Identification of phosphorylated form of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) as 46 kDa phosphoprotein in brain non-synaptic mitochondria overloaded by calcium

In our previous studies phosphorylation of several membrane-bound proteins in brain and liver mitochondria were found to be regulated by Ca²⁺ as a second messenger. One of the proteins, the 46 kDa phosphoprotein was found to be highly phosphorylated when Ca²⁺-induced permeability transition pore (mPTP) was opened in rat brain mitochondria (RBM). In the present study the 46 kDa phosphoprotein was identified as 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) after purification by 2D diagonal electrophoresis following mass spectrometric analysis and Western blot probed with anti-CNP antibody. CNPase was discovered in immunoprecipitates of mitochondria, phosphorylated under both conditions (control and with opened mPTP). Status phosphorylation of CNPase was found to be higher in the inmmunoprecipiates of calcium-overloaded RBM. The phospohoserine and phosphotyrosine residues were detected in phosphorylated 46 kDa band (CNPase) as well as in CNPase immunoprecipitates indicating possible participation of tyrosine and serine protein kinases in phosphorylation of CNPase in mitochondria. The levels of phospo-Ser and phospho-Tyr were increased in RBM with mPTP opened. It was found that CNPase substrate, 2′,3′-cAMP (5 μM) and, a non-competitive CNPase inhibitor, atractyloside (5 μM), were able to increase the level of CNPase phosphorylation in calcium-overloaded mitochondria, while CsA (mPTP blocker) was able to strong suppress the phosphorylation of the enzyme. Collectively, our results provide evidence that Ca²⁺-stimulated and mPTP-associated CNPase phosphorylation might be an important stage of mPTP regulation in mitochondria, revealing a new function of CNPase outside of myelin structure.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

Induction of permeability of the inner membrane of yeast mitochondria

The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a non-specific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochon-dria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca²⁺-dependent pores, neither the classical Ca²⁺/P_i-dependent, cyclosporin A-sensitive pore even under deenergization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca²⁺ concentrations (in the presence of the Ca²⁺ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Augmented O-GlcNAc signaling attenuates oxidative stress and calcium overload in cardiomyocytes

O-linked β-N-acetylglucosamine (O-GlcNAc) is an inducible, dynamically cycling and reversible post-translational modification of Ser/Thr residues of nucleocytoplasmic and mitochondrial proteins. We recently discovered that O-GlcNAcylation confers cytoprotection in the heart via attenuating the formation of mitochondrial permeability transition pore (mPTP) and the subsequent loss of mitochondrial membrane potential. Because Ca²⁺ overload and reactive oxygen species (ROS) generation are prominent features of post-ischemic injury and favor mPTP formation, we ascertained whether O-GlcNAcylation mitigates mPTP formation via its effects on Ca²⁺ overload and ROS generation. Subjecting neonatal rat cardiac myocytes (NRCMs, n ≥ 6 per group) to hypoxia, or mice (n ≥ 4 per group) to myocardial ischemia reduced O-GlcNAcylation, which later increased during reoxygenation/reperfusion. NRCMs (n ≥ 4 per group) infected with an adenovirus carrying nothing (control), adenoviral O-GlcNAc transferase (adds O-GlcNAc to proteins, AdOGT), adenoviral O-GlcNAcase (removes O-GlcNAc to proteins, AdOGA), vehicle or PUGNAc (blocks OGA; increases O-GlcNAc levels) were subjected to hypoxia–reoxygenation or H₂O₂, and changes in Ca²⁺ levels (via Fluo-4AM and Rhod-2AM), ROS (via DCF) and mPTP formation (via calcein-MitoTracker Red colocalization) were assessed using time-lapse fluorescence microscopy. Both OGT and OGA overexpression did not significantly (P > 0.05) alter baseline Ca²⁺ or ROS levels. However, AdOGT significantly (P < 0.05) attenuated both hypoxia and oxidative stress-induced Ca²⁺ overload and ROS generation. Additionally, OGA inhibition mitigated both H₂O₂-induced Ca²⁺ overload and ROS generation. Although AdOGA exacerbated both hypoxia and H₂O₂-induced ROS generation, it had no effect on H₂O₂-induced Ca²⁺ overload. We conclude that inhibition of Ca²⁺ overload and ROS generation (inducers of mPTP) might be one mechanism through which O-GlcNAcylation reduces ischemia/hypoxia-mediated mPTP formation.     

