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Increase in the asymmetry of light scattering diagram of myofibril suspensions resulting from their relaxation was shown to be not associated with essential alterations of the myofibrillar size. Optical changes caused by relaxation appear to be conditioned by the inner rearrangement of the myofibrillar structure.
The binding of [3H]ADP and [3H]adenyl-5'-yl-imidodiphosphate ([3H]AMP-PNP) to rabbit skeletal myofibrils was measured at 25 and 7 degrees C, mu = 0.12 M, using [14C]mannitol as a volume marker. We found that ADP bound to myosin heads in overlap with a binding constant of about 10(4) M-1, similar to the value we previously obtained in vitro with acto.S-1. The binding of AMP-PNP to myosin heads was measured both in and out of overlap. The affinity of AMP-PNP to the heads out of overlap was similar to that obtained in vitro with S-1 alone. The binding of AMP-PNP to the myosin heads in overlap was much weaker. We could fit these data with a binding constant of about 1 x 10(3) M-1, assuming a single population of cross-bridges and 1 mol of AMP-PNP bound per mol of myosin head. This value was reduced by a factor of 2 when we corrected for nonspecific binding. It was also possible to fit the data assuming two equal populations of cross-bridges with one of the populations binding AMP-PNP about 5-fold more strongly than the other population. Therefore, for at least half of the cross-bridges in overlap, the binding of AMP-PNP is almost as weak as the value of 3 x 10(2) M-1 we previously measured for the acto.S-1 complex in vitro (Biosca, J. A., Greene, L. E., and Eisenberg, E. (1986) J. Biol. Chem. 261, 9793-9800).
The immunochemical method has been proposed to indicate the adenin compounds in isolated myofibrils of striated muscle. Adenosine--containing antigen was prepared by coupling adenosine with bovine serum albumin (adenosine--BSA). Antibodies against adenosine--BSA were labelled by ferritin. The sites of the antigen antibody reaction were marked by localization of ferritin molecules. Ferritin antibodies were found to concentrate in the following sarcomere sites: M and Z bands, N-lines and terminal cisternae of sarcoplasmic reticulum near the Z-bands.
Ultrastructural changes have been studied in fast (EDL) and slow (SOL) isolated muscles of rats during Zenker's degeneration (ZD). The general pattern of ZD is similar in both the muscles and proceeds in the same way as in the fast skeletal muscles of amphibians: vacuolation of the T-system and of the sarcoplasmic reticulum occur prior to necrosis, and then contraction knots are formed. The latter detach from the rest of the fiber, and myofibrils degrade into clots of electron dense material. Differences in the development of ZD in this muscle are as follows: vacuolation is more strongly pronounced in EDL, contraction and supercontraction of sarcomeres being more characteristic of SOL. In this ZD region of both the muscles, additional membranes are found in some intracrystal spaces of mitochondria, this phenomenon occuring more frequently in SOL. Selective lysis of the Z-line during ZD has been detected only in single EDL Fibres.
The differentiation of the myofibrils and the intermediate filament system during postnatal development of the rat heart has been investigated. Several aspects of some of the structural proteins, that means the intermediate filament subunit skeletin, myosin, and the myofibrillar M-line proteins MM-creatine kinase and myomesin have been studied by using gel electrophoresis as well as enzyme and immunohistochemical techniques in combination with electron microscopy of both plastic and cryosectional material. We show that marked changes take place in the organization of the intermediate filament system and in the contractile apparatus, both in atria and in ventricles of the rat heart during postnatal development. In the newborn rats no dense myofibrillar M-bands were present in the M-region and the sarcomeric bands were irregular while in the four-week-old rats dense M-bands composed of a set of five crossbridges interconnecting the thick filaments were present. The sarcomeric bands were now regular. These observations are related to the presence of different isomyosins in the atria and in the ventricles of the newborn and the four-week-old rats, to the observation that MM-creatine kinase was only present in the M-region in the four-week old rats and to the physiological maturation of the heart.
Myocardial function can be modulated at the level of the sarcolemma, the intracellular Ca2+ stores, and the myofilaments. A way of following myofibrillar modulation of mechanochemical activity is by studying the Ca2+-dependent activation of myofibrils. In this study, the role of Mg2+ in the Ca2+ activation of myofibrillar ATPase was evaluated. The concentrations of both free Ca2+ and free Mg2+ were varied at a given concentration of MgATP (3.16 mM), ionic strength (0.12), and pH 7.0. The experimental ATPase-vs.-Ca2+ data were fit to the model-independent Hill equation and to Tawada's model of intertropomyosin cooperation. In the micromolar range, Mg2+ did not affect the Ca2+ sensitivity, whereas it was reduced at high Mg2+ (5 mM), corresponding to a shift of the activation curve to higher Ca2+ concentrations. Positive cooperativity (n = 2.4) and ATPase activity at saturating Ca2+ concentrations were affected only at low Mg2+. At 0.032 mM Mg2+, the positive cooperation was partially lost (n = 1.3), and maximum ATPase was reduced by 23%. The consequences of a change in the activation curves for the mechanogram are shown. The influence of other modulatory mechanisms on the activation properties was also analyzed on the basis of the Hill equation and the intertropomyosin cooperation model. Acute changes in contractility can arise from changes in the intracellular pH, in sarcomere length, and from phosphorylation of troponin I. Long-term modifications of cardiac performance involve genetic expression of different myosin isoenzymes. The relevance of these different modulatory mechanisms for myocardial function is shown.
