Rat Skeletal Muscle Mitochondria Research Articles

Fish oil supplementation during pregnancy attenuates sex-specific changes induced by perinatal maternal high-fat diet in mitochondria of rat skeletal muscle at weaning

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

PO-084 Research of HIIT Detraining on Mitochondria of Soleus Muscle Beclin1 and Bnip3 Contents in Aging Rats

Objective To observe the temporal variation of Beclin1 and Bnip3 protein in skeletal muscle aging degeneration by constructing the aged rat model, and to observe the effect of HIIT intervention on the changes of Beclin1 and Bnip3 protein and the relationship between the two. It provides a theoretical basis for the effect of exercise on the aging degeneration of skeletal muscle by affecting the level of mitochondrial autophagy.
 Methods 40 male Wistar rats aged 8 months were randomly divided into quiet control group (C) and HIIT intervention group (H). After the rats entered the animal room for one week of adaptive feeding and exercise, the rats in the C group did not exercise, and the H group exercise alternately based on the maximum oxygen uptake test results of the rats with the 70%-90%-50%VO2max intensity. Once every two weeks, the maximal oxygen uptake of the rats in group H and group C was tested. Group H underwent 50min/ days, 5 days / weeks, and lasted for 16 weeks. The rats in the two groups were randomly selected after the first VO2 test and eighth and sixteenth weeks after intervention. After anesthesia, blood was collected from the abdominal aorta and soleus tissue was obtained. The ROS activity in soleus muscle was tested by fluorescence enzyme labeling method. Isolation of mitochondria from soleus muscle using tissue mitochondria Isolation Kit, and the expression of Beclin1 and Bnip3 in the mitochondria of the soleus muscle was tested by Western blot. The Image Lab 4 software was used to collect the data of the protein test strip, and the SPSS 17 software was used to analyze the data. The results of the data analysis were presented in the form of mean standard deviation. In the process of protein strip analysis, the relative value of the protein content of each sample was obtained by the gray scale analysis method. The results of the first sampling were taken as the baseline value, and the ratio of the H group in the C group of 8 weeks and 16 weeks was obtained with the baseline value, that is, the relative value of the protein content. Then, repeated measurement of variance analysis was used to analyze the differences of different indicators at baseline level, 8 weeks and 12 weeks between group C and group H. The independent sample T test was used without interaction effect, and multivariate analysis of variance was used. A significant level of alpha =0.05 is set.
 Results (1) the content of ROS in skeletal muscle of rats was related to the process of natural aging (F=119.314, P < 0.001), and the level of ROS would rise with the process of natural collars (F=28.884, P=0.001; F=127.607, P < 0.001) through the comparison of the time points in the group C and the H group. At the same time, the level of ROS in group H was lower than that in group C, but there was no significant difference (P=0.310). And the interaction effect of time and exercise mode (HIIT) will not affect the result (F=0.814, P=0.477). But the growth rate of ROS in group H was lower than that in C group. ⑵Exercise, time change and their interaction did not affect the content of Beclin1 in rat skeletal muscle mitochondria (P > 0.05). ⑶The mitochondrial Bnip3 content in H group and C group was significantly different at 8 weeks (F=14.500, P=0.001), H group was significantly higher than that in C group, but there was no significant difference in mitochondrial Bnip3 content at the 16 week (F=0.090, P=0.767), and the Bnip3 content of skeletal muscle mitochondria changed with age (F=20.852, 0.001). The trend of H increased, but then decreased. There was a linear trend (F=6.950, P=0.005) between the level of mitochondrial Bnip3 content and the intergroup factors (time point changes) and the interaction between time and HIIT movement in rats.
 Conclusions With the process of aging, (1) The content of ROS in skeletal muscle of rats increased significantly, while long-term HIIT training could delay the increase, but the best exercise time was unknown. (2) There was no obvious change in Beclin1 content in skeletal muscle mitochondria of rats, and HIIT training had no obvious effect on it. However, the changes in mitochondrial Beclin1 content relative to the total Beclin1 content of skeletal muscle need to be further studied; (3) The content of Bnip3 in skeletal muscle mitochondria in rats is increased, and long-term HIIT training has a delayed effect.

