A QUANTITATIVE DESCRIPTION OF CORTISONE-INDUCED ALTERATIONS IN THE ULTRASTRUCTURE OF RAT LIVER PARENCHYMAL CELLS

A stereological comparison of the hepatic parenchymal cells from 125-g male rats given a daily injection for 6 days of either 5 mg of cortisone acetate or saline (controls) was carried out with both light and electron microscopy. Cortisone treatment results in an increase in average parenchymal cell cytoplasmic volume from 5100 to 5800 µ³ and a decrease in average nuclear diameter from 7.1 to 6.5 µ. The volume of the average mitochondrion is increased fourfold in midzonal and peripheral regions of hepatic lobules, and there is a decrease in the number of mitochondria per cell such that the total mitochondrial volume per cell remains approximately unchanged. The numbers of peroxisomes are reduced, while the numbers of lysosomes and lipid droplets are increased in all parts of the lobules. The average volume of glycogen is doubled in all cells. The areas of membranes of the smooth- and rough-surfaced endoplasmic reticulum are decreased to one-half and two-thirds of their control values, respectively. The effects of cortisone on these various structural elements is discussed with respect to steroid-related alterations in biochemical processes.     

Growth Dynamics of Mitochondria in Synchronized Chinese Hamster Cells

The increase in cell volume (from electronic cell sizing) and the apportionment of this volume amongst the nuclear, cytoplasmic, and mitochondrial subcellular compartments (from electron microscopy) were studied throughout the cell division cycle in partially synchronized cultures of Chinese hamster V79-S171 cells. Average whole cell volume was found to increase smoothly, consistent with the doubling in one generation of individual cell volume. Nuclear size increased in like fashion. Mean total mitochondrial volume and number of mitochondria per cell both showed a different kind of variation, most notably a significant decrease in G₁ and G₂ as compared with mid S. These results are therefore counter to a model of simple doubling of mitochondria either synchronously with the cell division cycle or asynchronously. Absolute mean values per cell for log phase Chinese hamster cells were also determined, as follows: whole cell volume, 710 μ³; nuclear volume, 190 μ³; total mitochondrial volume, 37.5 μ³; number of mitochondria per cell, 90.     

Dynamic Adaptation of Liver Mitochondria to Chronic Alcohol Feeding in Mice: BIOGENESIS,REMODELING, AND FUNCTIONAL ALTERATIONS*

Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD⁺ and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD⁺, the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver.     

Involvment of Cytosolic and Mitochondrial GSK-3β in Mitochondrial Dysfunction and Neuronal Cell Death of MPTP/MPP+-Treated Neurons

Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson''s disease (PD). 1-methyl-4-phenylpyridinium iodide (MPP⁺), the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3β (GSK-3β), a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3β in modulating MPP⁺-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP⁺ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3β, evidenced by the increased level of the active form of the kinase, i.e. GSK-3β phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3β partially localized within mitochondria in both neuronal cell models. Moreover, MPP⁺ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3β labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP⁺ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3β activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP⁺-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3β is a critical mediator of MPTP/MPP⁺-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3β activity might provide protection against mitochondrial stress-induced cell death.     

IFN‐β rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource‐demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN‐β is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN‐β induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN‐β signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria–endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN‐β in the Ifnb ^–/– model of Parkinson disease (PD) disrupts STAT5‐PGAM5‐Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN‐β rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN‐β activates mitochondrial fission in neurons through the pSTAT5/PGAM5/^S622Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.     

Oxygen consumption and the composition of skeletal muscle tissue after training and inactivation in the European woodmouse (Apodemus sylvaticus)

Summary In European woodmice the amount and intensity of daily activity was compared to oxygen uptake and to the potential for oxidative metabolism of heart and skeletal muscle. One group of animals was inactivated by exposition to light during night time; another group of animals was trained by enforced running on a treadmill. The oxidative potential of the muscle tissue was assessed by morphometry of capillaries and mitochondria. A novel sampling technique was used which allowed us to obtain morphological data related to single muscles, to muscle groups, and finally to whole body muscle mass.Reducing the spontaneous activity by ten fold had no effect on oxygen uptake nor on capillaries or mitochondria in locomotory muscles. Mitochondrial volume was reduced, however, in heart and diaphragm. Enforced running increased the weight specific maximal oxygen uptake significantly. It also increased the mitochondrial volume in heart and diaphragm as well as in M. tibialis anterior. Capillary densities were neither affected by training nor by inactivation. A significant correlation was found between the capillary density and the volume density of mitochondria in all muscles analysed morphometrically. For the whole skeletal muscle mass of a European woodmouse the inner mitochondrial membranes were estimated to cover 30 m². The oxygen consumption per unit time and per unit volume of muscle mitochondrion was found to be identical in all groups of animals (4.9 ml O₂ min^–1 cm^–3).Symbols S _v (im,m) surface area of inner mitochondrial membranes per unit mitochondrial volume - V _v (mt, f) volume density of mitochondria (mitochondrial volume per fiber volume) - V (mt) total mitochondrial volume - V (f) muscle volume - N _A (c, f) capillary density - (f) mean fiber cross-sectional area     