During limb regeneration of the salamander, Salamandra salamandra, muscles dedifferentiate and then mesenchymal cells appear, which subsequently, differentiate into muscle. Both in regenerates and in developing limbs of newts larvae mesenchymal cells begin to differentiate into myoblasts, which contain both thin (7 nm) and thick (14 nm) filaments, which are often associated with microtubules, developing a cytoskeletal network, which contribute directly to the shape of myoblasts and a linear alignment of the myofibrils. Both thin and thick filaments appear as a tandem near the periphery of the myoblasts, where myofibrilogenesis occurs. In the early stage of sarcomagenesis the appearance of "Z-bodies" was observed. They were intimately associated with the tandem of filaments thus forming the primary sarcomere units. Consequently the "Z-bodies" coalesce to form primitive Z-lines. The maturation of the primary sarcomeres occurs during fibrilogenesis and is manifested by the gradual thining of the Z-line and thickening of the M-line.
1. Z-line constituents contributing to the formation of the contraction bands in chicken myofibrils were investigated by using the rabbit calcium-activated factor (CAF). 2. CAF digestion hampered the formation of the contraction bands and increased the amount of soluble proteins which are apparently derived from the Z-line. Among several proteins released by CAF digestion, a 95,000 dalton component, which corresponds to alpha-actinin, was predominant, followed by a 220,000 dalton component and several other proteins. 3. An apparent inverse relationship between the formation of the contraction bands and the release of Z-line proteins was observed. It is concluded that the presence of Z-line, including at least the two components mentioned above, is essential for the formation of the contraction bands.
1) The contractile system consists of thick and thin filaments arranged side by side in a double network of hexagonal cross-section. 2) The thick filaments are principally made up of myosin and the thin ones of actin, tropomyosin and troponin. 3) Myosin is an enzyme catalysing the hydrolysis of ATP; actin increases the specific activity of this enzyme, converting it from a Ca+2 sensitive ATPase to a Mg+2 sensitive ATPase. 4) Hydrolysis of the last phosphoryl group of adenosine triphosphate (ATP) salts is the energy source for muscle contraction. 5) The adenosine diphosphate (ADP) salts, formed by ATP splitting, are rephosphorylated and reinjected into the myofibril.
The effects of cyclochlorotine (CC), a secondary metabolite of Penicillium islandicum, on cardiomyocytes and fibroblasts were investigated by fluorescent and electron microscopy. The in vitro application of CC on myocytes induced disruption of myofibrils and large accumulations of actin, myosin, alpha-actinin, and vinculin at the cellular processes. This agent also induced the formation of islands of myosin and alpha-actinin aggregates. Actin filament bundles in fibroblasts were either unaffected or disrupted completely, suggesting the presence of two, physiocochemically different, populations of fibroblasts. CC damage was dose-dependently reversible. Further studies will be required to explore the feasibility of using CC for analyzing the properties of contractile and/or cytoskeletal proteins.
Glycerol-extracted rabbit psoas muscle fibres were treated with a solution containing the head subunits of myosin. Interaction of the isolated myosin heads with actin filaments in situ was indicated by an increase in the density of the I-band and by an increased diameter of actin filaments alongside their whole length. We conclude that actin filaments are able to interact with a considerably larger number of myosin molecules than that available in the myofibril under in vivo conditions.
To understand the factors underlying the functional differences between atrial and ventricular tissues, intrinsic properties of myofibrils and mitochondria of atrial skinned fibers were compared with those of fibers from adult or immature (1 and 2 weeks old) ventricular muscle. Isometric mechanical parameters were determined at various calcium concentrations in fibers treated with Triton X-100 to solubilize all cellular membranes. Maximal active tension and stiffness measured at pCa 4.5, as well as calcium sensitivity, were not different in adult atria and ventricles. Both force and stiffness increased in adult ventricles, while calcium sensitivity diminished in adult ventricles, compared with immature muscles. Myofibrillar contractile kinetics, assessed by the rate constant of tension fall following quick stretches, were similar in adult atria (79.7 +/- 6.9 s-1) and ventricles (72.4 +/- 6.8 s-1) and higher in adult atria and ventricles than in immature ventricles (24.1 +/- 2.3 s-1 in 1-week-old rats and 49.3 +/- 4.2 s-1 in 2-week-old rats). Sensitivity of rigor tension development to MgATP in the presence and in the absence of phosphocreatine was not markedly different in the different tissues. Mitochondrial function was assessed in saponin-skinned fibers. Tissue oxidative capacities, expressed as nmol O2.min-1.mg-1 fiber dry weight, were lower in immature ventricles and atria than in adult ventricles. Creatine failed to stimulate respiration in ventricles of young rats and in adult atria, whereas a 74 +/- 10% increase in respiration was observed in adult ventricles. Since mitochondrial creatine kinase was present in adult atria, this suggests an absence of coupling between oxidative phosphorylation and mitochondrial creatine kinase in this tissue. Thus, adult atrial tissue differs from neonatal ventricular tissue but it exhibits contractile properties similar to adult ventricular properties and differs from adult ventricle mainly in metabolic properties.
