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1.
Monoclonal antibodies (mAbs), electrophoresis, immunoblotting, and immunohistochemistry were used to determine the molecular properties of cardiac myosin heavy chain (MHC) isoforms and the regions of the developing chicken heart in which they were expressed. Adult atria expressed three electrophoretically distinct MHCs that reacted specifically with mAbs F18, F59, or S58. During embryonic Days 2-4, when the atrial and ventricular chambers are forming, MHCs that reacted with mAbs F18, F59, or S58 were expressed in both the atria and ventricles. The atria continued to express MHCs that reacted with mAbs F18, F59, or S58 at all stages of development and in the adult. In the ventricles, expression of the MHCs reacting with these mAbs was found to be developmentally regulated. By embryonic Day 16, MHC(s) reacting with mAb F18 had disappeared from the developing ventricles, whereas MHCs reacting with S58 and F59 continued to be expressed throughout the ventricles. As development continued, MHC(s) reacting with S58 in the ventricle became restricted to expression in only the ventricular conducting system. MHC(s) reacting with F59 were expressed in both the ventricular myocytes and the ventricular conducting system throughout development and in the adult. Thus, in contrast to the embryonic chicken heart where at least three MHC isoforms were expressed in both the atria and ventricles, we found in the adult chicken heart that-at a minimum-three MHC isoforms were expressed in the atria, two MHC isoforms were expressed in the ventricular conducting system, and one MHC isoform in the ventricular myocardium. MHC isoform expression in the developing avian heart appears to be more complex than previously recognized.  相似文献   

2.
Monoclonal antibodies (McAb) against myosin from the pectoralis muscle of the adult chicken have been generated and shown to react specifically with the myosin heavy chain (MHC). The reactivities of two such McAbs with myosin from adult chicken atrial and ventricular myocardium were further analysed by immunoautoradiography, radioimmunoassay, and immunofluorescence microscopy. Monoclonal antibody MF 20 was found to bind both atrial and ventricular MHC and stain all striated muscle cells of the adult chicken heart. In contrast, McAb B1 bound specifically to atrial myocytes in immunofluorescence studies, while immunoautoradiography and radioimmunoassay demonstrated the specificity of this antibody for the atrial MHC. Upon reacting these McAbs with myosin isolated from embryonic hearts where definitive atria and ventricles were present, the same specificity of antibody binding was observed. Immunofluorescence studies demonstrated that all striated muscle cells of the embryonic heart contained MHCs recognized by MF 20, while only atrial muscle cells were bound by B1. When extracts of presumptive atrial and ventricular tissue were reacted with MF 20 and B1, significant reactivity of MF 20 was first observed at stage 10 in the presumptive ventricle and thereafter this McAb reacted with all regions of the developing myocardium. Binding of B1 was detected approximately 1 day later at stage 15 and was confined to atrial-forming tissues. These data demonstrate antigenic similarity between adult and embryonic MHC isolated from atrial myocardium and suggest the expression of an atrial-specific MHC early in the regional differentiation of the heart.  相似文献   

3.
Dissociated cells from neonatal rat atria and ventricles were cultured in monolayers for 3 days. Newly synthesized 35S-methionine labeled myosin light chain isoforms ALC-1, ALC-2 (atrial) and VLC-1, VLC-2 (ventricular) were identified on 2D gels, and their pattern of synthesis was compared to that of myocard fragments immediately after explanation. ALCs were synthesized in 5- to 10-fold excess over VLCs by atrial cultures, whereas the converse was true for ventricular cultures, with two exceptions: one third of the LC-1 synthesized by ventricular fragments was ALC-1, and dissociated atrial cells synthesized very little LC-2 of either isoform. The former finding corresponds to the relatively high proportion of ALC-1 in neonatal ventricular tissue. We conclude that the regional programme of LC isoform expression is basically retained after tissue explantation and even after dissociation and culturing of cardiac myocytes.  相似文献   

