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.     

Adult cardiac muscle cells in long-term serum-free culture: Myofibrillar organization and expression of myosin heavy chain isoforms

Summary A culture system for adult rat cardiac muscle cells has been established without exposure of cells to serum at any step of the procedure. The methodology has been standardized and optimized to obtain better quality and high yield of cells and culture. Subsequent to enzyme perfusion, the release of myocytes from enzyme-perfused tissues was carried out in enzyme-free Joklik's medium instead of exposing cells to proteolytic enzyme(s) as done previously. Approximately 5 million cylindrical muscle cells per ventricle were obtained. The culture medium contained Eagle's minimum essential medium with Earle's salts, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, selenium, norepinephrine, triiodothyronine (T₃), bovine serum albumin, nonessential amino acids, and ascorbic acid. The plating efficiency of the experimental cultures was comparable to that of the control cultures grown in the presence of serum. The cells in the serum-free medium contained myofibrillar and myosin isoforms characteristics of the adult myocytes. The cells underwent cellular reorganization comparable to that of the controls. The initial phase of reorganization involved the breakdown of myofibrils and extrusion of mitochondria, degraded myofibrils, and other cellular organelles. The latter phase of reorganization included myofibrillogenesis and organellogenesis resulting in the development of myofibrillar apparatus with cellular organelles. Myocytes were contractile throughout the culture period. Cardiac myocytes grown, in serum-free medium expressed the predominant myosin isoform V1 similar to their counterparts in vivo. T₃ is essential for the expression of isomyosin V1. This study demonstrates that adult cardiac muscle cells can be maintained in long-term serum-free culture from seeding to termination. The cells in serum-free conditions maintain at least two differentiated characteristics of adult myocytes investigated, namely, abundant organized myofibrils and predominant myosin isoform V1. This work is supported by grant DCB-8709594 from the National Science Foundation, Washington, DC     

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.     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult cardiac muscle cells.

The effect of a tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the expression of myosin heavy chain isoforms in cultured rat cardiac ventricular muscle cells was studied. The previous preliminary report [Claycomb WC (1988): "Biology of Isolated Adult Cardiac Myocytes." In Clark WA, Decker RS, Borg TK (eds): New York: Elsevier, pp 284-287] indicated that TPA turns off the expression of myosin heavy chain genes in cultured adult cardiac myocytes. Electrophoretic and immunocytochemical analyses were carried out in the present studies. The myosin heavy chain isoform profiles of cardiac myocytes exposed to TPA at concentrations of 50-250 ng/ml culture medium for varying periods were similar to those of controls that were grown in the absence of TPA, showing predominant isoform V1. Immunofluorescence microscopy with monoclonal antibodies to cardiac ventricular isomyosin revealed the structural organization of myosin in TPA-treated cells. The organization of myosin was variable among different myocytes and within a single myocyte. Immunofluorescence microscopy was extended to the examination of the organization of alpha-actinin which did not differ from that of myosin in some myocytes. In contrast to the previous report [Claycomb, 1988], this study has demonstrated that TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult ventricular cardiac muscle cells.     

Changes in myosin isozymes during development of chicken breast muscle

The patterns of myosin isozymes in embryonic and adult chicken pectoralis muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light chains and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, the predominant isozyme component in embryonic pectoralis myosin could be clearly distinguished from adult myosin isozymes. SDS-polyacrylamide gel electrophoresis indicated that the light chain composition of embryonic myosin was also different from that of adult myosin. The pattern of peptide fragments produced by myosin digestion with a-chymotrypsin differed significantly between embryonic and adult skeletal myosin. These results suggest that myosin in the embryonic pectoralis muscle is different in both light and heavy chain composition from myosin in the same adult tissue.     

Hereditary pituitary dwarfism in mice affects skeletal and cardiac myosin isozyme transitions differently

The dwarf mutation in mice interferes with the development of those anterior pituitary cells responsible for production of thyroid stimulating hormone, growth hormone, and prolactin. Myosin isozyme transitions in both cardiac and skeletal muscle were also found to be affected in this mutant. Electrophoresis of native myosins demonstrated that the fetal (V3) to adult (V1) ventricular cardiac isozyme transition was completely blocked in dwarf mice; in contrast, the neonatal to adult fast myosin transition in hind limb skeletal muscle was slowed but not totally inhibited. The persistence of neonatal myosin heavy chain for up to 55-75 d after birth in dwarf mice, as compared with 16 d in normal mice, was directly demonstrated by polypeptide and immunopolypeptide mapping. Morphological examination of 18-36-d-old dwarf skeletal muscles by optical and electron microscopy revealed a relative immaturity, but no signs of gross pathology were evident. Immunocytochemical analysis showed that the abnormal persistence of neonatal myosin occurs in most of the fibers. Multiple injections of thyroxine restored a normal isozyme complement to both cardiac and skeletal muscles within 11-15 d. Therefore, the effects of the dwarf mutation on myosin isozymes can be explained by the lack of thyroid hormone in these animals. Because the synthesis of growth hormone is not stimulated by thyroid hormone in dwarf mice as it would be in normal animals, these results demonstrate that thyroid hormone promotes myosin isozyme transitions independent of growth hormone production.     

Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells

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.     

Enzymatic comparisons between light chain isozymes of human cardiac myosin subfragment-1

Human cardiac ventricular myosin subfragment-1 (S-1) was prepared by chymotryptic digestion of myosin purified from adult and fetal hearts. The enzymatic properties of adult S-1 were compared to those of two light chain isozymes of fetal S-1 which were separated by ion-exchange chromatography. One fetal isozyme contained a light chain (LC) indistinguishable from the adult ventricular LC1 and the other fetal isozyme contained the LC1 variant that is a component of intact fetal myosin. The fetal isozymes had identical actin-activated Mg2+ ATPase rates at all actin concentrations, as well as the same K+EDTA, Ca2+, and Mg2+ATPase rates. Furthermore, both fetal isozymes had the same actin-activated Mg2+ATPase rates as S-1 purified from adult hearts. The K+EDTA and Ca2+ATPase rates of adult S-1 were only slightly different from those of fetal S-1. These observations are consistent with other available data suggesting that human fetal and adult ventricular myosin differ only in light chain content, not in heavy chain composition, and indicate that isozymic LC1 variation does not alter the steady-state ATPase rate of human cardiac S-1.     

DNA synthesis of adult mammalian cardiac muscle cells in long-term culture

Adult rat cardiac ventricular muscle cells were isolated and cultured in monolayer for 30-45 days. Most of the cardiac muscle cells undergo external and internal structural alterations, resembling embryonic/neonatal cardiac muscle cells in culture (Nag and Cheng, 1981; Nag et al., 1983). These cultured cells underwent DNA synthesis and mitosis as revealed by autoradiography studies that involved the exposure of the cells to [3H]-thymidine for 24 hr prior to the termination of the culture at selected intervals. During the first week of culture, cardiac muscle cells showed less than 5% labeled cells. The labeling index of myocytes attained a peak in the second week of culture, exhibiting approximately 23% labeled cells. The labeling indices of cardiac muscle cells declined over the period of 30 days of culture. During the end of the incubation period, approximately 4% of the myocytes were labeled. When the extent of the total cell population involved in DNA synthesis was examined by exposing the cells to [3H]-thymidine continuously for long periods of time, it was observed that approximately 26% of the cardiac muscle cells regained the capacity for DNA synthesis during 1-10 days of culture. From day 1 to day 14, approximately 29% of the total muscle cell population was labeled. When the cells were exposed to the radioactive isotope continuously for 30 days, approximately 31% of the cells incorporated radioactive isotope, showing their capacity for DNA synthesis. Approximately 90% of the cardiac muscle cells in long-term culture contained more than one nucleus. The nuclei were often observed in multiples of two. Labeled mitotic apparatus was observed in cardiac myocytes, indicating the replication of DNA, followed by karyokinesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myosin transitions in developing fast and slow muscles of the rat hindlimb

Abstract. Myosin isozymes from the slow soleus and fast EDL muscles of the rat hindlimb were analyzed by pyrophosphate gel electrophoresis, by peptide mapping of heavy chains, and by antibody staining. At the earliest stage examined, 20 days gestation, distinctions between the developing fast and slow muscles were seen by all these criteria; all fibers in the distal hindlimb reacted strongly with antibody to adult fast myosin. Some fibers also reacted with antibody to adult slow myosin; these fibers had a precise, axial distribution in the hindlimb. This pattern of staining which includes the entire soleus, foreshadows the adult distribution of slow fibers and may indicate that the specific pattern of innervation of the limb is already determined. In the early developing soleus there are four fetal and neonatal isozymes plus two isozymes present in equal proportions in the 'slow' area of the pyrophosphate gel. The mobility of these two slow isozymes decreases with maturity and the slowest moving isozyme gradually becomes the dominant species. Thus early diversity between the soleus and EDL is expressed by myosins which are distinct from the mature isozymes. The relative proportion of slow isozymes significantly increases with development and as this occurs the fetal and neonatal isozymes are progressively eliminated. Transiently at least one mature fast isozyme appears in the soleus. This is present at 15 days postpartum and probably correlates with the population of fast, type II fibers, which comprise 50% of this muscle cell population at 15 days. The EDL contained three fetal and neonatal isozymes and only one slow isozyme which does not change in mobility with age. Slow isozymes in the soleus and EDL are thus not identical. Each muscle underwent a unique series of changes until the adult pattern of isozymes and heavy chains was reached about one month postpartum.     

