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Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Published: July 16th, 2013



1Department of Cellular and Molecular Medicine, Laboratory of Molecular and Cellular Signaling, KU Leuven

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular communication and signaling are essential for the coordination of physiological processes in response to extracellular agonists at the tissue and whole-organ level 1,2 . The most direct way of intercellular communication is created by the occurrence of gap junctions. Gap junctions are plaques of gap junction channels, which are proteinaceous channels formed by the head-to-head docking of two connexin (Cx) hemichannels of adjacent cells 3,4 (Figure 1). Gap junctions allow the passage of small signaling molecules with a molecular weight of less than 1.5 kDa, including Ca2+ or IP3 5, ca....

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1. Isolation of Corneal Endothelial Cells

Before getting started: Isolate the cells from the fresh eyes, obtained from a local slaughterhouse, as soon as possible after enucleating the eye. Make sure that the eye was enucleated from a cow of maximal 18 months old, five minutes post mortem and preserved in Earle's Balanced Salt Solution - 1% iodine solution at 4 °C for transportation to the laboratory.

  1. Take the eye out of the Earle's Balanced Salt Solution - 1% iodine solution a.......

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All experiments are executed in compliance with all relevant guidelines, regulations and regulatory agencies and the protocol being demonstrated is performed under the guidance and approval of the animal care and use committee of the KU Leuven.

In bovine corneal endothelial cells (BCEC), functional gap junctions are expressed and both gap junctional intercellular communication and paracrine intercellular communication contribute significantly to intercellular communication in an interactive wa.......

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In this manuscript, we describe a simple method to measure intercellular Ca2+-wave propagation in monolayers of primary bovine corneal endothelial cells by providing a localized and controlled mechanical stimulation using a micropipette. Mechanically stimulated cells respond with a local increase in intracellular IP3 and Ca2+, both of which are essential intracellular signaling molecules that drive intercellular Ca2+-wave propagation 11,67 . IP3 is directl.......

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Research work performed in the laboratory was supported by grants from the Research Foundation - Flanders (FWO; grant numbers G.0545.08 and G.0298.11), the Interuniversity Attraction Poles Program (Belgian Science Policy; grant number P6/28 and P7/13) and is embedded in an FWO-supported research community. CDH is a post-doctoral fellow of the Research Foundation - Flanders (FWO). The authors are very grateful to all current and former members of the Laboratory of Molecular and Cellular Signaling (KU Leuven), Dr. SP Srinivas (Indiana University School of Optometry, USA), the laboratory of Dr. Leybaert (Ghent University) and of Dr. Vinken (VUB) who provided help....

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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalog Number Column1
Earle's Balanced Salt Solution (EBSS) Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany) 14155-048
Iodine Sigma-Aldrich (Deisenhofen, Germany) 38060-1EA
Dulbecco's Modified Eagle's Medium (DMEM) Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany) 11960-044
L-glutamine (Glutamax) Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany) 35050-038
Amphotericin-B Sigma-Aldrich (Deisenhofen, Germany) A2942
Antibiotic-antimycotic mixture Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany) 15240-096
Trypsin Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany) 25300-054
Dulbecco's PBS Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany) 14190-091
Fluo-4 AM Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany) F14217
ARL-67156 (6-N,N-Diethyl-b,g-dibromomethylene-D-ATP) Sigma-Aldrich (Deisenhofen, Germany) A265
Apyrase VI Sigma-Aldrich (Deisenhofen, Germany) A6410
Apyrase VII Sigma-Aldrich (Deisenhofen, Germany) A6535
Gap26 (VCYDKSFPISHVR) Custom peptide synthesis
Gap27 (SRPTEKTIFII) Custom peptide synthesis
Control Peptide (SRGGEKNVFIV) Custom peptide synthesis
siRNA1 Cx43 (sense: 5'GAAGGAGGAGGAACU-CAAAdTdT) Annealed siRNA was purchased at Eurogentec (Luik, Belgium)
siRNA2 Cx43 (sense: 5'CAAUUCUUCCUGCCGCAAUdTdT) Annealed siRNA was purchased at Eurogentec (Luik, Belgium)
siRNA scramble: scrambled sequence of siCx43-1 (sense: 5'GGUAAACG-GAACGAGAAGAdTdT) Annealed siRNA was purchased at Eurogentec (Luik, Belgium)
TAT-L2-H126K/I130N (TAT-DGANVDMKLKQNEIKKFKYGIEEHGK) Thermo Electron (Ulm, Germany)
Two chambered glass slides Laboratory-Tek Nunc (Roskilde, Denmark) 155380
Confocal microscope Carl Zeiss Meditec (Jena, Germany) LSM510
Piezoelectric crystal nanopositioner (Piezo Flexure NanoPositioner) PI Polytech (Karlsruhe, Germany) P-280
HVPZT-amplifier PI Polytech (Karlsruhe, Germany) E463 HVPZT-amplifier
Glass tubes (glass replacement 3.5 nanoliter) World Precision Instruments, Inc. Sarasota, Florida, USA 4878
Microelectrode puller Zeitz Instrumente (Munchen, Germany) WZ DMZ-Universal Puller

