Distribution of isomyosin in cultured cardiac myocytes as determined by monoclonal antibodies and adenosine triphosphatase activity

The distribution of isomyosin in cardiac muscle cells in culture has been investigated with monoclonal antibodies and Ca2+-activated myosin ATPase cytochemical staining. With immunofluorescent studies using monoclonal antibodies to isomyosins V1 and V3, the cardiac myocytes grown in a serum-free and thyroxine (T4)-free medium for 7 days contained a predominant population of cells which were strongly reactive to anti-V3 antibody. A small population of myocytes in this culture exhibited weak or no reaction to anti-V3 antibody. When cultures were exposed to anti-V1 antibody, the predominant cardiac myocyte population showed little or no reactivity to this antibody, whereas a small population of the myocytes were strongly reactive. The myosin ATPase staining reaction of the positive myocyte population was significantly less pronounced than that of the V3-negative population which showed a strong reaction. The staining pattern changed dramatically after exposure of cultured myocytes to thyroid hormone for 7 days. Most of the cells were found to react strongly with anti-V1 antibody, while some cells showed little reactivity and some were not stained at all. A small number of cardiac myocytes in this culture showed little or no reactivity to anti-V1 antibody but were strongly reactive to anti-V3 antibody. The predominant anti-V1-positive myocyte population exhibited strong myosin ATPase staining as compared to a smaller V3-positive myocyte population which showed very weak staining. The cytochemical results of ATPase staining in cardiac myocytes agreed well with ATPase activity as determined on pyrophosphate gels containing isomyosin derived from cultured cardiac myocytes with or without T4. This study has demonstrated that cultured myocytes contain a small population of muscle cells which is not responsive to thyroid hormone or to the lack of it.     

Myotoxic effects of clenbuterol in the rat heart and soleus muscle.

Myocyte-specific necrosis in the heart and soleus muscle of adult male Wistar rats was investigated in response to a single subcutaneous injection of the anabolic beta(2)-adrenergic receptor agonist clenbuterol. Necrosis was immunohistochemically detected by administration of a myosin antibody 1 h before the clenbuterol challenge and quantified by using image analysis. Clenbuterol-induced myocyte necrosis occurred against a background of zero damage in control muscles. In the heart, the clenbuterol-induced necrosis was not uniform, being more abundant in the left subendocardium and peaking 2.4 mm from the apex. After position (2.4 mm from the apex), dose (5 mg clenbuterol/kg), and sampling time (12 h) were optimized, maximum cardiomyocyte necrosis was found to be 1.0 +/- 0.2%. In response to the same parameters (i.e., 5 mg of clenbuterol and sampled at 12 h), skeletal myocyte necrosis was 4.4 +/- 0.8% in the soleus. These data show significant myocyte-specific necrosis in the heart and skeletal muscle of the rat. Such irreversible damage in the heart suggests that clenbuterol may be damaging to long-term health.     

Troponin T switching in the developing rat heart

A monoclonal antibody specific for cardiac troponin T has been used to investigate troponin changes during development in the rat heart. Specificity of the antibody was determined by immunoblot analysis with purified bovine cardiac troponin. In the rat heart, immunoblot analysis shows that anticardiac troponin T reacts with a 42.5-kDa band in fetal ventricles and with a 41-kDa band in adult ventricles. The faster migrating troponin T is present in traces in the fetal heart and increases markedly during the first 2 weeks after birth, concomitantly with the progressive decrease of the slower migrating form that is no longer detectable in the adult. The pattern of reactivity of the monoclonal antibody is not modified by alkaline phosphatase pretreatment, suggesting that the antibody is not specific for a phosphorylated epitope. Conditions known to affect cardiac myosin composition, such as hypothyroidism and hypertrophy secondary to systemic hypertension, do not change the troponin T isoform profile of adult rat ventricles. The expression and accumulation of the adult isoforms of troponin T are not suppressed by propylthiouracil treatment of pregnant and nursing rats.     

