The role of tropomyosin phosphorylation in muscle.

描述（由申请人提供）：本研究中提出的实验研究了心脏中的原肌球蛋白磷酸化（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其在细丝内的相互作用以影响收缩。迄今为止，由心脏应激引起的Tm的改变及其对收缩的影响是未知的。我们提出了三个目标：1）评估Tm-P在应激心脏。Tm-P将在用缺血/再灌注处理的离体灌注心脏中通过2-D凝胶的磷蛋白特异性染色和质谱法来表征。II）确定Tm-P对与肌动蛋白、肌钙蛋白以及在肌钙蛋白存在下与肌动蛋白结合的影响。Tm结合将通过共沉降进行，并在2-D凝胶上用密度测定法定量。III）确定Tm-P对心肌中力的发展的影响。将在用凝溶胶蛋白处理以选择性去除细丝并用肌动蛋白、肌钙蛋白和非磷酸化或磷酸化Tm重建的皮肤乳头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.