Biomedical subjects

M A Pellegrino

Publications and source records attributed to M A Pellegrino.

At least 19 recordsLinked to original sources

PubMed2005

Effects of resistance training on myosin function studied by the in vitro motility assay in young and older men.

It is generally believed that the maximum shortening velocity (V(o)) of a skeletal muscle fiber type does not vary unless a change in myosin heavy chain (MHC) isoform composition occurs. However, recent findings have shown that V(o) of a given fiber type can change after training, suggesting the hypothesis that the function of myosin can vary without a change in isoform. The present study addressed the latter hypothesis by studying the function of isolated myosin isoforms by the use of the in vitro motility assay (IVMA) technique. Four young (age 23-29 yr, YO) and four elderly men (age 68-82 yr, EL) underwent a 12-wk progressive resistance training program of the knee extensor muscles and to one pre- and one posttraining biopsy of the vastus lateralis muscle. The significant increase in one-repetition maximum posttraining in both YO and EL indicated that training was effective. After training, MHC isoform composition showed a shift from MHC(2X) toward MHC(2A) in YO and no shift in EL. The velocity of sliding (V(f)) of actin filaments on pure myosin isoforms extracted from single fibers was studied in IVMA. One hundred sixty IVMA samples were prepared from 480 single fibers, and at least 50 filaments were analyzed in each experiment. Whereas no training-induced change was observed in V(f) of myosin isoform 1 either in YO or in EL, a significant increase in V(f) of myosin isoform 2A after training was observed in both YO (18%) and EL (19%). The results indicate that resistance training can change the velocity of the myosin molecule.

Adaptation, Physiological↗

PubMed2003

Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils.

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca2+ (kACT), and the rate of tension redevelopment following a quick release and restretch (kTR), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (Pi) was abruptly increased (kPi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak-to-strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, kACT or kTR, suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa50 of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca2+ sensitivity and the cooperativity of the force-generating mechanism.

Animals↗

PubMed2003

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

Maximum shortening velocity (V(0)) 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 V(0) were compared with the values previously obtained in man and rat under identical experimental conditions. Significant differences in V(0) were found between fibres containing corresponding myosin isoforms in different species: as a general rule for each isoform V(0) 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 V(0) 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 (V(f)) 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 V(0) and V(f) 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.

Animals↗

PubMed2001

Differing ADP release rates from myosin heavy chain isoforms define the shortening velocity of skeletal muscle fibers.

To understand mammalian skeletal myosin isoform diversity, pure myosin isoforms of the four major skeletal muscle myosin types (myosin heavy chains I, IIA, IIX, and IIB) were extracted from single rat muscle fibers. The extracted myosin (1-2 microg/15-mm length) was sufficient to define the actomyosin dissociation reaction in flash photolysis using caged-ATP (Weiss, S., Chizhov, I., and Geeves, M. A. (2000) J. Muscle Res. Cell Motil. 21, 423-432). The ADP inhibition of the dissociation reaction was also studied to give the ADP affinity for actomyosin (K(AD)). The apparent second order rate constant of actomyosin dissociation gets faster (K(1)k(+2) = 0.17 -0.26 microm(-1) x s(-1)), whereas the affinity for ADP is weakened (250-930 microm) in the isoform order I, IIA, IIX, IIB. Both sets of values correlate well with the measured maximum shortening velocity (V(0)) of the parent fibers. If the value of K(AD) is controlled largely by the rate constant of ADP release (k(-AD)), then the estimated value of k(-AD) is sufficiently low to limit V(0). In contrast, [ATP]K(1)k(+2) at a physiological concentration of 5 mm ATP would be 2.5-6 times faster than k(-AD).

Adenosine Diphosphate↗

PubMed2001

Effects of the beta(2)-agonist clenbuterol on respiratory and limb muscles of weaning rats.

The aim of this study was to analyze the effects of chronic administration of the beta(2)-agonist clenbuterol (1.5 mg x kg(-1) x day(-1) for 4 wk in the drinking water) on respiratory (diaphragm and parasternal intercostal) and hindlimb (tibialis and soleus) muscles in young rats during postnatal development (21 to 49 postnatal days). The treatment resulted in very little stimulation of muscle growth. Significant slow-to-fast transitions in the expression of myosin heavy chain isoforms and significant increases in the myofibrillar ATPase activity were found in the diaphragm and soleus, whereas tibialis anterior and intercostal muscles did not show any significant fiber-type alteration. Decrease of oxidative enzyme activities and increase of glycolytic enzyme activities were also observed. It is concluded that whereas the growth stimulation is age dependent and only detectable in adult rats, the fiber-type transformation is also present in weaning rats and particularly evident in the soleus and diaphragm. The fiber-type transformation caused by clenbuterol might lead to an enhancement of contractile performance and also to a reduced resistance to fatigue.

Adenosine Triphosphatases↗

PubMed2000

ATP consumption and efficiency of human single muscle fibers with different myosin isoform composition.

