Role of desmin filaments in chicken cardiac myofibrillogenesis

Desmin filaments are muscle-specific intermediate filaments located at the periphery of the Z-discs, and they have been postulated to play a critical role in the lateral registration of myofibrils. Previous studies suggest that intermediate filaments may be involved in titin assembly during the early stages of myofibrillogenesis. In order to investigate the putative function of desmin filaments in myofibrillogenesis, rabbit anti-desmin antibodies were introduced into cultured cardiomyocytes by electroporation to perturb the normal function of desmin filaments. Changes in the assembly of several sarcomeric proteins were examined by immunofluorescence. In cardiomyocytes incorporated with normal rabbit serum, staining for alpha-actinin and muscle actin displayed the typical Z-line and I-band patterns, respectively, while staining for titin with monoclonal anti-titin A12 antibody, which labels a titin epitope at the A-I junction, showed the periodic doublet staining pattern. Staining for C-protein gave an amorphous pattern in early cultures and identified A-band doublets in older cultures. In contrast, in cardiomyocytes incorporated with anti-desmin antibodies, alpha-actinin was found in disoriented Z-discs and the myofibrils became fragmented, forming mini-sarcomeres. In addition, titin was not organized into the typical A-band doublet, but appeared to be aggregated. Muscle actin staining was especially weak and appeared in tiny clusters. Moreover, in all ages of cardiomyocytes tested, C-protein remained in the disassembled form. The present data suggest the essential role of desmin in myofibril assembly.     

Inhibitors arrest myofibrillogenesis in skeletal muscle cells at early stages of assembly

A three-step model for myofibrillogenesis has been proposed for the formation of myofibrils [Rhee et al., 1994: Cell Motil. Cytoskeleton 28:1-24; Sanger et al., 2002: Adv. Exp. Med. 481:89-105]: premyofibril to nascent myofibril to mature myofibril. We have found two chemically related inhibitors that will arrest development at both the first and second step. Cultured quail embryonic skeletal myoblasts were treated with ethyl methane sulfonate (EMS) or 2-aminoethyl-methanesulfonate (MTSEA+). When the myoblasts fused in the presence of either of these compounds, myosheets rather than myotubes formed. Treated cells were fixed and immunostained against multiple proteins commonly found in muscle cells. Protein expression and localization throughout the myosheet were similar to that of developing myotube tips. Cells treated with high concentrations of EMS (10 mM) stained for non-muscle myosin II, sarcomeric alpha-actinin, and tropomyosin. No zeugmatin (Z-band region of titin) or muscle myosin II antibody staining was detected in fibers in this treatment group. These fibers are comparable to premyofibrils in control myotubes. At lower concentrations of EMS (7.5 to 5 mM), fibers that formed stained for muscle myosin II and titin as well as for non-muscle myosin IIB, sarcomeric alpha-actinin, and tropomyosin. Muscle myosin II was in an unbanded pattern. These fibers are comparable to nascent myofibrils observed during normal myofibrillogenesis. Similar effects to those obtained by treating cells with EMS were obtained when we treated cultured cells with MTSEA+ (5 mM) and stained them with sarcomeric alpha-actinin. MTSEA+ is chemically related to EMS, and is a well-known inhibitor of ryanodine receptors in skeletal muscle cells. Some abnormalities such as nemaline-like rods and other protein aggregates also appear within the myosheet during EMS and MTSEA+ treatment. Removal of these two inhibitors of myofibrillogenesis allows the premyofibrils and nascent myofibrils to form mature myofibrils.     

