Rationale: 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.Objective: We explored the mechanism by which the GJA1-20k isoform is required for full-length Cx43 forward trafficking to intercalated discs.Methods and Results: 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.Conclusions: 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.

原理：将连接蛋白43（Cx43）传递到嵌入的椎间盘对于细胞间偶联至关重要，是一个连续而快速的过程。通过涉及微管公路的有针对性的传递途径，Cx43半通道的囊泡被有效地贩运到插入盘的粘附连接处。还已经确定肌动蛋白为Cx43的正向贩运提供了休息站，并且Cx43具有20kDa的内部翻译小C末端亚型（GJA1-20k），这是全长Cx43贩运所必需的，但机制未知。目的：我们探讨了全长Cx43正向转运至插层盘所需的GJA1-20k异构体的机制。方法和结果：使用体内AAV9介导的基因转移系统，我们在整只动物中证实了GJA1-20k在插入盘处明显增加了内源性心肌Cx43间隙连接斑的大小。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），而不会影响肌动蛋白的蛋白表达，并且GJA1-20k与肌动蛋白和微管蛋白均复合。我们还发现，F-肌动蛋白调节微管的组织，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，保留微管向细胞-细胞边界的轨迹。对于治疗意义，结论：内部翻译的GJA1-20k亚型可稳定肌动蛋白丝，从而引导Cx43微管运输机械的生长轨迹，增加Cx43半通道向心脏插层盘的递送。在预期的心肌缺血的情况下，外源性的GJA1-20k有助于维持细胞间的偶联。