Intracellular targeting of isoproteins in muscle cytoarchitecture.

Part of the muscle creatine kinase (MM-CK) in skeletal muscle of chicken is localized in the M-band of myofibrils, while chicken heart cells containing myofibrils and BB-CK, but not expressing MM-CK, do not show this association. The specificity of the MM-CK interaction was tested using cultured chicken heart cells as "living test tubes" by microinjection of in vitro generated MM-CK and hybrid M-CK/B-CK mRNA with SP6 RNA polymerase. The resulting translation products were detected in injected cells with isoprotein-specific antibodies. M-CK molecules and translation products of chimeric cDNA molecules containing the head half of the B-CK and the tail half of the M-CK coding regions were localized in the M-band of the myofibrils. The tail, but not the head portion of M-CK is essential for the association of M-CK with the M-band of myofibrils. We conclude that gross biochemical properties do not always coincide with a molecule's specific functions like the participation in cell cytoarchitecture which may depend on molecular targeting even within the same cellular compartment.

Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function.

The intracompartmental sorting and functional consequences of ectopic expression of the six vertebrate actin isoforms was investigated in different types of cultured cells. In transfected fibroblasts all isoactin species associated with the endogenous microfilament cytoskeleton, even though cytoplasmic actins also showed partial localization to peripheral submembranous sites. Functional and structural studies were performed in neonatal and adult rat cardiomyocytes. All the muscle isoactin constructs sorted preferentially to sarcomeric sites and, to a lesser extent, also to stress-fiber-like structures. The expression of muscle actins did not interfere with cell contractility, and did not disturb the localization of endogenous sarcomeric proteins. In sharp contrast, ectopic expression of the two cytoplasmic actin isoforms resulted in rapid cessation of cellular contractions and induced severe morphological alterations characterized by an exceptional outgrowth of filopodia and cell flattening. Quantitative analysis in neonatal cardiomyocytes indicated that the levels of accumulation of the different isoactins are very similar and cannot be responsible for the observed isoproteins-specific effects. Structural analysis revealed a remodeling of the cytoarchitecture including a specific alteration of sarcomeric organization; proteins constituting the sarcomeric thin filaments relocated to nonmyofibrillar sites while thick filaments and titin remained unaffected. Experiments with chimeric proteins strongly suggest that isoform specific residues in the carboxy-terminal portion of the cytoplasmic actins are responsible for the dominant negative effects on function and morphology.

Point mutations in human beta cardiac myosin heavy chain have differential effects on sarcomeric structure and assembly: an ATP binding site change disrupts both thick and thin filaments, whereas hypertrophic cardiomyopathy mutations display normal assembly.

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including beta cardiac myosin heavy chain (beta MHC). To determine whether point mutations in human beta MHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human beta MHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human beta MHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse beta MHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human beta MHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type beta MHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human beta MHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased beta MHC function.

The Mr 165,000 M-protein myomesin: a specific protein of cross-striated muscle cells.

The tissue specificity of chicken 165,000 M-protein, tentatively names "myomesin", a tightly bound component of the M-line region of adult skeletal and heart myofibrils, was investigated by immunological techniques. Besides skeletal and heart muscle, only thymus (known to contain myogenic cells) was found to contain myomesin. No myomesin could however, be detected in smooth muscle or any other tissue tested. This result was confirmed in vitro on several cultured embryonic cell types. Only skeletal and heart muscle cells, but not smooth muscle or fibroblast cells, showed the presence of myomesin. When the occurrence and the distribution of myomesin during differentiation of breast muscle cells in culture were studied by the indirect immunofluorescence technique, this protein was first detected in postmitotic, nonproliferating myoblasts in a regular pattern of fluorescent cross-striations. In electron micrographs of sections through young myotubes, it could be shown to be present within the forming H-zones of nascent myofibrils. In large myotubes the typical striation pattern in the M-line region of the myofibrils was observed. Synthesis of myomesin measured by incorporation of [35S]methionine into immunoprecipitable protein of differentiating cells increased sharply after approximately 48 h in culture, i.e., at the time when the major myofibrillar proteins are accumulated. No significant amounts of myomesin were, however, found in cells prevented from undergoing normal myogenesis by 5'-bromodeoxyuridine. The results indicate that myomesin (a) is a myofibrillar protein specific for cross-striated muscle, (b) represents a highly specific marker for cross-striated muscle cell differentiation and (c) might play an important role in myofibril assembly and/or maintenance.

