Trafficking Highways to the Intercalated Disc: New Insights Unlocking the Specificity of Connexin 43 Localization

Abstract

With each heartbeat, billions of cardiomyocytes work in concert to propagate the electrical excitation needed to effectively circulate blood. Regulated expression and timely delivery of connexin proteins to form gap junctions at the specialized cell–cell contact region, known as the intercalated disc, is essential to ventricular cardiomyocyte coupling. We focus this review on several regulatory mechanisms that have been recently found to govern the lifecycle of connexin 43 (Cx43), the short-lived and most abundantly expressed connexin in cardiac ventricular muscle. The Cx43 lifecycle begins with gene expression, followed by oligomerization into hexameric channels, and then cytoskeletal-based transport toward the disc region. Once delivered, hemichannels interact with resident disc proteins and are organized to effect intercellular coupling. We highlight recent studies exploring regulation of Cx43 localization to the intercalated disc, with emphasis on alternatively translated Cx43 isoforms and cytoskeletal transport machinery that together regulate Cx43 gap junction coupling between cardiomyocytes.     

Cell Junctions in the Specialized Conduction System of the Heart

Abstract

Anchoring cell junctions are integral in maintaining electro-mechanical coupling of ventricular working cardiomyocytes; however, their role in cardiomyocytes of the cardiac conduction system (CCS) remains less clear. Recent studies in genetic mouse models and humans highlight the appearance of these cell junctions alongside gap junctions in the CCS and also show that defects in these structures and their components are associated with conduction impairments in the CCS. Here we outline current evidence supporting an integral relationship between anchoring and gap junctions in the CCS. Specifically we focus on (1) molecular and ultrastructural evidence for cell–cell junctions in specialized cardiomyocytes of the CCS, (2) genetic mouse models specifically targeting cell–cell junction components in the heart which exhibit CCS conduction defects and (3) human clinical studies from patients with cell–cell junction-based diseases that exhibit CCS electrophysiological defects.     

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1 hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.     

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRPα and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.     

Giardia lamblia: Ultrastructural Study of the In Vitro Effect of Benzimidazoles

ABSTRACT Axenically grown Giardia lamblia trophozoites treated with low concentrations of the benzimidazole carbamates albendazole and mebendazole detach from glass culture tubes and lose viability. Scanning electron microscopic observations revealed that these drugs produce grotesque modifications of the cell shape of the parasite and disassembly of the adhesive disc. Transmission electron microscopy showed several stages of the fragmentation of adhesive discs with dispersion of microtubules and microribbons in the cytoplasm. Flagella appeared undamaged. In drug-treated trophozoites electron-dense precipitates were selectively deposited on microtubules and microribbons. The results indicate that the antigiardial effect of benzimidazoles is the result of binding to microtubules and subsequent alterations of the cytoskeleton. The electron microscopic observations also suggest that the drugs may bind to microribbon components of the adhesive disc, possibly giardin proteins.     

植物细胞工程在小麦抗赤霉病育种中的应用

小麦赤霉病是全球性小麦病害,严重影响小麦产量和品质,赤霉菌产生的毒素进一步威胁人畜安全,培育抗病品种是控制小麦赤霉病危害的根本途径。植物细胞工程技术可创造新的遗传变异、加快育种进程,已经广泛应用于小麦抗赤霉病育种。概述了体细胞无性系变异诱导、花药培养、小麦与玉米杂交培育加倍单倍体以及幼胚培养一年多代快速成苗等植物细胞工程技术研究进展,着重介绍了其在抗小麦赤霉病育种中的应用。最后对未来发展趋势做了展望,植物细胞工程结合分子育种技术将在小麦抗赤霉病品种培育中发挥更重要的作用。     

Optimization of 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester for Labeling Human Intervertebral Disc Cells in Vitro

We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 JJIM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 JJLM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultra-structural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies.     

