Whole-muscle and single-fibre contractile properties and myosin heavy chain isoforms in humans

 The contractile characteristics of three human muscle groups (triceps surae, quadriceps femoris and triceps brachii) of seven young male subjects were examined. The contractile properties were determined from electrically evoked isometric responses and compared with fibre type composition determined from needle biopsy samples. Fibre types were identified using myosin heavy chain (MHC) isoforms as molecular markers with gel electrophoresis (SDS-PAGE) and histochemical ATPase staining. Four contractile parameters (twitch time to peak torque, the maximal rate of torque development, frequency response and fatiguability) were found to be related to fibre type composition. From the biopsy samples, single muscle fibres were isolated and chemically skinned. Isometric tension (P _o) unloaded shortening velocity (V _o) and rate of tension rise (dP/dt) were determined. Each fibre was classified on the basis of its MHC isoform composition determined by SDS-PAGE. Fibres belonging to the same type showed identical contractile parameters regardless of the muscle of origin, except minor differences in P _o of the fast fibres and dP/dt of slow fibres. The results are in favour of the conclusion that fibre type composition, determined using MHC isoforms as markers, is the major determinant of the diversity of contractile properties among human muscle groups. Received: 26 December 1995 / Received after revision: 26 March 1996 / Accepted: 29 April 1996     

Expression of myosin heavy and light chains changes during pregnancy in the rat uterus

We investigated the in vivo expression of myosin heavy chains (MHC) and myosin light chains (MLC) in the rat uterus during pregnancy and post parturn (p. p.). According to their antigenic reactivity, we observed two smooth-muscle-specific MHC (SM-MHC) of 204 kDa and 200 kDa (SM1 and SM2 respectively) and one non-muscle-specific MHC band of 198 kDa (NM-MHC). Adult virgin female rats expressed SM1 and NM-MHC (65/35) but no SM2. During the first pregnancy NM-MHC expression decreased in favour of the SM1 form, the SM1/NM-MHC ratio being 82/12 after 20 days of pregnancy. SM2 is newly expressed in the first p. p. state, the SM1/SM2/NM-MHC ratio being 58/28/14 between 3 and 33 days p. p. During the second pregnancy, starting 34 days p. p., both SM2 and NM-MHC expression decreased, the SM1/SM2/NM-MHC ratio being 85/0/15 after 20 days of pregnancy. Two protein bands of approximately 154 kDa and 145 kDa (sodium dodecylsulphate electrophoresis), designated SMP1 and SMP2 respectively, with the same immunoreactivity as the SM-MHC were observed in vivo in the pregnant but not in the non-pregnant uterus. In addition, a Ca²⁺-independent protease, which uses MHC as substrate, is expressed in the pregnant but not in the non-pregnant rat uterus. Two isoforms of the 17-kDa MLC (LC_17a and LC_17b) exist in the rat uterus. Expression of the LC_17a isoform increased during the first pregnancy from 46% in virgin rats to 65% in uteri of rats 20 days pregnant. During the first p. p. state LC_17a expression decreased to 42% at 19 days p. p. then remained constant up to 33 days p. p. Again, during the second pregnancy LC_17a expression increased to the same extent as observed in the first pregnancy. Phosphorylation of the 20-kDa MLC forms of skinned uterus fibres derived from 6-days- and 20-days-pregnant rats (first and second pregnancy) increased upon Ca²⁺ activation but the phosphorylation levels were the same in both groups. As extrapolated from the force/velocity relation, the maximal shortening velocity (V _max) of chemically skinned uteri from 6-days pregnant-rats was 0.104 muscle length (ML) s^–1 and increased up to 0.201 ML s^–1 in uteri from 20-days-pregnant animals (first pregnancy). V _max remained high 2 days p. p. (0.2 ML s^–1) and decreased to 0.09 ML s^–1 19 days p. p. During the second pregnancy V _max was 0.1 ML for 6-days-pregnant uteri and increased up to 0.21 ML s^–1 after 20 days of pregnancy. Our results suggest a correlation between LC_17a expression and V _max rather than between MHC and V _max.     

