Intercellular Dye Transfer Research Articles

Preparation of myocardial patches from DiI-labeled rat bone marrow mesenchymal stem cells and neonatal rat cardiomyocytes contact co-cultured on polycaprolactone film

To provide better treatment of myocardial infarction, DiI-labeled bone marrow mesenchymal stem cells (BMSCs) were contact co-cultured with cardiomyocytes (CMs) on polycaprolactone (PCL) film to prepare myocardial patches. BMSCs from Sprague Dawley rats were isolated, cultured, and characterized for expression of surface markers by flow cytometry. CMs were isolated from suckling rats. After BMSCs were cultured for three generations, they were labeled with DiI dye. DiI-labeled BMSCs were co-cultured with CMs on PCL film in the experimental group, while CMs were replaced with the same amount of unlabeled BMSCs in the control group. After 24 h, cell growth was observed by light microscopy and cells were fixed for scanning electron microscopy (SEM). After 7 d of co-culture, cells were stained for immunofluorescence detection of myocardial markers cardiac troponin T (cTnT) and α-actin. Differentiation of BMSCs on PCL was observed by fluorescence microscopy. The efficiency of BMSC differentiation into CMs was analyzed by flow cytometry on the first and seventh days of co-culture. CMs were stained with calcein alone and contact co-cultured with DiI-labeled BMSCs on PCL film to observe intercellular dye transfer. Finally, cells were stained for immunofluorescence detection of connexin 43 (Cx43) expression and to observe the relationship between gap junctions and contact co-culture. BMSCs were identified by flow cytometry as strongly positive for CD90 and CD44H, and negative for CD11b/c and CD45. After co-culture for 24 h, cells were observed to have attached to PCL by light microscopy. Upon appropriate excitation, DiI-labeled BMSCs exhibited red fluorescence, while unlabeled CMs did not. SEM revealed a large number of cells on the PCL membrane and their cell state appeared normal. On the seventh day, some DiI-labeled BMSCs expressed cTnT and α-actin. Flow cytometry showed that the rate of stem cell differentiation in the experimental group was significantly higher than the control group on the seventh day (20.12% > 3.49%, P < 0.05). From the second day of co-culture, immunofluorescence staining for Cx43 revealed green fluorescent puncta in some BMSCs; from the third day of co-culture, a portion of BMSCs exhibited green fluorescence in dye transfer tests. Contact co-culture of DiI-labeled BMSCs and CMs on PCL film generated primary myocardial patches. The mechanism by which contact co-culture promoted differentiation of the myocardial patch may be related to gap junctions and gap junction-mediated intercellular signaling pathways.

Generation and Characterization of Immortalized Mouse Cortical Astrocytes From Wildtype and Connexin43 Knockout Mice.

We transduced mouse cortical astrocytes cultured from four litters of embryonic wildtype (WT) and connexin43 (Cx43) null mouse pups with lentiviral vector encoding hTERT and measured expression of astrocyte-specific markers up to passage 10 (p10). The immortalized cell lines thus generated (designated IWCA and IKOCA, respectively) expressed biomarkers consistent with those of neonatal astrocytes, including Cx43 from wildtype but not from Cx43-null mice, lack of Cx30, and presence of Cx26. AQP4, the water channel that is found in high abundance in astrocyte end-feet, was expressed at moderately high levels in early passages, and its mRNA and protein declined to low but still detectable levels by p10. The mRNA levels of the astrocyte biomarkers aldehyde dehydrogenase 1L1 (ALDH1L1), glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) remained relatively constant during successive passages. GS protein expression was maintained while GFAP declined with cell passaging but was still detectable at p10. Both mRNA and protein levels of glutamate transporter 1 (GLT-1) declined with passage number. Immunostaining at corresponding times was consistent with the data from Western blots and provided evidence that these proteins were expressed at appropriate intracellular locations. Consistent with our goal of generating immortalized cell lines in which Cx43 was either functionally expressed or absent, IWCA cells were found to be well coupled with respect to intercellular dye transfer and similar to primary astrocyte cultures in terms of time course of junction formation, electrical coupling strength and voltage sensitivity. Moreover, barrier function was enhanced in co-culture of the IWCA cell line with bEnd.3 microvascular endothelial cells. In addition, immunostaining revealed oblate endogenous Cx43 gap junction plaques in IWCA that were similar in appearance to those plaques obtained following transfection of IKOCA cells with fluorescent protein tagged Cx43. Re-expression of Cx43 in IKOCA cells allows experimental manipulation of connexins and live imaging of interactions between connexins and other proteins. We conclude that properties of these cell lines resemble those of primary cultured astrocytes, and they may provide useful tools in functional studies by facilitating genetic and pharmacological manipulations in the context of an astrocyte-appropriate cellular environment.