Optimization of 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester for Labeling Human Intervertebral Disc Cells in Vitro

We have assessed the utility of an intracellular fluorochrome, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), as a tracking label for human intervertebral disc cells in vitro. Although 5 JJIM provides adequate intracellular labeling for whole cell fluorescent microscopic identification of labeled cells, 20 JJLM was preferable for immunocytochemical localization of paraffin embedded labeled cells. Electron dense vesicles are seen at the ultra-structural level in labeled cells. Discrete vesicular labeling can also be observed in whole cell mounts viewed with fluorescence microscopy. Whole cells retain good label for 6 weeks. CFSE labeling is relatively easy, nontoxic to cells and nonradiocactive. Initial optimization of dose with specific cells types is recommended when confirmation of positive immunocytochemistry is needed for tissue engineering studies.     

Caco-2 Cell Line Used as an In Vitro Model to Study Cadmium Accumulation in Intestinal Epithelial Cells

¹⁰⁹Cd uptake was studied using the highly differentiated TC7 clone of Caco-2 cells as a model of human enterocyte function. Intracellular accumulation of 0.3 μm ¹⁰⁹Cd involved a rapid and a slow uptake phase, which resulted in complete equilibration (t _?= 17.3 ± 1.3 min) with an apparent in-to-out distribution ratio (α _e ) of 11.6 ± 0.8. The amplitude of the rapid phase (U ₀) and the rate of the slow phase (V) were similarly reduced in the less differentiated PF11 clone, but comparable α _e values were observed at equilibrium. In both clones, the t _? and α _e values increased and decreased, respectively, upon addition of unlabeled Cd to the uptake media. In TC7 cells, ¹⁰⁹Cd uptake at 1 min (U ₁) was unaffected by Ca concentrations four order of magnitude in excess, but both U ₀ and V demonstrated similar sensitivities to unlabeled Cd, Zn and sulfhydryl-reactive agents. Only U ₀ disappeared when EDTA was present in the wash solutions. U ₁ showed saturation kinetics and the data were found compatible with a model assuming rapid initial Cd binding and transport through a unique transport protein (K _m = 3.8 ± 0.7 μm). Cd efflux kinetics demonstrated partial reversibility in EDTA-containing solutions, suggesting that the taken up Cd might be both tightly and loosely bound to intracellular binding sites. However, the displacement of ¹⁰⁹Cd measured at 65 min failed to reveal this heterogeneity: the data were found compatible with a model equation assuming the presence of one class of high-capacity high-affinity binding sites. We conclude that a slow-transport fast-intracellular binding mechanism of Cd uptake best accounts for these results and that Cd transport most likely involves a carrier-type of protein unrelated to Ca absorption. Received: 19 January 1996/Revised: 23 January 1997     

组织工程心脏瓣膜的研究进展

组织工程心脏瓣膜(tissue engineering heart valve,TEHV)理论上能克服机械瓣及生物瓣的不足,具有广阔的发展前景。目前组织工程心脏瓣膜的研究主要集中在瓣膜支架材料的选取及制备、种子细胞的选择和种子细胞的种植及培养等三方面。本文将分别就这三方面研究进展进行介绍,分析目前存在的问题,并对其应用进行展望。     

内皮祖细胞在血管组织工程研究中的应用现状

组织工程血管是修复紫绀型先心病右心室流出道的潜在替代材料,实验研究表明外周血内皮祖细胞(endothelial progenitor cell,EPC)可作为构建组织工程血管的良好种子细胞。现阶段国内外对组织工程血管的研究方兴未艾,EPC在血管组织工程研究中的应用还处在体外培养或动物实验阶段,尚无临床应用报道。近来某些基础研究的成果对于新生血管的形成和EPC的自动募集机制有了合理的解释,其中缺氧被认为是重要的始动因素,这些研究成果也为EPC作为种子细胞应用于血管组织工程提供了理论基础。所以,阐明EPC的低氧诱导机制及其在血管组织工程的应用必将有助于复杂紫绀型先心病的外科治疗。本文主要介绍了目前该领域研究现状及相关研究基础的进展,并总结提出了在EPC研究和未来临床应用中需解决的问题。     

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.     

