Human Muscle Type Research Articles

Influence of sex and fiber type on the satellite cell pool in human skeletal muscle.

The repair, remodeling, and regeneration of myofibers are dependent on satellite cells (SCs), although, the distribution of SCs in different fiber types of human muscle remains inconclusive. There is also a paucity of research comparing muscle fiber characteristics in a sex-specific manner. Therefore, the aim of this study was to investigate fiber type-specific SC content in men and women. Muscle biopsies from vastus lateralis were collected from 64 young (mean age 27±5), moderately trained men (n=34) and women (n=30). SCs were identified by Pax7-staining together with immunofluorescent analyses of fiber type composition, fiber size, and myonuclei content. In a mixed population, comparable number of SCs was associated to type I and type II fibers (0.07±0.02 vs 0.07±0.02 SCs per fiber, respectively). However, unlike men, women displayed a fiber type-specific distribution, with SC content being lower in type II than type I fibers (P=.041). Sex-based differences were found specifically for type II fibers, where women displayed lower SC content compared to men (P<.001). In addition, positive correlations (r-values between 0.36-0.56) were found between SC content and type I and type II fiber size in men (P=.03 and P<.01, respectively), whereas similar relationships could not be detected in women. Sex-based differences were also noted for fiber type composition and fiber size, but not for myonuclei content. We hereby provide evidence for sex-based differences present at the myocellular level, which may have important implications when studying exercise- and training-induced myogenic responses in skeletal muscle.

Ether and Carbamate Derivatives of 3-quinuclidinol and 3- hydroxymethylquinuclidine: Synthesis and Evaluation as Nicotinic Ligands.

A lead quinuclidine-based nicotinic ligand (EQA) served as the basis for the design of novel compounds. A new series of 3-substituted quinuclidines was designed, synthesized and evaluated as nicotinic ligands. The goal was to improve affinity for nicotinic receptors in the CNS. Interatomic distance calculations were performed on the proposed compounds as well as, known nicotinic ligands. The proposed compounds were then synthesized, characterized and evaluated in in vitro assays as nicotinic receptor ligands. Compounds 9a and 9b were found to inhibit the specific binding of 3H-(S)-Nicotine with Ki values of 48 nM and 42 nM respectively, indicating high affinity interactions with the α4β2 subtype. Data suggest that several compounds act as partial agonists at CNS receptors with an efficacy between 28 and 40% and are potent partial activators of human muscle type receptors (α1β1γδ Emax= 80% that of 100 µM nicotine). Together these results indicate a partial agonism at muscle type receptors (ca. 40%) with no significant activation of rat ganglion-type receptors (α3β4*: asterisk indicates potential additional subunit that could partner to form the ganglionic receptor). The partial agonism inducing dopamine release from striatal synaptosomes (α4β2α6α4β2β3, and/or α6β2β3) suggest that these compounds may in addition be acting at the α4β2 and/or the α6β3* receptors. The partial agonists reported herein are interesting nicotinic ligands worthy of further investigation.

NMR Signal Assignments of Human Adenylate Kinase 1 (hAK1) and its R138A Mutant (hAK1R138A)

Adenylate kinase (AK) enzyme which acts as the catalyst of reversible high energy phosphorylation reaction between ATP and AMP which associate with energetic metabolism and nucleic acid synthesis and signal transmission. This enzyme has three distinct domains: Core, AMP binding domain (AMPbd) and Lid domain (LID). The primary role of AMPbd and LID is associated with conformational changes due to flexibility of two domains. Three dimensional structure of human AK1 has not been confirmed and various mutation experiments have been done to determine the active sites. In this study, AK1R138A which is changed arginine[138] of LID domain with alanine[138] was made and conducted with NMR experiments, backbone dynamics analysis and mo-lecular docking dynamic simulation to find the cause of structural change and substrate binding site. Synthetic human muscle type adenylate kinase 1 (hAK1) and its mutant (AK1R138A) were re-combinded with E. coli and expressed in M9 cell. Expressed proteins were purified and finally gained at 0.520 mM hAK1 and 0.252 mM AK1R138A. Multinuclear multidimensional NMR experiments including HNCA, HN(CO)CA, were conducted for amino acid sequence analysis and signal assignments of HSQC spectrum. Our chemical shift perturbation data is shown LID domain residues and around alanine[138] and per-turbation value(0.22ppm) of valine[179] is consid-ered as inter-communication effect with LID domain and the structural change between hAK1 and AK1R138A.