Visualization and quantification of dynamic intercellular coupling in human embryonic stem cells using single cell sonoporation

Gap junctions (GJs), which are proteinaceous channels, couple adjacent cells by permitting direct exchange of intracellular molecules with low molecular weights. GJ intercellular communication (GJIC) plays a critical role in regulating behaviors of human embryonic stem cells (hESCs), affecting their proliferation and differentiation. Here we report a novel use of sonoporation that enables single cell intracellular dye loading and dynamic visualization/quantification of GJIC in hESC colonies. By applying a short ultrasound pulse to excite single microbubbles tethered to cell membranes, a transient pore on the cell membrane (sonoporation) is generated which allows intracellular loading of dye molecules and influx of Ca2+ into single hESCs. We employ live imaging for continuous visualization of intercellular dye transfer and Ca2+ diffusion in hESC colonies. We quantify cell–cell permeability based on dye diffusion using mass transport models. Our results reveal heterogeneous intercellular connectivity and a variety of spatiotemporal characteristics of intercellular Ca2+ waves in hESC colonies induced by sonoporation of single cells.

The in-vitro effects of cAMP and cGMP modulators on inter-cellular dye transfer and gene expression levels in rat cumulus cell – oocyte complexes

Supplementation of in-vitro maturation medium with reagents that inhibit meiotic resumption whilst supporting normal function of cumulus cell–oocyte complexes (COC) is challenging. This study compared the in-vitro effects of synthetic and physiologically-relevant reagents on meiotic resumption, gap junction activity and gene expression of rat COC. Higher doses of forskolin reduced gap junction activity. Whilst addition of phosphodiesterase inhibitors initially promoted gap junction activity, this decreased with time in-vitro. Moreover despite oocytes remaining in meiotic arrest, there was a concomitant decline in expression of genes critical for oocyte maturation, and evidence of a reduction in overall transcription rate. Similarly, supplementing media with C-type natriuretic peptide and/or oestradiol delayed meiotic resumption and only initially maintained gap junction activity. In contrast, several key genes were stimulated and overall transcription rates remained constant with time in-vitro. In summary, supplementation of media with physiologically-relevant reagents may better enable normal functions of the COC.

Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated

Several Cx40 mutants have been identified in patients with atrial fibrillation (AF). We have been working to identify physiological or cell biological abnormalities of several of these human mutants that might explain how they contribute to disease pathogenesis. Wild type (wt) Cx40 or four different mutants (P88S, G38D, V85I, and L229M) were expressed by the transfection of communication-deficient HeLa cells or HL-1 cardiomyocytes. Biophysical channel properties and the sub-cellular localization and protein levels of Cx40 were characterized. Wild type Cx40 and all mutants except P88S formed gap junction plaques and induced significant gap junctional conductances. The functional mutants showed only modest alterations of single channel conductances or gating by trans-junctional voltage as compared to wtCx40. However, immunoblotting indicated that the steady state levels of G38D, V85I, and L229M were reduced relative to wtCx40; most strikingly, G38D was only 20–31% of wild type levels. After the inhibition of protein synthesis with cycloheximide, G38D (and to a lesser extent the other mutants) disappeared much faster than wtCx40. Treatment with the proteasomal inhibitor, epoxomicin, greatly increased levels of G38D and restored the abundance of gap junctions and the extent of intercellular dye transfer. Thus, G38D, V85I, and L229M are functional mutants of Cx40 with small alterations of physiological properties, but accelerated degradation by the proteasome. These findings suggest a novel mechanism (protein instability) for the pathogenesis of AF due to a connexin mutation and a novel approach to therapy (protease inhibition).

