title: Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription.
abstract: Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1-3week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2'-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell cycle after birth.
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Thomas V A
authors.initials: TV
authors.lastname: Murray
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Smyrnias
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Moritz
authors.initials: M
authors.lastname: Schnelle
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Rajesh K
authors.initials: RK
authors.lastname: Mistry
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Matteo
authors.initials: M
authors.lastname: Beretta
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Martin
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Narayana
authors.initials: N
authors.lastname: Anilkumar
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Shana M
authors.initials: SM
authors.lastname: de Silva
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK.
authors.forename: Ajay M
authors.initials: AM
authors.lastname: Shah
authors.affiliation: King's College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK. Electronic address: alison.brewer@kcl.ac.uk.
authors.forename: Alison C
authors.initials: AC
authors.lastname: Brewer
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D006332:Cardiomegaly / Q000235:genetics / Q000473:pathology; D002453:Cell Cycle / Q000235:genetics; D049109:Cell Proliferation; D017124:Conserved Sequence / Q000235:genetics; D056742:Cyclin D2 / Q000235:genetics* / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000201:enzymology*; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014158:Transcription, Genetic; D015854:Up-Regulation / Q000235:genetics
chemical_list: D056742:Cyclin D2; D016271:Proto-Oncogene Proteins c-myc; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Cardiomyocyte proliferation; Cyclin D2; ERK1/2; Nox4; Redox signalling; c-Myc