Study of age-dependent structural and functional changes of mitochondria in skeletal muscles and heart of naked mole rats (<Emphasis Type="Italic">Heterocephalus glaber</Emphasis>)

Morphometric analysis of mitochondria in skeletal muscles and heart of 6- and 60-month-old naked mole rats (Heterocephalus glaber) revealed a significant age-dependent increase in the total area of mitochondrial cross-sections in studied muscle fibers. For 6- and 60-month-old animals, these values were 4.8 ± 0.4 and 12.7 ± 1.8%, respectively. This effect is mainly based on an increase in the number of mitochondria. In 6-month-old naked mole rats, there were 0.23 ± 0.02 mitochondrial cross-sections per μm² of muscle fiber, while in 60-month-old animals this value was 0.47 ± 0.03. The average area of a single mitochondrial cross-section also increased with age in skeletal muscles–from 0.21 ± 0.01 to 0.29 ± 0.03 μm². Thus, naked mole rats show a drastic enlargement of the mitochondrial apparatus in skeletal muscles with age due to an increase in the number of mitochondria and their size. They possess a neotenic type of chondriome accompanied by specific features of mitochondrial functioning in the state of oxidative phosphorylation and a significant decrease in the level of matrix adenine nucleotides.     

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.     

Skeletal Muscle Type Comparison of Subsarcolemmal Mitochondrial Membrane Phospholipid Fatty Acid Composition in Rat

The phospholipid composition of membranes can influence the physiological functioning of the cell or subcellular organelle. This association has been previously demonstrated in skeletal muscle, where cellular or subcellular membrane, specifically mitochondria, phospholipid composition is linked to muscle function. However, these observations are based on whole mixed skeletal muscle analysis, with little information on skeletal muscles of differing fiber-type compositions. These past approaches that used mixed muscle may have misidentified outcomes or masked differences. Thus, the purpose of this study was to compare the phospholipid fatty acid composition of subsarcolemmal (SS) mitochondria isolated from slow-twitch postural (soleus), fast-twitch highly oxidative glycolytic locomotory (red gastrocnemius), and fast-twitch oxidative glycolytic locomotory (plantaris) skeletal muscles. The main findings of the study demonstrated unique differences between SS mitochondrial membranes from postural soleus compared to the other locomotory skeletal muscles examined, specifically lower percentage mole fraction of phosphatidylcholine (PC) and significantly higher percentage mole fraction of saturated fatty acids (SFA) and lower n6 polyunsaturated fatty acids (PUFA), resulting in a lower unsaturation index. We also found that although there was no difference in the percentage mole fraction of cardiolipin (CL) between skeletal muscle types examined, CL of soleus mitochondrial membranes were approximately twofold more SFA and approximately two-thirds less PUFA, resulting in a 20–30% lower unsaturation and peroxidation indices. Thus, the results of this study indicate unique membrane lipid composition of mitochondria isolated from different skeletal muscle types, a potential consequence of their respective duty cycles.     

N- and C-terminal amino acids of proteolipids from the rat brain and various other organs

N- and C-terminal amino acids of proteolipid proteins from the whole brain and some other organs were investigated. N-terminal amino acids were identified by the dansylation procedure. C-terminal amino acids were determined after the enzymatic hydrolysis with carboxy peptidases A and B with the following dansylation. Phenyl alanine and lysine were identified as C-terminal amino acids of the proteolipids from the whole brain and only lysine--as the C-terminal amino acid of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle. The corresponding N-terminal amino acids of the proteolipids from the whole brain were aspartic acid and glycine and of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle--only aspartic acid. A comparison of the data obtained with the previous ones has shown that in the brain there exist only two types of proteolipids--one characteristic of myelin, another-- of mitochondria, and in other organs--only one characteristic of mitochondria.     