Shear stress modulates the expression of the atheroprotective protein Cx37 in endothelial cells

High laminar shear stress (HLSS) is vasculoprotective partly through induction of Kruppel-like factor 2 (KLF2). Connexin37 (Cx37) is highly expressed in endothelial cells (ECs) of healthy arteries, but not in ECs overlying atherosclerotic lesions. Moreover, Cx37 deletion in apolipoprotein E-deficient (ApoE−/−) mice increases susceptibility to atherosclerosis. We hypothesized that shear stress, through KLF2 modulation, may affect Cx37 expression in ECs. Cx37 expression and gap-junctional intercellular (GJIC) dye transfer are prominent in the straight portion of carotid arteries of ApoE−/− mice, but are reduced at the carotid bifurcation, a region subjected to oscillatory flow. Shear stress-modifying vascular casts were placed around the common carotid artery of ApoE−/− mice. Whereas Cx37 expression was conserved in HLSS regions, it was downregulated to ~50% in low laminar or oscillatory flow regions. To study the mechanisms involved, HUVECs or bEnd.3 cells were exposed to flow in vitro. Cx37 and KLF2 expression were increased after 24h of HLSS. Interestingly, shear-dependent Cx37 expression was significantly reduced after silencing of KLF2. Moreover after exposure to simvastatin, a well-known KLF2 inducer, KLF2 binds to the Cx37 promoter region as shown by ChIP. Finally, GJIC dye transfer was highly reduced after KLF2 silencing and was increased after exposure to simvastatin. HLSS upregulates the expression of Cx37 in ECs by inducing its transcription factor KLF2, which increases intercellular communication. Therefore, this effect of shear stress on Cx37 expression may contribute to the synchronization of ECs and participate in the protective effect of HLSS.

Tuning Electrical Conduction Along Endothelial Tubes of Resistance Arteries Through Ca 2+ -Activated K + Channels

Electrical conduction through gap junction channels between endothelial cells of resistance vessels is integral to blood flow control. Small and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca)/IK(Ca)) initiate electrical signals in endothelial cells, but it is unknown whether SK(Ca)/IK(Ca) activation alters signal transmission along the endothelium. We tested the hypothesis that SK(Ca)/IK(Ca) activity regulates electrical conduction along the endothelium of resistance vessels. Freshly isolated endothelial cell tubes (60 μm wide; 1-3 mm long; cell length, ≈35 μm) from mouse skeletal muscle feed (superior epigastric) arteries were studied using dual intracellular microelectrodes. Current was injected (±0.1-3 nA) at site 1 while recording membrane potential (V(m)) at site 2 (separation distance=50-2000 μm). SK(Ca)/IK(Ca) activation (NS309, 1 μmol/L) reduced the change in V(m) along endothelial cell tubes by ≥50% and shortened the electrical length constant (λ) from 1380 to 850 μm (P<0.05) while intercellular dye transfer (propidium iodide) was maintained. Activating SK(Ca)/IK(Ca) with acetylcholine or SKA-31 also reduced electrical conduction. These effects of SK(Ca)/IK(Ca) activation persisted when hyperpolarization (>30 mV) was prevented with 60 mmol/L [K(+)](o). Conversely, blocking SK(Ca)/IK(Ca) (apamin+charybdotoxin) depolarized cells by ≈10 mV and enhanced electrical conduction (ie, changes in V(m)) by ≈30% (P<0.05). These findings illustrate a novel role for SK(Ca)/IK(Ca) activity in tuning electrical conduction along the endothelium of resistance vessels by governing signal dissipation through changes in membrane resistance. Voltage-insensitive ion channels can thereby tune intercellular electrical signaling independent from gap junction channels.

Nicotine effects on human endothelial intercellular communication via α4β2 and α3β2 nicotinic acetylcholine receptor subtypes