Mitochondrial bioenergetics deregulation caused by long-chain 3-hydroxy fatty acids accumulating in LCHAD and MTP deficiencies in rat brain: A possible role of mPTP opening as a pathomechanism in these disorders?

Long-chain 3-hydroxylated fatty acids (LCHFA) accumulate in long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (MTP) deficiencies. Affected patients usually present severe neonatal symptoms involving cardiac and hepatic functions, although long-term neurological abnormalities are also commonly observed. Since the underlying mechanisms of brain damage are practically unknown and have not been properly investigated, we studied the effects of LCHFA on important parameters of mitochondrial homeostasis in isolated mitochondria from cerebral cortex of developing rats. 3-Hydroxytetradecanoic acid (3 HTA) reduced mitochondrial membrane potential, NAD(P)H levels, Ca^2 + retention capacity and ATP content, besides inducing swelling, cytochrome c release and H₂O₂ production in Ca^2 +-loaded mitochondrial preparations_. We also found that cyclosporine A plus ADP, as well as ruthenium red, a Ca^2 + uptake blocker, prevented these effects, suggesting the involvement of the mitochondrial permeability transition pore (mPTP) and an important role for Ca^2 +, respectively. 3-Hydroxydodecanoic and 3-hydroxypalmitic acids, that also accumulate in LCHAD and MTP deficiencies, similarly induced mitochondrial swelling and decreased ATP content, but to a variable degree pending on the size of their carbon chain. It is proposed that mPTP opening induced by LCHFA disrupts brain bioenergetics and may contribute at least partly to explain the neurologic dysfunction observed in patients affected by LCHAD and MTP deficiencies.     

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.     

Synaptic and nonsynaptic mitochondria demonstrate a different degree of calcium-induced mitochondrial dysfunction

AimsSince variety in response to Ca²⁺-induced mitochondrial dysfunction in different neuronal mitochondrial populations is associated with the pathogenesis of several neurological diseases, we investigated the effects of Ca²⁺ overload on synaptic (SM) and nonsynaptic mitochondrial (NM) dysfunction and probed the effects of cyclosporin A (CsA), 4′-chlorodiazepam (CDP) and Ru360 on relieving mitochondrial damage.Main methodsSM and NM mitochondria were isolated from rats' brains (n = 5/group) and treated with various concentrations (5, 10, 100, and 200 μM) of Ca²⁺, with and without CsA (mPTP blocker), CDP (PBR/TSPO blocker) and Ru360 (MCU blocker) pretreatments. Mitochondrial function was determined by mitochondrial swelling, ROS production and mitochondrial membrane potential changes (ΔΨm).Key findingsAt 200-μM Ca²⁺, SM presented mitochondrial swelling to a greater extent than NM. At 100 and 200-μM Ca²⁺, the ROS production of SM was higher than that of NM and ΔΨm dissipation of SM was also larger. CsA, CDP and Ru360 could reduce ROS production of SM and NM with exposure to 200-μM Ca²⁺. However, only Ru360 could completely inhibit ROS generation in both SM and NM, whereas CsA and CDP could only partially reduce the ROS level in SM. Moreover, CsA and CDP pretreatments were not able to restore ΔΨm. However, Ru360 pretreatment could protect ΔΨm dissipation in both SM and NM, with complete protection observed only in NM.SignificanceOur findings suggested that mitochondrial calcium uniporter is a possible major pathway for calcium uptake in both mitochondrial populations. However, SM might have additional pathways involved in the calcium uptake.     

