Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle.
A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n=21). Total phosphorylation was 2.7 ± 0.07 mol Pi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n=19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the three sites was not random, but indicated positive and negative interactions between the three sites. Phosphorylation at Ser-282 was not proportional to the number of sites available. The 2P band contained 302 but not 273; the 3P band contained 273 but not 302.
Copeland
O'Neal
O
National Heart and Lung Institute, Imperial College London, London, SW3 6LY, UK.
Sadayappan
Sakthivel
S
Messer
Andrew E
AE
Steinen
Ger J M
GJ
van der Velden
Jolanda
J
Marston
Steven B
SB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D000099163:Myosin Binding Protein C
D000596:Amino Acids; D002352:Carrier Proteins; D010750:Phosphoproteins; D000099163:Myosin Binding Protein C