  1. Vinken, M., et al. Connexins and their channels in cell growth and cell death. Cell Signal. 18, 592-600 (2006).
  2. Mese, G., Richard, G., White, T. W. Gap junctions: basic structure and function. J. Invest. Dermatol. 127, 2516-2524 (2007).
  3. Bruzzone, R., White, T. W., Paul, D. L. Connections with connexins: the molecular basis of direct intercellular signaling. Eur. J. Biochem. 238, 1-27 (1996).
  4. White, T. W., Bruzzone, R., Paul, D. L. The connexin family of intercellular channel forming proteins. Kidney Int. 48, 1148-1157 (1995).
  5. Decrock, E., et al. Connexin-related signaling in cell death: to live or let die?. Cell Death Differ. 16, 524-536 (2009).
  6. Herve, J. C. Gap junctional complexes: from partners to functions. Prog. Biophys. Mol. Biol. 94, 1-4 (2007).
  7. Herve, J. C., Bourmeyster, N., Sarrouilhe, D., Duffy, H. S. Gap junctional complexes: from partners to functions. Prog. Biophys. Mol. Biol. 94, 29-65 (2007).
  8. Bruzzone, R., Barbe, M. T., Jakob, N. J., Monyer, H. Pharmacological properties of homomeric and heteromeric pannexin hemichannels expressed in Xenopus oocytes. J. Neurochem. 92, 1033-1043 (2005).
  9. Ebihara, L., Steiner, E. Properties of a nonjunctional current expressed from a rat connexin46 cDNA in Xenopus oocytes. J. Gen. Physiol. 102, 59-74 (1993).
  10. Evans, W. H., De Vuyst, E., Leybaert, L. The gap junction cellular internet: connexin hemichannels enter the signalling limelight. Biochem. J. 397, 1-14 (2006).
  11. Leybaert, L., Sanderson, M. J. Intercellular Ca2+ waves: mechanisms and function. Physiol. Rev. 92, 1359-1392 (2012).
  12. Sanderson, M. J., Charles, A. C., Dirksen, E. R. Mechanical stimulation and intercellular communication increases intracellular Ca2+ in epithelial cells. Cell Regul. 1, 585-596 (1990).
  13. Himpens, B., Stalmans, P., Gomez, P., Malfait, M., Vereecke, J. Intra- and intercellular Ca2+ signaling in retinal pigment epithelial cells during mechanical stimulation. Faseb J. 13, 63-68 (1999).
  14. Williams, K. K., Watsky, M. A. Bicarbonate promotes dye coupling in the epithelium and endothelium of the rabbit cornea. Curr. Eye Res. 28, 109-120 (2004).
  15. Hernandez Galindo, E. E., Theiss, C., Steuhl, K. P., Meller, D. Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on intact amniotic membrane. Exp Eye Res. 76, 303-314 (2003).
  16. Williams, K., Watsky, M. Gap junctional communication in the human corneal endothelium and epithelium. Curr. Eye Res. 25, 29-36 (2002).
  17. Anderson, S. C., Stone, C., Tkach, L., SundarRaj, N. Rho and Rho-kinase (ROCK) signaling in adherens and gap junction assembly in corneal epithelium. Invest. Ophthalmol. Vis. Sci. 43, 978-986 (2002).
  18. Joyce, N. C., Harris, D. L., Zieske, J. D. Mitotic inhibition of corneal endothelium in neonatal rats. Invest. Ophthalmol. Vis. Sci. 39, 2572-2583 (1998).
  19. Klepeis, V. E., Weinger, I., Kaczmarek, E., Trinkaus-Randall, V. P2Y receptors play a critical role in epithelial cell communication and migration. J. Cell Biochem. 93, 1115-1133 (2004).
  20. Klepeis, V. E., Cornell-Bell, A., Trinkaus-Randall, V. Growth factors but not gap junctions play a role in injury-induced Ca2+ waves in epithelial cells. J. Cell Sci. 114, 4185-4195 (2001).
  21. Laux-Fenton, W. T., Donaldson, P. J., Kistler, J., Green, C. R. Connexin expression patterns in the rat cornea: molecular evidence for communication compartments. Cornea. 22, 457-464 (2003).