Myofibrillogenesis in the first cardiomyocytes formed from isolated quail precardiac mesoderm

De novo assembly of myofibrils was investigated in explants of precardiac mesoderm from quail embryos to address a controversy about different models of myofibrillogenesis. The sequential expression of sarcomeric components was visualized in double- and triple-stained explants before, during, and just after the first cardiomyocytes began to beat. In explants from stage 6 embryos, cultured for 10 h, ectoderm, endoderm, and the precardiac mesoderm displayed arrays of stress fibers with alternating bands of the nonmuscle isoforms of alpha-actinin and myosin IIB. With increasing time in culture, mesoderm cells contained fibrils composed of actin, nonmuscle myosin IIB, and sarcomeric alpha-actinin. Several hours later, before beating occurred, both nonmuscle and muscle myosin II localized in some of the fibrils in the cells. Concentrations of muscle myosin began as thin bundles, dispersed in the cytoplasm, often overlapping one another, and progressed to small, aligned A-band-sized aggregates. The amount of nonmuscle myosin decreased dramatically when Z-bands formed, the muscle myosin became organized into A-bands, and the cells began beating. The sequential changes in protein composition of the fibrils in the developing muscle cells supports the model of myofibrillogenesis in which assembly begins with premyofibrils and progresses through nascent myofibrils to mature myofibrils.     

Myofibrillogenesis and formation of cell contacts mediate the localization of N-RAP in cultured chick cardiomyocytes

The expression of N-RAP was investigated in immuofluorescently stained embryonic chick cardiomyocyte cultures. After 1 day in culture, the cardiomyocytes were spherical and N-RAP, titin, alpha-actinin, and vinculin were all diffusely distributed. As the cardiomyocytes spread and formed myofibrils and cell contacts, N-RAP became localized to distinct areas in the cells. During myofibrillogenesis, N-RAP was found concentrated in premyofibrils. As the premyofibrils transformed into bundles of mature myofibrils, N-RAP became concentrated at the longitundal ends of the cells, and was not found in the mature sarcomeres. At sites of cell-cell contacts, N-RAP was localized to the cell junction even in cells without any significant myofibril formation. As the cell-cell contacts became more extensive and formed structures resembling the intercalated disks found in hearts, N-RAP became even more specifically concentrated at these junctions. The results show that myofibrillogenesis and cell contact formation can each independently target N-RAP to the longitudinal ends of cardiomyocytes.     

Early incorporation of obscurin into nascent sarcomeres: implication for myofibril assembly during cardiac myogenesis

Obscurin is a recently identified giant multidomain muscle protein whose functions remain poorly understood. The goal of this study was to investigate the process of assembly of obscurin into nascent sarcomeres during the transition from non-striated myofibril precursors to striated structure of differentiating myofibrils in cell cultures of neonatal rat cardiac myocytes. Double immunofluorescent labeling and high resolution confocal microscopy demonstrated intense incorporation of obscurin in the areas of transition from non-striated to striated regions on the tips of developing myofibrils and at the sites of lateral fusion of nascent sarcomere bundles. We found that obscurin rapidly and precisely accumulated in the middle of the A-band regions of the terminal newly assembled half-sarcomeres in the zones of transition from the continuous, non-striated pattern of sarcomeric α-actinin distribution to cross-striated structure of laterally expanding nascent Z-discs. The striated pattern of obscurin typically ended at these points. This occurred before the assembly of morphologically differentiated terminal Z-discs of the assembling sarcomeres on the tips of growing myofibrils. The presence of obscurin in the areas of the terminal Z-discs of each new sarcomere was detected at the same time or shortly after complete assembly of sarcomeric structure. Many non-striated fibers with very low concentration of obscurin were already immunopositive for sarcomeric actin and myosin. This suggests that obscurin may serve for organization and alignment of myofilaments into the striated pattern. The comparison of obscurin and titin localization in these areas showed that obscurin assembly into the A-bands occurred soon after or concomitantly with incorporation of titin. Electron microscopy of growing myofibrils demonstrated intense formation and integration of myosin filaments into the “open” half-assembled sarcomeres in the areas of the terminal Z–I structures and at the lateral surfaces of newly formed, terminally located nascent sarcomeres. This process progressed before the assembly of the second-formed, terminal Z-discs of new sarcomeres and before the development of ultrastructurally detectable mature M-lines that define the completion of myofibril assembly, which supports the data of immunocytochemical study. Abundant non-aligned sarcomeres in immature myofibrils located on the growing tips were spatially separated and underwent the transition to the registered, aligned pattern. The sarcoplasmic reticulum, the organelle known to interact with obscurin, assembled around each new sarcomere. These results suggest that obscurin is directly involved in the proper positioning and alignment of myofilaments within nascent sarcomeres and in the establishment of the registered pattern of newly assembled myofibrils and the sarcoplasmic reticulum at advanced stages of myofibrillogenesis. This paper is dedicated to the memory of Professor Pavel P. Rumyantsev (1927–1988), a pioneer in studies of cardiac muscle differentiation, who is a lasting inspiration to all who worked with him.     

Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin- binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.     

BDM (2,3-butanedione monoxime), an inhibitor of myosin-actin interaction, suppresses myofibrillogenesis in skeletal muscle cells in culture

During the initial phase of myofibrillogenesis in developing muscle cells, the majority of thin filaments lie parallel to, and exhibit correct polarity and spatial position with thick filaments, as in mature myofibrils. Since myosin is known to function as an accelerator of actin polymerization in vitro, it has been postulated that myosin-actin interaction is important in the initial phase of myofibrillogenesis. To clarify further the role of actin-myosin interaction in myofibril formation during development, BDM (2,3-butanedione 2-monoxime), an inhibitor of myosin ATPase, was applied to primary cultures of skeletal muscle to inhibit myosin activity during myofibrillogenesis, and myofibril formation was examined. When 10 mM BDM was added to the myotubes just after fusion and the cultures were maintained for a further 4 days, cross-striated myofibrils were scarcely observed by fluorescence microscopy when examined by staining with antibodies to actin, myosin, troponin and !-actinin, whereas in the control myotubes not exposed to BDM, typical sarcomeric structures were detected. Electron microscopy revealed a disorganized arrangement of myofilaments and incomplete sarcomeric structures in the BDM-treated myotubes. Thus, formation of cross-striated myofibrils was remarkably suppressed in the BDM-treated myotubes. When the myotubes cultured in BDM-containing media were transferred to control media, sarcomeric structures were formed in 2-3 days, suggesting that the inhibitory effect of BDM on myotubes is reversible. These results suggest that actin-myosin interaction plays a critical role in the early process of myofibrillogenesis.     

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the alpha- and gamma-isoforms of actin

The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.     

Ectopic expression and dynamics of TPM1alpha and TPM1kappa in myofibrils of avian myotubes

From the four known vertebrate tropomyosin genes (designated TPM1, TPM2, TPM3, and TPM4) over 20 isoforms can be generated. The predominant TPM1 isoform, TPM1alpha, is specifically expressed in both skeletal and cardiac muscles. A newly discovered alternatively spliced isoform, TPM1kappa, containing exon 2a instead of exon 2b contained in TPM1alpha, was found to be cardiac specific and developmentally regulated. In this work, we transfected quail skeletal muscle cells with green fluorescent proteins (GFP) coupled to chicken TPM1alpha and chicken TPM1kappa and compared their localizations in premyofibrils and mature myofibrils. We used the technique of fluorescence recovery after photobleaching (FRAP) to compare the dynamics of TPM1alpha and TPM1kappa in myotubes. TPM1alpha and TPM1kappa incorporated into premyofibrils, nascent myofibrils, and mature myofibrils of quail myotubes in identical patterns. The two tropomyosin isoforms have a higher exchange rate in premyofibrils than in mature myofibrils. F-actin and muscle tropomyosin are present in the same fibers at all three stages of myofibrillogenesis (premyofibrils, nascent myofibrils, mature myofibrils). In contrast, the tropomyosin-binding molecule nebulin is not present in the initial premyofibrils. Nebulin is gradually added during myofibrillogenesis, becoming fully localized in striated patterns by the mature myofibril stage. A model of thin filament formation is proposed to explain the increased stability of tropomyosin in mature myofibrils. These experiments are supportive of a maturing thin filament and stepwise model of myofibrillogenesis (premyofibrils to nascent myofibrils to mature myofibrils), and are inconsistent with models that postulate the immediate appearance of fully formed thin filaments or myofibrils.     

