Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening velocity in smooth muscle

The relation between the isoform distribution of the myosin 17 kDa essential light chain (LC₁₇) and the mechanical properties of smooth muscle was investigated. The relative content of the basic (LC_17b) and acidic (LC_17a) isoelectric variants of the 17 kDa myosin light chain was determined in different mammalian smooth muscle tissues. The relative content of LC_17b varied between muscles: rabbit rectococcygeus 0%, rabbit trachea 5%, guinea-pig taenia coli 21%, rat uterus 38%, rabbit aorta 56% and rat aorta 60%. The rate of tension development was determined following photolysis of cagedadenosine triphosphate (ATP) in skinned fibres activated with thiophosphorylation of the regulatory light chains. The half-time for force development was 0.67 s in rabbit rectococcygeus, 1.6 s in rabbit trachea, 1.13 s in guineapig taenia coli and 1.38 s in rabbit aorta. The maximal shortening velocity (v _max) was determined with the isotonic quick release technique in skinned fibre preparations activated with thiophosphorylation. v _max was 0.25 muscle lengths per second (ML/s) in rabbit rectococcygeus, 0.24 ML/s in rabbit trachea, 0.17 ML/s in guinea-pig taenia coli, 0.11 ML/s in rat uterus and 0.03 ML/s in rabbit aorta. The range of variation in v _max between muscles was larger than in the half-time for force development. The inverse relationship between v _max and the relative content of LC_17b in the investigated muscles suggests that the type of essential myosin light chain influences the v _max in smooth muscle.     

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     

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     

Effects of ageing and gender on contractile properties in human skeletal muscle and single fibres

Aim: The objective of this study is to improve our understanding of the mechanisms underlying the ageing‐ and gender‐related muscle weakness. Methods: Ageing‐ and gender‐related differences in regulation of muscle contraction have been studied in knee‐extensor muscles at the whole muscle and single muscle fibre levels in young and old sedentary men and women. In vivo knee‐extensor muscle function was measured at slow (30° s^?1) and faster (180 ° s^?1) speeds of movement. Maximum velocity of unloaded shortening (V₀) and maximum force normalized to cross‐sectional area (CSA) [specific tension (ST)] were measured in single ‘skinned’ skeletal muscle fibre segments. Results: Significant ageing‐ and gender‐related differences were observed in muscle torque. A 33–55% ageing‐related decline (P < 0.001) in maximum torque was observed irrespective of gender. At the single muscle fibre level, the ageing‐related decline in knee‐extensor muscle function was accompanied by a 20–28% decline in ST in muscle fibres expressing the type I MyHC isoform in both men and women, and a 29% decline in type IIa muscle fibre CSA, but the decreased fast‐twitch fibre size was restricted to the men. Furthermore, in both men and women, V₀ decreased in muscle cells expressing the type I and IIa MyHC isoforms. Conclusion: The present results provide evidence of specific ageing‐ and gender‐related differences in regulation of muscle contraction at the cellular level. It is suggested that these cellular changes have a significant impact on muscle function and the ageing‐related motor handicap.     

Scaling of skeletal muscle shortening velocity in mammals representing a 100,000-fold difference in body size

To fully understand the effect of scaling on skeletal muscle shortening velocity (V ₀), it is important to know which phenotypic characteristics drive the changes between species. The purpose of the current investigation was to compare the effects of body mass and femur length, as an estimate of total limb length, on V ₀ in species that cover a 100,000-fold range of body masses. Using the slack test procedure, V ₀ was determined for fibers expressing types I and IIa myosin heavy chain (MyHC) isoforms in the mouse, rat, dog, human, horse, and rhinoceros under identical experimental conditions. A significant scaling effect on V ₀ was detected when compared to body mass (type I fibers, r=0.95, p<0.01; type IIa fibers, r=0.83, p<0.05). However, the horse’s V ₀ for both fiber types was faster than the human’s, despite having a 5-fold greater body mass than the human. When V ₀ was scaled vs limb length, the strength of the relationships improved in fibers expressing both types I and IIa MyHC (r=0.98, p<0.001, and r=0.89, p<0.05, respectively) and scaled with the expected relationship, with the species with the shorter femur, the horse, having the faster V ₀. A similar effect can be seen with stride frequency scaling more closely with limb length than body mass. These results suggest that limb length, not body mass, is a more relevant factor driving the scaling effect on skeletal muscle shortening velocity.     

