Myosin Light Chain Pattern Research Articles

Changes in the biomechanical properties of neutrophils and endothelial cells during adhesion

AbstractThis study examined changes in the biomechanical properties of cultured pulmonary microvascular endothelial cells (ECs) and neutrophils induced by adhesion of neutrophils to these ECs. The biomechanical properties of cells were evaluated using magnetic twisting cytometry, which measures the angular rotation of ferromagnetic beads bound to cells through antibody ligation on application of a specified magnetic torque. Adhesion of neutrophils to 24-hour tumor necrosis factor-α (TNF-α)–treated ECs, but not to untreated ECs, induced an increase in EC stiffness within 2 minutes, which was accompanied by an increase and a reorganization of F-actin in ECs. A cell-permeant, phosphoinositide-binding peptide attenuated the EC stiffening response, suggesting that intracellular phosphoinositides are required. The stiffening response was not inhibited by ML-7, a myosin light-chain kinase inhibitor, or BAPTA, an intracellular Ca2+ chelator. Moreover, the phosphorylation pattern of the regulatory myosin light chains was unaltered within 15 minutes of neutrophil adherence. These data suggested that the EC stiffening response appeared not to be mediated by myosin light-chain–dependent mechanisms. Concomitantly, neutrophil adhesion to 24-hour TNF-α–treated ECs also induced changes in the biomechanical properties of neutrophils compared to neutrophils bound to untreated ECs. Taken together, these results demonstrated that neutrophil adhesion to TNF-α–treated ECs induces changes in the biomechanical properties of both cell types through actin cytoskeletal remodeling. These changes may modulate neutrophil transmigration across the endothelium during inflammation.

Adult fast myosin pattern and Ca 2+ -induced slow myosin pattern in primary skeletal muscle culture

A primary muscle cell culture derived from newborn rabbit muscle and growing on microcarriers in suspension was established. When cultured for several weeks, the myotubes in this model develop the completely adult pattern of fast myosin light and heavy chains. When Ca2+ ionophore is added to the culture medium on day 11, raising intracellular [Ca2+] about 10-fold, the myotubes develop to exhibit properties of an adult slow muscle by day 30, expressing slow myosin light as well as heavy chains, elevated citrate synthase, and reduced lactate dehydrogenase. The remarkable plasticity of these myotubes becomes apparent, when 8 days after withdrawal of the ionophore a marked slow-to-fast transition, as judged from the expression of isomyosins and metabolic enzymes, occurs.

In vitro myoblast to myotube transformations in the presence of leukemia inhibitory factor

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which exerts a variety of effects on many cell types including neuronal cells, and is a potent mitogen for myoblasts. At concentrations of 0.1–0.3 ng/ml, the peptide stimulates a maximal increase in cell number. LIF initiates a prolonged proliferative response lasting up to 13 days, when myoblasts are exposed to it in culture. LIF expression can be detected in vivo during development of limb muscle and in adult regenerating skeletal muscle tissue. Here, we studied the levels of expression of α-bungarotoxin-binding sites as a measure of acetylcholine receptors (AChRs), myosin light chain pattern and rate of myotube formation in fused, control and LIF-treated muscle cultures derived from mouse hind-limb muscles. We found that both the level of expression of AChRs and myosin light chain pattern, are normal, following exposure of the cells to LIF. There was no difference in the rate of myotube formation between LIF-stimulated and control myoblasts over a 10-fold concentration range (0.3–3.0 ng/ml) as determined by nuclei counts. Taken together, these results suggest that LIF, in combination with other cytokines, may act in vivo to stimulate rapid growth, without significant differentiation, during the early phases of myogenesis.

Effects of beta-guanidinopropionic acid-feeding on the patterns of myosin isoforms in rat fast-twitch muscle.