Effects of IgM from rheumatic fever patients on intracellular calcium levels of neonatal rat cardiac myocytes

Rheumatic fever (RF), a potential sequela of Streptococcus pyogenes pharyngitis, sometimes results in myocarditis and heart failure. Antibodies have been implicated in the pathogenesis of RF and anti-cardiac myosin antibody levels are elevated in RF patients. Since myocarditis is associated with altered cardiomyocyte calcium transients it was of interest to determine the direct effects of RF patient antibodies on calcium transients in cultured myocytes. RF patient polyclonal IgM treatment caused increased calcium retention by neonatal rat heart cells in vitro as determined with isotopically labeled calcium. Therefore, to further characterize this finding, calcium transients were evaluated by real time fluorescence spectroscopy and deconvolution imaging. RF patient polyclonal IgM produced increased calcium retention during the relaxation stage of the contraction cycle leading to a slowing of contraction rate, disorganized calcium transients, and eventual tetany. In contrast, calcium transient studies of cardiomyocytes following treatment with monoclonal anti-myosin antibodies revealed declining intracellular calcium levels, accompanied by disorganized transients and tetany. Treatment with both antibodies led to myocyte dysfunction and these novel findings suggest a role for antibodies in the pathogenesis of the myocarditis associated with rheumatic carditis.     

Immunofluorescent and immunogold replica studies of desmin distribution in cultured normal and cardiomyopathic hamster heart cells.

The architecture of desmin intermediate filament arrangements in cultured cardiomyocytes from heart of normal and cardiomyopathic hamsters was studied by immunofluorescent light microscopy and immunogold replica electron microscopy. Both polyclonal and monoclonal antidesmin antibodies were used in a biotin-streptavidin system. Immunofluorescent staining of normal and cardiomyopathic myocytes for desmin at 5 days in culture exhibited filamentous staining patterns with polyclonal antidesmin and a coarse punctate staining pattern with the monoclonal antibody. At 9 days in culture, most normal myocytes showed filamentous staining with the polyclonal antibody; many of the stained filaments were associated with Z lines. With the monoclonal antidesmin, these same cells exhibited a very fine 'spotty' staining pattern. These results suggest that the arrangements and immunoreactivities of intermediate filaments change during normal cardiac myocyte development. In cardiomyopathic cells, this pattern of rearrangement and immunoreactivity appears to be delayed or possibly nonexistent. The three-dimensional electron-microscopic observation of immunogold localization of desmin achieved by a deep-etching replica technique is made on both normal and cardiomyopathic cultured heart cells. Abnormalities of desmin filament arrangements in cardiomyopathic cells are confirmed.     

Analysis of muscle protein expression in polyethylene glycol-induced chicken: rat myoblast heterokaryons

Heterokaryons derived from polyethylene glycol-mediated fusion of myoblasts at different stages of development were used to investigate the transition of cells in the skeletal muscle lineage from the determined to the differentiated state. Heterokaryons were analyzed by immunofluorescence, using rabbit antibodies against the skeletal muscle isoforms of chicken creatine kinase and myosin, and a mouse monoclonal antibody that cross-reacts with chicken and rat skeletal muscle myosin. When cytochalasin B-treated rat L8(E63) myocytes (Konieczny S.F., J. McKay, and J. R. Coleman, 1982, Dev. Biol., 91:11-26) served as the differentiated parental component and chicken limb myoblasts from stage 23-26 or 10-12-d embryos were used as the determined, undifferentiated parental cell, heterokaryons exhibited a progressive extinction of rat skeletal muscle myosin during a 4-6-d culture period, and no precocious expression of chicken differentiated gene products was detected. In the reciprocal experiment, 85-97% of rat myoblast X chicken myocyte heterokaryons ceased expression of chicken skeletal muscle myosin and the M subunit of chicken creatine kinase within 7 d of culture. Extinction was not observed in heterokaryons produced by fusion of differentiated chicken and differentiated rat myocytes and thus is not due to species incompatibility or to the polyethylene glycol treatment itself. The results suggest that, when confronted in a common cytoplasm, the regulatory factors that maintain myoblasts in a proliferating, undifferentiated state are dominant over those that govern expression of differentiated gene products.     