Mitochondrial apoptotic signaling is elevated in cardiac but not skeletal muscle in the obese Zucker rat and is reduced with aerobic exercise

Mitochondrial apoptosis and apoptotic signaling modulations by aerobic training were studied in cardiac and skeletal muscles of obese Zucker rats (OZR), a rodent model of metabolic syndrome. Comparisons were made between left ventricle, soleus, and gastrocnemius muscles from OZR (n = 16) and aged-matched lean Zucker rats (LZR; n = 16) that were untrained (n = 8) or aerobically trained on a treadmill for 9 wk (n = 8). Cardiac Bcl-2 protein expression levels were approximately 50% lower in the OZR compared with the LZR, with no difference in either of the skeletal muscles. Bax protein expression levels were similar in skeletal muscles of the OZR compared with the LZR. Furthermore, mitochondrial apoptotic signaling was not different in skeletal muscles of OZR and LZR groups. However, there was an approximate sevenfold increase in the Bax protein accumulation in the myocardial mitochondrial-rich protein fraction of the OZR compared with the LZR. Additionally, there was an increase in cytosolic cytochrome c released from the mitochondria, caspase-9 and caspase-3 activity, with a corresponding elevation in DNA fragmentation in the cardiac muscles of the OZR compared with the LZR. Exercise training reduced cardiac Bax protein levels, the mitochondrial localization of Bax, cytosolic cytochrome c, caspase activity, and DNA fragmentation in cardiac muscles of the OZR after exercise, with no change in the skeletal muscles. These data show that mitochondrial apoptosis is elevated in the cardiac but not skeletal muscles of the OZR, but aerobic exercise training was effective in reducing cardiac mitochondrial apoptotic signaling.     

A monoclonal antibody against cytochrome c oxidase distinguishes cardiac and skeletal muscle mitochondria

The mitochondrial enzyme cytochrome c oxidase (COX) in eukaryotes consists of at least seven subunits, three of which (I-III) are encoded by mitochondrial DNA (mitDNA) and the others (IV-VII) by the nuclear genome. There is increasing evidence that COX in mammals exists in multiple tissue-specific forms, presumably specified by nuclearly encoded subunits. We performed immunologic studies in human cardiac and skeletal muscle, using a monoclonal antibody raised against subunit IV of COX purified from human cardiac muscle. In immunotitration studies, the antibody bound with high affinity to mitochondria from cardiac muscle, but reacted only weakly with mitochondria from skeletal muscle. Similarly, immunocytochemical studies showed prominent mitochondrial staining in frozen sections of heart, but no staining in sections of mature skeletal muscle. Although this antibody did not stain mitochondria in mature skeletal muscle, it clearly stained mitochondria in myoblasts and immature myotubes of human muscle cultures, suggesting that mitochondria in immature muscle cells are different from those in mature muscle, and similar to heart mitochondria. Immunotitration data using either native or denatured COX protein from heart or skeletal muscle showed similar immunoreactivity. These studies indicate that the epitope for recognition by this antibody is exposed in mitochondria from heart and immature muscle cells, but masked in mitochondria from mature skeletal muscle.     

Mitochondrial regular arrangement in muscle cells: a "crystal-like" pattern

The aim of this work was to characterize quantitatively the arrangement of mitochondria in heart and skeletal muscles. We studied confocal images of mitochondria in nonfixed cardiomyocytes and fibers from soleus and white gastrocnemius muscles of adult rats. The arrangement of intermyofibrillar mitochondria was analyzed by estimating the densities of distribution of mitochondrial centers relative to each other (probability density function). In cardiomyocytes (1,820 mitochondrial centers marked), neighboring mitochondria are aligned along a rectangle, with distance between the centers equal to 1.97 ± 0.43 and 1.43 ± 0.43 µm in the longitudinal and transverse directions, respectively. In soleus (1,659 mitochondrial centers marked) and white gastrocnemius (621 pairs of mitochondria marked), mitochondria are mainly organized in pairs at the I-band level. Because of this organization, there are two distances characterizing mitochondrial distribution in the longitudinal direction in these muscles. The distance between mitochondrial centers in the longitudinal direction within the same I band is 0.91 ± 0.11 and 0.61 ± 0.07 µm in soleus and white gastrocnemius, respectively. The distance between mitochondrial centers in different I bands is 3.7 and 3.3 µm in soleus and gastrocnemius, respectively. In the transverse direction, the mitochondria are packed considerably closer to each other in soleus than in white gastrocnemius, with the distance equal to 0.75 ± 0.22 µm in soleus and 1.09 ± 0.41 µm in gastrocnemius. Our results show that intermyofibrillar mitochondria are arranged in a highly ordered crystal-like pattern in a muscle-specific manner with relatively small deviation in the distances between neighboring mitochondria. This is consistent with the concept of the unitary nature of the organization of the muscle energy metabolism. confocal microscopy; quantitative analysis; cardiac and skeletal muscles; probability density function; unitary structure of cells     

