Karla Punkt

Region- and age-dependent variations of muscle fibre properties.

The cytophotometric-morphometrical analysis of extensor digitorum longus and soleus muscles of 2.5 and 18 months old rats revealed regional and age-dependent differences in fibre type distribution, fibre area and fibre type related-enzyme activities which characterize contractility and metabolic profile. Variations along the longitudinal axis from the origin to the insertion and along three transversal axes from superficial to deep were found dependent on the muscle investigated. For example, the fibres of extensor digitorum longus muscle showed increased contractile and glycolytic capacities near insertion and the fibres of soleus muscle increased oxidative capacity in its middle part. Furthermore, the contribution of the fibre type that is dominant in a muscle (fast-glycolytic fibre type in extensor digitorum longus and slow-oxidative fibre type in soleus muscle) to the total number of fibres increased from origin to insertion by 15 and 30%, respectively. Along the superficial-deep axes the oxidative capacity of all fibres increased, the most in fast fibres of the soleus muscle by approximately 50%. In soleus muscle, a decrease of cross areas of all fibre types from superficial to deep was found, correlating negatively with the succinate dehydrogenase activity of the fibres. In extensor digitorum longus muscle the change in cross areas of slow-oxidative and fast-oxidative glycolytic fibres was dependent on the position of the transversal axis in the muscle. The results suggest that distribution patterns of fibre types and the metabolic make up of individual muscle fibres are adapted on the basis of local functional demands. In both muscles, higher numbers and increased oxidative capacity of fast-glycolytic fibres were found during ageing, but variations from superficial to deeper regions were irrespective of age.

Nitric oxide synthase in human skeletal muscles related to defined fibre types

Skeletal muscle functions regulated by NO are now firmly established. However, the knowledge about the NO synthase (NOS) expression related to a defined fibre type in human skeletal muscles necessitates further clarification. To address this issue, we examined localization of NOS isoforms I, II and III, in human skeletal muscles employing immunocytochemical labeling with tyramide signal amplification complemented with enzyme histochemistry and Western blotting. The NOS immunoreactivity was related to fibre types of different classification systems: physiological classification into slow and fast, ATPase classification into I, IIA, IIAX, IIX, and physiological-metabolic classification into slow-oxidative (SO), fast-oxidative glycolytic (FOG) and fast-glycolytic (FG). We found a correlation of NOS I–III immunoreactivity to metabolic defined fibre types with strong expression in FOG fibres. This implies that NO as modulator of muscle function is involved in oxidative metabolism in connection with fast force development, which only occurs in FOG fibres. The NOS expression showed no correlation to ATPase fibre subtypes due to the metabolic heterogeneity of ATPase fibre types. Healthy and affected vastus medialis muscles after anterior cruciate ligament rupture revealed similar NOS expression level as shown by Western blotting with, however, different expression patterns related to the fibre types in affected muscles. This suggests an altered modulation of force development in the fibres of diseased muscles.

Changes of enzyme activities in the rat myocardium caused by experimental hypoxia with and without Ginkgo biloba extract EGb 761 pretreatment. A cytophotometrical study

Using cytophotometry activity changes of succinate dehydrogenase, glycerol-3-phosphate dehydrogenase and myofibrillar adenosine triphosphatase, were measured in the rat myocardium under normal and different experimental conditions. After hypoxia all enzyme activities were significantly decreased in comparison to the normal situation, and the alterations differed in both ventricles. Ginkgo biloba extract treatment over three months before exposition to hypoxia resulted in a lower inhibition of succinate dehydrogenase, a higher inhibition of glycerol-3-phosphate dehydrogenase and an unchanged activity of adenosine triphosphatase after hypoxia of 20 min. These results were interpreted as a protective effect of the Ginkgo biloba extract on the hypoxic myocardium.

Impact of angiotensin II on skeletal muscle metabolism and function in mice: contribution of IGF-1, Sirtuin-1 and PGC-1α.

