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Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle.

Author(s): Copeland O, Sadayappan S, Messer AE, Stienen GJ, van der Velden J, Marston SB

Affiliation(s): National Heart and Lung Institute, Imperial College London, London, SW3 6LY, UK.

Publication date & source: 2010-09-17, J Mol Cell Cardiol., [Epub ahead of print]

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 beta-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 (Ser273, 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+/-0.07molPi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C is unphosphorylated. Total phosphorylation levels are 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 show a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser273, Ser282 and Ser302 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 Ser282 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. Copyright (c) 2010. Published by Elsevier Ltd.

Page last updated: 2010-10-05

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