  22. Rae, J. L., Lewno, A. W., Cooper, K., Gates, P. Dye and electrical coupling between cells of the rabbit corneal endothelium. Curr. Eye Res. 8, 859-869 (1989).
  23. Watsky, M. A., Rae, J. L. Dye coupling in the corneal endothelium: effects of ouabain and extracellular calcium removal. Cell Tissue Res. 269, 57-63 (1992).
  24. Williams, K. K., Watsky, M. A. Dye spread through gap junctions in the corneal epithelium of the rabbit. Curr. Eye Res. 16, 445-452 (1997).
  25. D'hondt, C., Ponsaerts, R., Srinivas, S. P., Vereecke, J., Himpens, B. Thrombin inhibits intercellular calcium wave propagation in corneal endothelial cells by modulation of hemichannels and gap junctions. Invest. Ophthalmol. Vis. Sci. 48, 120-133 (2007).
  26. D'hondt, C., Ponsaerts, R., Srinivas, S. P., Vereecke, J., Himpens, B. Reduced intercellular communication and altered morphology of bovine corneal endothelial cells with prolonged time in cell culture. Curr. Eye Res. 34, 454-465 (2009).
  27. D'hondt, C., Srinivas, S. P., Vereecke, J., Himpens, B. Adenosine Opposes Thrombin-Induced Inhibition of Intercellular Calcium Wave in Corneal Endothelial Cells. Invest Ophthalmol. Vis. Sci. 48, 1518-1527 (2007).
  28. Gomes, P., Srinivas, S. P., Van Driessche, W., Vereecke, J., Himpens, B. ATP release through connexin hemichannels in corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 46, 1208-1218 (2005).
  29. Gomes, P., Srinivas, S. P., Vereecke, J., Himpens, B. ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 46, 104-113 (2005).
  30. Gomes, P., Srinivas, S. P., Vereecke, J., Himpens, B. Gap junctional intercellular communication in bovine corneal endothelial cells. Exp Eye Res. , (2006).
  31. Ponsaerts, R., et al. The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 49, 4816-4827 (2008).
  32. Ponsaerts, R., et al. RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System. PLoS ONE. 7, e42074 (2012).
  33. Ponsaerts, R., et al. Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity. Faseb J. 24, 4378-4395 (2010).
  34. Charles, A. Reaching out beyond the synapse: glial intercellular waves coordinate metabolism. Sci STKE. 2005, pe6 (2005).
  35. Laird, D. W. Life cycle of connexins in health and disease. Biochem. J. 394, 527-543 (2006).
  36. Kelsell, D. P., Dunlop, J., Hodgins, M. B. Human diseases: clues to cracking the connexin code. Trends Cell Biol. 11, 2-6 (2001).
  37. Pearson, R. A., Dale, N., Llaudet, E., Mobbs, P. ATP released via gap junction hemichannels from the pigment epithelium regulates neural retinal progenitor proliferation. Neuron. 46, 731-744 (2005).
  38. Klepeis, V. E., Weinger, I., Kaczmarek, E., Randall, V. T. P2Y receptors play a critical role in epithelial cell communication and migration. J. Cell Biochem. 93, 1115-1133 (2004).
  39. Cotrina, M. L., Lin, J. H., Lopez-Garcia, J. C., Naus, C. C., Nedergaard, M. ATP-mediated glia signaling. J. Neurosci. 20, 2835-2844 (2000).
  40. Burnstock, G., Williams, M. P2 purinergic receptors: modulation of cell function and therapeutic potential. J. Pharmacol. Exp. Ther. 295, 862-869 (2000).
  41. Schwiebert, E. M., Zsembery, A. Extracellular ATP as a signaling molecule for epithelial cells. Biochim. Biophys Acta. 1615, 7-32 (2003).
  42. Lazarowski, E. R., Boucher, R. C., Harden, T. K. Mechanisms of release of nucleotides and integration of their action as P2X- and P2Y-receptor activating molecules. Mol. Pharmacol. 64, 785-795 (2003).
  43. Dubyak, G. R., el-Moatassim, C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am. J. Physiol. 265, C577-C606 (1993).
