Stimulation of myofibrillar synthesis by exercise is mediated by more efficient translation of mRNA.
(9/1851)
Resistance exercises stimulate protein synthesis in human muscle, but the roles of changes in mRNA concentrations and changes in the efficiency of mRNA translation have not been defined. The present study was done to determine whether resistance exercise affects concentrations of total RNA, total mRNA, actin mRNA, or myosin heavy-chain mRNA (total and isoform specific). Eight subjects, 62-75 yr old, performed unilateral knee extensions at 80% of their one-repetition-maximum capacity on days 1, 3, and 6 of the study. On day 7, biopsies of exercised and nonexercised vastus lateralis muscles were obtained. Myofibrillar synthesis was determined by stable- isotope incorporation, and mRNA concentrations were determined by membrane hybridization and PCR-based methods. The exercise stimulated myofibrillar synthesis [30 +/- 6 (SE)%] without affecting RNA or mRNA concentrations. The effect of exercise on protein synthesis in individual subjects did not correlate with the effect on total RNA and mRNA concentrations. These data suggest that the stimulation of myofibrillar synthesis by resistance exercise is mediated by more efficient translation of mRNA. (+info)
Modulation by substrate concentration of maximal shortening velocity and isometric force in single myofibrils from frog and rabbit fast skeletal muscle.
(10/1851)
1. The effects of magnesium adenosine triphosphate (MgATP; also referred to as 'substrate') concentration on maximal force and shortening velocity have been studied at 5 C in single and thin bundles of striated muscle myofibrils. The minute diameters of the preparations promote rapid diffusional equilibrium between the bathing medium and lattice space so that during contraction fine control of substrate and product concentrations is achieved. 2. Myofibrils from frog tibialis anterior and rabbit psoas fast skeletal muscles were activated maximally by rapidly (10 ms) exchanging a continuous flux of pCa 8.0 for one at pCa 4.75 at a range of substrate concentrations from 10 microM to 5 mM. At high substrate concentrations maximal isometric tension and shortening velocity of both frog and rabbit myofibrils were very close to those determined in whole fibre preparations from the same muscle types. 3. As in frog and rabbit skinned whole fibres, the maximal isometric force of the myofibril preparations decreases as MgATP concentration is increased. The maximal velocity of unloaded shortening (V0) depends hyperbolically on substrate concentration. V0 extrapolated to infinite MgATP (3.6 +/- 0.2 and 0.8 +/- 0.03 l0 s-1 in frog and rabbit myofibrils, respectively) is very close to that determined directly at high substrate concentration. The Km is 210 +/- 20 microM for frog tibialis anterior and 120 +/- 10 microM for rabbit psoas myofibrils, values about half those found in larger whole fibre preparations of the same muscle types. This implies that measurements in whole skinned fibres are perturbed by diffusional delays, even in the presence of MgATP regenerating systems. 4. In both frog and rabbit myofibrils, the Km for V0 is about one order of magnitude higher than the Km for myofibrillar MgATPase determined biochemically in the same experimental conditions. This confirms that the difference between the Km values for MgATPase and shortening velocity is a basic feature of the mechanism of chemomechanical transduction in muscle contraction. (+info)
Assembly of thick filaments and myofibrils occurs in the absence of the myosin head.
(11/1851)
We investigated the importance of the myosin head in thick filament formation and myofibrillogenesis by generating transgenic Drosophila lines expressing either an embryonic or an adult isoform of the myosin rod in their indirect flight muscles. The headless myosin molecules retain the regulatory light-chain binding site, the alpha-helical rod and the C-terminal tailpiece. Both isoforms of headless myosin co-assemble with endogenous full-length myosin in wild-type muscle cells. However, rod polypeptides interfere with muscle function and cause a flightless phenotype. Electron microscopy demonstrates that this results from an antimorphic effect upon myofibril assembly. Thick filaments assemble when the myosin rod is expressed in mutant indirect flight muscles where no full-length myosin heavy chain is produced. These filaments show the characteristic hollow cross-section observed in wild type. The headless thick filaments can assemble with thin filaments into hexagonally packed arrays resembling normal myofibrils. However, thick filament length as well as sarcomere length and myofibril shape are abnormal. Therefore, thick filament assembly and many aspects of myofibrillogenesis are independent of the myosin head and these processes are regulated by the myosin rod and tailpiece. However, interaction of the myosin head with other myofibrillar components is necessary for defining filament length and myofibril dimensions. (+info)
Myofibrillogenesis in the developing chicken heart: assembly of Z-disk, M-line and the thick filaments.
