Heavy Myosin Research Articles

Identification of copper-responsive genes in an early life stage of the fathead minnow Pimephales promelas

While physiological changes associated with copper toxicity have been studied in adult fathead minnow, Pimephales promelas, little is known about the effect of copper on newly hatched larvae. As a result we initiated an investigation on the mechanism of copper toxicity in 24 h post-hatch larvae using gene expression changes to identify responsive genes. Fish were exposed to copper concentrations of 0, 50, 125 and 200 mug/L in a 48 h toxicity test. Total RNA from survivors was used in a differential display assay to screen for differentially expressed gene products. Altogether, 654 copper-responsive differentially expressed bands were collected. Database searches found homology for 261 sequences. One hundred and sixty-one bands were homologous to NCBI genes of known function, of which 69 were individual genes. The most abundant categories of functional genes responding to copper were involved in protein synthesis/translational machinery and contractile proteins. Twenty-one dose-responsive genes were measured for expression changes using real-time quantitative PCR. Differential gene expression was validated for 11 of 13 genes, when a 1.2 times qPCR difference between the copper and control samples was observed. Transcripts identified as titin, cytochrome b, fast muscle specific heavy myosin chain 4, fast muscle troponin I, proteasome 26S subunit and troponin T3a were induced over twofold. Differential display bands identified as 60S ribosomal proteins L27 and L12 were repressed approximately threefold. We conclude that copper exposure affects several cellular pathways in larval fathead minnows with protein synthesis, ribosome structure, and muscle contractile proteins being the most sensitive to this stress.

Abstract 1015: Human Heart Contains A Stem Cell Population With Mesenchymal Properties

Background Mesenchymal Stem Cells (MSC) were initially isolated from the bone marrow (BM) and identified as plastic adherent cells with a typical immunophenotype (negative for CD45, CD34, CD117 and HLA-DR and positive for CD105, CD90, CD44, CD73 and CD13), and able to differentiate into mesodermal lineages in vitro . Recently, MSC have been isolated from different adult tissues, i.e. liver, fat, kidney and thymus. Aim of the present study was to search for Cardiac Mesenchymal Stem Cells (C-MSC) in the adult human heart. Methods and Results : Auricle fragments were obtained from adult patients undergoing cardiac surgery. From these specimens we isolated a high proliferating plastic adherent cell population that, by FACS analysis, expressed mesenchymal markers (CD105, CD44, CD73, CD13 ) and was negative for hematopoietic markers (CD45, CD34) and HLA-DR. Further, in appropriate differentiating media, these cells exhibited osteogenic and adipogenic differentiation as cultures were positive for Oil Red O and Von Kossa staining used to mark intracellular lipid droplets and calcium salts deposits, respectively. Quantitative Real Time (qRT) -PCR analysis revealed that cells were negative for the early cardiac markers Nkx2.5 and Tbx5 and for the adult cardiac markers α-Myosin Heavy Chain and Myosin Light Chain-2a. Interestingly, in standard culture conditions, cells were positive for the early cardiac marker GATA4; in contrast, BM-MSC expressed GATA4 only after dexamethasone treatment. Additionally, when injected into the zebrafish blastula (about 100 –200 cells, n=70), C-MSC specifically homed in the cardiac region, as found at 24 –28 hpf (hours post fertilization). In contrast, transplanted BM-MSCs, were found not only in proximity of the heart, but throughout several mesodermal derivatives of the embryo (n=25). Conclusions: The adult human heart contains C-MSCs that can be easily expanded in vitro and may represent a useful population for autologous cell therapy of some cardiac diseases.

Abstract 1118: Novel Lentiviral Vectors Bearing the Cardiac-Specific Promoter of the Sodium-Calcium Exchanger (NCX1) Report Cardiogenic Differentiation in Stem Cells