  44. Blair, S. A., Kane, S. V., Clayburgh, D. R., Turner, J. R. Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease. Lab. Invest. 86, 191-201 (2006).
  45. Boudreault, F., Grygorczyk, R. Cell swelling-induced ATP release and gadolinium-sensitive channels. Am. J. Physiol. Cell Physiol. 282, C219-C226 (2002).
  46. Romanov, R. A., Rogachevskaja, O. A., Khokhlov, A. A., Kolesnikov, S. S. Voltage dependence of ATP secretion in mammalian taste cells. J. Gen. Physiol. 132, 731-744 (2008).
  47. Pelegrin, P., Surprenant, A. Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor. Embo J. 25, 5071-5082 (2006).
  48. Surprenant, A., Rassendren, F., Kawashima, E., North, R. A., Buell, G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science. 272, 735-738 (1996).
  49. D'hondt, C., et al. Pannexin channels in ATP release and beyond: an unexpected rendezvous at the endoplasmic reticulum. Cell Signal. 23, 305-316 (2011).
  50. Leybaert, L., et al. Connexin channels, connexin mimetic peptides and ATP release. Cell Commun. Adhes. 10, 251-257 (2003).
  51. Stout, C. E., Costantin, J. L., Naus, C. C., Charles, A. C. Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J. Biol. Chem. 277, 10482-10488 (2002).
  52. Verma, V., Hallett, M. B., Leybaert, L., Martin, P. E., Howard Evans, W. Perturbing plasma membrane hemichannels attenuates calcium signalling in cardiac cells and HeLa cells expressing connexins. Eur. J. Cell Biol. , (2008).
  53. Pharmacol, B. r. J. . 147, S172-S181 (2006).
  54. Slakey, L. L., Gordon, E. L., Pearson, J. D. A comparison of ectonucleotidase activities on vascular endothelial and smooth muscle cells. Ann. N.Y. Acad. Sci. 603, 366-378 (1990).
  55. Gordon, E. L., Pearson, J. D., Slakey, L. L. The hydrolysis of extracellular adenine nucleotides by cultured endothelial cells from pig aorta. Feed-forward inhibition of adenosine production at the cell surface. J. Biol. Chem. 261, 15496-15507 (1986).
  56. Moerenhout, M., Himpens, B., Vereecke, J. Intercellular communication upon mechanical stimulation of CPAE- endothelial cells is mediated by nucleotides. Cell Calcium. 29, 125-136 (2001).
  57. Ralevic, V., Burnstock, G. Receptors for purines and pyrimidines. Pharmacol. Rev. 50, 413-492 (1998).
  58. Edelhauser, H. F. The resiliency of the corneal endothelium to refractive and intraocular surgery. Cornea. 19, 263-273 (2000).
  59. George, A. J., Larkin, D. F. Corneal transplantation: the forgotten graft. Am. J. Transplant. 4, 678-685 (2004).
  60. Hong, S. J., Wu, K. Y., Wang, H. Z., Fong, J. C. Change of cytosolic Ca2+ mobility in cultured bovine corneal endothelial cells by endothelin-1. J. Ocul. Pharmacol. Ther. 19, 1-9 (2003).
  61. Crawford, K. M., MacCallum, D. K., Ernst, S. A. Histamine H1 receptor-mediated Ca2+ signaling in cultured bovine corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 33, 3041-3049 (1992).
  62. Crawford, K. M., MacCallum, D. K., Ernst, S. A. Agonist-induced Ca2+ mobilization in cultured bovine and human corneal endothelial cells. Curr. Eye Res. 12, 303-311 (1993).
  63. Srinivas, S. P., Yeh, J. C., Ong, A., Bonanno, J. A. Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors. Curr. Eye Res. 17, 994-1004 (1998).
  64. Satpathy, M., Gallagher, P., Jin, Y., Srinivas, S. P. Extracellular ATP opposes thrombin-induced myosin light chain phosphorylation and loss of barrier integrity in corneal endothelial cells. Exp Eye Res. 81, 183-192 (2005).