Energy state and myosin heavy chain isoforms in single fibres of normal and transforming rabbit muscles

 Energy-rich phosphates, [ATP]/[ADP_free] ratios, and the myosin heavy chain (MHC) complement were determined in single fibres from normal rabbit muscles, and in fibres isolated from tibialis anterior muscle undergoing fast-to-slow conversion by chronic low-frequency stimulation (CLFS). In normal muscles, energy-rich phosphate contents and [ATP]/[ADP_free] ratios could thus be assigned to different MHC-based fibre types. Phosphocreatine (PCr) contents and [ATP]/[ADP_free] ratios differed markedly between fast- and slow-twitch fibres, as well as within the fast fibre subtypes. Both magnitudes were approximately twofold higher in the fastest (type IIB) fibres as compared to the slowest (type I) fibres. According to PCr contents and [ATP]/[ADP_free] ratios pure and hybrid fibres were aligned in an order similar to that determined by their contractile properties and myofibrillar ATPase activities. CLFS for up to 30 days induced pronounced decreases in PCr and [ATP]/[ADP_free] which attained levels twofold lower than in normal slow-twitch fibres. In both normal and stimulated muscles, PCr and [ATP]/[ADP_free] ratios were correlated, indicating their equilibrium in the different fibre types. The relationship detected between MHC isoform expression and the [ATP]/[ADP_free] ratio suggests that the drastic and persistent depression of the cellular energy state may act as an important signal initiating fast-to-slow transformation processes in muscle fibres. Received: 26 June 1998 / Accepted: 31 July 1998     

Mechanical properties and myosin heavy chain isoform composition of skinned skeletal muscle fibres from a human biopsy sample

 Experiments were conducted to investigate the mechanics of contraction of chemically skinned muscle fibre segments of a biopsied sample of single human quadriceps muscle. Subsequently, the isoforms of the myosin heavy chain (MHC) were analysed by sodium dodecyl sulphate (SDS) gel electrophoresis.Of the 41 fibres, 26 contained MHCI (type I), 11 of the fibres contained MHCIIa (type IIA), and 4 of the fibres contained both MHCI and MHCIIa (of which MHCIIa was always slightly predominant (type IIC)). Distinct differences between fibre types were found in terms of the kinetics of force responses following stepwise length changes (order of velocity: IIA > IIC > I). The differences in maximal shortening velocity and in the kinetics of Ca²⁺-dependent activation were of the same order, but much less pronounced. Type I fibres had significantly greater fibre diameters than type IIA fibres. No significant differences were found among different fibre types in terms of isometric tension, resting sarcomere length or the length change needed to discharge the elasticity of maximally Ca²⁺-activated fibres (y _o value). The distribution of shortening velocity and kinetics of stretch activation values suggest that two muscle fibre subtypes may exist in human type I fibres. Received: 15 April 1997 / Received after revision 19 May 1997 / Accepted: 20 May 1997     

Favourable associations between the myosin heavy-chain and light-chain isoforms in human skeletal muscle

Histochemical and biochemical analyses were performed in order to examine the relationship between myosin light-chain (LC) isoforms and fibre-type distributions in whole human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis muscle in six healthy men, and analysed for the relative area occupied by each fibre type (percentage of fibre type area) and the molar ratio of each LC isoform. The percentage of type I fibre area was positively correlated with the molar ratio of slow LC (LC1s and LC2s) to total LC. The regression line was located below the line of unity. Also, the ratio of percentage of type II fibre area to that of type II fibre area was positively correlated with the molar ratio of the fast alkali LC LC1f to fast alkali LCs LC1f and LC3f. These results support previous study, having shown that in human skeletal muscle some type I fibres express various amounts of fast LC in addition to slow LC and suggest that fast myosin heavy-chain HCII a is favourably associated with LC1f, whereas HCIIb is favourably associated with LC3f.     