A carnitine/acylcarnitine translocase assay applicable to biopsied muscle specimens without requiring mitochondrial isolation.

A simple method for assaying the mitochondrial carnitine/acylcarnitine translocase of muscles that needs only few milligrams of fresh tissue is described. The procedure involves monitoring of the sulphobetaine (an inhibitor of the translocase)-sensitive acetylation of sub-saturating concentrations of carnitine in the medium, linked to the oxidation of [2-14C]pyruvate in the presence of malonate. Conditions affecting the reliability of the outlined procedure and the ancillary information to be collected, namely the activities of pyruvate oxidase system and carnitine acetyltransferase, for detecting possible deficiency of the translocase are described, together with data on the translocase activity in human skeletal muscle, in rat red and white skeletal muscles and in rat heart. The concepts outlined should allow development of assays of other mitochondrial transporters that also would require neither isolation of mitochondria nor availability of a large quantity of tissue, both of which are otherwise needed at present.     

Simvastatin induces impairment in skeletal muscle while heart is protected

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) are widely used to reduce plasma cholesterol concentration. However, statins are also known to induce various forms of muscular toxicity. We have previously shown that acute application of simvastatin on human skeletal muscle samples induced a cascade of cellular events originating from mitochondria and resulting in a global alteration of Ca2+ homeostasis. The present study was designed to further define the origin of the mitochondria impairment and to understand the apparent lack of deleterious effect on the heart. Using fluorescence imaging analysis and oxygraphy on human and rat skinned skeletal muscle samples, we show that the simvastatin-induced mitochondria impairment results from inhibition of the complex I of respiratory chain. Similar simvastatin-induced mitochondria impairment and alteration of Ca2+ homeostasis occur in permeabilized but not in intact ventricular rat cardiomyocytes. In intact rat skeletal muscle fibers from the flexor digitorum brevis muscle, the simvastatin-induced alteration of Ca2+ homeostasis is abolished when monocarboxylate transporter (MCT4) is inhibited. The impairment of complex I by simvastatin might be the primary step of its cellular deleterious effects leading to muscle fiber death. This mechanism is seen specifically in skeletal muscles. This specificity should be in part attributed to a preferential uptake of statins by MCT4 that is not expressed in cardiomyocytes.     

Gas—liquid chromatography of free amino acids in the hyaloplasm of rat cerebral,cerebellar and ocular tissues,and in skeletal and heart muscle

The paper deals with the composition of amino acids in the hyaloplasm of cerebral tissue, cerebellum, eyeball, heart muscle and skeletal muscles. The investigations performed showed that: the most numerous groups of peaks were obtained from heart muscle (45), cerebellar tissue (43), skeletal muscle (36), eyeball (29) and cerebral tissue (25); and the highest molar levels corresponded to those of tryptophan in skeletal muscle, heart and cerebellum, proline in the heart, valine in the eyeball, and aspartic acid in the brain. Weight ratios indicated high contents of histidine, tyrosine and phenylalanine in the tissues of the skeletal muscles, the heart and cerebellum.     

