Exercise-induced HSP27, HSP70 and MAPK responses in human skeletal muscle.

AIM: The present work examined protein and messenger RNA (mRNA) expression of intramuscular heat shock protein 27 (HSP27), heat shock cognate (HSC70) and HSP70 in human biceps brachii (BB) and vastus lateralis (VL) subsequent to two different exercises. METHODS: Untrained subjects performed 50 high-force eccentric contractions with their non-dominant BB and ran downhill (-10 degrees) for 30 min. The 48-h PX stress response was evaluated with immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Muscle damage was indicated indirectly at 48 h post-exercise (PX) [loss of mobility, muscle soreness and serum creatine kinase (CK) activity]. RESULTS: On the protein level, HSP27 and HSP70 increased significantly PX in the BB (384 and 227%, respectively; P < 0.01), but there were no significant HSP changes in the VL or in HSC70 in either muscle. The RT-PCR data complemented these findings: BB HSP27 and HSP70C mRNA levels increased (135 and 128%, respectively; P < 0.05); in the VL only HSP70B increased (206%; P < 0.05). Phosphorylation of e-jun NH2-terminal kinase (JNK) and extracellular regulated kinase (ERK) increased significantly in the BB (226 and 200%, respectively; P < 0.05) but not in the VL, indicating activation of these pathways only after the resistance exercise. CONCLUSION: These data indicate that the PX HSP and mitogen-activated protein kinase responses are exercise-specific and local, not systemic. Further, only the resistance exercise induced HSP expression (protein and mRNA) and JNK/ERK activation at 48 h PX, suggesting that these molecules may be important to long-term skeletal muscle adaptations such as hypertrophy.

The repeated bout effect and heat shock proteins: intramuscular HSP27 and HSP70 expression following two bouts of eccentric exercise in humans.

Exercise-induced damage significantly and predictably alters indirect indicators of muscle damage after one bout of damaging exercise but this response is dampened following a second bout of the same exercise performed 1-6 weeks later. Previously we have described a marked increase in the levels of heat shock proteins (HSPs) HSP27 and HSP70 in human biceps muscle following one bout of high-force eccentric exercise. The purpose of the present study was to examine the intramuscular HSP27 and HSP70 response following two identical bouts of exercise [bout 1 (B1) and bout 2 (B2), separated by 4 weeks] relative to indirect indices of muscle damage. Ten human subjects performed 50 high-force eccentric contractions with their non-dominant forearm flexors; muscle damage of the biceps brachii was evaluated 48 h post-exercise with indirect indices [serum creatine kinase (CK) activity, soreness, isometric maximal voluntary contraction (MVC) force and relaxed arm angle] and immunoblotting of high ionic strength muscle biopsy extracts for both HSPs. Not unexpectedly, the indirect indicators of damage changed dramatically and significantly (P < 0.01) after B1 but had a much smaller response after B2. The magnitude of the HSP response was the same after both bouts of exercise, though the control and exercised samples of B2 demonstrated a lower basal HSP expression. Thus, though both indirect and cellular indicators of exercise-induced muscle damage demonstrate an adaptation consequent to the first bout of exercise, these adaptations are quite different. It is possible that the lower basal HSP expression of the cellular response mediates the attenuation of damage associated with B2 as indicated by indirect indices.

A single bout of eccentric exercise increases HSP27 and HSC/HSP70 in human skeletal muscle.

Changes in heat shock proteins (HSPs), HSP27 and HSC/HSP70 were characterized in human biceps brachii muscle following damaging high-force eccentric exercise. Male and female volunteers performed a maximal eccentric resistance exercise with the elbow flexor muscles of the non-dominant arm known to be sufficient to cause substantial muscle damage. Protein extracts of biopsy tissue samples taken 48 h post-exercise were immunoblotted for HSC/HSP70 and HSP27. Densitometric analysis demonstrated that these proteins increased significantly (P < 0.01) in the damaged biceps brachii relative to the control arm. The HSC/HSP70 increased 1064% in the exercised sample while HSP27 increased by 234%. Although the literature reports a muscular heat shock response following aerobic, oxidative exercise, this is the first documentation of increases in protein expression of both HSC/HSP70 and HSP27 in human skeletal muscle in response to a single bout of resistance exercise.

Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata.

The presence and localization of actin and myosin have been examined in pollen tubes of Lilium longiflorum and Nicotiana alata. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy using various methods to preserve the pollen tubes; chemical fixation, rapid freeze fixation and freeze substitution (RF-FS) followed by rehydration or by embeddment in a methacrylate mixture, was performed to optimize preservation. Immunocytochemistry reaffirmed that actin is localized longitudinally in the active streaming lanes and near the cortical surface of the pollen tube. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus, whereas, myosin V was found in the vegetative cytoplasm in a punctate fashion representing smaller organelles. Myosin II subfragment 1 and light meromyosin were localized in a punctate fashion on the larger organelles throughout the vegetative cytoplasm. In addition, isolated generative cells and vegetative nuclei labeled only with the myosin I antibody. Competition studies indicated the specificity of the heterologous antibodies utilized in this study suggesting the presence of three classes of myosins in pollen. These results lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally through the pollen tube to the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally) that are involved in growth.

Myofibrillogenesis in rodent skeletal muscle in vitro: two pathways involving thick filament aggregates.

Thick filament aggregates play an important role in myofibrillogenesis in rodent skeletal muscle in vitro. This ultrastructural study describes these aggregates, shows their involvement in the process of myofibril formation, and correlates their appearance and function with current models of myofibrillogenesis. Initially, following myoblast fusion in normal mouse skeletal muscle in vitro, abundant stress fiber-like structures (SFLS) are found near the periphery of early myotubes. These undergo internal rearrangements, forming subcortical sarcomeres and early myofibrils. However, additional thick filaments are synthesized, and some join appositionally to the nascent myofibrils, increasing their diameter. More interiorly, this thick filament synthesis accelerates, with filaments aligning into aggregates resembling discrete A-bands, usually with M-lines and M-regions. The ends of these 'A-band' aggregates are infiltrated with ribosomes and capped by flocculent material. Ultimately, aggregates are incorporated into preexisting myofibrils or associate end-to-end to form new, parallel myofibrils, the flocculent material forming putative I-bands with diminished Z-lines and few thin filaments. As differentiation continues, Z-lines and thin filaments appear, forming true myofibrils. Dysgenic mouse skeletal muscle develops similarly, but when this non-contractile cell matures (i.e., generates action potentials), filaments and their organization break down. Cloned myogenic rat L5/A10 cells also follow this developmental pattern, but in mature, contracting myotubes, Z-lines remain irregular and thin filaments are reduced. In all three types of muscle developing in vitro, thick filament aggregates are a common and predominant feature and as such appear to constitute an additional or alternate pathway to previously described models of myofibrillogenesis.

Ubiquitin changes in human biceps muscle following exercise-induced damage.

Changes in ubiquitin levels were characterized in human biceps muscle following high-force eccentric exercise. Volunteers performed damaging eccentric-isokinetic actions of the biceps muscle with the non-dominant arm. Protein extracts of biopsy tissue samples taken two days post-exercise were run on SDS polyacrylamide gels, analyzed densitometrically and revealed a 64% higher level of a protein band at 12 kD. New monoclonal immunoblotting techniques identified the band as free ubiquitin. On these blots, free ubiquitin increased in the exercised sample by 55% over the control, and ubiquitin conjugates of varying molecular weights follow a similar pattern. The changes seen in both free and conjugated ubiquitin suggest that their increases are involved in the response to exercise-induced muscle damage.

Muscle protein changes following eccentric exercise in humans.

