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PubMed1993

Structural changes in Z-disks of skeletal muscle myofibrils during growth of chicken.

Postnatal structural changes in Z-disks of skeletal muscles of chicken from 2 to 35 wk after hatching were examined to elucidate how Z-disks develop from the embryonic to the mature stage. The mechanical strength of Z-disks of breast muscle (i.e. the ratio of the number of myofibrils to the total number of myofibrils and myofibrillar fragments composed of 1-4 sarcomeres, which were formed under the mechanical forces exerted during homogenization) increased from 66% at 2 wk of age to 75% at 10 wk, and leveled off at 76% at 25 wk. The Z-disks of leg muscle were stronger than those of breast muscle throughout growth. Measurements by electron microscope showed that the width of Z-disks increased during growth from 26 to 33 nm, 48 to 53 nm, and 89 to 101 nm, in white, intermediate, and red muscle fibers, respectively. It was proved that the configuration of Z-filaments, the structural backbone of Z-disks, takes its final shape within 2 wk after hatching; the immature Z-disks are then reinforced by the accumulation of amorphous matrix materials. These results confirm that the maturation of Z-disks is brought about by physical motion in muscle tissue and the accompanying development of tension.

Animals↗
PubMed1980

Extractability of actin and actin-like protein from myosin-removed myofibrils of skeletal muscle.

Extractability of 42,000 dalton components (42 K component) from myosin-removed myofibrils (called I-Z-I segments in this paper) under low-salt conditions was compared at pH 6.5 and pH 8.0. Kinetic and isoelectric focusing analysis of the 42 K components revealed that the 42 K component extracted at pH 8.0 was actin, which depolymerized from the end of the I-filaments of the I-Z-I segments. On the other hand, isoelectric focusing of the 42 K component extracted at pH 6.5 indicated two protein bands. The relative mobility of the minor band was virtually identical to that of actin. While the isoelectric point of the major band was more acidic than that of skeletal muscle actin. Therefore, it is concluded that there are at least two kinds of 42,000 dalton components in the myosin-removed myofibrils.

Actins↗
PubMed1981

Susceptibilities of various myofibrillar proteins to cathepsin B and morphological alteration of isolated myofibrils by this enzyme.

The abilities of cathepsin B purified from liver to degrade purified myofibrillar proteins, myosin, actin, troponin, tropomyosin, and alpha-actinin from rabbit skeletal muscle were studied. The amino acids or peptides liberated from these proteins by cathepsin B were determined quantitatively by fluorometry with o-phthalaldehyde, and qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At a molar ratio of cathepsin B to substrate of 1 : 100, the order of susceptibilities was myosin much greater than troponin greater than tropomyosin much greater than actin. alpha-Actinin was not degraded. Myosin heavy chain was degraded to several fragments with molecular weights of 175,000, 170,000, 160,000, and 145,000, whereas the light chains were scarcely degraded. Cathepsin B degraded troponin-T rapidly, and troponin-I more slowly, but did not degrade troponin-C. Troponin-T and troponin-I were degraded to three fragments with molecular weights of 30,000, 18,000, and 12,800. Tropomyosin was degraded slightly and its product had a molecular weight of 32,000. Actin was also degraded only slowly, and no liberated product could be detected. Morphological changes in myofibrils prepared from glycerinated psoas muscle of rabbit during incubation with cathepsin B were observed. Three notable phenomena were observed: 1) disappearance of the Z-band in the early stage of incubation, 2) disappearance of the M-line following loss of the Z-band, and 3) decrease in the density of the A-band after swelling of the myofibrils.

Actins↗
PubMed2005

Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril.

Calpain 1, a ubiquitous calcium-dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097-2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl(2) and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.

Animals↗
PubMed1985

Dependence of adenosine triphosphatase activity of rabbit psoas muscle fibres and myofibrils on substrate concentration.

