Interplay between troponin T phosphorylation and O-N-acetylglucosaminylation in ischaemic heart failure.
OBJECTIVE
Previous studies have reported that decreased serine 208 phosphorylation of troponin T (TnTpSer208) is associated with ischaemic heart failure (HF), but the molecular mechanisms and functional consequences of these changes are unknown. The aim of this study was to characterize the balance between serine phosphorylation and O-N-acetylglucosaminylation (O-GlcNAcylation) of TnT in HF, its mechanisms, and the consequences of modulating these post-translational modifications.


RESULTS
Decreased TnTpSer208 levels in the left ventricles of HF male Wistar rats were associated with reduced expression of PKCε but not of other cardiac PKC isoforms. In both isolated perfused rat hearts and cultured neonatal cardiomyocytes, the PKCε inhibitor εV1-2 decreased TnTpSer208 and simultaneously decreased cardiac contraction in isolated hearts and beating amplitude in neonatal cardiomyocytes (measured by atomic force microscopy). Down-regulating PKCε by silencing RNA (siRNA) also reduced TnTpSer208 in these cardiomyocytes, and PKCε-/- mice had lower TnTpSer208 levels than the wild-type. In parallel, HF increased TnT O-GlcNAcylation via both increased O-GlcNAc transferase and decreased O-GlcNAcase activity. Increasing O-GlcNAcylation (via O-GlcNAcase inhibition with Thiamet G) decreased TnTpSer208 in isolated hearts, while reducing O-GlcNAcylation (O-GlcNAc transferase siRNA) increased TnTpSer208 in neonatal cardiomyocytes. Mass spectrometry and NMR analysis identified O-GlcNAcylation of TnT on Ser190.


CONCLUSIONS
These data demonstrate interplay between Ser208 phosphorylation and Ser190 O-GlcNAcylation of TnT in ischaemic HF, linked to decreased activity of both PKCε and O-GlcNAcase and increased O-GlcNAc transferase activity. Modulation of these post-translational modifications of TnT may be a new therapeutic strategy in HF.
INSERM, U1167, 1 rue du Professeur Calmette, Lille, France Institut Pasteur de Lille, Lille, France University of Lille Nord de France, Lille, France.
Emilie
E
Dubois-Deruy
INSERM, U1167, 1 rue du Professeur Calmette, Lille, France Institut Pasteur de Lille, Lille, France University of Lille Nord de France, Lille, France.
Aude
A
Belliard
Inserm U1096, Rouen, France Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France.
Paul
P
Mulder
INSERM, U1167, 1 rue du Professeur Calmette, Lille, France Institut Pasteur de Lille, Lille, France University of Lille Nord de France, Lille, France.
Marion
M
Bouvet
University of Lille Nord de France, Lille, France CNRS UMR 8576, Villeneuve D'Ascq, France.
Caroline
C
Smet-Nocca
Bioimaging Center Lille Nord de France, Lille, France.
Sébastien
S
Janel
Institut Pasteur de Lille, Lille, France University of Lille Nord de France, Lille, France Bioimaging Center Lille Nord de France, Lille, France CNRS UMR 8204, INSERM U1019, Lille, France.
Frank
F
Lafont
INSERM, U1167, 1 rue du Professeur Calmette, Lille, France Institut Pasteur de Lille, Lille, France University of Lille Nord de France, Lille, France.
Olivia
O
Beseme
INSERM, U1167, 1 rue du Professeur Calmette, Lille, France Institut Pasteur de Lille, Lille, France University of Lille Nord de France, Lille, France Centre Hospitalier Régional et Universitaire de Lille, Lille, France.
Philippe
P
Amouyel
Inserm U1096, Rouen, France Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France.
Vincent
V
Richard
INSERM, U1167, 1 rue du Professeur Calmette, Lille, France Institut Pasteur de Lille, Lille, France University of Lille Nord de France, Lille, France florence.pinet@pasteur-lille.fr.
Florence
F
Pinet
D000818:Animals; D006333:Heart Failure / Q000378:metabolism*; D008297:Male; D009200:Myocardial Contraction; D017202:Myocardial Ischemia / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000502:physiology*; D010766:Phosphorylation; D051744:Protein Kinase C-epsilon / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D020107:Troponin T / Q000378:metabolism*
D020107:Troponin T; D017351:N-Acetylglucosaminyltransferases; C449880:O-GlcNAc transferase; C497257:Prkce protein, rat; D051744:Protein Kinase C-epsilon
Atomic force microscopy; Myocardial infarction; NMR; O-GlcNAcylation; Phosphorylation; Troponin T