| In the heart, gap junctions (GJs) form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all cells of the myocardium. In recently years, many experiments found that there was a uncoupling between cardiomyocytes (decreasing of gap junctional conductance) in ischemic heart. This uncoupling can isolate the cardiomyocytes of ischemic area from non-ischemic area, thus turning these ischemic cardiomyocytes into resting condition. However, uncoupling can also be harmful to the heart while it protect heart from further hurt. In the heart, GJ intercellular communication is the dominant factor in arrhythmogenesis. Decreased GJ coupling between cardiac myocytes may reduce the propagation velocity, and severe uncoupling can block the propagation of the impulse, which facilitates the genesis of reentry arrhythmias. On the other hand, the findings in ischemic myocardium also suggested there is a possibility for the reduction of the uncoupling, i.e., it is possible that the gap junction protein reduced in ischemic heart.In the present study, we used a immunofluorescence method to determine the connexin43 (Cx43) level hi cultured cardiomyocytes under normoxia and hypoxia, and a mathematical model to investigate the effects of altered coupling on the interaction of autorhythmic cells and neighboring myocytes. METHODSMethods for immunofluorescence.: The myocytes were fixed with 4% paraformaldehyde after treated with normoxia or hypoxia. Cx43 was labeled with a rabbit-anti-connexin43antibody followed by secondary antibody (FITC-conjugated goat anti-rabbit IgG). The cells were observed with Confocal microscopy and the fluorescence density of Cx43 was measured with Sigmascanpro.The model contained a variable number (1, 2, 4, 8 or 16) of autorhythmic cells (ARC) as the pacemaker. Forty quiescent but excitable cells (QC) were connected to the ARC. QCs were represented by the Luo-Rudy dynamic (LRd) model of the mammalian ventricular myocyte. In this model, the action potential was numerically constructed from ionic processes formulated on the basis of experimental data obtained mainly from guinea pig heart. ARCs were constructed according to a slightly modified LRd model, in which an inward current was activated during the phase 4 spontaneous stage to mimic ischemia/reperfusion injury. The basic cycle length of ARC in complete isolation from neighboring cells was 160 ms. GJ conductance between two adjacent ARCs was Gl, and between QCs, G2. Boundary conductance between ARC and QC was set at Gl. Gl could be varied from 1.2 uS to 0.009375 uS in different experiments, to mimic the reduced coupling under ischemia/reperfusion conditions. The simulation process was programmed with Visual C++ 6.0, and codes from Dr. Rudy's website were used to modify the single LRd model cell. Computations were run on 7 Pentium-Ill computers. Action potentials of cells at both ends of the chain were recorded and analyzed with Matlab (Ver. 5.1.0.421, The Math Works, Natick, MA). RESULTSIn immunofluorescence analysis, it was found that the Cx43 content in cultured neonatal rat ventriculat myocytes showed no difference between the control myocytes and myocytes treated with hypoxia for 3 hours. Density analysis also showed that there were no difference of fluorescence areas between control, cardiomyocytes treated with hypoxia for 3 hours andcardiomyocytes pretreated with 30 min hypoxia followed by 3 hours hypoxia.In model study, when Gjm was low, the action potential in ARC (cell-0) could not propagate. When Gjm was high, the excitation spread from cell-0 and propagated through all cells in the chain. The frequency of action potentials initiated in ARC increased with decreasing Gjm. Furthermore, the autorhythmicity of the ARC was suppressed with increasing Gjm. At the same Gjm, increasing the number of coupled ARCs elevated the frequency, while at higher Gjm the effect of coupled cell number on the frequency was more significant than that at lower Gjm. The larger the number of ARCs, the...
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