Muscle Fiber Myosin Research Articles

Effects of Short-Term Concentric vs. Eccentric Resistance Training on Single Muscle Fiber MHC Distribution in Humans

The purpose of this investigation was to determine the effects of a concentric vs. eccentric resistance training program on single muscle fiber myosin heavy chain (MHC) adaptations in humans. Fifteen sedentary, healthy males were divided into three groups: concentric training (CTG) (n = 6, 24.2 +/- 1.7 y, 181 +/- 2 cm, 82.5 +/- 4.6 kg), eccentric training (ETG) (n = 6, 23.7 +/- 1.6 y, 178 +/- 3 cm, 90.4 +/- 6.1 kg), and control (CTL) (n = 3, 23 +/- 1.5 y, 181 +/- 2 cm, 97 +/- 13.2 kg). The subjects performed 4 sets of 8 unilateral repetitions starting at 80 % of concentric 1-RM, 3 days/week for a total of 4 weeks. Subjects were tested pre- and post-training for concentric 1-RM. Muscle biopsies were obtained from the vastus lateralis pre- and post-training for determination of single fiber MHC isoform distribution using SDS-PAGE/silver staining (100 fibers analyzed/subject pre- and post-training). Fibers expressing more than one MHC isoform (i.e., hybrid fibers) were analyzed for relative MHC isoform proportions via densitometry. The training program resulted in a 19 % 1-RM strength gain for CTG (p < 0.05) with no change in ETG or CTL. MHC-IIx fibers decreased by 7 % in CTG (p < 0.05) and ETG had an 11 % increase in total hybrids (MHC-I/IIa + MHC-IIa/IIx) (p < 0.05). No other differences were noted in MHC distribution among the three groups. Densitometry analysis of hybrid fibers showed no change in relative MHC isoform proportions pre- to post-training for any group. These data suggest that the MHC distribution did not change dramatically as a result of 4 weeks of concentric vs. eccentric resistance training despite the increase in whole muscle strength from concentric muscle actions.

DSC and EPR study on AMP.PNP, BeFx and AlF4 containing myosin nucleotide complexes

Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated skeletal muscle fibres to study the effect of the binding of nucleotides and nucleotide analogues to myosin. The thermal unfolding of muscle fibres in rigor showed three discrete domain regions with thermal stability of 52.2, 58.8 and 67.8°C. AMP.PNP and ATP plus AlF3 or BeF2 affected markedly the transitions, which implies the strong interaction between AMP.PNP or nucleotide analogues and catalytic domain of myosin, and a partial dissociation of heads from actin. ADP.BeFx and states model the transition states of the ATP hydrolysis cycle which precede the powerstroke of the muscle fibres. Spectrum deconvolution on isothiocyanate-labelled fibres in AMP.PNP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The myosin heads which showed high degree of order were in the strongly binding ADP-state. The spectra in - and ADP.BeFx state reflected random orientation of labels with increased rotational mobility in comparison with rigor. The results suggest that myosin in muscle fibres in ADP.BeFx state exists in two forms.

EFFECTS OF CONCENTRIC VS. ECCENTRIC RESISTANCE TRAINING ON SINGLE MUSCLE FIBER MHC DISTRIBUTION IN YOUNG MEN

PURPOSE: The purpose of this investigation was to determine the effects of a concentric vs. eccentric short-term resistance training program on single muscle fiber myosin heavy chain (MHC) distribution in humans. METHODS: Fifteen sedentary, healthy males were divided into three groups: concentric training (CTG) (n = 6, 24.2 ± 1.7 y, 181 ± 2 cm, 82.5 ± 4.6 kg), eccentric training (ETG) (n = 6, 23.7 ± 1.6 y, 178 ± 3 cm, 90.4 ± 6.1 kg) and control (CTL) (n = 3, 23 ± 1.5 y, 181 ± 2 cm, 97 ± 13.2 kg). The subjects performed 4 sets of 8 unilateral repetitions starting at 80% of concentric 1-RM, 3 days/week for a total of 4 weeks. Subjects were tested pre- and post-training for concentric 1-RM. Muscle biopsies were obtained from the vastus lateralis pre- and post-training for determination of single fiber MHC isoform distribution using SDS-PAGE/silver staining (100 fibers analyzed/subject pre- and post-training). Fibers expressing more than one MHC isoform (i.e., hybrid fibers) were analyzed for relative MHC isoform proportions via densitometry. RESULTS: The training program resulted in a 19% 1-RM strength gain for CTG (p < 0.05), and no change in ETG or CTL. MHC-IIx fibers decreased by 7% in CTG (p < 0.05) and ETG had an 11% increase in total hybrids (MHC-I/IIa+MHC-I/IIa/IIx+MHC-IIa/IIx) (p < 0.05). No other differences were noted in MHC distribution in the 3 groups. Densitometry analysis of hybrid fibers showed no change in relative MHC isoform proportions pre- to post-training for any group. CONCLUSION: After 4 weeks of concentric vs. eccentric resistance training the MHC distribution did not change dramatically and appears to have contributed little to the change in whole muscle strength. Supported partially by a Ball State University Graduate Research Award

