** P<0.01 compared with ctrl by two-tailed unpaired Student's t-test. The presented images are representative of three separate experiments. Right, analysis of overlap coefficient R. Left, representative images Scale bar, 10 μm. PDI, an ER marker protein GM130, a Golgi marker protein. ( a, b) Immunofluorescence imaging of the co-localization of Cx43 with the ER and Golgi apparatus. ( e) Effects of Cx43 knockdown on LRP6 protein expression in NRVMs. ** P<0.01 compared with ctrl by one-way ANOVA with Bonferroni's post hoc test. Note that Lrp6 or Cx43 knockdown significantly reduces dye coupling among cardiomyocytes. Photographs are taken at 1 min after Lucifer yellow dye injection and injected cardiomyocytes are indicated by asterisks. ( d) Dye coupling in Lrp6 knockdown, Cx43 knockdown and control NRVMs. ( c) Immunofluorescence imaging of endogenous Cx43 gap junction in LRP6-deficient myocytes. *** P<0.001 compared with ctrl by one-way analysis of variance (ANOVA) with Bonferroni's post hoc test. Three independent Lrp6 siRNA sequences were employed. Top, typical western blots bottom, pooled data. ( a, b) Effects of Lrp6 knockdown on Cx43 protein levels in cultured NRVMs. These findings uncover a distinct role of LRP6 as a platform for intracellular protein trafficking. Accordingly, the hearts of conditional cardiac-specific Lrp6-knockout mice consistently exhibit overt reduction of Cx43 gap junction plaques without any abnormality in Wnt signalling and are predisposed to lethal arrhythmias. The defect in Cx43 gap junction resulting from LRP6 reduction is attributable to the defective traffic of de novo Cx43 proteins from the endoplasmic reticulum to the Golgi apparatus, leading to the lysosomal degradation of Cx43 proteins.
A deficiency in LRP6 disrupts Cx43 gap junction formation and thereby impairs the cell-to-cell coupling, which is independent of Wnt/β-catenin signalling. Cardiac LRP6 is spatially restricted to intercalated discs and binds to gap junction protein connexin 43 (Cx43). Here, we report the scaffold function of LRP6 in gap junction formation of cardiomyocytes. Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor in the canonical Wnt/β-catenin signalling.
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