4.
5.
A monoclonal antibody (anterior latissimus dorsi 58 [ALD58]; antimyosin heavy chain, MHC) directed against myosin from slow tonic muscle was found to react specifically with the striated muscle cells of the conductive system in the adult chicken heart. This monoclonal antibody was used to study the expression of myosin in the conductive system of the adult and developing heart. Using immunofluorescence microscopy with ALD58, muscle cells of the conductive system were demonstrated in both the atria and ventricles of the adult heart as previously shown by Sartore et al. (Sartore, S., S. Pierobon-Bormioli, and S. Schiafinno, 1978, Nature (Lond.), 274: 82-83). Radioactive myosin from adult atria and ventricles was precipitated with ALD58 and subjected to limited proteolysis and subsequent peptide mapping. Peptide maps of ALD58 reactive myosin from atria and ventricles were very similar, if not identical, but differed from peptide maps of ordinary atrial and ventricular myosin. The same antibody was used to study cardiac myogenesis in the chick embryo. When ALD58 was reacted with myosin isolated from atria and ventricles at selected stages of development in radioimmunoassays, reactivity was not observed until the last week of embryonic life (greater than 15 d of egg incubation). Thereafter concomitant and progressively increased reactivity was observed in atrial and ventricular preparations. Also, no ALD58 positive cells were observed in immunofluorescence studies of embryonic hearts until 17 d of egg incubation. Primary cell cultures of embryonic hearts also proved to be negative for this antibody. This study demonstrates that an epitope recognized by ALD58 associated with an antimyosin heavy chain of striated muscle cells of the adult heart conductive system is absent or present in only small amounts in the early embryonic heart.  相似文献   

6.
Two new monoclonal antibodies (McAbs), ALD-180 and ALD-88, produced against the myosin of the slow anterior latissimus dorsi (ALD) muscle of the chicken are described. Their specificity for myosin heavy chain (MHC) was established by radioimmunoassay, immunoautoradiography, and immunofluorescence. They were used in conjunction with McAbs MF-14 and MF-30 (which have been characterized previously to be directed against MHC of the fast skeletal muscle) to examine the developmental changes of the chicken ALD muscle. At the 16-day embryonic, early posthatch, and adult stages the ALD muscle fibers differed in their reaction pattern with the McAbs; at the embryonic stage all fibers reacted strongly with ALD-180 and weakly with ALD-88 and MF-30; at the early posthatch stage there was a checkerboard pattern with many fibers not reacting with any of these three McAbs; and at the adult stage all fibers reacted strongly with ALD-180 and ALD-88 and weakly with MF-30. The MF-14 antibody did not react with ALD muscle at any developmental stage. The mature pattern of immunoreactivity of the ALD muscle fibers with the antibodies was established only after 9 weeks posthatch, and during this 9-week period the immunofluorescence changes were nonsynchronous. Based on immunocytochemical evidence of changes in myosin isoform expression, this study clearly demonstrates a distinctive neonatal (early posthatch) stage in the development of the chicken slow muscle.  相似文献   

7.
Summary The fiber type composition of two fast muscles of the chicken, namely, adductor superficialis (AS) and pectoralis major (PM) was examined by the histochemical myosin ATPase staining and immunochemical techniques using monoclonal antibodies (McAbs). Two new McAbs produced against the myosin of the anterior latissimus dorsi (ALD) muscle of the chicken and named ALD-122 and ALD-83 were characterized to be specific for myosin heavy chain (MHC) and for myosin light chain-1 respectively. They were used in conjunction with previously reported McAbs specific for slow MHC (ALD-47), fast MHC (MF-14) and fast light chain-2 (MF-5). By the histochemical ATPase test most muscle fibers of AS and PM muscles reacted as IIA and IIB respectively. By immunofluorescent staining with the anti-MHC McAbs, ALD-122, and MF-14, the fibers of AS, muscle showed remarkable heterogeneity whereas PM muscle fibers reacted, uniformly. Differences in the myosin light chain composition of AS and PM muscles were also found by SDS-gel electrophoresis and immunoblot analysis with the anti-light chain McAb, ALD-83. The study clearly indicated that the histochemically homogenous (type IIA) AS muscle is composed of several subpopulations of fibers which differ in their myosin composition and that this heterogeneity of the muscle is not simply due to presence of variable amounts of slow myosin in its fibers.  相似文献   

