Biomedical subjects

M L Greaser

Publications and source records attributed to M L Greaser.

At least 55 records · Page 3Linked to original sources

PubMed1991

Substitution of cardiac troponin C into rabbit muscle does not alter the length dependence of Ca2+ sensitivity of tension.

1. The isometric length-tension relationship for cardiac muscle is generally steeper than for skeletal muscle in the physiological range of sarcomere lengths. Recent studies suggest that cardiac troponin C (cTnC) may have intrinsic properties that confer greater length-dependent changes in Ca2+ sensitivity of tension than for skeletal troponin C (sTnC). We tested this hypothesis by characterizing tension-pCa (pCa is -log[Ca2+]) relationships in rabbit skinned psoas muscle fibres at mean sarcomere lengths of 2.32 and 1.87 microns both before and after partial replacement of endogenous sTnC with cTnC. 2. In untreated control fibres, the mid-point (pCa50) of the tension-pCa relationship shifted to lower pCa by 0.15 +/- 0.02 pCa units, i.e. became less sensitive to Ca2+, when sarcomere length was reduced, and the relationship became steeper. 3. Partial extraction of endogenous sTnC and reconstitution with cTnC resulted in no change in the length-dependent shift of pCa50 when reconstitution with cTnC was more than 95% complete; however, when reconstitution was less than 95% complete, there were significant increases in the length-dependent shift in pCa50. 4. An increase in the length-dependent shift of pCa50 was also observed in fibres from which sTnC was partially extracted, but no cTnC was subsequently re-added. 5. We conclude that differences in type of TnC alone are not sufficient to explain differences between skeletal and cardiac muscles in the length dependence of Ca2+ sensitivity of tension.

Animals↗

PubMed1991

Distribution of capillaries in normal and ischemic turkey skeletal muscle.

Capillary to fiber relationship was studied in musculi biceps femoris and musculi pectoralis thoracicus from 20 normal and 10 ischemic turkeys. Capillary density, the number of capillaries surrounding a fiber, capillary to fiber ratio, intercapillary distance, and fiber area were measured. The muscles from the ischemic group had significantly lower (P less than .05) values for capillary density and capillary to fiber ratio and significantly higher intercapillary distance than those from the normal group. There was no evidence of larger muscle fiber size in the ischemic versus normal birds. It is suggested that the occurrence of muscle ischemia in the domestic turkey is due to alterations in capillarity and reduced vasodilatation, resulting from lack of exercise.

Animals↗

PubMed1991

Histopathological and ultrastructural alterations of turkey skeletal muscle.

The pectoralis thoracicus and biceps femoris muscles were obtained from 30 18-wk-old Large White turkey males at the time of slaughter. The birds had no observable mobility or postural problems, and the meat appeared normal. Samples were processed for histology, histochemistry, and electron microscopy. Degenerative features were found in muscle from 10 of the 30 birds. Four of the 10 had alterations in both muscles. The degenerative changes included scattered focal necrosis, hypercontraction of muscle fibers, infiltration by mononuclear cells, formation of fibrous tissue scars, and Z-band streaming. These structural alterations appear to be related to muscle ischemia.

Abattoirs↗

PubMed1990

Regulation of binding of subfragment 1 in isolated rigor myofibrils.

A steric-hindrance model has been used to explain the regulation of muscle contraction by tropomyosin-troponin complex. The regulation of binding was studied by microscopic observation of mixtures of fluorescent subfragment 1 (S1) with rigor myofibrils at different actin-to-S1 ratios and in the presence and absence of calcium. Procedures were adapted to protect the critical thiols of S1 before conjugation to thiol-specific fluorochromes, this giving fluorescent S1 with unaltered enzyme activity. S1 binding was greatest in the I band (except at the Z-lines) in the presence of calcium regardless of the [S1]. The patterns in the absence of calcium depended on the actin-to-S1 ratios: low [S1], binding in the myosin-actin overlap region; intermediate [S1], highest binding at the A-I junction; high [S1], greatest binding in the I-band. The two distinct binding patterns observed at low [S1] were demonstrated by dual-channel fluorescence microscopy when myofibrils were sequentially incubated with fluorescent S1 without calcium followed by a different fluorescent S1 with calcium. These observations support the concept of rigor activation of actin sites. The change in the pattern upon increasing [S1] without calcium demonstrate cooperative interactions along the thin filament. However, these interactions (under the conditions used without calcium) do not appear to extend over greater than 2-3 tropomyosin-troponin-7 actin functional units.

