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.     

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.     

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.     

Effects of low-resistance/high-repetition strength training in hypoxia on muscle structure and gene expression

To test the hypothesis that severe hypoxia during low-resistance/high-repetition strength training promotes muscle hypertrophy, 19 untrained males were assigned randomly to 4 weeks of low-resistance/high-repetition knee extension exercise in either normoxia or in normobaric hypoxia (FiO₂ 0.12) with recovery in normoxia. Before and after the training period, isokinetic strength tests were performed, muscle cross-sectional area (MCSA) measured (magnetic resonance imaging) and muscle biopsies taken. The significant increase in strength endurance capacity observed in both training groups was not matched by changes in MCSA, fibre type distribution or fibre cross-sectional area. RT-PCR revealed considerable inter-individual variations with no significant differences in the mRNA levels of hypoxia markers, glycolytic enzymes and myosin heavy chain isoforms. We found significant correlations, in the hypoxia group only, for those hypoxia marker and glycolytic enzyme mRNAs that have previously been linked to hypoxia-specific muscle adaptations. This is interpreted as a small, otherwise undetectable adaptation to the hypoxia training condition. In terms of strength parameters, there were, however, no indications that low-resistance/high-repetition training in severe hypoxia is superior to equivalent normoxic training.     

The relationship between muscle myosin ATP-ase activity and isometric endurance in untrained male subjects

Summary The influence of variations in muscle fibre composition on isometric endurance capacity was measured in 23 young healthy untrained male volunteers. After determination of the maximum voluntary force of contraction (MVC), subjects sustained to fatigue contractions at forces of 80%, 50% and 20% of MVC with a 5-min rest between each. A needle biopsy was obtained from m. vastus lateralis and used for histochemical determination of fibre composition based on myosin ATP-ase activity, and fibre are a based on succinate dehydrogenase (SDH) activity. Endurance times were 21±9 s (mean±SD) at 80% of MVC, 56±17 s at 50% of MVC and 203±89 s at 20% of MVC. A wide range of muscle fibre compositions was observed with Type I fibres accounting for 48.0±10.5% of the total, corresponding to 45.0±11.5% of the total muscle area. Muscle fibre composition, whether expressed as the proportions of the different fibre types present, or as the fraction of total muscle cross-sectional area occupied by each fibre type was not correlated with isometric endurance capacity at any of the three forces studied. Endurance time was also unrelated to MVC. In contrast to the results of previous studies where trained subjects were used, or where different muscle groups were compared, these results suggest that isometric endurance is not influenced by muscle fibre composition.     

The effect of varying the time of concentric and eccentric muscle actions during resistance training on skeletal muscle adaptations in women

This study investigated the effect of manipulating the time to complete both the concentric (CON) and eccentric (ECC) muscle actions during resistance training on strength, skeletal muscle properties and cortisol in women. Twenty-eight women (mean ± SE age = 24.3 ± 1.1 year) with strength training experience completed three training sessions per week for 9 weeks. Two sets of four lower body exercises (leg press, parallel squat, knee extension and knee flexion) were completed using 6–8 RM intensity. The long CON (LC) group performed the CON action for 6 s and the ECC action for 2 s, while the long ECC (LE) group completed the CON and ECC phases for 2 and 6 s, respectively. Both groups experienced significant increases in leg press CON only, ECC only and combined ECC and CON maximal strength (1 RM). Immunohistochemical analyses demonstrated that both types I and IIA vastus lateralis fibre areas significantly increased following LC training while only type I fibre area increased following LE training. There was a decrease in MHCIId(x) with a concomitant increase in MHCIIa (P < 0.05) in both groups. Twenty-four hour urinary cortisol significantly increased after LC training only. It was concluded that LC resistance training was more effective than LE for increasing both types I and IIA fibre area and cortisol when time under tension and intensity of muscle actions were matched between the two modes of resistance training in young healthy women.     

Calcineurin and heat-shock proteins modulation in clenbuterol-induced hypertrophied rat skeletal muscles