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.Key words: Bnip3, autophagy, cardiac myocytes, mitochondria, permeability transition pore, cyclophilin D     

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.     

The effect of complexes of precursors and modulators of coenzyme Q biosynthesis on the functional state of heart mitochondria of old rats

Aging is accompanied by changes in activity of electron-transport enzyme complexes in myocardial mitochondria of old rats and by increased sensitivity of the mitochondrial permeability transition pore (MPTP) to inductors of its opening (Ca²⁺ and phenylarsine oxide). We also observed activation of lipid and protein free-radical peroxidation processes. Administration of a complex of biologically active substances that included precursors and modulators of coenzyme Q biosynthesis (α-tocopherol acetate, 4-hydroxybenzoic acid, and methionine) caused the increase in coenzyme Q content, correction of functional activity of mitochondrial electron-transport chain enzyme complexes, the decrease in intensity of lipid and protein free-radical peroxidation in the heart mitochondria and the decrease in sensitivity of mitochondrial permeability transition pore to inductors of its opening. This complex may be recommended for treatment of mitochondrial dysfunction in various pathologies of cardiovascular system, including in aging.     

Translocator protein (18 kDa) ligand PK 11195 induces transient mitochondrial Ca2+ release leading to transepithelial Cl- secretion in HT-29 human colon cancer cells

Background information. TSPO (translocator protein), known previously as PBR (peripheral‐type benzodiazepine receptor), is a 18 kDa protein expressed in the mitochondrial membrane of a variety of tissues. TSPO has been reported to be over‐expressed in human colorectal tumours and cancer cell lines, but its function is not well characterized. Results. We investigated the expression and function of TSPO in the human colon cancer cells HT‐29. Immunohistochemical studies revealed that TSPO is localized in mitochondria, and its endogenous ligand, the polypeptide diazepam‐binding inhibitor, in the cytosol. Radioligand binding studies using the specific high‐affinity drug ligand [³H]PK 11195 and membrane fraction demonstrated saturable binding, with K_d and B_max values of 13.5±1.5 nM and 10.1±1.0 pmol/mg respectively. PK 11195 induced a rapid and transient dose‐dependent rise in intracellular [Ca²⁺], which was unaffected by extracellular Ca²⁺, but was blocked by the PTP (permeability transition pore) inhibitor, cyclosporin A, and by the TSPO partial agonist, flunitrazepam. Using HT‐29 clone 19A cell line, which forms cell monolayers, we demonstrated that TSPO ligand stimulated a Ca²⁺‐dependent transepithelial Cl^? secretion. This secretion was inhibited: (i) after removal of extracellular Cl^?; (ii) by apical addition of the Cl^? channel blocker NPPB [5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate]; and (iii) by basolateral addition of the Na⁺–K⁺–2Cl^? co‐transporter inhibitor bumetanide. Furthermore, the intracellular Ca²⁺ chelator BAPTA/AM [bis‐(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis(acetoxymethyl ester)] and cyclosporin A abolished the rise in PK 11195‐induced Cl^? secretion. Conclusions. These findings indicate that TSPO is located in mitochondrial membranes of HT‐29 and reveal that its activation induces a rise in cytosolic Ca²⁺, leading to the stimulation of Cl^? secretion.     

Cinnamon intake alleviates the combined effects of dietary-induced insulin resistance and acute stress on brain mitochondria

Insulin resistance (IR), which is a leading cause of the metabolic syndrome, results in early brain function alterations which may alter brain mitochondrial functioning. Previously, we demonstrated that rats fed a control diet and submitted to an acute restraint stress exhibited a delayed mitochondrial permeability transition pore (mPTP) opening. In this study, we evaluated the combined effects of dietary and emotional stressors as found in western way of life. We studied, in rats submitted or not to an acute stress, the effects of diet-induced IR on brain mitochondria, using a high fat/high fructose diet (HF²), as an IR inducer, with addition or not of cinnamon as an insulin sensitizer. We measured Ca²⁺ retention capacity, respiration, ROS production, enzymatic activities and cell signaling activation. Under stress, HF² diet dramatically decreased the amount of Ca²⁺ required to open the mPTP (13%) suggesting an adverse effect on mitochondrial survival. Cinnamon added to the diet corrected this negative effect and resulted in a partial recovery (30%). The effects related to cinnamon addition to the diet could be due to its antioxidant properties or to the observed modulation of PI3K-AKT-GSK3β and MAPK-P38 pathways or to a combination of both. These data suggest a protective effect of cinnamon on brain mitochondria against the negative impact of an HF² diet. Cinnamon could be beneficial to counteract deleterious dietary effects in stressed conditions.     

