Calcium containing particles in mitochondria of heart muscle cells as shown by cryo-ultramicrotomy and X-ray microanalysis

Summary Mitochondria of normal myocardial cells of the sand rat and the mouse as well as of the left ventricle of man, have been examined for their content of calcium. Ultrahistochemistry and X-ray microanalysis revealed two basically different inclusions: Osmiophilic mitochondrial granules and Spherical mitochondrial particles. Osmiophilic mitochondrial granules were found in conventionally fixed and plastic embedded tissues as well as in cryosections of chemically fixed and sucrose infused tissues. Such granules lacked inert electron density and probably consisted mainly of unsaturated lipids. X-ray spectra obtained from these tissues revealed no peaks for calcium. Spherical mitochondrial particles were present in dry-cut cryo-sections of N₂-frozen tissues not treated by fixatives and/or cryoprotectants. These particles were deeply electron dense in unstained, freeze-dried cryo-sections. They usually measured from 600Å–900Å in diameter in the normal myocardium of the sand rat and the mouse and from 250 Å–400Å in diameter in the left ventricular myocardium of man. Significant calcium peaks could be identified in the X-ray spectra of these particles, whereas none occurred in the analyses of other tissue regions. Potassium was detected with about equal frequency in the particles and in other parts of the tissue. On the basis of the inert electron density of the particles and their absence in chemically fixed tissues as well as of the results of the X-ray analysis, it is concluded that they contain precipitates of extremely labile ions of mitochondrial calcium.We should like to thank Mrs. Trine Jensen for skillful technical assistance. This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from the Norwegian Research Council for Science and the Humanities     

A correlative transmission and scanning electron microscopic study of the pigeon myocardial cell

Summary A comparative study of the pigeon ventricular myocardial cell has been performed by transmission electron microscopy (TEM) and by scanning electron microscopy (SEM). Three-dimensional access to the cell interior was obtained by cryo-fracturing paraffin-embedded tissue immersed in liquid nitrogen. The TEM studies revealed parallelly arranged myofibrils separated by rows of mitochondria. The sarcoplasmic reticulum is represented by a well-developed network of tubules which, at the Z- and H-band level of the sarcomere, expands to form belt-like cisternae. The cisternae at the Z-band level lie in close proximity to both myofilaments and mitochondria. Transverse tubules are absent and thus only peripheral couplings are present.SEM observations of the fractured tissue revealed the spatial relationship between the different cell organelles, the most important of these being the parallel myofibrils and the mitochondria. The conspicuous ridges transversing the myofibril at the Z-band level consist mainly of expanded Z-bands, but overlying SR-tubules also contribute to these ridges. Traces of the SR can sometimes be seen covering the myofibrils. The close proximity between the SR and the mitochondria was also confirmed in the SEM.Preparation and examination of SEM prepared tissue in the TEM confirmed that no essential damage or reorganization of cell organelles had taken place during the SEM procedure. On the other hand some shrinkage of the tissue, which was probably caused by critical point drying, was noticed.     

Regulation of mitochondrial bioenergetics by the non-canonical roles of mitochondrial dynamics proteins in the heart

Recent advancement in mitochondrial research has significantly extended our knowledge on the role and regulation of mitochondria in health and disease. One important breakthrough is the delineation of how mitochondrial morphological changes, termed mitochondrial dynamics, are coupled to the bioenergetics and signaling functions of mitochondria. In general, it is believed that fusion leads to an increased mitochondrial respiration efficiency and resistance to stress-induced dysfunction while fission does the contrary. This concept seems not applicable to adult cardiomyocytes. The mitochondria in adult cardiomyocytes exhibit fragmented morphology (tilted towards fission) and show less networking and movement as compared to other cell types. However, being the most energy-demanding cells, cardiomyocytes in the adult heart possess vast number of mitochondria, high level of energy flow, and abundant mitochondrial dynamics proteins. This apparent discrepancy could be explained by recently identified new functions of the mitochondrial dynamics proteins. These “non-canonical” roles of mitochondrial dynamics proteins range from controlling inter-organelle communication to regulating cell viability and survival under metabolic stresses. Here, we summarize the newly identified non-canonical roles of mitochondrial dynamics proteins. We focus on how these fission and fusion independent roles of dynamics proteins regulate mitochondrial bioenergetics. We also discuss potential molecular mechanisms, unique intracellular location, and the cardiovascular disease relevance of these non-canonical roles of the dynamics proteins. We propose that future studies are warranted to differentiate the canonical and non-canonical roles of dynamics proteins and to identify new approaches for the treatment of heart diseases. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

