Evidence of intercellular coupling between co‐cultured adult rabbit ventricular myocytes and myofibroblasts

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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.

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