title: Evidence of intercellular coupling between co-cultured adult rabbit ventricular myocytes and myofibroblasts.
abstract: Intercellular coupling between ventricular myocytes and myofibroblasts was studied by co-culturing adult rabbit ventricular myocytes with previously prepared layers of cardiac myofibroblasts. Intercellular coupling was examined by: (i) tracking the movement of the fluorescent dye calcein; (ii) immunostaining for connexin 43 (Cx43); and (iii) measurement of intracellular [Ca2+] ([Ca2+]i). The effects of stimulating ventricular myocytes on the underlying myofibroblasts was examined by confocal measurements of [Ca2+]i using fluo-3. When ventricular myocytes were preloaded with calcein and co-cultured with myofibroblasts for 24 h, calcein fluorescence was detected in 52+/-4% (n=8 co-cultures) of surrounding myofibroblasts. Treatment with the gap junction uncoupler heptanol significantly reduced the movement of calcein (12+/-3%, n=6 co-cultures). Immunostaining showed expression of Cx43 in co-cultured myofibroblasts and myocytes. Field stimulation of ventricular myocytes co-cultured with myofibroblasts increased myofibroblast [Ca2+]i, no response was observed after treatment with heptanol or stimulation of fibroblasts in the absence of ventricular myocytes. Action potential parameters of ventricular myocytes in co-culture were similar to control values. However, application of the hormone sphingosine-1-phosphate (S-1-P) to the co-culture caused a depolarization of ventricular myocytes to approximately -20 mV. Sphingosine-1-phosphate had no effect on ventricular myocytes alone. Voltage-clamp measurements of isolated myofibroblasts indicated that S-1-P activated a significant quasi-linear current with a reversal potential of approximately -40 mV. In conclusion, this study shows that stimulation of the ventricular myocyte influences the intracellular Ca2+ of the linked myofibroblast via connexons. These intercellular links also allow the myofibroblasts to influence the electrical activity of the myocyte. This work indicates the nature of the gap junction-mediated bi-directional interactions that occur between ventricular myocyte and myofibroblast.
authors.affiliation: Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Chilton
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Giles
authors.forename: Godfrey L
authors.initials: GL
authors.lastname: Smith
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D004396:Coloring Agents; D018031:Connexin 43 / Q000378:metabolism; D004594:Electrophysiology; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019850:Heptanol / Q000494:pharmacology; D007365:Intercellular Junctions / Q000187:drug effects / Q000502:physiology*; D008246:Lysophospholipids / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011817:Rabbits; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D004396:Coloring Agents; D018031:Connexin 43; D008246:Lysophospholipids; C060506:sphingosine 1-phosphate; D019850:Heptanol; D013110:Sphingosine; D002118:Calcium