Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles.

The objective of this study was to determine whether differences in pork tenderness and water-holding capacity could be explained by factors influencing calpain activity and proteolysis. Halothane-negative (HAL-1843 normal) Duroc pigs (n = 16) were slaughtered, and temperature and pH of the longissimus dorsi (LD), semimembranosus (SM), and psoas major (PM) were measured at 30 and 45 min and 1, 6, 12, and 24 h postmortem. Calpastatin activity; mu-calpain activity; and autolysis and proteolysis of titin, nebulin, desmin, and troponin-T were determined on muscle samples from the LD, SM, and PM at early times postmortem. Myofibrils from each muscle were purified to assess myofibril-bound (mu-calpain. Percentage drip loss was determined, and Warner-Bratzler shear (WBS) force was analyzed. Myosin heavy-chain (MHC) isoforms were examined using SDS-PAGE. The pH of PM was lower (P < 0.01) than the pH of LD and SM at 30 and 45 min and 1 h postmortem. The PM had a higher (P < 0.01) percentage of the MHC type IIa/IIx isoforms than the LD. The-LD had the greatest proportion of (P < 0.01) MHC IIb isoforms of any of the muscles. The PM had the lowest (P < 0.01) percentage of MHC IIb isoforms and a greater (P < 0.05) percentage of type I MHC isoforms than the LD and SM. The PM had less (P < 0.01) drip loss after 96 h of storage than the SM and LD. The PM had more desmin degradation (P < 0.01) than the LD and SM at 45 min and 6 h postmortem. Degradation of titin occurred earlier in the PM than the LD and SM. At 45 min postmortem, the PM consistently had some autolysis of mu-calpain, whereas the LD and SM did not. At 6 h postmortem, some autolysis of mu-calpain (80-kDa subunit) was observed in all three muscles. The rapid pH decline and increased rate of autolysis in the PM paralleled an earlier appearance of myofibril-bound mu-calpain. The SM had higher calpastatin activity (P < 0.05) at 45 min, 6 h, and 24 h and had higher WBS values at 48 h (P < 0.01) and 120 h (P < 0.05) postmortem than the LD. At 48 and 120 h postmortem, more degradation of desmin, titin, and nebulin were observed in the LD than in the SM. These results show that mu-calpain activity, mu-calpain autolysis, and protein degradation are associated with differences in pork tenderness and water-holding capacity observed in different muscles.

Synemin may function to directly link muscle cell intermediate filaments to both myofibrillar Z-lines and costameres.

Synemin is a large intermediate filament (IF) protein that has been identified in all types of muscle cells in association with desmin- and/or vimentin-containing IFs. Our previous studies (Bellin, R. M., Sernett, S. W., Becker, B., Ip, W., Huiatt, T. W., and Robson, R. M. (1999) J. Biol. Chem. 274, 29493-29499) demonstrated that synemin forms heteropolymeric IFs with major IF proteins and contains a binding site for the myofibrillar Z-line protein alpha-actinin. By utilizing blot overlay assays, we show herein that synemin also interacts with the costameric protein vinculin. Furthermore, extensive assays utilizing the Gal4 yeast two-hybrid system demonstrate interactions of synemin with desmin and vimentin and additionally define more precisely the protein subdomains involved in the synemin/alpha-actinin and synemin/vinculin interactions. The C-terminal approximately 300-amino acid region of synemin binds to the N-terminal head and central rod domains of alpha-actinin and the approximately 150-amino acid C-terminal tail of vinculin. Overall, these interactions indicate that synemin may anchor IFs to myofibrillar Z-lines via interactions with alpha-actinin and to costameres at the sarcolemma via interactions with vinculin and/or alpha-actinin. These linkages would enable the IFs to directly link all cellular myofibrils and to anchor the peripheral layer of myofibrils to the costameres.

Molecular characteristics and interactions of the intermediate filament protein synemin. Interactions with alpha-actinin may anchor synemin-containing heterofilaments.