Divergent differentiation during embryonal histogenesis of skeletal muscle tissue

Morphometric analysis of the developmental processes of the satellite cells and myosimplasts has been performed in embryonal histogenesis of the skeletal muscle tissue in 17 human fetuses 8-27 weeks of the intrauterine development. The sequence of death of some myoblasts in embryonal histogenesis is described in details. Basing on the data obtained, a conception on existance of muscular-proliferative units (MPU) in composition of the skeletal muscles is put forward. The amount of the MPU determines the whole number of muscle fibers in the muscle. The anlage of the MPU occurs as a result of divergent differentiation of the stem myogenic cells at early stages of myogenesis (myosimplasts and myotubes) from the cells commited to mutual fusion. The fund of these cells is determined by the number of myogenic elements that are at the state of the proliferative rest. One of the mechanisms regulating the number of the resting cells is the growth rate of the simplast lengthwise. The resting cells, appearing at late stages of myogenesis (of the muscle fibers), are the sources for development of the myosatellites in mature muscle fibers. In dying myotubes there is a sharp disturbance in growth processes lengthwise, in biosynthesis of contractile proteins, in correlation between the number of nuclei in the satellite cells and those of simplasts.     

Dietary coconut oil affects more lipoprotein lipase activity than the mitochondria oxidative capacities in muscles of preruminant calves

The presence of coconut oil in a milk replacer stimulates the growth rate of calves, suggesting a better oxidation of fatty acid in muscles. Because dietary fatty acid composition influences carnitine palmitoyltransferase I (CPT I) activity in rat muscles, this study was designed to examine the effects of a milk replacer containing either tallow (TA) or coconut oil (CO) on fatty acid utilization and oxidation and on the characteristics of intermyofibrillar (IM) and subsarcolemmal (SS) mitochondria in the heart and skeletal muscles of preruminant calves. Feeding CO did not affect palmitate oxidation rate by whole homogenates, but induced higher palmitate oxidation by IM mitochondria (+37%, P < 0.05). CPT I activity did not significantly differ between the two groups of calves. Heart and longissimus thoracis muscle of calves fed CO had higher lipoprotein lipase activity (+27% and 58%, respectively; P < 0.05) but showed no differences in fatty acid binding protein content or activity of oxidative enzymes. Whatever the muscle and the diet, IM mitochondria had higher respiration rates and enzyme activities than those of SS mitochondria (P < 0.05). Furthermore, CPT I activity of the heart was 28-fold less sensitive to malonyl-coenzyme A inhibition in IM mitochondria than in SS mitochondria. In conclusion, dietary CO marginally affected the activity of the two mitochondrial populations and the oxidative activity of muscles in the preruminant calf. In addition, this study showed that differences between IM and SS mitochondria in the heart and muscles were higher in calves than in other species studied so far.     

Immunocytochemical localization of annexin V (CaBP33), a Ca(2+)-dependent phospholipid- and membrane-binding protein, in the rat nervous system and skeletal muscles and in the porcine heart.

We investigated the ultrastructural localization of annexin V a Ca(2+)-dependent phospholipid- and membrane-binding protein in the nervous system, heart, and skeletal muscles. The results indicate that in the cerebellum the protein is restricted to glial cells, where it is found diffusely in the cytoplasm as well as associated with plasma membranes. Bergmann glial cell bodies and processes and astrocytes in the cerebellar cortex and oligodendrocytes in the cerebellar white matter displayed an intense immune reaction product. In sciatic nerves, the protein was exclusively found in Schwann cells with a subcellular localization similar to that seen in glial cells in the cerebellum. Pituicytes in the neurohypophysis were intensely immunostained, whereas axons were not. In the heart, annexin V was restricted to the sarcolemma, transverse tubules, and intercalated discs. In skeletal muscles the protein was localized to the sarcolemma and transverse tubules. No evidence for the presence of the protein in the sarcoplasm or in association with mitochondria, the sarcoplasmic reticulum, or contractile elements was obtained. The observation that plasma membranes in cells expressing annexin V have the protein associated with them is in agreement with previous data on Ca(2+)-dependent binding of the protein to brain and heart membranes, and on existence of both EGTA- and Triton X-100-extractable and resistant fractions of annexin V in these membranes. The present data support the hypothesis that annexin V might be involved in membrane trafficking and suggest a role for this protein in the regulation of cytoplasmic activities in glial cells.     

