González-Solá Maryví, Al-Khayat Hind A, Behra Martine, Kensler Robert W
Department of Anatomy and Neurobiology, University of Puerto Rico Medical School, San Juan, Puerto Rico.
National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, United Kingdom.
Biophys J. 2014 Apr 15;106(8):1671-80. doi: 10.1016/j.bpj.2014.01.050.
To understand how mutations in thick filament proteins such as cardiac myosin binding protein-C or titin, cause familial hypertrophic cardiomyopathies, it is important to determine the structure of the cardiac thick filament. Techniques for the genetic manipulation of the zebrafish are well established and it has become a major model for the study of the cardiovascular system. Our goal is to develop zebrafish as an alternative system to the mammalian heart model for the study of the structure of the cardiac thick filaments and the proteins that form it. We have successfully isolated thick filaments from zebrafish cardiac muscle, using a procedure similar to those for mammalian heart, and analyzed their structure by negative-staining and electron microscopy. The isolated filaments appear well ordered with the characteristic 42.9 nm quasi-helical repeat of the myosin heads expected from x-ray diffraction. We have performed single particle image analysis on the collected electron microscopy images for the C-zone region of these filaments and obtained a three-dimensional reconstruction at 3.5 nm resolution. This reconstruction reveals structure similar to the mammalian thick filament, and demonstrates that zebrafish may provide a useful model for the study of the changes in the cardiac thick filament associated with disease processes.
为了理解诸如心肌肌球蛋白结合蛋白-C或肌联蛋白等粗肌丝蛋白中的突变如何导致家族性肥厚型心肌病，确定心脏粗肌丝的结构很重要。斑马鱼的基因操作技术已经成熟，它已成为研究心血管系统的主要模型。我们的目标是开发斑马鱼作为哺乳动物心脏模型的替代系统，用于研究心脏粗肌丝及其组成蛋白的结构。我们已成功从斑马鱼心肌中分离出粗肌丝，采用了与哺乳动物心脏类似的方法，并通过负染色和电子显微镜分析了它们的结构。分离出的肌丝排列有序，具有X射线衍射预期的肌球蛋白头部42.9纳米准螺旋重复特征。我们对这些肌丝C区的电子显微镜图像进行了单颗粒图像分析，并获得了3.5纳米分辨率的三维重建。该重建显示出与哺乳动物粗肌丝相似的结构，并表明斑马鱼可能为研究与疾病过程相关的心脏粗肌丝变化提供一个有用的模型。