Evolution of Allosteric Citrate Binding Sites on 6-phosphofructo-1-kinase

As an important part of metabolism, metabolic flux through the glycolytic pathway is tightly regulated. The most complex control is exerted on 6-phosphofructo-1-kinase (PFK1) level; this control overrules the regulatory role of other allosteric enzymes. Among other effectors, citrate has been reported to play a vital role in the suppression of this enzyme's activity. In eukaryotes, amino acid residues forming the allosteric binding site for citrate are found both on the N- and the C-terminal region of the enzyme. These site has evolved from the phosphoenolpyruvate/ADP binding site of bacterial PFK1 due to the processes of duplication and tandem fusion of prokaryotic ancestor gene followed by the divergence of the catalytic and effector binding sites. Stricter inhibition of the PFK1 enzyme was needed during the evolution of multi-cellular organisms, and the most stringent control of PFK1 by citrate occurs in vertebrates. By substituting a single amino acid (K557R or K617A) as a component of the allosteric binding site in the C-terminal region of human muscle type PFK-M with a residue found in the corresponding site of a fungal enzyme, the inhibitory effect of citrate was attenuated. Moreover, the proteins carrying these single mutations enabled growth of E. coli transformants encoding mutated human PFK-M in a glucose-containing medium that did not support the growth of E. coli transformed with native human PFK-M. Substitution of another residue at the citrate-binding site (D591V) of human PFK-M resulted in the complete loss of activity. Detailed analyses revealed that the mutated PFK-M subunits formed dimers but were unable to associate into the active tetrameric holoenzyme. These results suggest that stricter control over glycolytic flux developed in metazoans, whose somatic cells are largely characterized by slow proliferation.

Characterization of human muscle type cofilin (CFL2) in normal and regenerating muscle.

Cofilins are actin binding proteins and regulate actin assembly in vivo. Numerous cofilin homologues have been characterized in various organisms including mammals. In mice, a ubiquitously expressed cofilin (CFL1) and a skeletal muscle specific cofilin (CFL2) have been described. In the present study, we identified and characterized a human CFL2 gene localized on chromosome 14, with high homology to murine CFL2. Furthermore, we provide evidence for differentially spliced CFL2 transcripts (CFL2a and CFL2b). CFL2b is expressed predominantly in human skeletal muscle and heart, while CFL2a is expressed in various tissues. Genetic defects of CFL2 were excluded for one human muscle disorder, the chromosome 14 linked distal myopathy MPD1, and shown to be only possible to be a rare cause of another, nemaline myopathy. In a mouse model of mechanically induced muscle damage the changes of cofilin expression were monitored during the first 10 days of regeneration, with dephosphorylated CFL2 being the major isoform at later stages of muscle regeneration. A similar predominance of dephosphorylated CFL2 was observed in chronically regenerating dystrophin-deficient muscles of Duchenne muscular dystrophy patients. Therefore, the CFL2 isoform may play an important role in normal muscle function and muscle regeneration.

Novel Expression of Equivocal Messages Containing Both Regions of Choline/Ethanolamine Kinase and Muscle Type Carnitine Palmitoyltransferase I

For characterization of the detailed gene structure of human muscle type carnitine palmitoyltransferase I (M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts. As a result, we found a cDNA clone containing a nucleotide sequence unexpected from the reported M-CPTI gene structure in the upstream region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase-beta (CK/EK-beta) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the CK/EK-beta gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of CK/EK-beta and M-CPTI. Furthermore, cDNAs containing both exons of these genes were detected by the polymerase chain reaction using the cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these results, the production and organization of these overlapping transcripts are discussed.