Analysis of compartmentation of ATP in skeletal and cardiac muscle using 31P nuclear magnetic resonance saturation transfer.

We have developed a model for the analysis of the forward creatine kinase reaction in muscle as measured by the nuclear magnetic resonance (NMR) technique of magnetization transfer. The model, accounting for the double-exponential behavior observed in some NMR magnetization transfer data, allows for the existence of two ATP pools, one that is NMR-visible (NMR-VIS) and another that is NMR-invisible (NMR-INVIS). We have applied the model to experimental data for the forward creatine kinase reaction in skeletal and cardiac muscles to study the dependence of the creatine kinase rate constants and fluxes on workload and to account for the differences between heart and skeletal muscle. The results suggest that an NMR-distinct ATP pool exists in both heart and skeletal muscles, and that phosphate exchange with this pool catalyzed by creatine kinase increases with increased workload. The results also agree with previously published estimates of the rates of mitochondrial translocase and net ATP synthesis obtained by traditional biochemical methods.     

一次性力竭运动对大鼠PHB1表达及线粒体功能的影响

目的：观察一次性力竭运动后大鼠脑、心、骨骼肌组织和线粒体中PHB1含量的变化及对大鼠线粒体功能的影响,探寻PHB1与线粒体功能和能量代谢的关系。方法：健康雄性SD大鼠40只,随机分为2组（n=20）：对照组和一次性力竭运动组,大鼠进行一次性急性跑台运动建立力竭运动模型。收集各组大鼠的心、脑和骨骼肌组织样品并提取线粒体,检测其呼吸功能和ROS的变化。用Western blot方法检测组织和线粒体中PHB1蛋白表达水平;用分光光度计检测各器官中ATP含量以及线粒体中复合体V活性（ATP合酶活性）。结果：①一次性力竭运动后脑、心肌、骨骼肌中ATP含量显著性降低;②一次性力竭运动后脑、心肌、骨骼肌线粒体中复合体V活性、RCR、ROS显著性降低,ST4均显著性升高,ST3无显著性差异。③一次性力竭运动后心、脑、骨骼肌线粒体中PHB1的表达显著性减少。④通过相关性分析得出：一次性力竭运动后心、脑、骨骼肌中ATP含量与心、脑、骨骼肌中复合体V活性呈正相关;心、脑、骨骼肌中ATP含量和心、脑骨骼肌中PHB1的表达呈正相关。结论：一次性力竭运动后,降低线粒体氧化磷酸化功能,使大鼠脑、骨骼肌线粒体内ROS生成增加,PHB1的表达、ATP含量和复合体V活性均下降。一次性力竭运动使得大鼠线粒体内PHB1表达降低,线粒体功能减弱,机体能量代谢降低。     

Detection and regulation of δ-aminolevulinic acid synthetase activity in rat skeletal muscle

The presence of δ-aminolevulinic acid synthetase (ALAS) in mitochondria obtained from rat skeletal muscles has been observed. Optimal conditions for the meausurement of this activity are described. The activity of skeletal muscle ALAS was investigated under conditions known to affect the activity of this enzyme in other tissues. ALAS activity in skeletal muscle mitochondria was decreased 55% by a 48-h fast. Treatment with dexamethasone did not reverse the effect of starvation on ALAS activity and did not change the activity in the fed controls. ALAS activity was decreased 56% in skeletal muscle mitochondria obtained from rats in which diabetes mellitus had been induced by streptozotocin. Administration of insulin to the diabetic animals partially reversed the effect of diabetes on skeletal muscle ALAS; however, administration of insulin to control animals caused a 21% decrease in skeletal muscle ALAS activity. By contrast, treatment with inducers of hepatic ALAS such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine had no effect on skeletal muscle ALAS. These results confirm our previous suggestion that ALAS activity is regulated in a tissue-specific manner.     