Chemomechanical transduction was studied in single fibers isolated from human skeletal muscle containing different myosin isoforms. Permeabilized fibers were activated by laser-pulse photolytic release of 1.5 mM ATP from p(3)-1-(2-nitrophenyl)ethylester of ATP. The ATP hydrolysis rate in the muscle fibers was determined with a fluorescently labeled phosphate-binding protein. The effects of varying load and shortening velocity during contraction were investigated. The myosin isoform composition was determined in each fiber by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At 12 degrees C large variations (three- to fourfold) were found between slow and fast (2A and 2A-2B) fibers in their maximum shortening velocity, peak power output, velocity at which peak power is produced, isometric ATPase activity, and tension cost. Isometric tension was similar in all fiber groups. The ATP consumption rate increased during shortening in proportion to shortening velocity. At 12 degrees C the maximum efficiency was similar (0.21-0.27) for all fiber types and was reached at a higher speed of shortening for the faster fibers. In all fibers, peak efficiency increased to approximately 0.4 when the temperature was raised from 12 degrees C to 20 degrees C. The results were simulated with a kinetic scheme describing the ATPase cycle, in which the rate constant controlling ADP release is sensitive to the load on the muscle. The main difference between slow and fast fibers was reproduced by increasing the rate constant for the hydrolysis step, which was rate limiting at low loads. Simulation of the effect of increasing temperature required an increase in the force per cross-bridge and an acceleration of the rate constants in the reaction pathway.

Actins↗

PubMed2000

Functional diversity between orthologous myosins with minimal sequence diversity.

To define the structural differences that are responsible for the functional diversity between orthologous sarcomeric myosins, we compared the rat and human beta/slow myosins. Functional comparison showed that rat beta/slow myosin has higher ATPase activity and moves actin filaments at higher speed in in vitro motility assay than human beta/slow myosin. Sequence analysis shows that the loop regions at the junctions of the 25 and 50 kDa domains (loop 1) and the 50 and 20 kDa domains (loop 2), which have been implicated in determining functional diversity of myosin heavy chains, are essentially identical in the two orthologs. There are only 14 non-conservative substitutions in the two myosin heavy chains, three of which are located in the secondary actin-binding loop and flanking regions and others correspond to residues so far not assigned a functional role, including two residues in the proximal S2 domain. Interestingly, in some of these positions the rat beta/slow myosin heavy chain has the same residues found in human cardiac alpha myosin, a fast-type myosin, and fast skeletal myosins. These observations indicate that functional and structural analysis of myosin orthologs with limited sequence diversity can provide useful clues to identify amino acid residues involved in modulating myosin function.

Actins↗

PubMed1999

Profound misregulation of muscle-specific gene expression in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by an insidious onset and progressive course. The disease has a frequency of about 1 in 20,000 and is transmitted in an autosomal dominant fashion with almost complete penetrance. Deletion of an integral number of tandemly arrayed 3.3-kb repeat units (D4Z4) on chromosome 4q35 is associated with FSHD but otherwise the molecular basis of the disease and its pathophysiology remain obscure. Comparison of mRNA populations between appropriate cell types can facilitate identification of genes relevant to a particular biological or pathological process. In this report, we have compared mRNA populations of FSHD and normal muscle. Unexpectedly, the dystrophic muscle displayed profound alterations in gene expression characterized by severe underexpression or overexpression of specific mRNAs. Intriguingly, many of the deregulated mRNAs are muscle specific. Our results suggest that a global misregulation of gene expression is the underlying basis for FSHD, distinguishing it from other forms of muscular dystrophy. The experimental approach used here is applicable to any genetic disorder whose pathogenic mechanism is incompletely understood.

Base Sequence↗

PubMed1999

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.

Biomechanical Phenomena↗

PubMed1999

Speeds of actin translocation in vitro by myosins extracted from single rat muscle fibres of different types.

As skeletal muscle fibres mostly express a single myosin isoform, they are a potential source of pure myosin isoforms. A technique is described that allows extraction and identification of pure myosin isoforms from single fibres, and testing of such myosins in an in vitro motility assay (IVMA). The results show that the extraction procedure does not alter myosin function and support the view that single fibres are reliable sources of purified myosin isoforms for IVMA.

Actins↗

PubMed1998

Thyroid hormone regulation of MHC isoform composition and myofibrillar ATPase activity in rat skeletal muscles.

Myosin heavy chain (MHC) isoform composition and Ca2+ Mg2+ dependent ATPase activity were determined in myofibrils prepared from skeletal muscles (diaphragm, soleus, plantaris and tibialis anterior) of euthyroid (C), hypothyroid (Tx) and hyperthyroid (T3) rats. Direct comparison between T3 and Tx gave an indication of the maximal effect of thyroid hormones. Significant differences in MHC-1 and MHC-2B proportions and in ATPase activity were found in all muscles. The difference in MHC-2A/X proportion was significant only in soleus, diaphragm and plantaris. When T3 and C were compared, significant variations in MHC isoform composition were found only in plantaris and diaphragm. The comparison between Tx and C showed significant differences in MHC isoform distribution and in ATPase activity in most muscles. The differences in ATPase activity among muscles and among thyroid states were consistent with those in MHC isoform distribution. From the correlations between ATPase activity and MHC isoform distribution the enzymatic activities of individual MHC isoforms were calculated. The results indicate that MHC isoform distribution is controlled by thyroid state in all skeletal muscles and that changes in MHC isoforms distribution are accompanied by proportional changes in ATPase activity.

