitochondrial Ca(2+) sequestration system using an experimental data set from isolated guinea pig cardiac mitochondria. The proposed mechanism elucidates this phenomenon and others in a mathematical framework and is integrated into a previously corroborated model of oxidative phosphorylation including the Na(+)/Ca(2+) cycle. The integrated model reproduces the Ca(2+) dynamics observed in both compartments of the isolated mitochondria respiring on pyruvate after a bolus of CaCl2 followed by ruthenium red and a bolus of NaCl. The model reveals why changes in mitochondrial Ca(2+) concentration of Ca(2+) loaded mitochondria appear significantly mitigated relative to the corresponding extra-mitochondrial Ca(2+) concentration changes after Ca(2+) efflux is initiated. The integrated model was corroborated by simulating the set-point phenomenon. The computational results support the conclusion that the Ca(2+) sequestration system is composed of at least two classes of Ca(2+) buffers. The first class represents prototypical Ca(2+) buffering, and the second class encompasses the complex binding events associated with the formation of amorphous calcium phosphate. With the Ca(2+) sequestration system in mitochondria more precisely defined, computer simulations can aid in the development of innovative therapeutics aimed at addressing the myriad of complications that arise due to mitochondrial Ca(2+) overload.
| S-EPMC3615037 | biostudies-literature
Project description:Long-lasting mitochondrial permeability transition pore (mPTP) openings damage mitochondria, but transient mPTP openings protect against chronic cardiac stress. To probe the mechanism, we subjected isolated cardiac mitochondria to gradual Ca(2+) loading, which, in the absence of BSA, induced long-lasting mPTP opening, causing matrix depolarization. However, with BSA present to mimic cytoplasmic fatty acid-binding proteins, the mitochondrial population remained polarized and functional, even after matrix Ca(2+) release caused an extramitochondrial free [Ca(2+)] increase to >10 μM, unless mPTP openings were inhibited. These findings could be explained by asynchronous transient mPTP openings allowing individual mitochondria to depolarize long enough to flush accumulated matrix Ca(2+) and then to repolarize rapidly after pore closure. Because subsequent matrix Ca(2+) reuptake via the Ca(2+) uniporter is estimated to be >100-fold slower than matrix Ca(2+) release via mPTP, only a tiny fraction of mitochondria (<1%) are depolarized at any given time. Our results show that transient mPTP openings allow cardiac mitochondria to defend themselves collectively against elevated cytoplasmic Ca(2+) levels as long as respiratory chain activity is able to balance proton influx with proton pumping. We found that transient mPTP openings also stimulated reactive oxygen species production, which may engage reactive oxygen species-dependent cardioprotective signaling.
| S-EPMC3186421 | biostudies-literature
Project description:IntroductionIncidents of myocardial infarction and sudden cardiac arrest vary with time of the day, but the mechanism for this effect is not clear. We hypothesized that diurnal changes in the ability of cardiac mitochondria to control calcium homeostasis dictate vulnerability to cardiovascular events.ObjectivesHere we investigate mitochondrial calcium dynamics, respiratory function, and reactive oxygen species (ROS) production in mouse heart during different phases of wake versus sleep periods.MethodsWe assessed time-of-the-day dependence of calcium retention capacity of isolated heart mitochondria from young male C57BL6 mice. Rhythmicity of mitochondrial-dependent oxygen consumption, ROS production and transmembrane potential in homogenates were explored using the Oroboros O2k Station equipped with a fluorescence detection module. Changes in expression of essential clock and calcium dynamics genes/proteins were also determined at sleep versus wake time points.ResultsOur results demonstrate that cardiac mitochondria exhibit higher calcium retention capacity and higher rates of calcium uptake during sleep period. This was associated with higher expression of clock gene Bmal1, lower expression of per2, greater expression of MICU1 gene (mitochondrial calcium uptake 1), and lower expression of the mitochondrial transition pore regulator gene cyclophilin D. Protein levels of mitochondrial calcium uniporter (MCU), MICU2, and sodium/calcium exchanger (NCLX) were also higher at sleep onset relative to wake period. While complex I and II-dependent oxygen utilization and transmembrane potential of cardiac mitochondria were lower during sleep, ROS production was increased presumably due to mitochondrial calcium sequestration.ConclusionsTaken together, our results indicate that retaining mitochondrial calcium in the heart during sleep dissipates membrane potential, slows respiratory activities, and increases ROS levels, which may contribute to increased vulnerability to cardiac stress during sleep-wake transition. This pronounced daily oscillations in mitochondrial functions pertaining to stress vulnerability may at least in part explain diurnal prevalence of cardiac pathologies.