Background: Cardiosphere-derived resident cardiac stem cells (CDCs) are readily isolated from adult hearts and confer functional benefit in animal models of heart failure. To study cardiogenic differentiation in CDCs, we developed a method to genetically label and selectively enrich for cells that have differentiated down the cardiomyocyte lineage. Methods and Results: Lentiviral vectors achieved significantly higher transduction efficiency (>90%) and longer-term transgene expression in human CDCs than any of the nine adeno-associated viral serotypes tested (AAV 1– 6, 2.5, 8, 9). To define the best reporter of cardiogenic differentiation, five cardiac-specific promoters (sodium-calcium exchanger [NCX1], L-type calcium channel α-subunit, α-myosin heavy chain, troponin I and myosin ventricular light chain) were subcloned upstream of GFP in lentiviral vectors. Cardiac specificity was assessed by transducing neonatal rat ventricular myocytes (NRVMs), HEK293, cardiac fibroblasts, skeletal myoblasts, endothelial and vascular smooth muscle cells with each vector and then measuring GFP fluorescence by flow cytometry. The cardiac NCX1-promoter conveyed the highest degree of cardiac specificity, with expression limited to NRVMs. At a multiplicity of infection of 35, NCX1-GFP vectors did not affect cell death or apoptosis of CDCs. Expression persisted for up to 6 months in vitro . NCX1-GFP positive CDCs subpopulations, were FACS-sorted and expanded in vitro , demonstrating enhanced expression of a variety of cardiac markers by real-time PCR. The utility of lentiviral vectors bearing the cardiac NCX1-promoter was further exemplified by treating CDCs with cardiogenic sulfonylhydrazones (SHZ). SHZ potently activated a NCX1-firefly luciferase reporter (211 ± 21% greater than control cells) and upregulated cardiac transcripts (including BNP, troponin I, α-tropomyosin, GATA-4). Conclusion: Lentiviral vectors carrying the NCX1 promoter represent a useful tool for genetically marking stem cells and their progeny that have differentiated down the cardiomyocyte lineage. The ability to selectively enrich for cardiomyocytes and their precursors has potential relevance for the development of cell-based therapies.

The urethral striated sphincter in adult male rat

This study reports the morphology of the urethral sphincter in adult male rats, mainly the histological aspects, the features of the endplates, and the heavy myosin chain distribution in the striated fibres. First, the prostate is entirely out of the striated sphincter, which is surprising when compared to man. Second, the urethral striated sphincter consists of two lateral fascicles separated by an anterior and a posterior strip of connective tissue, which extend from the prostatic urethra (i.e. the part of the urethra which runs though the prostate) to the bulb of the penis. An additional third fascicle of striated muscle (SM) covers the caudal part of the anterior connective strip of the membranous urethra (i.e. the urethra which extends from its prostatic part to the bulb of the penis). In the membranous urethra, the striated sphincter surrounds directly the urethral lumen without intercalated smooth muscle. In urethral cross sections, the endplates detected by alpha-bungarotoxin, which binds to nicotinic receptors, are clustered in the postero-lateral part of the lateral fascicles. The cross-sectional area of the urethral striated fibres shows a bimodal distribution: the largest fibres are located at the periphery of the sphincter and these fibres express only fast myosin heavy chains (MHC) as shown by immunochemistry. The smallest fibres are less numerous and are situated near the lumen co-expressing fast and slow MHC. All the striated fibres express desmin and dystrophin as SM fibres do. Taken together, these results suggest that the urethral striated fibres in male rat present the same characteristics as those of the skeletal muscles. The predominance of fast fibres is consistent with phasic contractions playing a role not only during micturition and urinary continence but also probably during ejaculation.

Fine-tuning of cross-bridge kinetics in cardiac muscle of rat and mouse by myosin light chain isoforms

Cross-bridge kinetics underlying stretch-induced force transients was studied in cardiac muscle strips with different myosin heavy chain (MHC) and myosin light chain (MLC) isoforms. The force transients were induced by stepwise stretches of maximally Ca(2+)-activated skinned muscle strips. The MHC and MLC isoforms were analyzed by electrophoreses after the mechanical experiments. Muscle strips of euthyroid rats and mice exclusively containing alpha-MHC were used. In addition, muscle strips of hyper- and hypothyroid rats containing different combinations of MHC and MLC isoforms were used. The thyroid hormone is known to alter the expression of MHC but not of MLC isoforms. In muscle strips containing exclusively alpha-MHC, atrial MLC isoforms (all atria of rats and mice) were associated with about 30% faster kinetics than ventricular MLC isoforms (ventricles of hyperthyroid rats and some muscle strips of ventricles of euthyroid rats and mice). On the other hand, in muscle strips containing exclusively ventricular MLC isoforms, alpha-MHC (ventricles of hyperthyroid rats) was associated with about 2.6 times faster kinetics than beta-MHC (ventricles of hypothyroid rats). We conclude that the MLC isoforms fine-tune cross-bridge kinetics, which underlies stretch-induced force transients, whereas the MHC isoforms mainly determine this kinetics. The effect of MLC isoforms on the cross-bridge kinetics may partially contribute to the faster twitch contraction in atria than in ventricles. Furthermore, it may play a role in various cardiomyopathies where atrial MLC isoforms are partially expressed in ventricles or ventricular MLC isoforms are partially expressed in atria.