Myomesin and M-protein: expression of two M-band proteins in pectoral muscle and heart during development.

The expression of the myofibrillar M-band proteins myomesin and M-protein was studied in chicken pectoral muscle and heart during differentiation using monoclonal antibodies in a double-antibody sandwich enzyme-linked immunosorbent assay, immunoblotting, and immunocytochemistry. In presumptive pectoral muscle, myomesin accumulated first, increasing from 2% of the adult concentration at day 7 to 70% by day 16 in ovo. M-protein accumulation lagged 6-7 d behind that of myomesin attaining only 40% of the adult concentration in ovo. The molecular masses of myomesin (185 kD) and M-protein (165 kD) remained constant during embryogenesis. In cultured myogenic cells the accumulation and M-band localization of myomesin preceded that of M-protein by 1.5 d. Chicken heart was shown, in addition to M-protein, to contain unique isoforms of myomesin. In hearts of 6 d embryos, a 195-kD myomesin isoform was the major species; throughout development, however, a transition to a mixture of 195 and 190 kD was observed, the latter being the major species in the adult tissue. During heart differentiation the initial accumulation of myomesin again preceded that of M-protein, albeit on an earlier time scale than in pectoral muscle with M-protein reaching adult proportions first.

A new 185,000-dalton skeletal muscle protein detected by monoclonal antibodies.

The M line, which transverses the center of the thick filament region of skeletal muscle sarcomeres, appears to be a complex array of multiple structural elements. To date, two proteins have definitely been shown to be associated with the M line. They are MM-CK, localized in the M 4,4' substriations, and a 165,000-dalton (164 kd) protein, referred to as both M-protein and myomesin. Here we report the positive identification of a third M-line protein of 185 kd. In the course of making monoclonal antibodies (mAbs) against a 165-kd fraction, we also obtained mAbs that bound to the M line of isolated myofibrils as detected by indirect immunofluorescence, but recognized a protein band of 185 kd in immunoblotting experiments with either the original immunogen or low ionic strength myofibril extracts as antigenic targets. The evidence that the 185- and 165-kd proteins are distinct protein species is based on the separation of the two proteins into discrete peaks by ion exchange chromatography, the distinctive patterns of their degradation products, and non-cross-reactivity of any of seven mAbs. These mAbs recognize three unique antigenic determinants on the 185-kd molecule and at least two and probably four sites on the 165-kd molecule as determined from competitive binding and immunofluorescence experiments. To resolve the problem of multiple nomenclature for the 165-kd protein, the 185-kd protein will be referred to as myomesin and the 165-kd protein as M-protein.

Alterations at the intercalated disk associated with the absence of muscle LIM protein.

In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain- containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.

Single-chip microelectronic system to interface with living cells.

A high degree of connectivity and the coordinated electrical activity of neural cells or networks are believed to be the reason that the brain is capable of highly sophisticated information processing. Likewise, the effectiveness of an animal heart largely depends on such coordinated cell activity. To advance our understanding of these complex biological systems, high spatiotemporal-resolution techniques to monitor the cell electrical activity and an ideally seamless interaction between cells and recording devices are desired. Here we present a monolithic microsystem in complementary metal oxide semiconductor (CMOS) technology that provides bidirectional communication (stimulation and recording) between standard electronics technology and cultured electrogenic cells. The microchip can be directly used as a substrate for cell culturing, it features circuitry units per electrode for stimulation and immediate cell signal treatment, and it provides on-chip signal transformation as well as a digital interface so that a very fast, almost real-time interaction (2 ms loop time from event recognition to, e.g., a defined stimulation) is possible at remarkable signal quality. The corresponding spontaneous and stimulated electrical activity recordings with neuronal and cardiac cell cultures will be presented. The system can be used to, e.g., study the development of neural networks, reveal the effects of neuronal plasticity and study cellular or network activity in response to pharmacological treatments.

N-cadherin is essential for retinoic acid-mediated cardiomyogenic differentiation in mouse embryonic stem cells.