Administration of beta-guanidinopropionic acid (beta-GPA) to rats as 1% of their diet for 6 weeks led to an accumulation of beta-GPA and beta-GPA-phosphate and to a depletion of creatine and phosphocreatine in the fast-twitch plantaris muscle. Adenosine triphosphate concentration was also decreased. Electrophoretic analyses were performed to investigate the effects of beta-GPA on the patterns of fast (FM) and slow (SM) isomyosins, myosin heavy chain (HC) isoforms and myosin light chain (LC) isoforms. The relative concentrations of fast isomyosins FM1 and FM2 decreased, whereas slow isomyosin SM increased. The increase in slow isomyosin corresponded to an increase in the relative concentration of the slow myosin HCI. The changes of the myosin light chain pattern consisted of increases in the relative concentrations of the two slow isoforms, LC1sb and LC2s, and decreases in the fast isoforms LC2f and LC3f. These results demonstrate that beta-GPA administration, leading to a depletion in energy-rich phosphates and a reduced phosphorylation potential, has an impact on myosin isoform expression in rat fast-twitch skeletal muscle.

Fast myosin light chain expression in chicken muscles studied by in situ hybridization.

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.

Myosin light chain patterns in histochemically typed single fibers of the rat skeletal muscle

1. 1. This study conducted to investigate the relationship between histochemical fiber types and myosin light chain patterns in rat single muscle fibers. 2. 2. The hybrid of fast and slow light chains was observed in type I and II fibers of the soleus type II fibers of the red portion of lateral head of the gastrocnemius muscles. 3. 3. We also observed 7 types of light chain composition. Of the 7 types, 5 types were explainable by assuming the coexistence of isomyosins with either fast or slow light chains. However, the other 2 types could not be accounted for without hypothesizing the presence of isomyosins with promiscuous light chain distribution.

Arrest of developmental conversion of type II to type I fibres after suspension hypokinesia

The effects of hypokinesia and of the lack of gravity on muscle fibres, fibre type composition and myosin light chain pattern, as well as on muscle mechanoreceptors were investigated in the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles of young growing and adult rats after suspension hypokinesia (SH) of their hind limbs. The animals were suspended by their tail so that their hind limbs were relieved of their normal weight-bearing function for 3-6 weeks. In normal 3- to 4-week-old rats the SOL contained about 50% type I fibres and their percentage increased up to about 80% until the 10th week, with simultaneous reduction of type IIA fibres. After 3 to 6 weeks of suspension treatment maintained from 3- to 4-week-old rats up to 6 to 10 weeks of age, the SOL still only contained about 50% of type I fibres. The content of fast LC1 and LC2 in the SOL of 6-week-old rats after 3 weeks of suspension was higher than that of control litter-mates reflecting the higher occurrence of IIA fibres in the suspended solei. No changes in fibre type composition were observed after SH performed in adult rats. SH thus leads, in young animals, to the arrest of conversion of type IIA to type I fibres resulting in the persistence of the fibre type composition and of the myosin light chain pattern corresponding to those present in the SOL at the time of the onset of suspension. In both young and adult rats, SH markedly decreased the mass and the mean cross-sectional area of the SOL, mainly due to the severe atrophy of type I fibres. We observed no signs indicating conversion of type I back to type IIA muscle fibres due to the SH either in young or adult animals. In contrast to profound changes in the SOL, no significant differences were found in the EDL in any of the parameters studied. No changes in the investigated parameters of muscle spindles and tendon organs were observed after SH, performed either in young or in adult rats. We thus conclude that SH leads to muscle atrophy and that it influences mainly or exclusively type I fibres in muscles with a postural function such as the SOL.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium sensitivity and myosin light chain pattern of atrial and ventricular skinned cardiac fibers from patients with various kinds of cardiac disease