Postnatal expression of myosin heavy chains in muscle spindles of the rat

Summary The immunocytochemical expression of two myosin isoforms in intrafusal muscle fibers was examined in soleus muscles of neonatal (zero to six days postpartum) and adult rats. Monoclonal antibodies specific for myosin heavy chains of the slow-tonic anterior latissimus dorsi (ALD58) and fast-twitch pectoralis (MF30) muscles of the chicken were used. In adults ALD58 bound to the intracapsular regions of bag₁ and bag₂ fibers and MF30 bound to the intracapsular regions of bag₂ and chain fibers. The extracapsular regions of intrafusal fibers and all extrafusal fibers did not react to ALD58 or MF30. Bag₁ and bag₂ fibers of neonatal rats expressed immature myosin patterns but chain fibers did not. The adult pattern of immunoreactivity of intrafusal fibers developed by the fourth postnatal day, when the patterns of motor but not sensory innervation in the spindle are still immature. Data suggest that the expression and maintenance of the specific anti-myosin immunoreactivity of intrafusal fibers during postnatal development of rat spindles is dependent upon sensory but not motor innervation. Moreover, afferents might regulate the gene expression responsible for synthesis of myosins isoforms specific to intrafusal fibers only in those myonuclei located within the capsule, but not in the myonuclei in extracapsular regions of intrafusal fibers.     

Changes in the myosin heavy chain isoform profile of the triceps brachii muscle following 12 weeks of resistance training

The purpose of this investigation was to determine whether 12 weeks of resistance training, which increased arm girth (5%) and forearm extensor strength (39%), also altered the myosin heavy chain (MHC) characteristics of the triceps brachii muscle. Fifteen healthy, active men volunteered to participate under experimental (n = 11) or control (n = 4) conditions. The experimental group completed four sets of eight to 12 repetitions for each exercise (i.e. triceps pushdown, close grip bench press, triceps kickbacks and biceps curl) with loads of between 70–75% of one repetition maximum (1RM) three times a week. The inter-set and inter-exercise recovery period was only 90 s. Skeletal muscle tissue was removed from the triceps brachii muscle prior to (W0) and following 4 (W4), 8 (W8) and 12 (W12) weeks of the investigation. Samples were analysed for MHC isoform content using 6% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). MHC isoform composition in the control group did not change significantly. However, the percentage of MHC type IIb isoform decreased significantly from W0 to W4 and again from W4 to W12 [W0: 39.7 (9.2); W12: 29.2 (8.2)%] in the experimental condition. The increments in MHC type IIa [W0: 34.0 (9.9); W12: 41.5 (10.4)] and type I [W0: 26.3 (7.9); W12: 29.3 (9.6)] isoforms were not significant for the experimental group. However, the effect size (ES) transformation of changes in types IIa MHC content was moderate (ES = 0.75). Changes in MHC isoform content were not significantly correlated with changes in 1RM strength for the triceps pushdown exercise. These data indicated that resistance training rapidly, and in an ongoing manner, changed the contractile protein profile of trained skeletal muscle. However, changes in MHC isoform composition in the first 12 weeks of training were not implicated in the development of 1RM triceps pushdown strength.     

Effects of strength, endurance and combined training on myosin heavy chain content and fibre-type distribution in humans

This study investigated the effect of strength training, endurance training, and combined strength plus endurance training on fibre-type transitions, fibre cross-sectional area (CSA) and MHC isoform content of the vastus lateralis muscle. Forty volunteers (24 males and 16 females) were randomly assigned to one of four groups: control (C), endurance training (E), strength training (S), or concurrent strength and endurance training (SE). The S and E groups each trained three times a week for 12 weeks; the SE group performed the same S and E training on alternate days. The development of knee extensor muscle strength was S>SE>E (P<0.05) and has been reported elsewhere. The reduction in knee extensor strength development in SE as compared to S corresponded to a 6% increase in MHCIIa content (P<0.05) in SE at the expense of the faster MHCIId(x) isoform (P<0.05), as determined by electrophoretic analyses; reductions in MHCIId/x content after S or E training were attenuated by comparison. Both S and SE induced three- to fourfold reductions (P<0.05) in the proportion of type IIA/IID(X) hybrid fibres. S also induced fourfold increases in the proportion of type I/IIA hybrid fibres within both genders, and in a population of fibres expressing a type I/IID(X) hybrid phenotype within the male subjects. Type I/IIA hybrid fibres were not detected after SE. Both S and SE training paradigms induced similar increases (16–19%, P<0.05) in the CSA of type IIA fibres. In contrast, the increase in CSA of type I fibres was 2.9-fold greater (P<0.05) in S as compared to SE after 12 weeks. We conclude that the interference of knee extensor strength development in SE versus S was related to greater fast-to-slow fibre-type transitions and attenuated hypertrophy of type I fibres. Data are given as mean (SEM) unless otherwise stated.     