Abdominal Muscles↗

PubMed1990

Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers.

Various functional roles for myosin light chain 2 (LC2) have been suggested on the basis of numerous and predominantly in vitro biochemical studies. Using skinned fibers from rabbit psoas muscle, the present study examines the influence of partial removal of LC2 on isometric tension, stiffness, and maximum velocity of shortening at various levels of activation by Ca2+. Isometric tension, stiffness, and velocity of shortening were measured at pCa values between 6.6 and 4.5 (a) in a control fiber segment, (b) in the same fiber segment after partial removal of LC2, and (c) after recombination with LC2. The extraction solution contained 20 mM EDTA, 20 or 50 mM KCl, and either imidazole or PO4(2-) as a pH buffer (pH 7.0). The amount of LC2 extracted varied with the temperature, duration of extraction, and whether or not troponin C (0.5 mg/ml) was added to the extraction solution. Extraction of 20-40% LC2 resulted in increased active tensions in the range of pCa's between 6.6 and 5.7, but had no effect upon maximum tension. The tension-pCa relationship was left-shifted to lower [Ca2+] by as much as 0.2 pCa units after LC2 extraction. At low concentrations of Ca2+, an increase in stiffness proportional to the increase in tension was observed. Readdition of LC2 to these fiber segments resulted in a return of tension and stiffness to near control values. Stiffness during maximal activation was unaffected by partial extraction of LC2. LC2 extraction was shown to uniformly decrease (by 25-30%), the velocity of shortening during the high velocity phase but it did not significantly affect the low velocity phase of shortening. This effect was reversed by readdition of purified LC2 to the fiber segments. On the basis of these findings we conclude that LC2 may modulate the number of cross-bridges formed during Ca2+ activation and also the rate of cross-bridge detachment during shortening. These results are consistent with the idea that LC2 may modulate contraction via an influence upon the conformation of the S1-S2 hinge region of myosin.

Animals↗

PubMed1989

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins.

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.

Animals↗

PubMed1989

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the alpha- and gamma-isoforms of actin.

The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.

Actins↗

PubMed1989

Incidence of microscopically detectable degenerative characteristics in skeletal muscle of turkey.

1. The incidence of microscopically detectable degenerative characteristics in 5 skeletal muscles (m. pectoralis thoracicus, m. supracoracoideus, m. biceps femoris, m. semitendinosus, m. femorotibialis medius) of turkeys was investigated. 2. Samples were obtained from 30 Large White turkey males 14, 16 and 18 weeks old. Hyaline degeneration, infiltration of mononuclear cells and necrotic fibres were observed. 3. Individual fibres varied greatly in size and muscle fibre nuclei were often shrunken and pyknotic. 4. Weak and/or uniform reaction for Ca++-ATPase and SDH in all types of muscle fibres and loss of alkaline phosphatase activity in cell membranes were noted. A positive reaction for acid phosphatase occurred in regions of perivascular infiltration and in necrotic muscle fibres. The majority of muscle fibres possessed high activity for phosphorylase a and b. 5. Based on the use of fluorescein alpha-bungarotoxin conjugate, motor end-plates appeared to be morphologically intact. Direct immunofluorescence with anti-chicken IgG showed positive reaction in muscle fibres undergoing necrosis and in the involved connective tissue. 6. Degenerative changes varied with age and were most marked in the oldest birds. 7. Because gross degenerative symptoms were absent from both the birds and the meat from them, the condition appears to be either different from or a precursor to the degenerative myopathy characterised by other authors.