Since previous in vitro experiments revealed that nicotine can impair endothelial intercellular communication via the downregulation of connexin43 (Cx43), we wanted to find out which nicotinic acetylcholine receptors are involved in the molecular mechanism of communication failure. Cultured human endothelial cells were exposed to 1μM nicotine for 5days. Intercellular communication was measured using dye transfer study with/without subtype-specific nicotinic acetylcholine receptor (nAChR) inhibitors. Reverse transcriptase (RT)-PCR was used to further investigate the regulation of nAChR subtypes. Electron microscopy together with MAP LC3-II western blot was used to investigate possible autophagy processes. In cultured human endothelial cells, nicotine decreased the Cx43 protein amount as shown by western blot and immunohistochemistry; however, together with an unaltered mRNA expression as shown by RT-PCR. The nicotine-induced Cx43 downregulation functionally impaired intercellular dye transfer, which could be prevented by mecamylamine, κ-bungarotoxin, lobeline, and dihydro-β-erythroidine but not α-bungarotoxin, indicating that the nAChR subtypes α4β2 and α3β2 but not α7 are involved in signal cascade. RT-PCR analysis revealed that nicotine exposure resulted in the upregulation of α3 and β4 and the downregulation of α4-nAChR, while α7- and β2-nAChR-mRNA expressions remained unaltered. Furthermore, nicotine increased total protein ubiquinylation and proteasome activity as was shown by immunohistochemistry and peptide degradation analysis. Evidence of enhanced autophagic processes was assured by the occurrence of autophagic vacuoles in transmission electron microscopy and enhanced formation of MAP LC3-II in western blot. Reduced intercellular endothelial communication together with programmed cell death helps to explain the toxic effect of nicotine leading to endothelial dysfunction. The nAChR involved in the impairment of intercellular communication seem to be α4β2 and α3β2 but not α7.

New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes

The use of carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure lymphocyte proliferation by flow cytometry has become one of the most widely utilised assays for assessing lymphocyte responses. The properties of CFSE make it ideal for such a task, covalently labelling cells with a long-lived fluorescence of high intensity and low variance with minimal cell toxicity. No dye in the last 20years has been capable of replicating CFSE in these respects. However, currently CFSE is limited to following a maximum of 7 cell divisions and is not compatible for use with ubiquitously available fluorescein conjugates or other fluorescent molecules with spectral properties similar to fluorescein, such as EGFP. Here we characterise two new fluorescent dyes for measuring lymphocyte proliferation, Cell Trace Violet (CTV) and Cell Proliferation Dye eFluor 670 (CPD), which have different excitation and emission spectra to CFSE and, consequently, are compatible with fluorescein conjugates. We found that while both CTV and CPD can label cells to a high fluorescence intensity, which is long-lived and has low variability and low toxicity and makes them ideal for long-term tracking of non-dividing lymphocytes in vivo, CTV offers possibly the best available alternative to CFSE in the analysis of cell divisions. We also describe how intercellular dye transfer and cell autofluorescence can affect division resolution with the three different dyes and describe labelling conditions for the three dyes that produce ultra-bright lymphocytes for in vivo tracking studies and allow up to 11 cell divisions to be detected when using CFSE and CTV as the fluorescent dyes.

Gap junction assembly: roles for the formation plaque and regulation by the C-terminus of connexin43

Using an established gap junction (GJ) assembly system with experimentally reaggregated cells, we analyzed "formation plaques" (FPs), apparent sites of GJ assembly. Employing freeze-fracture electron microscopy methods combined with filipin labeling of sterols and immunolabeling for connexin43 (Cx43), we demonstrated that FPs constitute distinct membrane "domains" and that their characteristic 10-nm particles contain connexin43, thus representing precursors (i.e., GJ hemichannels) engaged in assembly. Analysis of FPs in new systems-HeLa and N2A cells-resolved questions surrounding several key but poorly understood steps in assembly, including matching of FP membranes in apposed cells, reduction in the separation between FP membranes during assembly, and the process of particle aggregation. Findings also indicated that "docking" of GJ hemichannels occurs within FP domains and contributes to reduction of intermembrane separation between FPs. Other experiments demonstrated that FPs develop following a major C-terminal truncation of Cx43 (M257), although assembly was delayed. Particle aggregation also occurred at lower densities, and densities of particles within developing GJ aggregates failed to achieve full-length levels. With regard to regulation, inhibition of assembly following protein kinase C activation failed to occur in the M257 truncation mutants, as measured by intercellular dye transfer. However, several C-terminal serine mutations failed to disrupt inhibition.

