title: Ischaemia-induced autophagy leads to degradation of gap junction protein connexin43 in cardiomyocytes.
abstract: GJIC (gap junction intercellular communication) between cardiomyocytes is essential for synchronous heart contraction and relies on Cx (connexin)-containing channels. Increased breakdown of Cx43 has been often associated with various cardiac diseases. However, the mechanisms whereby Cx43 is degraded in ischaemic heart remain unknown. The results obtained in the present study, using both HL-1 cells and organotypic heart cultures, show that simulated ischaemia induces degradation of Cx43 that can be prevented by chemical or genetic inhibitors of autophagy. Additionally, ischaemia-induced degradation of Cx43 results in GJIC impairment in HL-1 cells, which can be restored by autophagy inhibition. In cardiomyocytes, ubiquitin signals Cx43 for autophagic degradation, through the recruitment of the ubiquitin-binding proteins Eps15 (epidermal growth factor receptor substrate 15) and p62, that assist in Cx43 internalization and targeting to autophagic vesicles, via LC3 (light chain 3). Moreover, we establish that degradation of Cx43 in ischaemia or I/R (ischaemia/reperfusion) relies upon different molecular players. Indeed, degradation of Cx43 during early periods of ischaemia depends on AMPK (AMP-activated protein kinase), whereas in late periods of ischaemia and I/R Beclin 1 is required. In the Langendorff-perfused heart, Cx43 is dephosphorylated in ischaemia and degraded during I/R, where Cx43 degradation correlates with autophagy activation. In summary, the results of the present study provide new evidence regarding the molecular mechanisms whereby Cx43 is degraded in ischaemia, which may contribute to the development of new strategies that aim to preserve GJIC and cardiac function in ischaemic heart.
authors.affiliation: *Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Azinhaga de Sta Comba, 3000-354 Coimbra, Portugal.
authors.forename: Tania
authors.initials: T
authors.lastname: Martins-Marques
authors.affiliation: *Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Azinhaga de Sta Comba, 3000-354 Coimbra, Portugal.
authors.forename: Steve
authors.initials: S
authors.lastname: Catarino
authors.affiliation: *Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Azinhaga de Sta Comba, 3000-354 Coimbra, Portugal.
authors.forename: Monica
authors.initials: M
authors.lastname: Zuzarte
authors.affiliation: *Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Azinhaga de Sta Comba, 3000-354 Coimbra, Portugal.
authors.forename: Carla
authors.initials: C
authors.lastname: Marques
authors.affiliation: †Laboratory of Physiology, Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Azinhaga de Sta Comba, 3000-354 Coimbra, Portugal.
authors.forename: Paulo
authors.initials: P
authors.lastname: Matafome
authors.affiliation: *Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Azinhaga de Sta Comba, 3000-354 Coimbra, Portugal.
authors.forename: Paulo
authors.initials: P
authors.lastname: Pereira
authors.affiliation: *Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Azinhaga de Sta Comba, 3000-354 Coimbra, Portugal.
authors.forename: Henrique
authors.initials: H
authors.lastname: Girão
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D001343:Autophagy; D000071186:Beclin-1; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation / Q000235:genetics; D059748:Proteolysis; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
chemical_list: D055372:AMP-Activated Protein Kinases; D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; D002135:Calcium-Binding Proteins; D018031:Connexin 43; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D025801:Ubiquitin; C115010:Becn1 protein, mouse; C512829:Becn1 protein, rat; C082729:Eps15 protein, mouse; C526966:Eps15 protein, rat; C543076:GJA1 protein, mouse; C580592:Gja1 protein, rat; C526109:Anxa3 protein, rat; C480860:Map1lc3b protein, mouse