Effects of pale, normal, and dark chicken breast meat on microstructure, extractable proteins, and cooking of marinated fillets

The effects of chicken breast meat lightness value (L*) on microstructure, protein extraction, and marinating and tumbling was investigated. Pale soft, and exudative (PSE) meat (L* = 57.7, pH 5.72) showed significantly lower salt soluble protein extraction with less heavy myosin chains compared with dark, firm, and dry (DFD) meat (L* = 44.8, pH 6.27). The PSE meat showed larger intercellular spaces among muscle fibers and bundles compared with normal and DFD meat. Marinated and tumbled PSE breast fillets had higher unbound brine compared with the other meats. Further cooking resulted in lower yield and higher shear force values for the PSE meat compared with normal and DFD fillets.

826 Magnetic resonance imaging allows early noninvasive assessment of left ventricular remodelling after myocardial infarction in rats

826 Magnetic resonance imaging allows early noninvasive assessment of left ventricular remodelling after myocardial infarction in rats

Human single masseter muscle fibers contain unique combinations of myosin and myosin binding protein C isoforms.

Striated craniofacial and limb muscles differ in their embryological origin, regulatory program during myogenesis, and innervation. In an attempt to explore the effects of these differences on the striated muscle phenotype in humans, the expression of myosin and myosin-associated thick filament proteins were studied at the single fiber level both in the human jaw-closing masseter muscle and in two limb muscles (biceps brachii and quadriceps femoris muscles). In the masseter, unique combinations of myosin heavy chain (MyHC) and myosin binding protein C (MyBP-C) isoforms were observed at the single fiber level. Compared to the limb muscles, the MyHC isoform expression was more complex in the masseter while the opposite was observed for MyBP-C. In limb muscles, a coordinated expression of three MyHC and three MyBP-C isoforms were observed, i.e., single fibers contained one or two MyHC isoforms, and up to three MyBP-C isoforms. Also, the relative content of the different MyBP-C isoforms correlated with the MyHC isoform expression. In the masseter, on the other hand, up to five different MyHC isoforms could be observed in the same fiber, but only one MyBP-C isoform was identified irrespective MyHC isoform expression. This MyBP-C isoform had a migration rate similar to the slow MyBP-C isoform in limb muscle fibers. In conclusion, a unique myofibrillar protein isoform expression was observed in the human masseter muscle fibers, suggesting significant differences in structural and functional properties between muscle fibers from human masseter and limb muscles.

Myofibrillar proteins in white muscle of the developing African catfish Heterobranchus longifilis (Siluriforms, Clariidae)

Developmental changes in myofibrillar protein composition were investigated in the myotomal muscle of the African catfish, Heterobranchus longifilis (Clariidae), by several electrophoretic techniques. The main muscle fibres of larvae and the fast-white muscle fibres of juvenile and adult fish were found to express distinct myosin heavy chain and myosin light chain 2 (LC2) isoforms. Three myosin LC2 chains were successively detected, differing by their isoelectric points. In contrast, the alkali light chains remained qualitatively and quantitatively unchanged during fish growth. Actin, α-tropomyosin, and troponin-C (TN-C) were also similar in larval, juvenile, and adult white muscle, but an additional larval tropomyosin isoform was found in the first developmental stages. Two isoforms of troponin-T (TN-T) and troponin-I (TN-I) were synthesised in the course of fish growth. Transition from the larval to the adult isoform was much faster for TN-T than for TN-I. Slow-red muscle myofibrils from adult H. longifilis showed no common component (except actin) with larval, juvenile, or adult fast-white muscle myofibrils. Red myofibrils displayed a single TN-T and a single TN-I isoform, but two isoforms of TN-C. The myofibrillar protein isoforms synthesised at any given developmental stage almost certainly reflect changes in the functional requirements of swimming muscles in the course of fish development.

Immunocytochemical investigations of muscle differentiation in the Atlantic herring (Clupea harengus: Teleostei)

The myotomes in yolk-sac larvae of the Atlantic herring (Clupea harengus: Teleostei) contain a single layer of small-diameter superficial muscle fibres surrounding an inner mass of around 280 larger-diameter muscle fibres. The fraction of muscle fibre volume occupied by mitochondria is dependent on temperature, and in larvae reared at 8°C was 41% for the superficial fibres, and 25% for the inner muscle fibres. The inner muscle fibres of larvae share some myofibrillar proteins with adult white muscle, but contain unique isoforms of myosin heavy chains, troponin T, troponin I and myosin light chain 2. A monoclonal antibody has been produced which is specific to myosin light chain 3 (MLC3). Immunocytochemical studies have shown that the expression of MLC3 is switched off in the superficial muscle fibres at the start of metamorphosis when larvae reach 28–30 mm total length (TL). Metamorphosis to the juvenile stage is complete in fish 35–40 mm TL and is also associated with the development of gill filaments and the production of presumptive slow muscle fibres which form externally to the larval superficial muscle fibres in the region of the lateral line nerve.