Purification and partial characterization of creatine kinase from electric organ of Electrophorus electricus (L.)

The present investigation deals with the purification and the partial characterization of the soluble creatine kinase (CK) isoenzyme, isolated from the electric organ electrocyte of Electrophorus electricus (L.). Purification was performed by precipitation of the enzyme in the crude extract with ammonium sulfate (80%). The precipitate obtained was analyzed on an ion exchange column of diethylaminoethyl cellulose-52 (DEAE) followed by gel filtration on Superose 12 in a Fast Protein Liquid Chromatography (FPLC) system. Electrophoretic mobility of the active peak confirmed previous results identifying the hybrid isoenzyme MB in the eletrocyte cytoplasm. Electrocyte CK is a dimeric enzyme with two identical subunits of approximately 40 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The sequence analysis of the N-terminal peptide (14 amino acids) of the 40 kDa subunit showed homology with other CK enzymes from electric fish ( Torpedo) and human muscle type CK.

The 5′-untranslated region of the human muscle acylphosphatase mRNA has an inhibitory effect on protein expression

The cDNA of the human muscle type acylphosphatase was isolated and characterized. The mRNA presents a very long 5′-untranslated region, covering the first half of the molecule: 175 bases of this part were cloned and prediction of the possible secondary structure showed that a very stable stem-loop structure could be formed in that region. Moreover, an additional AUG triplet was found upstream of the start codon of the protein, defining an open reading frame of 60 codons which overlapped that of acylphosphatase. The possible regulatory effect on translation of this part of the mRNA molecule was studied by means of transient transfection experiments: a 10-fold decrease in the expression of a reporter protein and a dramatic decrease in the corresponding mRNA was observed, due to the presence of the 5′-untranslated region of acylphosphatase mRNA. Mutagenesis of the upstream AUG triplet eliminated mRNA instability, leading to the hypothesis that the product of the upstream open reading frame could play a role in this mechanism.

Structural features of the gene encoding human muscle type carnitine palmitoyltransferase I

We isolated a human muscle type of carnitine palmitoyltransferase I (CPTI-M) genomic clone and determined its entire nucleotide sequence. By comparison of the nucleotide sequence of the genomic clone with that of cDNA, we determined the intron/exon junctions. For detection of the exon(s) in the 5′-region of the CPTI-M gene, we isolated cDNA clones corresponding to the 5′-region of its transcript by 5′-rapid amplification of cDNA ends (5′-RACE method). Results showed two alternative exons, 1A and 1B, that do not encode amino acids in the 5′-region of the human CPTI-M gene. The gene encoding human CPTI-M was found to consist of two 5′-non-coding exons, 18 coding exons and one 3′-non-coding exon spanning approximately 10 kbp. Furthermore, on analysis of the 5′-flanking region, a putative gene encoding a `choline kinase homologue' was found to be located only about 300 bp upstream from exon 1A of the human CPTI-M gene. Comparison of the gene structure of human CPTI-M with the reported partial gene structure of human liver type CPTI (CPTI-L) showed that the intron insertion sites were completely conserved in these two genes.

Isolation and characterization of cDNA and genomic clones encoding human muscle type carnitine palmitoyltransferase I

With a cDNA probe encoding rat muscle type carnitine palmitoyltransferase I (CPTI), we isolated cDNA and genomic clones encoding the human homologue and deduced the primary structure of human muscle type CPTI. By Northern analysis, we confirmed the dominant expression of this isoform in heart and skeletal muscle.

Histoenzymatic profile of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord.