Myosin isozyme transitions occurring during the postnatal development of the rat soleus muscle

The myosin isozymes present in the developing rat soleus muscle from 1 week to 6 weeks after birth were investigated using biochemical and immunological methods. Electrophoresis of native myosin reveals that adult slow myosin is present in the soleus as early as 1 week after birth. At this time, embryonic and neonatal myosin can also be demonstrated. Using an immunotransfer technique, the presence of slow myosin heavy chain can be demonstrated at all time points examined whereas neonatal myosin heavy chain diminishes in quantity between 2 and 3 weeks, and is undetectable in the adult soleus. Specific polyclonal antibodies were prepared to embryonic, neonatal, and adult fast and slow myosins. Immunocytochemistry reveals a cellular heterogeneity at all stages examined. Different combinations of myosin isozymes can be found in the soleus fibers depending on the stage of development; these results suggest therefore that myosin isozyme transitions are occurring. Approximately half the fibers contain embryonic and slow myosin at 1 week after birth; these fibers subsequently contain only slow myosin. A second group of fibers contains embryonic and neonatal myosin at 1 week and most of them subsequently accumulate adult fast myosin. A portion of this latter group begins to acquire slow myosin from 4 weeks of age. These data are interpreted to suggest that a preprogrammed sequence of myosin isozymes is embryonic----neonatal----adult fast. At any time during development of an individual fiber, induction of slow myosin accumulation and repression of other types can occur.     

Cell-specific properties of type V and type IX adenylyl cyclase isozymes in 293T cells and embryonic chick ventricular myocytes

The cDNAs for types V and IX adenylyl cyclases were cloned from a chicken heart library and expressed in 293T cells (plasmid transfection) and in embryonic chick ventricular myocytes (adenovirus infection). Expression of type V or IX cyclases in 293T cells resulted in increases in basal and isoproterenol (ISO)-stimulated cAMP levels, whereas the expression of type V, but not type IX, cyclase increased forskolin (FK)-stimulated cAMP levels. Expression of type V cyclase in cardiac myocytes increased basal and FK-stimulated cAMP levels, variably increased ISO-stimulated cAMP levels, and decreased the content of beta-adrenergic receptors (betaARs). The expression of type IX cyclase in cardiac myocytes increased basal and ISO-elevated cAMP levels and, surprisingly, increased the cAMP-elevating effect of FK. The finding that FK responses are increased in cardiac myocytes but not in 293T cells expressing the type IX cyclase suggests that the host cell influences the properties of the type IX isozyme.     

Different distributions of microtubules, desmin filaments and isomyosins during the onset of cardiac hypertrophy in the rat

To elucidate the role of the cytoskeleton in the development of adult heart, microtubules and intermediate filaments of desmin were studied in young and adult rat heart myocytes during the onset of growth, after mechanical overloading induced by aortic stenosis. Such overloading is known to cause heart hypertrophy by stimulating overall protein synthesis, and to initiate a shift in myosin isozymes. For this study, we used double immunolabelling of isolated myocytes with specific antibodies raised against tubulin, desmin, and the two main isomyosins V1 and V3. Whereas desmin remained unchanged, tubulin was redistributed in arrays parallel to the long axis of the myocytes, and was densest around the nuclei. Alterations in the microtubule pattern were observed very early after aortic stenosis, during the onset of heart growth; they were transitory, and did not occur simultaneously in all myocytes. Chronological examination of myocytes labelling with both antitubulin and anti V3 myosin clearly suggested that the transitory alteration in the microtubule pattern was an early event preceding the change in the expression of the myosin gene. Results, observed in young rats, in which mitosis is stimulated by overloading, and in adult rats, exhibiting no mitosis, showed that microtubules are involved in the development of cells in which mitosis does not occur. This work provides the first evidence of a correlation in functional adult heart, between the reorganization of cytoplasmic microtubules and the onset of growth.     

Hormonal regulation of myosin heavy chain and alpha-actin gene expression in cultured fetal rat heart myocytes

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.     

