Myosin binding protein C phosphorylation in normal, hypertrophic and failing human heart muscle

Phosphorylation of myosin binding protein C (MyBP-C) was investigated in intraventricular septum samples taken from patients with hypertrophic cardiomyopathy undergoing surgical septal myectomy. These samples were compared with donor heart muscle, as a well-characterised control tissue, and with end-stage failing heart muscle. MyBP-C was partly purified from myofibrils using a modification of the phosphate-EDTA extraction of Hartzell and Glass. MyBP-C was separated by SDS-PAGE and stained for phosphoproteins using Pro-Q Diamond followed by total protein staining using Coomassie Blue. Relative phosphorylation level was determined from the ratio of Pro-Q Diamond to Coomassie Blue staining of MyBP-C bands as measured by densitometry. We compared 9 myectomy samples and 9 failing heart samples with 9 donor samples. MyBP-C phosphorylation in pathological muscle was lower than in donor (myectomy 40 +/- 2% of donor, P<0.0001; failing 45 +/- 3% of donor, P<0.0001). 6 myectomy samples were identified with MYBPC3 mutations, one with MYH7 mutation and two remained unknown, but there was no correlation between MYBPC3 mutation and MyBP-C phosphorylation level.In order to determine the number of phosphorylated sites in human cardiac MyBP-C samples, we phosphorylated the recombinant MyBP-C fragment, C0-C2 (1-453) with PKA using P-gamma 32-ATP up to 3.5 mol Pi/mol C0-C2. This measurement of phosphorylation was used to calibrate measurements of phosphorylation in SDS-PAGE using Pro-Q Diamond stain. The level of phosphorylation in donor heart MyBP-C was calculated to be 4.6 +/- 0.6 mol Pi/mol and 2.0 +/- 0.3 mol Pi/mol in myectomy samples.We conclude that MyBP-C is a highly phosphorylated protein in vivo and that diminished MyBP-C phosphorylation is a feature of both end-stage heart failure and hypertrophic cardiomyopathy. (C) 2008 Elsevier Inc. All rights reserved.