Ultrastructural Characteristics of the In Vitro Cell Cycle of the Protozoan Pathogen of Oysters, Perkinsus marinus

Ultrastructural characteristics of vegetative and zoosporangial stages of cultured Perkinsus marinus, a pathogen of the eastern oyster, Crassostrea virginica, were examined by transmission electron microscopy. An axenic cell culture was propagated from infected Chesapeake Bay oyster hemolymph. Different stages of the in vitro cell cycle, including schizonts and different size trophonts, were examined. Trophonts had spherical nuclei with wide perinuclear spaces, mitochondria with tubular cristae, and vacuoles with vacuoplasts. There were micropores on the inside of cell walls. A tubular network in the cytoplasm connected lomasomes to vacuoles, and contained vacuoplast precursor material. Vacuoplasts and precursor material diminished when cell cultures were not fed, suggesting a function in metabolite storage. Cells divided by schizogony or binary fission. Daughter cells in a schizont were not alike, and may specialize for different functions. Some of the daughter cells in a schizont died. Some hypnospores, directly isolated from infected oyster hemolymph enlarged in Ray's fluid thioglycollate medium, and were induced to zoosporulate. Zoosporangia contained varicose, hypha-like structures, whose apical tips gave rise to prezoospores. Ultrastructural characteristics of the vegetative and zoosporangial stages did not resemble any apicomplexan parasites other than members of the genus Perkinsus.     

In Vitro Study of a Novel Nanogold-Collagen Composite to Enhance the Mesenchymal Stem Cell Behavior for Vascular Regeneration

Novel nanocomposites based on type I collagen (Col) containing a small amount (17.4, 43.5, and 174 ppm) of gold nanoparticles (AuNPs, approximately 5 nm) were prepared in this study. The pure Col and Col-AuNP composites (Col-Au) were characterized by the UV-Vis spectroscopy (UV-Vis), surface-enhanced raman spectroscopy (SERS) and atomic force microscopy (AFM). The interaction between Col and AuNPs was confirmed by infrared (IR) spectra. The effect of AuNPs on the biocompatibility of Col, evaluated by the proliferation and reactive oxygen species (ROS) production of mesenchymal stem cells (MSCs) as well as the activation of monocytes and platelets, was investigated. Results showed that Col-Au had better biocompatibility than Col. Upon stimulation by vascular endothelial growth factor (VEGF) and stromal derived factor-1α (SDF-1α), MSCs expressed the highest levels of αvβ3 integrin/CXCR4, focal adhesion kinase (FAK), matrix metalloproteinase-2 (MMP-2), and Akt/endothelial nitric oxide synthase (eNOS) proteins when grown on the Col-Au (43.5 ppm) nanocomposite. Taken together, Col-Au nanocomposites may promote the proliferation and migration of MSCs and stimulate the endothelial cell differentiation. These results suggest that Col-Au may be used to construct tissue engineering scaffolds for vascular regeneration.     

A Collagen Substrate Enhances the Sealing Capacity of Tight Junctions of A6 Cell Monolayers

A6 cells, a kidney derived epithelial cell line, when cultured either on a collagen-coated substrate or on polycarbonate substrate without collagen form confluent monolayers that are similar in cell density and overall morphology. However, the transepithelial electrical resistance (TER) of monolayers grown on the collagen-coated substrate is ninefold higher than that of monolayers grown without collagen. A comparative freeze-fracture study showed that this large difference in TER is not related to the length or number of tight junction strands but to differences in the specific conductance of individual strands. This conductance was obtained considering the TER, the linear junctional density and the mean number of tight junction strands. We estimated the specific linear conductance of the tight junction strands to be 2.56 × 10⁻⁷ S/cm for cells grown on collagen and 30.3 × 10⁻⁷ S/cm for the cells grown without collagen. We also examined changes in distribution and phosphorylation states of the zonula occludens associated protein, ZO-1, during monolayer formation. Immunocytochemistry reveals that the distribution of ZO-1 follows a similar time course and pattern independent of the presence or absence of collagen. While the amount of ZO-1 expression is identical in cells grown on both substrates, this protein is phosphorylated to a greater extent during the initial stages of confluence in cells cultured on collagen. We suggest that the phosphorylation levels of ZO-1 in A6 cells at the early stages of monolayer formation may determine the final molecular structure and specific conductance of the tight junctions strands. Received: 18 September 1996/Revised: 17 June 1997     