Synemin is a cytoskeletal protein originally identified as an intermediate filament (IF)-associated protein because of its colocalization and copurification with the IF proteins desmin and vimentin in muscle cells. Our sequencing studies have shown that synemin is an unusually large member (1,604 residues, 182,187 Da) of the IF protein superfamily, with the majority of the molecule consisting of a long C-terminal tail domain. Molecular interaction studies demonstrate that purified synemin interacts with desmin, the major IF protein in mature muscle cells, and with alpha-actinin, an integral myofibrillar Z-line protein. Furthermore, expressed synemin rod and tail domains interact, respectively, with desmin and alpha-actinin. Analysis of endogenous protein expression in SW13 clonal lines reveals that synemin is coexpressed and colocalized with vimentin IFs in SW13.C1 vim+ cells but is absent in SW13.C2 vim- cells. Transfection studies indicate that synemin requires the presence of another IF protein, such as vimentin, in order to assemble into IFs. Taken in toto, our results suggest synemin functions as a component of heteropolymeric IFs and plays an important cytoskeletal cross-linking role by linking these IFs to other components of the cytoskeleton. Synemin in striated muscle cells may enable these heterofilaments to help link Z-lines of adjacent myofibrils and, thereby, play an important role in cytoskeletal integrity.

The effects of mono-ADP-ribosylation on desmin assembly-disassembly.

Previous studies have shown that desmin, the muscle-specific intermediate filament protein, is a substrate for the endogenous muscle arginine-specific mono-ADP-ribosyltransferase and that ADP-ribosylation inhibits assembly of desmin into intermediate filaments (Huang et al., Exp. Cell Res. 226, 147-153, 1996). In this paper, the effects of mono-ADP-ribosylation on assembly and disassembly of desmin intermediate filaments were further characterized. First, it was found that ADP-ribosylated desmin does not coassemble with unmodified desmin and has no effect on assembly of unmodified desmin. Second, incubation of assembled desmin filaments with mono-ADP-ribosyltransferase and NAD+ results in disassembly of the filaments. Finally, the structural components of the attached ADP-ribose moiety responsible for altering the assembly of desmin into filaments were investigated by a stepwise cleavage of ADP-ribose with snake venom phosphodiesterase and alkaline phophatase, followed by analysis of assembly. The reactions catalyzed by these two enzymes were established using a desmin peptide as a substrate. Our results show that ribosylated desmin, but not phosphoribosylated desmin, was able to self-assemble into intermediate filaments, suggesting that the presence of a phosphate group is needed to alter desmin's assembly ability.

Properties of the novel intermediate filament protein synemin and its identification in mammalian muscle.

We examined specific properties of highly purified synemin (230 kDa), recently identified as a novel intermediate filament (IF) protein, from avian smooth muscle. Soluble synemin in 10 mM Tris-HCl, pH 8.5, appears as approximately 11-nm-diameter globular structures by negative-stain and low-angle shadow electron microscopy. Chemical crosslinking and SDS-PAGE analysis indicate that soluble synemin molecules contain two 230-kDa subunits. The pH- and ionic strength-dependent solubility properties of synemin are similar to those of the type III IF protein desmin, but under physiological-like conditions in which desmin self-assembles into long approximately 10-nm-diameter IFs, synemin self-associates into complex, approx 15- to 25-nm-diameter globular structures. Calpain digestion demonstrated that synemin is extremely proteolytically labile. Western blot analysis, with monospecific polyclonal antibodies against avian synemin, shows the presence of the reactive 230-kDa synemin band in samples of adult avian skeletal, cardiac, and smooth muscle and of two reactive bands at approximately 225 kDa (major) and approximately 195 kDa in adult porcine skeletal, cardiac, and smooth muscle. Partial purification of synemin from porcine smooth muscle also resulted in fractions highly enriched in the approximately 225- and approximately 195-kDa polypeptides. Conventional immunofluorescence and immunoconfocal microscopy of isolated myofibrils and of frozen sections also demonstrated, for the first time, that synemin is present in all three adult porcine muscle cell types and is colocalized with desmin in skeletal and cardiac muscle cells at the myofibrillar Z-lines.