In the present study, the Ca 2+-sensitivity and myosin light chain patterns of skinned fibers of right atrium and left papillary muscles of 27 patients suffering from mitral valve disease (MVD, moderate heart failure), ischemic cardiomyopathy (ICM, severe heart failure), dilated cardiomyopathy (DCM, severe heart failure), and coronary heart disease (CHD, no heart failure, no atrial hypertrophy) were investigated. Myosin light chains of both chemically skinned and intact samples were studied by two-dimensional gel electrophoresis (2D-PAGE). Ca 2+-sensitivity of ventricular fibers was about 0.14 pCa-units higher than that of atrial fibers in all groups except dilated cardiomyopathy where this difference was markedly diminished (only 0.06 pCa-units). Generally, Ca 2+-sensitivity of skinned ventricular fibers was the same among the different heart diseases. Skinned atrial fibers from patients with dilated cardiomyopathy, however, were significantly (about 0.08 pCa-units) more sensitive for Ca 2+ than those of the other groups (coronary heart disease, mitral valve disease or ischemic cardiomyopathy) which showed similar Ca 2+-tension relationships. Ventricle-specific P-light chain forms could be observed in atrial samples from patients of all groups, whereas no atrium-specific light chain forms were detectable in any ventricular sample. It is concluded that there is no difference in Ca 2+-sensitivity of the ventricular contractile elements of the human heart in different heart diseases. In atrial myocardium, there is an increased Ca 2+-sensitivity of skinned fibers from hearts with dilated cardiomyopathy which is probably related to an elevation of right atrial pressure.

Altered expression of myosin light-chain isoforms in chronically stimulated fast-twitch muscle of the rat.

Fast-twitch tibialis anterior muscle of the rat was chronically stimulated for periods of 18 days, 28 days and 56 days. Changes in the myosin light-chain (LC) pattern consisted in an increase in LC1f, concomitant with a decrease in LC3f. In contrast to previous findings in chronically stimulated fast-twitch tibialis anterior muscle of the rabbit, no substantial increases occurred in the slow myosin light-chain isoforms. In vivo labeling using [35S]methionine incorporation revealed differences in relative turnover between the fast myosin light chains. The relative turnover of the fast myosin light chains appeared to increase in normal muscle in the order LC2f less than LC1f less than LC3f. As judged from [35S]methionine incorporation, the changes in light-chain tissue content mainly resulted from altered synthesis rates. However, in the case of LC3f the decrease in protein content could not only be explained by a reduced synthesis, but, additionally, appeared to be due to enhanced degradation. Parvalbumin, which was included in the present study, was also found to decrease in the stimulated muscle. However, its decrease appeared to result primarily from reduced synthesis.

Myosin ATPase activity during avian cardiac and skeletal muscle development

The myosin ATPase activity and myosin light chain composition in developing chick heart and skeletal muscles were studied and compared. Embryonic myosin was purified and characterized from day 7 to day 19 of embryogenesis. Embryonic cardiac myosin generally showed the same Ca 2+-activated myosin ATPase activity level as the adult value. In comparison, pooled pectoralis and hindlimb skeletal muscles from day 10 through day 19 showed myosin ATPase activities that were all significantly less than the adult counterpart. The myosin light chain pattern of embryonic cardiac myosin remained relatively constant like the myosin ATPase activity, whereas developmental changes were observed in skeletal myosin light chains.

Modifications in the histochemical and biochemical changes in tenotomized rat soleus by denervation.

Response of tenotomized rat soleus muscle to denervation performed at different time intervals, has been investigated. Tenotomized muscles showed typical central core lesions seven days post-operatively. These were not observed in m-ATPase stained sections of simultaneously denervated and tenotomized muscles, and muscles denervated 24 h after tenotomy. Central core lesions were not prevented in muscles denervated 28 h after tenotomy, indicating that tenotomy effects responsible for central lesions are completed by this time. Myosin light chain pattern of muscles denervated 28 h after tenotomy, and tenotomized only were similar showing increased LC3/LC1 ratio. Simultaneously denervated and tenotomized muscle however showed all the three light chains relatively equal in quantity. The results suggest that elimination of neural activation within 28 h prevents myofibrillar loss and minimized other changes which occur due to tenotomy.

Fast to slow transition induced by experimental myotonia in rat EDL muscle.

