Manoel Luis Costa

2D and 3D-organized cardiac cells shows differences in cellular morphology, adhesion junctions, presence of myofibrils and protein expression.

Cardiac cells are organized in vivo in a complex tridimensional structural organization that is crucial for heart function. While in vitro studies can reveal details about cardiac cell biology, usually cells are grown on simplified two-dimensional (2D) environments. To address these differences, we established a cardiac cell culture composed of both 2D and three-dimensional (3D)-organized cells. Our results shows significant differences between the two culture contexts in relation to the overall morphology of the cells, contraction ability, proliferation rate, presence of intercellular adhesion structures, organization of myofibrils, mitochondria morphology, endoplasmic reticulum contents, cytoskeletal filaments and extracellular matrix distribution, and expression of markers of cardiac differentiation. Cardiac cells grown in 2D-context displayed a flattened and well spread shape, were mostly isolated and their cytoplasm was filled with a large network of microfilaments and microtubules. In contrast, 3D-cells were smaller in size, were always in close contact with each other with several cellular junctions, and displayed a less conspicuous cytoskeletal network. 3D-cells had more mitochondria and myofibrils and these cells contract spontaneously more often than 2D-cells. On the other hand, endoplasmic reticulum membranes were present in higher amounts in 2D-cells when compared to 3D-cells. The expression of desmin, cadherin and alpha-actinin was higher in 3D-aggregates compared to 2D-spread cells. These findings indicate that the tridimensional environment in which the cardiac cells are grown influence several aspects of cardiac differentiation, including cell adhesion, cell shape, myofibril assembly, mitochondria contents and protein expression. We suggest that the use of this cardiac culture model, with 2D and 3D-context cells, could be useful for studies on the effects of different drugs, or growth factors, giving valuable information on the biological response of cells grown in different spatial organizations.

Some distinctive features of zebrafish myogenesis based on unexpected distributions of the muscle cytoskeletal proteins actin, myosin, desmin, α-actinin, troponin and titin

The current myofibrillogenesis model is based mostly on in vitro cell cultures and on avian and mammalian embryos in situ. We followed the expression of actin, myosin, desmin, alpha-actinin, titin, and troponin using immunofluorescence microscopy of zebrafish (Danio rerio) embryos. We could see young mononucleated myoblasts with sharp striations. The striations were positive for all the sarcomeric proteins. Desmin distribution during muscle maturation changes from dispersed aggregates to a perinuclear concentration to striated afterwards. We could not observe desmin-positive, myofibrillar-proteins-negative cells, and we could not find any non-striated distribution of sarcomeric proteins, such as stress fiber-like structures. Some steps, like fusion before striation, seem to be different in the zebrafish when compared with the previously described myogenesis sequences.

Cholesterol depletion by methyl-β-cyclodextrin enhances myoblast fusion and induces the formation of myotubes with disorganized nuclei

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-β-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric α-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and β-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.

Intermediate filament proteins in TPA-treated skeletal muscle cells in culture

SummaryThe cocarcinogenic phorbol ester 13-tetradecanoyl-O-phorbol acetate selectively and reversibly inhibits the ongoing differentiation programme of chick muscle cells in culture. 13-tetradecanoyl-O-phorbol acetate promptly blocks spontaneous contractions in mature myotubes and induces them to retract, forming giant myosacs and concurrently stress fibre-like structures are assembled. Using indirect immunofluorescence to localise desmin, the muscle specific intermediate filament protein, it was shown that its distribution is longitudinally oriented in mature myotubes. In myosacs, desmin has a reticular pattern although not as linearly oriented as in control myotubes. Using gel electrophoresis of control and 13-tetradecanoyl-O-phorbol acetate treated cell extracts, three major protein bands were observed with molecular weight of 43, 50 and 55 kDa. They migrate as actin, desmin and vimentin, respectively. The 50 kDa and 55 kDa proteins were expressed more in 13-tetradecanoyl-O-phorbol acetate-treated cells. The 50 kDa band was confirmed as desmin by immunoblotting using anti-chicken desmin antibody. Two-dimensional gel electrophoresis analysis showed the appearance of more acidic isoforms of the 50 and 55 kDa proteins 13-tetradecanoyl-O-phorbol in acetate-treated cells. The 43 kDa protein was seen as three distinct isoforms in control cells and as only two isoforms in 13-tetradecanoyl-O-phorbol acetate-treated cells.

