Characterization of human carbonic anhydrase III from skeletal muscle

A third form of human carbonic anhydrase (CA III), found at high concentrations in skeletal muscle, has been purified and characterized. This isozyme shows relatively poor hydratase and esterase activities compared to the red cell isozymes, CA I and CA II, but is similar to these isozymes in subunit structure (monomer) and molecular size (28,000). CA III is liable to posttranslational modification by thiol group interaction. Monomeric secondary isozymes, sensitive to beta-mercaptoethanol, are found in both crude and purified material and can be generated in vitro by the addition of thiol reagents. Active dimeric isozymes, generated apparently by the formation of intermolecular disulfide bridges, also occur but account for only a small proportion of the total protein and appear only when the concentration of CA III is particularly high.     

Characterization of primary human skeletal muscle cells from multiple commercial sources

There is a significant unmet need for safe, anabolic muscle therapies to treat diseases and conditions associated with severe muscle weakness and frailty. The identification of such therapies requires appropriate cell-based screening assays to select compounds for further development using animal models. Primary human skeletal muscle cells have recently become available from a number of commercial vendors. Such cells may be valuable for studying the mechanisms that direct muscle differentiation, and for identifying and characterizing novel therapeutic approaches for the treatment of age- and injury-induced muscle disorders. However, only limited characterization of these cells has been reported to date. Therefore, we have examined four primary human muscle cell preparations from three different vendors for their capacity to differentiate into multinucleated myotubes. Two of the preparations demonstrated robust myotube formation and expressed characteristic markers of muscle differentiation. Furthermore, these myotubes could be induced to undergo morphological atrophy- and hypertrophy-like responses, and atrophy could be blocked with an inhibitor of myostatin signaling, a pathway that is known to negatively regulate muscle mass. Finally, the myotubes were efficiently infected with recombinant adenovirus, providing a tool for genetic modification. Taken together, our results indicate that primary human muscle cells can be a useful system for studying muscle differentiation, and may also provide tools for studying new therapeutic molecules for the treatment of muscle disease.     

Leptin receptors in human skeletal muscle.

Human skeletal muscle expresses leptin receptor mRNA; however, it remains unknown whether leptin receptors (OB-R) are also expressed at the protein level. Fourteen healthy men (age = 33.1 +/- 2.0 yr, height = 175.9 +/- 1.7 cm, body mass = 81.2 +/- 3.8 kg, body fat = 22.5 +/- 1.9%; means +/- SE) participated in this investigation. The expression of OB-R protein was determined in skeletal muscle, subcutaneous adipose tissue, and hypothalamus using a polyclonal rabbit anti-human leptin receptor. Three bands with a molecular mass close to 170, 128, and 98 kDa were identified by Western blot with the anti-OB-R antibody. All three bands were identified in skeletal muscle: the 98-kDa and 170-kDa bands were detected in hypothalamus, and the 98-kDa and 128-kDa bands were detected in thigh subcutaneous adipose tissue. The 128-kDa isoform was not detected in four subjects, whereas in the rest its occurrence was fully explained by the presence of intermuscular adipose tissue, as demonstrated using an anti-perilipin A antibody. No relationship was observed between the basal concentration of leptin in serum and the 170-kDa band density. In conclusion, a long isoform of the leptin receptor with a molecular mass close to 170 kDa is expressed at the protein level in human skeletal muscle. The amount of 170-kDa protein appears to be independent of the basal concentration of leptin in serum.     

Characterization of diverse forms of myosin heavy chain expressed in adult human skeletal muscle.

In an attempt to define myosin heavy chain (MHC) gene organization and expression in adult human skeletal muscle, we have isolated and characterized genomic sequences corresponding to different human sarcomeric MHC genes (1). In this report, we present the complete DNA sequence of two different adult human skeletal muscle MHC cDNA clones, one of which encodes the entire light meromyosin (LMM) segment of MHC and represents the longest described MHC cDNA sequence. Additionally, both clones provide new sequence data from a 228 amino acid segment of the MHC tail for which no protein or DNA sequence has been previously available. One clone encodes a "fast" form of skeletal muscle MHC while the other clone most closely resembles a MHC form described in rat cardiac ventricles. We show that the 3' untranslated region of skeletal MHC cDNAs are homologous from widely separated species as are cardiac MHC cDNAs. However, there is no homology between the 3' untranslated region of cardiac and skeletal muscle MHCs. Isotype-specific preservation of MHC 3' untranslated sequences during evolution suggests a functional role for these regions.     