Differential mitochondrial roles for α-synuclein in DRP1-dependent fission and PINK1/Parkin-mediated oxidation

Mitochondria are highly dynamic organelles with strict quality control processes that maintain cellular homeostasis. Within axons, coordinated cycles of fission-fusion mediated by dynamin related GTPase protein (DRP1) and mitofusins (MFN), together with regulated motility of healthy mitochondria anterogradely and damaged/oxidized mitochondria retrogradely, control mitochondrial shape, distribution and size. Disruption of this tight regulation has been linked to aberrant oxidative stress and mitochondrial dysfunction causing mitochondrial disease and neurodegeneration. Although pharmacological induction of Parkinson’s disease (PD) in humans/animals with toxins or in mice overexpressing α-synuclein (α-syn) exhibited mitochondrial dysfunction and oxidative stress, mice lacking α-syn showed resistance to mitochondrial toxins; yet, how α-syn influences mitochondrial dynamics and turnover is unclear. Here, we isolate the mechanistic role of α-syn in mitochondrial homeostasis in vivo in a humanized Drosophila model of Parkinson’s disease (PD). We show that excess α-syn causes fragmented mitochondria, which persists with either truncation of the C-terminus (α-syn^1–120) or deletion of the NAC region (α-syn^ΔNAC). Using in vivo oxidation reporters Mito-roGFP2-ORP1/GRX1 and MitoTimer, we found that α-syn-mediated fragments were oxidized/damaged, but α-syn^1–120-induced fragments were healthy, suggesting that the C-terminus is required for oxidation. α-syn-mediated oxidized fragments showed biased retrograde motility, but α-syn^1–120-mediated healthy fragments did not, demonstrating that the C-terminus likely mediates the retrograde motility of oxidized mitochondria. Depletion/inhibition or excess DRP1-rescued α-syn-mediated fragmentation, oxidation, and the biased retrograde motility, indicating that DRP1-mediated fragmentation is likely upstream of oxidation and motility changes. Further, excess PINK/Parkin, two PD-associated proteins that function to coordinate mitochondrial turnover via induction of selective mitophagy, rescued α-syn-mediated membrane depolarization, oxidation and cell death in a C-terminus-dependent manner, suggesting a functional interaction between α-syn and PINK/Parkin. Taken together, our findings identify distinct roles for α-syn in mitochondrial homeostasis, highlighting a previously unknown pathogenic pathway for the initiation of PD.Subject terms: Mechanisms of disease, Parkinson''s disease     

Formation of enlarged mitochondria in a liver cell line in response to a synthetic glucocorticoid

For a number of years it has been recognized that glucocorticoids cause alterations in liver cell morphology (6, 9). Several investigators have shown that in liver in vivo mitochondria can be enlarged to many times their normal volume by treatment with cortisone (13, 15). There is a concomitant decrease in mitochondrial number, and the results of Kimberg and Loeb suggest that this is due to mitochondrial fusion (7). However, the exact mechanism whereby mitochondrial volume is altered and whether in fact cortisone is the direct causal agent are not known due to the complexity of studying these questions in a whole animal system. We have found that dexamethasone sodium phosphate (dex), a synthetic glucocorticoid, causes the formation of enlarged mitochondria in a liver cell line RLC-GAI, which grows in defined medium. In this paper we present our observations on the amount of enlargement that occurs after 5 days of treatment. The formation of enlarged mitochondria is reversible upon removal of the hormone from the medium, and we have attempted to determine whether "mitochondrial" or "nonmitochondrial" inhibitors are more effective in blocking the return of mitochondria to their normal size when the hormone is removed.     