Transgenic Expression of the Formin Protein Fhod3 Selectively in the Embryonic Heart: Role of Actin-Binding Activity of Fhod3 and Its Sarcomeric Localization during Myofibrillogenesis

Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain.     

Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

Smooth muscle cells use an actin-myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.     

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. I. Presence of immunofluorescent titin spots in premyofibril stages

Our initial attempts to immunolabel intact myocardial walls of 4-12 somite stage chick embryos were hindered by the presence of the cardiac jelly that covers the inner myocardial wall surface and prevents the access of antibodies to that surface. We overcame this difficulty by treating the specimens with hyaluronidase, which made the cardiac jelly permeable to the antibodies. An additional nonionic detergent treatment made the two or more cell layers of the myocardial wall accessible to the antibodies from both surfaces of the wall. Specimens treated in this manner were fluorescently labeled with antibodies to titin, myosin, or actin or with NBD-phallacidin for F-actin and examined as whole mount preparations or cut into semithin sections after resin embedding. These preparations and sections revealed that titin, a putative scaffolding protein of sarcomeres, is present in a punctate state and also in a diffuse form throughout the cytoplasm of cardiac myocytes in the premyofibril stages (4-7 somite stages) as well as in the early stages of myofibril formation. We interpreted the punctate and diffuse states to represent an aggregated state of several titin molecules and a dispersed state of individual titin molecules, respectively. In the 4-7 somite cardiac primodia, myosin and actin show only a uniform labeling throughout the cytoplasm of the myocytes. These observations are in contrast to a previous report that titin and myosin are tightly linked during in vitro skeletal myofibrillogenesis (Hill, C. S., S. Duran, Z. Ling, K. Weber, and H. Holtzer, 1986, J. Cell Biol., 103:2185-2196). In the 8-11 somite stage hearts, the number of individual titin spots rapidly reduces, while the number of myofibrils with periodically aligned titin spots increases, which strongly suggests that the titin spots are incorporated into the newly arising myofibrils. Titin spots were seen as doublets only after titin spots were incorporated into the first myofibrils. However, the fact that the distance between the components of the narrowest doublet was close to the resolution limit of the light microscope left open the possibility that undiscernible doublets of submicroscopic separations might exist in the premyofibril stages. The myosin labeling revealed the sarcomeric periodicity in an earlier stage of myofibril development than the F- actin labeling. In addition, we made two morphogenic observations.(ABSTRACT TRUNCATED AT 400 WORDS)     

ALP and MLP distribution during myofibrillogenesis in cultured cardiomyocytes

The Z-line is a multifunctional macromolecular complex that anchors sarcomeric actin filaments, mediates interactions with intermediate filaments and costameres, and recruits signaling molecules. Antiparallel alpha-actinin homodimers, present at Z-lines, cross-link overlapping actin filaments and also bind other cytoskeletal and signaling elements. Two LIM domain containing proteins, alpha-actinin associated LIM protein (ALP) and muscle LIM protein (MLP), interact with alpha-actinin, distribute in vivo to Z-lines or costameres, respectively, and, when absent, are associated with heart disease. Here we describe the behavior of ALP and MLP during myofibrillogenesis in cultured embryonic chick cardiomyocytes. As myofibrils develop, ALP and MLP are observed in distinct distribution patterns in the cell. ALP is coincident with alpha-actinin from the first stage of myofibrillogenesis and co-distributes with alpha-actinin to Z-lines and intercalated discs in mature myofibrils. Interestingly, we also demonstrate using ALP-GFP transfection experiments and an in vitro binding assay that the ALP-alpha-actinin binding interaction is not required to target ALP to the Z-line. In contrast, MLP localization is not co-incident with that of alpha-actinin until late stages of myofibrillogenesis; however, it is present in premyofibrils and nascent myofibrils prior to the incorporation of other costameric components such as vinculin, vimentin, or desmin. Our observations support the view that ALP function is required specifically at actin anchorage sites. The subcellular distribution pattern of MLP during myofibrillogenesis suggests that it functions during differentiation prior to the establishment of costameres.     

