MODULATION OF CALCIUM CONTROL OF CARDIAC MYOFIBRILS

心脏肌原纤维钙控制的调节

• 批准号: 2215537
• 负责人: R John Solaro
• 金额: $ 19.78万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2215537
• 关键词: acid base balance; acidity /alkalinity; actins; adenosine diphosphate; adenosinetriphosphatase; affinity chromatography; biological signal transduction; calcium binding protein; calcium channel; calcium metabolism; cations; fluorescent dye /probe; homeostasis; hydrogen; hypoxia; ion transport; ischemia; isomer; laboratory rabbit; laboratory rat; lactates; magnesium; membrane permeability; muscle relaxants; muscle stimulant; muscle tension; myocardium; myofibrils; myosins; nucleotides; phosphorylation; protein isoforms; protein signal sequence; stretch reflex; troponin

Experiments proposed here test the hypothesis that an important element in intrinsic and extrinsic regulation of cardiac function is modulation of the process by which Ca2+ - binding to TNC controls the actin-myosin reaction. A general objective is to know how and when the Ca-signalling mechanism is altered by i) the chemical and mechanical state of the myofilaments and ii) the population of isoforms of myofilament proteins. The aims are: (1) To know whether shifts in isoform population of myofilament proteins other than TNC influence the length dependence of myofibrillar Ca2+ -activation; (2) To determine if shifts in isoforms of myofilament proteins influence the effect of actin-cross-bridge interactions on TNC Ca2+ -binding and conformation; (3) To determine the influence of shifts in myofilament isoform population on the effect of chemical mileau (pH, Pi) on myofilament response to Ca2+; (4) To know how changes in phosphorylation of isoforms of TNT and TNI affect myofilament activity and regulation; (5) To measure the altered myofilament response to Ca2+ in intact heart preparations with known isoform population of myofilament proteins. The approach to these aims involves the use of perfused hearts, of myofilaments at various levels of organization containing isoforms of TNT and TNI. Isoforms are identified by PAGE and immunoblots. Ca-binding is measured directly to TNC in skinned fibers and reconstituted preparations. Fluorescent probes attached to TNC and TNT are used as reporters of Ca2+ -binding and conformational changes. Actin-cross-bridge reactions are controlled by changes in length, by changes in concentrations of heavy meromyosin in reconstituted preparations, and by conditions altering weak and strong binding states. TNT is phosphorylated i) in skinned fibers and reconstituted preparations using TNT kinase and protein kinase C and ii) in intact muscle by stimulation of the protein kinase C pathway in preparations treated with 32P. Myofilament Ca2+ -response in intact preparations is measured with the aequorin technique by plotting steady light against steady force in preparations stimulated tetanically in different Ca2+ concentrations. These studies provide insight into the mechanism of altered myofilament response to Ca2+ and its potential significance in myocardial physiology, pathology and pharmacology.

这里提出的实验检验了以下假设 心脏功能的内在和外在调节是调制 Ca2+与TNC结合控制肌动蛋白 - 肌球蛋白反应的过程。 一个一般目标是知道CA签名机制的方式以及何时 由i）改变肌膜的化学和机械状态和II） 肌丝蛋白的同工型种群。 目的是：（1） 知道其他其他肌丝蛋白的同工型群体是否转移其他 比TNC影响肌原纤维Ca2+激活的长度依赖性； （2）确定肌丝蛋白的同工型是否影响 肌动蛋白 - 桥桥相互作用对TNC Ca2+结合的影响 构象； （3）确定肌丝的转移的影响 同工型人群对化学米（pH，pi）对肌丝的影响 对Ca2+的响应； （4）知道如何变化的同工型的磷酸化 TNT和TNI会影响肌丝活动和调节； （5）测量 改变了对Ca2+的肌丝的反应。 已知的肌丝蛋白的同工型种群。 这些方法 目的涉及使用灌注心，各个层面的肌丝 包含TNT和TNI同工型的组织。 同工型是 通过页面和免疫印迹识别。 CA结合直接测量到TNC 在皮肤纤维和重构制剂中。 荧光探针 连接到TNC和TNT作为Ca2+结合的记者， 构象变化。 肌动蛋白 - 交叉桥反应受到控制 长度的变化，通过重霉素浓度的变化 重组准备工作，以及改变弱和强大的条件 结合状态。 TNT是磷酸化的i）在皮肤纤维中 使用TNT激酶和蛋白激酶C和II的重构制剂 通过刺激蛋白激酶C途径的完整肌肉 用32便士处理的制剂。 完整的肌丝Ca2+反应 通过绘制稳定来测量制剂 在制剂中对稳定力的照明在三刺中刺激 不同的Ca2+浓度。 这些研究提供了有关 肌丝对Ca2+的反应改变的机制及其潜力 心肌生理，病理学和药理学的重要性。