Integrin expression in developing smooth muscle cells.

We studied the specific expression patterns and distributions of alpha1 and beta1 integrin subunits, the major cell adhesion receptors in smooth muscle, in developing smooth muscle cells from 16-, 18-, and 20-day embryonic gizzards and from 1- and 7-day post hatch chick gizzards by SDS-PAGE, immunoblotting, and immunoelectron microscopy. Antibodies raised against alpha1 and beta1 integrins isolated from avian gizzards were used as probes. Gels and blots showed that the amount of alpha1 and beta1 integrins increased as age increased, with major increases at 1 and 7 days post hatch. Image analysis of immunoelectron micrographs demonstrated that statistically significant labeling increases occurred between embryonic Days 16 and 18, between embryonic Day 20 and 1 day post hatch, and between 1 day and 7 days post hatch. Immunolabeling with both anti-alpha1 and anti-beta1 integrin was prominent at membrane-associated dense plaques (MADPs) and at filament anchoring regions at cell ends. This indicates that alpha1 and beta1 integrin expression coincides temporally with the intracellular proliferation and reorientation of myofilaments. The similarity in distribution patterns of alpha1 and beta1 integrins during development suggests that the two integrin subunits are synchronously expressed during development and do not appear sequentially. (J Histochem Cytochem 46:119-125, 1998)

Regulatory role of arginine-specific mono(ADP-ribosyl)transferase in muscle cells.

Earlier we demonstrated that meta-iodobenzylguanidine (MIBG), a specific inhibitor of arginine mono-ADP-ribosylation blocks proliferation and differentiation of chick skeletal myogenic cells in culture (Exp. Cell Res., 1992, 201:33-42). Membrane fractions from 4-day, myotube cultures of embryonic chick muscle cells were incubated with 32P-NAD+. Several proteins were labeled, but labeling of two hands of about 53 and 36 kDa appeared to be due to arginyl ADP-ribosylation. Immunoprecipitation with D3 monoclonal antibody to the intermediate filament protein desmin, SDS-PAGE and autoradiography demonstrated that the 53 kDa band contained desmin, and that this desmin is ADP-ribosylated by the endogenous arginine-specific mono(ADP-ribosyl)transferase (Exp. Cell Res., 1996, in press). Desmin is the muscle-specific intermediate filament protein, and it appears to be one of the first muscle-specific proteins expressed during terminal myogenic differentiation. We have examined whether desmin can be ADP-ribosylated in muscle cells by use of polyclonal antibodies for ADP-ribosylated arginyl residues. We have found that soluble desmin is present in 5-6 day myogenic cell cultures and that this desmin contains ADP-ribose, demonstrating that desmin is ADP-ribosylated in skeletal muscle cells. We also found that purified avian desmin contains antigenic material that reacts with these antibodies. In both cases, NaCl had no effect on the reactivity, but NH2OH did, which is consistent with an arginine-ADPR linkage. In summary, these results suggest that ADP-ribosylation is an important regulatory mechanism in differentiating muscle cells, and that the intermediate filament protein desmin is an important substrate for modification in muscle cells.

Molecular characteristics of the novel intermediate filament protein paranemin. Sequence reveals EAP-300 and IFAPa-400 are highly homologous to paranemin.

Paranemin was initially found to copurify with the intermediate filament (IF) proteins vimentin and desmin from embryonic chick skeletal muscle and was described as an IF-associated protein (IFAP). We have purified paranemin from embryonic chick skeletal muscle, prepared antibodies, and demonstrated that they label at the Z-lines of both adult avian and porcine cardiac and skeletal muscle myofibrils. We determined the cDNA sequence of paranemin by immunoscreening a lambdagt22A cDNA library from embryonic chick skeletal muscle. Northern blot analysis revealed a single transcript of 5.3 kilobases, which is much smaller than predicted from the size of paranemin (280 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The derived amino acid sequence of paranemin (1,606 residues; 178,161 kDa) contains the conserved IF rod domain (308 amino acids), which has highest homology to the rod domains of nestin and tanabin. Thus, paranemin is an IF protein rather than an IFAP. Sequence analysis also revealed that the partial cDNA sequences of two proteins, namely EAP-300 and IFAPa-400, are almost identical to regions of the cDNA sequence of paranemin. The complete paranemin cDNA was expressed in a cell line (SW13) with, and without, detectable cytoplasmic IFs. Antibody labeling of these cells suggests that paranemin does not form IFs by itself, but rather is incorporated into heteropolymeric IFs with vimentin.

