Myosin in primary cultures of hamster heart cells.

Primary cultures of neonatal hamster heart cells contained a heterogeneous population of cell types. Muscle cells represented one such type, and could be identified by their ability to contract spontaneously in culture and by their morphology when examined directly by phase contrast microscopy or after staining with phosphotungstic acid-hematoxylin. Overgrowth of muscle cells by more rapidly dividing nonmuscle cells was prevented by treatment of the cultures with the mitotic inhibitor, 1-β-d-arabinofuranosylcytosine. The muscle cells increased their specific content of myosin over a period of 5 days in culture. They were then, however, relatively immature when compared with the predominating cells in 1-day-old hamster hearts. The structural and enzymatic properties of purified myosin from the cultures were not different from those of the isoenzyme type of this protein normally found in neonatal hamster hearts, and nonmuscle cells contributed little of the total myosin.     

Histochemical demonstration of myosin Ca2+-ATPase accumulation in primary cultures of skeletal and heart muscle cells

Summary A modified histochemical technique is described for the improved detection of myosin Ca²⁺-ATPase activity in single muscle cells in culture. The method was used to demonstrate the increase in myosin Ca²⁺-ATPase activity in differentiating chick skeletal muscle cells. Functional muscle cells were also positively identified in the heterogeneous cell population of primary hamster heart cell cultures. An age-dependent increase in the number of cells with high levels of myosin ATPase activity in mitotically arrested heart cell cultures was shown. Maturation of individual muscle cells could thus be evaluated.     

Effects of 5-bromo-2''-deoxyuridine on beating heart cell cultures from neonatal hamsters.

1. Primary heart cell cultures from neonatal hamsters yielded a heterogeneous cell population, containing muscle cells undergoing progressive differentiation, as well as non-muscle cells. 2. Addition of 5-bromo-2'-deoxyuridine, at an early stage, to such cultures enhanced the formation of beating sheets of differentiated muscle cells. Accumulation of myosin heavy chains and creatine kinase also occurred in the presence of the analogue. 3. To obtain these effects, the analogue had to be added during the initial rapid growth phase of the cells. Division of the treated cells then ceased when the cell numbers had approximately doubled. 4. Similar results were obtained with other inhibitors of DNA synthesis. Thus improved muscle cell cultures can be obtained by preventing non-muscle cells from overgrowing the cultures. 5. One effect caused only by 5-bromo-2'-deoxyuridine was a large increase in the Ca2+-stimulated ATPase (adenosine triphosphatase) activity which sedimented at low ionic strength. This increase was not due to a greater content of myofibrillar myosin, or to myosin isoenzyme changes, because purified myosin prepared from treated and untreated cultures did not exhibit the increased Ca2+-stimulated ATPase activity.     

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.     

Immunochemical analysis of myosin heavy chains in the developing chicken heart

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.     

The use of 5-bromodeoxyuridine and irradiation for the estimation of the myoblast and myocyte content of primary rat heart cell cultures

A method for killing dividing cells (Puck and Kao, '67) was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo-natal rat heart. Cells were cultivated in BUdR (5-bromodeoxyuridine) 10(-4) M for 3 days and then irradiated with long UV light. The selective elimination of dividing cells led to a loss of myosin Ca2+-activated ATPase in the cultures. This indicates the presence of dividing cells which contain myosin. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1-day postnatal rats and 48% in cultures from 4-day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages.     

A new state of cardiac myosin with very slow ATP turnover: a potential cardioprotective mechanism in the heart

The mechanisms that control cardiac contractility are complex. Recent work we conducted in vertebrate skeletal muscle identified a new state of myosin, the super-relaxed state (SRX), which had a very low metabolic rate. To determine whether this state also exists in cardiac muscle we used quantitative epi-fluorescence to measure single nucleotide turnovers by myosin in bundles of relaxed permeable rabbit ventricle cells. We measured two turnover times—one compatible with the normal relaxed state, and one much slower which was shown to arise from myosin heads in the SRX. In both skeletal and cardiac muscle, the SRX appears to play a similar role in relaxed cells, providing a state with a very low metabolic rate. However, in active muscle the properties of the SRX differ dramatically. We observed a rapid transition of myosin heads out of the SRX in active skeletal fibers, whereas the population of the SRX remained constant in active cardiac cells. This property allows the SRX to play a very different role in cardiac muscle than in skeletal muscle. The SRX could provide a mechanism for decreasing the metabolic load on the heart, being cardioprotective, particularly in time of stress such as ischemia.     