Changes in cytoplasmic and lysosomal enzyme activities in cultured rat heart cells: the relationship to cell differentiation and cell population in culture

Postnatal rat heart cells in culture enriched with respect to muscle cells were obtained by either high density seeding or by the replating technique. [3H]Thymidine incorporation to DNA and the enzymatic pattern of cytoplasmic and lysosomal enzymes have been studied as a function of the culture's age, of seeding density, and replating. It was shown that replating maintains predominance of myocyte population for at least 2 wk in culture; heavy seeding density allows homogeneous myocyte population for the 1st wk in culture; and the enzyme profile of the culture may serve as an indicator for the type of cell population in culture and its state of differentiation.     

Expression of myoblast and myocyte antigens in relation to differentiation and the cell cycle

Cell cycle parameters and expression of myoblast and myocyte antigens were investigated during exponential growth and during the differentiation phase of rat L8( E63 ) myoblasts by an integrated approach involving microspectrophotometry with DNA fluorochromes, [3H]thymidine autoradiography, and immunofluorescent staining with monoclonal antibodies. In addition to the majority of cells which are recruited into myotubes, two distinct populations of mononucleate cells were resolved in cultures of rat myoblasts undergoing differentiation. These mononucleate cells consist of (1) a population of proliferating cells with a prolonged G1 transit time; (2) a population of non-proliferating cells which remain arrested in G1 for more than 72 h. The latter group was examined with respect to the expression of two marker antigens recognized by two monoclonal antibodies: antibody B58 reacts with a macromolecular component present in undifferentiated myoblasts but not in mature myotubes, and antibody XMlb reacts with a muscle-specific isoform of myosin. All four possible combinations of expression of these antigens by single cells were found: B58 +XM1b -, B58 +XM1b +, B58 - XM1b -, and B58 - XMlb +. The implication of these findings with respect to the transition from the proliferative to the differentiative phase of myogenesis is discussed.     

Evidence for new forms of cardiac myosin heavy chains in mechanical heart overloading and in ageing

Heavy chains of myosin rods and subfragment 1 were isolated from normal hearts and from mechanically overloaded hearts of young and older rats. These myosin heavy-chain fragments were cleaved by cyanogen bromide or partially proteolysed by pronase and by chymotrypsin after denaturation with sodium dodecyl sulfate. The peptides, analyzed by electrophoresis on a one-dimensional polyacrylamide slab gel, varied depending on the origin of the cardiac myosin heavy chains. Some bands present in the peptide patterns of the normal heart of young rats were missing from the pattern of greatly hypertrophied hearts and vice versa. We conclude that mechanical overloading of the heart stimulates the synthesis of cardiac myosin 'isozyme' with a heavy-chain primary structure which is different from that observed in the normal heart of young rat. The patterns from myosin heavy-chain peptides from the hearts of older rats were different from those for peptides from young rat hearts; these results also indicate the presence of a new myosin heavy chain specific to ageing. No difference was detected between the peptide patterns of heavy chains isolated from hypertrophied hearts of young and older rats, and those isolated from normal hearts of older rats.     