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.     

Nonmuscle myosin, force maintenance, and the tonic contractile phenotype in smooth muscle

Recent studies have demonstrated that nonmuscle (NM) myosin II forms filaments and can generate and maintain force in smooth muscle tissue [Lofgren et al. (2003) J Gen Physiol 121:301–310; Morano et al. (2000) Nat Cell Biol 2:371–375]. To further investigate the mechanical contribution of NM myosin to force maintenance during smooth muscle contraction, we utilized a selective inhibitor of the NM myosin ATPase, blebbistatin [Straight et al. (2003) Science 299:1743–1747]. Force and myosin light chain (MLC₂₀) phosphorylation were measured during KCl stimulation of small strips of intact mouse bladder and aorta at 22°C. The bladder strips contracted with a typical phasic force response, characterized by a large, rapid, transient increase in force followed by a decline to a lower, steady-state level. The addition of blebbistatin did not alter the peak force, but decreased force maintenance. KCl depolarization of aortic strips resulted in a tonic contraction; force increased to a sustained steady state. Similar to the bladder tissue, blebbistatin substantially decreased the steady-state force in the aorta. Blebbistatin did not influence the MLC₂₀ phosphorylation transient in either tissue type. Additionally, blebbistatin did not change the maximum shortening velocity (V _max) during KCl depolarization of the aorta. Our results also suggest that NMIIA and NMIIB isoforms are differentially expressed. The expression of NMIIA is more prominent in the bladder, while NMIIB expression is predominant in the aorta. These results suggest that NM myosin contributes to the mechanism of force maintenance in smooth muscle, and could suggest that the expression of NMIIB is a factor for determining the tonic contractile phenotype.     

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.     

Correlation between percentage fiber type area and myosin heavy chain content in human skeletal muscle

Histochemical methods are routinely used to delineate skeletal muscle fiber types. In the present investigation, this qualitative determination of fiber type composition was compared to the electrophoretically determined myosin heavy chain (MHC) content from a large number of human muscle biopsy samples. Biopsies were taken from the vastus lateralis muscle at the beginning and every 2 weeks during 8 weeks of highi-ntensity resistance training from men (n = 13) and woman (n = 8). Muscle was also extracted from nontraining men (n = 7) and women (n = 5) at the same periods. Six muscle fiber types (I, IC, IIAC, IIA, IIAB, and IIB) were determined using basic myofibrillar adenosine triphosphatase histochemistry. Cross-sectional areas were determined for the three major fiber types (I, IIA, and IIB) and used to calculate the percentage area of these types. Electrophoretic techniques were used to separate and quantify the percentage MHC content in these same biopsy samples, and these data were then used to compare with the percentage fiber type area. Correlation analyses suggest a relationship between the histochemically assessed percentage fiber type area and the electrophoretically assessed MHC content in human limb musculature. However, because of possible histochemical misclassification of some fibers (especially in trained muscle) both techniques may be essential in yielding important information about fiber type composition and possible fiber type transformations.     

Ageing alters the myosin heavy chain composition of single fibres from human skeletal muscle