Contraction forces developed by cardiomyocytes are transmitted across the plasma membrane through end-to-end connections between the myocytes, called intercalated disks, which enable the coordinated contraction of heart muscle. A component of the intercalated disk, the adherens junction, consists of the cell adhesion molecule, N-cadherin. Embryos lacking N-cadherin die at mid-gestation from cardiovascular abnormalities. We have evaluated the role of N-cadherin in cardiomyogenesis using N-cadherin-null mouse embryonic stem (ES) cells grown as embryoid bodies (EBs) in vitro. Myofibrillogenesis, the spatial orientation of myofibers, and intercellular contacts including desmosomes were normal in N-cadherin-null ES cell-derived cardiomyocytes. The effect of retinoic acid (RA), a stage and dose-dependent cardiogenic factor, was assessed in differentiating ES cells. all-trans (at) RA increased the number of ES cell-derived cardiomyocytes by approximately 3-fold (at 3 x 10(-9) M) in wt EBs. However, this effect was lost in N-cadherin-null EBs. In the presence of supplemented at-RA, the emergence of spontaneously beating cardiomyocytes appeared to be delayed and slightly less efficient in N-cadherin-null compared with wt and heterozygous EBs (frequencies of EBs with beating activity at 5 days: 54+/-18% vs. 96+/-0.5%, and 93+/-7%, respectively; peak frequencies of EBs with beating activity: 83+/-8% vs. 96+/-0.5% and 100%, respectively). In conclusion, cardiomyoyctes differentiating from N-cadherin-null ES cells in vitro show normal myofibrillogenesis and intercellular contacts, but impaired responses to early cardiogenic effects mediated by at-RA. These results suggest that N-cadherin may be essential for RA-induced cardiomyogenesis in mouse ES cells in vitro.

Different domains of the M-band protein myomesin are involved in myosin binding and M-band targeting.

Myomesin is a 185-kDa protein located in the M-band of striated muscle where it interacts with myosin and titin, possibly connecting thick filaments with the third filament system. By using expression of epitope-tagged myomesin fragments in cultured cardiomyocytes and biochemical binding assays, we could demonstrate that the M-band targeting activity and the myosin-binding site are located in different domains of the molecule. An N-terminal immunoglobulin-like domain is sufficient for targeting to the M-band, but solid-phase overlay assays between individual N-terminal domains and the thick filament protein myosin revealed that the unique head domain contains the myosin-binding site. When expressed in cardiomyocytes, the head domains of rat and chicken myomesin showed species-specific differences in their incorporation pattern. The head domain of rat myomesin localized to a central area within the A-band, whereas the head domain of chicken myomesin was diffusely distributed in the cytoplasm. We therefore conclude that the head domain of myomesin binds to myosin but that this affinity is not sufficient for the restriction of the domain to the M-band in vivo. Instead, the neighboring immunoglobulin-like domain is essential for the precise incorporation of myomesin into the M-band, possibly because of interaction with a yet unknown protein of the sarcomere.

Study of the mechanical properties of myomesin proteins using dynamic force spectroscopy.

Myomesin is the most prominent structural component of the sarcomeric M-Band that is expressed in mammalian heart and skeletal muscles. Like titin, this protein is an intracellular member of the Ig-fibronectin superfamily, which has a flexible filamentous structure and which is largely composed of two types of domain that are similar to immunoglobulin (Ig)-like and fibronectin type III (FNIII) domains. Several myomesin isoforms have been identified, and their expression patterns are highly regulated both spatially and temporally. Particularly, alternative splicing in the central part of the molecule gives rise to an isoform, EH (embryonic heart)-myomesin, containing a serine and proline-rich insertion with no well-defined secondary structure, the EH segment. EH-myomesin represents the major myomesin isoform at embryonic stages of mammalian heart and is rapidly down-regulated around birth, but it is re-expressed in the heart of patients suffering from dilated cardio-myopathy. Here, in order to facilitate a better understanding of the physiological, and possibly pathological, functions of myomesin proteins, we explore the mechanical stability, elasticity and force-driven structural changes of human myomesin's sub-molecular segments using single-molecule force spectroscopy and protein engineering. We find that human myomesin molecules are composed of modules (Ig and FNIII), that are designed to withstand force and we demonstrate that the human cardiac EH segment functions like an additional elastic stretch in the middle part of the EH-myomesin and behaves like a random coil. Consequently myomesin isoforms (proteins with or without the EH segment) have different elastic properties, the EH-myomesin being the more compliant one. These findings imply that the compliance of the M-band increases with the amount of EH-myomesin it contains. So, we provide the evidence that not only titin but also other sarcomeric proteins have complicated visco-elastic properties depending on the contractile parameters in different muscle types.

Complete nucleotide and encoded amino acid sequence of a mammalian myosin heavy chain gene. Evidence against intron-dependent evolution of the rod.