Biochemical characteristics of functionally active actin-like protein from liver mitochondria

The actin-like protein with a molecular weight of 42 kDa was obtained from the preparation of freshly isolated mitochondria of the rat liver using the method of immobilized DNAse affinity chromatography. The inhibitory ability of the isolated protein with respect to pancreatic DNAse I was the same as that of muscular actin. The native structure of the mitochondria protein is confirmed by the data of spectral analysis and its ability to globular-fibrillar transformation with an increased ionic strength of the solution. The polymerization ability as well as a stimulating effect of the actin-like protein of mitochondria on the ATPase activity of myosin is much less pronounced as compared to actin of skeletal muscles.     

A high-caloric diet rich in soy oil alleviates oxidative damage of skeletal muscles induced by dexamethasone in chickens

Objective: Glucocorticoids (GCs) can induce oxidative damage in skeletal muscles. The purpose of this study was to demonstrate a high caloric (HC) diet rich in soy oil would change the oxidative stress induced by a GC.

Methods: The effect of dexamethasone (DEX) and HC diet on oxidative stress in plasma, skeletal muscles (M. pectoralis major, PM; M. biceps femoris, BF), and mitochondria were determined. The biomarkers of oxidative damage and antioxidative enzyme activity were determined. The fatty acid profile of muscles and the activities of complex I and II in mitochondria were measured.

Results: The results showed that DEX increased the concentrations of oxidative damage markers in plasma, muscles, and mitochondria. The activity of complex I was significantly suppressed by DEX. DEX-chickens had higher proportions of polyunsaturated fatty acids and lower proportions of monounsaturated fatty acids in the PM. A HC diet decreased the levels of oxidative damage biomarkers in plasma, muscles, and mitochondria. The interaction between DEX and diet suppressed the activities of complex I and II in HC-chickens.

Discussion: Oxidative damage in skeletal muscles and mitochondria was the result of GC-induced suppression of the activity of mitochondrial complex I. A HC diet improved the antioxidative capacity and reduced the oxidative damage induced by the GC.     

Expression and activity of cyclooxygenase isoforms in skeletal muscles and myocardium of humans and rodents.

Conflicting data have been reported on cyclooxygenase (COX)-1 and COX-2 expression and activity in striated muscles, including skeletal muscles and myocardium, in particular it is still unclear whether muscle cells are able to produce prostaglandins (PGs). We characterized the expression and enzymatic activity of COX-1 and COX-2 in the skeletal muscles and in the myocardium of mice, rats and humans. By RT-PCR, COX-1 and COX-2 mRNAs were observed in homogenates of mouse and rat hearts, and in different types of skeletal muscles from all different species. By Western blotting, COX-1 and -2 proteins were detected in skeletal muscles and hearts from rodents, as well as in skeletal muscles from humans. Immunoperoxidase stains showed that COX-1 and -2 were diffusely expressed in the myocytes of different muscles and in the myocardiocytes from all different species. In the presence of arachidonic acid, which is the COX enzymatic substrate, isolated skeletal muscle and heart samples from rodents released predominantly PGE(2). The biosynthesis of PGE(2) was reduced between 50 and 80% (P < 0.05 vs. vehicle) in the presence of either COX-1- or COX-2-selective blockers, demonstrating that both isoforms are enzymatically active. Exogenous PGE(2) added to isolated skeletal muscle preparations from rodents did not affect contraction, whereas it significantly fastened relaxation of a slow type muscle, such as soleus. In conclusion, COX-1 and COX-2 are expressed and enzymatically active in myocytes of skeletal muscles and hearts of rodents and humans. PGE(2) appears to be the main product of COX activity in striated muscles.