Induction of smooth muscle cells in the fibrous capsule of human hepatocellular carcinoma but not in the septa of hepatic cirrhosis

 We examined the expression of smooth muscle cytoskeleton in spindle-shaped cells in the capsule of hepatocellular carcinoma (HCC) and the septa of liver cirrhosis (LC). Serial sections of livers resected from 11 patients were stained with monoclonal antibodies against vimentin, desmin, smooth muscle actin (1A4, HHF35, CGA7) and smooth muscle myosin heavy chain isoforms (SM1, SM2). Capsular spindle-shaped cells exhibited a cytoskeletal feature indicative of intermediately differentiated smooth muscle cells. Computer-assisted morphometry revealed that the proportions of 1A4-, HHF35-, CGA7- and SM1- positive areas to vimentin-positive area were 88.0±11.0%, 50.8±17.4%, 25.3±16.4% and 19.4±12.4% (n=11) in main tumours and 86.6±9.4%, 50.9±18.7%, 21.1±12.3% and 17.6±9.7% (n=12) in daughter tumours, indicating that spindle-shaped cells are heterogeneous in cytoskeletal expression. Septal spindle-shaped cells in LC lacked the cytoskeletal proteins specific to differentiated smooth muscle cells (CGA7, SM1, SM2 and desmin). Electron microscopically, capsular spindle-shaped cells contained more microfilaments and less rough endoplasmic reticulum than do septal cells. Intermediately differentiated smooth muscle cells are induced in the capsule of HCC but not in the septa of LC, suggesting a role for stromal interaction by tumour cells in the induction of smooth muscle cells. Received: 23 July 1998 / Accepted: 20 December 1998     

Gender- and age-related differences in the regulatory influence of thyroid hormone on the contractility and myosin composition of single rat soleus muscle fibres

 The effects of 4 weeks of thyroid hormone (3,5,3′-triiodothyronine, T₃) treatment on the myosin isoform composition and maximum velocity of unloaded shortening (V ₀) of single soleus muscle fibres of young (3–6 months) and old (20–24 months) female (149 fibres) and male (200 fibres) rats were studied. Gender-related differences in the up-regulation of fast myosin heavy chain (MyHC) and myosin light chain (MyLC) isoforms were observed. In the female hyperthyroid rats, pure type I fibres and fibres co-expressing type I and type IIA MyHC (type I/IIA fibres) predominated. Some fibres expressed an α cardiac-like MyHC isoform either purely (α cardiac-like fibre type) or in co-expression with IIA MyHC (α cardiac-like/IIA fibre type). In the male hyperthyroid rats, on the other hand, all fibres were either type I/IIA or type I/IIAX. The relative quantities of fast MyLC isoforms in type I/IIA and type I/IIAX fibres was higher in female than in male hyperthyroid rats. V ₀ was similar in male and female control rats, and decreased with age in both genders (P<0.001). After T₃ treatment, the average V ₀ increased (P<0.001) in females with a concomitant up-regulation of fast MyHC and fast MyLC isoforms irrespective of age. The average V ₀ of the pooled fibres was higher (P<0.001) in female than in male hyperthyroid rats at both ages. In conclusion, gender- and age-related differences were observed in the regulatory influence of 4 weeks’ T₃ treatment on myosin isoform composition and V ₀ in soleus fibres. These differences are presumably related to an interaction of thyroid and sex hormones in the regulation of myosin gene expression. Received: 10 March 1998 / Received after revision 3 June 1998 / Accepted: 14 August 1998     