Characterization of ADP-ribosylation sites on desmin and restoration of desmin intermediate filament assembly by de-ADP-ribosylation .

Desmin is an intermediate filament protein that can be ADP-ribosylated by arginine-specific mono(ADP-ribosyl) transferase. Stoichiometric modification of desmin by the transferase causes inhibition of assembly of desmin into 10-nm intermediate filaments (Huang et al., 1993, Biochem. Biophys. Res. Commun. 197, 570-577). In this work, the sites of modification that can affect disassembly have been identified. ADP-ribosylated desmin (1.2 mol ADP-ribose/mol desmin) was digested with lysyl endopeptidase followed by trypsin. Two ADP-ribosylated peptides were obtained, sequenced by Edman degradation, and analyzed by the use of matrix-assisted laser desorption/ionization mass spectrometry. Arginines 48 and 68 of desmin's head domain were shown to be sites of modification, with arginine 48 the major ADP-ribosylation site. ADP-ribosylated desmin (4 mol ADP-ribose/mol desmin) was treated with ADP-ribosylarginine hydrolase. Removal of more than three ADP-ribose groups results in partial restoration of desmin's ability to form intermediate filaments. It is necessary to remove all ADP-ribose groups from desmin to restore its complete ability to form intermediate filaments. The fact that the effect of ADP-ribosylation on the filament-forming properties of desmin is fully reversible suggests that ADP-ribosylation alone is responsible for the changes noted in desmin.

Target proteins for arginine-specific mono(ADP-ribosyl) transferase in membrane fractions from chick skeletal muscle cells.

In a previous study, we found that a specific inhibitor of cellular arginine-specific mono(ADP-ribosyl) transferase, meta-iodobenzylguanidine (MIBG), reversibly inhibited both proliferation and differentiation of cultured embryonic chick primary muscle myoblasts. In addition, we observed that arginine-specific ADP-ribosyltransferase activity increased with muscle-cell differentiation in cultures. Therefore, muscle-cell cultures, especially the 96-h myotube cultures that contain the highest levels of ADP-ribosyltransferase, were used as a working system to determine the cellular protein substrates for arginine-specific ADP-ribosyltransferase. When membrane fractions extracted from 96-h chick myotubes were incubated with [32P]NAD at 30 degrees C for 30 min, only a few proteins were labeled. The labeling of two proteins of 36 and 56 kDa was inhibited by the presence of an arginine-specific mono(ADP-ribosyl) transferase inhibitor, MIBG, and by novobiocin. To prove that these proteins are indeed the targets for arginine-specific mono(ADP-ribosyl)ation, active recombinant muscle ADP-ribosyltransferase was incubated with membrane proteins under the same conditions. ADP-ribosylation of these two membrane proteins, as seen in the endogenous reactions, was also catalyzed by the added muscle transferase and was also inhibited by MIBG and novobiocin. By using antibody specific for desmin for immunoprecipitation and immunoblot analysis, we found that a 56-kDa protein associated with the membrane of myotubes is desmin. Our results showed that incorporation of isotope into this protein band from [32P]NAD is due to ADP-ribosylation of desmin.

Synemin contains the rod domain of intermediate filaments.