Experimental myotonia was induced by feeding rats with 20,25-diazacholesterol for up to 8 months. Histochemical analysis of myotonic extensor digitorum longus (EDL) muscle showed a progressive decrease of type IIB fibres and a concomitant increase of type IIA and type I fibres. A transient hypertrophy of type IIA fibres was observed 6 months after beginning the treatment. Analysis of the pattern of myosin light chains of single fibres from EDL showed that myotonia caused a progressive decrease of fibres showing a pure fast myosin light chain pattern and an increase of fibres showing coexistence of fast and slow myosin light chains (intermediate fibres). Only a small percentage of intermediate fibres showed coexistence of fast and slow myosin heavy chains. Myotonic fibres presented an increased sensitivity to caffeine which approached that of normal soleus fibres. Furthermore, sarcoplasmic reticulum (SR) vesicles isolated from hind limb fast muscles of myotonic rats demonstrated a decrease of Ca2+-dependent ATPase and Ca2+-transport activities as well as a decrease of immunoreactivity with anti-rabbit SR fast Ca2+-ATPase antibody. These results suggest that the increased electrical activity brought about by 20,25-diazacholesterol-induced myotonia, caused a fast to slow transition in the phenotypic expression of myosin and sarcoplasmic reticulum proteins.

Changes of energy metabolism, myosin light chain composition, lactate dehydrogenase isozyme pattern and fibre type distribution of denervated fast-twitch muscle from rabbit after low frequency stimulation.

The influence of low frequency (8-10 Hz) electrical stimulation on denervated fast-twitch muscle from rabbit was investigated. Prolonged direct stimulation of denervated muscle resulted in higher oxidative enzyme activities. Furthermore, single fibre analyses for succinate dehydrogenase showed a more uniform distribution of activity in stimulated-denervated muscle when compared to normal muscle. As was also the case following stimulation of innervated muscle, glycolytic enzymes were decreased in activity and the LDH-isozyme pattern was also shifted towards heart type. No change of the myosin light chain pattern could be observed after 56 days of stimulation.

Electrophoretic and Ultrastructural Analysis of the Rabbit’s Striated External Urethral Sphincter

The male rabbit’s external urethral sphincter was investigated with O’Farrell’s 2-dimensional electrophoretic analysis of myosin light chains and electron microscopy. Its pattern of myosin light chains was different from that of the soleus (predominantly slow twitch muscle) but was very similar to that of the psoas (predominantly fast twitch muscle). Ultrastructurally it was shown to be red muscle resembling the soleus. Therefore the fiber type of the rabbit’s external urethral sphincter was determined to be the red (fast) type.

Changes of myosin and its ATPase in “neuronally” and “mechanically” contralateral muscles after cross-reinnervation in normal and capsaicin-treated rats

The extensor digitorum longus (EDL) muscle was cross-reinnervated by the soleus (SOL) nerve in normal and neonatally capsaicin-treated rats. After 5 months the muscles were investigated for their myofibrillar ATPase reaction and their myosin light and heavy chain composition. Besides the well known transformation of the cross-reinnervated EDL toward a slow muscle, muscular changes were also found in the contralateral leg. Although these changes were hardly detectable in the EDL muscle, a remarkable (70 to 100%) reduction of the fast type IIA fiber population was found in the SOL. The decrease of the number of IIA fibers (compared with time-matched controls) was paralleled by corresponding changes in the myosin light and heavy chain patterns. After the cross-reinnervation of a muscle, two kinds of contralaterality must be distinguished. In the experiments reported the cross-reinnervated EDL muscle remains “mechanically” contralateral to the EDL muscle of the other leg, while it becomes “neuronally” contralateral to the SOL muscle. Our results are interpreted as a symmetric “slowing down” of these “neuronally” contralateral muscles. Neonatal capsaicin treatment that decreased considerably the number of unmyelinated group IV afferent fibers did not influence the outcome of these experiments.

Effects of chronic stimulation on the metabolic heterogeneity of the fibre population in rabbit tibialis anterior muscle.

Chronic indirect stimulation (10 Hz) was performed on rabbit tibialis anterior muscle. Long-term stimulation (52-140 days) produced a transformation of the fast tibialis anterior into a slow red muscle as judged from the histochemistry of myofibrillar actomyosin ATPase, the pattern of myosin light chains and the thorough rearrangement of the enzyme activity pattern of energy metabolism. Activity levels of citrate synthetase (CS), malate dehydrogenase (MDH), succinate dehydrogenase (SDH), 3-hydroxy-acyl-CoA dehydrogenase (HAD), and lactate dehydrogenase (LDH) were determined quantitatively by either microbiochemical assays (CS, MDH, HAD and LDH) on microdissected, single fibres or by kinetic microphotometry on cross-sectioned fibres (SDH). The activity profiles of these enzymes displayed pronounced scattering in the fibre population of the unstimulated muscle. Despite a several fold increase in the activities of CS, MDH, SDH and HAD and a pronounced decrease in LDH, chronic stimulation failed to abolish the metabolic heterogeneity of the fibre population. It is possible that chronic indirect stimulation cannot produce uniformity of fibres because of continuing diverse natural activity of the motor units.