The rate of hydrolysis of adenosine triphosphate (ATP) by chemically skinned rabbit muscle fibres was measured as a function of Mg ATP concentration in the range 5 microM to 5 mM. Pyruvate kinase and lactate dehydrogenase were used to link adenosine diphosphate formation to oxidation of nicotinamide adenine dinucleotide which was followed by the change in absorption at 340 nm. The ATPase rate of a fully activated fibre (pCa = 4.5) increased monotonically with Mg ATP concentration in a manner that could be readily fitted by a hyperbola. At 15 degrees C, pH 7 and an ionic strength of 0.2 M the rate at saturating Mg ATP (Vm) was 1.78 +/- 0.2 s-1 per myosin head (mean +/- S.D.; n = 6) and the Mg ATP concentration needed for half the maximal rate (Km) was 16.6 +/- 2 microM. The ATPase of fibres that had been stabilized by cross-linking with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) was also investigated. Cross-linking did not significantly affect the Vm or Km and these fibres proved useful for investigating the adequacy of the pyruvate kinase activity for regenerating hydrolysed ATP. Myofibrils were cross-linked with EDC or glutaraldehyde to prevent shortening. Their ATPase properties were investigated: the values of Vm were 0.85 +/- 0.18 (mean +/- S.D.; n = 14) and 0.82 +/- 0.05 s-1 (n = 6) and of Km were 18.0 +/- 2.8 and 12.4 +/- 2.4 microM respectively. The values of Vm and Km for EDC cross-linked myofibrils were fairly insensitive to ionic strength, the Km decreasing 40% and the Vm increasing 50% for a change from 0.2 to 0.3 M. This slight dependence on ionic strength is considered in relation to the ionic strength dependence of the elementary rate constants of the actomyosin subfragment-1 ATPase cycle.

Adenosine Triphosphatases↗
PubMed1988

Increase by trifluoperazine in calcium sensitivity of myofibrils in a skinned fibre from frog skeletal muscle.

1. Since it has been demonstrated that trifluoperazine (TFP) increases the affinity for Ca2+ of troponin C as well as calmodulin, the effect of TFP was examined on the Ca2+-induced tension in mechanically skinned fibres isolated from frog skeletal muscle and on Ca2+-dependent ATPase activity of myofibrils from similar frog skeletal muscle. 2. Lower concentrations of TFP increased the Ca2+ sensitivity of myofibrils without a change in the maximum tension, giving rise to a less steep tension-pCa relationship. This effect was reversible although thorough washes were necessary. The drug also enhanced myofibrillar ATPase activity, not only at low Ca2+ concentrations but also at saturating high Ca2+ concentrations. The increased affinity of troponin C for Ca2+ is difficult to accept as the sole explanation for the stimulatory effect of TFP. 3. Half of the maximum stimulating effect was obtained between 10 and 30 microM-TFP, which is similar to the reported apparent inhibition constant (Ki) for calmodulin-dependent enzyme reactions. However, the stimulating effect of TFP cannot be attributed to its inhibition of calmodulin because of the finding that this effect was independent of Ca2+. Earlier published results (e.g. Klee & Vanaman, 1982) also support this conclusion. 4. Studies on myofibrillar ATPase activity suggest that the stimulating effect of TFP is not identical in its underlying action with those of caffeine and quercetin, which are also known as Ca2+-sensitizing drugs, having a similar eventual effect on tension development. 5. Higher concentrations of TFP decreased the maximum tension induced by high concentrations of Ca2+, while enhancing the tension in the presence of low concentrations of Ca2+. Analogous findings for ATPase activity were also made. TFP concentration for half the maximum depression was about 10 times higher than that for half the maximum stimulation. This suggests that different site(s) are involved in the stimulatory and inhibitory effects of TFP, although there may be some sites in common. 6. Discussion favours the stimulating effects of TFP as being caused considerably by the affected molecular interactions among myosin, actin, tropomyosin and troponin.