Vascular cell adhesion molecule-1 up-regulation and phenotypic modulation of vascular smooth muscle cells predate mononuclear infiltration in transplant arteriopathy

Objective: Expression of embryonic myosin heavy chain isoforms and vascular cell adhesion molecule-1 by neointimal vascular smooth muscle cells are independent indicators of atherosclerotic plaque development in both human beings and experimental animal models. We examined the chronologic change in smooth muscle cell myosin heavy chain isoforms, vascular cell adhesion molecule-1 expression, and mononuclear cell infiltration in a carotid arterial transplant model to ascertain whether similar phenotypic changes would occur in transplant arteriopathy. Methods: Transplanted rabbit carotid arteries were examined at 7, 14, 21, and 35 days (n = 5, 7, 6, and 5, respectively). Lesion progression and the prevalence of smooth muscle cell myosin heavy chain isoforms, T-lymphocytes, macrophages, and vascular cell adhesion molecule-1 expression were evaluated immunohistochemically by computerized image analysis. Results: In this carotid arterial transplant model, the intima/media area ratio increased significantly by 35 days ( P = .01) as cell density decreased ( P = .01), suggesting extracellular matrix elaboration. Intimal smooth muscle cells expressing embryonic phenotypes were seen as early as 7 days, a phenotypic change that predated mononuclear cell infiltration of the graft by at least 7 days. By 35 days, up to 70% of intimal smooth muscle cells expressed the embryonic phenotype, coinciding with the transition from inflammatory to chronic lesions. Although, in early lesions, vascular cell adhesion molecule-1 was identified on luminal endothelium overlying mononuclear infiltrates, in advanced lesions vascular cell adhesion molecule-1 was identified primarily on intimal vascular smooth muscle cells. Conclusions: Overall, these vascular smooth muscle cell changes mark important early events in transplant arteriopathy that may not be ameliorated by immunosuppressive regimens in routine use.

Renal vascular walls in patients with preeclampsia superimposed on essential hypertension

This study was performed to clarify the relationship between changes in contractile proteins in renal vascular walls and the prognosis of hypertension during pregnancy. Twenty preeclamptic patients underwent renal biopsies after delivery and were divided into the following three groups: group I, patients with persistent hypertension after delivery (n = 7; mean age, 34.8 ± 1.4 years [SE]); group II, patients who became normotensive after delivery and hypertensive again during follow-up (n = 5; mean age, 34.8 ± 1.6 years), and group III, patients who became normotensive after delivery (n = 8; mean age, 28.0 ± 1.0 years). We also examined age-matched healthy controls (group IV; n = 7; mean age, 34.9 ± 1.5 years). Renal biopsy specimens were immunohistochemically stained by the avidin-biotinylated peroxidase complex method using antimonoclonal smooth muscle cell myosin heavy chain isoform antibodies (SM-1, SM-2) and antimonoclonal α-smooth muscle cell actin antibody (actin). We estimated and semiquantitatively scored the degree of staining in each section. In interlobular arteries, SM-1, SM-2, and actin staining in group I were significantly reduced compared with group IV (SM-1, SM-2, P < 0.05; actin, P < 0.01). In afferent arterioles (Afs), SM-1, SM-2, and actin staining were reduced in group I. SM-2 staining in group I was significantly reduced compared with the other three groups (versus group II, P < 0.05; versus groups III and IV, P < 0.01). These findings suggest that phenotypic changes in vascular smooth muscle cells (especially the disappearance of SM-2 in Afs) reflect the stage of underlying essential hypertension and can predict from the change in hypertension during pregnancy whether it will persist after delivery.

Network of Vascular-Associated Dendritic Cells in Intima of Healthy Young Individuals

In earlier studies, our group has established a new "immunological" hypothesis for atherogenesis supported by experimental and clinical studies showing that inflammatory immunological reactions against heat shock protein 60 initiate the development of atherosclerosis. In the present study, we describe the discovery of a so-far-unknown network of dendritic cells in the innermost layer of arteries, the intima, but not veins of healthy humans and rabbits. The number of these dendritic cells is comparable to that of Langerhans cells in the skin, and dendritic cells show a similar phenotype (CD1a(+) S-100(+) lag(+) CD31(-) CD83(-) CD86(-) and no staining for von Willebrand factor or smooth muscle cell myosin). These vascular-associated dendritic cells accumulate most densely in those arterial regions that are subjected to major hemodynamic stress by turbulent flow conditions and are known to be predisposed for the later development of atherosclerosis. These results open new perspectives for the activation of the immune system within the arterial wall.