Impaired membrane targeting and aberrant cellular localization of human Cx26 mutants associated with inherited recessive hearing loss

Conclusion: This study demonstrated that five Cx26 mutations (R32H, S199F, 572delT, 631-632delGT, and Y155X) affect gap junction (GJ) functions by causing impaired membrane targeting and aberrant cellular localization, and one mutation (R165W) leads to a constriction of the channel pore with no dye coupling. Objective: To investigate the pathogenetic roles of six recessive Cx26 mutations (p.R32H, p.R165W, p.S199F, c.572delT, c.631-632delGT, and p.Y155X), which have not been functionally analyzed in vitro. Methods: The six mutants and wild-type Cx26 (wtCx26) were cloned into the EcoRI and SalI sites of pEGFP-N1 vector. We transfected the seven constructs into HeLa cells, followed by analysis of their protein expression using the western blot method, study of the protein localizations and gap junction-plaques on the cytomembrane under confocal microscopy, and assessment of the dye coupling of the mutated GJ channels by intercellular dye transfer experiment. Results: p.R165W targeted the cytomembrane and formed GJ-like structures in adjacent HeLa cells, causing null dye coupling. The mutants (p.R32H, p.S199F, c.572delT, c.631-632delGT, and p.Y155X) failed to reach the cell surface, and perfectly co-localized with endoplasmic reticulum (ER) throughout the cells.

In Vivo Evaluation of a Biologically Active Cardiomyocyte Seeded Scaffold

Background: While direct cell injections have been the main focus of cell-based therapies for chronic heart failure (CHF), newer approaches utilizing tissue engineered scaffolds have evolved. In CHF, we have shown that a 3-dimensional fibroblast construct (3DFC) increases myocardial blood flow and promotes angiogenesis but does not reverse maladaptive left ventricular (LV) remodeling. To attenuate remodeling requires repopulating the infarcted wall with new viable cells. We propose a neonatal cardiomyocyte seeded 3DFC (NC-3DFC) to salvage damaged myocardium and improve LV function. Methods: Cardiomyocytes are isolated from 24-48 hour old neonatal Sprague Dawley rat hearts then differentially plated in Ham's F-12 with 100mg/ml BSA, seeded onto the 3DFC, and implanted 3 weeks after left coronary artery ligation. Hemodynamic and echocardiographic studies are performed 3 weeks later. Connexin formation of NC-3DFC cultured for 6 days were evaluated using single cell dye injections. Results: The NC-3DFC contracts in a consistent rhythmic and directional fashion, spontaneously beating at 43 ± 3 bpm with a mean displacement of 1.3 ± 0.3 mm and contraction velocity of 0.8 ± 0.2 mm/sec (N = 10). Additionally, the NC-3DFC, can be electrically paced at near physiological rates, (270 ± 30 bpm). In rats with ejections fraction (EF) ≤ 35% at 3 weeks post MI, NC-3DFC implanted 3 weeks post MI increases (p < 0.05) EF from 31% ± 2 to 39% ± 1, cardiac index from 0.46 ± 0.05 to 0.61 ± 0.06 mL/(g min), and fractional shortening from 13% ± 1 to 17% ± 1, while end diastolic pressure decreases from 24 ± 2 to 14 ± 3 mmHg. Functional connexins are present at 48 hours and display increased connectivity through 6 days. Single cell dye injections show immediate intercellular dye transfer that is blocked by halothane, demonstrating that connexins are responsible for the cell-to-cell communication. Conclusion: Neonatal cardiomyocytes seeded and co-cultured onto biodegradable 3DFC allows for cellular survival, communication, and electrical coupling with resultant improvement in LV function in rats with CHF. This newly seeded NC-3DFC scaffold is a novel cell delivery system to potentially treat heart failure.

Dominant connexin26 mutants associated with human hearing loss have trans-dominant effects on connexin30

Dominant mutations in GJB2, the gene encoding the human gap junction protein connexin26 (Cx26), cause hearing loss. We investigated whether dominant Cx26 mutants interact directly with Cx30. HeLa cells stably expressing nine dominant Cx26 mutants, six associated with non-syndromic hearing loss (W44C, W44S, R143Q, D179N, R184Q and C202F) and three associated with hearing loss and palmoplantar keratoderma (G59A, R75Q and R75W), individually or together with Cx30, were analyzed by immunocytochemistry, co-immunoprecipitation, and functional assays (scrape-loading and/or fluorescence recovery after photobleaching). When expressed alone, all mutants formed gap junction plaques, but with impaired intercellular dye transfer. When expressed with Cx30, all mutants co-localized and co-immunoprecipitated with Cx30, indicating they likely co-assembled into heteromers. Furthermore, 8/9 Cx26 mutants inhibited the transfer of neurobiotin or calcein, indicating that these Cx26 mutants have trans-dominant effects on Cx30, an effect that may contribute to the pathogenesis of hearing loss.