Characterization of skeletal muscle insulin receptor

The insulin receptor from rat skeletal muscle was characterized. Treatment of muscle membranes with the photoactive insulin analog, 125I[N-epsilonB29-monoazidobenzoyl]-insulin revealed a single protein band of 135,000 Da, the alpha subunit. Iodination of total membrane protein followed by Triton X-100 solubilization and immunoprecipitation demonstrated the presence of a protein band of 90,000 Da, the beta subunit, together with a protein band of 190,000 Da, which may be the receptor precursor. In partially purified receptor preparations, the beta subunit exhibited dose-dependent, insulin-stimulated phosphorylation with incorporation of phosphate solely into tyrosine residues, which was also observed in the 190,000-Da receptor precursor. Purified plasma membranes contained a large amount of insulin-degrading activity which had to be inactivated prior to performing insulin-binding studies. If degradation of insulin was not prevented, apparent enhanced binding in the presence of unlabeled insulin was observed.     

Cytological differentiation of human fetal skeletal muscle.

The ultrastructural differentiation of several different muscles was investigated in human fetuses ranging in age from 13 weeks to neonatal. At approximately 16 weeks of gestation cell cluster containing both myotubes and satellite cells lie enclosed by a newly formed basal lamina and show evidence of fusion. The development of organelles is evident in myoblasts, proceeds as the cells transform into myofibers, and continues in the neonate. Filament synthesis occurs primarily in the cell periphery where thin filaments appear to align themselves in relations to parallel arrays of ribosome-studded thick filaments: Z line formation follows the appearance of thin filaments. Intermediate filaments, approximately 10-12 nm thick, were also consistently observed in perinuclear regions and distal to filament assembly. Although sarcoplasmic reticulum (SR) development is closely related to fibril formation, connections between Z lines and SR are not consistent, thus supporting the conclusion that SR does not evoke the formation of the Z line. Bristlecoated vesicles appear to be the precursors of elements of the SR, possibly the lateral sacs. Development of the transverse tubules, as invaginations of the sarcolemma, is closely associated with the formation of lateral sacs since the latter occur along the sarcolemma as soon as transverse tubules appear. Cytological differentiation is similar, though not identical, in several different muscles. During the last trimester muscle fibers show some evidence of diversity mainly of variation in Z line width. In gerneral the results suggest that the sequence and stages of human myogenesis are similar to those of other species.     

Myosin and actomyosin from human skeletal muscle.

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (+/-2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37 degrees C. Activation of the Mg-ATPase activity of Ca2+-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 muM Ca2+ concentration (CaEGTA binding constant equals 4.4 - 10(5) at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6-9 range, the Ca2+-ATPase activity of the subfragment 1 was 1.8- and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6-10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca2+-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca2+ is similar to that of rabbit fast or slow muscle.     

Early depression of Ankrd2 and Csrp3 mRNAs in the polyribosomal and whole tissue fractions in skeletal muscle with decreased voluntary running

The wheel-lock (WL) model for depressed ambulatory activity in rats has shown metabolic maladies ensuing within 53-173 h after WL begins. We sought to determine if WL beginning after 21-23 days of voluntary running in growing female Wistar rats affected the mRNA profile in the polyribosomal fraction from plantaris muscle shortly following WL. In experiment 1, WL occurred at 0200 and muscles were harvested at 0700 daily at 5 h (WL5h, n = 4), 29 h (WL29h, n = 4), or 53 h (WL53h, n = 4) after WL. Affymetrix Rat Gene 1.0 ST Arrays were used to test the initial question as to whether WL affects mRNA occupancy on skeletal muscle polyribosomes. Using a false discovery rate of 15%, no changes in mRNAs in the polyribosomal fraction were observed at WL29h and eight mRNAs (of over 8,200 identified targets) were altered at WL53h compared with WL5h. Interestingly, two of the six downregulated genes included ankyrin repeat domain 2 (Ankrd2) and cysteine-rich protein 3/muscle LIM protein (Csrp3), both of which encode mechanical stretch sensors and RT-PCR verified their WL-induced decline. In experiment 2, whole muscle mRNA and protein levels were analyzed for Ankrd2 and Csrp3 from the muscles of WL5h (4 original samples + 2 new), WL29h (4 original), WL53h (4 original + 2 new), as well as WL173 h (n = 6 new) and animals that never ran (SED, 4-5 new). Relative to WL5h controls, whole tissue Ankrd2 and Csrp3 mRNAs were lower (P < 0.05) at WL53h, WL173h, and SED; Ankrd2 protein tended to decrease at WL53h (P = 0.054) and Csrp3 protein was less in WL173h and SED rats (P < 0.05). In summary, unique early declines in Ankrd2 and Csrp3 mRNAs were identified with removal of voluntary running, which was subsequently followed by declines in Csrp3 protein levels during longer periods of wheel lock.     

In vivo specific tension of human skeletal muscle.