This study characterized changes in the protein composition of human muscle tissue after eccentric exercise. Four subjects performed 70 maximum eccentric, isokinetic actions of the forearm flexors with one arm. The other arm served as control. A biopsy of the biceps muscle of each arm was taken 2 days after exercise when muscles were very sore (mean = 8.0; 1 = normal; 10 = very, very sore), and muscle damage was documented by a mean decrease of 0.2 radians in the relaxed elbow angle. Proteins from the biopsy tissue were solubilized in a high ionic strength buffer containing several proteolytic inhibitors. Protein concentrations of the extracts were determined and identical amounts loaded onto sodium dodecyl sulfate (SDS) polyacrylamide gels (7.5, 12.5, and 17.5%). Densitometric analysis of the Coomassie brilliant blue stained gels revealed alterations in the amounts of three protein bands in the exercised tissue relative to the control. These changes were in the linear portion of the graph of absorbance versus protein amount. Wilcoxon's signed rank test showed the first two of the following bands to increase significantly in amount (P less than 0.062). The average percentage changes [mean (SEM)] for these bands were 63 (21), 39 (5), and 82 (35). The corresponding molecular weights determined from known standards were 76300 (860), 33200 (310), and 12000 (80) daltons, respectively. These changes imply that the increased synthesis, decreased degradation, or some combination thereof, of these three proteins may be necessary for the repair or regeneration response to exercise-induced muscle damage.

Immunochemical and immunocytochemical identification of a myosin heavy chain polypeptide in Nicotiana pollen tubes.

A myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes using monoclonal anti-myosin antibodies. The epitopes of these antibodies were found to reside on the myosin heavy chain head and rod portion and were, therefore, designated anti-S-1 (myosin S-1) and anti-LMM (light meromyosin). On Western blots of the total soluble pollen tube proteins, both anti-S-1 and anti-LMM label a polypeptide of approximately 175,000 Mr. Immunofluorescence microscopy shows that both antibodies yield numerous fluorescent spots throughout the whole length of the tube, often with an enrichment in the tube tip. These fluorescent spots are thought to represent vesicles and/or organelles in the pollen tubes. In addition to this common pattern, anti-S-1 stains both the generative cell and the vegetative nuclear envelope. The different staining patterns of the nucleus between anti-S-1 and anti-LMM may be caused by some organization and/or anchorage state of the myosin molecules on the nuclear surface that differs from those on the vesicles and/or organelles.

The control of myocardial contraction with skeletal fast muscle troponin C.

The present study describes experiments on the myocardial trabeculae from the right ventricle of Syrian hamsters whose troponin C (TnC) moiety was exchanged with heterologous TnC from fast skeletal muscle of the rabbit. These experiments were designed to help define the role of the various classes of Ca2+-binding sites on TnC in setting the characteristic sensitivities for activations of cardiac and skeletal muscles. Thin trabeculae were skinned and about 75% of their troponin C extracted by chemical treatment. Tension development on activations by Ca2+ and Sr2+ was found to be nearly fully blocked in such TnC extracted preparations. Troponin C contents and the ability to develop tension on activations by Ca2+ and Sr2+ was permanently restored after incubation with 2-6 mg/ml purified TnC from either rabbit fast-twitch skeletal muscle (STnC) or the heart (CTnC, cardiac troponin C). The native (skinned) cardiac muscle is characteristically about 5 times more sensitive to activation by Sr2+ than fast muscle, but the STnC-loaded trabeculae gave response like fast muscle. Attempts were also made to exchange the TnC in psoas (fast-twitch muscle) fibers, but unlike cardiac muscle tension response of the maximally extracted psoas fibers could be restored only with homologous STnC. CTnC was effective in partially extracted fibers, even though the uptake of CTnC was complete in the maximally extracted fibers. The results in this study establish that troponin C subunit is the key in setting the characteristic sensitivity for tension control in the myocardium above that in the skeletal muscle. Since a major difference between skeletal and cardiac TnCs is that one of the trigger sites (site I, residues 28-40 from the N terminus) is modified in CTnC and has reduced affinity for Ca2+ binding, the possibility is raised that this site has a modulatory effect on activation in different tissues and limits the effectiveness of CTnC in skeletal fibers.

Evidence for dysfunction in the regulation of cytosolic Ca2+ in excitation-contraction uncoupled dysgenic muscle.