Aging↗

PubMed1989

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle.

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady-state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast-twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e-kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585-600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

Myosin heavy chain composition of single cells from avian slow skeletal muscle is strongly correlated with velocity of shortening during development.

We have determined the myosin heavy chain (MHC) composition (using a sensitive sodium dodecyl sulfate-polyacrylamide gel electrophoresis system) and the maximal velocity of shortening (Vmax) of single cells from neonatal and adult chicken anterior latissimus dorsi (ALD) muscles. In addition, the MHC, myosin light chain, and regulatory protein (i.e., troponin and tropomyosin subunits) compositions of bundles of ALD fibers were determined at late embryonic, neonatal, and adult ages. At young ages, there are two MHCs in ALD muscle, SM1 and SM2, with SM1 decreasing in relative amount with increasing age, as shown previously by others. The mean Vmax of single fibers also decreases from neonatal to adult ages. A strong quantitative correlation is demonstrated between the specific MHC composition and Vmax among individual cells of the ALD muscle at several ages. Since virtually no changes occur in the regulatory protein and myosin light chain compositions of the ALD muscle between late embryonic and adult ages, it appears that the MHC composition of an individual cell in this muscle is the primary determinant of the maximal shortening velocity. These results are the first to illustrate the functional significance of the developmental transition in myosin heavy chain composition of an avian slow skeletal muscle, consistent with our previous findings on mammalian muscle.

Animals↗

PubMed1988

Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin.

Cardiac myofibrillogenesis was examined in cultured chick cardiac cells by immunofluorescence using antibodies against titin, actin, tropomyosin, and myosin. Primitive cardiomyocytes initially contained stress fiber-like structures (SFLS) that stained positively for alpha actin and/or muscle tropomyosin. In some cases the staining for muscle tropomyosin and alpha actin was disproportionate; this suggests that the synthesis and/or assembly of these two isoforms into the SFLS may not be stoichiometric. The alpha actin containing SFLS in these myocytes could be classified as either central or peripheral; central SFLS showed developing sarcomeric titin while peripheral SFLS had weak titin fluorescence and a more uniform stain distribution. Sarcomeric patterns of titin and myosin were present at multiple sites on these structures. A pair of titin staining bands was clearly associated with each developing A band even at the two or three sarcomere stage, although occasional examples of a titin band being associated with a half sarcomere were noted. The appearance of sarcomeric titin patterns coincided or preceded sarcomere periodicity of either alpha actin or muscle tropomyosin. The early appearance of titin in myofibrillogenesis suggests it may have a role in filament alignment during sarcomere assembly.

Actins↗

PubMed1988

Variations in contractile properties of rabbit single muscle fibres in relation to troponin T isoforms and myosin light chains.

1. The maximal velocity of shortening (Vmax), tension-pCa relationships and the contractile and regulatory protein composition were determined in single, chemically skinned fibres from adult rabbit plantaris muscles. 2. Three groups of fibres were identified based on their protein compositions. One group had exclusively the slow-type myosin heavy chain (MHC) and myosin light chains (LC) and had low velocities. Another group of fibres had mixtures of fast-type and slow-type MHCs and LCs and had intermediate shortening velocities. The third group of fibres had fast-type myosin heavy and light chains and high velocities. 3. The low-velocity fibres had a mean velocity (+/- S.E.M.) of 0.86 +/- 0.03 muscle lengths/s (ML/s) at 15 degrees C. The remaining fibres formed a continuum with respect to Vmax from 1.37 to 3.94 ML/s. These results indicate that a much greater diversity exists among single fibres from adult mammalian skeletal muscle than previously recognized. The intermediate- and high-velocity fibres formed a continuum (from slow to fast) with respect to the amount of myosin light chain 3 (LC3). That is, Vmax increased with the relative LC3 content in single fibres in the intermediate- and high-velocity groups in a quantitative, statistically significant manner. 4. Three isoforms of fast-type troponin T were identified among the intermediate- and high-velocity fibres. These fibres also contained fast-type troponin C and troponin I. As was the case with the relative LC3 content, these fibres also formed a continuum with respect to the relative proportions of the three isoforms of fast-type troponin T. It appears that different isoforms of troponin T are responsible for a slightly higher Ca2+ sensitivity of tension development in the high-velocity fibres compared to the intermediate fibres. The continuum in troponin T isoform composition paralleled an increase in Vmax among these fibres. 5. The low-velocity fibres had the highest Ca2+ sensitivity of the three groups and had exclusively the slow-type isoforms of the regulatory proteins in the troponin complex. 6. The co-ordinated variations in troponin T and LC3 compositions among the intermediate- and high-velocity fibres are discussed as a possible means for the further differentiation of the contractile properties of the fibres in these two groups, beyond that provided by myosin heavy chain isoforms alone.

