Monica Canepari

Force‐velocity properties of human skeletal muscle fibres: myosin heavy chain isoform and temperature dependence.

1. A large population (n = 151) of human skinned skeletal muscle fibres has been studied. Force‐velocity curves of sixty‐seven fibres were obtained by load‐clamp manoeuvres at 12 degrees C. In each fibre maximum shortening velocity (Vmax), maximum power output (Wmax), optimal velocity (velocity at which Wmax is developed, Vopt), optimal force (force at which Wmax is developed, Popt), specific tension (Po/CSA, isometric tension/cross‐sectional area) were assessed. Unloaded shortening velocity (Vo) was also determined at 12 degrees C in a different group (n = 57) of fibres by slack‐test procedure. 2. All fibres used for mechanical experiments were characterized on the basis of the myosin heavy chain (MHC) isoform composition by sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis and divided into five types: type I (or slow), types IIA and IIB (or fast), and types I‐IIA and IIA‐IIB (or mixed types). 3. Vmax, Wmax, Vopt, Popt, Vopt/Vmax ratio, Po/CSA and Vo were found to depend on MHC isoform composition. All parameters were significantly lower in type I than in the fast (type IIA and IIB) fibres. Among fast fibres, Vmax, Wmax, Vopt and Vo were significantly lower in type IIA and than in IIB fibres, whereas Popt, Po/CSA and Vopt/Vmax were similar. 4. The temperature dependence of Vo and Po/CSA was assessed in a group of twenty‐one fibres in the range 12‐22 degrees C. In a set of six fibres temperature dependence of Vmax was also studied. The Q10 (5.88) and activation energy E (125 kJ mol‐1) values for maximum shortening velocity calculated from Arrhenius plots pointed to a very high temperature sensitivity. Po/CSA was very temperature dependent in the 12‐17 degrees C range, but less dependent between 17 and 22 degrees C.

The effect of ageing and immobilization on structure and function of human skeletal muscle fibres

Biopsy samples were taken from vastus lateralis muscle of seven young (YO, age 30.2 ± 2.2 years), and seven elderly (EL, age 72.7 ± 2.3 years) subjects and two elderly subjects whose right leg had been immobilized for 3.5 months (EL‐IMM, ages 70 and 75). The following main parameters were studied: (1) myosin heavy chain (MHC) isoform distribution of the samples, determined by SDS‐PAGE; (2) cross‐sectional area (CSA), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n= 593) of single skinned muscle fibres, classified on the basis of MHC isoform composition determined by SDS‐PAGE; (3) actin sliding velocity (Vf) on pure myosin isoforms determined by in vitro motility assays; (4) myosin concentration in single fibres determined by quantitative SDS‐PAGE. MHC isoform distribution was shifted towards fast isoforms in EL and to a larger extent in EL‐IMM. In EL and, more consistently, in EL‐IMM we observed a higher percentage of hybrid fibres than in YO, and noted the presence of MHC‐neonatal and of unusual hybrid fibres containing more than two MHC isoforms. Po/CSA significantly decreased in type 1 and 2A fibres in the order YO → EL → EL‐IMM. Vo of type 1 and 2A fibres was significantly lower in EL and higher in EL‐IMM than in YO, i.e. immobilization more than counteracted the age‐dependent decrease in Vo. The latter phenomenon was not observed for Vf. Vf on myosin 1 was lower in both EL and EL‐IMM than in YO. Vf on myosin 2X was lower in EL than in YO, and a similar trend was observed for myosin 2A. Myosin concentration decreased in type 1 and 2A fibres in the order YO → EL → EL‐IMM and was linearly related to the Po/CSA values of corresponding fibre types from the same subjects. The experiments suggest that (1) myosin concentration is a major determinant of the lower Po/CSA of single fibres in ageing and especially following immobilization and (2) ageing is associated with lower Vo of single fibres due to changes in the properties of myosin itself, whereas immobilization is associated with higher Vo in the absence of a change in myosin function.

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 (Po) unloaded shortening velocity (Vo) 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 Po 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.

Myofibrillar ATPase activity during isometric contraction and isomyosin composition in rat single skinned muscle fibres.