The incorporation of cholesterol into inner mitochondrial membranes and its effect on lipid phase transition

Incubations of rat liver inner mitochondrial membranes with liposomes prepared from $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) and cholesterol resulted in a considerable enrichment of the cholesterol composition of these membranes. This enrichment is not accompanied by an alteration in the membrane phospholipid content or fatty acid composition. The exogenous cholesterol appears to be integrated into the membrane structure because it has effects consistent with the known properties of this sterol in other natural and artificial membrane systems.Differential scanning calorimetry on both intact membranes and extracted lipids showed that as the ratio of cholesterol to phospholipid was increased, the endotherm corresponding to the lipid phase transition was reduced. Freeze-fracture electron microscopy of the native membranes showed that intramembranous particles are randomly distributed above the phase transition temperature. Below this temperature large smooth areas, believed to correspond to lipid in the gel state from which proteins have been excluded, can be observed. In the presence of high concentrations of cholesterol the fracture faces observed below the lipid transition temperature show no regions of phase segregation, an observation consistent with previous studies using pure lipids where cholesterol was observed to prevent the lipid undergoing a cooperative phase transition.The results are discussed in terms of the observed low concentrations of cholesteorl in normal liver inner mitochondrial membranes and the distribution of cholesterol within the liver cells.     

Localized energization of the mitochondrial inner membrane by ATP

Studies were made to determine whether the energy-dependent binding of ethidium to the mitochondrial inner membrane reflects the membrane potential or the energization of localized regions of the membrane.The number of binding sites of ethidium in mitochondria energized with ATP was 72 nmol/mg protein and decreased with increase in the amount of the ATPase system (F₁ · F_o) inactivated by oligomycin. These findings clearly show that the energy-dependent binding of ethidium to the mitochondrial inner membrane energized with ATP does not reflect the membrane potential, in good accord with the previous conclusion (Higuti, T., Yokota, M., Arakaki, N., Hattori, A. and Tani, I. (1978) Biochim. Biophys. Acta 503, 211–222), but that ethidium binds to localized regions of the energized membrane that are directly affected by ATPase (F₁), reflecting the localized energization of the membrane by ATP.     

Fusion of mitochondrial inner membranes by electric fields produces inside-out vesicles: Visualization by freeze-fracture electron microscopy

The fusion of vesicular-shaped mitochondrial inner membranes was observed by a new approach which combines freeze-fracture electron microscopy and electric field-induced fusion. Results show that membrane events caused by the exposure to the electric field can be time-coordinated with sample freezing for subsequent analysis by freeze-fracture electron microscopy.     

Effects of ionic strength and sulfhydryl reagents on the binding of creatine phosphokinase to heart mitochondrial inner membranes

The concept that creatine phosphokinase is bound to the outer surface of the heart mitochondrial inner membrane originated from observations that the enzyme is retained by water-swollen heart mitochondria and by digitonintreated heart mitochondria suspended in isotonic sucrose. The present study establishes that digitonin-treated mitochondria release creatine phosphokinase in isotonic KCl, and other investigators have reported an identical response for the water-swollen organelles. These observations suggest that mitochondrial creatine phosphokinase is not bound to the outer surface of the inner membrane at a site adjacent to the adenine nucleotide translocase under physiologic conditions.     