Effect of Muscular Activity on the Turnover Rate of Actin and Myosin Heavy and Light Chains in Different Types of Skeletal Muscle

In endurance-trained rats the myosin HC have a more rapid turnover rate than in control animals in all three types of skeletal muscle fibres, but in sprint-trained rats only in fast-glycolytic and fast-oxidative-glycolytic fibres. A comparison of the actin turnover rate in sprint- and endurance-trained animals shows that in both groups there are more rapid turnover rates in all types of muscle fibres in comparison with control rats. During long-lasting exhaustive exercise the turnover rate of myosin HC decreases. Twelve hours after exercise, myosin LC 2 and 3, and actin have a more rapid turnover than in control rats. Changes in the turnover rate of muscle contractile proteins during exercise and the recovery period after exercise reflect the functional conditions of the contractile apparatus in different types of skeletal muscle fibres and have a physiological significance.

Study of proteolysis during the processing of a dry fermented pork sausage

Dry pork sausage was formulated, fermented and dried for 41 days. Gross composition was determined and proteolysis was monitored by assaying the following N fractions: water-soluble nitrogen (WSN), salt-soluble nitrogen (SSN), ultrafiltration (UF) permeates of WSN and SSN, phosphotungstic acid (PTA)-soluble and -insoluble fractions and free amino acids. Meat and sausages, water- and salt-insoluble components, UF-retentates of WSN and SSN were assessed by SDS-PAGE at different stages of ripening. The amount of WSN, WSN permeate, PTA-soluble N and free amino acids increased during processing, while the SSN and PTA-insoluble N decreased. The electrophoretic studies demonstrated that proteolysis of the heavy myosin chain, α-actinin and actin was most prominent. The increased insolubility of meat proteins was confirmed by SDS-PAGE.

The mechanism of skeletal muscle myosin ATPase. The mechanism of ADP interaction.

The mechanism of interaction between ADP and the myosin active center has been studied using a transient kinetic technique. The results show that the interaction of ADP with the myosin active center is a homogeneous process independent of the association state of the active centers; namely, whether ADP interacts with the monomeric myosin subfragment-1, or with the dimeric forms heavy meromyosin and myosin. The kinetics of the interaction conforms to a simple two-step reaction mechanism for ADP binding. The kinetic and thermodynamic constants for this mechanism have been determined. In addition, analysis of the binding isotherm indicates that the two active sites in heavy meromyosin and myosin function as identical and independent sites.

Immunochemically Detected Structural Difference in the Heavy Chains of Chicken Breast Muscle Myosin Isozymes<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

Antisera against the isozymes of heavy meromyosin from chicken breast muscle myosin were prepared by immunizing rabbits. Both antisera against heavy meromyosin containing g1 light chain (HMM(g1) and that containing g3 light chain (HMM(g3)) reacted with both heavy meromyosin isozymes and myosin, but not with whole light chain mixture. This indicates that only antibodies against the heavy chain of heavy meromysin were elicited. The antisera were applied successively to two columns, one coupled with subfragment-2 and the other with heterologous subfragment-1 isozymes. When the antiserum against HMM(g3) was absorbed with subfragment-1 containing g1 (S-1(g1)), it did not react with HMM(g1), but reacted with HMM(g3). On the other hand, when the antiserum against HMM(g1) was absorbed with subfragment-1 containing g3 (S-1(g3)), it lost its ability to react with both heavy meromyosin isozymes. This indicates the presence of a specific antibody against the heavy chain of HMM(g3). The head portion of myosin containing g3 may hold an unique antigenic determinant which is not present in the head of a myosin containing g1.

Immunochemical localization of contractile proteins in mammalian meiotic chromosomes.

