MODULATION OF CALCIUM CONTROL OF CARDIAC MYOFIBRILS

心脏肌原纤维钙控制的调节

• 批准号: 3336762
• 负责人: R John Solaro
• 金额: $ 18.92万
• 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/3336762
• 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 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节 Ca2+ 与 TNC 结合的过程控制肌动蛋白-肌球蛋白反应。 总体目标是了解 Ca 信号传导机制如何以及何时发挥作用 i) 肌丝的化学和机械状态以及 ii) 改变 肌丝蛋白亚型的群体。 目标是： (1) 了解肌丝蛋白同工型群体是否发生其他变化 TNC 影响肌原纤维 Ca2+ 激活的长度依赖性； (2) 确定肌丝蛋白异构体的变化是否影响 肌动蛋白跨桥相互作用对 TNC Ca2+ 结合的影响 构象； (3) 确定肌丝移动的影响 异构体群体对化学环境（pH、Pi）对肌丝影响的影响 对Ca2+的反应； (4) 了解亚型的磷酸化如何变化 TNT 和 TNI 影响肌丝活性和调节； (5) 测量 改变完整心脏制剂中肌丝对 Ca2+ 的反应 已知的肌丝蛋白亚型群。 处理这些问题的方法 目标涉及在不同层面使用灌注心脏和肌丝 含有 TNT 和 TNI 亚型的组织。 同工型是 通过 PAGE 和免疫印迹鉴定。 直接测量 TNC 的 Ca 结合 存在于带皮纤维和重构制剂中。 荧光探针 连接到 TNC 和 TNT 用作 Ca2+ 结合的报告基因 构象变化。 肌动蛋白跨桥反应由以下因素控制 长度的变化，通过改变重meromyosin的浓度 重组制剂，并通过改变弱和强的条件 绑定状态。 TNT 在带皮纤维中被磷酸化，并且 使用 TNT 激酶和蛋白激酶 C 重构制剂和 ii) 通过刺激蛋白激酶 C 通路来完整肌肉 用32P处理的制剂。 肌丝 Ca2+ 反应完整 通过绘制稳定的水母发光蛋白技术来测量制剂 光对稳定力的准备刺激强直 不同的Ca2+浓度。 这些研究提供了对 改变肌丝对 Ca2+ 反应的机制及其潜力 在心肌生理学、病理学和药理学中具有重要意义。