Calmodulin dependent NAD-kinase is associated with both the outer and inner mitochondrial membranes in maize roots

Maize root mitochondrial have been subfractionated after osmotic rupture. A calcium-calmodulin-dependent NAD-kinase activity has been shown to be present in both inner and outer membrane fractions. Cytochrome c-reductase activities are also associated with outer and inner membrane fractions but whereas the former is entirely insensitive to 50 mol·1^-1 antimycin A the latter is reduced by 60% in its presence. This residual antimycin A-insensitive cytochrome c-reductase activity cosediments with the major portion of NAD-kinase activity and equilibrates in sucrose gradients at densities around 1.146 g·cm^-3. Rate zonal centrifugation with renografin allows an excellent separation of both cytochrome c-reductase and NAD-kinase activities. We have no evidence for allocating NAD-kinase activity to endo- or plasma membranes.Abbreviations CCO cytochrome c-oxidase - CCR cytochrome c-reductase - IDPase inosine diphosphatase - IMM inner mitochondrial membrane(s) - OMM outer mitochondrial membrane(s)     

Ultrastructure of barley aleurone cells as shown by freeze-etching

Summary Aleurone tissue from non-germinated or germinating barley seeds, as well as from isolated aleurone layers imbibed with water or gibberellic acid, was frozen directly in Freon and investigated by the freeze-etching technique. Aleurone grains remained spherical under all conditions, although the volume increased on hydration. They contained both protein crystalloids and globoids (phytin or lipid) embedded in the matrix. Globoids appeared to be membrane-enclosed. Spherosomes were evidently enclosed within a normal membrane, and they were in close contact with the membrane of aleurone grains, covering the surfaces of the latter. A highly-ordered particle structure was seen on some preparations of the plasmalemma. During germination no alterations were observed in membrane structure.This work was partly supported by a grant from S.O.T.A. (Société coopérative pour l'Achat du Tabac indigène).     

Cardiac-specific overexpression of thioredoxin 1 attenuates mitochondrial and myocardial dysfunction in septic mice

Sepsis-induced myocardial dysfunction is associated with increased oxidative stress and mitochondrial dysfunction. Current evidence suggests a protective role of thioredoxin-1 (Trx1) in the pathogenesis of cardiovascular diseases. However, it is unknown yet a putative role of Trx1 in sepsis-induced myocardial dysfunction, in which oxidative stress is an underlying cause. Transgenic male mice with Trx1 cardiac-specific overexpression (Trx1-Tg) and its wild-type control (wt) were subjected to cecal ligation and puncture or sham surgery. After 6, 18, and 24 h, cardiac contractility, antioxidant enzymes, protein oxidation, and mitochondrial function were evaluated. Trx1 overexpression improved the average life expectancy (Trx1-Tg: 36, wt: 28 h; p = 0.0204). Sepsis induced a decrease in left ventricular developed pressure in both groups, while the contractile reserve, estimated as the response to β-adrenergic stimulus, was higher in Trx1-Tg in relation to wt, after 6 h of the procedure. Trx1 overexpression attenuated complex I inhibition, protein carbonylation, and loss of membrane potential, and preserved Mn superoxide dismutase activity at 24 h. Ultrastructural alterations in mitochondrial cristae were accompanied by reduced optic atrophy 1 (OPA1) fusion protein, and activation of dynamin-related protein 1 (Drp1) (fission protein) in wt mice at 24 h, suggesting mitochondrial fusion/fission imbalance. PGC-1α gene expression showed a 2.5-fold increase in Trx1-Tg at 24 h, suggesting mitochondrial biogenesis induction. Autophagy, demonstrated by electron microscopy and increased LC3-II/LC3-I ratio, was observed earlier in Trx1-Tg. In conclusion, Trx1 overexpression extends antioxidant protection, attenuates mitochondrial damage, and activates mitochondrial turnover (mitophagy and biogenesis), preserves contractile reserve and prolongs survival during sepsis.     