Disturbance of mitochondrial functions provoked by the major long-chain 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies in skeletal muscle

The pathogenesis of the muscular symptoms and recurrent rhabdomyolysis that are commonly manifested in patients with mitochondrial trifunctional protein (MTP) and long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiencies is still unknown. In this study we investigated the effects of the major long-chain monocarboxylic 3-hydroxylated fatty acids (LCHFA) accumulating in these disorders, namely 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, on important mitochondrial functions in rat skeletal muscle mitochondria. 3HTA and 3HPA markedly increased resting (state 4) and decreased ADP-stimulated (state 3) and CCCP-stimulated (uncoupled) respiration. 3HPA provoked similar effects in permeabilized skeletal muscle fibers, validating the results obtained in purified mitochondria. Furthermore, 3HTA and 3HPA markedly diminished mitochondrial membrane potential, NAD(P)H content and Ca2+ retention capacity in Ca2+-loaded mitochondria. Mitochondrial permeability transition (mPT) induction probably underlie these effects since they were totally prevented by cyclosporin A and ADP. In contrast, the dicarboxylic analogue of 3HTA did not alter the tested parameters. Our data strongly indicate that 3HTA and 3HPA behave as metabolic inhibitors, uncouplers of oxidative phosphorylation and mPT inducers in skeletal muscle. It is proposed that these pathomechanisms disrupting mitochondrial homeostasis may be involved in the muscle alterations characteristic of MTP and LCHAD deficiencies.

The thioredoxin and glutathione-dependent H2O2 consumption pathways in muscle mitochondria: Involvement in H2O2 metabolism and consequence to H2O2 efflux assays

The most common methods of measuring mitochondrial hydrogen peroxide production are based on the extramitochondrial oxidation of a fluorescent probe such as amplex ultra red (AUR) by horseradish peroxidase (HRP). These traditional HRP-based assays only detect H2O2 that has escaped the matrix, raising the potential for substantial underestimation of production if H2O2 is consumed by matrix antioxidant pathways. To measure this underestimation, we characterized matrix consumers of H2O2 in rat skeletal muscle mitochondria, and developed specific means to inhibit these consumers. Mitochondria removed exogenously added H2O2 (2.5µM) at rates of 4.7 and 5.0nmol min−1 mg protein−1 when respiring on glutamate+malate and succinate+rotenone, respectively. In the absence of respiratory substrate, or after disrupting membranes by cycles of freeze-thaw, rates of H2O2 consumption were negligible. We concluded that matrix consumers are respiration-dependent (requiring respiratory substrates), suggesting the involvement of either the thioredoxin (Trx) and/or glutathione (GSH)-dependent enzymatic pathways. The Trx-reductase inhibitor auranofin (2µM), and a pre-treatment of mitochondria with 35µM of 1-chloro-2,4-dintrobenzene (CDNB) to deplete GSH specifically compromise these two consumption pathways. These inhibition approaches presented no undesirable “off-target” effects during extensive preliminary tests. These inhibition approaches independently and additively decreased the rate of consumption of H2O2 exogenously added to the medium (2.5µM). During traditional HRP-based H2O2 efflux assays, these inhibition approaches independently and additively increased apparent efflux rates. When used in combination (double inhibition), these inhibition approaches allowed accumulation of (endogenously produced) H2O2 in the medium at a comparable rate whether it was measured with an end point assay where 2.5µM H2O2 is initially added to the medium or with traditional HRP-based efflux assays. This finding confirms that a high degree of inhibition of all matrix consumers is attained with the double inhibition. Importantly, this double inhibition of the matrix consumers allowed revealing that a large part of the H2O2 produced in muscle mitochondria is consumed before escaping the matrix during traditional HRP-based efflux assays. The degree of this underestimation was substrate dependent, reaching >80% with malate, which complicates comparisons of substrates for their capacity to generate H2O2 in normal conditions i.e. when matrix consumers are active. Our results also urge caution in interpreting changes in H2O2 efflux in response to a treatment; when HRP-based assays are used, large changes in apparent H2O2 efflux may come from altered capacity of the matrix consumers.