Cryptic epitope identified in rat and human cardiac myosin S2 region induces myocarditis in the Lewis rat

Myocarditis is a common cause of dilated cardiomyopathy leading to heart failure. Chronic stages of myocarditis may be initiated by autoimmune responses to exposed cardiac Ags after myocyte damage. Cardiac myosin, a heart autoantigen, induced experimental autoimmune myocarditis (EAM) in susceptible animals. Although cardiac myosin-induced myocarditis has been reported in Lewis rats, the main pathogenic epitope has not been identified. Using overlapping synthetic peptides of the S2 region of human cardiac myosin, we identified an amino acid sequence, S2-16 (residues 1052-1076), that induced severe myocarditis in Lewis rats. The myocarditic epitope was localized to a truncated S2-16 peptide (residues 1052-1073), which contained a sequence identical in human and rat cardiac myosin. The S2-16 peptide was not myocarditic for three other strains of rats, in which the lack of myocarditis was accompanied by the absence of strong S2-16-specific lymphocyte responses in vitro. For Lewis rats, S2-16 was characterized as a cryptic epitope of cardiac myosin because it did not recall lymphocyte and Ab responses after immunization with cardiac myosin. Lymphocytes from S2-16 immunized rats recognized not only S2-16, but also peptides in the S2-28 region. Furthermore, peptide S2-28 was the dominant epitope recognized by T cells from cardiac myosin immunized rats. S2-16 was presented by Lewis rat MHC class II molecules, and myocarditis induction was associated with an up-regulation of inflammatory cytokine production. S2-16-induced EAM provides a defined animal model to investigate mechanisms of EAM and modulation of immune responses to prevent autoimmune myocarditis.     

Expression of TASK-1, a pH-sensitive twin-pore domain K(+) channel,in rat myocytes

We have investigated the expression of TASK-1, a pH-sensitive, twin-pore domain K(+) channel in the rat heart. A mammalian cell line of Chinese hamster ovary cells (CHO), transfected with a plasmid containing mouse TASK-1, demonstrated the specificity of the anti-TASK-1 antibody. TASK-1 expression in cardiac tissue was initially demonstrated by Western blot and then localized by immunofluorescence. In single rat ventricular myocytes, strong staining of the TASK-1 protein was located at the intercalated disks and across the cell in a striated pattern, corresponding to the transverse axial tubular network (T tubules). In contrast, single rat atrial myocytes were stained at the intercalated disks with a weak punctate, striated pattern corresponding to underdeveloped T tubules. Also, formamide was used to induce the detubulation of ventricular myocytes, which enabled confirmation that TASK-1 protein expression occurs in T tubules. Consistent with this, RT-PCR revealed the expression of TASK-1 mRNA in total RNA from both the ventricles and atria. In this study, we conclusively demonstrated that TASK-1 protein and mRNA were expressed in rat atrial and ventricular tissue. The extensive distribution of TASK-1 shown to exist within myocyte membranes may provide a potential future target for antiarrhythmic drugs.     

Localization of cytoplasmic and skeletal myosins in developing muscle cells by double-label immunofluorescence

Antibodies to a cytoplasmic myosin, rat lymphoma myosin, and to rat skeletal myosin were prepared in rabbits and shown to be specific for their corresponding antigens. The two antibodies did not cross-react. The skeletal myosin antibody was directly labeled with rhodamine, and the cytoplasmic myosin antibody was detected by indirect immunofluorescence with fluorescein-labeled goat anti-rabbit antibody. The two antibodies were used to examine developing rat muscle cultures for the presence and location of the antigens. The antibody to cytoplasmic myosin reacted with multinucleated myotubes and with all the mononucleated cells in the culture. The antibody to skeletal myosin reacted with myotubes and with a small fraction of the mononucleated cells. In the myotubes, the cytoplasmic myosin appeared to be localized primarily in two structures: fine stress fibers, often visible also by phase microscopy and present predominantly in the ends of the cells, and in a submembranous rim all along the cell's border. In addition, a diffuse fluorescence within the cells was observed. The skeletal myosin was localized in the central part of the myotubes in sarcomeres or in fibers without periodicities and was excluded from the ends of the myotubes. When the same cells were doubly stained with the two antibodies, the complementary distribution of the two isozymes was very clear. There was also a narrow region of overlap of staining, with cytoplasmic myosin present in some stress fibers that appeared to be continuous with fibrous elements containing skeletal myosin. Myotubes that rounded up with cytochalasin B or with trypsin displayed a diffuse distribution of both isozymes. When these cells were allowed to respread into extended configurations, the location of the two myosins were essentially the same as in untreated cells. The ability of myotubes to adhere to the surface and to move in culture may be related to the presence of cytoplasmic myosin. Our results show that in myotubes and myoblasts the two isozymes differ sufficiently to be localized in distinct regions of the cell and to be sorted out into different structures, even after the cytoplasmic contents have been reshuffled. The cell can, by some unknown mechanism, distinguish the two myosins.     