We have screened a lambda gt11 cDNA library from chicken gizzard muscle with polyclonal antiserum to avian synemin. Immunopositive clones were characterized and sequenced. Computer searches identified primarily intermediate filament (IF) proteins as being homologous to the synemin clones. Synemin's sequence contained all structural features characteristic of the rod domain of IF proteins: (a) its length (approximately 310 amino acids), (b) the heptad repeat pattern of hydrophobic residues of coiled-coil proteins, (c) subdomain structure of the IF rod, by which helical subdomains (1A, 1B, 2A, and 2B) are separated by three short non-helical linker regions, and (d) several potential intrahelical ion pairs along the sequence. We also confirmed the presence of the IF rod domain at the protein level by immunoblotting a proteolytic digest of synemin by using a monoclonal antibody specific to the rod domain of IFs.

Substructure of cytoplasmic dense bodies and changes in distribution of desmin and alpha-actinin in developing smooth muscle cells.

The substructure of assembling cytoplasmic dense bodies (CDBs) and changes in the distribution of desmin and alpha-actinin during development of smooth muscle were studied in gizzard samples from 10- and 16-day embryos and from 1- and 7-day post-hatch chickens. CDBs in these cells lack the density of CDBs in mature or adult smooth muscle cells and, thus, allow observations of the changes inside CDBs. The random filament orientation seen in younger embryonic cells is first modified to include relatively small patches of IFs that are somewhat straighter and are approaching a side-by-side arrangement. As development proceeds, the IFs in these arrays become straighter, are parallel over longer lengths of the IFs and later acquire the density characteristic of mature CDBs. Anti-desmin labeling in embryonic 10- and 16-day cells showed that desmin intermediate filaments (IFs) were located in the myofilament compartment but were concentrated in or near assembling CDBs. Anti-desmin labeling shifted to the perimeter of CDBs after hatching. Cross sections, longitudinal sections, and stereo pairs all show that IF profiles are present inside unlabeled assembling CDBs. Anti-alpha-actinin labeling was directly on CDBs and was often associated with the cross-connecting filaments (CCFs) (average diameter of 2-3nm) inside CDBs. We propose, based on these data, that desmin IFs, alpha-actinin-containing CCFs, and actin filaments are the principal components of the substructure of assembling CDBs. We also present a proposed model for CDB assembly.

ADP-ribosylation of the intermediate filament protein desmin and inhibition of desmin assembly in vitro by muscle ADP-ribosyltransferase.

Arginine-specific mono(ADP-ribosyl)transferase purified from rabbit skeletal muscle catalyzes stoichiometric ADP-ribosylation of the intermediate filament protein, desmin. In contrast, cholera toxin catalyzes a much lower level of ADP-ribosylation of desmin. Modification results in potent inhibition of desmin's ability to assemble into filaments. Phosphorylation of desmin by the catalytic subunit of cAMP dependent protein kinase is also inhibited by ADP-ribosylation. ADP-ribosylation site(s) are located within the N-terminal head domain of desmin.

Molecular characterization of avian muscle titin.

Titin is an approximately 3000-kDa polypeptide that constitutes a set of elastic filaments that connect thick filaments to the Z-line in vertebrate striated muscle myofibrils. To characterize the primary structure of titin, three overlapping cDNA clones comprising 2.4 kilobases of avian muscle titin coding sequence were obtained from a cDNA library constructed from embryonic chick cardiac muscle RNA size-selected for large transcripts. Expression of one cDNA clone in Escherichia coli produced a fusion protein that reacted specifically with titin antibodies, and titin antiserum affinity-purified against this fusion protein reacted specifically with titin on immunoblots of chicken cardiac and skeletal muscle myofibrils. Indirect immunofluorescence localization with the fusion protein-specific antibodies demonstrated that the cDNA sequence was from the region of titin located in the myofibrillar A-band adjacent to the A/I junction. Derived amino acid sequences demonstrated a repeating pattern of fibronectin type III and immunoglobulin C2 motifs, as shown previously for a portion of rabbit skeletal muscle titin located in the central region of the A-band and for other myofibrillar proteins that bind to myosin. Differences between the rabbit and chicken titin sequences included a unique, serine-rich region in one motif, which represents a potential phosphorylation site. This is the first report of sequence information for avian titin from a previously uncharacterized portion of the titin molecule.