Factors controlling embryonic heart cell proliferation in serum-free synthetic media

Summary Embryonic chick cardiac cell cultures, plated on collagen-coated dishes, containing serum-free synthetic media proliferate actively. The basic medium contained Ham's F12 nutrient mixture, fetuin, ascorbic acid, and bovine serum albumin. This medium was supplemented with various combinations of factors; endothelial cell growth supplement (ECGS), epidermal growth factor (EGF), insulin (I), transferrin (T), selenium (S), hydrocortisone, and thyroxine or supplemented alone. Basic medium supplemented with ECGS alone contributes to the highest final cell density among all other factors used in various combinations or alone. The final cell density of the control culture with 2% fetal bovine serum was higher than those of all experimental cultures and an additional control culture grown in the basic medium. Combinations of factors without ECGS do not promote significant cell proliferation. Thyroxine is required to induce optimal differentiation and contractility of cardiac myocytes in vitro. Fibronectin and laminin did not show any more influence than collagen did on the growth and maintenance of cardiac myocytes in serum-free media. The proportion of cardiac muscle cells in ECGS-containing media was higher than those in other experimental media and control media with the exception of ECGS and ITS-containing medium that showed lower proportion of cardiac myocytes than that of serum-containing medium on Days 3 and 5. The profiles of incorporation of [³H]thymidine into DNA of heart cells in experimental and control cultures showed a peak in incorporation values within the first week of culture and subsequently declined. Autoradiography studies revealed that cardiac myocytes in culture supplemented with ECGS alone attained a peak in labeling index on Day 1 with approximately 62% labeled cells. Subsequently, the labeling indices declined. Cardiac myocytes grown in media without ECGS showed significantly lower labeling indices than those in ECGS-containing media. This study has demonstrated the influence of ECGS, EGF and ITS in promoting the growth of cardiac myocytes and also in contributing to the maintenance of contractile cardiac myocytes in serum-free, long-term culture. The influence of ECGS on heart cell proliferation is considered to be superior to that of EGF and ITS. This study was supported in part by a grant HL-25482 from the National Heart Lung and Blood Institute and a grant from the American Heart Association of Michigan.     

Myosin isozyme changes in the heart following constriction of the ascending aorta of a 25-day old rat

When a constricting band is placed around the ascending aorta of young (25-day old) rats, all chambers of the heart eventually produce hypertrophy. Both the left and right ventricles show strong shifts toward an isozyme pattern in which V3 is predominant, similar to that seen in models where hypertrophy is induced in adult rats. The hypertrophied atria however, show no detectable change in the native myosin isozymes or the light chain subunits.     

Regenerating adult chicken skeletal muscle and satellite cell cultures express embryonic patterns of myosin and tropomyosin isoforms

Regenerating areas of adult chicken fast muscle (pectoralis major) and slow muscle (anterior latissimus dorsi) were examined in order to determine synthesis patterns of myosin light chains, heavy chains and tropomyosin. In addition, these patterns were also examined in muscle cultures derived from satellite cells of adult fast and slow muscle. One week after cold-injury the regenerating fast muscle showed a pattern of synthesis that was predominately embryonic. These muscles synthesized the embryonic myosin heavy chain, beta-tropomyosin and reduced amounts of myosin fast light chain-3 which are characteristic of embryonic fast muscle but synthesized very little myosin slow light chains. The regenerating slow muscle, however, showed a nearly complete array of embryonic peptides including embryonic myosin heavy chain, fast and slow myosin light chains and both alpha-fast and slow tropomyosins. Peptide map analysis of the embryonic myosin heavy chains synthesized by regenerating fast and slow muscles showed them to be identical. Thus, in both muscles there is a return to embryonic patterns during regeneration but this return appears to be incomplete in the pectoralis major. By 4 weeks postinjury both regenerating fast and slow muscles had stopped synthesizing embryonic isoforms of myosin and tropomyosin and had returned to a normal adult pattern of synthesis. Adult fast and slow muscles yielded a satellite cell population that formed muscle fibers in culture. Fibers derived from either population synthesized the embryonic myosin heavy chain in addition to alpha-fast and beta-tropomyosin. Thus, muscle fibers derived in culture from satellite cells of fast and slow muscles synthesized a predominately embryonic pattern of myosin heavy chains and tropomyosin. In addition, however, the satellite cell-derived myotubes from fast muscle synthesized only fast myosin light chains while the myotubes derived from slow muscle satellite cells synthesized both fast and slow myosin light chains. Thus, while both kinds of satellite cells produced embryonic type myotubes in culture the overall patterns were not identical. Satellite cells of fast and slow muscle appear therefore to have diverged from each other in their commitment during maturation in vivo.     

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.