Role of M-line proteins in sarcomeric titin assembly during cardiac myofibrillogenesis

A rat polyclonal anti-M-line protein antiserum and three mouse monoclonal anti-titin antibodies (E2, F3, and A12) were used to study the spatiotemporal relationship between M-line proteins and titin during myofibril assembly in cultured chicken cardiomyocytes by immunofluorescence microscopy. In day 2 cultures, M-line proteins and titin were detected as punctate staining in most cardiomyocytes, which possessed many nonstriated fibrils. At a late stage (day 3 cultures), M-line proteins were incorporated into dot-like structures along nonstriated fibrils, while titin staining was continuous on these structures. As development progressed, M-line proteins were registered in periodic pattern in the mid-A band. In cardiomyocytes from day 5 cultures, the titin bands were separated by an unstained region, and achieved their adult doublet pattern. Thus, the organization of titin in the sarcomere appears to occur later than that of M-line proteins in the M-line. Our morphological data indicate that the early registration of M-line proteins in primitive myofibrils may guide titin filament alignment via interaction between M-line proteins and titin. In order to investigate the role of M-line proteins in the assembly of titin filaments, anti-M-line protein or anti-titin antibodies were introduced into cultured cardiomyocytes by electroporation to functionally bind the respective proteins, and the profile of myofibril assembly was examined. Cardiomyocytes from day 2–3 cultures with incorporated anti-M-line protein antibodies became shrunk, and exhibited defective myofibrillar assembly, as shown by the failure of titin to assemble into a typical sarcomeric pattern. Incorporation of anti-titin antibody E2, which recognizes the M-line end domain of titin, resulted in the failure of M-line proteins organized into the M-line structure, as shown by random, sporadic staining with anti-M-line protein antibody. These studies confirm the essential role of M-line proteins in the organization of titin filaments in the sarcomere and that the interaction between titin and M-line proteins is crucial to the formation of the M-line structure. J. Cell. Biochem. 71:82–95, 1998. © 1998 Wiley-Liss, Inc.     

Myofibrillogenesis in skeletal muscle cells in the presence of taxol

We address the controversy of whether mature myofibrils can form in the presence of taxol, a microtubule-stabilizing compound. Previous electron microscopic studies reported the absence of actin filaments and Z-bands in taxol-treated myocytes [Antin et al., 1981: J Cell Biol 90:300-308; Toyoma et al., 1982: Proc Natl Acad Sci USA 79:6556-6560]. Quail skeletal myoblasts were isolated from 10-day-old embryos and grown in the presence or absence of taxol. Taxol inhibited the formation of multinucleated elongated myotubes. Myocytes cultured in the continual presence of taxol progressed from rounded to stellate shapes. Groups of myocytes that were clustered together after the isolation procedure fused in the presence of taxol but did not form elongated myotubes. Actin filaments and actin-binding proteins were detected with several different fluorescent probes in all myofibrils that formed in the presence of taxol. The Z-bands contained both alpha-actinin and titin, and the typical arrays of A-Bands were always associated with actin filaments in the myofibrils. Myofibril formation was followed by fixing cells each day in culture and staining with probes for actin, muscle-specific alpha-actinin, myosin II, nebulin, troponin, tropomyosin, and non-muscle myosin II. Small linear aggregates of alpha-actinin or Z-bodies, premyofibrils, were detected at the edges of the myocytes and in the arms of the taxol-treated cells and were always associated with actin filaments. Non-muscle myosin II was detected at the edges of the taxol-treated cells. Removal of the taxol drug led to the cells assuming a normal compact elongated shape. During the recovery process, additional myofibrils formed at the spreading edges of these elongated and thicker myotubes. Staining of these taxol-recovering cells with specific fluorescent reagents reveals three different classes of actin fibers. These results are consistent with a model of myofibrillogenesis that involves the transition of premyofibrils to mature myofibrils.     