The myosin heavy chain composition of single fibres (n = 1088) was analysed with an electrophoretic technique in biopsy material from m. vastus lateralis (n = 5) and m. biceps brachii (n = 4) of young (23-31 years old) and elderly men (68-70 years old). In m. vastus lateralis, elderly subjects had a higher proportion of fibres showing a coexistence of myosin heavy chain types I and IIa (20 +/- 3% vs 8 +/- 1%, P less than 0.05) and of myosin heavy chain types IIa and IIb (33 +/- 2% vs 12 +/- 4%, P less than 0.05). In contrast, the young subjects had a higher proportion of fibres containing only myosin heavy chain type I (50 +/- 5% vs 33 +/- %, P less than 0.05) and type IIa (26 +/- 3% vs 12 +/- 2%, P less than 0.05). A similar pattern of myosin heavy chain expression was found in single fibres from m. biceps brachii, with the exception that the elderly subjects had a lower proportion of fibres with coexistence of types IIa and IIb (23 +/- 1% vs 34 +/- 2%, P less than 0.05) and a higher proportion of fibres containing only myosin heavy chain type IIa (25 +/- 5% vs 12 +/- 2%, P less than 0.05). Three fibres from m. biceps brachii contained all three isoforms. These results indicate that coexistence of myosin heavy chain isoforms in single fibres is present in skeletal muscles of young adults, and that there is an increased occurrence of this phenomenon with ageing.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the ability of 8-bromoadenosine triphosphate to support contractility of vertebrate skeletal muscle fibers

Previous studies using solubilized fragments of myosins have shown that an ATP analogue, 8-bromoadenosine triphosphate (8-Br-ATP) is a poor substrate for fast skeletal myosin isoform. We further characterized the analogue by using vertebrate skeletal muscle fibers. In the absence of calcium, the rate of 8-Br-ATP hydrolysis by fibers was higher than that for ATP, but it kept the fibers relaxed. The X-ray diffraction patterns of fibers relaxed by 8-Br-ATP were also indistinguishable from those of fibers relaxed by ATP, but higher concentrations were needed to keep the fibers relaxed. In the presence of calcium, the fibers exhibited force development and active shortening to varying extents. Although some of the energy for the observed contractility could be ascribed to the trace ATP in the reagents, the fibers activated in 8-Br-ATP performed much more mechanical work than expected from the energy of the trace ATP alone. The results suggest that most of the hydrolytic products of 8-Br-ATP dissociate from myosin prematurely, but a small fraction of myosin with these products does enter the calcium-dependent work-producing pathway and complete the normal process of chemo-mechanical conversion.     

缺氧及缺氧-复合运动大鼠比目鱼肌肌球蛋白重链组成变化

 目的： 观察缺氧及缺氧复合运动条件下大鼠比目鱼肌肌球蛋白重链(MHC)异构体组成的变化。方法: Wistar大鼠随机分为4组：平原对照组、缺氧组、平原运动组和缺氧复合运动组。缺氧复合运动组大鼠持续暴露于模拟海拔5 000 m高原5周，每天降至4 000 m高原进行游泳运动1 h（6 d/week），运动结束后回升至5 000 m；缺氧组大鼠同时在低压舱内相同海拔高度饲养，但不进行游泳运动；平原运动组和平原对照组在舱外同时饲养，其中平原运动组每天进行游泳运动1 h（6 d/week）。在末次运动结束后24 h处死大鼠，分离后肢比目鱼肌。用含30%甘油的SDS-PAGE电泳分离比目鱼肌MHC异构体，观察MHC异构体组成变化。结果: 平原游泳运动大鼠比目鱼肌重量指数增加，而缺氧复合运动组与平原对照比较无显著差异；大鼠比目鱼肌中段MHC主要由Ⅰ(78％)、Ⅱa(22％)2种异构体组成，缺氧组比目鱼肌MHC由Ⅱa向Ⅰ型转变，单纯运动和缺氧复合运动组与缺氧组变化趋势相同，但缺氧复合运动组显著低于单纯运动组。结论: 慢性缺氧和运动训练后，大鼠比目鱼肌肌纤维MHC组成向能量利用效率更高的慢收缩肌纤维Ⅰ型转变，提示慢性缺氧和缺氧复合运动后比目鱼肌能量利用效率增加。     

Effect of electrical stimulation and exercise on the phosphorylation state of myosin light chains from fish skeletal muscle