A comparative study of myosin and its subunits in adult and neonatal-rat hearts and in rat heart cells from young and old cultures.

A possible explanation for the decrease in myosin Ca2+-dependent ATPase activity as rat heart cells age in culture is presented. The subunit structure and enzyme kinetics of myosin from adult and neonatal rat hearts and from rat heart cells of young and old cultures are compared. These studies indicate that the loss in Ca-ATPase activity of myosin from older cultures was an intrinsic property of the myosin itself. Myofibrillar fractions from the indicated four sources showed no qualitative or quantitative differences in electrophoretic patterns. Myosin from older cultures was more sensitive to alkaline denaturation than was myosin from younger cultures, as indicated by its more accelerated loss of K+(EDTA)-dependent ATPase activity after 10 min of incubation at pH 10. Furthermore, myosin from older cultures was more temperature-sensitive, as indicted by a more rapid loss of Ca-ATPase with decrease in assay temperature. It is suggested that there is either a change in conformation of myosin molecules at or near the active site of the enzyme or alternatively there is a change in light chain 1-light chain 2 and/or light-chain-heavy-chain interaction(s) in the myosin molecules under study.     

Characterization of a myosin heavy chain in the conductive system of the adult and developing chicken heart

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.     

In vitro studies of beating heart cells in culture. XVI. The effect of a heart stable serum fraction on the level of myosin adenosinetriphosphatase

A heat stable serum factor of low molecular weight maintains the myosin ATPase activity of cultured rat heart cells. Its action is directly on the heart cells and it does not act by selection of heart muscle cells. It has no effect on heart muscle creatine phosphokinase or lactic dehydrogenase nor on myosin in skeletal muscle cultures.     

Incorporation of nascent myosin heavy chains into thick filaments of cardiac myocytes in thyroid-treated rabbits

A monoclonal antibody (mAb 37) specific for alpha-myosin heavy chain (alpha-MHC) is used to follow the spatial and temporal incorporation of alpha-MHC into rabbit left ventricular myocytes. The expression of the two adult cardiac MHC genes, alpha and beta, is regulated by manipulating the thyroid hormone level of the animal. 10 wk on a propylthiouracil diet down-regulates expression of alpha-MHC to near 0%. alpha-MHC gene expression is up-regulated by injecting L- triiodothyronine (100 micrograms/kg per d) for 1-4 d. This protocol provides a means by which to follow the redistribution pattern of alpha- MHC within the myocyte in vivo. A uniform distribution of immunofluorescent signal is seen within every myocyte throughout the left ventricle. Ultracryomicrotomy without fixation is used to obtain sections for immunogold-electron microscopy. To quantify the immunogold method the density of gold-labeled antibody per unit of area tissue is determined for various regions of the sarcomere. Tissue from normal and 2-wk baby has a uniform distribution of gold density along the length of the A band. The average gold density of the A band increases with days of thyroid injection from 38 +/- 4 grains/micron 2 (n = 2 animals) (mean +/- SE) at day 1 to 182 +/- 59 grains (n = 2 animals) at day 4. There is a nonuniform incorporation of the newly synthesized alpha-MHC within the A band of thyroid-treated animals since 50% more of the alpha-MHC is found at the end of the A band while the center of the A band has 40% less than the average alpha-MHC content (grains/micron 2, n = 7 animals). These results support a thick filament assembly model that allows every myosin in a thick filament to be exchanged with new myosin. However, in the intact functioning myocyte, there is greater exchange of new myosin at the ends than in the central region of the thick filament.     