Subcellular compartmentalization of myosin isoforms in embryonic chick heart ventricle myocytes during cytokinesis

Embryonic chick heart ventricle myocytes retain the ability to alternate between proliferation and functional differentiation. A cytoplasmic isoform of myosin is present in cleavage furrows of various nonmuscle cells during cytokinesis, whereas one or more of the cardiac myosin isoforms are localized in sarcomeres of beating cardiomyocytes. Antibodies were employed to reveal the subcellular localizations of cytoplasmic and cardiac myosin isoforms in embryonic chick ventricle cardiomyocytes during cytokinesis. Monoclonal anticytoplasmic myosin antibodies were prepared against myosin purified from brains of 1-day-posthatched chickens and shown to react with chick brain myosin heavy chain by Western blots and/or ELISA tests. One monoclonal antibrain myosin antibody also cross-reacted with chick cardiac myosin but not with skeletal or smooth muscle myosins. Two antichick cardiac myosin monoclonal antibodies and one antichick skeletal myosin polyclonal antibody that cross-reacts with cardiac myosin were employed to identify cardiac sarcomeric myosin. Cells were isolated from day 8 embryonic chick heart ventricles, enriched for myocytes, grown in vitro for 3 days, and then examined by immunofluorescence techniques. Monoclonal antibodies against cytoplasmic myosin preferentially localized in the cleavage furrows of both cardiofibroblasts and cardiomyocytes in all stages of cytokinesis. In contrast, antibodies that recognize cardiac myosin were distributed throughout cardiomyocytes during early stages of cytokinesis, but became progressively excluded from the furrow area during middle and late stages of cytokinesis. These data suggest that in cells that contain both cytoplasmic and sarcomeric myosin isoforms, only cytoplasmic myosin isoforms are mobilized to from the contractile ring for cytokinesis.     

The translation of chick skeletal muscle poly (A)-containing mRNA in primary heart muscle cells in culture

Chick skeletal muscle mRNA (40 μg/0.8 ml) in phosphate-buffered saline was added to 60 mm petri dishes containing a monolayer of primary heart muscle cells. After 30 minutes absorption the cultures were supplemented with complete medium and the incubation continued in a humidified CO₂-incubator. Sucrose density gradient analysis of the absorbed RNA showed no degradation. This skeletal muscle mRNA was translated in primary heart muscle cells in culture into functional proteins as indicated by the linear increase in the accumulation of acetylcholine receptors as well as a similar increase in creatine kinase activity. In addition, the synthesis of the three unique light chains of skeletal muscle myosin (SLC₁, SLC₂, SLC₃) in these primary heart muscle cells was also demonstrable.     

Skeletal muscle contractile protein function is preserved in human heart failure

Skeletal muscle weakness is a common finding in patients with chronic heart failure (CHF). This functional deficit cannot be accounted for by muscle atrophy alone, suggesting that the syndrome of heart failure induces a myopathy in the skeletal musculature. To determine whether decrements in muscle performance are related to alterations in contractile protein function, biopsies were obtained from the vastus lateralis muscle of four CHF patients and four control patients. CHF patients exhibited reduced peak aerobic capacity and knee extensor muscle strength. Decrements in whole muscle strength persisted after statistical control for muscle size. Thin filaments and myosin were isolated from biopsies and mechanically assessed using the in vitro motility assay. Isolated skeletal muscle thin-filament function, however, did not differ between CHF patients and controls with respect to unloaded shortening velocity, calcium sensitivity, or maximal force. Similarly, no difference in maximal force or unloaded shortening velocity of isolated myosin was observed between CHF patients and controls. From these results, we conclude that skeletal contractile protein function is unaltered in CHF patients. Other factors, such as a decrease in total muscle myosin content, are likely contributors to the skeletal muscle strength deficit of heart failure.     