Adenosine Triphosphatases↗
PubMed1981

Analysis of chromosome movement in crane fly spermatocytes by ultraviolet microbeam irradiation of individual chromosomal spindle fibres. II. Action spectra for stopping chromosome movement and for blocking ciliary beating and myofibril contractions.

Chromosome-to-pole movement in crane fly spermatocytes was temporarily blocked by ultraviolet light focussed to a 4-micrometer-diameter spot on single chromosomal spindle fibres. Since similar irradiation of the interzonal region did not alter chromosome-to-pole movement, this effect was specific to spindle fibres. The action spectrum for blocking chromosome movement in this specific way had two peaks, one at 270 nm and one at 290 nm. To block movement, irradiations with 280-nm-wavelength light required two to four times more energy than irradiations with 270- or 290-nm-wavelength light. Action spectra were obtained for blocking ciliary beating and for blocking myofibril contraction. The action spectrum for blocking ciliary beating had a broad peak, between 260 nm and 280 nm, whilst that for blocking myofibril contraction had two peaks, at 270 and 290 nm, just like that for blocking chromosome movement. We discuss the similarities and differences in the various action spectra, and we compare the action spectra to absorption spectra of spindle components and to other action spectra (e.g., that for depolymerizing actin-containing filaments). Absorption spectra were obtained for ultraviolet light passing through spindle fibres as well as for ultraviolet light passing through the interzone.

Animals↗
PubMed1983

Energy transport from mitochondria to myofibril by a creatine phosphate shuttle in cardiac cells.

In hyperpermeable cardiac cells, in which the surface membrane has been made highly permeable to small molecules and ions, resting tension increases when the concentration of ATP falls below 200 microM. Peak resting tension occurs in 10 microM ATP and equals 60% of maximum Ca-activated force in 5 mM ATP. The mitochondria in hyperpermeable cells can maintain an ATP concentration above 200 microM if supplied with O2, substrate, ADP, and inorganic phosphate (Pi). Removal of ATP from the bathing solution does not increase resting tension as long as creatine phosphate is present. However O2, substrate, and Pi cannot lower resting tension in the absence of ATP and creatine phosphate. These results are interpreted as evidence for adenine nucleotide tightly bound to the myofibrils and a creatine phosphate shunt of energy from the mitochondria to the myofibrils.

Adenosine Diphosphate↗
PubMed1978

Endotoxin and myocardial failure: role of the myofibril and venous return.

The effects of gramnegative endotoxin-induced myocardial failure in the pentobarbital-anesthetized dog were examined by monitoring its influence on cardiac myofibrillar ATPase activity. Myofibrils were isolated from endo- and epicardial portions of the left ventricular wall. ATPase activities were determined in animals treated with 4 mg/kg endotoxin and monitored 5 h, in animals monitored for 5 h without endotoxin (controls), and in animals implanted with a unilateral femoral shunt and given endotoxin. No differences were seen in the activities between the endo- and epicardial portions of any preparation. Activity was significantly depressed in endotoxemic animals. Increasing venous return by 313 +/- 71 ml/min significantly increased coronary flow by reducing coronary vascular resistance and prevented any observed depression of myofibrillar ATPase activity. In in vitro studies, adding endotoxin directly to a myofibril preparation did not modify normal activity. It appears that the mechanical and myofibrillar dysfunctions are due to the action of endotoxin at sites not associated with the actomyosin ATPase, but may be due to the production of an intermediary agent in concert with a decreased venous return.

Adenosine Triphosphatases↗
PubMed2005

Myosin-binding protein C phosphorylation, myofibril structure, and contractile function during low-flow ischemia.