To examine the changes in heat shock proteins (HSPs) and calcineurin (CaN), a calcium/calmodulin regulated protein phosphatase, in hypertrophied rat skeletal muscles, adult male Wistar rats were administered clenbuterol (CLB, 30 mg l^–1 in drinking water), a 2-agonist, for 4 weeks. Compared to controls, CLB-treated rats had significantly larger body (10%) and relative (to body weight) soleus (Sol, 16%), plantaris (Plt, 32%) and gastrocnemius (Gast, 27%) weights. Immunohistochemically classified fast fibers were hypertrophied in the Sol (64%), Plt (62%), and deep (d, 70%) and superficial (s, 44%) regions of the Gast, whereas slow fibers were hypertrophied only in the Plt (47%). The percentage of fast fibers in the Sol increased from 10% to 21%. The myosin heavy chain (MHC) isoform composition shifted from slow to fast in the Sol (increase in the percentage of type IIa MHC and de novo synthesis of type IIx MHC) and Gast-d (de novo synthesis of type IIb MHC) and to the faster isoforms in the Plt (increase in the percentage of type IIb MHC). Hsp72 and Hsp90 levels in CLB-treated rats were 52% and 50% lower in the Sol and 44 and 41% lower in the Gast-d, respectively, than in control rats. In control rats, the relative content of CaN was: Sol>Gast-d>Plt>Gast-s, and CLB treatment enhanced the CaN content by 1.4-, 1.2-, 5.0-, and 3.3-fold, respectively. These results indicate that the down-regulation of HSPs in the Sol and Gast-d was closely related to a decrease in the slow phenotype, and that the relative up-regulation of CaN among the muscles/regions was closely related to the selective hypertrophy of fast fibers in the CLB-treated rats.     

Effect of anabolic/androgenic steroids on myosin heavy chain expression in hindlimb muscles of male rats

We have examined the effect of male sex hormones on the myosin heavy chain (MHC) expression of the soleus and extensor digitorum longus (EDL) muscles. Young male adult Wistar rats were treated over a 25-day period with either oil (CON, n = 8), nandrolone (nortestosterone decanoate, NAN, n = 8), nandrolone combined with endurance exercise (treadmill running, NAN+EXE, n = 8), or were castrated (CAS, n = 8). The MHC composition of the soleus and EDL muscles was measured by electrophoresis. Castration and treatment with nandrolone had no effect (P > 0.05) on the relative levels of MHC in the soleus and EDL. In contrast, in NAN+EXE rats, the relative level of MHC-1 increased [slow isoform; mean (SD) 97.6 (4.7)% in NAN+EXE rats, versus 86.5 (7.5)% in CON rats] and MHC-2a decreased [fast isoform; 2.4 (4.7)% in NAN+EXE, versus 13.5 (7.5)% in CON rats], only in the soleus (P < 0.05). In conclusion, it appears that endogenous anabolic/androgenic steroids are not essential for the maintenance of the MHC expression of fast- and slow-twitch muscles in the young adult male rat. In addition, nandrolone combined with endurance exercise induced a shift from a fast to a slower MHC phenotype of the slow-twitch muscle. Accepted: 27 July 1999     

mRNA expression of fibroblast growth factors and hepatocyte growth factor in rat plantaris muscle following denervation and compensatory overload

We addressed the question of whether hypertrophy induced by compensatory overload differs according to innervation status, and how fibroblast growth factors (FGF) and hepatocyte growth factor (HGF) mRNAs are expressed in the rat plantaris muscle during overload (OL) and/or denervation. Male Wistar rats were divided into four groups (Normal-Cont, Normal-OL, Denervated-Cont, and Denervated-OL). according to the plantaris denervation and/or overload. Three weeks later, plantaris weight in Denervated-Cont and Denervated-OL was significantly lower than in the Normal-Cont. The muscle weights in the Normal-OL were higher than in the Normal-Cont. The muscle weights in the Denervated-OL were higher than in the Denervated-Cont. Three days after the treatment, FGF-2, FGF-6, FGF-7 and HGF mRNAs in the Normal-OL were significantly higher than those in the Normal-Cont. FGF-2, FGF-6, FGF-7 and HGF mRNAs in the Denervated-OL were also significantly higher after 3 days than those in the Denervated-Cont. After 7 days, FGF-2, FGF-5, FGF-6, FGF-7 and HGF mRNAs were significantly higher in the Normal-OL than those in the Normal-Cont. At 21 days, FGF-1, FGF-6 and HGF mRNA levels were significantly increased. In the Denervated-OL, FGF-2, FGF-7 and HGF mRNAs at 7 days, and FGF-2 mRNA at 21 days were significantly higher than those in the Denervated-Cont. FGF-2 and FGF-6 mRNA levels decreased significantly following denervation; however, FGF-1, FGF-5, FGF-7 and HGF mRNA levels increased and maintained this increase for the 21-days treatment period. Muscle hypertrophy was thus induced by compensatory overload irrespective of innervation status, possibly in association with certain FGFs and HGF. The differential mRNA expression patterns of FGFs and HGF observed following compensatory overload and/or denervation suggest distinct roles for individual FGFs and HGF in muscle hypertrophy and/or atrophy.     

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     

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.     