1. Myofibrillar ATPase activity, isometric tension (Po) and unloaded shortening velocity (Vo) were determined in single skinned fibres isolated from rat hindlimb muscles during maximal calcium activation at 12 degrees C. In each fibre, myosin heavy chain (MHC) isoforms were identified using electrophoresis and immunocytochemistry. ATPase activity was determined spectrophotometrically from NADH oxidation in a coupled enzyme assay. 2. On the basis of their MHC isoform composition, the fibres (n = 102) were divided into five groups containing the slow isoform, I MHC, or one of the fast isoforms, IIB MHC, IIA MHC, IIX MHC, or a mixture of the latter three. ATPase activity was significantly higher in IIB than in 2X and IIA fibres (0.230 +/‐ 0.010, 0.178 +/‐ 0.023 and 0.168 +/‐ 0.026 nmol mm‐3 s‐1, respectively). Mixed fibres had intermediate values. ATPase activity in slow fibres was considerably less (0.045 +/‐ 0.006 nmol mm‐3 s‐1). 3. The ratio between ATPase activity and Po, i.e. tension cost, was found to be 2.90 +/‐ 0.09, 2.56 +/‐ 0.14, 1.89 +/‐ 0.22, 1.52 +/‐ 0.13 and 0.66 +/‐ 0.004 pmol ATP nM‐1 mm‐1 s‐1 in IIB, mixed, IIX, IIA and slow fibres, respectively. All the differences were statistically significant except that between IIA and IIX fibres. 4. Within each group of fibres with the same MHC composition, ATPase activity was found to correlate with Po, but not Vo. However, ATPase activity was found to correlate with Vo when all the fibre types were pooled together. 5. In thirty‐seven fast fibres the MLC ratio, i.e. the proportion of the fast alkali light chain isoform, MLC3f, to the amount of the regulatory light chain, MLC2f, was determined. IIB fibres had the highest proportion of MLC3f and IIA fibres, the lowest. 6. A multiple regression analysis, used to distinguish between the effects of MHC and MLC composition, showed that ATPase activity was insensitive to the MLC ratio, whereas it had a significant impact on Vo. 7. The results obtained in this study indicate that in rat skeletal muscle fibres: (a) ATPase activity during isometric contractions and tension cost are strongly dependent on MHC isoform composition, and (b) there is no evidence that the alkali MLC ratio is a determinant of ATPase activity.

Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human muscles

Maximum shortening velocity (V0) was determined in single fibres dissected from hind limb skeletal muscles of rabbit and mouse and classified according to their myosin heavy chain (MHC) isoform composition. The values for rabbit and mouse V0 were compared with the values previously obtained in man and rat under identical experimental conditions. Significant differences in V0 were found between fibres containing corresponding myosin isoforms in different species: as a general rule for each isoform V0 decreased with body mass. Myosin isoform distributions of soleus and tibialis anterior were analysed in mouse, rat, rabbit and man: the proportion of slow myosin generally increased with increasing body size. The diversity between V0 of corresponding myosin isoforms and the different myosin isoform composition of corresponding muscles determine the scaling of shortening velocity of whole muscles with body size, which is essential for optimisation of locomotion. The speed of actin translocation (Vf) in in vitro motility assay was determined with myosins extracted from single muscle fibres of all four species: significant differences were found between myosin isoforms in each species and between corresponding myosin isoforms in different species. The values of V0 and Vf determined for each myosin isoform were significantly correlated, strongly supporting the view that the myosin isoform expressed is the major determinant of maximum shortening velocity in muscle fibres.

Fibre types in skeletal muscles of chronic obstructive pulmonary disease patients related to respiratory function and exercise tolerance

This study aimed to investigate the relationship between skeletal muscle, fibre type composition, functional respiratory impairment and exercise tolerance in patients with moderate to severe chronic obstructive pulmonary disease (COPD). A group of 22 COPD patients and 10 healthy control subjects were studied. In COPD patients, vital capacity (VC) and forced expiratory volume in one second (FEV1) were reduced to 79% and 51%, respectively. Diffusion indices (transfer factor of the lung for carbon monoxide (TL,CO) and carbon monoxide transfer coefficient (KCO)) were also reduced. Arterial oxygen tension (Pa,O2) was normal or slightly altered. A maximal exercise test was performed and anaerobic threshold was calculated. Muscle samples from vastus lateralis were obtained by needle biopsy. Myosin heavy chain (MHC) and light chain (MLC) isoforms were separated by gel electrophoresis and quantified by densitometry. MHC isoforms were considered as molecular markers of fibre types. The proportion of the fast MHC-2B isoform was increased in COPD patients. TL,CO, KCO, VC and FEV1 were positively correlated with slow MHC isoform content. TL,CO and KCO were also negatively correlated with the content of the fast MHC-2B isoform. No correlation was found between exercise parameters and MHC isoform composition. The co-ordinated expression between MHC and MLC isoforms was altered in COPD patients. We conclude that reduced oxygen availability, probably in combination with muscle disuse, may determine muscle alterations in chronic obstructive pulmonary disease patients. The altered correlations between myosin heavy chain and light chain isoforms suggest that co-ordinated protein expression is lost in chronic obstructive pulmonary disease muscles.

Specific contributions of various muscle fibre types to human muscle performance: an in vitro study

Human skeletal muscle fibres can be divided in five groups: 1, 1-2A, 2A, 2A-2B and 2B, by using myosin heavy chain (MHC) isoforms as molecular markers. This study aimed to define the contribution of each fibre type to the contractile performance of human muscles. Single fibre segments were dissected from bioptic samples of vastus lateralis and chemically skinned. Force-velocity properties, including isometric tension (P0), maximal shortening velocity (Vmax), maximum power output (Wmax) and the velocity at which Wmax is reached (Vopt), were determined at maximum calcium activation. Among these parameters Wmax showed the largest range of variation: about nine times between 2B and slow fibres. Vopt also showed large (about four times) and significant variations between fibre types. Force development at submaximum calcium activation was studied and force-pCa curves were obtained for each fibre type. Calcium sensitivity was greater in 2B than in 2A and in slow fibres. The slope of the force-pCa curve was greater in fast than in slow fibres. At the end of the experiment the MHC isoform composition of each fibre segment was determined by gel electrophoresis. The functional properties of each fibre type are discussed in the light of the motor unit recruitment mechanism to understand their possible physiological role.