In this study, we estimated the specific tensions of soleus (Sol) and tibialis anterior (TA) muscles in six men. Joint moments were measured during maximum voluntary contraction (MVC) and during electrical stimulation. Moment arm lengths and muscle volumes were measured using magnetic resonance imaging, and pennation angles and fascicular lengths were measured using ultrasonography. Tendon and muscle forces were modeled. Two approaches were followed to estimate specific tension. First, muscle moments during electrical stimulation and moment arm lengths, fascicular lengths, and pennation angles during MVC were used (data set A). Then, MVC moments, moment arm lengths at rest, and cadaveric fascicular lengths and pennation angles were used (data set B). The use of data set B yielded the unrealistic specific tension estimates of 104 kN/m(2) in Sol and 658 kN/m(2) in TA. The use of data set A, however, yielded values of 150 and 155 kN/m(2) in Sol and TA, respectively, which agree with in vitro results from fiber type I-predominant muscles. In fact, both Sol and TA are such muscles. Our study demonstrates the feasibility of accurate in vivo estimates of human muscle intrinsic strength.     

Acoustic myography for investigating human skeletal muscle fatigue.

Sounds produced during voluntary isometric contractions of the quadriceps muscle were studied by acoustic myography (AMG) in five healthy adults. With the subject seated, isometric force, surface electromyography (EMG), and AMG were recorded over rectus femoris, and the EMG and AMG signals were integrated (IEMG and IAMG). Contractions lasting 5 s each were performed at 10, 25, 50, 60, 75, and 100% of maximum voluntary contraction (MVC) force. Fatigue was then induced by repeated voluntary contractions (10 s on, 10 s off) at 75% MVC until only 40% MVC could be sustained. After 15 min of rest, the different force levels were again tested in relation to the fresh MVC. Both before and after fatiguing activity the relationships between force and IEMG [r = 0.99 +/- 0.01 (SD), n = 10] and force and IAMG (r = 0.98 +/- 0.02) were linear. After activity, however, the slopes of the regression lines for force and IEMG increased (P less than 0.01) but those for force and IAMG remained the same (P greater than 0.05). The present results clarify the relationship between AMG and isometric force in fatigued muscle without the problem of fatigue-induced tremor, which hampered previous studies of prolonged activity. This study contributes to the validation of AMG and shows that it is a potentially useful method for noninvasive assessment of force production and fatigue. Further studies to establish the origin of AMG activity are required before AMG can be accepted for use in neuromuscular physiology or rehabilitation.     

Characterization of smooth muscle differentiation of purified human skeletal muscle‐derived cells

The purpose of this study is to characterize the smooth muscle differentiation of purified human muscle‐derived cells (hMDCs). The isolation and purification of hMDCs were conducted by modified preplate technique and Dynal CD34 cell selection. Smooth muscle cell differentiation was induced by the use of smooth muscle induction medium (SMIM) and low‐serum medium. The gene expressions at the mRNA and protein levels of undifferentiated and differentiated hMDCs were tested by RT‐PCR, Western blot and immunofluorescence studies. Western blot and immunofluorescence studies demonstrated the purified hMDCs cultured in SMIM for 4 weeks and expressed significant amount of smooth muscle myosin heavy chain (MHC) and α‐smooth muscle actin (ASMA). The cells cultured in low‐serum medium for 4 weeks also expressed ASMA, while the control group did not. RT‐PCR analysis showed increased gene expression of smooth muscle markers, such as ASMA, Calponin, SM22, Caldesmon, Smoothelin and MHC when purified hMDCs were exposed to SMIM for 2 and 4 weeks when compared to the controls. In conclusion, we confirmed the smooth muscle differentiation capability of purified hMDCs. The gene expression of smooth muscle differentiation of purified hMDCs was characterized. These cells may be potential biomaterials for human tissue regeneration.     

Lectin histochemistry of human skeletal muscle

Biotinyl derivatives of seven plant lectins-concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin I (RCA I), Ulex europeus agglutinin I (UEA I), soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), and wheat germ agglutinin (WGA)-were bound to cryostat sections of biopsied normal human muscle and visualized with avidin-horseradish peroxidase conjugates. A distinct staining pattern was observed with each lectin. The most general staining was observed with Con A, RCA I, and WGA, which permitted strong visualization of the plasmalemma-basement membrane unit, tubular profiles in the interior of muscle fibers, blood vessels, and connective tissue. PNA gave virtually no intracellular staining, while SBA and UEA I selectively stained blood vessels. DBA was unique in providing good visualization of myonuclei. In each case, lectin staining could be blocked by appropriate sugar inhibitors. Neuraminidase pretreatment of the cryostat sections altered the pattern of staining by all lectins except UEA I and Con A; staining with RCA I became stronger and that with WGA became less intense, while staining with PNA, SBA and DBA became stronger and more generalized, resembling that of RCA I. These effects of neuraminidase pretreatment are in conformity with the known structure of the oligosaccharide chains of membrane glycoproteins and specificities of the lectins involved.