The complete nucleotide sequence and exon/intron structure of the rat embryonic skeletal muscle myosin heavy chain (MHC) gene has been determined. This gene comprises 24 X 10(3) bases of DNA and is split into 41 exons. The exons encode a 6035 nucleotide (nt) long mRNA consisting of 90 nt of 5' untranslated, 5820 nt of protein coding and 125 nt of 3' untranslated sequence. The rat embryonic MHC polypeptide is encoded by exons 3 to 41 and contains 1939 amino acid residues with a calculated Mr of 223,900. Its amino acid sequence displays the structural features typical for all sarcomeric MHCs, i.e. an amino-terminal "globular" head region and a carboxy-terminal alpha-helical rod portion that shows the characteristics of a coiled coil with a superimposed 28-residue repeat pattern interrupted at only four positions by "skip" residues. The complex structure of the rat embryonic MHC gene and the conservation of intron locations in this and other MHC genes are indicative of a highly split ancestral sarcomeric MHC gene. Introns in the rat embryonic gene interrupt the coding sequence at the boundaries separating the proteolytic subfragments of the head, but not at the head/rod junction or between the 28-residue repeats present within the rod. Therefore, there is little evidence for exon shuffling and intron-dependent evolution by gene duplication as a mechanism for the generation of the ancestral MHC gene. Rather, intron insertion into a previously non-split ancestral MHC rod gene consisting of multiple tandemly arranged 28-residue-encoding repeats, or convergent evolution of an originally non-repetitive ancestral MHC rod gene must account for the observed structure of the rod-encoding portion of present-day MHC genes.

Developmental expression of creatine kinase isoenzymes in chicken growth cartilage.

We have shown previously that creatine kinase (CK) activity is required for normal development and mineralization of chicken growth cartilage and that expression of the cytosolic isoforms of CK is related to the biosynthetic and energy status of the chondrocyte. In this study, we have characterized changes in isoenzyme activity and mRNA levels of CK (muscle-specific CK, M-CK; brain-type CK, B-CK; and mitochondrial CK subunits, MiaCK and MibCK) in the growth plate in situ and in chondrocyte culture systems that model the development/maturation program of the cartilage. The in vitro culture systems analyzed were as follows: tibial chondrocytes, which undergo hypertrophy; embryonic cephalic and caudal sternal chondrocytes, which differ from each other in their mineralization response to retinoic acid; and long-term micromass cultures of embryonic limb mesenchymal cells, which recapitulate the chondrocyte differentiation program. In all systems analyzed, B-CK was found to be the predominant isoform. In the growth plate, B-CK expression was highest in the most calcified regions, and M-CK was less abundant than B-CK in all regions of the growth plate. In tibial chondrocytes, an increase in B-CK expression was seen when the cells became hypertrophic. Expression of B-CK increased slightly over 15 days in mineralizing, retinoic acid-treated cephalic chondrocytes, but it decreased in nonmineralizing caudal chondrocytes, while there was little expression of M-CK. Interestingly, in limb mesenchyme cultures, significant M-CK expression was detected during chondrogenesis (days 2-7), whereas hypertrophic cells expressed only B-CK. Finally, expression of MiaCK and MibCK was low both in situ and in vitro. These observations suggest that the CK genes are differentially regulated during cartilage development and maturation and that an increase in CK expression is important in initiating chondrocyte maturation.

Effect of okadaic acid on protein phosphorylation patterns of chicken myogenic cells with special reference to creatine kinase.

Okadaic acid and other agents affecting cellular phosphorylation and dephosphorylation processes profoundly changed the phosphoprotein pattern of 32Pi-labelled chicken embryonic skeletal muscle cells. The phosphorylation states of proteins in the lower molecular weight range were especially increased. Immunoprecipitation of cellular extracts with anti-creatine kinase antibodies enabled us to identify creatine kinase (CK) phosphoproteins. B-CK was phosphorylated after treating the cultures with 1-oleoyl-2-acetyl-sn-glycerol, dibutyryl-cAMP, okadiac acid and combinations thereof, but not with 1,2-dioleoyl-sn-glycerol. M-CK was also shown to be phosphorylated. The results indicated that in vivo, CK isoforms in muscle are subjected to control mediated by phosphorylation and dephosphorylation processes.

Phosphorylation of chicken brain-type creatine kinase affects a physiologically important kinetic parameter and gives rise to protein microheterogeneity in vivo.