8.
Monoclonal antibodies (McAbs) specific for the C-proteins of chicken pectoralis major and anterior latissimus dorsi (ALD) muscles have been produced and characterized. Antibody specificity was demonstrated by solid phase radioimmunoassay (RIA), immunoblots, and immunofluorescence cytochemistry. Both McAbs MF-1 (or MF-21) and ALD-66 bound to myofibrillar proteins of approximately 150,000 daltons; the former antibody reacted with pectoralis but not ALD myofibrils, whereas the latter recognized ALD but not pectoralis myofibrils. Chromatographic elution of the antigens from DEAE-Sephadex, and their distribution in the A-band, support the conclusion that both of these antibodies recognize variant isoforms of C-protein. Since both McAbs react with a protein of similar molecular weight in the A-band of all myofibrils of the posterior latissimus dorsi (PLD) muscle, we suggest that either another isoform of C-protein exists in the PLD muscle or both pectoralis and ALD-like isoforms coexist in the A-bands of PLD muscle.  相似文献   

9.
By means of 3H-thymidine autoradiography DNA replicative activity has been studied in cultured atrial and ventricular myocytes, and non-muscle cells from hearts of 2-week-old rats (age when cell proliferation in the myocardium is already significantly depressed). PAS-reaction was used as a cytochemical marker of cardiomyocytes: atrial myocytes are richer in glycogen than ventricular cells. Labeling indices of atrial myocytes after a 24 hour exposure to 3H-thymidine were higher than ventricular ones: on day 6 of culturing--47 and 5%, and on day 11-34 and 8%, respectively. After 10 days of culturing the number of binucleated atrial myocytes, non-typical for atrial myocardium in vivo, increased by 25-40% as compared with 8-13% on days 2-3 in culture. In 10-day cultures, 3- and 4-nucleated atrial myocytes were observed. Both mononucleated and binucleated atrial and ventricular myocytes incorporated 3H-thymidine. To find out whether the deeper inhibition of replicative activity in ventricular myocytes influences fibroblasts and endothelial cells from ventricles, the proliferative activity of non-muscle cells was studied. Non-muscle cells, both in atrial and ventricular cultures, behaved as a totally proliferating population (labeling indices on the 6th day are about 75-90%) and their growth rate decreased during the formation of the contact-inhibited monolayer. These cells, contrary to myocytes, are predominantly mononucleated in all the periods studied. The deeper depression of replication in ventricular myocytes appears to be related with their higher level of differentiation as compared to myocytes of the atrial myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

10.
Summary We have demonstrated that atrial natriuretic peptide-like immunoreactivity is stored and secreted by ventricular and atrial myocytes in dissociated cell culture preparations from the heart of newborn rat. Culture preparations were maintained in either foetal calf serum-supplemented medium 199 or in hormone-supplemented, serum-free medium 199. The presence of atrial natriuretic peptidelike immunoreactivity in the cultured myocytes was demonstrated at both light-and electron-microscopical levels. Release of atrial natriuretic peptide-like immunoreactivity into the culture medium was measured by radioimmunoassay; molecular forms of the stored and secreted peptide were determined by gel column chromatography. The atrial natriuretic peptide-like immunoreactivity of cultured atrial and ventricular myocytes was concentrated in the perinuclear cytoplasm and was localised to electron-dense secretory granules. The number of immunoreactive ventricular myocytes and the intensity of their immunofluorescence changed with time in culture and was higher in cultures in foetal calf serum-supplemented medium than in serum-free medium. Gamma-atrial natriuretic peptide was stored and released by cultured atrial and ventricular myocytes, but was broken down to alpha-atrial natriuretic peptide in the growth medium. This process was foetal calf serum-independent, since it occurred in both the media used, indicating that cardiac myocytes in culture may release a factor that cleaves gamma-atrial natriuretic peptide to form alphaatrial natriuretic peptide.  相似文献   