Evidence for distinct phosphorylatable myosin light chains in avian heart and slow skeletal muscle

In mammalian organisms the regulatory or phosphorylatable myosin light chains in heart and slow skeletal muscle have been shown to be identical and presumable constitute the product of a single gene. We analyzed the expression of the avian cardiac myosin light chain (MLC) 2-A in heart and slow skeletal muscle by a combination of experimental approaches, e.g., two-dimensional gel electrophoresis of the protein and hybridization of mRNA to specific MLC 2-A sequences cloned from chicken. The investigations have indicated that, unlike in mammals, in avian organisms the phosphorylatable myosin light chains from heart and slow skeletal muscle are distinct proteins and therefore products of different genes. The expression of MLC 2-A is restricted to the myocardium and no evidence was found that it is shared with slow skeletal muscle.     

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.     

X-ray diffraction patterns from mammalian heart muscle

We have obtained light and X-ray diffraction patterns from trabecular and papillary muscles of various mammalian hearts in the living resting state and in rigor. Equatorial X-ray diffraction patterns from living muscles show the 1,0 and 1,1 reflections from a hexagonal lattice of filaments. The lattice spacing varies with sarcomere length over the observable range (2·0 to 2·5 μm) in such a manner that the lattice volume remains constant. In the living resting state the 1,0 reflection is stronger than the 1,1 reflection, whereas in rigor the 1,1 reflection is almost as strong as the 1,0 reflection. These intensity changes are similar to those found in vertebrate skeletal muscle, suggesting that the mechanism of cross-bridge attachment to actin is similar in both muscles.Two types of meridional X-ray diffraction pattern were observed in muscles in different conditions. One type, obtained from dead or glycerol-extracted muscles or from muscles treated with iodoacetate, showed a strong actin-related pattern but only a weak pattern associated with myosin. This type of pattern was similar to that from vertebrate skeletal muscle in rigor. The other type, obtained from living, resting muscle, showed a weaker actin pattern but a stronger myosin pattern. The myosin pattern included layer-line reflections associated with projections from the thick filaments. This second type of pattern was similar to that from resting vertebrate skeletal muscle, but the layer lines were weaker. The weakness of the myosin layer lines may indicate that part of the high resting tension found in heart muscle arises from a small amount of actin-myosin interaction in the resting state. Such interaction could provide a mechanism for varying the diastolic length of heart muscle and thereby the diastolic volume of the heart.     

Myosin types and fiber types in cardiac muscle. III. Nodal conduction tissue

The sinoatrial (SA) and atrioventricular (AV) nodes are specialized centers of the heart conduction system and are composed of muscle cells with distinctive morphological and electrophysiological properties. We report here results of immunofluorescence and immunoperoxidase studies on the bovine heart showing that a large number of SA and AV nodal cells share a distinct type of myosin heavy chain (MHC) which is not found in other myocardial cells and can thus be used as a cell-type-specific marker. The antibody used in this study was raised against fetal skeletal myosin and reacted with fetal skeletal but not with adult skeletal MHCs. Both atrial and ventricular fibers, as well as fibers of the ventricular conduction tissue were unlabeled by this antibody. Specific reactivity was exclusively seen in most cells in the central portions of the SA and AV nodes and rare cells in perinodal areas. However, a number of nodal cells, particularly those located in the peripheral nodal regions, were unreactive with this antibody. The myosin composition of nodal tissues was also explored using two antibodies reacting specifically with alpha-MHC, the predominant atrial isoform, and beta-MHC, the predominant ventricular isoform. Most nodal cells were reactive for alpha-MHC and a number of them also for beta-MHC. Variation in reactivity with the two antibodies was also observed in perinodal areas: at these sites a population of large fibers reacted exclusively for beta-MHC. These findings point to the existence of muscle cell heterogeneity with respect to myosin composition both in nodal and perinodal tissues.     

Human heart muscle proteins: two-dimensional electrophoretic analysis in embryos

Human myocardium proteins synthesized in the course of heart muscle development have been analyzed by two-dimensional electrophoresis. The quantitative change was found in representation of the main retractable proteins in the course of the heart muscle formation (the light myosin chains, tropomyosin, etc.). Four polymorphous variants of myocardium proteins were found, one of which is, possibly connected with the defect in heart development.     

