Norepinephrine inhibits intercellular coupling in rat cardiomyocytes by ubiquitination of connexin43 gap junctions

Abstract

G α q -stimulation reduces intercellular coupling within 10 min via a decrease in the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), but the mechanism is unknown. Here we show that uncoupling in rat cardiomyocytes after stimulation of α -adrenergic G α q -coupled receptors with norepinephrine is prevented by proteasomal and lysosomal inhibitors, suggesting that internalization and possibly degradation of connexin43 (Cx43) is involved. Uncoupling was accompanied by increased Triton X-100 solubility of Cx43, which is considered a measure of the non-junctional pool of Cx43. However, inhibition of the proteasome and lysosome further increased solubility while preserving coupling, suggesting that communicating gap junctions can be part of the soluble fraction. Ubiquitination of Cx43 was also increased, and Cx43 co-immunoprecipitated with the ubiquitin ligase Nedd4. Conclusions : Norepinephrine increases ubiquitination of Cx43 in cardiomyocytes, possibly via Nedd4. We suggest that Cx43 is subsequently internalized, which is preceded by acquired solubility in Triton X-100, which does not lead to uncoupling per se .

Managing the complexity of communication: regulation of gap junctions by post-translational modification

“…Reduced PIP2 levels inhibit gap junctional coupling in both fibroblasts (van Zeijl et al, 2007) and cardiomyocytes (Hofgaard et al, 2008). Stimulation of cultured cardiomyocytes from neonatal rats with noradrenaline or angiotensin II decrease cell coupling and action potential conduction velocity mediated via a decrease in PIP2 levels (Hofgaard et al, 2008) and in a later study it was demonstrated that noradrenaline induced uncoupling was associated with ubiquitination of Cx43, possibly via Nedd4, and subsequent internalization of Cx43 (Mollerup et al, 2011). …”

“…The majority of the SUMOylated Cx43 was found in the Triton X-100 soluble fraction (Kjenseth et al, 2012), suggesting that Cx43 were not organized in functional gap junctions as gap junction plaques are Triton X-100 insoluble (Musil and Goodenough, 1991; VanSlyke and Musil, 2000). However, gap junctions seem to aquire Triton solubility before internalization (Sirnes et al, 2008) and work in cultured cardiomyocytes show that inhibition of the proteasome and lysosome increase both Triton solubility of Cx43 and intercellular coupling simultaneously, indicating that communicating gap junctions can be part of the soluble fraction (Mollerup et al, 2011). …”

Heart ischemia results in connexin43 ubiquitination localized at the intercalated discs

“…For example, it was previously shown that degradation of cardiac beta/slow myosin heavy chain ( /slow MHC) and MHCIIa occurs by the proteasome, with the involvement of the E3 ligases MuRF1 and MuRF3 [10]. Although Nedd4 was reported to interact with Cx43 in neonatal rat cardiomyocytes [12], its involvement in Cx43 remodeling following heart ischemia has never been assessed. Results presented in Fig.1B show that, both in control and ischemic conditions, Nedd4 is detected at IDs, where it co-localizes with Cx43.…”

Connexin 43 and CaV1.2 Ion Channel Trafficking in Healthy and Diseased Myocardium

“…Nedd4 was the first described E3 ubiquitin ligase to be implicated in regulating Cx43 ubiquitination, internalization and autophagic degradation through a mechanism involving recruitment of the endocytotic adaptor Eps15 (epidermal growth factor receptor substrate 15) and the autophagic receptor p62 in cell lines 66, 71 . In neonatal rat cardiomyocytes, Nedd4 has also been reported to interact with Cx43 possibly regulating its ubiquitination and internalization in response to norepinephrine 72 . Further evidence implicating the role of Nedd4 in regulating Cx43 in myocytes was recently reported showing that only under basal conditions, silencing of Nedd4 in the HL-1 mouse atrial cell line led to increased Cx43 protein with a decrease in its ubiquitination levels 64 .…”