Electron microscopy morphology of the mitochondrial network in human cancer

Mitochondria have been implicated in the process of carcinogenesis, which includes alterations of cellular metabolism and cell death pathways. The aim of this review is to describe and analyze the electron microscopy morphology of the mitochondrial network in human cancer. The structural mitochondrial alterations in human tumors are heterogeneous and not specific for any neoplasm. These findings could be representing an altered structural and functional mitochondrial network. The mitochondria in cancer cells, independently of histogenesis, predominantly are seen with lucent-swelling matrix associated with disarrangement and distortion of cristae and partial or total cristolysis and with condensed configuration in minor scale. Mitochondrial changes are associated with mitochondrial-DNA mutations, tumoral microenvironment conditions and mitochondrial fusion–fission disequilibrium. Functionally, the structural alterations suppose the presence of hypoxia-tolerant and hypoxia-sensitive cancer cells. Possibly, hypoxia-tolerant cells are related with mitochondrial condensed appearance and are competent to produce adequate amount of ATP by mitochondrial respiration. Hypoxia-sensitive cells are linked with lucent-swelling and cristolysis mitochondria profile and have an inefficient or null oxidative phosphorylation, which consequently use the glycolytic pathway to generate energy. Additionally, mitochondrial fragmentation is associated with apoptosis; however, alterations in the mitochondrial network are linked with the reduction in sensitivity to apoptosis induces and/or pro-apoptotic conditions. Pharmacological approaches designed to act on both glycolysis and oxidative phosphorylation can be considered as a new approach to selectively kill cancer cells.     

Sulforaphane inhibits mitochondrial permeability transition and oxidative stress

Exposure of mitochondria to oxidative stress and elevated Ca²⁺ promotes opening of the mitochondrial permeability transition pore (PTP), resulting in membrane depolarization, uncoupling of oxidative phosphorylation, and potentially cell death. This study tested the hypothesis that treatment of rats with sulforaphane (SFP), an activator of the Nrf2 pathway of antioxidant gene expression, increases the resistance of liver mitochondria to redox-regulated PTP opening and elevates mitochondrial levels of antioxidants. Rats were injected with SFP or drug vehicle and liver mitochondria were isolated 40 h later. Respiring mitochondria actively accumulated added Ca²⁺, which was then released through PTP opening induced by agents that either cause an oxidized shift in the mitochondrial redox state or directly oxidize protein thiol groups. SFP treatment of rats inhibited the rate of pro-oxidant-induced mitochondrial Ca²⁺ release and increased expression of the glutathione peroxidase/reductase system, thioredoxin, and malic enzyme. These results are the first to demonstrate that SFP treatment of animals increases liver mitochondrial antioxidant defenses and inhibits redox-sensitive PTP opening. This novel form of preconditioning could protect against a variety of pathologies that include oxidative stress and mitochondrial dysfunction in their etiologies.     

Sirtuin 3 regulates mitochondrial protein acetylation and metabolism in tubular epithelial cells during renal fibrosis

Proximal tubular epithelial cells (TECs) demand high energy and rely on mitochondrial oxidative phosphorylation as the main energy source. However, this is disturbed in renal fibrosis. Acetylation is an important post-translational modification for mitochondrial metabolism. The mitochondrial protein NAD⁺-dependent deacetylase sirtuin 3 (SIRT3) regulates mitochondrial metabolic function. Therefore, we aimed to identify the changes in the acetylome in tubules from fibrotic kidneys and determine their association with mitochondria. We found that decreased SIRT3 expression was accompanied by increased acetylation in mitochondria that have separated from TECs during the early phase of renal fibrosis. Sirt3 knockout mice were susceptible to hyper-acetylated mitochondrial proteins and to severe renal fibrosis. The activation of SIRT3 by honokiol ameliorated acetylation and prevented renal fibrosis. Analysis of the acetylome in separated tubules using LC–MS/MS showed that most kidney proteins were hyper-acetylated after unilateral ureteral obstruction. The increased acetylated proteins with 26.76% were mitochondrial proteins which were mapped to a broad range of mitochondrial pathways including fatty acid β-oxidation, the tricarboxylic acid cycle (TCA cycle), and oxidative phosphorylation. Pyruvate dehydrogenase E1α (PDHE1α), which is the primary link between glycolysis and the TCA cycle, was hyper-acetylated at lysine 385 in TECs after TGF-β1 stimulation and was regulated by SIRT3. Our findings showed that mitochondrial proteins involved in regulating energy metabolism were acetylated and targeted by SIRT3 in TECs. The deacetylation of PDHE1α by SIRT3 at lysine 385 plays a key role in metabolic reprogramming associated with renal fibrosis.Subject terms: Protein-protein interaction networks, End-stage renal disease     