Animals↗

PubMed1988

Functional significance of myosin transitions in single fibers of developing soleus muscle.

The maximal velocity of shortening and myosin heavy chain (MHC) composition of single, chemically skinned fibers from neonatal and adult rat soleus muscles were examined to determine the relationship between these parameters during slow muscle development in the rat. In addition, the MHC composition of bundles of fibers from soleus muscles at the same ages was studied. The MHC compositions were examined using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The results from the bundles of fibers indicate that from 3 days to 5 mo postnatal, the rat soleus contains predominantly MHCs that migrate in the vicinity of the MHC from adult slow muscle. From 14 days to 2 mo postnatal, there are also significant amounts of additional MHCs that comigrate on SDS gels with those characteristic of adult rat fast muscle. All the fibers studied at 3 and 7 days postnatal and at 5 mo and the majority of fibers from 14 days to 2 mo postnatal had relatively low shortening velocities. A few fibers from the latter group had significantly higher velocities. The faster fibers at each age had greater amounts of the MHCs that comigrate with the adult fast-type MHC on SDS gels. Thus the velocity of shortening of single fibers from the rat soleus muscle appears to be related to MHC composition during postnatal development.

Animals↗

PubMed1988

Rat and avian myofibers having similar innervation share antigenic determinants.

The antigenic relationship between extrafusal myofiber types of avian and rat muscles was investigated. Antibodies specific for myosin heavy-chain isozymes of chicken fast-twitch myofibers (anti-twitch) or chicken slow-tonic myofibers (anti-tonic) were used in the immunohistochemical examination of embryonic and adult rat muscle. Anti-twitch antibodies reacted with chicken fast-twitch myofibers, with all extrafusal myofibers of adult rat and with embryonic rat myofibers. Anti-tonic antibodies reacted with chicken slow-tonic myofibers and with embryonic rat myofibers, but not with any extrafusal myofibers of adult rat. All intrafusal myofibers of adult rat reacted with both antibodies. However, individual intrafusal myofibers reacted most strongly with one or the other of the antibodies and weakly with the remaining antibody, thereby segregating the intrafusal myofibers into two classes. These results indicate shared antigenic determinants between the myosin heavy chains of avian and rat twitch myofibers and between the myosin heavy chains of avian tonic myofibers and embryonic rat myofibers. Therefore, antigenically related myosin heavy-chain isozymes are present in avian and rat myofibers having similar patterns of innervation (twitch or tonic). This tonic vs twitch comparison presents a unique perspective for the immunochemical analysis of myosin isozymes, compared with the traditional division based on speed of contraction, slow or fast.

Animals↗

PubMed1987

Accumulation of newly synthesized myosin heavy chain during thyroxine-induced myofiber type transition.

Slow-tonic myofiber to fast-twitch myofiber conversion was induced in chickens by feeding thyroxine. Incorporation of newly synthesized myosin heavy chain (MHC) into myofibers and myofibrils was followed by immunofluorescence with antibodies specific for fast-twitch MHC and slow-tonic MHC. Presence of more than one type of myosin heavy chain was detected in thyroxine-induced transitional myofibers of chicken pectoralis. Myofibers undergoing a transition were histochemically identical to immunologically cross-reacting, transitional myofibers of normal anterior latissimus dorsi. Newly synthesized MHC appeared to be incorporated uniformly across the cross sectional area of transitional myofibers and incorporated homogeneously into each sarcomere of transitional myofibrils. These observations are consistent with a theory of continuous protein exchange between myofibrillar protein and a non-myofibrillar protein pool, such that every sarcomere of a myofibril, and every myofibril of a myofiber, would be turned over simultaneously.

