GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated Discs.
GJA1-20k Arranges Actin to Guide Cx43 Delivery to Cardiac Intercalated Discs.
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DOI：
10.1161/circresaha.117.311955
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发表时间：
2017-10-13
影响因子：
20.1
通讯作者：
Shaw RM
中科院分区：
文献类型：
--
作者：
Basheer WA;Xiao S;Epifantseva I;Fu Y;Kleber AG;Hong T;Shaw RM
连接蛋白43 （Cx43）传递到插入椎间盘是一个连续和快速的过程，对细胞间偶联至关重要。通过涉及微管高速公路的靶向递送途径，Cx43半通道的囊泡被有效地运输到嵌入盘的粘附连接。还发现肌动蛋白为Cx43向前转运提供休息站，并且Cx43具有20kDa的内部翻译小c端异构体（GJA1-20k），这是全长Cx43转运所必需的，但机制未知。我们探索了GJA1-20k异构体在全长Cx43向前运输到插入椎间盘中所必需的机制。通过体内aav9介导的基因转移系统，我们在全动物中证实GJA1-20k显著增加内源性心肌间隔椎间盘Cx43间隙连接斑块的大小。在微模式细胞配对系统中，我们发现外源性GJA1-20k表达稳定了丝状肌动蛋白（F-actin）而不影响肌动蛋白的表达，并且GJA1-20k与肌动蛋白和微管蛋白结合。我们还发现f -肌动蛋白调节微管组织，因为用低剂量的latrunculin a （LatA）抑制肌动蛋白聚合会破坏微管对细胞-细胞连接的靶向。GJA1-20k保护肌动蛋白丝免受LatA破坏，保持微管轨迹到细胞-细胞边界。在治疗意义上，我们发现事先在体内将aav9介导的GJA1-20k基因递送到心脏可以保护Cx43定位到间插椎间盘免受急性缺血性损伤。内部翻译的GJA1-20k异构体稳定肌动蛋白丝，引导Cx43微管运输机制的生长轨迹，增加Cx43半通道到心脏间插盘的输送。外源性GJA1-20k有助于在预期的心肌缺血情况下维持细胞-细胞偶联。
Delivery of connexin 43 (Cx43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubule highways, vesicles of Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has also been identified that actin provides rest stops for Cx43 forward trafficking, and that Cx43 has a 20kDa internally translated small C-terminus isoform (GJA1-20k) which is required for full-length Cx43 trafficking, but by an unknown mechanism. We explored the mechanism by which the GJA1-20k isoform is required for full-length Cx43 forward trafficking to intercalated discs. Using an in-vivo AAV9-mediated gene transfer system, we confirmed in whole animal that GJA1-20k markedly increases endogenous myocardial Cx43 gap junction plaque size at the intercalated discs. In micropatterned cell pairing systems, we found that exogenous GJA1-20k expression stabilizes filamentous actin (F-actin) without affecting actin protein expression, and that GJA1-20k complexes with both actin and tubulin. We also found that F-actin regulates microtubule organization as inhibition of actin polymerization with a low dose of latrunculin A (LatA) disrupts the targeting of microtubules to cell-cell junctions. GJA1-20k protects actin filament from LatA disruption, preserving microtubule trajectory to the cell-cell border. For therapeutic implications, we found that prior in vivo AAV9-mediated gene delivery of GJA1-20k to the heart protects Cx43 localization to the intercalated discs against acute ischemic injury. The internally translated GJA1-20k isoform stabilizes actin filaments which guides growth trajectories of the Cx43 microtubule trafficking machinery, increasing delivery of Cx43 hemichannels to cardiac intercalated discs. Exogenous GJA1-20k helps to maintain cell-cell coupling in instances of anticipated myocardial ischemia.