Phosphorylation/O-GlcNAc interplay in heart failure: pathophysiological roles and interest as biomarkers – PHOGIHF

Heart Failure (HF), especially secondary to myocardial infarction, still represents a major health concern worldwide. Improvement of HF treatment requires both to improve our knowledge on the molecular mechanisms of the disease and to development relevant biomarkers that may predict the outcome. Following this dual objective, both mechanistic and diagnostic, we initiated studies based on differential proteomics aimed at assessing, in a rat model of post-MI HF, the cardiac protein changes associated with chronic HF, and especially the post-translational modifications (PTM) of various contractile proteins, and at correlating these changes with the severity of adverse left ventricular (LV) remodeling and LV dysfunction.
In parallel to the protein studies that allowed us to identify 27 differentially expressed proteins, we started studying PTM by assessing changes in phosphorylation and O-GlcNAcylation, two dynamic, reversible and often mutually exclusive processes involved in many cellular functions. We compared the phosphoproteome of LV in normal- and 2 month-HF rats using a combination of 2D electrophoresis and Pro-Q®Diamond staining. Among the 29 different phosphoproteins identified, we focused on proteins constituting the myofilaments :Troponin T (TnT), myosin light chain-3 (MLC-3), myosin light chain-2 (MLC-2), a1-tropomyosin (TMa-1), desmin and g-actin and the chaperone protein : aB-crystallin and Hsp 70. For each described proteins, we validated the modulation of phosphorylation using specific antibodies against the proteins and determined the aminoacid involved in the phosphorylation modulation. This analysis was pursued in more details with Tnt, for which the decrease in phosphorylation on serine208 revealed in the experimental model was confirmed in patients post IdM, and which forms the basis of a recent patent, and might present a major diagnostic value.
Based on these promising results, our aim is now to better understand the role of these phosphorylation and O-GlCNAcylation processes in contractile cardiac defects in experimental HF. Several experimental models will be used: the model of MI induced by left coronary ligation, and a model of pressure overload hypertrophy (aortic stenosis), both in rats and mice, as well the isolated perfused heart (Langendorf), in order to correlate the PTM of the selected proteins, or their modulation, to the changes in cardiac contractility. For each protein, we will determine if they bear O-GlcNAc residues and in the positive case, we will determine if there is interplay between phosphorylation and O-GlcNAcylation in HF. We will characterize the enzymes involved in the phosphorylation (kinases and phosphatases) and O-GlcNAcylation (O-GlcNAc transferase (OGT) and O- GlcNAcase) interplay. Finally, concerning specifically Tnt, our production of specific antibodies for the phosphorylated form of the protein will allow to correlate the level of this phosphorylated form with the severity of HF, by tissue and cellular staining. This will help to study the role of PTM of TnT in the interaction with other myofilament proteins.
The strength of the collaboration of the two groups is that they possess complementary expertise in experimental HF models and in proteomics. The former ANR project granted between the same two partners was fruitful with several publications and a patent.