STRUCTURAL CHANGES IN ISOLATED LIVER MITOCHONDRIA OF RATS DURING ESSENTIAL FATTY ACID DEFICIENCY

Liver mitochondria isolated in 0.44 M sucrose from rats deficient in essential fatty acids (EFA) oxidized citrate, succinate, α-ketoglutarate, glutamate, and pyruvate at a faster rate than did mitochondria isolated from normal rats; however, the oxidation of malate, caprylate, and β-hydroxybutyrate was not significantly increased. The mitochondria from deficient rats exhibited an increased ATPase activity and extensive structural damage as revealed by electron microscope examination of thin sections. An increase in citrate oxidation and ATPase activity, together with some structural damage, could be demonstrated as early as the 4^th week in rats on a fat-free diet. Saturated fat in the diet did not prevent the change in mitochondrial structure but accelerated its appearance. Both the biochemical and structural defects could be reversed within three weeks after feeding deficient rats a source of EFA. In the presence of a phosphate acceptor the effect of EFA deficiency on substrate oxidation was largely eliminated. A trend toward a reduced efficiency of oxidative phosphorylation was noted in mitochondria from EFA-deficient rats, but significant uncoupling was found only in the case of citrate, β-hydroxybutyrate, and glutamate in the presence of malonate. Together with the increased ATPase activity, the uncoupling of phosphorylation could account for the poor respiratory control found with the deficient preparation. However, EFA deficiency was without effect on the respiration of liver slices, which supports the belief that the observed changes in oxidation and phosphorylation are an artifact resulting from damage sustained by the deficient mitochondria during their isolation.     

NIK promotes metabolic adaptation of glioblastoma cells to bioenergetic stress

Cancers, including glioblastoma multiforme (GBM), undergo coordinated reprogramming of metabolic pathways that control glycolysis and oxidative phosphorylation (OXPHOS) to promote tumor growth in diverse tumor microenvironments. Adaptation to limited nutrient availability in the microenvironment is associated with remodeling of mitochondrial morphology and bioenergetic capacity. We recently demonstrated that NF-κB-inducing kinase (NIK) regulates mitochondrial morphology to promote GBM cell invasion. Here, we show that NIK is recruited to the outer membrane of dividing mitochondria with the master fission regulator, Dynamin-related protein1 (DRP1). Moreover, glucose deprivation-mediated metabolic shift to OXPHOS increases fission and mitochondrial localization of both NIK and DRP1. NIK deficiency results in decreased mitochondrial respiration, ATP production, and spare respiratory capacity (SRC), a critical measure of mitochondrial fitness. Although IκB kinase α and β (IKKα/β) and NIK are required for OXPHOS in high glucose media, only NIK is required to increase SRC under glucose deprivation. Consistent with an IKK-independent role for NIK in regulating metabolism, we show that NIK phosphorylates DRP1-S616 in vitro and in vivo. Notably, a constitutively active DRP1-S616E mutant rescues oxidative metabolism, invasiveness, and tumorigenic potential in NIK^−/− cells without inducing IKK. Thus, we establish that NIK is critical for bioenergetic stress responses to promote GBM cell pathogenesis independently of IKK. Our data suggest that targeting NIK may be used to exploit metabolic vulnerabilities and improve therapeutic strategies for GBM.Subject terms: Cancer metabolism, Energy metabolism, Cancer metabolism     

DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA

Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK^– osteosarcoma cells that had been depleted of mtDNA (ρ⁰) or not (wt). Despite the total absence of mtDNA in the ρ⁰ cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in ρ⁰ relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase γ activities were more affected, 65 and 45% lower, respectively, in ρ⁰ mitochondria. Overall BER activity in lysates was also about 65% reduced in ρ⁰ mitochondria. To identify the limiting deficiencies in BER of ρ⁰ mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase γ. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase γ. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in ρ⁰ mitochondria. While nuclear BER protein levels and activities were generally not altered in ρ⁰ cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in ρ⁰ cells, and not a general dysfunction of ρ⁰ cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.     

