The role of tropomyosin phosphorylation in muscle.

描述（由申请人提供）：本研究提出的实验研究了心脏原肌球蛋白磷酸化（Tm- p）被心脏应激改变的假设，并且Tm的这种电荷修饰改变了它在细丝内的相互作用，从而影响收缩。迄今为止，由心脏应激引起的Tm的改变及其对收缩的影响尚不清楚。我们提出了三个目标：1)评估应激心脏中的Tm-P。Tm-P将在缺血/再灌注处理的离体灌注心脏中通过二维凝胶和质谱的磷酸化蛋白特异性染色来表征。II)确定Tm-P与肌动蛋白、肌钙蛋白结合的影响，以及在肌钙蛋白存在的情况下与肌动蛋白结合的影响。Tm结合将通过共沉淀进行，并通过密度测定法在二维凝胶上进行定量。2)测定Tm-P对心肌力量发展的影响。用凝胶选择性地去除细丝，并用肌动蛋白、肌钙蛋白和非磷酸化或磷酸化的Tm进行重建，在剥皮的乳头状束中测量钙激活张力。从这些实验中获得的知识将为Tm-P的调节机制及其在健康和疾病中对心肌收缩的影响提供新的见解。

DESCRIPTION (provided by applicant): The experiments proposed in this study investigate the hypothesis that tropomyosin phosphorylation (Tm-P) in the heart is altered by cardiac stress and this charge modification of Tm alters it's interactions within the thin filament to affect contraction. To date the modification of Tm resulting from cardiac stress and its effect on contraction are unknown. We propose three aims: I) To evaluate Tm-P in the stressed heart. Tm-P will be characterized in isolated perfused hearts treated with ischemia/reperfusion by phosphoprotein specific staining of 2-D gels and mass spectrometry. II) To determine the effect of Tm-P on binding to actin, troponin and to actin in the presence of troponin. Tm binding will be carried out by co-sedimentation and quantified on 2-D gels with densitometry. Ill) To determine the effect of Tm-P on the development of force in cardiac muscle. Calcium activated tension will be measured in skinned papillary bundles treated with gelsolin to selectively remove the thin filament and reconstituted with actin, troponin and non-phosphorylated or phosphorylated Tm. The knowledge gained from these experiments will provide novel insight into the regulatory mechanism of Tm-P and its effect on cardiac muscle contraction in health and disease.