Smooth muscle heavy myosin and actin have been detected in mouse and rat meiotic chromosomes, by indirect immunofluorescence performed on testis cryostat sections and isolated germ cells. Both contractile proteins are detectable in the nuclei of meiotic cells during the first prophase. The appearance and disappearance time of myosin and actin, however, is not synchronous. While actin is visible in small spots from resting to late diplotene spermatocytes, myosin appears as filaments in the primary spermatocytes from the zygotene to the early state of diplotene. The number of myosin filaments in the pachytene spermatocytes corresponds to the number of bivalent chromosomes, whereas actin spots constantly outnumber the pairing chromosomes by two units. These immunochemical observations suggest that the two contractile proteins are associated with the synaptonemal complex (SC). Myosin seems to be associated with the central region of the SC, while actin is present in its basal knob which is in connection with the nuclear membrane. The difference in number between myosin filaments and actin spots appears to be related to the peculiar behaviour of the pairing sex chromosomes. The presence of contractile proteins in the nuclei of primary spermatocytes seems to suggest that they might play a role in the process of pairing of homologous chromosomes.

Use of antibodies against dodecylsulfate-denatured heavy meromyosins to probe structural differences between muscular myosin isoenzymes.

Heavy meromyosin, a tryptic myosin fragment, was purified from rabbit fast twitch muscles and rat cardiac ventricles. Both types of heavy meromyosin were denatured by sodium dodecylsulfate and used to immunize guinea-pigs after chromatography on Sephadex G-10 to remove excess dodecylsulfate. Micro-complement fixation analysis showed that the antisera were specific to a denatured configuration of heavy meromyosin and myosin, and hardly recognized the native proteins. Cross-reactions performed with both rabbit skeletal and rat cardiac antisera indicated that the antigenic structures of denatured myosins varied according both to species (man, rabbit, rat or mouse), and to muscle-type (red skeletal slow twitch, while skeletal fast twitch, cardiac atria or cardiac ventricles). Denatured heavy meromyosin chromatography on Sephadex G-200 in the presence of 0.1% sodium dodecylsulfate enabled separation of several polypeptides groups. Of these, a polypeptide of Mr 29000 was the most reactive and exhibited the same immunological specificities as the whole myosin molecule. The use of antibodies against denatured heavy meromyosin in conjunction with micro-complement fixation therefore provides a discriminant means, not only for estimating the structural relationship between several myosin isoenzymes, but also for localizing constant and variable regions in the heavy chains of these isoenzymes.

Detection of antibodies specific to sodium dodecyl sulfate-treated proteins

Heavy meromyosin (HMM) denatured by sodium dodecyl sulfate (SDS) was injected into guinea pigs, either in the presence of 1 mg SDS/mg protein or after chromatography on Sephadex G-10 to remove detergent excess. Antigen-antibody interactions were analyzed by the microcomplement fixation technique. When HMM was injected in the presence of excess of SDS, the microcomplement fixation curves exhibited two maxima; one was specific to the random coil configuration of heavy meromyosin or myosin, and the other was common to several SDS-protein complexes. The latter peak disappeared when the excess SDS was removed from the immunogen by chromatography. Results showed the presence of antibodies directed either against SDS or against the non-specific SDS protein link.

Immunochemical and biochemical analyses of myosin light chains from normal and denervated cardiac muscle

Rabbit antisera were prepared against the combined low molecular weight subunits (the light chains) of canine cardiac myosin. The antisera reacted specifically with the light chains and with purified myosin. There were no immunochemical reactions with the heavy myosin chains. The antisera were then used to test preparations of light chains isolated from hearts maintained in a denervated condition for 12, 4, or 2 weeks prior to killing the animals. Denervation was accomplished by mediastinal neural ablation. The light chains prepared from the denervated hearts all reacted with the antisera and showed immunological identity with the light chains from control muscle. The specific adenosine triphosphatase activity of myosin prepared from hearts denervated for 12, 4, or 2 weeks exhibited a marked deviation from control activity. We concluded that, although cardiac denervation did not affect the immunological properties of myosin light chains, the biological activity of cardiac myosin was greatly reduced after 2 weeks of denervation and recovered somewhat after 4 weeks.

Snythesis of polypeptides with the properties of myosin light chains direct by RNA extracted from muscle cultures.

Polyadenylylated RNA, extracted from differentiating primary cultures of rat muscle and the myogenic cell line L8, directs the synthesis of polypeptides in the wheat germ cell-free system which comigrate with myosin light chains under several electrophoretic conditions. The peptides also associate specifically with heavy myosin subunits during dissociation-reassociation treatment. Intact cells of primary skeletal muscle cultrues and of the myogenic line synthesize predominantly two ligh chains. RNA extracted from primary muscle cultures directs the synthesis of a third polypeptide in the cell-free system, similar to the third light chain found in myosin extracted from adult rat thigh muscle. Products of cell-free systems directly by RNA extracted from fibroblasts, reticulocytes, and myeloma cells did not contain detectable amounts of similar polypeptides.