Phosphocreatine as a marker of contractile activity in single muscle fibres

ATP and phosphocreatine (PCr) were measured in randomly selected single fibres from control, 1- and 8-day low-frequency-stimulated rabbit tibialis anterior muscles. The fibres were classified according to their myosin heavy chain (MHC) complement as type I, IIA or IID. In 1-day stimulated muscle, which has previously been shown to exhibit a steep decline in force output, two fibre populations could be distinguished according to either normal or markedly depressed PCr levels. The fibre population exhibiting normal PCr levels encompassed a major fraction (65%) of type IID fibres and a minor fraction (35%) of IIA fibres. The population with reduced PCr levels comprised type I fibres (@50% reduced), the majority of type IIA fibres (@80% reduced), and a minor fraction of type IID fibres (@70% reduced). Levels of ATP were unaltered in type I and IIA fibres, but were @ 20% reduced in those IID fibres that exhibited low PCr levels. Assuming that those fibres that displayed reduced PCr levels were contracting, the IID and IIA fibres with normal PCr levels were regarded as metabolically recovering, non-contracting fibres. As previously shown, these fibres are transiently refractory during the early phase of low-frequency stimulation. After 8 days of chronic low-frequency stimulation, when force was shown to rise again, most fibres appeared more uniform with regard to reduced PCr and ATP levels. Our results suggest that PCr can be used as a sensitive measure of the degree of activity in single-fibre studies. Received: 14 January 1999 / Received after revision and accepted 21 April 1999     

Myosin heavy chain isoform transition in ageing fast and slow muscles of the rat

Sugiura , T., Matoba , H., Miyata , H., Kawai , Y. & Murakami , N. 1992. Myosin heavy chain isoform transition in ageing fast and slow muscles of the rat. Acta Physiol Scand 144 , 419423. Received 26 August 1 991 , accepted 3 December 1991. ISSN 0001–6772. Laboratory of Biomechanics and Physiology, Faculty of Liberal Arts, Yamaguchi University, Yamaguchi, Faculty of Integrated Arts and Sciences, Tokushima University, Tokushima, and Department of Physiology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan. Using gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis, myosin heavy chain (MHC) isoforms were studied in the extensor digitorum longus (EDI,) and the soleus muscles of male Wistar rats at different ages ( 5, 10 , 20 weeks, 1 and 2 years). In the EDL muscle, four types of MHC isoforms were observed in all age groups. There was an increase in the percentage of HCIId and a concomitant decrease in the percentage of HCIIb with increasing age. No significant difference was observed in the percentages of HCI and HCIIa isoforms in all the age groups. In contrast, the soleus muscle contained two MHC isoforms, HCI and HCIIa. There was an increase in the percentage of HCI and a concomitant decrease in the percentage of HCIIa with increasing age. These results suggest that age-related changes in the MHC isoforms in both the fast-twitch EDI, and the slow-twitch soleus muscles are one factor underlying the age-related decrease in the speed of muscle contraction.     

Distribution of fast myosin heavy chain-based muscle fibres in the gluteus medius of untrained horses: mismatch between antigenic and ATPase determinants

The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in muscle biopsies from the gluteus medius of adult untrained horses by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies and standard myofibrillar ATPase (mATPase) histochemistry. Percutaneous needle biopsies were taken at 3 depths (20, 40 and 60 mm) from 4 4-y-old Andalusian stallions. The percentage of ‘pure’ I MHC fibres increased whereas that for pure IIX MHC fibres decreased from the most superficial to the deepest sampling site. Within the fast fibres, types IIA and IIAX MHC-classified fibres were proportionately more abundant in the deepest sampling site than in the superficial region of the muscle. The immunohistochemical and histochemical characterisation of a large number of single fibres (n=1375) was compared and correlated on a fibre-to-fibre basis. The results showed that 40% of the fibres analysed were pure type I (expressing only MHC-I); they showed correct matching between their antigenic and mATPase determinants. In contrast, within the fast fibres, a considerable proportion of fibres were found showing a mismatch between their immunohistochemical and mATPase profiles. The most common mismatched fibre phenotypes comprised fibres displaying coexpression of both fast MHCs when analysed by immunocytochemistry, but showing an mATPase profile similar to typical IIX fibres (moderate mATPase reaction after preincubation at pH 4.4). Considered altogether, the total mismatched fibres represented only 4.2% of the whole fast fibre population in the superficial region of the muscle, but their proportion increased to 15.6% and 38.4% in the middle and deep regions, respectively, of gluteus medius. It is concluded that a considerable number of hybrid fast MHC IIAX fibres are present in the gluteus medius of untrained horses, suggesting that equine type II fibres have probably been misclassified in numerous previous publications based on the use of histochemistry alone. This has important implications in attempts to study the physiological properties of fast fibre types adequately in horses.     