Animals↗

PubMed1986

Isolation and characterization of a new 40-kilodalton protein from bovine cardiac muscle.

A new protein having a subunit weight of 40,000 has been purified from myosin-extracted bovine cardiac myofibrils. Its amino acid composition and isoelectric point are distinct from actin, eu-actinin, and a variety of sarcoplasmic proteins of similar size. Affinity-purified antibodies made to this protein only react with a single 40-kDa protein band from cardiac myofibrils on immunoblots. The anti-40-kDa protein also shows cross-reactivities with cardiac myofibrils from rabbits, rats, and chickens. Immunofluorescence studies demonstrate that the 40-kDa protein is localized at the Z-bands of cardiac myofibrils and at the intercalated discs. The antibody did not react with skeletal muscle myofibrils by immunofluorescence or immunoblotting. It appears that the 40-kDa protein may play a role in the strong attachments between adjacent myofibrils in cardiac muscle.

Actinin↗

PubMed1986

Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C.

Binding of Ca2+ to the troponin C (TnC) subunit of troponin is necessary for tension development in skeletal and cardiac muscles. Tension was measured in skinned fibers from rabbit skeletal muscle at various [Ca2+] before and after partial substitution of skeletal TnC with cardiac TnC. Following substitution, the tension-pCa relationship was altered in a manner consistent with the differences in the number of low-affinity Ca2+-binding sites on the two types of TnC and their affinities for Ca2+. The alterations in the tension-pCa relationship were for the most part reversed by reextraction of cardiac TnC and readdition of skeletal TnC into the fiber segments. These findings indicate that the type of TnC present plays an important role in determining the Ca2+ dependence of tension development in striated muscle.

Animals↗

PubMed1986

Effects of partial extraction of troponin complex upon the tension-pCa relation in rabbit skeletal muscle. Further evidence that tension development involves cooperative effects within the thin filament.

Partial extraction of troponin C (TnC) decreases the Ca2+ sensitivity of tension development in mammalian skinned muscle fibers (Moss, R. L., G. G. Giulian, and M. L. Greaser. 1985. Journal of General Physiology. 86:585), which suggests that Ca2+-activated tension development involves molecular cooperativity within the thin filament. This idea has been investigated further in the present study, in which Ca2+-insensitive activation of skinned fibers from rabbit psoas muscles was achieved by removing a small proportion of total troponin (Tn) complexes. Ca2+-activated isometric tension was measured at pCa values (i.e., -log[Ca2+]) between 6.7 and 4.5: (a) in control fiber segments, (b) in the same fibers after partial removal of Tn, and (c) after recombination of Tn. Tn removal was accomplished using contaminant protease activity found in preparations of LC2 from rabbit soleus muscle, and was quantitated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning densitometry. Partial Tn removal resulted in the development of a Ca2+-insensitive active tension, which varied in amount depending on the duration of the extraction, and concomitant decreases in maximal Ca2+-activated tensions. In addition, the tension-pCa relation was shifted to higher pCa values by as much as 0.3 pCa unit after Tn extraction. Readdition of Tn to the fiber segments resulted in the reduction of tension in the relaxing solution to control values and in the return of the tension-pCa relation to its original position. Thus, continuous Ca2+-insensitive activation of randomly spaced functional groups increased the Ca2+ sensitivity of tension development in the remaining functional groups along the thin filament. In addition, the variation in Ca2+-insensitive active tension as a function of Tn content after extraction suggests that only one-third to one-half of the functional groups within a thin filament need to be activated for complete disinhibition of that filament to be achieved.

Animals↗