King's British Heart Foundation Centre, King's College London, London SE5 9NU, United Kingdom.
Mol Cell Proteomics. 2010 Mar;9(3):497-509. doi: 10.1074/mcp.M900275-MCP200. Epub 2009 Dec 27.
Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were "skinned," and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56alpha. Immunoblot analysis of myocyte fractions confirmed that beta-adrenergic stimulation by isoproterenol decreased the B56alpha content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56alpha from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following beta-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.
肌球蛋白纤维蛋白负责心脏收缩。然而，肌球蛋白亚基组至今尚未被全面分析。在本研究中，从啮齿动物心脏中分离出心肌细胞，并使用内皮素-1 和异丙肾上腺素（肌球蛋白纤维蛋白磷酸化的强效诱导剂）刺激这些细胞。随后，对心肌细胞进行“去皮质”处理，并使用配备电子转移解离的高质量精度离子阱串联质谱仪（LTQ Orbitrap XL）分析肌球蛋白亚基组。正如预期的那样，少数肌球蛋白纤维蛋白构成了总蛋白质量的大部分，其中几个已知的磷酸化位点通过电子转移解离得到了证实。在心脏肌球蛋白亚基组中还鉴定出了 600 多种其他蛋白质，包括激酶和磷酸酶亚基。肌球蛋白纤维蛋白的蛋白质组比较表明，异丙肾上腺素处理改变了蛋白磷酸酶 2A 调节亚基 B56alpha 的亚细胞定位。对心肌细胞分数的免疫印迹分析证实，β-肾上腺素能刺激异丙肾上腺素降低了肌球蛋白纤维蛋白部分的 B56alpha 含量，而肌球蛋白磷酸酶靶亚基同工型 1 和 2（MYPT1 和 MYPT2）的含量没有显著变化。此外，免疫标记和共聚焦显微镜显示这些蛋白质的空间重新分布，即在 Z 盘和 M 带区域失去 B56alpha，但在β-肾上腺素能刺激后，与肌球蛋白纤维蛋白的 A 带区域的 MYPT1/2 关联增加。总之，我们提出了第一张去皮心肌细胞的蛋白质组学综合数据集，并证明了蛋白质组学揭示蛋白质组成动态变化的潜力，这些变化可能有助于神经激素对肌球蛋白纤维蛋白收缩的调节。