Exendin-4 Protects Mitochondria from Reactive Oxygen Species Induced Apoptosis in Pancreatic Beta Cells

Objective

Mitochondrial oxidative stress is the basis for pancreatic β-cell apoptosis and a common pathway for numerous types of damage, including glucotoxicity and lipotoxicity. We cultivated mice pancreatic β-cell tumor Min6 cell lines in vitro and observed pancreatic β-cell apoptosis and changes in mitochondrial function before and after the addition of Exendin-4. Based on these observations, we discuss the protective role of Exendin-4 against mitochondrial oxidative damage and its relationship with Ca²⁺-independent phospholipase A2.

Methods

We established a pancreatic β-cell oxidative stress damage model using Min6 cell lines cultured in vitro with tert-buty1 hydroperoxide and hydrogen peroxide. We then added Exendin-4 to observe changes in the rate of cell apoptosis (Annexin-V-FITC-PI staining flow cytometry and DNA ladder). We detected the activity of the caspase 3 and 8 apoptotic factors, measured the mitochondrial membrane potential losses and reactive oxygen species production levels, and detected the expression of cytochrome c and Smac/DLAMO in the cytosol and mitochondria, mitochondrial Ca₂-independent phospholipase A2 and Ca²⁺-independent phospholipase A2 mRNA.

Results

The time-concentration curve showed that different percentages of apoptosis occurred at different time-concentrations in tert-buty1 hydroperoxide- and hydrogen peroxide-induced Min6 cells. Incubation with 100 µmol/l of Exendin-4 for 48 hours reduced the Min6 cell apoptosis rate (p<0.05). The mitochondrial membrane potential loss and total reactive oxygen species levels decreased (p<0.05), and the release of cytochrome c and Smac/DLAMO from the mitochondria was reduced. The study also showed that Ca²⁺-independent phospholipase A2 activity was positively related to Exendin-4 activity.

Conclusion

Exendin-4 reduces Min6 cell oxidative damage and the cell apoptosis rate, which may be related to Ca²-independent phospholipase A2.     

A Reaction-Diffusion Model of ROS-Induced ROS Release in a Mitochondrial Network

Loss of mitochondrial function is a fundamental determinant of cell injury and death. In heart cells under metabolic stress, we have previously described how the abrupt collapse or oscillation of the mitochondrial energy state is synchronized across the mitochondrial network by local interactions dependent upon reactive oxygen species (ROS). Here, we develop a mathematical model of ROS-induced ROS release (RIRR) based on reaction-diffusion (RD-RIRR) in one- and two-dimensional mitochondrial networks. The nodes of the RD-RIRR network are comprised of models of individual mitochondria that include a mechanism of ROS-dependent oscillation based on the interplay between ROS production, transport, and scavenging; and incorporating the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and Ca²⁺ handling. Local mitochondrial interaction is mediated by superoxide (O₂ ^.−) diffusion and the O₂ ^.−-dependent activation of an inner membrane anion channel (IMAC). In a 2D network composed of 500 mitochondria, model simulations reveal ΔΨ_m depolarization waves similar to those observed when isolated guinea pig cardiomyocytes are subjected to a localized laser-flash or antioxidant depletion. The sensitivity of the propagation rate of the depolarization wave to O₂^.− diffusion, production, and scavenging in the reaction-diffusion model is similar to that observed experimentally. In addition, we present novel experimental evidence, obtained in permeabilized cardiomyocytes, confirming that ΔΨ_m depolarization is mediated specifically by O₂ ^.−. The present work demonstrates that the observed emergent macroscopic properties of the mitochondrial network can be reproduced in a reaction-diffusion model of RIRR. Moreover, the findings have uncovered a novel aspect of the synchronization mechanism, which is that clusters of mitochondria that are oscillating can entrain mitochondria that would otherwise display stable dynamics. The work identifies the fundamental mechanisms leading from the failure of individual organelles to the whole cell, thus it has important implications for understanding cell death during the progression of heart disease.     

DIRECT COUNTING AND SIZING OF MITOCHONDRIA IN SOLUTION

Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 µ aperture is 0.48 µ diameter, or 0.056 µ³ particle volume The mean volume of rat liver mitochondria was 0.42 µ³ or 0.93 µ in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 10³, and per gram of rat liver was about 11 x 10¹⁰. These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 µ³ and from rat kidney cortex, 0.23 µ³. These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a ÷ a₃), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement