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 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 (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 27 +/- 007 molPi/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 (0 601 +/- 28% 1P 27 8 +/- 2 8% 2P 4 8 +/- 2 0% 3P 37 +/- 1 2% 4P 28 +/- 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 (C) 2010 Elsevier Ltd All rights reserved