We examined histoenzymatic characteristics of human muscle fibers grown in monolayer culture and innervated de novo in culture for 60-90 days by fetal rat spinal cord neurons. Serial cryostat cross sections were obtained using a freshly frozen sandwich of adult rat muscle and cultured human muscle. An advanced degree of morphologic and histoenzymatic maturation of cultured human muscle was reached after innervation. In contrast to aneurally cultured human muscle fibers, the innervated muscle fibers were smaller in diameter and had myonuclei preferentially located at the periphery of the fiber. The innervated fibers contained a well-developed intermyofibrillar network revealed by the NADH-TR and SDH reactions. Phosphorylase activity was strong to moderate in most muscle fibers. Although most of the innervated cultured muscle fibers were still not fully differentiated into two histochemical fiber types because they had strong ATPase activity after both alkaline and acid preincubation, a few of them had an ATPase profile similar to type 2 fibers in human adult muscle and had reciprocal staining with phosphorylase and NADH-TR reactions. This is the first evidence of differentiation into different histochemical fiber types of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord.

Degradation of branched-chain amino acids and 2-oxo acids in human and rat muscle

Studies were made at physiological substrate concentrations on the transamination and oxidative decarboxylation of branched-chain amino acids and on the oxidation of branched-chain 2-oxo acids in homogenates, 600 g supernatants, and mitochondria of various types of human skeletal muscle and of rat m. quadriceps and liver. l-Carnitine increased the oxidation of 4-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate in mitochondria of human muscle, but to a lesser degree than it did in mitochondria of rat muscle. It had no effect on the oxidation of 3-methyl-2-oxopentanoate. Half-maximal rates of α-decarboxylation of the 2-oxo acids were observed at similar concentrations in muscle mitochondria of man and rat, but the maximal rates were much higher in rat muscle mitochondria. Rates of transamination of valine and leucine were about similar in four types of human muscle, but lower than those in rat m. quadriceps. The oxidation rate of the branched-chain 2-oxo acids appears to vary for the different types of human muscle, but is markedly lower than that in rat muscle. In homogenates of human muscle CO 2 was only produced from 4-methyl-2-oxopentanoate by oxidative decarboxylation and the oxidation appears to proceed no further.

Cytochrome c oxidase activity and fatty acid oxidation in various types of human muscle

Cytochrome c oxidase activity, carnitine concentration and oxidation rates of pyruvate and palmitate were determined in homogenates of various types of human skeletal muscle. Cytochrome c oxidase activity appeared to be closely related to the pyruvate oxidation rate, but its correlation with palmitate oxidation was less distinct. Trunk muscles oxidize less palmitate and have a lower cytochrome c oxidase activity per mg homogenate protein than leg muscles; soleus muscle biopsies showed higher activities than those of other leg muscles. Based on cytochrome c oxidase activity no large differences are found in palmitate oxidation rate between various types of human muscle. Cytochrome c oxidase activity and palmitate oxidation rate of muscles do not show an age dependency. The carnitine concentration is similar in all kinds of human skeletal muscle.

Diagnosis of cancer by radioimmunoassay of muscle type aldolase.

A specific radioimmunoassay was developed for the quantification of human muscle type aldolase (M-ALD) in human serum. The method utilized the double antibody radioimmunoassay technique using radioiodinated M-ALD, chicken antibody to M-ALD and rabbit antibody to chicken IgG. The serum M-ALD values of 41 normal healthy subjects ranged from 130 to 210 ng/ml, average 171 +/- 39 ng/ml. In 76 noncancer hospital patients, excluding patients with muscle diseases, the serum M-ALD values ranged from 125 to 220 ng/ml, average 164 +/- 52 ng/ml. In contrast, 129 cancer patients had high serum M-ALD values of 586 +/- 926 ng/ml, ranging from 85 to 5,800 ng/ml. Eighty-six percent of the cancer patients showed serum M-ALD concentrations over the normal range. The measurement of M-ALD values in the serum of patients by radioimmunoassay may be a valuable laboratory test in the diagnosis and follow-up of cancer patients.