Animals↗

PubMed1997

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 degrees 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.

Adenosine Triphosphatases↗

PubMed1997

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.

Adult↗

PubMed1997

Contractile properties and fiber type distribution of quadriceps muscles in adults with childhood-onset growth hormone deficiency.

Adults with GH deficiency (GHD) report weakness and fatigability. The origin of such symptoms is still debated. This work aimed to clarify whether weakness and fatigability depend on impairment of skeletal muscle contractile capacity. Five males with childhood-onset GHD (age +/- SE, 29.6 +/- 1.9) and 13 age- and sex-matched controls were enrolled in the study. Quadriceps muscle cross-sectional area (CSA), strength, twitch characteristics, and fatigue index of voluntary and electrically evoked contractions were determined in vivo in all subjects. Fiber type distribution and CSA of identified types of skeletal fibers were determined on needle biopsy samples of the vastus lateralis muscle of all subjects. Fiber type distribution was assessed on the basis of myosin heavy chain (MHC) isoform composition determined by electrophoresis on polyacrylamide gels. Fiber CSA was determined on cross-cryosections of fiber bundles immunostained by monoclonal antibodies against MHC isoforms. Absolute values of strength and fiber CSA of quadriceps were significantly lower in patients affected by GHD than in controls. However, once strength and fiber CSA were normalized for quadriceps CSA and subject height, respectively, differences disappeared. No difference was found between GHD patients and controls for quadriceps muscle twitch characteristics, fatigue index, and fiber type distribution. The results reported here suggest that weakness and fatigability in childhood-onset GHD do not have a skeletal muscle origin.

Adult↗

PubMed1996

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.

Adult↗

PubMed1996

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.

Adult↗

PubMed1996

Adaptations of masticatory muscles to a hyperpropulsive appliance in the rat.

The plasticity of masticatory muscles was studied by comparing rats that were wearing a protrusive appliance and were kept on a liquid diet with two control groups: (1) pair-fed rats and (2) rats that had free access to ordinary pelleted food. The animals were 45 days old at the beginning of the experiment and were studied for a period of 20 days. Three jaw muscles with different functions were examined: masseter, temporalis, and digastric. Muscle fiber composition was determined (1) by fiber counting after staining with four monoclonal antibodies, which were able to recognize the four major myosin heavy chain (MHC) isoforms and therefore four fiber types (I, IIA, IIX, IIB) and (2) by electrophoresis on 6% polyacrylamide gels. The comparison between free-diet rats and pair-fed rats showed that the change from a hard pelleted diet to a liquid diet caused a shift in fiber type and MHC distribution, characterized by an increase of IIB MHC in temporalis and digastric muscles but not in the masseter muscle. The comparison between pair-fed rats and rats wearing appliances showed on the contrary a decrease in IIB MHC and an increase in IIA and IIX MHC. The results support the conclusions that (1) rat jaw muscles can quickly adapt to functional demand changing their fiber type composition, (2) the changes appear restricted inside the fast fiber population, and (3) fiber-type changes caused by dietary variation are not less than those caused by orthodontic intervention and must be taken into account to assess the effect of the appliance correctly.

Adaptation, Physiological↗

PubMed1984

Analysis of the repertoire of anti-HLA antibodies with anti-idiotypes to a murine anti-HLA-A2,A28 monoclonal antibody.

Xenoantibodies to idiotypes of the anti-HLA-A2,A28 MoAb CR11 -351 were isolated from an antiserum raised in rabbit #81 by immunizations with purified MoAb CR11 -351. The purification procedure involved absorption with insolubilized mouse immunoglobulins and monoclonal antibodies and affinity chromatography on insolubilized MoAb CR11 -351. Two antibody populations were identified in the xenoantibody preparation #81: one recognizes a recurrent idiotope expressed by the MoAb CR10 -215, CR10 -402, Q1/28, Q6 /64, and 6/31 to monomorphic determinants of HLA-A,B antigens and by the MoAb CR10 -343, CR11 -462, and Q5/6 to human Ia antigens. The other antibody population recognizes a private idiotope. Neither idiotope was detected on the anti-HLA-A2,A28 variant (A28) MoAbs BB7 .2, MA2 .2, and PA2.1, on the anti-HLA-A2,B17 MoAb MA2 .1 and on antibody populations in conventional anti-HLA-A2,A28 antisera. The idiotopes were also not detected on the anti-HLA-A2,A28 MoAb A2,A28 M1 which recognizes a determinant spatially close to that identified by the MoAb CR11 -351. The idiotope(s) recognized by the xenoantibodies #81 may be located in the combining site of the MoAb CR11 -351, since its incubation with the anti-idiotype antibodies specifically blocks the reactivity with lymphoid cells with the appropriate HLA phenotype.

Animals↗