Skeletal muscle hypertrophy and structure and function of skeletal muscle fibres in male body builders

Needle biopsy samples were taken from vastus lateralis muscle (VL) of five male body builders (BB, age 27.4 ± 0.93 years; mean ±s.e.m.), who had being performing hypertrophic heavy resistance exercise (HHRE) for at least 2 years, and from five male active, but untrained control subjects (CTRL, age 29.9 ± 2.01 years). The following determinations were performed: anatomical cross‐sectional area and volume of the quadriceps and VL muscles in vivo by magnetic resonance imaging (MRI); myosin heavy chain isoform (MHC) distribution of the whole biopsy samples by SDS‐PAGE; cross‐sectional area (CSA), force (Po), specific force (Po/CSA) and maximum shortening velocity (Vo) of a large population (n= 524) of single skinned muscle fibres classified on the basis of MHC isoform composition by SDS‐PAGE; actin sliding velocity (Vf) on pure myosin isoforms by in vitro motility assays. In BB a preferential hypertrophy of fast and especially type 2X fibres was observed. The very large hypertrophy of VL in vivo could not be fully accounted for by single muscle fibre hypertrophy. CSA of VL in vivo was, in fact, 54% larger in BB than in CTRL, whereas mean fibre area was only 14% larger in BB than in CTRL. MHC isoform distribution was shifted towards 2X fibres in BB. Po/CSA was significantly lower in type 1 fibres from BB than in type 1 fibres from CTRL whereas both type 2A and type 2X fibres were significantly stronger in BB than in CTRL. Vo of type 1 fibres and Vf of myosin 1 were significantly lower in BB than in CTRL, whereas no difference was observed among fast fibres and myosin 2A. The findings indicate that skeletal muscle of BB was markedly adapted to HHRE through extreme hypertrophy, a shift towards the stronger and more powerful fibre types and an increase in specific force of muscle fibres. Such adaptations could not be fully accounted for by well known mechanisms of muscle plasticity, i.e. by the hypertrophy of single muscle fibre (quantitative mechanism) and by a regulation of contractile properties of muscle fibres based on MHC isoform content (qualitative mechanism). Two BB subjects took anabolic steroids and three BB subjects did not. The former BB differed from the latter BB mostly for the size of their muscles and muscle fibres.

Autophagy impairment in muscle induces neuromuscular junction degeneration and precocious aging.

Summary The cellular basis of age-related tissue deterioration remains largely obscure. The ability to activate compensatory mechanisms in response to environmental stress is an important factor for survival and maintenance of cellular functions. Autophagy is activated both under short and prolonged stress and is required to clear the cell of dysfunctional organelles and altered proteins. We report that specific autophagy inhibition in muscle has a major impact on neuromuscular synaptic function and, consequently, on muscle strength, ultimately affecting the lifespan of animals. Inhibition of autophagy also exacerbates aging phenotypes in muscle, such as mitochondrial dysfunction, oxidative stress, and profound weakness. Mitochondrial dysfunction and oxidative stress directly affect acto-myosin interaction and force generation but show a limited effect on stability of neuromuscular synapses. These results demonstrate that age-related deterioration of synaptic structure and function is exacerbated by defective autophagy.

Chemo‐mechanical energy transduction in relation to myosin isoform composition in skeletal muscle fibres of the rat

1 ATP consumption and force development were determined in single skinned muscle fibres of the rat at 12 °C. Myofibrillar ATPase consumption was measured photometrically from NADH oxidation which was coupled to ATP hydrolysis. Myosin heavy chain (MHC) and light chain (MLC) isoforms were identified by gel electrophoresis. 2 Slow fibres (n= 14) containing MHCI and fast fibres (n= 18) containing MHCIIB were compared. Maximum shortening velocity was 1.02 ± 0.63 and 3.05 ± 0.23 lengths s−1, maximum power was 1.47 ± 0.22 and 9.59 ± 0.84 W l−1, and isometric ATPase activity was 0.034 ± 0.003 and 0.25 ± 0.01 mM s−1 in slow and in fast fibres, respectively. 3 In fast as well as in slow fibres ATP consumption during shortening increased above isometric ATP consumption. The increase was much greater in fast fibres than in slow fibres, but became similar when expressed relative to the isometric ATPase rate. 4 Efficiency was calculated from mechanical power and free energy change associated with ATP hydrolysis. Maximum efficiency was larger in slow than in fast fibres (0.38 ± 0.04 versus 0.28 ± 0.03) and was reached at a lower shortening velocity. 5 Within the group of fast fibres efficiency was lower in fibres which contained more MLC3f. We conclude that both MHC and essential MLC isoforms contribute to determine efficiency of chemo‐mechanical transduction.