Studies on muscular dystrophy: A comparison by physical and chemical means of the normal human ATP-creatine transphosphorylases (creatine kinases) with those from tissues of duchenne muscular dystrophy

The four human Duchenne dystrophic isoenzymes (M-M, M-B, B-B, from the muscle and B-B from the brain) of ATP-creatine transphosphorylase (S. A. Kuby, H. J. Keutel, K. Okabe, H. K. Jacobs, F. Ziter, D. Gerber, and F. H. Tyler, 1977, J. Biol. Chem. 252, 8382–8390) have now been compared physically and chemically with their normal human counterparts ( viz., with the three isoenzymes, M-M, M-B, B-B, 2). All isoenzymes proved to be composed of two noncovalently linked polypeptide chains, by sedimentation equilibrium analyses in the presence and absence of disruptive agents. In the presence of 2-mercaptoethanol at 0.16(Γ/2), pH 7.8, the two native muscle types yielded identical values for s 20,w, concentration dependencies, and molecular weight, and similarly for the brain types (from the brain). But the human brain type proved to be slightly heavier than the muscle type ( viz. 88,400 vs 85,900). All of the isoenzymes showed similar electrophoretic behavior between their several counterparts between pH 5–8, except perhaps between pH 8–10, where small differences appeared. The three native normal human isoenzymes, as well as the dystrophic human isoenzymes (M-M from the muscle and B-B from the brain) all contain 2 reactive sulfhydryl groups per mole or 1 per polypeptide chain of these two-chain proteins, which may be titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs 2); and under acidic conditions, quantitative titrations with 4,4′-dithiodipyridine yield a total of 10 -SH groups per mole of each brain type and 8 -SH groups per mole of muscle type, in the case of man, dystrophic man, calf, and rabbit. The kinetics of reactions between Nbs 2 and the sulfhydryl groups of all three normal human isoenzymes and two dystrophic human isoenzymes have been measured under several sets of denaturing conditions. A comparison of their reactive calculated second-order velocity constants reveal significant differences between these three normal human isoenzymes, but the k second order values for the reactions of the sulfhydryl groups of the dystrophic M-M and B-B with Nbs 2, when compared with their normal counterparts, gave identical values in the presence of 7.3 m urea or 1.8% laurylsulfate, from which it may be inferred that very similar, if not identical, environments surround these two sets of sulfhydryl groups. A comparison of the amino acid compositions of the normal human muscle type and brain type with the human dystrophic M-M and B-B (from the brain) reveal essentially identical values for the muscle types but nearly identical values for the brain types, with a few differences. Their respective tryptic peptide maps have been compared of the S-carboxy-methylated proteins (alkylated with iodo[2- 14C]acetic acid at the two exposed -SH groups per mole). Thus, the muscle types, normal and dystrophic, yield identical maps, but the brain types nearly identical maps, with a few significant differences. Isolation of the tryptic tridecapeptide from the S-carboxymethylated normal human and dystrophic human dimeric muscle-type ATP-creatine transphosphorylases, labeled at the single exposed SH group per polypeptide chain with iodo[2- 14C]acetate, yielded the following sequence for both proteins: ValLeuThrCys(CH 2COOH)ProSerAsnLeuGlyThr GlyLeuArg [where Cys(CH 2COOH) is S-carboxymethyl cysteine]. This sequence showed remarkable homology with a few other equivalent peptides reported to be derived from the exposed SH group of other ATP-creatine transphosphorylases. In conclusion, there does not appear to be a mutation in the structural genes for the muscle-type creatine kinases detectable by the analyses presented here. However, the brain types warrant further investigation.

Studies on adenosine triphosphate transphosphorylases: Isolation and several properties of the crystalline calf ATM-AMP transphosphorylases (adenylate kinases) from muscle and liver and some observations on the rabbit muscle adenylate kinase

