Abstract 952: Upregulated P21-activated Kinases Contribute To Dephosphorylation Of Connexin 43 Through Enhanced PP2A Activity In The Failing Rabbit And Human Heart
Cardiac P21-activated kinases (PAK1, 2, & 3) are a family of serine/threonine protein kinases expressed in heart that can enhance activity of protein phosphatase 2A (PP2A). In an arrhythmogenic rabbit model of nonischemic heart failure (HF), we demonstrated a 2.5-fold increase in the amount of PP2A co-localized with connexin 43 (Cx43) contributing to enhanced Cx43 dephosphorylation and reduced cell coupling in left ventricular (LV) myocytes. Moreover, we recently discovered in rabbit LV that both PAK1 and PAK2 were increased in HF, that PAKs co-localized with Cx43 protein, and that with HF, co-localized PAK2 was increased. In the present studies, we further explore the role of PAKs on Cx43 dephosphorylation in the failing rabbit and human heart. PAK1 overexpression (by adenovirus encoding constitutively-activated PAK1, AdPAK1) enhanced total PP2A activity by 115% and resulted in a 138% increase in the nonphosphorylated Cx43 in control rabbit myocytes (n=3,3, p<0.05, vs. AdLacZ-infected cells). We confirmed this in a HEK293 cell line with wild-type Cx43 stably expressed (HEK293-Cx43). Our data demonstrated a 49% increase in dephosphorylated Cx43 associated with an elevated PP2A activity (34%) in AdPAK1-infected HEK293-Cx43 cells (n=4,4, p<0.05, vs. AdLacZ controls). These results support a role for PAK modulation of Cx43 phosphorylation through PP2A. Finally, to validate our findings of PAK upregulation in HF rabbits, we determined PAK1, 2, &3 protein expression and their interactions with Cx43 (by immunoblotting and co-immunoprecipitation) in LV from patients with idiopathic dilated cardomyopathy (IDCM) and from nonfailing (NF) human hearts. We found PAK1, 2, & 3 proteins were increased 65%, 111%, and 86% in IDCM LV (n=7,7, p<0.0001 vs. NF), respectively. Moreover, PAK1, 2, & 3 co-immunoprecipitated with Cx43 antibody in nonfailing human LV and, with IDCM, co-localized PAK1 & 2 were increased by 53% and 33% (n=3,3, p<0.05), respectively, while PAK3 was unchanged. Thus, PAKs and PP2A are integral components of a macromolecular complex with cardiac Cx43, and upregulated PAK proteins regulate the level of dephosphorylated Cx43 by increasing PP2A activity in HF. PAKs may represent a novel therapeutic target to modulate altered Cx43 phosphorylation in HF.