Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle
Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle
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DOI：
10.1016/j.yjmcc.2010.09.007
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发表时间：
2010-12-01
影响因子：
5
通讯作者：
Marston, Steven B.
中科院分区：
文献类型：
--
作者：
Copeland, O'Neal;Sadayappan, Sakthivel;Marston, Steven B.
MyBP-C在心肌的一个独特的特性是它有多个磷酸化网站主要由PKA MyBP C磷酸化中扮演着重要的角色在调制收缩性的细胞反应,β肾上腺素能刺激体外研究显示MyBP-C可以在丝氨酸磷酸化273 282 302和307(鼠标序列),但很少有人知道MyBP C水平磷酸化或当前网站以来,心肌磷酸化方法是有限的在特异性和不定量调查的使用磷酸亲和力SDS页面一起共抗MyBP C抗体和一系列磷酸化网站特定抗体的主要网站(Ser 273 -282和-302年)与这些新开发的方法我们能够做个详细的定量分析MyBP-C磷酸化在心脏组织原位我们发现MyBP C是高度磷酸化在非失败的人类心脏或鼠标心脏三羟甲基氨基甲烷和液(捐赠)tetra-phosphorylated物种支配和不到10%的MyBP C是磷酸化(0 9 3 + / - 1% 1 p 13 4 + / - 2 7% 2 p 10 5 + / - 3 3% 3 p 28 7 + / - 3 7% 4 p 36 7% 4 + / - 2 n = 21)总磷酸化27 + / - 007 molPi /摩尔MyBP C相比之下在失败的心和肌切除术HCM患者大多数样本MyBP-C磷酸化总磷酸化水平是23%的不正常心脏肌原纤维(0 601 + / - 28% 1 p 27个8 + / - 2 8% 2 4 8 + / - 2 0% 3 p 37 + / - 1 2% 4 p 28 + / - 13% n = 19)和39%的正常肌切除术样本网站特定抗体显示出独特的分布格局的磷酸化网站多个273年物种我们发现磷酸化丝氨酸的磷酸化水平Ser 282和Ser - 302都出现在4 p C群MyBP但都不重要1 p乐队表明必须有至少一个其他网站MyBP-C磷酸化在人类心脏磷酸化的模式三个网站并不是随机的Ser-282位点的磷酸化与可用位点的数量不成比例，2P带包含302但不包含273,3P带包含273但不包含302 (C) 2010 Elsevier Ltd All rights reserved
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