Targeted disruption of nebulette protein expression alters cardiac myofibril assembly and function

To evaluate nebulette's role in cardiac myofibrils, cardiomyocytes expressing green fluorescent protein (GFP)-nebulette constructs were monitored for their ability to contract and myofilament protein distribution was analyzed. Cells expressing full-length GFP-nebulette appear unaffected and exhibit normal beating frequencies. Expression of the GFP linker and SH3 results in loss of the endogenous nebulette and tropomyosin; however, Z-line and thick filaments are undisturbed. Cells expressing either of these domains have dramatically reduced beating frequencies, consistent with the loss of thin filament proteins. This loss was inhibited by the addition of protease inhibitors during culturing. The GFP repeat domain disrupts both myofibrillogenesis and contraction in spreading cardiomyocytes, whereas introduction of this protein into well-spread cardiomyocytes results in localization at the Z-line and a 50% reduction in beating frequency. Ultimately, these cells form bundles containing the GFP repeat and many myofilament proteins. Interestingly, butanedione monoxime inhibition of contraction inhibited the formation of these bundles. These results show that the GFP-nebulette domains have a dominant-negative effect on the distribution and function of the sarcomeric proteins. Taken together with the observation that nebulette colocalizes with alpha-actinin in the pre-, nascent, and mature myofibrils, our data demonstrate the importance of this cardiac-specific nebulin isoform in myofibril organization and function.     

Myofibril assembly is linked with vinculin, alpha-actinin, and cell-substrate contacts in embryonic cardiac myocytes in vitro

The relationship of nascent myofibrils with the accumulation of adhesion plaque proteins and the formation of focal cell contacts was studied in embryonic chick cardiac myocytes in vitro. The cultures were double-stained with various combinations of the specific antiactin drug phalloidin and antibodies against vinculin, alpha-actinin, connectin (titin), myosin heavy chain, fibronectin, and desmin and examined under fluorescence and interference reflection microscopy. In the areas of myofibril assembly, vinculin and alpha-actinin plaques were formed at the ventral sarcolemmae. These areas overlapped with the sites of cell-to-substrate focal contacts and extracellular fibronectin. Because the myofibrils always ran in a straight line between these sites, polarized lines appeared to be generated within the cells in response to their physical (e.g., stress) and/or biochemical environment (e.g., adhesion plaque proteins). The possible presence of other factors cannot be ruled out for the proper alignment of myofibrils. As soon as myofibrils came to span between these adhesion sites, they exhibited typically mature cross-striated characteristics. Thus, the formation of these inferred lines has some relation to, or is in fact necessary for, the maturation of myofibrils, in addition to the directional arrangement of sarcomeric proteins. Additionally, synthesis and distribution of myosin and connectin were tightly linked during early developmental (premyofibril and myofibril) stages. The spatial deployment of desmin was not coupled with vinculin. Thus, connectin and desmin do not appear to form the initial scaffold of sarcomeres.     

Scaffolds and chaperones in myofibril assembly: putting the striations in striated muscle

Sarcomere assembly in striated muscles has long been described as a series of steps leading to assembly of individual proteins into thick filaments, thin filaments and Z-lines. Decades of previous work focused on the order in which various structural proteins adopted the striated organization typical of mature myofibrils. These studies led to the view that actin and α-actinin assemble into premyofibril structures separately from myosin filaments, and that these structures are then assembled into myofibrils with centered myosin filaments and actin filaments anchored at the Z-lines. More recent studies have shown that particular scaffolding proteins and chaperone proteins are required for individual steps in assembly. Here, we review the evidence that N-RAP, a LIM domain and nebulin repeat protein, scaffolds assembly of actin and α-actinin into I-Z-I structures in the first steps of assembly; that the heat shock chaperone proteins Hsp90 & Hsc70 cooperate with UNC-45 to direct the folding of muscle myosin and its assembly into thick filaments; and that the kelch repeat protein Krp1 promotes lateral fusion of premyofibril structures to form mature striated myofibrils. The evidence shows that myofibril assembly is a complex process that requires the action of particular catalysts and scaffolds at individual steps. The scaffolds and chaperones required for assembly are potential regulators of myofibrillogenesis, and abnormal function of these proteins caused by mutation or pathological processes could in principle contribute to diseases of cardiac and skeletal muscles.     