In noncontracting, dysgenic murine muscle, excitation is uncoupled from contraction. To test whether the gene lesion is expressed as a defect in the regulation of the intracellular free Ca2+ levels, cultured normal and dysgenic muscle at various stages of development (proliferative myoblasts, early, late, and mature myotubes) were exposed to increasing increments (0.5-mM steps) of extracellular Ca2+ in ionophore A23187-Ca2+-EGTA-buffered media. Normal and dysgenic muscle at all stages (except myoblast) displayed contractures at approximately 500 microM free Ca2+ and higher. Experiments using finer increments of Ca2+ and different ionophore concentrations indicated an external Ca2+ threshold for contracture at 265 microM Ca2+ for early and late myotubes and 47-78 microM for mature normal and dysgenic myotubes. Low extracellular concentrations of calcium (14 microM and 0.76 nM) caused elongation of both normal and dysgenic myotubes. Mature cells were depolarized by exposure to increasing extracellular K+ and monitored by intracellular recording; normal and dysgenic myotubes showed similar reductions in membrane potentials. Depolarization to -35 mV elicited contractures in normal myotubes, but even depolarization to -9 mV in dysgenic cells elicited no response. Thus steady-state depolarization of dysgenic muscle does not cause contractures, which can, however, be elicited by increasing the intracellular free Ca2+. These results offer new evidence for a possible defect in the regulation of Ca2+ levels in dysgenic muscle.

Macrophages contain at least two myosins.

A number of motile functions of macrophages are thought to be mediated by myosin. We have observed that myosin from rabbit alveolar macrophages is heterogeneous with respect to its 20 kDa light chain: two species of 20 kDa light chain are identified by one-dimensional and two-dimensional polyacrylamide gel electrophoresis, in a ratio of 2:1. Native myosin, analyzed on non-denaturing gels, is also composed of two species, in a ratio of 2:1. These results indicate that macrophages contain at least two different myosins, which might have different physiological functions.

Osteosarcoma cells in tissue culture. III. Actin filament distribution.

Microfilaments, especially actin, have been demonstrated in a variety of noncontractile cells. In earlier studies, the quantity as well as the distribution of these microfilaments has been used to differentiate normal murine fibroblasts from virally transformed cells. This study examines these differences in cells from spontaneously occurring, human osteosarcomas and normal human fibroblasts by transmission election microscopy and heavy meromyosin (HMM) decoration. Both the tumor cells and the normal fibroblasts were found to have subcortical bands of 7-nm microfilaments that labelled with HMM and were 150-300 nm in thickness. There was a central reticular pattern of microfilaments predominantly composed of 7-nm filaments in the fibroblasts but with a larger proportion of 10-nm filaments in the osteosarcoma cells. Arrowhead formation was present after mild Triton X-100 extraction and HMM decoration on only the 7-nm microfilaments, in both types of cells. Differences in the quantity of 10-nm filaments between cells in culture from spontaneously occurring human sarcomas and normal fibroblasts may account for differential surface responses, e.g., contact inhibition and saturation density. Unlike some evidence from viral transformation models, the data from this study do not support the hypothesis that tumorgenicity is linked mechanistically to decreases in polymerized cellular actin, at least not in cells from spontaneously occurring human sarcomas. The cellular behavior of human sarcomas, both in vitro and in vivo, may be a manifestation of differences both in structural proteins and cell surface proteins.

Changes in myosin and myosin light chain kinase during myogenesis.

Myosins and myosin light chain kinases have been isolated from a cloned line of myoblasts (L5/A10) as this cell line undergoes differentiation toward adult muscle. At least three myosin isozymes were obtained during this developmental process. Initially a nonmuscle type of myosin was found in the myoblasts. The molecular weights of the myoblast light chains were 20 000 and 15 000. Myosin isolated from early myotubes had light chains with molecular weights of 20 000 and 19 500. Myosin isolated from myotubes which contained sarcomeres had light chains with molecular weights of 23 000, 18 500, and 16 000. This last myosin was similar in light chain complement to adult rat thigh muscle. Two forms of the myosin light chain kinase activity were detected: a calcium-independent kinase in the myoblasts and a calcium-dependent kinase in the myotubes with sarcomeres. No myosin light chain kinase activity was detected in the early myotubes.

The cytoskeleton and cell movement: general considerations.

The cytoskeletal proteins, actin, myosin, tubulin, dynein, and their associated proteins, are discussed selectively with regards to their biochemical and structural properties. Particular emphasis is placed on the comparison of non-muscle proteins to their muscle counterparts, and on the various mechanisms for regulating actin polymerization, actin-myosin interaction, and tubulin polymerization. This review as well as the bibliography accompanying it is selective. An attempt is made to stress the most recent findings and to emphasize those areas which appear to hold the greatest promise for future research.

