Novel Phosphorylation Sites And Reduced Phosphorylation Levels In Human Cardiac Mybp-C From Failing Hearts

Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain protein associated with the thick filaments. Phosphorylation of cMyBP-C is a regulator of cardiac contractility and it is known that the phosphorylation status of cMyBP-C is altered during heart failure (HF). There are several identified phosphorylated residues, including the regulatory phosphorylation sites Ser275, Ser284 and Ser304. Methods: Comparison of the extent of MyBP-C phosphorylation status in failing (N=10) and non-failing (N=10) human left ventricle (LV) tissue. The myofilament subproteome was extracted using the ‘IN Sequence’ method prior to analysis with some samples dephosphorylated using alkaline phosphatase as control. Sample analysis was carried out using gel- (e.g. phos-tag) and mass spectrometry (MS)-based methods. Results: Using an MS-based phospho-peptide (Ti02 chromatography) enrichment strategy we identified novel phosphorylation sites at residues Ser286 and Thr290, located in the M-motif. These sites were as well observed in MyBP-C from LV canine hearts. A novel site at residue Thr1109, the titin binding domain C9, was only observed in the samples without phospho-enrichment. Our results indicate a marked reduction in cMyBP-C phosphorylation at residues Ser284, Ser286, Thr290 and Thr1109 in end-stage HF compared to the non-failing group. Conclusion: This study provides evidence for novel phosphorylation sites on cMyBP-C both in human and canine hearts and reduced phosphorylation levels in the end-stage failing heart.