The thermal unfolding of myosin in skeletal muscle myofibrils was studied by differential scanning calorimetry (DSC). In the absence of nucleotide two major transitions with Tm of 52 degrees C and 58 degrees C, and a minor transition with Tm of 19 degrees C were detected. The unfolding can be characterized with a total enthalpy of -90 +/- 6.1 mJ/g protein. The major transition with Tm of 58 degrees C was independent of the presence of nucleotide and orthovanadate (Vi), and it can be assigned to the unfolding of the alpha-helical rod part of myosin and partly to actin. In the presence of MgADP, the minor transition shifted to higher temperature, indicating changes between the heavy chain of subfragment-1 and the LC-2 light chain. The transition with Tm of 52 degrees C exhibited a significant broadening and a small shift to lower temperature. It indicates an internal domain or segmental rearrangement of the myosin motor. Upon addition of MgADP and Vi, a shift to higher temperature was observed for the lower major transition, evidencing that with trapped ADP and Vi the intermolecular interactions stabilized the myosin head region.
C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A.
Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K(m) for exogenous ADP in regulation of respiration was equal to 300-400 microM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 microM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20-30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems - with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.
To investigate the roles of cross-bridge dissociation and cross-bridge-induced thin filament activation in the time course of muscle relaxation, we initiated force relaxation in single myofibrils from skeletal muscles by rapidly (approximately 10 ms) switching from high to low [Ca(2+)] solutions. Full force decay from maximal activation occurs in two phases: a slow one followed by a rapid one. The latter is initiated by sarcomere "give" and dominated by inter-sarcomere dynamics (see the companion paper, Stehle, R., M. Krueger, and G. Pfitzer. 2002. Biophys. J. 83:2152-2161), while the former occurs under nearly isometric conditions and is sensitive to mechanical perturbations. Decreasing the Ca(2+)-activated force preceding the start of relaxation does not increase the rate of the slow isometric phase, suggesting that cycling force-generating cross-bridges do not significantly sustain activation during relaxation. This conclusion is strengthened by the finding that the rate of isometric relaxation from maximum force to any given Ca(2+)-activated force level is similar to that of Ca(2+)-activation from rest to that given force. It is likely, therefore, that the slow rate of force decay in full relaxation simply reflects the rate at which cross-bridges leave force-generating states. Because increasing [P(i)] accelerates relaxation while increasing [MgADP] slows relaxation, both forward and backward transitions of cross-bridges from force-generating to non-force-generating states contribute to muscle relaxation.
Kinetics of force development and relaxation after rapid application and removal of Ca(2+) were measured by atomic force cantilevers on subcellular bundles of myofibrils prepared from guinea pig left ventricles. Changes in the structure of individual sarcomeres were simultaneously recorded by video microscopy. Upon Ca(2+) application, force developed with an exponential rate constant k(ACT) almost identical to k(TR), the rate constant of force redevelopment measured during steady-state Ca(2+) activation; this indicates that k(ACT) reflects isometric cross-bridge turnover kinetics. The kinetics of force relaxation after sudden Ca(2+) removal were markedly biphasic. An initial slow linear decline (rate constant k(LIN)) lasting for a time t(LIN) was abruptly followed by an ~20 times faster exponential decay (rate constant k(REL)). k(LIN) is similar to k(TR) measured at low activating [Ca(2+)], indicating that k(LIN) reflects isometric cross-bridge turnover kinetics under relaxed-like conditions (see also. Biophys. J. 83:2142-2151). Video microscopy revealed the following: invariably at t(LIN) a single sarcomere suddenly lengthened and returned to a relaxed-type structure. Originating from this sarcomere, structural relaxation propagated from one sarcomere to the next. Propagated sarcomeric relaxation, along with effects of stretch and P(i) on relaxation kinetics, supports an intersarcomeric chemomechanical coupling mechanism for rapid striated muscle relaxation in which cross-bridges conserve chemical energy by strain-induced rebinding of P(i).
Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.