Establishment of human corneal epithelial cells stably expressing human connexin43

Corneal epithelial cells communicate with each other through gap junctions. Whereas this property is retained in corneal epithelial cells in primary culture, it is often lost in immortalized epithelial cells. However, the life span of primary cultured corneal epithelial cells is short and the availability of human tissue for their preparation is limited. To examine the role of the gap-junction protein connexin43 (Cx43) in human corneal epithelial cells, we set out to establish an immortal human corneal epithelial cell line that stably expresses this protein. An expression vector encoding human Cx43 fused to enhanced green fluorescent protein (EGFP) was constructed and introduced by transfection into SV40-immortalized human corneal epithelial (HCE) cells. Stable transfectants were isolated by selection with the antibiotic G418. The expression and localization of the Cx43-EGFP fusion protein were examined by immunoblot analysis and fluorescence microscopy, respectively, and gap-junctional intercellular communication was monitored on the basis of dye coupling. HCE cells stably expressing Cx43-EGFP manifested intercellular dye transfer, whereas those stably expressing EGFP alone did not. Cx43-EGFP localized to the interfaces of neighboring cells. Stable expression of Cx43-EGFP in HCE cells did not affect the expression of keratins 3 and 12, which is a characteristic of corneal epithelial cells, but it did inhibit cell proliferation. We have established an HCE cell line that stably expresses human Cx43 and forms functional gap junctions. These cells may prove useful for studies of the role of gap junctions in the human corneal epithelium.

Assaying dynamic cell–cell junctional communication using noninvasive and quantitative fluorescence imaging techniques: LAMP and infrared-LAMP

This protocol describes a fluorescence imaging assay, local activation of molecular fluorescent probes (LAMP), for measuring rates of intercellular dye transfer across gap junction channels in intact living cells. The LAMP method consists of four steps: (i) loading cells with a cell-permeable and photo-activatable fluorophore, NPE-HCCC2/AM (acetoxymethyl ester); (ii) locally photolyzing a caged coumarin in one cell of a coupled cell pair to release the parent fluorophore, HCCC2; (iii) imaging cell-cell transfer of HCCC2; and (iv) analyzing rates of dye diffusion. Compared with other methods available for measuring junctional coupling, the LAMP method offers a number of advantages, including noninvasiveness, ease of quantification of coupling strength, good temporal resolution and compatibility with multicolor imaging. Moreover, as the LAMP assay can be carried out multiple times in the same coupled cell pairs, changes in molecular permeability of connexin channels can be tracked by comparing rates of dye transfer. Finally, NPE-HCCC2 and HCCC2 have high two-photon uncaging and excitation efficiency, respectively, enabling two-photon uncaging and imaging to be combined to examine cell coupling in three dimensions (infrared-LAMP assay). It takes roughly 3 h or 4 h to complete a LAMP or an infrared-LAMP assay, respectively.

Cardiovascular Imaging Using Two-Photon Microscopy

Two-photon excitation microscopy has become the standard technique for high resolution deep tissue and intravital imaging. It provides intrinsic three-dimensional resolution in combination with increased penetration depth compared to single-photon confocal microscopy. This article will describe the basic physical principles of two-photon excitation and will review its multiple applications to cardiovascular imaging, including second harmonic generation and fluorescence laser scanning microscopy. In particular, the capability and limitations of multiphoton microscopy to assess functional heterogeneity on a cellular scale deep within intact, Langendorff-perfused hearts are demonstrated. It will also discuss the use of two-photon excitation-induced release of caged compounds for the study of intracellular calcium signaling and intercellular dye transfer.

