Takanari Hiroki, Bourgonje Vincent J A, Fontes Magda S C, Raaijmakers Antonia J A, Driessen Helen, Jansen John A, van der Nagel Roel, Kok Bart, van Stuijvenberg Leonie, Boulaksil Mohamed, Takemoto Yoshio, Yamazaki Masatoshi, Tsuji Yukiomi, Honjo Haruo, Kamiya Kaichiro, Kodama Itsuo, Anderson Mark E, van der Heyden Marcel A G, van Rijen Harold V M, van Veen Toon A B, Vos Marc A
Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Utrecht, The Netherlands Department of Cardiovascular Research, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan Department of Pathophysiology, Oita University School of Medicine, Yufu, Japan.
Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Utrecht, The Netherlands.
Cardiovasc Res. 2016 Sep;111(4):410-21. doi: 10.1093/cvr/cvw173. Epub 2016 Jun 29.
In healthy hearts, ventricular gap junctions are mainly composed by connexin43 (Cx43) and localize in the intercalated disc, enabling appropriate electrical coupling. In diseased hearts, Cx43 is heterogeneously down-regulated, whereas activity of calmodulin/calcium-calmodulin protein kinase II (CaM/CaMKII) signalling increases. It is unclear if CaM/CaMKII affects Cx43 expression/localization or impulse propagation. We analysed different models to assess this.
AC3-I mice with CaMKII genetically inhibited were subjected to pressure overload (16 weeks, TAC vs. sham). Optical and epicardial mapping was performed on Langendorff-perfused rabbit and AC3-I hearts, respectively. Cx43 subcellular distribution from rabbit/mouse ventricles was evaluated by immunoblot after Triton X-100-based fractionation. In mice with constitutively reduced CaMKII activity (AC3-I), conduction velocity (CV) was augmented (n = 11, P < 0.01 vs. WT); in AC3-I, CV was preserved after TAC, in contrast to a reduction seen in TAC-WT mice (-20%). Cx43 expression was preserved after TAC in AC3-I mice, though arrhythmias and fibrosis were still present. In rabbits, W7 (CaM inhibitor, 10 µM) increased CV (6-13%, n= 6, P< 0.05), while susceptibility to arrhythmias decreased. Immunoconfocal microscopy revealed enlarged Cx43 cluster sizes at intercalated discs of those hearts. Total Cx43 did not change by W7 (n= 4), whereas Triton X-100 insoluble Cx43 increased (+21%, n= 4, P< 0.01). Similar findings were obtained in AC3-I mouse hearts when compared with control, and in cultured dog cardiomyocytes. Functional implication was shown through increased intercellular coupling in cultured neonatal rat cardiomyocytes.
Both acute and chronic CaM/CaMKII inhibition improves conduction characteristics and enhances localization of Cx43 in the intercalated disc. In the absence of fibrosis, this reduced the susceptibility for arrhythmias.
在健康心脏中，心室间隙连接主要由连接蛋白43（Cx43）组成，并定位于闰盘，实现适当的电偶联。在患病心脏中，Cx43呈异质性下调，而钙调蛋白/钙 - 钙调蛋白蛋白激酶II（CaM/CaMKII）信号通路的活性增加。尚不清楚CaM/CaMKII是否影响Cx43的表达/定位或冲动传导。我们分析了不同模型以评估这一点。
对CaMKII基因被抑制的AC3 - I小鼠进行压力超负荷处理（16周，主动脉缩窄术与假手术对照）。分别在Langendorff灌注的兔心脏和AC3 - I心脏上进行光学和心外膜标测。通过基于Triton X - 100分级分离后的免疫印迹法评估兔/小鼠心室中Cx43的亚细胞分布。在CaMKII活性持续降低的小鼠（AC3 - I）中，传导速度（CV）增加（n = 11，与野生型相比P < 0.01）；在AC3 - I小鼠中，主动脉缩窄术后CV得以保留，而主动脉缩窄术处理的野生型小鼠CV降低（-20%）。在AC3 - I小鼠中，主动脉缩窄术后Cx43表达得以保留，尽管仍存在心律失常和纤维化。在兔心脏中，W7（钙调蛋白抑制剂，10 μM）使CV增加（6 - 13%，n = 6，P < 0.05），同时心律失常易感性降低。免疫共聚焦显微镜显示这些心脏闰盘处Cx43簇大小增大。W7处理后总Cx43未改变（n = 4），而Triton X - 100不溶性Cx43增加（+21%，n = 4，P < 0.01）。与对照相比，在AC3 - I小鼠心脏以及培养的犬心肌细胞中也获得了类似结果。通过培养的新生大鼠心肌细胞中细胞间偶联增加显示了其功能意义。
急性和慢性抑制CaM/CaMKII均可改善传导特性并增强Cx43在闰盘中的定位。在无纤维化的情况下，这降低了心律失常的易感性。