Cholesterol depletion by methyl-β-cyclodextrin enhances cell proliferation and increases the number of desmin-positive cells in myoblast cultures.

Skeletal myogenesis comprises myoblast replication and differentiation into striated multinucleated myotubes. Agents that interfere with myoblast replication are important tools for the understanding of myogenesis. Recently, we showed that cholesterol depletion by methyl-β-cyclodextrin (MCD) enhances the differentiation step in chick-cultured myogenic cells, involving the activation of the Wnt/β-catenin signaling pathway. However, the effects of cholesterol depletion on myoblast replication have not been carefully studied. Here we show that MCD treatment increases cell proliferation in primary chick myogenic cell cultures. Treatment of myogenic cells with the anti-mitotic reagent cytosine arabinoside, immediately following cholesterol depletion, blocks the MCD-induced effects on proliferation. Cholesterol depletion induced an increase in the number of desmin-positive mononucleated cells, and an increase in desmin expression. MCD induces an increase in the expression of the cell cycle regulator p53 and the master switch gene MyoD1. Treatment with BIO, a specific inhibitor of GSK3β, induced effects similar to MCD on cell proliferation; while treatment with Dkk1, a specific inhibitor of the Wnt/β-catenin pathway, neutralized the effects of MCD. These findings indicate that rapid changes in the cholesterol content in cell membranes of myoblasts can induce cell proliferation, possibly by the activation of the Wnt/β-catenin signaling pathway.

Cell adhesion in zebrafish myogenesis: distribution of intermediate filaments, microfilaments, intracellular adhesion structures and extracellular matrix.

To overcome the limitations of in vitro studies, we have been studying myogenesis in situ in zebrafish embryos, at a sub-cellular level. While in previous works we focused on myofibrillogenesis and some aspects of adhesion structures, here we describe in more detail cell adhesion structures and interactions among cytoskeletal components, membrane and extracellular matrix during zebrafish muscle development. We studied the intermediate filaments, and we describe the full range of desmin distribution in zebrafish development, from perinuclear to striated, until its deposition around the intersomite septa of older somites. This adhesion structure, positive for desmin and actin, has not been previously observed in myogenesis in vitro. We also show that actin is initially located in the intersomite septum region whereas it is confined to the myofibrils later on. While actin localization changes during development, the adhesion complex proteins vinculin, paxillin, talin, dystrophin, laminin and fibronectin always appear exclusively at the intersomite septa, and appear to be co-distributed, even though the extracellular proteins accumulates before the intracellular ones. Contrary to the adhesion proteins, that are continuously distributed, desmin and sarcomeric actin form triangular aggregates among the septa and the cytoskeleton. We studied the cytoskeletal linker plectin as well, and we show that it has a distribution similar to desmin and not to actin. We conclude that the in situ adhesion structures differ from their in vitro counterparts, and that the actual zebrafish embryo myogenesis is quite different than that which occurs in in vitro systems.

Chondroitin sulfate and keratan sulfate are the major glycosaminoglycans present in the adult zebrafish Danio rerio (Chordata-Cyprinidae)

The zebrafish Danio rerio (Chordata-Cyprinidae) is a model organism frequently used to study the functions of proteoglycans and their glycosaminoglycan (GAG) chains. Although several studies clearly demonstrate the participation of these polymers in different biological and cellular events that take place during embryonic development, little is known about the GAGs in adult zebrafish. In the present study, the total GAGs were extracted from the whole fish by proteolytic digestion, purified by anion-exchange chromatography and characterized by electrophoresis after degradation with specific enzymes and/or by high-performance liquid chromatography (HPLC) analysis of the disaccharides. Two GAGs were identified: a low-molecular-weight chondroitin sulfate (CS) and keratan sulfate (KS), corresponding to ∼80% and 20% of the total GAGs, respectively. In the fish eye, KS represents ∼ 80% of total GAGs. Surprisingly, no heparinoid was detected, but may be present in the fish at concentrations lower than the limit of the method used. HPLC of the disaccharides formed after chondroitin AC or ABC lyase degradation revealed that the zebrafish CS is composed by ΔUA-1→3-GalNAc(4SO4) (59.4%), ΔUA-1→3-GalNAc(6SO4) (23.1%), and ΔUA-1→3-GalNAc (17.5%) disaccharide units. No disulfated disaccharides were detected. Immunolocalization on sections from zebrafish retina using monoclonal antibodies against CS4- or 6-sulfate showed that in the retina these GAGs are restricted to the outer and inner plexiform layers. This is the first report showing the presence of KS in zebrafish eye, and the structural characterization of CS and its localization in the zebrafish retina. Detailed information about the structure and tissue localization of GAGs is important to understand the functions of these polymers in this model organism.