Myosin heavy chain isoforms influence surface EMG parameters: a study of the trapezius muscle in cleaners with and without myalgia and in healthy teachers

The present study had two aims: (1) to investigate potential differences in proportion of myosin heavy chain isoforms in the trapezius muscle between female cleaners with and without trapezius myalgia and healthy teachers, and (2) to elucidate if myosin heavy chain composition and surface electromyogram (EMG) during an isokinetic endurance test of shoulder flexors are significantly related. The subjects were divided into three groups: (1) 25 female cleaners with diagnosed work-related trapezius myalgia; (2) 25 female cleaners without work-related trapezius myalgia; (3) a control group of 21 healthy female teachers who were not exposed to repetitive or static muscle work. All subjects performed a test involving150 forward flexions using an isokinetic dynamometer. During the test surface EMG was recorded from the trapezius muscle. Muscle samples were obtained from the descending part of the trapezius muscle. The fibre type area percentage based on ATPase staining and the proportions of different MHC isoforms as determined by gel electrophoresis. The trapezius was dominated by MHC I (71%), followed by MHC IIA (22–24%), and MHC IIX (5–7%); no significant differences between the three groups of subjects were found. The proportion of MHC I correlated negatively with MHC IIA (r=–0.78; P<0.001) and MHC IIX (r=–0.53; P<0.001). Significant correlations existed between the proportion of the MHC isoforms and the corresponding proportion of the fibre type area; 27–45% of the variance was explained. The multivariate analysis of the three groups of subjects revealed that MHC variables showed intercorrelations with EMG (both amplitude and frequency variables). However, the directions of the relationships differed among the three groups; the cleaners with myalgia and the teachers showed the greatest similarities in patterns. Intrinsic muscle properties appear to influence the frequency content of the EMG, which is in contrast to some of the theoretical models of the EMG. Our data could suggest that monotonous work such as professional cleaning can change the relationships between muscle structure and the frequency content of the EMG. Electronic Publication     

Effects of eccentric and concentric training on capillarization and myosin heavy chain contents in rat skeletal muscles after hindlimb suspension

We studied the effects of different protocols of post-disuse rehabilitation on angiogenesis and myosin heavy chain (MHC) content in rat hindlimb muscles after caudal suspension. Thirty female Wistar rats were divided into five groups: (1) Control I, (2) Control II, (3) Suspended, (4) Suspended trained on declined treadmill, and (5) Suspended trained on flat treadmill. Fragments of the soleus and tibialis anterior (TA) muscles were frozen and processed by electrophoresis and immunohistochemistry (CD31 antibody). Hindlimb suspension caused reduction of capillary/fiber (C/F) ratios and contents of MHC type I (MHCI) in the soleus in parallel to increased capillary density. Flat treadmill protocols increased the content of the MHCI isoform. The C/F ratio was increased by concentric training after hypokinesis, but was not modified by eccentric training, which caused a greater reduction of capillary density compared to the other protocols. In the TA muscle, hindlimb suspension caused a non-significant increase in capillary density and C/F ratio with limited changes in MHC. The present data demonstrate that the different training protocols adopted and the functional performance of the muscles analyzed caused specific changes in capillarization and in the content of the various MHC types.     

Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

 The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca²⁺], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22° C. In the maximally thiophosphorylated muscle neither OA (10 μM) nor Ca²⁺ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca²⁺] in the presence of 0.5 μM calmodulin, both force and the maximal shortening velocity (V _max) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, V _max was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca²⁺] and at varied [Ca²⁺]. The relation between force and V _max when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca²⁺]. The results show that Ca²⁺ and OA do not influence force or V _max in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining V _max. The finding that the relationship between force and V _max was similar when activation was altered with OA and Ca²⁺ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres. Received: 1 December 1995 / Received after revision: 20 June 1996 / Accepted 12 July 1996     

Effects of hypobaric hypoxia on histochemical fibre-type composition and myosin heavy chain isoform component in the rat soleus muscle