Activation of the renin-angiotensin-aldosterone system and increased levels of angiotensin II (Ang-II) occurs in numerous cardiovascular diseases such as chronic heart failure (CHF). Another hallmark in CHF is a reduced exercise tolerance with impaired skeletal muscle function. The aim of this study was to investigate in an animal model the impact of Ang-II on skeletal muscle function and concomitant molecular alterations. Mice were infused with Ang-II for 4 weeks. Subsequently, skeletal muscle function of the soleus muscle was assessed. Expression of selected proteins was quantified by qRT-PCR and Western blot. Infusion of Ang-II resulted in a 33% reduction of contractile force, despite a lack of changes in muscle weight. At the molecular level an increased expression of NAD(P)H oxidase and a reduced expression of Sirt1, PGC-1α and IGF-1 were noticed. No change was evident for the ubiquitin E3-ligases MuRF1 and MafBx and α-sarcomeric actin expression. Cytophotometrical analysis of the soleus muscle revealed a metabolic shift toward a glycolytic profile. This study provides direct evidence of Ang-II-mediated, metabolic deterioration of skeletal muscle function despite preserved muscle mass. One may speculate that the Ang-II-mediated loss of muscle force is due to an activation of NAD(P)H oxidase expression and a subsequent ROS-induced down regulation of IGF-1, PGC-1α and Sirt1.

Nitric oxide synthase isoforms I, III and protein kinase-Ctheta in skeletal muscle fibres of normal and streptozotocin-induced diabetic rats with and without Ginkgo biloba extract treatment.

The expression of nitric oxide synthase (NOS) isoforms I, III and protein kinase-Cθ (PKCθ) in rat vastus lateralis muscle was demonstrated immunohistochemically and then correlated to the physiological metabolic fibre types: SO (slow-oxidative), FOGI, FOGII (fast-oxidative glycolytic; I more glycolytic, II more oxidative), and FG (fast-glycolytic). NOS expression in muscles from different experimental groups (normal and diabetic rats, with and without Ginkgo biloba extract treatment) was assayed by Western blotting. Generally, NOS I and PKCθ were co-expressed in fibres with predominantly oxidative metabolism (SO, FOGII). This suggests an interplay of PKCθ and NOS I in nitric oxide production by oxidative fibres. NOS III was more highly expressed in fibres with predominantly glycolytic metabolism (FOGI, FG). A somewhat lower NOS I immunoreactivity was also found in NOS III positive fibres suggesting that NOS III and NOS I are co-expressed in these fibres. Western blotting revealed that NOS I as well as NOS III expression in the vastus lateralis muscle was down-regulated in diabetes and increased after Ginkgo biloba extract treatment. These effects may be associated with a diminished glucose uptake by myocytes of diabetic muscles and with an improved muscle function after Ginkgo biloba treatment.

The correlation of cytophotometrically and biochemically measured enzyme activities: Changes in the myocardium of diabetic and hypoxic diabetic rats, with and without Ginkgo biloba extract treatment

Changes of enzyme activities in the myocardium of rats from 6 different experimental groups (normal rats, diabetic rats, hypoxic diabetic rats, each with and without Ginkgo biloba extract treatment) were measured by using both cytophotometric and biochemical methods. The activity of succinate dehydrogenase, a marker of oxidative capacity, and of menadione-dependent glycerol-3-phosphate dehydrogenase and total lactate dehydrogenase, both markers of glycolytic capacity were measured to characterize changes of the metabolic profile in myocardium. A strong correlation between cytophotometric and biochemical data were found by linear regression analysis, justifying the use of cytophotometrical enzyme activity measurements in cells of organized tissue, where biochemistry cannot provide topographical information. The comparison of the results obtained from the different groups revealed the following: Enzyme activities in the myocardium of rats with streptozotocin-induced diabetes were significantly increased by 10-30% as compared to the normal myocardium. This effect was interpreted as a metabolic compensation of the diabetic heart with reduced performance. When diabetic rats were exposed to acute hypoxia of 20 min duration, enzyme activities decreased under the normal level, to 56% of the succinate dehydrogenase activity, to 87% of glycerol-3-phosphate dehydrogenase activity and to 69% of lactate dehydrogenase activity. Treatment of rats with the oxygen radical scavenger Ginkgo biloba extract (EGb 761) over 3 months resulted primarily in an increase by 10% of oxidative capacity and in a decrease by 30% of glycolytic capacity. Under diabetic conditions a shift to more glycolytic metabolism was observed by increasing the glycolytic activity by 39% and remaining the oxidative activity.