Role of cell division in the cytodifferentiation of rat heart cells in culture

BrdU and irradiation with visible light eliminates dividing cells from rat heart cell cultures. The lethal effect of light on the cultures is dependent upon the prior integration of BrdU into the DNA. Elimination of dividing cells is shown by the decreased uptake of ³H TdR in the remaining cells. Cultures in which the dividing cells were eliminated displayed a loss in myosin ATPase activity and a decrease in the rate of myosin ATPase activity increase, normally seen in control cultures. These results are consistent with the existence of cells at three stages of development; premyoblasts, which divide and contain no myosin; myoblasts which divide and contain myosin; and myocytes, which cannot divide and contain myosin. The results also indicate that the increase in myosin ATPase activity normally seen in heart cell cultures is a result of an increase in myosin in fully developed cells and in the increase in the number of cells capable of synthesizing myosin.     

Expression of myosin isoenzymes in cardiac-muscle cells in culture.

Myosin isoenzyme profiles of rat and chicken embryonic cardiac myocytes were studied during differentiation and growth in vitro by native-gel electrophoresis and assay of Ca2+-activated ATPase. The electrophoretic pattern of myosin extracted from 18-day-embryonic-rat myocytes after 7 days in culture exhibits three isoenzyme bands, V1, V2 and V3, of which the slow-migrating V3 is predominant. This resembles the isoenzyme profiles from 18-20-day-embryonic ventricles in vivo. However, the isoenzyme profile of the 7-day-old culture differs from that of its counterpart in vivo, as well as from that of the young and adult rat ventricles, the last two containing the predominant fast-migrating component, V1. When embryonic cardiac myocytes were grown in vitro for 7 days in a medium containing a physiological concentration of L-thyroxine (T4), myosin isoenzyme profiles of these cells shifted to the adult form, with isoenzyme V1 predominating after day 4 of culture. The 7-day-old intact embryonic-chicken ventricles and isolated myocytes showed a single myosin isoenzyme band after 7 days of culture that resembles the pattern seen for the adult chicken. T4 had no effect on the electrophoretic mobility of this isoenzyme pattern. ATPase activity of isoenzyme V1 in cultured rat myocytes treated with T4 was comparable with that of V1 in the untreated adult heart. This study demonstrates that ATPase activity of the chicken myosin isoenzyme is significantly lower than that of isoenzyme V1, but is comparable with that of rat V3. This study shows that the expression of myosin isoenzyme profiles in cultured rat cardiac myocytes does not fully represent the situation in vivo. Physiological concentrations of T4 can modulate the predominant foetal-type isoenzyme V3 to the adult type V1 in cultured embryonic-rat cardiac myocytes within a brief period.     

Power output is linearly related to MyHC content in rat skinned myocytes and isolated working hearts

The amount of work the heart can perform during ejection is governed by the inherent contractile properties of individual myocytes. One way to alter contractile properties is to alter contractile proteins such as myosin heavy chain (MyHC), which is known to demonstrate isoform plasticity in response to disease states. The purpose of this study was to examine myocyte functionality over the complete range of MyHC expression in heart, from 100% alpha-MyHC to 100% beta-MyHC, using euthyroid and hypothyroid rats. Peak power output in skinned cardiac myocytes decreased as a nearly linear function of beta-MyHC expression during maximal (r2 = 0.85, n = 44 myocyte preparations) and submaximal (r2 = 0.82, n = 31 myocyte preparations) Ca2+ activation. To determine whether single myocyte function translated to the level of the whole heart, power output was measured in working heart preparations expressing varied ratios of MyHC. Left ventricular power output of isolated working heart preparations also decreased as a linear function of increasing beta-MyHC expression (r2 = 0.82, n = 34 myocyte preparations). These results demonstrate that power output is highly dependent on MyHC expression in single myocytes, and this translates to the performance of working left ventricles.