BACKGROUND: Contractile dysfunction develops in the chronically instrumented canine myocardium after bouts of low-flow ischemia and persists after reperfusion. The objective of this study is to identify whether changes in the phosphorylation state of myosin-binding protein C (MyBP-C) are a potential cause of dysfunction. METHODS AND RESULTS: During low-flow ischemia, MyBP-C is dephosphorylated, and the number of actomyosin cross-bridges in the central core of the sarcomere decreases as thick filaments dissemble from the periphery of the myofibril. During reperfusion, MyBP-C remains dephosphorylated, and its degradation is accelerated. CONCLUSIONS: Dephosphorylation of MyBP-C may initiate changes in myofibril thick filament structure that decrease the interaction of myosin heads with actin thin filaments. Limiting the formation of actomyosin cross-bridges may contribute to the contractile dysfunction that is apparent after low-flow ischemia. Breakdown of MyBP-C during reperfusion may prolong myocardial stunning.

Actins↗
PubMed1981

Postnatal development of the M-band in rat cardiac myofibrils.

Cardiac muscle fibers in rats at 1 and 5 days after birth showed little evidence of M-bands. An ultrastructural analysis of myofibrils failed to demonstrate dense M-band material in longitudinal sections or M-bridges in transverse sections of sarcomeres. M-bands began to increase in number after 5 days of postnatal life and were present in 60% of all sarcomeres at 11 days of age. Polypeptides with molecular weights of 190,000 (Ma) and 175,000 (Mb) were obtained by polyacrylamide-sodium dodecyl sulfate (SDS) gel electrophoresis of myofibril preparations. These two proteins were found in both 1- and 11-day-old rats and were considered to be specific components of the M-band. The densitometric analysis demonstrated that Ma and mb polypeptides increased approximately 2-fold during the interval from 1 to 11 days after birth. These structural and biochemical changes of the M-band material in myocytes appear to be related to the maturation of contractile function in the young heart.

Animals↗
PubMed1996

Binding of cytosolic proteins to myofibrils in ischemic rat hearts.

Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is alpha B-crystallin. The binding of the two cytosolic proteins to myofibrils was confirmed by immunofluorescence analysis on cryosections of ischemic hearts. In vitro studies showed that acidosis was sufficient to induce the binding of alpha B-crystallin, whereas the inhibition of ATP depletion prevented the binding of GAPDH. Thiol oxidation is unlikely to promote GAPDH binding, since perfusion with iodoacetate under aerobic conditions or treatment of homogenates with N-ethylmaleimide or diamide failed to induce GAPDH association with the myofibrils. These changes of the myofibrillar proteins could be considered as intracellular markers of the evolution of the ischemic damage. In addition, the binding of the 23-kD peptide might be involved in alterations of contractility.

Amino Acid Sequence↗
PubMed1996

Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils.

Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H2O2) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100-treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca(2+)-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pre-treated with 1 mmo1/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 mumol/L per 30 mIU/mL) or H2O2. pMgATP-rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the absence of PCr, pMgATP50, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03 +/- 0.17 (n = 21) to 4.22 +/- 0.22 (n = 4) after 25 minutes of incubation in the presence one of 30 mIU/mL. XO and 100 mumol/L xanthine or to 4.04 +/- 0.1 (n = 11) after incubation in the presence of 2.5 mmol/L H2O2. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP50 when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H2O2 induced on increase in Ca2+ sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H2O2 only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion.

Animals↗
PubMed2001

Interaction between PEVK-titin and actin filaments: origin of a viscous force component in cardiac myofibrils.

The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B-cardiac PEVK was expressed in Escherichia coli and tested-along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains-for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca(2+)] reversed the PEVK effect. PEVK concentrations >/=10 microgram/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0 degrees C, but more strongly at 30 degrees C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca(2+)-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.

Actin Cytoskeleton↗
PubMed2002

Transient association of titin and myosin with microtubules in nascent myofibrils directed by the MURF2 RING-finger protein.

