Vinculin directly binds zonula occludens-1 and is essential for stabilizing connexin 43 containing gap junctions in cardiac Myocytes

Abstract

Vinculin (Vcl) links actin filaments to integrin and cadherin-based cellular junctions. Zonula occludens-1 (ZO-1) binds connexin-43 (Cx43), cadherin and actin. Vcl and ZO-1 anchor the actin cytoskeleton to the sarcolemma. Since loss of Vcl from cardiomyocytes caused mal-distribution of Cx43 and predisposed cardiomyocyte-specific Vcl knockout mice with preserved heart function to arrhythmic sudden death, we hypothesized that Vcl and ZO-1 would interact and that loss of this interaction would destabilize gap junctions. Results: Vcl, Cx43 and ZO-1 colocalized at the intercalated disc (ICD). Loss of cardiomyocyte Vcl caused parallel loss of ZO-1 from ICDs. Vcl co-immunoprecipitated Cx43 and ZO-1, and directly bound ZO-1 in yeast two-hybrid studies. Vcl gene excision in neonatal mouse cardiomyocytes caused reduction of Vcl transcript and protein expression, leading to: 1) decreased protein expression of Cx43, ZO-1, talin, and β1D-integrin, 2) reduced PI3K activation, 3) increased activation of Akt, Erk1, and Erk2, and 4) cardiomyocyte necrosis. Summary: This is the first study showing a direct interaction between Vcl and ZO-1 and illustrates how Vcl plays a critical role in stabilizing gap junctions and myocyte integrity.