Kinetics of the non-specific calcium leak from non-mitochondrial calcium stores in permeabilized A7r5 cells.
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ABSTRACT:
SUBMITTER: Missiaen L
PROVIDER: S-EPMC1217562 | biostudies-other | 1996 Aug
REPOSITORIES: biostudies-other
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Project description:In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca2+, mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca2+ overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca2+-sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.
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Project description:The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling.
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Project description:Sirolimus (rapamycin) is an immunosuppressive drug used in transplantation. One of its major side effects is the increased risk of diabetes mellitus; however, the exact mechanisms underlying such association have not been elucidated. Here we show that sirolimus impairs glucose-stimulated insulin secretion both in human and murine pancreatic islets and in clonal β cells in a dose- and time-dependent manner. Importantly, we demonstrate that sirolimus markedly depletes calcium (Ca2+) content in the endoplasmic reticulum and significantly decreases glucose-stimulated mitochondrial Ca2+ uptake. Crucially, the reduced mitochondrial Ca2+ uptake is mirrored by a significant impairment in mitochondrial respiration. Taken together, our findings indicate that sirolimus causes depletion of intracellular Ca2+ stores and alters mitochondrial fitness, eventually leading to decreased insulin release. Our results provide a novel molecular mechanism underlying the increased incidence of diabetes mellitus in patients treated with this drug.
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Project description:Cardiac disease is associated with deleterious emission of mitochondrial reactive oxygen species (mito-ROS), as well as enhanced oxidation and activity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor (RyR2). The transfer of Ca2+ from the SR via RyR2 to mitochondria is thought to play a key role in matching increased metabolic demand during stress. In this study, we investigated whether augmented RyR2 activity results in self-imposed exacerbation of SR Ca2+ leak, via altered SR-mitochondrial Ca2+ transfer and elevated mito-ROS emission. Fluorescent indicators and spatially restricted genetic ROS probes revealed that both pharmacologically and genetically enhanced RyR2 activity, in ventricular myocytes from rats and catecholaminergic polymorphic ventricular tachycardia (CPVT) mice, respectively, resulted in increased ROS emission under β-adrenergic stimulation. Expression of mitochondrial Ca2+ probe mtRCamp1h revealed diminished net mitochondrial [Ca2+] with enhanced SR Ca2+ leak, accompanied by depolarization of the mitochondrial matrix. While this may serve as a protective mechanism to prevent mitochondrial Ca2+ overload, protection is not complete and enhanced mito-ROS emission resulted in oxidation of RyR2, further amplifying proarrhythmic SR Ca2+ release. Importantly, the effects of augmented RyR2 activity could be attenuated by mitochondrial ROS scavenging, and experiments with dominant-negative paralogs of the mitochondrial Ca2+ uniporter (MCU) supported the hypothesis that SR-mitochondria Ca2+ transfer is essential for the increase in mito-ROS. We conclude that in a process whereby leak begets leak, augmented RyR2 activity modulates mitochondrial Ca2+ handling, promoting mito-ROS emission and driving further channel activity in a proarrhythmic feedback cycle in the diseased heart.
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Project description:BackgroundDiabetes and obesity are associated with an increased risk of arrhythmia and sudden cardiac death. Abnormal lipid accumulation is observed in cardiomyocytes of obese and diabetic patients, which may contribute to arrhythmia, but the mechanisms are poorly understood. A transgenic mouse model of cardiac lipid overload, the peroxisome proliferator-activated receptor-γ (PPARg) cardiac overexpression mouse, has long QT and increased ventricular ectopy.ObjectiveThe purpose of this study was to evaluate the hypothesis that the increase in ventricular ectopy during cardiac lipid overload is caused by abnormalities in calcium handling due to increased mitochondrial oxidative stress.MethodsVentricular myocytes were isolated from adult mouse hearts to record sparks and calcium transients. Mice were implanted with heart rhythm monitors for in vivo recordings.ResultsPPARg cardiomyocytes have more frequent triggered activity and increased sparks compared to control. Sparks and triggered activity are reduced by mitotempo, a mitochondrial-targeted antioxidant. This is explained by a significant increase in oxidation of RyR2. Calcium transients are increased in amplitude, and sarcoplasmic reticulum (SR) calcium stores are increased in PPARg cardiomyocytes. Computer modeling of the cardiac action potential demonstrates that long QT contributes to increased SR calcium. Mitotempo decreased ventricular ectopy in vivo.ConclusionDuring cardiac lipid overload, mitochondrial oxidative stress causes increased SR calcium leak by oxidizing RyR2 channels. This promotes ventricular ectopy, which is significantly reduced in vivo by a mitochondrial-targeted antioxidant. These results suggest a potential role for mitochondrial-targeted antioxidants in preventing arrhythmia and sudden cardiac death in obese and diabetic patients.
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