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     

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     

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     

Postnatal transitions in myosin heavy chain isoforms of the rabbit superficial masseter and digastric muscle

We investigated the early (< 8 weeks) and late (> 8 weeks) postnatal development of the fibre type composition and fibre cross-sectional area in the superficial masseter and digastric muscle of male rabbits. It was hypothesized, first, that due to the transition between suckling and chewing, during early postnatal development the increase in the proportion of slow fibre types and in fibre cross-sectional areas would be larger in the masseter than in the digastric; and second, that due to the supposed influence of testosterone during late postnatal development, the proportion of slow fibre types in both muscles would decrease. Fibre types were classified by immunostaining according to their myosin heavy chain (MyHC) content. The proportion of slow fibre types significantly increased in the masseter, from 7% at week 1 to 47% at week 8, and then decreased to 21% at week 20, while in the digastric it increased from 5% in week 1 to 19% at week 8 and remained the same thereafter. The changes in the proportion of fast fibre types were the opposite. The remarkable increase and decrease in the proportion of slow fibre types in the masseter was attributed predominantly to MyHC-cardiac alpha fibres. During early development, the cross-sectional area of all fibres in both muscles increased. However, only the fast fibre types in the masseter continued to grow further after week 8. Before weaning, the fast fibre types in the digastric were larger than those in the masseter, but after week 8, they became larger in the masseter than in the digastric. In adult animals, masseter and digastric had the same percentage of fast fibre types, but these fibres were almost twice as large in masseter as in digastric.     

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.     

Nonuniform myosin expression along single fibers of chronically stimulated and contralateral rabbit tibialis anterior muscles

Using a combined histochemical and biochemical technique, single fiber analyses were performed on chronically stimulated and contralateral tibialis anterior (TA) muscles of the rabbit. The major fiber population (60%) in 30 days stimulated TA was transforming fibers (type IIC). Some of these fibers displayed a nonuniform distribution of histochemically assessed myofibrillar actomyosin ATPase (mATPase) activity. This heterogeneity of mATPase activity along the fibers was verified in longitudinal sections and by microphotometric evaluation of mATPase staining intensities in serial cross-sections. Biochemical analyses of single fiber segments revealed that these C fibers not only coexpressed fast- and slow-myosin subunits but did so nonuniformly along their length. The distribution of fast- and slow-myosin subunits in these fibers was not random but focal. Variations in myosin expression were also observed in some of the C fibers in the contralateral TA. As opposed to the transforming fibers in the stimulated TA, heterogeneities of mATPase activity and myosin subunits in these contralateral C fibers were less focal and more gradual. These findings suggest that muscle fibers in chronically stimulated TA and the contralateral muscle do not transform synchronously or uniformly along their length.     

Cortisone-induced changes in myosin heavy chain distribution in respiratory and hindlimb muscles

In this study the effects of administration of cortisone acetate (100 mg kg^-1 body weight subcutaneously for 11 days) on distribution and cross-sectional area of different fibre types of rat skeletal muscles were investigated. Diaphragm, parasternal intercostal (PI), extensor digitorum longus (EDL) and soleus muscles were examined in cortisone treated animals (CA) in comparison with ad libitum controls (CTRL) and pair-fed (PF) controls. Four fibre types (I or slow and IIA, IIX, IIB or fast) were identified on the basis of their myosin heavy chain composition using a set of monoclonal antibodies. In CA rats the reduction of cross-sectional area was above 30% in IIX fibres of diaphragm, IIB fibres of PI and in all fast fibres of EDL. In all muscles slow fibres were spared from atrophy. Significant variations in fibre type distribution were found in the muscles of CA rats when compared to CTRL. The percentage of IIB fibres decreased in EDL, PI and diaphragm. This decrease was accompanied by an increase in the percentage of IIA fibres in the same muscles. No changes in the percentage of slow fibres and of fast IIX fibres were observed in EDL, PI and diaphragm of CA rats in comparison with CTRL. In soleus of CA rats the proportion of IIA fibres was lower than in CTRL. In EDL of PF rats atrophy of IIA fibres and changes in fibre type distribution were similar to those observed in CA rats. In diaphragm, PI and soleus of PF rats no significant decrease in fibre cross-sectional area nor significant changes in fibre distribution were found in comparison with CTRL rats.     

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     

Myosin heavy chain isoform transformation in single fibres from m. vastus lateralis in spinal cord injured individuals: Effects of long-term functional electrical stimulation (FES)

The myosin heavy chain (MHC) composition of single fibres from m. vastus lateralis of five spinal-cord-injured (SCI) individuals was analysed by Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) before, and after 6 and 12 months of functional electrical stimulation (FES)-training, administrated for 30 min three times per week. Prior to FES training 37.2% of the fibres contained only MHC HB, 21.2% only MHC IIA, and 40.7% co-expressed MHC IIA and MHC IIB. After 6 months of FES-training the number of fibres containing only MHC IIB was reduced to 2.6% (P < 0.05), the number of fibres containing only MHC IIA was increased to 44.3 (P < 0.05), and the number of fibres co-expressing MHC IIA and MHC HB was 50.9% (ns). After 12 months almost all fibres (91.2%,P < 0.05) contained only MHC IIA. The number of fibres containing only MHC IIB was 2.3 % and the fibres co-expressing MHC HA and HB had decreased to 4.6% (P < 0.05). The amount of fibres containing only MHC I never exceeded 0.5%. Likewise, the number of fibres co-expressing MHC I and MHC IIA was below 2% throughout the study period. In total, the MHC composition of 1596 single fibres was determined. This study shows that FES-training of paralysed human skeletal muscle administrated over a prolonged period of time, can lead to a marked switch in MHC expression from about equal amounts of MHC HA and MHC HB to an almost total dominance of MHC HA.     