Evolutionary features of chondriome divergence in Triticum (wheat) and Aegilops shown by RFLP analysis of mitochondrial DNAs

The first comprehensive analysis was made of restriction fragment length polymorphism (RFLP) of the mitochondrial (mt) DNA of two related genera, Triticum (wheat) and Aegilops. This led to clarification of the nature of mtDNA variability and the inference of the phylogeny of the mitochondrial genomes (=chondriome). Forty-six alloplasmic lines and one euplasmic line of common wheat (2n = 42, genomes AABBDD) carrying plasmons (cytoplasmic genomes) of 47 accessions belonging to 33 species were used. This consisted of nearly all the Triticum and Aegilops species. RFLP analysis, carried out with seven mitochondrial gene probes (7.0 kb in total) in combination with three restriction endonucleases, found marked variation: Of the 168 bands detected, 165 were variable (98.2%), indicative that there is extremely high mtDNA variability in these genera. This high variability is attributed to the variation present in the intergenic regions. Most of the variation was between chondriomes of different plasmon types; only 8 bands (4.8%) between those of the same plasmon types were variable, evidence of clear chondriome divergence between different plasmon types. The first comprehensive phylogenetic trees of the chondriome were constructed on the basis of genetic distances. All but 1 of the polyploids had chondriomes closely related to those of 1 putative parent, indicative of uniparental chondriome transmission at the time of polyploid formation. The chondriome showed parallel evolutionary divergence to the plastome (chloroplast genome). Use of a minimum set of 3 mtDNA probe-enzyme combinations is proposed for tentative plasmon type identification and the screening of new plasmon types in those genera. Received: 20 March 1999 / Accepted: 22 June 1999     

Energization of mitochondrial inner membranes caused by l-malate

It was found that 0.06 μg antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c₁ completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.     

SIRT1 activation by curcumin pretreatment attenuates mitochondrial oxidative damage induced by myocardial ischemia reperfusion injury

Ischemia reperfusion (IR) injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Silent information regulator 1 (SIRT1), a type of histone deacetylase, contributes to IRI. Curcumin (Cur) is a strong natural antioxidant and is the active component in Curcuma longa; Cur has protective effects against IRI and may regulate the activity of SIRT1. This study was designed to investigate the protective effect of Cur pretreatment on myocardial IRI and to elucidate this potential mechanism. Isolated and in vivo rat hearts and cultured neonatal rat cardiomyocytes were subjected to IR. Prior to this procedure, the hearts or cardiomyocytes were exposed to Cur in the absence or presence of the SIRT1 inhibitor sirtinol or SIRT1 siRNA. Cur conferred a cardioprotective effect, as shown by improved postischemic cardiac function, decreased myocardial infarct size, decreased myocardial apoptotic index, and several biochemical parameters, including the up-regulation of the antiapoptotic protein Bcl2 and the down-regulation of the proapoptotic protein Bax. Sirtinol and SIRT1 siRNA each blocked the Cur-mediated cardioprotection by inhibiting SIRT1 signaling. Cur also resulted in a well-preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase activity, and decreased formation of mitochondrial hydrogen peroxide and malondialdehyde. These observations indicated that the IR-induced mitochondrial oxidative damage was remarkably attenuated. However, this Cur-elevated mitochondrial function was reversed by sirtinol or SIRT1 siRNA treatment. In summary, our results demonstrate that Cur pretreatment attenuates IRI by reducing IR-induced mitochondrial oxidative damage through the activation of SIRT1 signaling.     