Use of 18O-labelled leucine and phenylalanine to measure protein turnover in muscle cell cultures and possible futile cycling during aminoacylation.

Amino acids labelled with 18(O) on both carboxy oxygen atoms have the potential for use as non-recyclable tracers to measure protein turnover. During protein synthesis one of the labelled oxygen atoms is removed, and thus release of the mono-labelled amino acid could be used to determine proteolysis. Primary cultures of embryonic-chick skeletal-muscle cells were used to test the use of 18(O2)-labelled Leu to measure proteolysis. For 9-day cultures, prelabelled on days 2-8 with medium containing one-half the Leu as [18O2]Leu and one-half as [2H3]Leu, release of [18(O)]Leu was less than 50% that of [2H]Leu over 24 h, suggesting a loss of the 18O label by a mechanism other than protein synthesis. Medium containing [18(O2)]Leu, [2H3]Leu, [18O2]Phe and [13C]Phe was then incubated with 9-day cultures to compare the rate of loss of the 18(O)-label from Leu and Phe with the rate of uptake of the non-carboxy-oxygen-labelled amino acids. Results for Leu demonstrated an 81% loss of the 18(O) label compared with a 33% decrease in [2H]Leu over 12 h. Loss of the 18(O) label was four times as great for Leu as for Phe. Loss of the 18(O) label was not decreased by addition of cycloheximide or by addition of a 3-fold excess of Ile, Val and Tyr; thus the loss of label was not due to protein synthesis alone or to misbinding to incorrect tRNAs. Infusion of the isotopes into pigs showed that the 18(O) label of Leu was not lost during transamination to alpha-ketoisocaproate (alpha-oxoisohexanoate). The most probable explanation is that the 18(O) label is lost as a result of the enzymic deacylation of tRNA, that this process is substantially faster for Leu than for Phe, and that this represents a potentially costly futile cycle for Leu.

Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture.

Primary cultures of embryonic chick skeletal myogenic cells were used as an experimental model to examine the possible role of mono(ADP-ribosyl)ation reactions in myogenic differentiation. Initial studies demonstrated arginine-specific mono(ADP-ribosyl)transferase activity in the myogenic cell cultures. We then examined the effect of a novel inhibitor of cellular arginine-specific mono(ADP-ribosyl)transferases, meta-iodobenzylguanidine (MIBG), on differentiation of cultured embryonic chick skeletal myoblasts. MIBG reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (15-60 microM) concentrations of MIBG blocked myoblast fusion, the differentiation-specific increase in creatine phosphokinase activity, and both DNA and protein accumulation in myogenic cell cultures. Meta-iodobenzylamine, an analog of MIBG missing the guanidine group, had no effect. Low concentrations of methylglyoxal bis-guanylhydrazone, a substrate for cholera toxin with a higher Km than MIBG, also had no effect, but higher concentrations reversibly inhibited fusion. These findings suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis. In addition, the total arginine-specific mono(ADP-ribosyl)transferase activity increased with differentiation in the myogenic cell cultures, and this increase was also blocked by MIBG treatment. Because high levels of activity were found in the membrane fraction derived from later, myotube cultures, the membrane fraction from 96-h cultures was incubated with [32P]NAD+ and subjected to electrophoresis and autoradiography. Three proteins, migrating at 21, 20, and 17 kDa, that were ADP-ribosylated in the absence, but not the presence, of MIBG were identified. These proteins may be endogenous substrates for this enzyme.

Paraneoplastic IgG striational autoantibodies produced by clonal thymic B cells and in serum of patients with myasthenia gravis and thymoma react with titin.

