Change in ß-catenin localization suggests involvement of the canonical Wnt pathway in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease with high prevalence in the Boxer dog population. It is characterized by replacement of the myocardium with fatty or fibro-fatty tissue. Several mechanisms for the development of ARVC have been suggested, including dysfunction of the canonical Wnt pathway, which is linked to many cellular functions, including growth and differentiation of adipocytes. HYPOTHESIS: Wnt pathway dysfunction is involved in the development of ARVC in the Boxer as evidenced by mislocalization of ß-catenin, an integral Wnt pathway modulator, and striatin, a known Wnt pathway component. ANIMALS: Five dogs without ARVC and 15 Boxers with ARVC were identified by 24-hour Holter monitoring and histopathologic examination of the heart. METHODS: Right ventricular samples were collected and examined using confocal microscopy, Western blots, and quantitative (q) PCR. RESULTS: Confocal microscopy indicated that ß-catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern in hearts without ARVC. In hearts affected with ARVC, both ß-catenin and striatin were colocalized with the endoplasmic reticulum (ER) marker calreticulin. Western blots indentified a 50% increase in the amount of ß-catenin in ARVC samples. No change in ß catenin mRNA was detected using qPCR. CONCLUSIONS: Our data suggest that trafficking of Wnt pathway proteins from the ER to their proper location within the cell is inhibited in Boxers with ARVC. These results suggest that disturbances in the Wnt pathway may play a role in the development of ARVC in the Boxer.

Identification of C-terminal domain residues involved in protein kinase A-mediated potentiation of kainate receptor subtype 6.

Glutamate receptors are the major excitatory receptors in the vertebrate CNS and have been implicated in a number of physiological and pathological processes. Previous work has shown that glutamate receptor function may be modulated by protein kinase A (PKA)-mediated phosphorylation, although the molecular mechanism of this potentiation has remained unclear. We have investigated the phosphorylation of specific amino acid residues in the C-terminal cytoplasmic domain of the rat kainate receptor subtype 6 (GluR6) as a possible mechanism for regulation of receptor function. The C-terminal tail of rat GluR6 can be phosphorylated by PKA on serine residues as demonstrated using [gamma-32P]ATP kinase assays. Whole cell recordings of transiently transfected human embryonic kidney (HEK) 293 cells showed that phosphorylation by PKA potentiates whole cell currents in wildtype GluR6 and that removal of the cytoplasmic C-terminal domain abolishes this potentiation. This suggested that the C-terminal domain may contain residue(s) involved in the PKA-mediated potentiation. Single mutations of each serine residue in the C-terminal domain (S815A, S825A, S828A, and S837A) and a truncation after position 855, which removes all threonines (T856, T864, and T875) from the domain, do not abolish PKA potentiation. However, the S825A/S837A mutation, but no other double mutation, abolishes potentiation. These results demonstrate that phosphorylation of the C-terminal tail of GluR6 by PKA leads to potentiation of whole cell response, and the combination of S825 and S837 in the C-terminal domain is a vital component of the mechanism of GluR6 potentiation by PKA.