A comparative study of the myosin light chain kinases from myoblast and muscle sources. Studies on the kinases from proliferative rat myoblasts in culture, rat thigh muscle, and rabbit skeletal muscle.

Myosin light chain kinases have been isolated from rat thigh and rabbit skeletal muscle and cultured rat myoblasts. From these preparations, two types of kinases can be distinguished: calcium-dependent and calcium-independent. Both types of kinases can phosphorylate isolated P-light chains of myosin from several sources (skeletal muscle, cardiac muscle, and platelet). Data are shown which support the phosphorylation of the same site on the non-muscle P-light chains by both types of kinases. The rates of these reactins are, however, different for the two types of kinases. Kinetic analysis of the myoblast kinase shows differing affinities for various P-light chains (non-muscle greater than cardiac greater than skeletal). In the proliferative rat myoblast, phosphorylation of myosin is a prerequisite for actin activation of the myosin ATPase activity.

Assays of the metabolic viability of single giant mitochondria. Experiments with intact and impaled mitochondria.

Single giant mitochondria isolated from mice fed cuprizone were assayed for their metabolic viability. Two tests were devised. One test optically detected the accumulation of calcium phosphate within the mitochondria under massive loading conditions (including the presence of succinate and ATP). The accumulation corresponds to a test of energy coupling from either electron transport or the hydrolysis of ATP since it is blocked by either antimycin A or oligomycin. The other assay tested for the production of ATP from ADP and Pi, using myofibrils. Myofibrils prepared from glycerinated rabbit psoas muscle contract only in the presence of ATP and not in the presence of ADP. Myofibrillar contraction is unaffected by the presence of antimycin A or oligomycin. However, myofibrils in the presence of mitochondria that are phosphorylating ADP to ATP do contract. This contraction is blocked by antimycin A and/or oligomycin. Hence, the ATP which causes myofibrillar contraction is produced by oxidative phosphorylation. At low mitochondrial concentration, only the myofibrils in close proximity with mitochondria contract in the presence of ADP. Therefore the assay can be used to test the viability of individual mitochondria. Individual giant mitochondria were found to be viable, using both of these assays. Comparable results were obtained in mitochondria impaled with microelectrodes. The potentials and resistances were unaffected by concomitant calcium phosphate accumulation or oxidative phosphorylation.

Membrane potentials and resistances of giant mitochondria. Metabolic dependence and the effects of valinomycin.

The membrane potentials and resistances of giant mitochondria from mice fed cuprizone have been studied. They were found to correspond approx. 10-20 mV, positive inside, and 2 M omega, respectively. These properties were found to be independent of the metabolic state. The microelectrodes were in the inner mitochondrial space since (a) the potentials in the presence of valinomycin depended on the K+ concentration of the medium and magnitude of the K+ diffusion potentials was consistent with the presence of a high internal concentration of K+, (b) almost identical results were obtained with mitochondria from which the external membrane had been removed and the cristae were evaginated, and (c) punch-through experiments, in which the microelectrodes were advanced until they emerged through the other side of the mitochondria, showed an identical membrane potential both in the presence and in the absence of valinomycin. The potentials were stable under a variety of conditions and showed no sign of decay of membrane leakiness. Detailed evidence that the impaled mitochondria are metabolically viable will be presented in a separate publication.

Membrane potential of mitochondrial measured with microelectrodes.

The membrane potentials of giant mitochondria from cuprizone-fed mice were found to be independent of metabolic state. Experiments are described in which the presence of the microelectrodes in the inner mitochondrial space, and the metabolic viability of the impaled mitochonidra, are validated.

Donnan potential of rabbit skeletal muscle myofibrils I: electrofluorochromometric detection of potential.

The fluorescence of the dye CC-6 [(3-hexyl-2-(3-hexyl-2-benzoxazolinylidene)-1-propenyl)-benzoxazolium iodide] has been shown to indicate Donnan potentials in rabbit skeletal muscle myofibrils. These results are in agreement with previously published work in which the potentials were measured with microelectrodes on glycerol-extraced muscle fibers. The magnitude of the Donnan potential of the myofibrils has been shown to be dependent on the state (rigor or relaxed) of the system.