A soluble and active form of Wnt-3a protein is involved in myogenic differentiation after cholesterol depletion

Cholesterol is one of the major lipids of plasma membranes. Recently, we have shown that cholesterol depletion by methyl‐β‐cyclodextrin (MβCD) induces the activation of the Wnt/β‐catenin pathway and enhances myogenic differentiation. Here, we show that MβCD‐conditioned media accelerates myogenesis in a similar way as MβCD does, suggesting that the effects induced by MβCD could be caused by soluble factors present in the culture medium. Soluble Wnt‐3 protein is significantly enhanced in MβCD‐conditioned medium. Wnt‐3a‐enriched media induces myogenesis as much as MβCD does, whereas Wnt‐5a‐enriched media inhibits. We suggest that Wnt‐3a is involved in the myogenic induction observed after cholesterol depletion.

Cytoskeletal and cellular adhesion proteins in zebrafish (Danio rerio) myogenesis

The current myogenesis and myofibrillogenesis model has been based mostly on in vitro cell culture studies, and, to a lesser extent, on in situ studies in avian and mammalian embryos. While the more isolated artificial conditions of cells in culture permitted careful structural analysis, the actual in situ cellular structures have not been described in detail because the embryos are more difficult to section and manipulate. To overcome these difficulties, we used the optically clear and easy to handle embryos of the zebrafish Danio rerio. We monitored the expression of cytoskeletal and cell-adhesion proteins (actin, myosin, desmin, alpha-actinin, troponin, titin, vimentin and vinculin) using immunofluorescence microscopy and video-enhanced, background-subtracted, differential interference contrast of 24- to 48-h zebrafish embryos. In the mature myotome, the mononucleated myoblasts displayed periodic striations for all sarcomeric proteins tested. The changes in desmin distribution from aggregates to perinuclear and striated forms, although following the same sequence, occurred much faster than in other models. All desmin-positive cells were also positive for myofibrillar proteins and striated, in contrast to that which occurs in cell cultures. Vimentin appeared to be striated in mature cells, while it is developmentally down-regulated in vitro. The whole connective tissue septum between the somites was positive for adhesion proteins such as vinculin, instead of the isolated adhesion plaques observed in cell cultures. The differences in the myogenesis of zebrafish in situ and in cell culture in vitro suggest that some of the previously observed structures and protein distributions in cultures could be methodological artifacts.

Reentrant spiral waves of spreading depression cause macular degeneration in hypoglycemic chicken retina

Spreading depression (SD), a slow diffusion-mediated self-sustained wave of depolarization that severely disrupts neuronal function, has been implicated as a cause of cellular injury in a number of central nervous system pathologies, including blind spots in the retina. Here we show that in the hypoglycemic chicken retina, spontaneous episodes of SD can occur, resulting in irreversible punctate lesions in the macula, the region of highest visual acuity in the central region of the retina. These lesions in turn can act as sites of origin for secondary self-sustained reentrant spiral waves of SD that progressively enlarge the lesions. Furthermore, we show that the degeneration of the macula under hypoglycemic conditions can be prevented by blocking reentrant spiral SDs or by blocking caspases. The observation that spontaneous formation of reentrant spiral SD waves leads to the development of progressive retinal lesions under conditions of hypoglycemia establishes a potential role of SD in initiation and progression of macular degeneration, one of the leading causes of visual disability worldwide.