Endovascular hypothermia improves post-resuscitation myocardial dysfunction by increasing mitochondrial biogenesis in a pig model of cardiac arrest

The aim of the study was to investigate the effects of endovascular hypothermia on mitochondrial biogenesis in a pig model of prolonged cardiac arrest (CA). Ventricular fibrillation was electrically induced, and animals were left untreated for 10 min; then after 6min of cardiopulmonary resuscitation (CPR), defibrillation was attempted. 25 animals that were successfully resuscitated were randomized into three groups: Sham group (SG, 5, no CA), normal temperature group (NTG, 5 for 12 h observation and 5 for 24 h observation), and endovascular hypothermia group (EHG, 5 for 12 h observation and 5 for 24 h observation). The core temperatures (T_c) in the EHG were maintained at 34 ± 0.5 °C for 6 h by an endovascular hypothermia device (Coolgard 3000), then actively increased at the speed of 0.5 °C per hour during the next 6 h to achieve a normal body temperature, while T_c were maintained at 37.5 ± 0.5 °C in the NTG. Cardiac and mitochondrial functions, the quantification of myocardial mitochondrial DNA (mtDNA), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), nuclear respiratory factor (NRF)-1, and NRF-2 were examined. Results showed that myocardial and mitochondrial injury and dysfunction increased significantly at 12 h and 24 h after CA. Endovascular hypothermia offered a method to rapidly achieve the target temperature and provide stable target temperature management (TTM). Cardiac outcomes were improved and myocardial injuries were alleviated with endovascular hypothermia. Compared with NTG, endovascular hypothermia significantly increased mitochondrial activity and biogenesis by amplifying mitochondrial biogenesis factors’ expressions, including PGC-1α, NRF-1, and NRF-2. In conclusions, endovascular hypothermia after CA alleviated myocardial and mitochondrial dysfunction, and was associated with increasing mitochondrial biogenesis.     

Intercellular mitochondrial transfer as a means of revitalizing injured glomerular endothelial cells

BACKGROUNDRecent studies have demonstrated that mesenchymal stem cells (MSCs) can rescue injured target cells via mitochondrial transfer. However, it has not been fully understood how bone marrow-derived MSCs repair glomeruli in diabetic kidney disease (DKD).AIMTo explore the mitochondrial transfer involved in the rescue of injured glomerular endothelial cells (GECs) by MSCs, both in vitro and in vivo.METHODS In vitro experiments were performed to investigate the effect of co-culture with MSCs on high glucose-induced GECs. The transfer of mitochondria was visua lized using fluorescent microscopy. GECs were freshly sorted and ultimately tested for apoptosis, viability, mRNA expression by real-time reverse transcri ptase-polymerase chain reaction, protein expression by western blot, and mitochondrial function. Moreover, streptozotocin-induced DKD rats were infused with MSCs, and renal function and oxidative stress were detected with an automatic biochemical analyzer and related-detection kits after 2 wk. Kidney histology was analyzed by hematoxylin and eosin, periodic acid-Schiff, and immunohistochemical staining.RESULTSFluorescence imaging confirmed that MSCs transferred mitochondria to injured GECs when co-cultured in vitro. We found that the apoptosis, proliferation, and mitochondrial function of injured GECs were improved following co-culture. Additionally, MSCs decreased pro-inflammatory cytokines [interleukin (IL)-6, IL-1β, and tumor necrosis factor-α] and pro-apoptotic factors (caspase 3 and Bax). Mitochondrial transfer also enhanced the expression of superoxide dismutase 2, B cell lymphoma-2, glutathione peroxidase (GPx) 3, and mitofusin 2 and inhibited reactive oxygen species (ROS) and dynamin-related protein 1 expression. Furthermore, MSCs significantly ameliorated functional parameters (blood urea nitrogen and serum creatinine) and decreased the production of malondialdehyde, advanced glycation end products, and ROS, whereas they increased the levels of GPx and superoxide dismutase in vivo. In addition, significant reductions in the glomerular basement membrane and renal interstitial fibrosis were observed following MSC treatment.CONCLUSIONMSCs can rejuvenate damaged GECs via mitochondrial transfer. Additionally, the improvement of renal function and pathological changes in DKD by MSCs may be related to the mechanism of mitochondrial transfer.