Cardiac Myosin-Binding Protein C: Molecular Mechanisms Governing Cardiac Contractility

心肌肌球蛋白结合蛋白C(cMyBP-C)是一种肌节粗丝相关蛋白 恢复正常的心脏结构和功能。CMyBP-C的突变强调了cMyBP-C的重要性 是肥厚型心肌病的主要原因。尽管它是心脏收缩能力的关键调节器， CMyBP-C调节肌动球蛋白力和运动生成的分子机制远未见报道 当然。虽然cMyBP-C的N-末端结构域可以与肌动蛋白和肌球蛋白头部区域结合，但尚不清楚 这些结合伙伴中的哪些是生理上相关的，以及这些结合伙伴的相互作用 通过直接影响肌动球蛋白的产生或间接通过改变钙离子来调节心肌收缩能力 依赖细丝激活。与β反应发生cMyBP-C的N端的磷酸化- 肾上腺素能刺激，磷酸化可以提供cMyBP-C功能可调性的测量，以便 增强心脏收缩能力。为了解决这些问题，我们提出了两个具体目标。目标1将测试 磷酸化调控cMyBP-C N-末端结构域结构影响其结合的假说 伙伴相互作用(即细丝和肌球蛋白头部区域)。我们将使用一种新的质谱学 技术和原子力显微镜表征cMyBP-C N末端的分子力学 由于磷酸化或突变而发生结构改变。这些结构对功能的影响 将在心肌肌原纤维和天然粗丝的背景下表征扰动，以确定 CMyBP-C只在存在于粗丝中的地方起作用，以及它是否能将心肌肌球蛋白隔离成 储备了超放松的肌球蛋白头部。因此，我们将测量的位置和时间进程 单个心肌原纤维的荧光-ATP翻转和天然粗丝在心肌细胞中的作用力 激光诱捕表达磷酸化和结合伴侣去除突变体的转基因小鼠的制备 CMyBP-C。在目标2中，我们将创建基于DNA的“设计者”粗丝纳米管，以定义空间 CMyBP-C与其肌球蛋白和肌动蛋白结合的粗丝中通常存在的关系 合作伙伴是cMyBP-C运作模式的关键决定因素。这些DNA纳米管将使精致的 表达的cMyBP-C和人β-心肌肌球蛋白在纳米管表面的纳米空间定位 相互之间的关系。通过这种新的方法，我们可以将cMyBP C对肌动球蛋白运动的调节分配给 肌球蛋白头部和/或细丝的结合，通过细丝运动和力产生来评估 使用激光陷阱。通过对cMyBP-C功能的了解和理解，这些集体 研究，针对cMyBP-C结合伙伴相互作用的靶向治疗可能会被开发出来 调节和改善衰竭心脏的心功能。

Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric thick filament associated protein that is essential to normal cardiac structure and function. The importance of cMyBP-C is emphasized by mutations to cMyBP-C being a leading cause of hypertrophic cardiomyopathy. Despite being a key regulator of cardiac contractility, the molecular mechanism by which cMyBP-C modulates actomyosin force and motion generation is far from certain. Although cMyBP-C's N-terminal domains can bind to actin and the myosin head region, it is not known which of these binding partners is physiologically relevant and whether these binding partner interactions modulate cardiac contractility by directly affecting actomyosin power generation or indirectly by altering Ca2+- dependent thin filament activation. With phosphorylation of cMyBP-C's N terminus occurring in response to β- adrenergic stimulation, phosphorylation may offer a measure of cMyBP-C functional tunability in order to enhance cardiac contractility. To address these questions, we propose two specific aims. Aim 1 will test the hypothesis that phosphorylation modulates cMyBP-C's N-terminal domain structure to influence its binding partner interactions (i.e. thin filament and myosin head region). We will use a novel mass-spectrometry technique and atomic force microscopy to characterize the molecular mechanics of cMyBP-C's N terminus that has been structurally altered due to phosphorylation or mutagenesis. The functional impact of these structural perturbations will be characterized in the context of cardiac myofibrils and native thick filaments to determine if cMyBP-C operates only where it exist in the thick filament and whether it can sequester cardiac myosin into a reserve pool of super-relaxed myosin heads. Thus, we will measure the location and time course of fluorescent-ATP turnover in single cardiac myofibrils and the force generated by native thick filaments in the laser trap in preparations from transgenic mice expressing phosphorylation and binding partner ablated mutant cMyBP-C. In Aim 2 we will create DNA-based “designer” thick filament nanotubes to define how the spatial relationships that normally exist in the thick filament between cMyBP-C and its myosin and actin binding partners are critical determinants of cMyBP-C's modes of operation. These DNA-nanotubes will allow exquisite nanometer spatial positioning of expressed cMyBP-C and human β-cardiac myosin on the nanotube surface relative to each other. By this novel approach we can assign cMyBP C's modulation of actomyosin motility to binding of the myosin head and/or thin filament, as assessed by both thin filament motility and force generation using the laser trap. With the knowledge and understanding of cMyBP-C function derived from these collective studies, targeted therapies directed at cMyBP-C binding partner interactions may be developed to help modulate and to improve cardiac performance in the failing heart.