The effect of androgen status on skeletal muscle myosin heavy chain mRNA and protein levels in rats recovering from immobilization

Immobilization rapidly alters skeletal muscle. The aim of the present study was to determine whether testosterone administration or, in contrast, hypogonadism affects the recovery of muscle mass and myosin heavy chain (MHC) profile at both the mRNA and protein level, after 1 week of immobilization. Male rats were assigned to one of five groups: control (C), hindlimb-immobilized (IMM), and recovery (REC; where animals were allowed 2 weeks of free cage-activity after immobilization). The recovery group was further divided to eugonadal (REC-C), castrated (REC-GDX), and a testosterone-treated (REC-T). In all groups except REC-T, the body masses after immobilization were smaller than in C, although after immobilization the body mass in REC-T recovered at a slower rate than in the other two REC groups. The gastrocnemius mass and the amount of type IIa MHC mRNA decreased during immobilization, but the control levels were regained after recovery. The amount of type IIb mRNA was reduced in REC-GDX compared to C and IMM. The changes in the relative distribution of MHC mRNA were in line with these results. After recovery, the proportion of type IIx MHC protein increased and type IIb protein decreased, although in REC-T the changes were not statistically significant. The proportion of type IIa MHC protein increased only in REC-GDX. In summary, during recovery from immobilization it seems that muscle mass increases and the MHC mRNA and protein profile tend to change toward a slower phenotype, primarily as a result of the decrease in type IIb MHC. However, these changes occur rather independently of the testosterone status. Accepted: 9 June 2000     

伊贝沙坦对血管紧张素Ⅱ所致心肌细胞蛋白质合成和肌球蛋白重链表达的影响

目的 观察伊贝沙坦对血管紧张素Ⅱ（AngⅡ）所致心肌细胞中蛋白质合成速率及肌球蛋白重链（MHC）基因表达改变的影响.方法 以AngⅡ及伊贝沙坦分别或同时作用于培养的细胞.采用放射性同位素[^3H]-leu掺入法检测培养心肌细胞蛋白质合成速率.应用荧光定量PCR方法检测心肌细胞心房利钠肽因子（ANF）以及α-MHC、β-MHC的表达.结果 AngⅡ处理使心肌细胞中[^3H]-Leu掺人增加（P＜0.05）,同时ANF mRNA的表达明显高于正常（P＜0.05）;α-MHC mRNA的表达显著低于正常（P＜0.05）,而β-MHC mRNA的表达显著高于正常（P＜0.05）,α-MHC/β-MHC的比值下降（P＜0.05）.当伊贝沙坦与AngⅡ共同作用于培养的心肌细胞时,与AngⅡ组比较,[^3H]-Leu的掺入明显下降（P＜0.05）,与正常组比无统计学意义（P＞0.05）;同时ANF的表达下降,与正常组比无统计学意义（P＞0.05）;心肌细胞中α-MHC的表达明显增高（P＜0.05）,而β-MHCmRNA的表达显著降低（P＜0.05）,α-MHC/β-MHC的比值上升（P＜0.05）.结论 伊贝沙坦能抑制AngⅡ所致的心肌细胞肥大和细胞中α-MHC向β-MHC表达的转换.     

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.     

The potential protective role of taurine against 5-fluorouracil-induced nephrotoxicity in adult male rats

Nephrotoxicity is common with the use of the chemotherapeutic agent 5-Fluorouracil (5-FU). The current study aimed to investigate the probable protective effect of taurine (TAU) against 5-FU-induced nephrotoxicity in rats using biochemical, histological and ultrastructural approaches. Twenty-four rats were equally divided into control, TAU, 5-FU and 5-FU + TAU groups.5-FU significantly elevated levels of blood urea nitrogen (BUN), creatinine, and uric acid; while it reduced activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Also, 5-FU induced significant elevation in malondialdehyde (MDA) levels accompanied with marked decline in γ-glutamyltranspeptidase (GGT) and alkaline phosphatase (AP) levels in kidney tissues. These biochemical alterations were accompanied by histopathological changes marked by destruction of the normal renal structure, in addition to ultrastructural alterations represented by thickened and irregular glomerular basement membranes, congested glomerular capillaries, damaged lining fenestrated endothelium, mesangial cells hyperplasia with expanded mesangial matrix, and distorted podocyte’s processes. Also, the proximal (PCT) and distal (DCT) convoluted tubules showed thickened basement membranes, destructed apical microvilli and loss of basal infoldings of their epithelial cells.Administration of TAU to 5-FU-treated rats reversed most of the biochemical, histological, and ultrastructural alterations. These results indicate that TAU has a protective effect against 5-FU-induced nephrotoxicity.     

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