Production of superoxide/hydrogen peroxide by the mitochondrial 2-oxoadipate dehydrogenase complex

In humans, mutations in dehydrogenase E1 and transketolase domain containing 1 (DHTKD1) are associated with neurological abnormalities and accumulation of 2-oxoadipate, 2-aminoadipate, and reactive oxygen species. The protein encoded by DHTKD1 has sequence and structural similarities to 2-oxoglutarate dehydrogenase, and the 2-oxoglutarate dehydrogenase complex can produce superoxide/H2O2 at high rates. The DHTKD1 enzyme is hypothesized to catalyze the oxidative decarboxylation of 2-oxoadipate, a shared intermediate of the degradative pathways for tryptophan, lysine and hydroxylysine. Here, we show that rat skeletal muscle mitochondria can produce superoxide/H2O2 at high rates when given 2-oxoadipate. We identify the putative mitochondrial 2-oxoadipate dehydrogenase complex as one of the sources and characterize the conditions that favor its superoxide/H2O2 production. Rates increased at higher NAD(P)H/NAD(P)+ ratios and were higher at each NAD(P)H/NAD(P)+ ratio when 2-oxoadipate was present, showing that superoxide/H2O2 was produced during the forward reaction from 2-oxoadipate, but not in the reverse reaction from NADH in the absence of 2-oxoadipate. The maximum capacity of the 2-oxoadipate dehydrogenase complex for production of superoxide/H2O2 is comparable to that of site IF of complex I, and seven, four and almost two-fold lower than the capacities of the 2-oxoglutarate, pyruvate and branched-chain 2-oxoacid dehydrogenase complexes, respectively. Regulation by ADP and ATP of H2O2 production driven by 2-oxoadipate was very different from that driven by 2-oxoglutarate, suggesting that site AF of the 2-oxoadipate dehydrogenase complex is a new source of superoxide/H2O2 associated with the NADH isopotential pool in mitochondria.

Mitochondrial reactive oxygen species production by fish muscle mitochondria: Potential role in acute heat-induced oxidative stress.

Acute heat challenge is known to induce cell-level oxidative stress in fishes. Mitochondria are well known for the capacity to make reactive oxygen species (ROS) and as such are often implicated as a source of the oxidants associated with this thermally-induced oxidative stress. This implication is often asserted, despite little direct data for mitochondrial ROS metabolism in fishes. Here we characterize mitochondrial ROS metabolism in three Actinopterygian fish species at two levels, the capacity for superoxide/H2O2 production and the antioxidant thiol-reductase enzyme activities. We find that red muscle mitochondria from all three species have measurable ROS production and respond to different assay conditions consistent with what might be anticipated; assuming similar relative contributions from difference ROS producing sites as found in rat skeletal muscle mitochondria. Although there are species and assay specific exceptions, fish mitochondria may have a greater capacity to produce ROS than that found in the rat when either normalized to respiratory capacity or determined at a common assay temperature. The interspecific differences in ROS production are not correlated with thiol-based antioxidant reductase activities. Moreover, mimicking an acute in vivo heat stress by comparing the impact of increasing assay temperature on these processes in vitro, we find evidence supporting a preferential activation of mitochondrial H2O2 production relative to the increase in the capacity of reductase enzymes to supply electrons to the mitochondrial matrix peroxidases. This supports the contention that mitochondria may be, at least in part, responsible for the ROS that lead to oxidative stress in fish tissues exposed to acute heat challenge.

Sites of Superoxide and Hydrogen Peroxide Production by Muscle Mitochondria Assessed ex Vivo under Conditions Mimicking Rest and Exercise

The sites and rates of mitochondrial production of superoxide and H2O2 in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity (e.g. the outer quinol site in complex III (site IIIQo) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site IF) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H2O2 using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site IQ) and the flavin site in complex II (site IIF) each account for about a quarter of the total measured rate of H2O2 production. Site IF, site IIIQo, and perhaps site EF in the β-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site IF dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H2O2 in vivo.

Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria

Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H2O2. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H2O2 directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H2O2 production at a fairly high rate of about 300pmol H2O2·min−1·mg protein−1 when oxidation of ubiquinol is prevented and complex II is uninhibited. This H2O2 production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site IIF of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H2O2 production (rotenone to inhibit sites in complex I (site IQ and, indirectly, site IF), myxothiazol to inhibit site IIIQo in complex III, and malonate plus atpenin A5 to inhibit site IIF in complex II), dihydroorotate dehydrogenase generates superoxide/H2O2, at a small but significant rate (23pmol H2O2·min−1·mg protein−1), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H2O2 directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.

Sites of superoxide and hydrogen peroxide production during fatty acid oxidation in rat skeletal muscle mitochondria

H2O2 production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H2O2 production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H2O2 production was dominated by complex II (44% from site IIF in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site IF). With added carnitine, H2O2 production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site IIIQo) and 25% from site IF. Thus complex II (site IIF in the forward reaction) is a major source of H2O2 production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H2O2 production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site IIF in the reverse reaction). About half the rest was from site IF, but a significant portion, ∼40pmol H2O2·min−1·mg protein−1, was not from complex I, II, or III and was attributed to the proteins of β-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was ∼200pmol H2O2·min−1·mg protein−1 under conditions of compromised antioxidant defense and reduced ubiquinone pool. Thus complex II and the ETF system both contribute to H2O2 productionduring fatty acid oxidation under appropriate conditions.

Mitochondrial Complex II Can Generate Reactive Oxygen Species at High Rates in Both the Forward and Reverse Reactions

Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.

Evidence for Two Sites of Superoxide Production by Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I)

Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD(+)-reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH(2) oxidation via the Q cycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD(+) redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I(Q)).

Stanozolol treatment decreases the mitochondrial ROS generation and oxidative stress induced by acute exercise in rat skeletal muscle

Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.

Hydrogen peroxide efflux from muscle mitochondria underestimates matrix superoxide production – a correction using glutathione depletion

The production of H(2)O(2) by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H(2)O(2). However, matrix enzymes such as the glutathione peroxidases can consume H(2)O(2) and compete with efflux of H(2)O(2), causing an underestimation of superoxide production. To assess this underestimate, we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90% as a consequence of pretreatment with 1-chloro-2,4-dintrobenzene (CDNB). The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H(2)O(2), consistent with decreased peroxidase capacity, but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H(2)O(2) formation from matrix-directed superoxide by up to two-fold from several sites of production, as defined by substrates and electron transport inhibitors, over a wide range of control rates, from 0.2-2.5 nmol H(2)O(2) min(-1) mg protein(-1). Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submitochondrial particles. The data indicate that the increased H(2)O(2) efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic correction curve was constructed, making H(2)O(2) efflux a more quantitative measure of matrix superoxide production. For rat muscle mitochondria, the correction equation was: CDNB-pretreated rate = control rate + [1.43 x (control rate)]/(0.55 + control rate). These results have significant ramifications for the rates and topology of superoxide production by isolated mitochondria.

Synthesis and characterization of cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA

The measurement of acyl-CoA dehydrogenase activities is an essential part of the investigation of patients with suspected defects in fatty acid oxidation. Multiple methods are available for the synthesis of the substrates used for measuring acyl-CoA dehydrogenase activities; however, the yields are low and the products are used without purification. In addition, the reported characterization of acyl-CoAs focuses on the CoA moiety, not on the acyl group. Here we describe the synthesis of three medium-chain acyl-CoAs from mixed anhydrides of the fatty acids using an aqueous–organic solvent mixture optimized to obtain the highest yield. First, cis-4-decenoic acid and 2,6-dimethylheptanoic acid were prepared (3-phenylpropionic acid is commercially available). These were characterized by gas chromatography/mass spectrometry (GC/MS), 1H nuclear magnetic resonance (NMR), and 13C NMR. Then cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA were synthesized. These were then purified by ion exchange solid-phase extraction using 2-(2-pyridyl)ethyl-functionalized silica gel, followed by reversed-phase semipreparative high-performance liquid chromatography with ultraviolet detection (HPLC–UV). The purified acyl-CoAs were characterized by analytical HPLC–UV followed by data-dependent tandem mass spectrometry (MS/MS) analysis on the largest responding MS mass (HPLC–UV–MS–MS/MS) and 13C NMR. The yields of the purified acyl-CoAs were between 75% and 78% based on coenzyme A trilithium salt (CoASH). Acyl-CoA dehydrogenase activities were measured in rat skeletal muscle mitochondria using, as substrates, the synthesized cis-4-decenoyl-CoA, 3-phenylpropionyl-CoA, and 2,6-dimethylheptanoyl-CoA. These results were compared with the results using our standard substrates butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA.