| S-EPMC8240107 | biostudies-literature
Project description:Ca2+ influx into mitochondria is mediated by the mitochondrial calcium uniporter (MCU), whose identity was recently revealed as a 40-kDa protein that along with other proteins forms the mitochondrial Ca2+ uptake machinery. The MCU is a Ca2+-conducting channel spanning the inner mitochondrial membrane. Here, deletion of the MCU completely inhibited Ca2+ uptake in liver, heart, and skeletal muscle mitochondria. However, in brain nonsynaptic and synaptic mitochondria from neuronal somata/glial cells and nerve terminals, respectively, the MCU deletion slowed, but did not completely block, Ca2+ uptake. Under resting conditions, brain MCU-KO mitochondria remained polarized, and in brain MCU-KO mitochondria, the electrophoretic Ca2+ ionophore ETH129 significantly accelerated Ca2+ uptake. The residual Ca2+ uptake in brain MCU-KO mitochondria was insensitive to inhibitors of mitochondrial Na+/Ca2+ exchanger and ryanodine receptor (CGP37157 and dantrolene, respectively), but was blocked by the MCU inhibitor Ru360. Respiration of WT and MCU-KO brain mitochondria was similar except that for mitochondria that oxidized pyruvate and malate, Ca2+ more strongly inhibited respiration in WT than in MCU-KO mitochondria. Of note, the MCU deletion significantly attenuated but did not completely prevent induction of the permeability transition pore (PTP) in brain mitochondria. Expression level of cyclophilin D and ATP content in mitochondria, two factors that modulate PTP induction, were unaffected by MCU-KO, whereas ADP was lower in MCU-KO than in WT brain mitochondria. Our results suggest the presence of an MCU-independent Ca2+ uptake pathway in brain mitochondria that mediates residual Ca2+ influx and induction of PTP in a fraction of the mitochondrial population.
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Project description:Ryanodine receptor channels at calcium release sites of cardiac myocytes operate on the principle of calcium-induced calcium release. In vitro experiments revealed competition of Ca2+ and Mg2+ in the activation of ryanodine receptors (RyRs) as well as inhibition of RyRs by Mg2+. The impact of RyR modulation by Mg2+ on calcium release is not well understood due to the technical limitations of in situ experiments. We turned instead to an in silico model of a calcium release site (CRS), based on a homotetrameric model of RyR gating with kinetic parameters determined from in vitro measurements. We inspected changes in the activity of the CRS model in response to a random opening of one of 20 realistically distributed RyRs, arising from Ca2+/Mg2+ interactions at RyR channels. Calcium release events (CREs) were simulated at a range of Mg2+-binding parameters at near-physiological Mg2+ and ATP concentrations. Facilitation of Mg2+ binding to the RyR activation site inhibited the formation of sparks and slowed down their activation. Impeding Mg-binding to the RyR activation site enhanced spark formation and speeded up their activation. Varying Mg2+ binding to the RyR inhibition site also dramatically affected calcium release events. Facilitation of Mg2+ binding to the RyR inhibition site reduced the amplitude, relative occurrence, and the time-to-end of sparks, and vice versa. The characteristics of CREs correlated dose-dependently with the effective coupling strength between RyRs, defined as a function of RyR vicinity, single-channel calcium current, and Mg-binding parameters of the RyR channels. These findings postulate the role of Mg2+ in calcium release as a negative modulator of the coupling strength among RyRs in a CRS, translating to damping of the positive feedback of the calcium-induced calcium-release mechanism.
| S-EPMC8821920 | biostudies-literature
Project description:The kinetics of uptake of Ca(2+) by rat heart mitochondria were studied by a spectrophotometric method with Arsenazo III indicator. The exponential rate coefficients measured with or without added phosphate increase with the amount of Ca(2+) added up to about 24mum. Evidence is given that the effect is attributable to a combination of formation of chelates at low concentrations to act as Ca(2+) buffers, with co-transport of substrate to provide more respiratory fuel. The inhibitory effect of Mg(2+) depends on the Ca(2+) concentration, so with a constant [Mg(2+)] the low concentrations of Ca(2+) are most inhibited, and the rate coefficients are still more Ca(2+)-dependent. Ca(2+) uptake is slowed by local anaesthetics such as butacaine and dibucaine, and also by propranolol and palmitoyl-CoA. After an uptake, the release of Ca(2+) was investigated. The spontaneous release involves an initially slow and small appearance of free Ca(2+) and is followed by an auto-accelerated phase. The release is accompanied by a gradual decrease in internal ATP; it is initiated by palmitoyl-CoA (reversed by carnitine), by lysophosphatidylcholine, by Na(+) salts (reversed by oligomycin) and by K(+) salts added to a K(+)-free medium containing valinomycin. The process is probably a response to an increased energy load imposed on the mitochondria by the various conditions, which include the spontaneous action of phospholipase activated by traces of Ca(2+). The problem of how much mitochondrial activity is participating in normal heart Ca(2+) turnover is discussed, and experiments showing only 7-14% exchange of the mitochondrial Ca(2+) occurring in vivo in 10 or 20min are reported.
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Project description:Rods and cones use intracellular Ca2+ to regulate many functions, including phototransduction and neurotransmission. The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.
| S-EPMC7525533 | biostudies-literature