11.
The goal of this study was to test the hypothesis that the relative amounts of the cardiac myosin heavy chain (MHC) isoforms MHC-alpha and MHC-beta change during development and transition to heart failure in the human myocardium. The relative amounts of MHC-alpha and MHC-beta in ventricular and atrial samples from fetal (gestational days 47--110) and nonfailing and failing adult hearts were determined. The majority of the fetal right and left ventricular samples contained small relative amounts of MHC-alpha (mean < 5% of total MHC). There was a small significant decrease in the level of MHC-alpha in the ventricles between 7 and 12 wk of gestation. Fetal atria expressed predominantly MHC-alpha (mean > 95%), with MHC-beta being detected in most samples. The majority of adult nonfailing right and left ventricular samples had detectable levels of MHC-alpha ranging from 1 to 10%. Failing right and left ventricles expressed a significantly lower level of MHC-alpha. MHC-alpha comprised approximately 90% of the total MHC in adult nonfailing left atria, whereas the relative amount of MHC-alpha in the left atria of individuals with dilated or ischemic cardiomyopathy was approximately 50%. The differences in MHC isoform composition between fetal and nonfailing adult atria and between fetal and nonfailing adult ventricles were not statistically significant. We concluded that the MHC isoform compositions of fetal human atria are the same as those of nonfailing adult atria and that the ventricular MHC isoform composition is different between adult nonfailing and failing hearts. Furthermore, the marked alteration in atrial MHC isoform composition, associated with cardiomyopathy, does not represent a regression to a pattern that is uniquely characteristic of the fetal stage.  相似文献   

12.
Thyroid hormone regulates the expression of ventricular myosin isoenzymes by causing an accumulation of alpha-myosin heavy chain (MHC) mRNA and inhibiting expression of beta-MHC mRNA. However, the mechanism of thyroid hormone action has been difficult to examine in vivo because of its diverse actions. Accordingly, hormonal control of expression of six MHC isoform mRNAs and cardiac and skeletal alpha-actin mRNAs was studied in primary cultures of fetal rat heart myocytes grown in defined medium. The results indicate that in the absence of thyroid hormone, cultured heart cells express predominantly beta-MHC and cardiac alpha-actin mRNAs. Addition of 3,5,3'-triiodo-L-thyronine (T3) caused a rapid induction of alpha-MHC mRNA and decreased beta-MHC mRNA levels without affecting the skeletal muscle MHC mRNAs. There was an almost parallel change in the myosin isoenzymes. Cardiac alpha-actin mRNA levels were transiently increased by T3 treatment, but skeletal alpha-actin was unaffected. Elimination of insulin and epithelial growth factor from the medium did not alter the effects of T3 on cardiac MHC mRNA expression. Addition of various adrenergic agents to the medium had no appreciable effect on cardiac MHC mRNA expression despite the presence of functionally coupled alpha- and beta-adrenergic receptors. Addition of steroid hormones, muscarinic agents, and glucagon to the medium also had no effect. Thus, under defined conditions, T3 is able to regulate MHC gene expression at a pretranslational level without the need for other exogenous factors.  相似文献   