Obscurin regulates the organization of myosin into A bands

Obscurin is a giant sarcomeric protein composed of adhesion modules and signaling domains. It surrounds myofibrils at the level of the Z disk and the M line. To study the role of obscurin during myofibrillogenesis, we used adenovirus-mediated gene delivery to overexpress part of its COOH terminus in primary cultures of postnatal day 1 (P1) skeletal myotubes. Examination of the subcellular distribution of a number of sarcomeric proteins revealed that the organization of myosin into A bands was dramatically reduced. Myosin assembled into A bands normally in mock- or control-infected P1 myotubes. Overexpression of the COOH terminus of obscurin did not affect the organization of other sarcomeric markers, including actin, -actinin, titin, and myomesin. Assembly of myomesin into nascent M lines in treated myotubes suggests that these structures can form independently of A bands. Immunoblot analysis indicated that there was a small (20%) but consistent decrease in the amount of myosin expressed in cells infected with the COOH terminus of obscurin. Coimmunoprecipitation experiments in which we used adult skeletal muscle homogenates demonstrated that obscurin exists in a complex with myosin. Thus our findings suggest that the COOH-terminal region of obscurin interacts with sarcomeric myosin and may play a critical role in its ability to assemble into A bands in striated muscle. titin; myofibrillogenesis; sarcomere; M line; muscle     

Dynamics of obscurin localization during differentiation and remodeling of cardiac myocytes: obscurin as an integrator of myofibrillar structure.

Obscurin is a newly identified giant muscle protein whose functions remain to be elucidated. In this study we used high-resolution confocal microscopy to examine the dynamics of obscurin localization in cultures of rat cardiac myocytes during the assembly and disassembly of myofibrils. Double immunolabeling of neonatal and adult rat cells for obscurin and sarcomeric alpha-actinin, the major protein of Z-lines, demonstrated that, during myofibrillogenesis, obscurin is intensely incorporated into M-band areas of A-bands and, to a lesser extent, in Z-lines of newly formed sarcomeres. Presarcomeric structural precursors of myofibrils were intensely immunopositive for alpha-actinin and, unlike mature myofibrils, weakly immunopositive or immunonegative for obscurin. This indicates that most of the obscurin assembles in developing myofibrils after abundant incorporation of alpha-actinin and that massive integration of obscurin occurs at more advanced stages of sarcomere assembly. Immunoreactivity for obscurin in the middle of A-bands and in Z-lines of sarcomeres bridged the gaps between individual bundles of newly formed myofibrils, suggesting that this protein appears to be directly involved in their primary lateral connection and registered alignment into larger clusters. Close sarcomeric localization of obscurin and titin suggests that they may interact during myofibril assembly. Interestingly, the laterally aligned striated pattern of obscurin formed at a stage when desmin, traditionally considered as a molecular linker responsible for the lateral binding and stabilization of myofibrils at the Z-bands, was still diffusely localized. During the disassembly of the contractile system in adult myocytes, disappearance of the cross-striated pattern of obscurin preceded the disorganization of registered alignment and intense breakdown of myofibrils. The cross-striated pattern of desmin typical of terminally differentiated myocytes disappeared before or simultaneously with obscurin. During redifferentiation, as in neonatal myocytes, sarcomeric incorporation of obscurin closely followed that of alpha-actinin and occurred earlier than the striated arrangement of desmin intermediate filaments. The presence of obscurin in the Z-lines and its later assembly into the A/M-bands indicate that it may serve to stabilize and align sarcomeric structure when myosin filaments are incorporated. Our data suggest that obscurin, interacting with other muscle proteins and possibly with the sarcoplasmic reticulum, may have a role as a flexible structural integrator of myofibrils during assembly and adaptive remodeling of the contractile apparatus.