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 (gamma32)P-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.
Cardiovascular Science, NHLI, Imperial College London, London, United Kingdom.
Adam M
AM
Jacques
O'Neal
O
Copeland
Andrew E
AE
Messer
Clare E
CE
Gallon
Katie
K
King
William J
WJ
McKenna
Victor T
VT
Tsang
Steven B
SB
Marston
D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006333:Heart Failure / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D000099163:Myosin Binding Protein C
D002352:Carrier Proteins; D009218:Myosins; D000099163:Myosin Binding Protein C