13.
Monoclonal antibodies ( McAbs ) have been generated against a preparation of intermediate filament proteins (IFP) from adult chicken gizzard. Two antibodies, D3 and D76 , have been characterized in detail. They bind specifically to desmin but recognize different epitopes. In the adult chicken, both McAbs produced equivalent immunofluorescent staining patterns, reacting in frozen sections with all forms of muscle tissue, including vascular smooth muscle, but with no other tissue types. In isolated skeletal myofibrils and in longitudinal frozen sections of cardiac and skeletal muscle, desmin was detected with both McAbs at the Z-band and in longitudinally-oriented filament bundles between myofibrils. In contrast to these results in the adult, the intermediate filaments (IF) of embryonic cardiac myocytes in primary cultures were decorated only with McAb D3, whereas McAb D76 was completely unreactive with these cells. Similarly, frozen sections through the heart at early stages of embryonic chick development (Hamburger-Hamilton stages 17-18) revealed regions of myocytes, identified by double immunofluorescence with myosin-specific McAbs , that were unstained with McAb D76 even though similar regions were stained by McAb D3. That McAb D76 reacted with desmin in all adult cardiac myocytes but not with all embryonic heart cells indicates that embryonic and adult cardiac IF are immunologically distinct and implies a conversion in IF immunoreactivity during cardiac development.  相似文献   

14.
In cardiac ventricular myocytes, Na current is generated mainly by the cardiac NaV1.5 isoform, but the presence of "neuronal" Na channel isoforms in the heart has been demonstrated recently. In this study, we quantified the density and sub-cellular distribution of cardiac and neuronal channel isoforms in rat ventricular myocytes. INa was recorded using the patch clamp technique in control and detubulated myocytes. Detubulation reduced cell capacitance (by approximately 29%) but maximum conductance was not altered (1.94+/-0.15, 14 control vs 1.98+/-0.19 nS/pF, 17 detubulated myocytes). The kinetic properties of INa were similar in both cell types suggesting good voltage control of surface and t-tubule membranes. We calculated Na channel densities assuming the sub-cellular current localization we recently provided (neuronal isoform: approximately 11% of total sarcolemmal current, approximately 3% of cell surface, and approximately 31% of t-tubule current). Single channel conductances were assumed to be 2.2 and 2.5 pS for the cardiac and neuronal isoforms, respectively, after accounting for the use of low Na concentration. We calculated that the density of the cardiac Na channel isoform is relatively constant (in channels/microm2: approximately 11 in total sarcolemma, approximately 13 at the cell surface, approximately 10 at the t-tubules). In contrast, neuronal Na channel isoforms are concentrated at the t-tubules (in channels/microm2: approximately 1 in total sarcolemma, approximately 0.3 at the cell surface, approximately 2.5 at the t-tubules). We conclude that, in contrast to skeletal muscle in which Na channel density is higher at the cell surface than the t-tubules, in ventricular cardiac myocytes the sub-cellular distribution of Na channel density is relatively homogeneous (approximately 13 channels/microm2).  相似文献   

15.
P19 embryonal carcinoma cells are multipotential stem cells that differentiate into striated muscle as well as some other cell types when aggregated and exposed to dimethyl sulfoxide (DMSO). Immunofluorescence experiments using monospecific antibodies indicated that the majority of muscle cells were mononucleate and contained four myosin isoforms normally found in cardiac muscle; atrial and ventricular myosin heavy chains, ventricular myosin light chain 1, and atrial myosin light chain 2. Northern blot analysis of RNA isolated from differentiating cultures indicated that cardiac actin and skeletal actin mRNAs were expressed at similar levels and with identical kinetics during the differentiation of P19-derived myocytes. These results demonstrate that most of the P19-derived myocytes are of the cardiac type and suggest that they closely resemble the cells of the early embryonic myocardium.  相似文献   

16.
Alpha- and beta-myosin heavy chain (MHC), the two MHC isoforms expressed in the mammalian heart, differ quantitatively in their enzymatic activities. The MHC composition of the heart can change dramatically in response to numerous stimuli, leading to the hypothesis that changes in cardiac function can be caused by myosin isoform shifts. However, this hypothesis has remained unproven because the stimuli used to generate these shifts are complex and accompanied by many additional physiological changes, including alterations in cardiac mass and geometry. Adult mouse ventricles normally express only alpha-MHC (the faster motor). To determine whether genetic alteration of the MHC isoform composition in the adult mouse heart would result in changes in cardiac chamber mass and contractility, we established transgenic mouse lines that express a Myc-tagged beta-MHC molecule (the slower motor) in adult ventricular tissue, one of which expresses 12% of its myosin as the transgene. There is no evidence of hypertrophy, induction of hypertrophic markers, and no histopathology. Myofibrillar Ca(2+)-activated ATPase activity is decreased by 23%, and Langendorff preparations demonstrate a significant 15% decrease in systolic function in transgenic hearts. These results suggest that even small shifts in the myosin isoform composition of the myocardium can result in physiologically significant changes in cardiac contractility and could be relevant to cardiovascular disease.  相似文献   

