Fast V G, Darrow B J, Saffitz J E, Kléber A G
Department of Physiology, University of Berne, Switzerland.
Circ Res. 1996 Jul;79(1):115-27. doi: 10.1161/01.res.79.1.115.
The role of tissue discontinuities in anisotropic impulse propagation was assessed in two-dimensional anisotropic monolayers of neonatal rat myocytes cultured on a growth-directing substrate of collagen. Activation spread and distribution of maximal upstroke rate of rise (Vmax) of the action potential were measured with an optical system using a voltage-sensitive fluorescent dye (RH-327) and a 10x10 photodiode array with a spatial resolution ranging from 7 to 15 microns. Activation maps were compared with the cellular architecture and the distribution of gap junctions obtained from immunostaining the gap junction protein connexin43 (Cx43). Four types of structures were studied: (1) dense cell cultures, (2) cultures with anisotropic intercellular clefts of variable size, (3) discontinuities created by inclusion of nonmyocyte cells, and (4) discontinuities resulting from nonuniform expression of gap junctions. In dense monolayers, activation spread was continuous with microinhomogeneities in both longitudinal and transverse directions. The average cell dimensions in such monolayers were smaller than in adult canine myocardium. However, the degree of cellular anisotropy (length-to-width ratio of 5.3 +/- 1.4) and connectivity were similar. The presence of small intercellular clefts (less than one cell in length) did not disturb the general pattern of transverse or longitudinal activation spread, but it was associated with wave front microcollisions during transverse propagation and a concomitant increase of Vmax beyond the cleft. Long intercellular clefts caused discontinuous transverse propagation. Conduction velocity and Vmax decreased significantly at narrow isthmuses formed by closely apposed clefts, rendering such sites susceptible for conduction block. In contrast Vmax increased when the wave front faced the borders of the clefts. Nonmyocyte cells were electrically connected to myocytes and served as sinks for electrotonic currents, thereby producing localized conduction slowing and a decrease in Vmax. Localized inhomogeneity in Cx43 distribution correlated accurately with circumscribed conduction block and changes in Vmax. Our results provide direct experimental evidence that the cellular structure and gap junction distribution correlate with action potential propagation and distribution of Vmax. We suggest that in tissue with a nonuniform anisotropy, connective tissue separating fiber bundles or sites of inhomogeneous connexin distribution may represent predilective sites for block in transverse direction.
在以胶原蛋白为生长导向基质培养的新生大鼠心肌细胞二维各向异性单层中，评估了组织不连续性在各向异性冲动传播中的作用。使用电压敏感染料（RH - 327）和空间分辨率为7至15微米的10×10光电二极管阵列的光学系统，测量动作电位的激活传播和最大上升速率（Vmax）的分布。将激活图与通过对缝隙连接蛋白连接蛋白43（Cx43）进行免疫染色获得的细胞结构和缝隙连接分布进行比较。研究了四种类型的结构：（1）密集细胞培养物，（2）具有大小可变的各向异性细胞间裂隙的培养物，（3）通过包含非心肌细胞产生的不连续性，以及（4）由于缝隙连接不均匀表达导致的不连续性。在密集单层中，激活传播是连续的，在纵向和横向都存在微观不均匀性。此类单层中的平均细胞尺寸小于成年犬心肌中的细胞尺寸。然而，细胞各向异性程度（长宽比为5.3±1.4）和连通性相似。存在小的细胞间裂隙（长度小于一个细胞）不会干扰横向或纵向激活传播的总体模式，但它与横向传播期间的波前微碰撞以及裂隙处Vmax的伴随增加有关。长的细胞间裂隙导致横向传播不连续。在由紧密相邻的裂隙形成的狭窄峡部处，传导速度和Vmax显著降低，使这些部位易发生传导阻滞。相反，当波前面对裂隙边界时，Vmax增加。非心肌细胞与心肌细胞电连接，并作为电紧张电流的汇，从而导致局部传导减慢和Vmax降低。Cx43分布的局部不均匀性与局限性传导阻滞和Vmax变化准确相关。我们的结果提供了直接的实验证据，表明细胞结构和缝隙连接分布与动作电位传播和Vmax分布相关。我们认为，在具有不均匀各向异性的组织中，分隔纤维束的结缔组织或连接蛋白分布不均匀的部位可能代表横向阻滞的偏好部位。