Ischemic Postconditioning Reduces NMDA Receptor Currents Through the Opening of the Mitochondrial Permeability Transition Pore and KATP Channel in Mouse Neurons

Ischemic postconditioning (PostC) is known to reduce cerebral ischemia/reperfusion (I/R) injury; however, whether the opening of mitochondrial ATP-dependent potassium (mito-K_ATP) channels and mitochondrial permeability transition pore (mPTP) cause the depolarization of the mitochondrial membrane that remains unknown. We examined the involvement of the mito-K_ATP channel and the mPTP in the PostC mechanism. Ischemic PostC consisted of three cycles of 15 s reperfusion and 15 s re-ischemia, and was started 30 s after the 7.5 min ischemic load. We recorded N-methyl-d-aspartate receptors (NMDAR)-mediated currents and measured cytosolic Ca²⁺ concentrations, and mitochondrial membrane potentials in mouse hippocampal pyramidal neurons. Both ischemic PostC and the application of a mito-K_ATP channel opener, diazoxide, reduced NMDAR-mediated currents, and suppressed cytosolic Ca²⁺ elevations during the early reperfusion period. An mPTP blocker, cyclosporine A, abolished the reducing effect of PostC on NMDAR currents. Furthermore, both ischemic PostC and the application of diazoxide potentiated the depolarization of the mitochondrial membrane potential. These results indicate that ischemic PostC suppresses Ca²⁺ influx into the cytoplasm by reducing NMDAR-mediated currents through mPTP opening. The present study suggests that depolarization of the mitochondrial membrane potential by opening of the mito-K_ATP channel is essential to the mechanism of PostC in neuroprotection against anoxic injury.

     

Physiological roles of the mitochondrial permeability transition pore

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca²⁺ exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F₁F_O ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.     

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

Impairment of mitochondria function and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9–20)-, mid-age (48–60)-, and >60 (62–76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H₂O₂ toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca²⁺ [Ca²⁺]_c, and mitochondrial Ca²⁺ [Ca²⁺]_m levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5′,6,6′-tetrachloro 1,1′3,3′-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca²⁺]_c, and increased sequestration of [Ca²⁺]_m in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.     

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.     

Mitochondrial function in permeabilized cardiomyocytes is largely preserved in the senescent rat myocardium

The aging heart is characterized by a progressive decline in contractile function and diastolic relaxation. Amongst the factors implicated in these changes is a progressive replacement fibrosis secondary to cardiomyoctye death, oxidative damage, and energetic deficit, each of which may be secondary to impaired mitochondrial function. Here, we performed an in-depth examination of mitochondrial function in saponin-permeabilized cardiomyocyte bundles, a preparation where all mitochondria are represented and their structure intact, from young adult (YA) and senescent (SEN) rats (n = 8 per group). When accounting for increased fibrosis (+19%, P<0.01) and proportional decrease in citrate synthase activity in the SEN myocardium (−23%, P<0.05), mitochondrial respiration and reactive oxygen species (H₂O₂) emission across a range of energized states was similar between age groups. Accordingly, the abundance of electron transport chain proteins was also unchanged. Likewise, except for CuZnSOD (−37%, P<0.05), the activity of antioxidant enzymes was unaltered with aging. Although time to mitochondrial permeability transition pore (mPTP) opening was decreased (−25%, P<0.05) in the SEN heart, suggesting sensitization to apoptotic stimuli, this was not associated with a difference in apoptotic index measured by ELISA. Collectively, our results suggest that the function of existing cardiac ventricular mitochondria is relatively preserved in SEN rat heart when measured in permeabilized cells.     