Dissociation of superoxide production by mitochondrial complex I from NAD(P)H redox state

The relationship between the rate of superoxide production by complex I and NAD(P)H redox state was investigated in rat skeletal muscle mitochondria. A high rate of superoxide production was observed during succinate oxidation; the rate during pyruvate oxidation was over fourfold lower. However, the NAD(P)H pool was significantly less reduced during succinate oxidation than during pyruvate oxidation. We conclude that there is no unique relationship between superoxide production by complex I and the reduction state of the NAD(P)H pool. Our data suggest that less than 10% of the superoxide originates from the flavin site during reverse electron transport from succinate.

Quantification of carbonylated proteins in rat skeletal muscle mitochondria using capillary sieving electrophoresis with laser‐induced fluorescence detection

Carbonyl-modified proteins are markers of oxidative damage. Here, we report a new method for detecting and quantifying carbonylated proteins by capillary sieving electrophoresis (CSE) with LIF detection (CSE-LIF). Alexa 488 hydrazide is used for the specific labeling of carbonyls while 3-(2-furoyl) quinoline-2-carboxaldehyde (FQ) is used for protein labeling. BSA subjected to metal-catalyzed oxidation is used to optimize the labeling reactions, confirm the separation power of CSE, and characterize the response of the LIF detector. The method is capable of detecting femtomole (fmol) amounts of carbonyls in proteins with molecular masses ranging from 26 to 30 kDa. Using this method, we determined that mitochondrial proteins isolated from skeletal muscle contains 2.1 +/- 0.1 (average +/- SD; n = 3) nmol carbonyl/mg protein. The methodology described here should be compatible with the analysis of single cells and needle biopsies taken from oxidative stress animal models.

Mitochondrial phospholipids of rat skeletal muscle are less polyunsaturated than whole tissue phospholipids: Implications for protection against oxidative stress1

The fatty acid composition of phospholipids is an important determinant of membrane function. Although the mitochondria play a pivotal role in skeletal muscle function, the fatty acid composition of their individual phospholipids has not been examined. The purpose of this study was to determine the fatty acid profile of each phospholipid in rat skeletal muscle mitochondria and compare it with that of the whole muscle. Lipids were extracted from the gastrocnemius muscles of 10 Wistar rats, and phospholipids were separated by thin-layer chromatography. The fatty acid composition of each phospholipid was then determined by gas chromatography. The same procedure was applied to a mitochondrial preparation from these muscles. We found that the fatty acid composition of the individual mitochondrial phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, cardiolipin, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, and lysophosphatidyl choline) and of the total mitochondrial phospholipids differed markedly (P < 0.05) from the fatty acid composition of the corresponding whole muscle phospholipids. Notably, the mitochondrial phospholipids had higher percentages of MUFA [13.9 (2.1) vs. 10.3 (0.9)] and lower percentages of PUFA [34.8 (4.3) vs. 39.5 (5.2)] and n6 fatty acids [25.0 (2.5) vs. 27.6 (2.5)]. Overall, the mitochondrial phospholipids had a lower unsaturation index than whole muscle phospholipids [135 (20) vs. 161 (26)]. Because PUFA are susceptible to peroxidation, unlike saturated fatty acids and MUFA, we propose that the low polyunsaturation of mitochondrial phospholipids is the result of selective pressure toward membranes that are more resistant to oxidative damage by reactive oxygen species produced in their vicinity. The negative effect of the low polyunsaturation on membrane fluidity may be counterbalanced by the higher percentage of MUFA and the known low cholesterol content of mitochondrial membranes.