Assembly of muscle sarcomeres is a complex dynamic process and involves a large number of proteins. A growing number of these have regulatory functions and are transiently present in the myofibril. We show here that the novel tubulin-associated RING/B-box protein MURF2 associates transiently with microtubules, myosin and titin during sarcomere assembly. During sarcomere assembly, MURF2 first associates with microtubules at the exclusion of tyrosinated tubulin. Then, MURF2-labelled microtubules associate transiently with sarcomeric myosin and later with A-band titin when non-striated myofibrils differentiate into mature sarcomeres. Finally, MURF2 labelled microtubules disappear from the sarcomere after the incorporation of myosin filaments and the elongation of titin. This suggests that the incorporation of myosin into nascent sarcomeres and the elongation of titin require an active, microtubule-dependent transport process and that MURF2-associated microtubules play a role in the alignment and extension of nascent sarcomeres. MURF2 is expressed in at least four isoforms, of which a 27 kDa isoform is cardiac specific. A C-terminal isoform is generated by alternative reading frame use, a novelty in muscle proteins. In mature cardiac sarcomeres, endogenous MURF2 can associate with the M-band, and is translocated to the nucleus. MURF2 can therefore act as a transient adaptor between microtubules, titin and nascent myosin filaments, as well as being involved in signalling from the sarcomere to the nucleus.

Actins↗
PubMed2000

Fast skeletal muscle isoforms exhibit the highest incorporation level into myofibrils and stress fibers among members of myosin alkali light chain isoform family.

Isoproteins of myosin alkali light chain (LC) were co-expressed in cultured chicken cardiomyocytes and fibroblasts and their incorporation levels into myofibrils and stress fibers were compared among members of the LC isoform family. In order to distinguish each isoform from the other, cDNAs of LC isoforms were tagged with different epitopes. Expressed LCs were detected with antibodies to the tags and their distribution was analyzed by confocal microscopy. In cardiomyocytes, the incorporation level of LC into myofibrils was shown to increase in the order from nonmuscle isoform (LC3nm), to slow skeletal muscle isoform (LC1sa), to slow skeletal/ventricular muscle isoform (LC1sb), and to fast skeletal muscle isoforms (LC1f and LC3f). Thus, the hierarchal order of the LC affinity for the cardiac myosin heavy chain (MHC) is identical to that obtained in the rat (Komiyama et al., 1996. J. Cell Sci., 109: 2089-2099), suggesting that this order may be common for taxonomic animal classes. In fibroblasts, the affinity of LC for the nonmuscle MHC in stress fibers was found to increase in the order from LC3nm, to LC1sb, to LC1sa, and to LC1f and LC3f. This order for the nonmuscle MHC is partly different from that for the cardiac MHC. This indicates that the order of the affinity of LC isoproteins for MHC varies depending on the MHC isoform. Further, for both the cardiac and nonmuscle MHCs, the fast skeletal muscle LCs exhibited the highest affinity. This suggests that the fast skeletal muscle LCs may be evolved isoforms possessing the ability to associate tightly with a variety of MHC isoforms.

Animals↗
PubMed1987

Alterations in fine structures of myofibrils and structural proteins in patients with dilated cardiomyopathy--studies with biopsied heart tissues.

Ultrastructural and biochemical alterations in myofibrils (Mf) were studied in biopsied myocardial tissues in 11 patients with dilated cardiomyopathy (DCM) and compared with those in non-hypertrophic control and secondary hypertrophic (SHT) heart muscles. Transverse diameters of biopsied cardiac myocytes increased significantly in both of SHT and DCM, and ultrastructural changes were similar in quality in both of them. Volume densities of Mf were 61.1 and 59.9% on average in control and SHT myocardial cells, respectively, and they were not significantly different. But in DCM volume density was significantly less (49.8% in left ventricular myocytes), and inverse relation between that and diameter of cardiac myocytes was observed (p less than 0.01). Electrophoretic pattern and relative composition of major structural proteins from control and SHT heart muscles were similar and statistically insignificant. In DCM, relative contents of myosin heavy chain and alpha-actinin decreased significantly and distinctively in all of cases suggesting primary degradation of myofibrils.

Biopsy↗