Development of heart cells in culture: studies using an affinity purified antibody to a myosin light chain

Cultured neonatal rat heart cells can be used to study the factors that regulate cardiac contractility and myocyte development in vitro. An antibody to the 26,000 dalton light chain of myosin (MLC1), has been produced and purified on a Sepharose 4B affinity column prepared with rat heart myosin. When primary cultures of myocytes are studied by indirect immunofluorescence using this antibody a predictable pattern of myofibrillar structure is observed to develop over 72 h. This myosin cytoskeleton is highly organized and the myosin fibrils exhibit cross striations. The antibody does not stain non-muscle heart cells and there is no evidence for myocyte division in culture. The qualitative immunofluorescent pattern of myosin organization is the same in both spontaneously beating and in non-contracting cells.     

Cardiac-muscle hypertrophy. Differentiation and growth of the heart cell during development.

An experimental model for the study of the control of tissue growth by cell proliferation (hyperplasia) and cell enlargement (hypertropht) is proposed. The model is constructed on the basis of the fact that, when DNA replication and cell proliferation cease in cardiac muscle during neonatal development, subsequent growth of the ventricular tissue is due to cell enlargement. The time period during development when this change in growth pattern occurs is documented with the cessation of DNA replication and the synthesis and accumulation of myosin as biochemical parameters. It is suggested that adrenergic innervation of the heart may be the physiological signal that changes the growth pattern of the muscle from hyperplasia to hypertrophy.     

Monoclonal antibodies distinguish titins from heart and skeletal muscle

Murine monoclonal antibodies specific for titin have been elicited using a chicken heart muscle residue as antigen. The three antibodies T1, T3, and T4 recognize both bands of the titin doublet in immunoblot analysis on polypeptides from chicken breast muscle. In contrast, on chicken cardiac myofibrils two of the antibodies (T1, T4) react only with the upper band of the doublet indicating immunological differences between heart and skeletal muscle titin. This difference is even more pronounced for rat and mouse. Although all three antibodies react with skeletal muscle titin, T1 and T4 did not detect heart titin, whereas T3 reacts with this titin both in immunofluorescence microscopy and in immunoblots. Immunofluorescence microscopy of myofibrils and frozen tissues from a variety of vertebrates extends these results and shows that the three antibodies recognize different epitopes. All three titin antibodies decorate at the A-I junction of the myofibrils freshly prepared from chicken skeletal muscle and immunoelectron microscopy using native myosin filaments demonstrates that titin is present at the ends of the thick filaments. In chicken heart, however, antibodies T1 and T4 stain within the I-band rather than at the A-I junction. The three antibodies did not react with any of the nonmuscle tissues or permanent cell lines tested and do not decorate smooth muscle. In primary cultures of embryonic chicken skeletal muscle cells titin first appears as longitudinal striations in mononucleated myoblasts and later at the myofibrillar A-I junction of the myotubes.     

Detection of a ventricular-specific myosin heavy chain in adult and developing chicken heart

In the present study, a monoclonal antibody (McAb), ALD19, generated against myosin of slow tonic muscle, was shown to react with the heavy chain of ventricular myosin in the adult chicken heart. With this antibody, it was possible to detect a ventricular-specific myosin during myocardial differentiation and to show that the epitope recognized by ALD19 was present from the earliest stages of ventricular differentiation and maintained throughout development only in the ventricle. A second McAb, specific for atrial myosin heavy chain (MHC) (Gonzalez-Sanchez, A., and D. Bader, 1984, Dev. Biol., 103:151-158), was used as a control to detect an atrial-specific myosin in the caudal portion of the developing heart at Hamburger-Hamilton stage 15. It was found that the appearance of ventricular MHC predated the expression of atrial MHC by approximately 1 d in ovo and that specific MHCs were always differentially distributed. While a common primordial MHC may be present in the early heart, this study showed the tissue-specific expression of a ventricular MHC during the initial stages of heart development and its differential accumulation throughout development.