This paper investigates the phosphorylation of fish muscle myosin following electrical stimulation and exercise. Purified myosin isolated from the fast myotomal muscle of rainbow trout (Salmo gairdneri) shows three light chains on SDS polyacrylamide gels of molecular weights 16,000 (LC1) 18,000 (LC2) and 24,000 (LC3). The 18,000 dalton light chain (LC2) is capable of being phosphorylated and dephosphorylated in vitro by respectively rabbit myosin light chain kinase and phosphatase.The native phosphorylation state of fast muscle myosin has been investigated in rainbow trout and dogfish (Scyliorhinus canicula) using a technique involving freeze-clamping in liquid nitrogen (–159°C) and extraction of a light chain fraction in 5 M guanidine-HCl/ethanol. The two forms of the LC2 light chain, can be resolved in 8 M urea polyacrylamide gels at pH 8.6.Fast muscle from anaesthetised trout contained 0.16 moles phosphorylated LC/mole LC2. Electrical stimulation of isolated muscle under various conditions (1–10 s isometric tetani, 5–500 Hz) did not result in a significant increase in the phosphorylated form of the LC2 light chain. Qualitatively similar results were obtained for isolated fast muscles from dogfish (Scyliorhinus canicula). In five trout subject to strenuous exercise, two fish showed a slight increase in myosin phosphorylation (0.32 moles P LC/mole LC2) and three, no significant change (0.20 moles P LC/mole LC2).The lack of correlation between the phosphorylation state of LC2 light chain and electrical stimulation indicates that, unlike rabbit and frog skeletal muscle myosin, phosphorylation is not an integral part of the excitation-contraction cycle in fish muscle.     

Changes in fibre-type composition and myosin heavy-chain IId isoform in rat soleus muscle during recovery period after hindlimb suspension

We examined the changes in myosin heavy-chain (HC) isoforms and fibre-type composition in rat soleus muscle using both myosin adenosine triphosphatase staining and sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE) analyses during the recovery period after 4 weeks of hindlimb suspension. Although there was no change in type IIc fibres after the suspension, an increase in this type of fibres was observed during the 1- to 4-week recovery period. The increase in type Ilc fibres was considered to be due to a shift from type Ila to IIc fibres. The SDS-PAGE analysis revealed the presence of the HC IId isoform, which was not observed in the control muscle, after a 4-week hindlimb suspension. The HC IId isoform gradually decreased over 3 weeks of recovery and disappeared in the 4th week of recovery after the suspension. These results suggest that the hypogravity conditions induced by hindlimb suspension stimulated the synthesis of the HC IId isoform, whereas an increase in mechanical load to the muscle accelerated the degradation of the HC IId isoform and the synthesis of type Ilc fibres during the recovery period after hindlimb suspension.     

Exercise training induces transitions of myosin isoform subunits within histochemically typed human muscle fibres

Fibre type composition based on histochemical myosin ATPase reaction was studied in cross section of biopsies from the vastus lateralis muscle of men. In addition, protein composition as well as peptide patterns of isolated myosin heavy chains were examined in batches of individually classified fibres from the same biopsies. High intensity endurance training during 8 weeks induces significant decreases by 31–70% of the type IIB fibre population in 3 of 4 subjects (in one case no change was observed). These decreases were offset by corresponding increases in either type I or type IIA fibres with the type IIC fibres remaining always below 3%. A total of 13 professional cyclists with training periods over several years have a 20 times lower content of type IIB fibres than 4 sedentary controls and a concomitant high content of 80% of type I fibres. The content of type I and type IIB fibres of 8 sprinter athletes did almost not differ from that of controls. Thus the type IIB fibres respond most sensitively with a decrease to aerobic endurance training. Since both type IIA and IIB fibres were identical in protein composition containing the same fast variety of myosin light chains and heavy chains as well as troponin-I, their interconversion could not be seen at the molecular level. However, the slow variety of myosin light chains and of troponin-I started accumulating after 8 weeks of training in type IIA fibres. Furthermore, the myosin heavy chain isoform started shifting by producing new peptide patterns that resemble the digestion pattern of slow myosin heavy chains in fibres which still classified as type IIA. These changes on the molecular level in type IIA fibres mark the beginning of their transition over the intermediate and variable type IIC fibres, towards the slow type I fibre.Abbreviations VLM vastus lateralis muscle - maximal oxygen uptake capacity - MVD total mitochondrial volume density - I slow-twitch fibre type - IIA IIB, fast-twitch fibre types - IIC intermediate fibre type - HC myosin heavy chain - LC myosin light chain - TN-I troponin-I - ATPase adenosine 5-triphosphatase (EC 3.6.1.3) - SAV-8 Staphylococcus aureus V-8 proteinase (EC 3.4.21.19) - SDS sodium dodecyl sulfate - kD kilo Dalton     

