Dilation of human atria: Increased diffusion restrictions for ADP,overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.     

Sarcoplasmic reticulum vesicles embedded in agarose gel exhibit propagating calcium waves

In different cell types, activation of signal transduction pathways leads to the generation of calcium oscillations and/or waves. Due to this important impact for cellular function, calcium waves are the subject of intensive investigations. To study interactions of cell organelles with no influence of the cell membrane, sarcoplasmic reticulum (SR) vesicles and well-coupled mitochondria were reconstituted. For the first time, we demonstrate the generation and propagation of calcium waves in a suspension of sarcoplasmic reticulum vesicles, embedded in an agarose gel. The propagation dynamics resemble those of calcium waves in living cells. Moreover, the addition of well-coupled mitochondria leads to more pronounced and significantly faster propagating waves, demonstrating the importance of the mitochondrial Ca(2+) transport. The experimental and simulation results indicate the resemblance of the in vitro system to an excitable medium.     

Effects of cyclosporine A and its immunosuppressive or non-immunosuppressive derivatives [D-Ser]8-CsA and Cs9 on mitochondria from different brain regions

We studied the functional properties of isolated brain mitochondria (BM) prepared from total rat brain (BM(total)) or from cerebral subregions under basal and Ca(2+) overload conditions in order to evaluate the effects of cyclosporine A (CsA) in a regiospecific manner. CsA-induced effects were compared with those of two derivatives-the none-immunosuppressive [O-(NH(2)(CH2)(5)NHC(O)CH(2))-D-Ser](8)-CsA (Cs9) and its congener, the immunosuppressive [D-Ser](8)-CsA. The glutamate/malate-dependent state 3 respiration of mitochondria (state 3(glu/mal)) differed in region-specific manner (cortex > striatum = cerebellum > substantia nigra > hippocampus), but was significantly increased by 1μM CsA (+21±5%) in all regions. Ca(2+) overload induced by addition of 20μM Ca(2+) caused a significant decrease of state 3(glu/mal) (-45 to -55%) which was almost completely prevented in the presence of 1μM CsA, 1μM Cs9 or 1μM [D-Ser](8)-CsA. Mitochondrial Ca(2+) accumulation thresholds linked to permeability transition (PT) as well as the rate and completeness of mitochondrial Ca(2+) accumulation differed between different brain regions. For the first time, we provide a detailed, regiospecific analysis of Ca(2+)-dependent properties of brain mitochondria. Regardless of their immunosuppressive impact, CsA and its analogues improved mitochondrial functional properties under control conditions. They also preserved brain mitochondria against Ca(2+) overload-mediated PT and functional impairments. Since Cs9 does not mediate immunosuppression, it might be used as a more specific PT inhibitor than CsA.     

Mitochondrial respiratory rates and activities of respiratory chain complexes correlate linearly with heteroplasmy of deleted mtDNA without threshold and independently of deletion size

To clarify the importance of deleted protein and tRNA genes on the impairment of mitochondrial function, we performed a quantitative analysis of biochemical, genetic and morphological findings in skeletal muscles of 16 patients with single deletions and 5 patients with multiple deletions of mtDNA. Clinically, all patients showed chronic progressive external ophthalmoplegia (CPEO). The size of deletions varied between 2.5 and 9 kb, and heteroplasmy between 31% and 94%. In patients with single deletions, the citrate synthase (CS) activity was nearly doubled. Decreased ratios of pyruvate- and succinate-dependent respiration were detected in fibers of all patients in comparison to controls. Inverse and linear correlations without thresholds were established between heteroplasmy and (i) CS referenced activities of the complexes of respiratory chain, (ii) CS referenced maximal respiratory rates, (iii) and cytochrome-c-oxidase (COX) negative fibers. In patients with single and multiple deletions, all respiratory chain complexes as well as the respiratory rates were decreased to a similar extent. All changes detected in patients with single deletions were independent of deletion size. In one patient, only genes of ND5, ND4L as well as tRNA(Leu(CUN)), tRNA(Ser(AGY)), and tRNA(His) were deleted. The pronounced decrease in COX activity in this patient points to the high pathological impact of these missing tRNA genes. The activity of nuclear encoded SDH was also significantly decreased in patients, but to a lesser extent. This is an indication of secondary disturbances of mitochondria at CPEO.In conclusion, we have shown that different deletions cause mitochondrial impairments of the same phenotype correlating with heteroplasmy. The missing threshold at the level of mitochondrial function seems to be characteristic for large-scale deletions were tRNA and protein genes are deleted.     

The influence of the cytosolic oncotic pressure on the permeability of the mitochondrial outer membrane for ADP: implications for the kinetic properties of mitochondrial creatine kinase and for ADP channelling into the intermembrane space

Summary Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed.     

Effects of extramitochondrial ADP on permeability transition of mouse liver mitochondria

Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 microM CAT and 75 microM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 microM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 microM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.     

Complex effects of 17β-estradiol on mitochondrial function

Existing literature on estradiol indicates that it affects mitochondrial functions at low micromolar concentrations. Particularly blockade of the permeability transition pore (PTP) or modulation of the enzymatic activity of one or more complexes of the respiratory chain were suspicious. We prepared mitoplasts from rat liver mitochondria (RLM) to study by single-channel patch-clamp techniques the PTP, and from rat astrocytes to study the potassium BK-channel said to modulate the PTP. Additionally, we measured respiration of intact RLM. After application of 17β-estradiol (βE) our single-channel results reveal a transient increase of activity of both, the BK-channel and the PTP followed by their powerful inhibition. Respiration measurements demonstrate inhibition of the Ca(2+)-induced permeability transition, as well, though only at higher concentrations (≥30μM). At lower concentrations, we observed an increase of endogenous- and state 2-respiration. Furthermore, we show that βE diminishes the phosphorylating respiration supported by complex I-substrates (glutamate/malate) or by the complex II-substrate succinate. Taken together the results suggest that βE affects mitochondria by several modes, including partial inhibition of the activities of ion channels of the inner membrane and of respiration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Reduced Basal Autophagy and Impaired Mitochondrial Dynamics Due to Loss of Parkinson's Disease-Associated Protein DJ-1

Background

Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson''s disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined.

Methodology/Principal Findings

Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2.

Conclusions/Significance

We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson''s disease.