Single rabbit stomach smooth muscle cell myosin heavy chain SMB expression and shortening velocity.

Isolated single smooth muscle cells (SMCs) from different regions of the rabbit stomach were used to determine a possible correlation between unloaded shortening velocity and smooth muscle (SM) myosin heavy chain (MHC) S1 head isoform composition (SMA, no head insert; SMB, with head insert). alpha-Toxin-permeabilized isolated single cells were maximally activated to measure unloaded shortening velocity and subsequently used in an RT-PCR reaction to determine the SMA/SMB content of the same cell. SM MHC SMA and SMB isoforms are uniquely distributed in the stomach with cells from the fundic region expressing little SMB (38.1 +/- 7.3% SMB; n = 16); cells from the antrum express primarily SMB (94.9 +/- 1.0% SMB; n = 16). Mean fundic cell unloaded shortening velocity was 0.014 +/- 0.002 cell lengths/s compared with 0.036 +/- 0.002 for the antrum cells. Unloaded shortening velocity in these cells was significantly correlated with their percent SMB expression (r2 = 0.58). Resting cell length does not correlate with the percent SMB expression (n = 32 cells). Previously published assays of purified or expressed SMA and SMB heavy meromyosin show a twofold difference in actin filament sliding speed in in vitro motility assays. Extrapolation of our data to 0-100% SMB would give a 10-fold range of shortening velocity, which is closer to the approximately 20-fold range reported from various SM tissues. This suggests that mechanisms in addition to the MHC S1 head isoforms regulate shortening velocity.

Binding of Nucleotides at the Active Site Modulates the Local and Global Conformation of Myosin in Muscle Fibres

Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated muscle fibres to study the effect of the binding of nucleotides (ADP and AMP⋅PNP) to myosin. The thermal unfolding of muscle fibres showed three discrete domain regions with thermal stabilities of 52.2, 58.8 and 67.8°C. AMP⋅PNP markedly affected the transitions, implying the strong interaction between AMP⋅PNP and catalytic domain, and partial dissociation of heads from actin. ADP produced only small changes in transition temperatures.

Effect of Oxygen Free Radicals on Myosin in Muscle Fibres. A DSC and EPR study

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation transfer EPR) were used to study the motional dynamics and segmental flexibility of myosin in muscle fibres in the presence of free radical generating system. Muscle fibre bundles isolated from psoas muscle of rabbit were spin-labelled with maleimide-and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules decreased with time following a single exponential curve. MgADP and MgATP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. The analysis of the EPR spectra of spin-labelled and oriented fibres showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the expense of the disordered population. This suggests a transition of myosin heads from weak-binding state into strong-binding state. DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves.

Synthesis of a spin-labeled photoaffinity ATP analogue, and its use to specifically photolabel myosin cross-bridges in skeletal muscle fibers.

A spin-labeled photoaffinity ATP analogue 3'(2')-O-[4-[4-oxo-(4-amido-2,2,6,6-tetramethyl-piperidino-1-oxyl)]-benz oyl]benzoyl adenosine 5'-triphosphate (SL-Bz2ATP) was synthesized and used to photolabel myosin in muscle fibers. Previous work has shown that 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP) photolabeled Ser-324 of the 50 kDa tryptic fragment of skeletal S1 heavy chain. In this work, [alpha-32P]SL-Bz2ATP was hydrolyzed and trapped as the diphosphate analogue with Co2+ and orthovanadate at the active site of myosin in rabbit psoas muscle fibers. After UV irradiation, the myosin heavy chain was the only protein band found to be significantly photolabeled as assayed by gel electrophoresis and radioactivity counting. The labeling was localized after brief trypsin digestion by SDS-PAGE to be on the 50 kDa tryptic fragment of the S1 heavy chain. Ca. 35% of the myosin in fibers was covalently photolabeled. The fibers photolabeled with SL-Bz2ATP had the same active tension and maximum shortening velocity as the control fibers. The resulting spin label on myosin was too mobile to report the orientation of the heads in fibers. Nonetheless, this is the first work to show the feasibility of utilizing active site binding and photoaffinity labeling to place covalent spectroscopic probes at the myosin active site in fibers with high specificity and yield without affecting mechanical function.

Effect of oxygen free radicals on myosin in muscle fibres

Experiments were performed on glycerol-extracted muscle fibres prepared from psoas muscle of rabbit in the presence of hydroxyl free radical generating system. Short irradiation of spin-labelled muscle fibres by UV light showed the interaction of probe molecules with oxygen free radicals. The intensity of the EPR signal from maleimide or isothiocyanate spin labels attached to the essential thiol groups decreased following irradiation. Oxygen free radicals affected the rate constant of the transition AM.ADP.V i→AM.ADP in the ATP hydrolysis cycle. It was found that the essential –SH groups of myosin were involved in the oxidation of sulphydryls by Ce(IV). Ce(IV) complexed to nitrilotriacetic acid in the presence of spin trap produced long-lived free radicals located partly on SH-1 sulphydryls.