Histochemical fibre-type composition and myosin heavy chain isoform component in the soleus muscle were studied in normoxic rats at postnatal ages of 5, 10, 15, and 20 weeks and in rats exposed to hypobaric hypoxia (460 torr) for 5 weeks from postnatal ages of 5, 10, and 15 weeks. The increase in the percentage of type I fibres and the concomitant decrease in that of type IIa fibres in the soleus muscle of normoxic rats were observed until 15 weeks of age. On the other hand, no change in the fibre-type composition of the muscle during postnatal development was observed in hypoxic rats, irrespective of the age at which they were exposed to hypoxia. The changes in the myosin heavy chain isoform component (MHC I and MHC IIa) of the muscle during postnatal development and by hypoxia corresponded well with those in the muscle fibre-type composition. It is concluded that hypobaric hypoxia inhibits the growth-related shift of muscle fibre-types from type IIa to type I and of myosin heavy chain isoforms from MHC IIa to MHC I in the rat soleus muscle, and that there are no changes in the muscle fibre-type composition or the myosin heavy chain isoform component caused by hypoxia after the shifts in these parameters which occur during postnatal development are completed.     

Alterations of basement membrane in di-isopropanolnitrosamine- induced carcinogenesis of the rat thyroid gland: an immunohistochemical study

Alterations of basement membrane (BM) in di-isopropanolnitrosamine (DIPN)-induced carcinogenesis of the rat thyroid gland were examined by means of immunohistochemical localization of collagen type IV (CN-IV), laminin (LN), and fibronectin (FN) in pre-nodular and nodular thyroid lesions, correlating with the morphogenesis and proliferative activity of these lesions. Adult male rats of the Wistar strain were injected s.c. in the back with DIPN, and the thyroid glands were removed at the 15th and 30th week of treatment. Each of 133 thyroid lesions was histochemically analyzed. The follicular epithelial BM as revealed by CN-IV and LN was discontinued or completely lost during the progression of thyroid lesions from pre-nodular to nodular lesions and finally overt carcinomas. At the same time, the BM of vascular endothelial cells demonstrated a loss of dense capillary networks of follicles, a sinusoidal dilatation and, predominantly in carcinomas, development of interstitial-type blood vessels. However, FN, which was hardly stained in the normal thyroid tissue, was remarkably deposited in the interstitium of invasive carcinomas. These observations strongly suggested that alterations of BM structure play a key role in the morphogenesis of rat thyroid tumors, and that the expression of FN is an important step in the invasive growth of thyroid tumors. Received: 12 October 1999 / Accepted: 30 December 1999     

Clenbuterol-induced fiber type transition in the soleus of adult rats

This study examined the effects of 6 weeks of treatment with theβ ₍₂₎-adrenoceptor agonist, clenbuterol, on the soleus muscle of adult female Sprague-Dawley rats. Animals (4 months old) were divided into two groups: clenbuterol treated (CL,n=7) (2 mg kg⁻¹ body mass injected subcutaneously every other day), and control (CON,n=7) (injected with isotonic saline). Post-treatment body weights were ≈ 5% greater in the CL group compared to CON (P<0.05). Polyacrylamide gel electrophoresis (SDS-PAGE) of soleus myofibrillar protein indicated a clenbuterol-induced decrease (P<0.05) in the relative percentage of type I myosin heavy chain (MHC) with a concomitant increase (P<0.05) in type IIdx MHC, while the proportion of type IIa MHC was unaffected. ATPase fiber typing revealed increases (P<0.05) in the proportion of type II fibers expressed both as a percentage of total fiber number and total cross-sectional area (CSA). Finally, mean type II fiber CSA was ≈25% greater (P<0.05) in the CL groups as compared to the CON group. These data indicate that clenbuterol treatment results in alterations in the MHC phenotype and an increased proportion of type II fiber CSA in the soleus of adult rats. These observations were due to an increase in the total number of type II fibers, as well as hypertrophy of these fibers. Thus, the relative increase in the number of histochemically determined type II fibers and the emergence of the normally unexpressed type IIdx MHC isoform in the soleus suggest a clenbuterol-induced transition of muscle fiber phenotype as well as selective hypertrophy of the type 11 fibers.