Effects of veratrine and veratridine on oxygen consumption and electrical membrane potential of isolated rat skeletal muscle and liver mitochondria

We have previously shown that veratrine, a mixture of alkaloids known as Veratrum alkaloids, produces skeletal muscle toxicity, and there is evidence that veratrine interferes with the energetics of various systems, including cardiomyocytes and synaptosomes. In this work, we explored the effects of veratrine and veratridine, a component of this mixture, in rat skeletal muscle mitochondria and compared the results with those seen in liver mitochondria. Veratrine and veratridine alkaloids caused a significant concentration-dependent decrease in the rate of state 3 respiration, respiratory control (RCR) and ADP/O ratios in isolated rat skeletal muscle mitochondria (RMM), but not in rat liver mitochondria (RLM) supported by either NADH-linked substrates or succinate. The oxygen consumption experiments showed that RMM were more susceptible to the toxic action of Veratrum alkaloids than RLM. The addition of veratrine (250 μg/ml) to RMM caused dissipation of the mitochondrial electrical membrane potential during succinate oxidation, but this effect was totally reversed by adding ATP. These results indicate that there are chemical- and tissue-specific toxic effects of veratrine and veratridine on mitochondrial respiratory chain complexes. Identification of the specific respiratory chain targets involved should provide a better understanding of the molecular mechanisms of the toxicity of these agents.

Effects of veratrine on skeletal muscle mitochondria: Ultrastructural, cytochemical, and morphometrical studies

The alkaloid veratrine is a lipid-soluble neurotoxin, which target voltage-gated Na+ channels for their primary action. Recently, we showed that this alkaloid may cause myonecrosis and evidences suggest mitochondria as one of its cell targets. Herein, we investigate the effects caused by variable concentration of veratrine (250 and 550 microg/mL) on mitochondrial oxygen consumption, respiratory chain enzymes activities, and ultrastructure, combining electron microscopy with cytochemical and biochemical approaches. The results showed different sort of ultrastructural changes, both in isolated and intramuscular mitochondria. Veratrine decreased mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH-d), succinic dehydrogenase (SDH), and cytochrome oxidase (COX) activities, significantly and dose-dependently inhibited the state 3 respiration rate, respiratory control ratio (RCR), and ADP/O on isolated rat skeletal muscle mitochondria, whereas state 4 was unaffected. A tendency of increase in mitochondria diameter was seen with 250 microg/mL veratrine. We conclude that the alkaloid would probably act on mitochondrial membrane phospholipid configuration, which would explain the changes observed.

Long-term caloric restriction increases UCP3 content but decreases proton leak and reactive oxygen species production in rat skeletal muscle mitochondria

Calorie restriction (CR) without malnutrition increases life span and delays the onset of a variety of diseases in a wide range of animal species. However, the mechanisms responsible for the retardation of aging with CR are poorly understood. We proposed that CR may act, in part, by inducing a hypometabolic state characterized by decreased reactive oxygen species (ROS) production and mitochondrial proton leak. Here, we examine the effects of long-term CR on whole animal energetics as well as muscle mitochondrial energetics, ROS production, and ROS damage. CR was initiated in male FBNF1 rats at 6 mo of age and continued for 12 or 18 mo. Mean whole body VO2 was 34.6 (P < 0.01) and 35.6% (P < 0.001) lower in CR rats than in controls after 12 and 18 mo of CR, respectively. Body mass-adjusted VO2 was 11.1 and 29.5% lower (both P < 0.05) in CR rats than in controls after 12 and 18 mo of CR. Muscle mitochondrial leak-dependent (State 4) respiration was decreased after 12 mo compared with controls; however, after 18 mo of CR, there were slight but not statistically significant differences. Proton leak kinetics were affected by 12 mo of CR such that leak-dependent respiration was lower in CR mitochondria only at protonmotive force values exceeding 170 mV. Mitochondrial H2O2 production and oxidative damage were decreased by CR at both time points and increased with age. Muscle UCP3 protein content increased with long-term CR, consistent with a role in protection from ROS but inconsistent with the observed decrease or no change in proton leak.