Myosin heavy chain turnover during cardiac mass changes by glucocorticoids.

One aim of this investigation was to determine whether the cardiac enlargement observed with glucocorticoid treatment is temporary or remains a permanent adaptation if steroid treatment is prolonged. A second aim was to study whether myosin heavy chain (MHC) synthesis rates are coordinated with the cardiac mass responses. Female rats received either a vehicle (1% aqueous carboxymethyl cellulose in saline) or hydrocortisone 21-acetate for 1, 3, 7, 11, and 15 days. Peak cardiac enlargement (10-15%) was observed after 7 days of hormone treatment in two separate series of experiments. The enlargement was maintained through 11 days of steroid injections but by 15 days had declined toward control levels. MHC synthesis measurements were performed by constant infusion of [3H]leucine. Leucine specific activities were similar among precursor pools (intracellular, extracellular, and leucyl-tRNA) and did not vary with steroid treatments. Fractional synthesis rates of ventricular MHC (%/day) did not change during the period of increase in ventricular mass but were reduced to 56-59% of controls (-11/19.5) at 7 and 11 days of treatment, when ventricular mass increases were highest. MHC breakdown (%/day) was reduced to approximately 60% (-11.5/18.7) of controls at 7 and 11 days. Changes in total protein synthesis, which was measured in isolated perfused hearts, were similar to the MHC responses and indicated that the alterations in MHC synthesis are synchronized with the hormonal effects on total protein metabolism. These results demonstrate that peak cardiac enlargement is not maintained with long-term glucocorticoid treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myosin heavy chain isoforms in postnatal muscle development of mice

In this study, using a high-resolution gel electrophoresis technique, we have characterized the myosin heavy chain composition in different skeletal muscle of the mouse during postnatal development. The pattern of myosin heavy chain expression was studied in four hind limb muscles, the diaphragm, the tongue and the masseter. All of these muscles displayed the usual sequential transitions from embryonic to neonatal and to adult myosin heavy chain isoforms but more interestingly these transitions occur with a distinct chronology in the different muscles. In addition, our results demonstrated a transitory pattern of expression for certain adult myosin heavy chain isoforms in the soleus and the tongue. In the soleus muscle IIB and in the tongue IIA myosin heavy chain isoforms were detected only for a short time during postnatal life. Our results demonstrate that muscles of the mouse with different functions are subjected to a distinct programs of myosin isoform transitions during postnatal muscle development. This study describes new data which will help us to understand both postnatal muscle development in transgenic mouse muscles as well as in muscle pathology.     

Myosin heavy chain isoform composition in striated muscle after denervation and self-reinnervation

The total content of myosin heavy chains (MHC) and their isoform pattern were studied by biochemical methods in the slow-twitch (soleus) and fast-twitch (extensor digitorum longus) muscles of adult rat during atrophy after denervation and recovery after self-reinnervation. The pattern of fibre types, in terms of ultrastructure, was studied in parallel. After denervation, total MHC content decreased sooner in the slow-twitch muscle than in the fast-twitch. The ratio of MHC-1 and the MHC-2B isoforms to the MHC-2A isoform decreased in the slow and the fast denervated muscles, respectively. After reinnervation of the slow muscle, the normal pattern of MHC recovered within 10 days and the type 1 isoform increased above the normal. In the reinnervated fast muscle, the 2B/2A isoform ratio continued to decrease. Traces of the embryonic MHC isoform, identified by immunochemistry, were found in both denervated and reinnervated slow and fast muscles. A shift in fibre types was similar to that found in the MHC isoforms. Within 2 months of recovery a tendency to normalization was observed. The results show that (a) MHC-2B isoform and the morphological characteristics of the 2B-type muscle fibres are susceptible to lack of innervation, similar to those of type 1, (b) during muscle recovery induced by reinnervation the MHC isoforms and muscle fibres shift transiently to type 1 in the soleus and to type 2A in the extensor digitorum longus muscles, and (c) the embryonic isoform of MHC may appear in the adult skeletal muscles if innervation is disturbed.     

Myosin heavy chain isoform diversity in smooth muscle is produced by differential RNA processing

We have isolated and characterized two distinct myosin heavy chain cDNA clones from a neonatal rat aorta cDNA library. These clones encode part of the light meromyosin region and the carboxyl terminus of smooth muscle myosin heavy chain. The two rat aorta cDNA clones were identical in their 5' coding sequence but diverged at the 3' coding and in a portion of the 3' untranslated regions. One cDNA clone, RAMHC21, encoded 43 unique amino acids from the point of divergence of the two cDNAs. The second cDNA clone, RAMHC 15, encoded a shorter carboxyl terminus of nine unique amino acids and was the result of a 39 nucleotide insertion. This extra nucleotide sequence was not present in RAMHC21. The rest of the 3' untranslated sequences were common to both cDNA clones. Genomic cloning and DNA sequence analysis demonstrated that an exon specifying the 39 nucleotides unique to RAMHC15 mRNA was present, together with the 5' upstream common exons in the same contiguous stretch of genomic DNA. The 39 nucleotide exon is flanked on either side by two relatively large introns of approximately 2600 and 2700 bases in size. RNase protection analysis indicated that the two corresponding mRNAs were coexpressed in both vascular and non-vascular smooth muscle tissues. This is the first demonstration of alternative RNA processing in a vertebrate myosin heavy chain gene and provides a novel mechanism for generating myosin heavy chain protein diversity in smooth muscle tissues.     

