Kofron C M, Kim T Y, King M E, Xie A, Feng F, Park E, Qu Z, Choi B-R, Mende U
Cardiovascular Research Center, Cardiovascular Institute, Rhode Island Hospital and Alpert Medical School of Brown University, Providence, Rhode Island; and.
Department of Medicine, University of California, Los Angeles, California.
Am J Physiol Heart Circ Physiol. 2017 Oct 1;313(4):H810-H827. doi: 10.1152/ajpheart.00181.2017. Epub 2017 Jul 14.
Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, G-mediated signaling was selectively enhanced in CFs by Gα adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMP). Enhanced G signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gα in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive G activation in CFs also depolarized RMP from -33 to -20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMP and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias. In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced G signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.
已知心脏成纤维细胞（CFs）在体内和二维体外培养中可调节心肌细胞（CMs）的功能。本研究检测了在三维（3-D）微组织环境中CF激活对CM电活动调节的影响。使用一个无支架的3-D平台，其中散布着新生大鼠心室CMs和CFs，在与CMs共接种于非粘附水凝胶之前，通过Gα腺病毒感染选择性增强CFs中的G介导信号。3天后，通过信号检测、组织学染色、定量PCR、蛋白质免疫印迹、用电压或钙敏感染料进行光学标测以及微电极记录CF静息膜电位（RMP）对微组织进行分析。CFs中增强的G信号增加了微组织大小以及促纤维化和促肥大标志物。CFs中组成型活性Gα的表达延长了CM动作电位持续时间（延长33%）和上升时间（延长31%），延长了钙瞬变持续时间（延长98%）和上升时间（延长65%），并基于去极化诱导的自律性导致异常电活动。CFs中组成型G激活还使RMP从-33 mV去极化至-20 mV，并增加了连接蛋白43和连接蛋白45的表达。计算模型表明，在3-D环境中升高的RMP以及CMs与CFs之间增加的细胞间耦合可能导致自律性。总之，我们的数据表明，单独的CF激活能够改变CMs的动作电位和钙瞬变特征，导致致心律失常的电活动。我们的结果还强调了细胞间相互作用普遍存在的3-D环境的重要性，强调了3-D组织中CF激活在调节CM电生理和心律失常中起重要作用。在一个降低心脏成纤维细胞基线激活但能实现细胞间、旁分泌和细胞-细胞外基质相互作用的三维微组织模型中，我们证明，通过增强G信号选择性激活心脏成纤维细胞，这是患病心脏中的一种病理生理触发因素，可调节心肌细胞电活动，导致致心律失常的自律性。