The role of myosin light chain kinase-dependent phosphorylation of myosin light chain in phorbol ester-induced contraction of rabbit aorta

 We investigated the role of 20 kDa myosin light chain (MLC20) phosphorylation in contractions following protein kinase C (PKC) activation by 12-deoxyphorbol-13-isobutyrate (DPB) in rabbit aortae. DPB induced a sustained contraction and phosphorylation of MLC20 independent of a change in cytosolic Ca²⁺ ([Ca²⁺]_i). Phosphorylation on Ser¹⁹ of MLC20, which is a target site of MLC kinase (MLCK), was 9.2 ± 5.1% and 22.3 ± 4.9% of the phosphorylation caused by KCl, at 5 and 30 min of application of DPB, respectively. When KCl-precontracted muscles were rinsed with Ca²⁺-free, EGTA solution, [Ca²⁺]_i rapidly declined, MLC20 was dephosphorylated and the tension decreased. If DPB was present in the Ca²⁺-free solution, the relaxation and the dephosphorylation of either total MLC20 or Ser¹⁹ were inhibited. The phospholipase A₂ inhibitor ONO-RS-082 partially antagonized the effects of DPB on the tension and the MLC20 dephosphorylation. In Ca²⁺-free solution, DPB induced a contraction smaller than that in normal solution without an increase in MLC20 phosphorylation, and the contraction was also sensitive to ONO-RS-082. These results suggest that a part of MLC20 phosphorylation following PKC activation is due to inhibition of MLC20 phosphatase and the phosphorylation is responsible for the contraction. Furthermore, a mechanism independent of [Ca²⁺]_i and phosphorylation may play a significant role in the PKC-dependent contraction. The involvement arachidonic acid is suggested, not only in the inhibition of dephosphorylation but also in the Ca²⁺-independent regulation of contractile proteins. Received: 13 January 1997 / Accepted: 22 July 1997     

Co-existence of myosin heavy chain I and IIa isoforms in human skeletal muscle fibres with endurance training

The myosin heavy chain (MHC) composition of single fibres from m. vastus lateralis was analysed by one-dimensional electrophoresis and immunoblotting in three groups of young men with distinct difference in physical activity patterns. No major co-existence of MHC isoforms was found in the group with some daily physical activity. In the very sedentary group, however, 19±5% (P<0.05) of the fibres exhibited coexistence of MHC type IIa and IIb. Further, in the endurance trained group co-existence of MHC type I and IIa was manifested in 36±4% (P<0.05) of the fibres. Disuse and extreme usage of muscle both give rise to an elevation in co-expression of MHC isoforms in single muscle fibres but of markedly different combination of isoforms.     

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.     

Myosin polymorphism in single fibers of chronically stimulated rabbit fast-twitch muscle

Rabbit tibialis anterior (TA) muscles were indirectly stimulated (10 Hz, 24 h/d) for 30 d and 60 d and single fibers were analysed using a combined histochemical and biochemical technique (Staron and Pette 1986, 1987a, b). After 30 d of chronic stimulation there was a pronounced increase in the normally rare (0.5%) C fiber population (i.e., fibers containing slow- and fast-myosins in varying ratios). At this time, C fibers amounted to almost 60% of the total population. In the 60 d stimulated muscles, the major population (98%) consisted of an atypical type I_t fiber. This fiber type which was not detectable in normal TA muscle, differed histochemically and biochemically from type I fibers. It contained the slow-myosin light chains LC1s and LC2s, the heavy chain HCI, and, in addition, high concentrations of the fast-myosin alkali light chain LC1f and possibly traces of a heavy chain with an electrophoretic mobility comparable with that of the fast-myosin heavy chain HCIIa. These I_t fibers were occasionally observed in the unstimulated, contralateral TA muscles which also contained an increased population of C fibers (1.3–6.3%). Although the transformation even after 60 d of chronic stimulation was incomplete, these changes demonstrate the ability of muscle fibers to adapt in a specific manner to altered functional demands brought about by an altered stimulus pattern. In addition, the pronounced heterogeneity of the fiber population undergoing transformation indicates a nonuniform response to a uniform stimulus pattern.     