(12/1851)
Myofibrillogenesis in situ was investigated by confocal microscopy of immunofluorescently labelled whole mount preparations of early embryonic chicken heart rudiments. The time-course of incorporation of several components into myofibrils was compared in triple-stained specimens, taken around the time when beating starts. All sarcomeric proteins investigated so far were already expressed before the first contractions and myofibril assembly happened within a few hours. No typical stress fibre-like structures or premyofibrils, structures observed in cultured cardiomyocytes, could be detected during myofibrillogenesis in the heart. Sarcomeric proteins like (&agr;)-actinin, titin and actin were found in a defined localisation pattern even in cardiomyocytes that did not yet contain myofibrils, making up dense body-like structures. As soon as the heart started to beat, all myofibrillar proteins were already located at their exact position in the sarcomere. The maturation of the sarcomeres was characterised by a short delay in the establishment of the pattern for M-line epitopes of titin with respect to Z-disk epitopes and the incorporation of the M-line component myomesin, which preceded that of myosin binding protein-C. Thus dense body-like structures, made up of titin, (&agr;)-actinin and actin filaments serve as the first organised complexes also during myofibrillogenesis in situ and titin functions as a ruler for sarcomere assembly as soon as its C termini have become localised. We suggest that assembly of thin and thick filament occurs independently during myofibrillogenesis in situ and that myomesin might be important for integrating thick filaments with the M-line end of titin. (+info)
UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle.
(13/1851)
The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils. (+info)
A filamentous cytoskeleton in vertebrate smooth muscle fibers.
(14/1851)
There are three classes of myofilaments in vertebrate smooth muscle fibers. The thin filaments correspond to actin and the thick filaments are identified with myosin. The third class of myofilaments (100 A diam) is distinguished from both the actin and the myosin on the basis of fine structure, solubility, and pattern of localization in the muscle fibers. Direct structural evidence is presented to show that the 100A filament constitute an integrated filamentous network with the dense bodies in the sarcoplasm, and that they are not connected to either the actin or myosin filaments. Examination of (a) isolated dense bodies, (b) series of consecutive sections through the dense bodies, and (c) redistributed dense bodies in stretched muscle fibers supports this conclusion. It follows that the 100-A filaments complexes constitute a structrally distinct filamentous network. Analysis of polyacrylamide gels after electrophoresis of cell fractions that are enriched with respect to the 100-A filaments shows the presence of a new muscle protein with a molecular weight of 55,000. This protein can form filamentous segments that closely resemble in structure the native, isolated 100-A filaments. The results indicate that the filamentous network has a structure and composition that distinguish it from the actin and myosin in vertebrate smooth muscle. (+info)
Cross bridge-dependent activation of contraction in cardiac myofibrils at low pH.
(15/1851)
Striated muscle contracts in the absence of calcium at low concentrations of MgATP ([MgATP]), and this has been termed rigor activation because rigor cross bridges attach and activate adjacent actin sites. This process is well characterized in skeletal muscle but not in cardiac muscle. Rigor cross bridges are also thought to increase calcium binding to troponin C and play a synergistic role in activation. We tested the hypothesis that cross bridge-dependent activation results in an increase in contractile activity at normal and low pH values. Myofibrillar ATPase activity was measured as a function of pCa and [MgATP] at pH 7.0, and the data showed that, at pCa values of >/=5.5, there was a biphasic relationship between activity and [MgATP]. Peak activity occurred at 10-50 microM MgATP, and [MgATP] for peak activity was lower with increased pCa. The ATPase activity of rat cardiac myofibrils as a function of [MgATP] at a pCa of 9.0 was measured at several pH levels (pH 5.4-7.0). The ATPase activity as a function of [MgATP] was biphasic with a maximum at 8-10 microM MgATP. Lower pH did not result in a substantial decrease in myofibrillar ATPase activity even at pH 5.4. The extent of shortening, as measured by Z-line spacing, was greatest at 8 microM MgATP and less at both lower and higher [MgATP], and this response was observed at all pH levels. These studies suggest that the peak ATPase activity associated with low [MgATP] was coupled to sarcomere shortening. These results support the hypothesis that cross bridge-dependent activation of contraction may be responsible for contracture in the ischemic heart. (+info)
Cellular and molecular remodeling in a heart failure model treated with the beta-blocker carteolol.
(16/1851)
Broad-breasted white turkey poults fed furazolidone developed dilated cardiomyopathy (DCM) characterized by ventricular dilatation, decreased ejection fraction, beta1-receptor density, sarcoplasmic reticulum (SR) Ca2+-ATPase, myofibrillar ATPase activity, and reduced metabolism markers. We investigated the effects of carteolol, a beta-adrenergic blocking agent, by administrating two different dosages (0.01 and 10.0 mg/kg) twice a day for 4 wk to control and DCM turkey poults. At completion of the study there was 59% mortality in the nontreated DCM group, 55% mortality in the group treated with the low dose of carteolol, and 22% mortality in the group treated with the high dose of carteolol. Both treated groups showed a significant decrease in left ventricle size and significant restoration of ejection fraction and left ventricular peak systolic pressure. Carteolol treatment increased beta-adrenergic receptor density, and the high carteolol dose restored SR Ca2+-ATPase and myofibrillar ATPase activities, along with creatine kinase, lactate dehydrogenase, aspartate transaminase, and ATP synthase activities, to normal. These results show that beta-blockade with carteolol improves survival, reverses contractile abnormalities, and induces cellular remodeling in this model of heart failure. (+info)