Imaging dynamic cell-cell junctional coupling in vivo using Trojan-LAMP

To study the physiological regulation and function of cell-cell gap junction communication in vivo, we developed a bioconjugate of caged dye, named dextran-CANPE-HCC, for imaging cell coupling in small model organisms. In vitro, the compound was photolyzed efficiently with robust fluorescence enhancement. Dextran-CANPE-HCC delivered into Caenorhabditis elegans oocytes was retained in cells throughout development. Using local uncaging, we photolyzed dextran-CANPE-HCC to release the small HCC dye and imaged the dynamics of intercellular dye transfer through gap junction channels, a technique we named Trojan-local activation of molecular fluorescent probes (LAMP). Early during embryonic development, the pattern of cell coupling undergoes dramatic remodeling and imaging revealed that the germ cell precursors, P2, P3 and P4, were isolated from the somatic cell communication compartment. As dextran-CANPE-HCC is chemically and metabolically stable, Labeled worms showed very bright signal upon photoactivation after hatching, which allowed us to examine cell coupling in living worms noninvasively.

Src Utilizes Cas to Block Gap Junctional Communication Mediated by Connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.

The antiarrhythmic peptide rotigaptide (ZP123) increases gap junction intercellular communication in cardiac myocytes and HeLa cells expressing connexin 43

We investigated the effects of rotigaptide (ZP123), a stable hexapeptide with antiarrhythmic properties, on gap junction mediated intercellular communication in contracting rat neonatal cardiac myocytes, HL-1 cells derived from cardiac atrium and in HeLa cells transfected with cDNA encoding Cx43-GFP, Cx32-GFP, Cx26-GFP, wild-type Cx43 or wild-type Cx26. Intercellular communication was monitored before and after treatment with rotigaptide following microinjection of small fluorescent dyes (MW<1 kDa). The communication-modifying effect of rotigaptide was confined to cells expressing Cx43 since the peptide had no effect on dye transfer in HeLa cells expressing Cx32-GFP, Cx26-GFP or wild-type Cx26. In contrast, HeLa cells expressing Cx43-GFP exposed to 50 nM rotigaptide for 5 h showed a 40% increase in gap junction mediated communication. Rotigaptide (50 nM) increased intercellular dye transfer in myocytes and atrial HL-1 cells, where Cx43 is the dominant connexin. However, it caused no change in cell beating rates of cardiac myocytes. Western blot analysis showed that rotigaptide did not modify the overall level of Cx43 expression and changes in the phosphorylation status of the protein were not observed.We conclude that the effects of rotigaptide were confined to cells expressing Cx43.

Human Adult Bone Marrow Mesenchymal Stem Cells Repair Experimental Conduction Block in Rat Cardiomyocyte Cultures

We evaluated whether human adult bone marrow-derived mesenchymal stem cells (hMSCs) could repair an experimentally induced conduction block in cardiomyocyte cultures. Autologous stem cell therapy is a novel treatment option for patients with heart disease. However, detailed electrophysiological characterization of hMSCs is still lacking. Neonatal rat cardiomyocytes were seeded on multi-electrode arrays. After 48 h, abrasion of a 200- to 450-microm-wide channel caused conduction block. Next, we applied adult hMSCs (hMSC group, n = 8), human skeletal myoblasts (myoblast group, n = 7), rat cardiac fibroblasts (fibroblast group, n = 7), or no cells (control group, n = 7) in a channel-crossing pattern. Cross-channel electrical conduction was analyzed after 24 and 48 h. Intracellular action potentials of hMSCs and cardiomyocytes were recorded. Immunostaining for connexins and intercellular dye transfer (calcein) assessed the presence of functional gap junctions. After creation of conduction block, two asynchronously beating fields of cardiomyocytes were present. Application of hMSCs restored synchronization between the two fields in five of eight cultures after 24 h. Conduction velocity across hMSCs (0.9 +/- 0.4 cm/s) was approximately 11-fold slower than across cardiomyocytes (10.4 +/- 5.8 cm/s). No resynchronization occurred in the myoblast, fibroblast, or control group. Intracellular action potential recordings indicated that conduction across the channel presumably occurred by electrotonic impulse propagation. Connexin-43 was present along regions of hMSC-to-cardiomyocyte contact, but not along regions of cardiomyocyte-to-myoblast or cardiomyocyte-to-fibroblast contact. Calcein transfer from cardiomyocytes to hMSCs was observed within 24 h after co-culture initiation. Human mesenchymal stem cells are able to repair conduction block in cardiomyocyte cultures, probably through connexin-mediated coupling.