  65. Srinivas, S. P., et al. Cell volume response to hyposmotic shock and elevated cAMP in bovine trabecular meshwork cells. Exp. Eye Res. 78, 15-26 (2004).
  66. D'hondt, C., Ponsaerts, R., De Smedt, H., Bultynck, G., Himpens, B. Pannexins, distant relatives of the connexin family with specific cellular functions. Bioessays. 31, 953-974 (2009).
  67. Boitano, S., Dirksen, E. R., Sanderson, M. J. Intercellular propagation of calcium waves mediated by inositol trisphosphate. Science. 258, 292-295 (1992).
  68. De Vuyst, E., et al. Intracellular calcium changes trigger connexin 32 hemichannel opening. EMBO J. 25, 34-44 (2006).
  69. De Vuyst, E., et al. Ca2+ regulation of connexin 43 hemichannels in C6 glioma and glial cells. Cell Calcium. 46, 176-187 (2009).
  70. Weissman, T. A., Riquelme, P. A., Ivic, L., Flint, A. C., Kriegstein, A. R. Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex. Neuron. 43, 647-661 (2004).
  71. Iyer, S., Deutsch, K., Yan, X., Lin, B. Batch RNAi selector: a standalone program to predict specific siRNA candidates in batches with enhanced sensitivity. Computer Methods and Programs in Biomedicine. 85, 203-209 (2007).
  72. Stehberg, J., et al. Release of gliotransmitters through astroglial connexin 43 hemichannels is necessary for fear memory consolidation in the basolateral amygdala. Faseb J. 26, 3649-3657 (2012).
  73. Evans, W. H., Bultynck, G., Leybaert, L. Erratum to: Manipulating Connexin Communication Channels: Use of Peptidomimetics and the Translational Outputs. J. Membr. Biol. 245, 451 (2012).
  74. Majumder, P., et al. ATP-mediated cell-cell signaling in the organ of Corti: the role of connexin channels. Purinergic Signal. 6, 167-187 (2010).
  75. Carvalho, A. C., et al. affects intracellular Ca2+ stores and induces Ca2+ wave propagation. Cell Death Differ. 11, 1265-1276 (2004).
  76. Torres, A., et al. Extracellular Ca2+ acts as a mediator of communication from neurons to glia. Sci. Signal. 5, ra8 (2012).
  77. Decrock, E., et al. Transfer of IP(3) through gap junctions is critical, but not sufficient, for the spread of apoptosis. Cell Death Differ. 19 (3), 947-957 (2012).
  78. Beltramello, M., Piazza, V., Bukauskas, F. F., Pozzan, T., Mammano, F. Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness. Nat. Cell Biol. 7 (1,4,5), 63-69 (2005).
  79. Bukauskas, F. F., Bukauskiene, A., Verselis, V. K. Conductance and permeability of the residual state of connexin43 gap junction channels. J. Gen. Physiol. 119, 171-186 (2002).
  80. Bukauskas, F. F., Verselis, V. K. Gap junction channel gating. Biochim. Biophys. Acta. 1662, 42-60 (2004).
  81. Dahl, G. Where are the gates in gap junction channels?. Clin. Exp. Pharmacol. Physiol. 23, 1047-1052 (1996).
  82. Retamal, M. A., Schalper, K. A., Shoji, K. F., Bennett, M. V., Saez, J. C. Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential. Proc. Natl. Acad. Sci. U.S.A. 104, 8322-8327 (2007).
  83. Shibayama, J., et al. Effect of charge substitutions at residue his-142 on voltage gating of connexin43 channels. Biophys. J. 91, 4054-4063 (2006).
  84. Desplantez, T., Verma, V., Leybaert, L., Evans, W. H., Weingart, R. Gap26, a connexin mimetic peptide, inhibits currents carried by connexin43 hemichannels and gap junction channels. Pharmacological Research: The Official Journal of the Italian Pharmacological Society. 65, 546-552 (2012).
  85. Delmar, M. Gap junctions as active signaling molecules for synchronous cardiac function. J. Cardiovasc. Electrophysiol. 11, 118-120 (2000).

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