Comparative effects of hindlimb suspension and exercise on skeletal muscle myosin isozymes in rats

Summary The purpose of this study was to ascertain the time course of changes, whilst suspending the hindlimb and physical exercise training, of myosin light chain (LC) isoform expression in rat soleus and vastus lateralis muscles. Two groups of six rats were suspended by their tails for 1 or 2 weeks, two other groups of ten rats each were subjected to exercise training on a treadmill for 9 weeks, one to an endurance training programme (1-h running at 20 m · min^–1 5 days · week^–1), and the other to a sprint programme (30-s bouts of running at 60 m · min^–1 with rest periods of 5 min). At the end of these experimental procedures, soleus and vastus lateralis superficialis muscles were removed for myosin LC isoform determination by two-dimensional gel electrophoresis. Hindlimb suspension for 2 weeks significantly increased the proportion of fast myosin LC and decreased slow myosin LC expression in the soleus muscle. The pattern of myosin LC was unchanged in the vastus lateralis muscle. Sprint training or endurance training for 9 weeks increased the percentage of slow myosin LC in vastus lateralis muscle, whereas soleus muscle myosin LC was not modified. These data indicate that hindlimb suspension influences myosin LC expression in postural muscle, whereas physical training acts essentially on phasic muscle. There were no differences in myosin LC observed under the influence of sprint- or endurance-training programme.     

肌球蛋白轻链激酶介导内皮细胞屏障功能变化的研究进展

<正>血管内皮细胞是连续被覆于全身血管内膜的一层细胞群,不仅构成一道半选择通透性屏障,还合成和分泌多种生物活性物质,维持正常的心血管功能。血管内皮细胞的完整和屏障功能完好对于维持心血管稳态有至关重要的作用。内皮细胞受损所致屏障功能下降是血管病变的始动环节,如脑血管内皮连接受损导致的脑水肿[1]。肌球蛋白轻链激酶(myosin light chain kinase,MLCK)是第1个被发现的依赖     

Contractile properties and myosin heavy chain isoform composition in single fibre of human laryngeal muscles

In the present study we aimed to determine the functional properties and the myosin heavy chain (MHC) isoform composition of single chemically skinned fibres from the vocal muscle of four adult men (age: 55–67 years). Single fibres, dissected from the bioptic samples, were chemically skinned and isometric tension (P ₀) and maximal shortening velocity (V ₀) were measured at pCa 4.6. MHC and myosin light chain (MLC) composition of fibre segments and MHC distribution of the biopsy samples were analysed by SDS–poly-acrylamide gel electrophoresis (SDS—PAGE) and densitometry. Four MHC isoforms (1, 2A, 2X and a fourth isoform, provisionally called L) and five MLC isoforms (MLC1s, MLC1f, MLC3f, MLC2f, MLC2s) were identified. The major findings of this study were: (1) fast MHC isoforms (in particular MHC-2A) and fast fibres were predominant, (2) one-third of the fibres were mixed or hybrid, i.e. expressed more than one MHC isoform, (3) V ₀ and P ₀ values were determined by the MHC isoform composition and mixed fibres showed functional properties which were intermediate between pure fibres; MHC-L was associated with V ₀ values similar to those of MHC-2A, (4) compared with limb muscles, V ₀ values of laryngeal fibres were similar to those of limb muscle fibres containing the same MHC isoform whereas P ₀ values were lower for slow and fast 2X fibres and similar for fibres expressing MHC-2A.