Interplay between troponin T phosphorylation and O-N-acetylglucosaminylation in ischaemic heart failure

Aims 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. Methods and results Decreased TnTpSer208 levels in the left ventricles of HF male Wistar rats were associated with reduced expression of PKC epsilon but not of other cardiac PKC isoforms. In both isolated perfused rat hearts and cultured neonatal cardiomyocytes, the PKC epsilon inhibitor epsilon V1-2 decreased TnTpSer208 and simultaneously decreased cardiac contraction in isolated hearts and beating amplitude in neonatal cardiomyocytes (measured by atomic force microscopy). Down-regulating PKC epsilon by silencing RNA (siRNA) also reduced TnTpSer208 in these cardiomyocytes, and PKC epsilon-/- 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. Conclusion These data demonstrate interplay between Ser208 phosphorylation and Ser190 O-GlcNAcylation of TnT in ischaemic HF, linked to decreased activity of both PKC epsilon 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.