Abstract 1092: Membrane Localization and Novel Phosphorylation of Connexin43 in Heart Failure
Heart failure (HF) is associated with a decrease in conduction velocity (CV) and an increase in fatal arrhythmias. We have previously reported a decrease in expression and lateralization of connexin 43 (Cx43) in HF. The mechanism by which Cx43 redistribution occurs in HF is unknown. Lipid rafts (LR) are triton-insoluble, buoyant cholesterol-enriched membrane sub-domains, involved in protein trafficking. We hypothesize that LR targeting of Cx43 in HF underlies its subcellular redistribution. In ventricular lysates from canine HF induced by tachypacing there is a decrease in triton-insoluble Cx43 compared with controls (N=5 per group, p=0.01). In sucrose gradient density centrifugation, hyperphosphorylated (P2) Cx43 is enriched in the buoyant caveolin-3 containing LR fraction (Fig⇓). Hypophosphorylated Cx43 (P0) identified by a S368 dephosphorylated Cx43 antibody (dCx43) is present exclusively in the non-LR (NLR) fraction. In HF dCx43 increases in abundance and is located at the intercalated disk region in immunofluorescence studies. Using immunoprecipitation followed by phosphopeptide enrichment and liquid chromatography coupled tandem mass spectrometry we have identified 6 phosphorylated residues in P2 (T326, S330, 365, 373, 306, 297) in both control and HF, of which T326, S297 and S306 are novel. In P0, S373 was phosphorylated in control lysates whereas S365 and T326 were phosphorylated in HF.
Hyperphosphorylated Cx43 preferentially localizes to LR.
HF is associated with a decrease in triton-insoluble Cx43 consistent with modified phosphorylation-dependent LR targeting and is a possible mechanism of altered Cx43 membrane localization in HF.