In addition to the two monomer subunits of chicken brain-type creatine kinase (B-CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb* was identified by chromatofocussing in the presence of 8 M urea (Quest et al., ). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D-gels with three minor species (Bb1-3), initially identified in immunoprecipitated, [35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [32P]-gamma-ATP, Bb1-3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb* is identical to Bb1-3 and represents phosphorylated derivatives of Bb. B-CK dimer populations from different tissues were separated by ion-exchange chromatography and the Km values of the resulting fractions were determined under phospho-creatine (CP)-limiting conditions. In fractions containing only Bb and Bb* two kinetically different enzyme species were detected (Km values for CP = 1.6 mM and 0.8 mM), while fractions containing B-CK dimers composed of the major Ba and Bb monomers, but no Bb*, were homogeneous in this respect (Km for CP = 1.6 mM). Phosphorylation of Bb to yield Bb* is concluded to reduce the Km of B-CK dimers for CP by about 50%. This Km shift is within the range of CP concentrations found in tissues expressing the B-CK isoform and may therefore be of physiological relevance.

Fast myosin light chain expression in chicken muscles studied by in situ hybridization.

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.

Fiber type-specific distribution of M-band proteins in chicken muscle.

The functions of two myofibrillar proteins, myomesin (Mr 185,000) and M-protein (Mr 165,000), associated with the M-band are as yet unknown. To extend our knowledge of these proteins, we have examined chicken striated muscles with fast and slow contractile properties, e.g., pectoralis major, PLD, ALD, medial adductor, and lateral adductor, to determine the expression and isoform composition of myomesin and M-protein in various muscles and fiber types. The high molecular weight M-band proteins were characterized and quantitated using monoclonal antibodies in immunoblotting and double-antibody sandwich ELISA. Fiber specificity was determined by immuno- and enzyme histochemistry. In addition to the previously reported Mr 195,000 and 190,000 isoforms of myomesin in heart [Grove et al. (1985): J Cell Biol 101:1431], the Mr 185,000 myomesin in skeletal muscles may represent different isoforms in fast and slow muscles on the basis of distinctive degradation patterns. M-protein has the same molecular weight in striated chicken muscles and degradation patterns indicate only one isoform. The low quantities of M-protein in slow muscles were shown to be due to the absence of M-protein in two of the generally recognized slow fiber types, types I and III. Thus, M-protein was present only in fast type II fibers, whereas myomesin was ubiquitous in all fiber types. Whatever the causal relationship, M-protein appears to function in fast motor units composed of type II fibers.

Mass production of embryoid bodies in microbeads.

Embryonic stem cells (ESC) are totipotent cells that can differentiate into a large number of different cell types. Stem cell-derived, differentiated cells are of increasing importance as a potential source for non-proliferating cells (e.g., cardiomyocytes or neurons) for future tissue engineering applications. Differentiation of ESC is initiated by the formation of embryoid bodies (EB). Current protocols for the generation of EB are either of limited productivity or deliver EB with a large variation in size and differentiation state. To establish an efficient and robust EB production process, we encapsulated mouse ESC into alginate microbeads using various microencapsulation technologies. Microencapsulation and culturing of ESC in 1.1% alginate microbeads gives rise to discoid colonies, which further differentiate within the beads to cystic EB and later to EB containing spontaneously beating areas. However, if ESC are encapsulated into 1.6% alginate microbeads, differentiation is inhibited at the morula-like stage, so that no cystic EB can be formed within the beads. ESC colonies, which are released from 1.6% alginate microbeads, can further differentiate to cystic EB with beating cardiomyocytes. Extended supplementation of the growth medium with retinoic acid promotes differentiation to smooth muscle cells.

Three-dimensional analysis and visualization of myofibrillogenesis in adult cardiomyocytes by confocal microscopy.

Confocal light microscopy has found its place among the standard analytical tools in cell and molecular biology. When combined with techniques such as immunofluorescence or fluorescent in situ hybridization, the spatial distribution of individual biological components can be traced within cells and tissues and, under certain circumstances, even with living samples. In this article, advanced 3D visualization techniques have been applied to analyze the distribution of myofibrillar proteins in cultured adult rat cardiomyocytes. By combining confocal immunofluorescence microscopy with specially designed three-dimensional visualization, we have obtained images which are similar to those obtained with the scanning electron microscope. The subcellular distribution of proteins expressed after transfection of cDNA is monitored in the cultured heart cells. The expressed proteins are distinguished from their endogenous counterparts by the use of an epitope tagging technique. The described methods are suitable to specifically monitor the behavior of several closely related isoprotein mutants in cell or tissue preparations.