17.
Monoclonal antibodies (McAbs) specific for the fast (MF-1) and slow (ALD-66) isoforms of C-protein from chicken skeletal muscle have been produced and characterized. Using these antibodies it was possible to demonstrate that skeletal muscles of varying fiber type express different isoforms of this protein and that in the posterior latissimus dorsi muscle both isoforms are co-expressed in the same myofiber (17, 18). Since we had shown that both isoforms were present in all sarcomeres, it was feasible to test whether the two isoforms co- distributed in the same 43-nm repeat within the A-band, thereby establishing a minimum number of C-proteins per repeat in the thick filaments. Here we describe the ultrastructural localization of C- protein in myofibers from three muscle types of the chicken using these same McAbs. We observed that although C-protein was present in a 43-nm repeat along the filaments in all three muscles, there were marked differences in the absolute number and position occupied by the different isoforms. Since McAbs MF-1 and ALD-66 decorated the same 43- nm repeats in the A-bands of the posterior latissimus dorsal muscle, we suggest that at least two C-proteins can co-localize at binding sites 43 nm apart along thick filaments of this muscle.  相似文献   

18.
A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent.  相似文献   

19.
Atrial and ventricular cardiac muscle cells isolated from the adult squirrel monkey Saimiri sciureus were cultured and characterized by light and transmission electron microscopy (TEM). Freshly isolated cells were striated and cylindrical in shape and, when placed in culture, rounded up and lost their highly organized morphology. With prolonged time in culture, they spread out on the surface of the culture flask and reacquired many of the internal ultrastructural characteristics of their in vivo atrial and ventricular cardiac muscle cell counterparts. Autoradiographic experiments indicated that both atrial and ventricular myocytes synthesized DNA when grown in culture. In some binucleated atrial cells only one nucleus became labelled. These studies show that it is now possible to culture cardiac muscle cells isolated from an adult primate.  相似文献   

20.
cDNA probes and antibodies for TGF-beta s 1, 2, 3, and 4 were used to study the expression of these different TGF-beta isoforms in cultured chicken embryo chondrocytes and cardiac myocytes, as well as in developing cartilage and heart tissues. TGF-beta s 2, 3, and 4 mRNAs, but not TGF-beta 1 mRNA, were detected in cultured chondrocytes and myocytes. Expression of TGF-beta s 2 and 4 mRNAs increased with age, while expression of TGF-beta 3 mRNA was independent of age in chondrocytes cultured from 12- to 17-day-old embryos. In contrast, expression of TGF-beta s 2, 3, and 4 mRNAs was constitutive in myocytes cultured from 7- to 9-day-old embryonic hearts; expression of TGF-beta s 3 and 4 mRNAs increased, while expression of TGF-beta 2 mRNA remained unchanged in myocytes from 10-day-old embryos. Immunoprecipitation studies demonstrated expression of TGF-beta in both the conditioned media and the cell lysates of metabolically labeled chondrocyte and myocyte cell cultures. Immunohistochemical staining of cultured chondrocytes and myocytes and of cartilage and heart tissues of developing chicken embryos with antibodies specific for each TGF-beta isoform showed immunoreactive TGF-beta s 1, 2, 3, and 4. Our results demonstrate coordinate expression of these four TGF-beta isoforms in chicken embryo chondrocytes and myocytes, both in vitro and in vivo, with expression of TGF-beta s 2, 3, and 4 mRNA and protein more prominent than that of TGF-beta 1.  相似文献   

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