Myosin turnover in cultured muscle fibers relaxed by tetrodotoxin

It is possible to maintain chick embryo muscle fibers in culture for several weeks. During this time the fibers mature and undergo spontaneous contractions. Contractions may be abolished by tetrodotoxin. When normal and tetrodotoxin (TTX) cultures are compared for myosin accumulation it is found that the relaxed or tetrodotoxin-treated cultures fail to accumulate myosin after about 4 days. Rates of myosin synthesis in normal and relaxed cultures are, however, identical. Failure to accumulate myosin in relaxed cultures is associated with a 30–40% increase in the rate of turnover of myosin heavy chains compared with normal, contracting cultures. It is suggested that one basis for hypotrophy is to be found in a post-translational mechanism for regulating myosin heavy chain (MHC) turnover.     

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle

Summary Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).     

Myosin heavy chain composition of muscle spindles in human biceps brachii.

Data on the myosin heavy chain (MyHC) composition of human muscle spindles are scarce in spite of the well-known correlation between MyHC composition and functional properties of skeletal muscle fibers. The MyHC composition of intrafusal fibers from 36 spindles of human biceps brachii muscle was studied in detail by immunocytochemistry with a large battery of antibodies. The MyHC content of isolated muscle spindles was assessed with SDS-PAGE and immunoblots. Four major MyHC isoforms (MyHCI, IIa, embryonic, and intrafusal) were detected with SDS-PAGE. Immunocytochemistry revealed very complex staining patterns for each intrafusal fiber type. The bag(1) fibers contained slow tonic MyHC along their entire fiber length and MyHCI, alpha-cardiac, embryonic, and fetal isoforms along a variable part of their length. The bag(2) fibers contained MyHC slow tonic, I, alpha-cardiac, embryonic, and fetal isoforms with regional variations. Chain fibers contained MyHCIIa, embryonic, and fetal isoforms throughout the fiber, and MyHCIIx at least in the juxtaequatorial region. Virtually each muscle spindle had a different allotment of numbers of bag(1), bag(2) and chain fibers. Taken together, the complexity in intrafusal fiber content and MyHC composition observed indicate that each muscle spindle in the human biceps has a unique identity.     

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle

Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).     

Myosin heavy chain composition of single fibres from normal human muscle.

Electrophoretic analysis in the presence of 33% glycerol of purified myosin from normal human muscle shows three distinct protein bands which are identified as type 1, 2B, and 2A myosin heavy chain (MHC) isoforms by affinity-purified polyclonal antibodies. Analysis of MHC of single human muscle fibres shows that human muscles contain a large population of fibres showing the coexistence of type 2A and 2B MHC.     

Myosin heavy chain isoforms regulate muscle function but not myofibril assembly.

Myosin heavy chain (MHC) is the motor protein of muscle thick filaments. Most organisms produce many muscle MHC isoforms with temporally and spatially regulated expression patterns. This suggests that isoforms of MHC have different characteristics necessary for defining specific muscle properties. The single Drosophila muscle Mhc gene yields various isoforms as a result of alternative RNA splicing. To determine whether this multiplicity of MHC isoforms is critical to myofibril assembly and function, we introduced a gene encoding only an embryonic MHC into Drosophila melanogaster. The embryonic transgene acts in a dominant antimorphic manner to disrupt flight muscle function. The transgene was genetically crossed into an MHC null background. Unexpectedly, transformed flies expressing only the embryonic isoform are viable. Adult muscles containing embryonic MHC assemble normally, indicating that the isoform of MHC does not determine the dramatic ultrastructural variation among different muscle types. However, transformed flies are flightless and show reduced jumping and mating ability. Their indirect flight muscle myofibrils progressively deteriorate. Our data show that the proper MHC isoform is critical for specialized muscle function and myofibril stability.     

Myosin heavy chain composition in the rat diaphragm: effect of age and exercise training.