ADAMTS-4 and Biglycan are Expressed at High Levels and Co-Localize to Podosomes During Endothelial Cell Tubulogenesis In Vitro

Proteolysis of the extracellular matrix influences vascular growth. We examined the expression of ADAMTS-1, -4, and -5 metalloproteinases and their proteoglycan substrates versican, decorin, and biglycan as human umbilical vein endothelial cells (HUVECs) formed tubes within type I collagen gels in vitro. Tubulogenic and control HUVEC cultures expressed low levels of ADAMTS-1 and -5 mRNAs, but ADAMTS-4 mRNA was relatively abundant and was significantly elevated (as was ADAMTS-4 protein) in tubulogenic cultures versus controls. Immunocytochemistry revealed ADAMTS-4 in f-actin- and cortactin-positive podosome-like puncta in single cells and mature tubes. Tubulogenic and control cultures expressed low levels of versican and decorin mRNAs; however, peak levels of biglycan mRNA were 400- and 16,000-fold that of versican and decorin, respectively. Biglycan mRNA was highest at 3 hr, declined steadily through day 7 and, at 12 hr and beyond, was significantly lower in tubulogenic cultures than in controls. Western blots of extracellular matrix from tubulogenic cultures contained bands corresponding to biglycan and its cleavage products. By immunocytochemistry, biglycan was found in the pericellular matrix surrounding endothelial tubes and in cell-associated puncta that co-localized with ADAMTS-4 and cortactin. Collectively, our results suggest that ADAMTS-4 and its substrate biglycan are involved in tubulogenesis by endothelial cells.     

Use of a Tetrazolium-based Cell Proliferation Assay to Measure Effects of In Vitro Conditions on Perkinsus marinus (Apicomplexa) Proliferation

ABSTRACT. Because the in vitro cell cycle of the apicomplexan oyster pathogen Perkinsus marinus generates cell populations heterogeneous for size and typified by aggregation, both turbidimetric and counting methods for determining population densities and proliferation rates are inaccurate or cumbersome. We show that a commercial, tetrazolium-based cell proliferation assay yields a soluble formazan chromophore upon intracellular reduction by P. marinus . at a rate proportional to cell population biovolume. Using this assay system, we have 1) defined selected culture system parameters which maximize P. marinus in vitro proliferation, 2) assessed selected chemosensitivities, and 3) standardized the assay system for quantification of densities and doubling times of populations propagated with our optimized system. Growth was supported by four tested base media and was maximized in 1:1 DME/Ham's F-12. Temperatures of 10–40° C permitted growth, which was maximized at 35° C. pH 6.0–8.5 permitted growth, which was maximized at 7.0–7.5. Osmolalities of 340–1,930 mOsm supported growth, which was maximized at 790 mOsm. Serum supplements from 1–10% (v/v) did not enhance log phase growth, but enhanced stationary phase metabolic activity in proportion to concentration. Our isolate (ATCC 50439) has a 13 h log phase doubling time when propagated under optimized conditions: 28° C, 800 mOsm, pH 7.0, 1:1 DME/Ham's F-12 medium, 5% (v/v) FBS. It is tolerant of antibacterial agents at concentrations commonly used in vertebrate tissue culture, but is inhibited by several antimycotics at similar concentrations.     

Molecular Characterization of Equine APRIL and its Expression Analysis During the Adipogenic Differentiation of Equine Adipose-Derived Stem Cell In Vitro

A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753 bp, encoding a 250-amino acid protein with the typical TNF structure domain. During the two weeks’ adipogenic differentiation of equine ASC, although the protein expression of APRIL and TACI had an insignificant change, that of BCMA increased significantly. Moreover, with the addition of recombinant protein His₆-sAPRIL, a reduced differentiation of equine ASC toward adipocyte was detected. These results may provide the basis for investigating the role of APRIL in ASC adipogenic differentiation.