Correlation of dystrophin–glycoprotein complex and focal adhesion complex with myosin heavy chain isoforms in rat skeletal muscle

Aim: The dystrophin–glycoprotein complex (DGC) and focal adhesion complex (FAC) are transmembrane structures in muscle fibres that link the intracellular cytoskeleton to the extracellular matrix. DGC and FAC proteins are abundant in slow‐type muscles, indicating the structural reinforcement which play a pivotal role in continuous force output to maintain posture for long periods. The aim of the present study was to examine the expression of these structures across fast‐type muscles containing different myosin heavy chain (MHC) isoform patterns which reflect the fatigue‐resistant characteristics of skeletal muscle. Methods: We measured the expression of dystrophin and β1 integrin (representative proteins of DGC and FAC respectively) in plantaris, extensor digitorum longus, tibialis anterior, red and white portions of gastrocnemius, superficial portion of vastus lateralis and diaphragm, in comparison with soleus (SOL) and cardiac muscle from rats. Results: The expression of dystrophin and β1 integrin correlated positively with the percentage of type I, IIa and IIx MHC isoforms and negatively with that of type IIb MHC isoform in fast‐type skeletal muscles, and their expression was abundant in SOL and cardiac muscle. Conclusion: Our results support the idea that DGC and FAC are among the factors that explain the fatigue‐resistant property not only of slow‐type but also of fast‐type skeletal muscles.     

The effect of inhibition of myosin light chain kinase by Wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle

 To investigate the role of myosin light chain kinase (MLCK) in phasic contractions of intact smooth muscle, we have applied Wortmannin, an MLCK inhibitor, to strips of guinea-pig ureter. Simultaneous measurements of electrical activity, intracellular [Ca²⁺] ([Ca²⁺]_i)and phasic force showed that Wortmannin (1–4 μM) abolishes force with little or no change in [Ca²⁺]_i and electrical activity. High-K⁺-induced force production was also abolished by Wortmannin. The effects of Wortmannin were dose dependent – at lower concentrations (100 nM) Wortmannin reduced phasic contractility by 40–50%. It also significantly increased the delay between the Ca²⁺ peak and force production. These data show that, in phasic smooth muscle, inhibition of MLCK causes contraction to fail, despite normal electrical activity and Ca²⁺ transients. Our results also indicate that Wortmannin has no secondary effects and that other means of producing force, independent of myosin phosphorylation, are negligible in this tissue. The increased lag between the rise of Ca²⁺ and force production when MLCK is inhibited was surprising and suggests that post-phosphorylation steps may play a larger role in the delay than was previously considered. Received: 4 June 1998 / Recived after revision: 3 July 1998 / Accepted: 3 July 1998     

Differential effects of a K+ channel agonist and Ca2+ antagonists on myosin light chain phosphorylation in relaxation of endothelin-1-contracted tracheal smooth muscle

 Smooth muscle contraction and relaxation are generally considered to be associated with phosphorylation and dephosphorylation of the 20-kDa regulatory myosin light chain (LC₂₀). Thus, contractions of lamb tracheal smooth muscle induced by Bay K 8644 and relaxed by calcium channel blockers (verapamil, D-600 and nitrendipine) are accompanied by an increase and decrease, respectively, of LC₂₀ phosphorylation. Similarly, endothelin-1 (ET-1) induces a sustained contraction, which is coupled with elevated LC₂₀ phosphorylation and reversed by LC₂₀ dephosphorylation after application of a potassium channel agonist (EMD 52692). In contrast, calcium channel blockers relax ET-1-induced contraction without any dephosphorylation of myosin light chains (MLC), suggesting that MLC phosphatase is inhibited in this case. Obviously, MLC dephosphorylation is not a prerequisite for smooth muscle relaxation. The variable relationship between MLC phosphorylation and force during relaxation suggests that there are mechanisms other than MLC phosphorylation that are important for regulation of contraction and relaxation in smooth muscle. Received: 24 July 1996 / Received after revision and accepted: 28 October 1996     

Identification of two novel mutations in the ventricular regulatory myosin light chain gene (MYL2) associated with familial and classical forms of hypertrophic cardiomyopathy

 Five disease genes encoding sarcomeric proteins and associated with familial and classical forms of hypertrophic cardiomyopathy have been determined since 1989. In 1996 two other genes encoding ventricular regulatory and essential myosin light chains were shown to be associated with a particular phenotype of the disease characterized by mid left ventricular obstruction. The aim of the present study was to search for mutations in the ventricular regulatory myosin light chain gene (MYL2), located on chromosome 12q23q24.3, in a panel of 42 probands presenting a classical phenotype of familial hypertrophic cardiomyopathy. Single-strand conformation polymorphism analysis was used to search for mutations in the coding segments of the MYL2 gene, and the abnormal products were sequenced. Two novel missense mutations, Phe18Leu in exon 2 and Arg58Gln in exon 4 were identified in three unrelated families. None of the affected patients had hypertrophy localized only at the level of the papillary muscle with mid left ventricular obstruction. By analysis of genetic recombinations, one of these mutations identified in a large family allowed us to refine the localization of the MYL2 gene on the genetic map, in an interval of 6 cM containing six informative microsatellite markers. In conclusion, we show that mutations in the MYL2 gene may be involved in familial and classical forms of hypertrophic cardiomyopathy, and we provide new tools for the genetic analysis of patients with familial hypertrophic cardiomyopathy. Received: 12 August 1997 / Accepted: 20 November 1997