Gamma irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle.

Mouse extensor digitorum longus (EDL) muscle was subjected to a dose of gamma irradiation that causes reproductive death of satellite cells and/or to chronic compensatory overload, achieved by removal of the distal portion of the tibialis anterior muscle. Four weeks later the mass, fiber type percentage, and fiber size of the EDL muscle were measured. Both the irradiated + overloaded and the irradiated only EDL muscles were significantly lighter and contained significantly smaller fibers than untreated muscle or muscle subjected to chronic overload only. Overload muscle, whether irradiated or not, had a larger percentage of type IIx fibers and a smaller percentage of type IIb fibers than muscle that had not been overloaded. The results confirm that satellite cell proliferation is a prerequisite for muscle hypertrophy induced by synergist incapacitation, but it appears not to be required for the maintenance of, or change in, normal muscle fiber myosin heavy chain phenotype expression.

Catalytic cooperativity induced by sulfhydryl1-labeling of myosin filaments

Modifications of SH1 groups on isolated myosin subfragment 1 (S-1) and myosin in muscle fibers affect differently the acto-S-1 ATPase and the fiber properties. Consistent with the findings of earlier work on fibers, the modification of SH1 groups in relaxed myofibrils with phenylmaleimide caused a loss of their shortening. This loss paralleled the decrease in the Vmax of extracted myosin but was not linear with the extent of SH1 labeling. Strikingly, the decrease in Vmax of S-1 prepared from the modified myofibrils was directly proportional to the extent of SH1 labeling. The specificity of SH1 labeling in myofibrils was verified by ATPase activities, thiol titrations, radiolabeling experiments, and comparisons to myosin labeled on SH1 in solution. To test for intermolecular interactions in the myosin filaments and their contribution to the differences between S-1 and myosin, the catalytic properties of copolymers of myosin were examined. Copolymers of myosin and rod minifilaments were formed in 5 mM citrate-Tris (pH 8.0) buffer, and their homogeneity was verified by sedimentation velocity analysis. The inhibition of actomyosin ATPase by rod particles was related to the decrease in the Km value. When rod particles were replaced in these minifilaments by SH1-modified myosin, the ATPase of the copolymers was increased over that of the combined ATPases of the individual filaments. The actomyosin ATP turnover rates on the unmodified heads were increased severalfold by the modified heads.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple binding reactivities of an IgG1 mouse monoclonal antibody raised against the extracellular matrix glycoprotein Gp 115.

An IgG1 mouse monoclonal antibody that recognized five different polypeptides from chick and man is described. The antibody was raised against the elastin associated chick glycoprotein gp 115. In addition to the immunizing antigen, antibody 106D6 bound to chick smooth muscle cell myosin and to the alpha, beta and gamma chains of human fibrinogen. Cross reaction was demonstrated by radioimmunobinding, immunofluorescence and immunoblotting experiments. The immunoblotting assays were carried out using identical amounts of the various antigens to exclude the possibility that relevant quantitative differences in their concentration might result in non-specific binding.

Cytotoxicity of lymphocytes from melanoma patients against autologous tumor cells and its potentiationin vitro

Enzymatic activity of cells, antigenic cellular markers and extracellular matrix of the hyperplastic intima of the aorta and carotid arteries was investigated in non-specific aorto-arteritis by immunomorphological and histochemical techniques. The cells of subendothelial layer of thickened arterial intima contained smooth muscle cell myosin, gave positive reactions to myosin ATP-ase and revealed high activity of thiamine pyrophosphatase. Fibronectin and type IV and V collagen were located in close proximity to these cells. The data obtained make it possible to consider these cells as modified smooth muscle cells. Type III collagen was the prevalent type of extracellular matrix of the thickened intima. A great number of blood vessels of the capillary and precapillary types have been found to penetrate into the intima from the adventitia. A possible role of pericytes surrounding newly formed capillaries as the precursors of subendothelial cell population in the hyperplastic intima is discussed.

Formula omitted] in myosin during muscle development

N G, N G- Dimethylarginine ( unsym - DMA ) has now been found to be present in myosin prepared from developing leg muscle. Neither monomethylarginine nor sym - DMA (N G N ′ G- dimethylarginine ) could be detected in our preparations. Cultured muscle cell myosin contains up to four residues of this amino acid per 5 × 10 5 grams of protein. Myosins from leg muscle of chick embryos and neonatal rats contained less than two residues of unsym - DMA , while none was detected in adult chicken or rat myosins. Cardiac myosins from developing as well as mature animals lacked or contained very little unsym - DMA . Actin was devoid of this amino acid.