Increases in aerobic capacity in both young and senescent rats consequent to endurance exercise training are now known to occur not only in locomotor skeletal muscle but also in diaphragm. In the current study the effects of aging and exercise training on the myosin heavy chain (MHC) composition were determined in both the costal and crural diaphragm regions of female Fischer 344 rats. Exercise training [treadmill running at 75% maximal oxygen consumption (1 h/day, 5 day/wk, x 10 wk)] resulted in similar increases in plantaris muscle citrate synthase activity in both young (5 mo) and old (23 mo) trained animals (P < 0.05). Computerized densitometric image analysis of fast and slow MHC bands revealed the ratio of fast to slow MHC to be significantly higher (P < 0.005) in the crural compared with costal diaphragm region in both age groups. In addition, a significant age-related increase (P < 0.05) in percentage of slow MHC was observed in both diaphragm regions. However, exercise training failed to change the relative proportion of slow MHC in either the costal or crural region.     

Myosin heavy chain pattern in the Akhal-Teke horses

This study investigates the myosin heavy chain (MyHC) isoform composition in the gluteus medius muscle of the Akhal-Teke horses using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Fifteen horses aged between 1.5 and 23.5 years were used in this study and divided into three age groups: 1.5 to 4 (n = 6), 9 to 13 (n = 5) and 18.5 to 23.5 years (n = 4). The average content of the MyHC I isoform was 11.72 ± 1.07% (variation between individuals: 7.09% to 20.14%). The relative content of the MyHC IIa and IIx isoforms was subsequently 38.20 ± 1.46% (30.73% to 48.78%) and 50.07 ± 1.10% (43.8% to 56.78%) from the total MyHC. The MyHC pattern in the skeletal muscles of the Akhal-Teke horses shows that the muscles of these horses have a high capacity both for endurance and speed.     

Myosin alkali light chain and heavy chain variations correlate with altered shortening velocity of isolated skeletal muscle fibers

This study examines the myosin isozyme heterogeneity (in terms of both alkali light chains and myosin heavy chains) among skeletal muscle fibers of the rabbit and correlates these isozyme differences with the differences in a contractile property, the velocity of unloaded shortening, of the fibers. The mean velocities of unloaded shortening (pCa 4.3; 12 degrees C) were as follows: psoas IIb fibers, 2.07 fiber lengths/s (n = 25); tibialis anterior (IIb) fibers, 1.63 fiber lengths/s (n = 18); vastus intermedius IIa fibers, 0.98 fiber lengths/s (n = 15); fibers (IIa) from chronically stimulated tibialis anterior, 0.86 fiber lengths/s (n = 16). Peptide maps of the myosins showed that the myosin heavy chains of the two groups of IIb fibers were indistinguishable from each other, but different from the heavy chains of the IIa fibers. However, the difference in maximal shortening velocity of the two groups of IIb fibers was correlated with a difference in the alkali light chain ratio deduced from the intensity ratio of myosin isoforms separated by gel electrophoresis under nondenaturing conditions. The vastus intermedius (IIa) and chronically stimulated tibialis anterior (IIa) fibers were indistinguishable in terms of either velocities of unloaded shortening or myosin isozyme contents. Soleus fibers contained only slow-twitch myosin. Thus, among fibers that contained a variety of myosin isozymes, differences in shortening velocities were correlated with the alkali light chain ratio, myosin heavy chain type, or a combination of both.     

Myosin heavy chain expression in embryonic cardiac cell cultures

Chick embryonic heart cell isolates and monolayer cultures were prepared from atria and ventricles at selected stages of cardiac development. The cardiac myocytes were assayed for myosin heavy chain (MHC) content using monoclonal antibodies (McAbs) specific in the heart for atrial (B-1), ventricular (ALD-19), or conductive system (ALD-58) isoforms. Using immunofluorescence microscopy or radioimmunoassay, MHC accumulation was measured before plating and at 48 hr or 7 days in culture. Reproducible changes in MHC antigenicity were observed by 7 days in both atrial and ventricular cultures. The changes were stage dependent and tissue specific but generally resulted in a decreased reactivity with the tissue specific MHC McAbs. In addition, the isoform recognized by ALD-58, characteristic of the conductive system cells in vivo, was never present in cultured myocytes. These results indicate that MHC isoforms produced in vivo may be replaced in monolayer cultures by an isoform(s) not recognized by our tissue specific MHC McAbs. This suggests that the intrinsic program of cardiac myogenesis, within cardiac myocytes, may not be sufficient to establish and maintain differential expression of tissue specific MHC in monolayer cell culture.     

Myosin light chain phosphorylation in intact human muscle

The phosphate content of the fast (LC2F) and two slow (LC2S and LC2S1) phosphorylatable light chains (P-light chains) in myosin isolated from biopsy samples of rested human vastus lateralis muscle averaged 0.21, 0.28 and 0.25 mol of phosphate per mol of P-light chain, respectively. Following a 10 s maximal contraction, phosphate content was increased by almost 2-fold in the fast and two slow P-light chains. After prolonged, moderate cycling activity phosphate content was only slightly increased in the three P-light chains. These data suggest that, unlike animal skeletal muscle, myosin light chain kinase and phosphatase activities are similar in human fast and slow muscle fibres.