Effects on skeletal muscle fibres of diabetes andGinkgo biloba extract treatment

Combined cytophotometric and morphometric analysis of muscle fibre properties and myosin heavy chain electrophoresis were performed on extensor digitorum longus and soleus muscles from healthy rats and rats with streptozotocin-induced diabetes. Moreover, the protective effect of Ginkgo biloba extract, a potent oxygen radical scavenger, on diabetic muscles was investigated. Changes in fibre type-related enzyme activities, fibre type distribution, fibre cross areas and myosin isoforms were found. In muscles of diabetic rats, a metabolic shift was measured mainly in fibres with oxidative metabolism. Fast-oxidative glycolytic fibres showed a shift to more glycolytic metabolism and about a third transformed into fast-glycolytic fibres. Slow-oxidative fibres became more oxidative. Fibre atrophy was measured in diabetic muscles dependent on fibre type and muscle. Different fibre types atrophied to a different degree. Therefore, a decreased area percentage of slow fibres and an increased area percentage of fast fibres of the whole muscle cross section in both muscles were found. This is supported by reduced slow and increased fast myosin heavy chain isoforms. These alterations of diabetic muscle fibres could be due to less motion of diabetic rats and diabetic neuropathy. After treatment with Ginkgo biloba extract, enzyme activities were increased mainly in oxidative fibres of diabetic muscles, which was interpreted as protective effect. Generally, the soleus muscle with predominant oxidative metabolism was more vulnerable to diabetic alterations and Ginkgo biloba extract treatment than the extensor digitorum longus muscle with predominant glycolytic metabolism.

Histophotometry--the method of choice in quantifying dehydrogenase histochemistry.

Succinate, malate, and lactate dehydrogenase were demonstrated histochemically and measured histophotometrically in the heart and skeletal muscle (m. extensor digitorum longus and m. soleus) of rats at different ages. To prove the value of histophotometry, the enzymes of the tissues were estimated biochemically. The gel film technique cannot sufficiently prevent the diffusion of the soluble enzymes (malate-, lactate dehydrogenase) out of the tissue sections. Because of the different mobility, various isoenzymes, histophotometry cannot give reliable results. But, as far as membrane-bound dehydrogenases (succinate dehydrogenase) are concerned, histophotometry is the method of choice for basic measurements as in routine practical work, especially with tissues where the enzyme activities are heterogeneously distributed, e.g. in different types of muscle fibres in skeletal muscles.

Specific Metabolic Properties of Rat Oculorotatory Extraocular Muscles Can Be Linked to Their Low Force Requirements

PURPOSE To gain insight into the metabolic pathways of oculorotatory extraocular muscle (EOM) fiber types at the cellular level to explain their high fatigue resistance, rapid contraction, and low force output. METHODS In consecutive sections of adult rat EOMs, the cross-sectional area (CSA) was calculated, and the activities of succinate-dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPDH) were measured by quantitative histochemistry of different fiber types classified by the myofibrillar adenosine triphosphatase (mATPase) staining pattern. RESULTS In the orbital regions, type 1 (fast) fibers were present, showing small CSA, medium SDH, and low GPDH activity. The type 2 (slow) fibers exhibited extremely small CSA and low SDH and GPDH activity. In the global region, fast types 3 to 5 fibers were found, forming a continuum with an inverse correlation between CSA and enzyme activity. SDH and GPDH activity showed an unusual positive relationship in contrast to the skeletal muscle fiber types. The type 6 fibers were slow forming a close and clearly separated group with medium CSA and extremely low SDH and low GPDH activity. CONCLUSIONS Muscle fibers in adult rat EOMs show unique metabolic properties not seen in other skeletal muscles, covering their extraordinary functional demands. It can be speculated that the EOMs embedded within the orbit material do not need to perform with high force, and therefore they could develop extensive systems that ensure both fatigue resistance (many mitochondria) and fast contraction with high mATPase activity (a well-developed sarcotubular system).

Nitric oxide synthase in muscular dystrophies: a re-evaluation

Duchenne and Becker muscular dystrophies (DMD and BMD) are associated with decreased total nitric oxide (NO). However, mechanisms leading to NO deficiency with consequent muscle-cell degeneration remain unknown. To address this issue, we examined skeletal muscles of DMD and BMD patients for co-expression of NO synthase (NOS) with nitrotyrosine and transcription factor CREB, as well as with enzymes engaged in NO signaling. Employing immunocytochemical labeling, Western blotting and RT-PCR, we found that, in contrast to the most commonly accepted view, neuronal NOS was not restricted to the sarcolemma and that muscles of DMD and BMD patients retained all three NOS isoforms with an up-regulation of the inducible NOS isoform, CREB and nitrotyrosine. We suggest that enhanced nitrotyrosine immunostaining in muscle fibers as well as in the vasculature of DMD and BMD specimens reflects massive oxidative stress, resulting in withdrawal of NO from its regular physiological course via the scavenging actions of superoxides.