Detailed procedures are described for the large-scale isolation of crystalline calf muscle and calf liver adenylate kinase isoenzymes applicable to frozen tissue. Proposed modifications in the large-scale isolation of the crystalline rabbit muscle adenylate kinase are also described. Although a great deal of homology exists between the muscle types, calf and rabbit, as revealed by their amino acid compositions (differing in only 10 out of 193 residues), the calf muscle adenylate kinase acts as a potent antigen in the rabbit. A description for the purification (ca. 600-fold) from the rabbit antiserum of the anti-enzyme globulin is given. The anti-enzyme shows a remarkable specificity in that it is largely unreactive as an inhibitor toward the calf liver-type enzyme, but is a powerful inhibitor of the calf muscle type (or human muscle type) with a kinetically evaluated dissociation constant of ca. 3 × 10 −14 m 2 (for the case of a stoichiometry of 2 Ag to 1 Ab), a second-order k ⋍ 5 × 10 6 M −1 · min −1 at 30 ° C , and an Arrhenius energy of activation of ca. 6.3 kcal/mol. Dodecyl sulfate polyacrylamide gel electrophoresis revealed only a single heavy chain ( M r, ~42,000) and a single light chain ( M r, ~25,000) of the antibody, which together with studies employing 3.5% stacking gels lead to a molecular weight of approximately 134,000 for the native antibody. Similarly, by dodecyl sulfate polyacrylamide gel electrophoreses, only single bands were obtained for the calf liver and rabbit muscle adenylate kinase ( M r, ca. 21,000–23,000) and the calf liver adenylate kinase ( M r, ca. 25,000). Sedimentation velocity of the calf muscle-type and calf liver-type proteins yielded single sedimenting boundaries, with s 0 20,w ⋍ 2.1 6 and 2.3 7 S, respectively. Estimates of the molecular weights of the native structure of the calf isoenzymes by sedimentation equilibrium gave M ̄ w = 21,200 ± 200 for the calf muscle enzyme and 25,600 ± 200 for the calf liver adenylate kinase. Electrophoresis by liquid boundary of the rabbit muscle enzyme at protein concentrations of 5–8 mg/ml resulted in a nonideal system, as a result largely of charge effects and interactions with buffer components. Similarly, isoelectric points determined by isoelectric focusing were very dependent on the protein load, but could be extrapolated to zero protein to approximately p I 0 of 10.6. However, electrophoresis on cellulose acetate at microgram to nanogram levels of protein under conditions where electroendosmosis seemed absent did permit an accurate estimate of the p I 0 by a plot of p I app vs ( Γ 2 ) 1 2 to a p I 0 = 10.6 0. This value is now in good agreement with an estimate made earlier from its amino acid composition (T. A. Mahowald et al., 1962, J. Biol. Chem. 237, 1138–1145). By this technique, the p I app values at 0.05 ( Γ 2 ) for the calf isoenzymes were estimated and found to differ only slightly (viz., 9.6 for the liver adenylate kinase vs 10.1 for the muscle myokinase). While inhibition of the rabbit and calf muscle adenylate kinases (which are apparently cytoplasmic enzymes) by Ap 5A [p 1,p 5-di(adenosine-5′)pentaphosphate] was very significant at even 10 −8 m and similar to that previously reported for the rabbit muscle adenylate kinase ( Lienhard and Secemski, 1973, J. Biol. Chem. 248, 1121–1123), inhibition of the liver-type adenylate kinase (which is apparently a mitochondrial enzyme) required almost 10 −6 m (Ap 5A) for similar percentage inhibitions. For the forward reactions (i.e., MgATP 2− + AMP 2− →), Ap 5A acts as a competitive inhibitor with respect to either substrate with K i ⋍ (0.5−1) × 10 −8 M for both of the muscle enzymes, but yields a K i ⋍ (3−8) × 10 −7 M for the liver enzyme. However, in the reverse direction (MgADP − + ADP 3− →), inhibition by Ap 5A appears to be noncompetitive with respect to either substrate, with a K i ⋍ (0.6−2) × 10 −8 M for the muscle enzymes, but only (3–4) × 10 −6 m for the liver enzyme. Thus, although Ap 5A may act in part as a transition state analog, the explanation for its “multisubstrate inhibition” (Lienhard and Secemski, see above) may also lie in the structure of its metal chelates, e.g., of Mg 2(Ap 5A) −.