Denervated skeletal muscle displays discoordinate regulation for the synthesis of several myofibrillar proteins.

Synthesis patterns of myosin heavy- and light-chain isoforms, tropomyosin and troponin, have been studied in chicken fast muscle denervated at both neonatal and adult stages. Denervated neonatal muscle does not synthesize the adult myosin heavy-chain isoform at the time of denervation, but it does synthesize the adult isoform several months after denervation. Thus, innervation does not appear to be necessary for the normal sequential replacement of embryonic and neonatal myosin heavy chain by the adult variant. Nerve is required, however, for the regulation of tropomyosin and troponin expression. Normally the pectoralis major muscle represses synthesis of both beta-tropomyosin and leg-type troponin T during late embryonic development. After denervation, however, the muscle relaxes its ongoing repression of these proteins and significant amounts of both beta-tropomyosin and leg-type troponin T are synthesized by the muscle. Denervation also results in an altered pattern of myosin light-chain synthesis so that the ratio of fast light-chain 3/fast light-chain 1 decreases. Similar results are found in muscle denervated at the adult stage. In denervated muscle, therefore, synthesis of these myofibrillar proteins is not coordinated: ongoing isoform shifts proceed to express an adult pattern of myosin heavy chain while tropomyosin, troponin, and myosin light-chain patterns appear to revert to embryonic configurations.

Ipsi- and contralateral fibre transformations by cross-reinnervation. A principle of symmetry.

Cross-reinnervation of rabbit soleus muscle by the peroneal nerve induces a 90% transformation of slow into fast fibres. These changes are reflected in corresponding transformations of the enzyme activity pattern of energy metabolism, the isozyme pattern of lactate dehydrogenase and, in confirmation of previous results (Srihari et al. 1981), transitions from a slow to a fast type myosin light chain pattern. The transformation process appears to be complete after 6 months. Similar changes, although less extensive are also found in the soleus muscle of the contralateral leg. Fibre type transitions in the contralateral muscle are not accompanied by fibre type grouping, as seen in the cross-reinnervated muscle and therefore these changes appear to result from a transformation of the motor units themselves. This phenomenon is interpreted as a compensatory process in maintaining symmetry within the neuromotor system.

Changes in transcriptional activity of chronically stimulated fast twitch muscle

mRNAs extracted from rabbit soleus, normal and 28-day, indirectly stimulated tibialis anterior muscles were translated in an in vitro system. Analysis for translation products by 2-dimensional electrophoresis showed fast myosin light chains in tibialis anterior, and slow myosin light chains in soleus muscle. The stoichiometry of the in vitro translated light chain varies from that seen in normal fast and slow twitch muscles. The stimulated muscle contained mRNA coding, both for fast and slow myosin light chains, although the pattern of slow myosin light chains appears not to be complete at this point of time of the transformation process.

Enzyme activities, histochemistry and myosin light chain pattern in extraocular muscles of rabbit.

A study was performed on enzyme activities, myosin light chain pattern and histochemical composition of extraocular muscles (EOM) of rabbit. The results were compared with those from slow-twitch soleus and fast-twitch psoas limb muscles. EOM were found to contain myosin light chains of both slow and fast skeletal muscles, with the latter being predominant. The activities of the enzymes regulating glycolysis and lactic acid fermentation were found to lie between those measured in soleus and psoas muscles, whereas the activities of the enzymes involved in oxidative metabolism were found to be very similar or even higher than in soleus muscle. All five isozymes of lactate dehydrogenase (LDH 1-5) were found in EOM. The predominant species was LDH-3 illustrating that this pattern is uncharacteristic of either slow soleus or fast psoas muscles. The complex structural and functional characteristics of EOM might result from the observed heterogeneity of fibres.