Effect of peripheral benzodiazepine receptor (PBR/TSPO) ligands on opening of Ca<Superscript>2+</Superscript>-induced pore and phosphorylation of 3.5-kDa polypeptide in rat brain mitochondria

The effect of nanomolar concentrations of PBR/TSPO ligands—Ro 5-4864, PK11195, and PPIX—on Ca²⁺-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 μM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [γ-³²P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca²⁺ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of F_oF₁-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of F_oF₁-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca²⁺- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.     

ADP Protects Cardiac Mitochondria under Severe Oxidative Stress

ADP is not only a key substrate for ATP generation, but also a potent inhibitor of mitochondrial permeability transition pore (mPTP). In this study, we assessed how oxidative stress affects the potency of ADP as an mPTP inhibitor and whether its reduction of reactive oxygen species (ROS) production might be involved. We determined quantitatively the effects of ADP on mitochondrial Ca²⁺ retention capacity (CRC) until the induction of mPTP in normal and stressed isolated cardiac mitochondria. We used two models of chronic oxidative stress (old and diabetic mice) and two models of acute oxidative stress (ischemia reperfusion (IR) and tert-butyl hydroperoxide (t-BH)). In control mitochondria, the CRC was 344 ± 32 nmol/mg protein. 500 μmol/L ADP increased CRC to 774 ± 65 nmol/mg protein. This effect of ADP seemed to relate to its concentration as 50 μmol/L had a significantly smaller effect. Also, oligomycin, which inhibits the conversion of ADP to ATP by F₀F₁ATPase, significantly increased the effect of 50 μmol/L ADP. Chronic oxidative stress did not affect CRC or the effect of 500 μmol/L ADP. After IR or t-BH exposure, CRC was drastically reduced to 1 ± 0.2 and 32 ± 4 nmol/mg protein, respectively. Surprisingly, ADP increased the CRC to 447 ± 105 and 514 ± 103 nmol/mg protein in IR and t-BH, respectively. Thus, it increased CRC by the same amount as in control. In control mitochondria, ADP decreased both substrate and Ca²⁺-induced increase of ROS. However, in t-BH mitochondria the effect of ADP on ROS was relatively small. We conclude that ADP potently restores CRC capacity in severely stressed mitochondria. This effect is most likely not related to a reduction in ROS production. As the effect of ADP relates to its concentration, increased ADP as occurs in the pathophysiological situation may protect mitochondrial integrity and function.     

Age-dependent changes of mitochondrial functions in Ca<Superscript>2+</Superscript>-induced opening of permeability transition pore

Mitochondria are intracellular organelles, which provide cells with energy and participate in multiple processes of cell vital functions. Within one of the numerous theories of aging, dysfunction of mitochondria is considered to lead to tissue degeneration and induce the initial stage in developing of degenerative diseases. Since mitochondria play a clue role in apoptosis/necrosis processes, it was suggested that dysfunction of mitochondria observed under aging is related with disturbance of programmed cell death regulation. In the present study, a comparative examination of parameters of the functional states of mitochondria isolated from young (2–3-months old) and old (20–22-months old) rats under conditions of opening of unselective pore (PTP, permeability transition pore) has been performed. Ca²⁺ accumulation rate in mitochondria isolated from old rats was found to be decreased by 25–30%, threshold calcium concentration was lowered to 50%, and the swelling of mitochondria loaded by calcium was stimulated 3–4-fold. Production of reactive oxygen species (ROS) has been also determined in these mitochondria. In old mitochondria superoxide anion level was increased. In addition, H₂O₂ content was found to be 2 times higher in mitochondria with PTP opened. Using electron microscopy method, a decreased amount of cristae in mitochondria was revealed under aging.