Autoantibodies with heterogeneous specificities for contractile elements of striated muscle are found in 80 to 90% of patients who have myasthenia gravis (MG) with thymoma. The stimulus for production of these paraneoplastic striational autoantibodies (StrAb) is unknown. One approach to understanding their association with MG and thymoma is to define the antigenic specificities of monoclonal StrAb secreted by B cell lines established from patients who have MG and thymoma. Here, we report the isolation from a single patient of two independent thymic B cell clones secreting StrAb of IgG isotype. In immunoblots, both StrAb bound monospecifically to an antigen of human skeletal muscle that comigrated with the high molecular weight protein, titin. The pattern of immunofluorescence staining yielded by both antibodies in cultured human muscle cells was similar to that produced by rabbit polyclonal anti-titin antibodies. Each monoclonal antibody bound to a different region of the sarcomere in stretched myofibrils; these corresponded to sites previously reported to bind murine anti-titin monoclonal antibodies. The pattern of sarcomere immunostaining produced by combining the two human monoclonal antibodies was indistinguishable from that produced by serum IgG from the patient whose thymus yielded the B cell clones. Thus, the monoclonal antibodies appear to identify two epitopes of titin that are recognized by IgG StrAb in serum. The finding that IgG anti-titin autoantibodies are restricted to serum of MG patients who have thymoma suggests that titin is a major specificity of IgG StrAb. Our additional finding that anti-titin IgG binds to striated elements in medullary myoid cells of the human thymus supports the hypothesis that StrAb represent an intrathymic B cell immune response that is initiated by autoantigens that are rendered immunogenic for helper T cells in the course of noeplastic transformation to thymoma.

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells.

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.

Effect of porcine stress syndrome on the solubility and degradation of myofibrillar/cytoskeletal proteins.

This study examined the effect of stress classification (stress-positive, stress-carrier, stress-negative) of pigs on selected properties of postmortem muscle, including protein solubility and degradation of proteins such as titin. Longissimus muscle samples were removed 45 min postslaughter, divided into samples, and stored at 0 to 2 degrees C for analysis at 0, 1, 3, 5, and 7 d postmortem. Whole-muscle samples (homogenates) and purified myofibrils were prepared from each sample for analysis by SDS-PAGE. A portion of each muscle sample also was extracted 1) with a low-ionic-strength solution to obtain a sarcoplasmic protein fraction and 2) with two different high-ionic-strength solutions to obtain a myofibrillar/cytoskeletal protein fraction for measurement of protein solubility and for analysis of extracts by SDS-PAGE. No significant differences were observed between muscle from stress-negative and stress-carrier animals in this study. Sarcoplasmic (P less than .05) and myofibrillar/cytoskeletal (P less than .01) protein solubility was lower in muscle samples from stress-positive animals than in muscle samples from stress-carrier and stress-negative animals at all postmortem times studied. The high molecular weight protein titin was degraded more slowly postmortem in muscle from stress-positive than in muscle from stress-negative animals, as observed by SDS-PAGE analysis of whole-muscle samples (homogenates) an myofibrils. The combination of lowered protein solubility and reduced rate of postmortem degradation of structural proteins such as titin may explain, at least in part, the reduced quality and protein functionality of muscle from stress-positive pigs.

Determination of the critical concentration required for desmin assembly.

The critical concentration required for filament assembly in vitro from highly purified desmin was determined by both turbidity and centrifugation assays. Assembly was done in the presence of 2 mM-Ca2+, 2 mM-Mg2+ or 150 mM-Na+ at 2, 22 and 37 degrees C. Similar values for critical concentration were obtained by both assays. As temperature increased, critical concentration decreased for each cation. The critical concentration was lowest in the presence of Ca2+ at 2, 22 and 37 degrees C, but was highest in the presence of 150 mM-Na+ at 2 degrees C. Negative staining showed that supernatants from the centrifugation assays contained protofilaments, protofibrils and short particles (less than 300 nm), but pellets contained long filaments (greater than 1 micron) with an average diameter of 10 nm. As the temperature increased, both the average diameter and average length of particles in the supernatant increased. Thermodynamic analysis indicated that hydrophobic interactions were dominant during desmin assembly, but that ionic interactions might also be involved. Our results demonstrated that the specific cation and temperature and temperature-cation interactions all are important in assembly of desmin intermediate filaments.