TNI ISOFORM SWITCHING AND CALCIUM ION CONTROL IN THE HEART

钙离子对肌丝活性的调节是至关重要的 兴奋-收缩耦合中的行列式。人们普遍认为， 肌丝异构体在正常发育和病理过程中的变化 导致该系统对胞浆[Ca~(2+)]_i的响应发生显著变化。 肌钙蛋白I(TnI)亚型转换被认为是一个中心角色 在新生儿和成人心脏对肌力调节的不同反应中 介导剂降低收缩装置对钙的敏感性 胎儿心脏表达慢抽动的骨骼肌TnI亚型 (TNIS)。出生后，成人心脏亚型TnIc成为排他性的 心脏中存在TnI亚型。PI已经研究了分子 克隆介导TnI异构体在心脏中缓慢表达的机制 人类tnix基因及其多种转录元件的鉴定 参与骨骼肌和心肌特异性基因的表达，并具有 在成人体内发现转基因报告基因的小室特异性差异 心肌。在体内的基因表达已经通过分析 转基因小鼠和报告基因直接注射成人 心肌。研究人员现在将在体内应用这些基因转移 TnI亚型调控分子机制的研究进展 心脏切换术并评估TnI亚型的后果 转而使用钙基肌丝功能。(I)新增行数 转基因小鼠将被分析以评估个体的作用 转录元件在时空基因调控中的作用 心脏；(Ii)瞬时转染原代心肌细胞，凝胶迁移率移动 化验和DNA足迹研究将被用来确定因素 调节新生儿和成人心脏中的TnIs基因；(Iii)空 突变将被引入小鼠的心脏和缓慢的骨骼中 肌肉TnI亚型基因以评估这些基因敲除的后果 突变对心肌细胞分化和钙激活的影响 来自胚胎干细胞；(Iv)解决功能重要性 先前确定的TnI相互作用和磷酸化结构域， 含TnIc间嵌合cDNA构建的表达载体 而Tnis亚型将在成年大鼠心脏中表达，并与一种 DNA直接注射标记质粒。肌细胞将从 注射心脏，以及杂合TnI蛋白对钙的影响 将对基于肌丝的调节进行评估。这是一个综合性的 利用体外和体内方法检测分子的研究 细丝蛋白基因表达调控机制研究进展 成熟心脏中TnI亚型转换的生理后果。

The calcium-based regulation of myofilament activity is a crucial determinant in excitation-contraction coupling. It is broadly seen that myofilament isoform switching during normal development and in pathology leads to marked changes in response of this system to cytosolic [CA2+]i. Troponin I (TnI) isoform switching has been implicated as a central player in the differential response of the neonatal versus adult heart to inotropi agents that mediate decreased Ca2+ sensitivity of the contractile apparatus The fetal heart expresses the slow-twitch skeletal muscle isoform of TnI (TnIs). After birth, the adult cardiac isoform, TnIc, becomes the exclusiv TnI isoform present in the heart. The PI has investigated the molecular mechanisms mediating slow TnI isoform expression in the heart by cloning the human TnIx gene and identifying multiple transcriptional elements involved in skeletal versus cardiac muscle-specific gene expression and has found chamber-specific differences in transgene reporter genes into adult myocardium. In vivo gene expression has been examined by analysis of transgenic mice and direct injection of reporter genes into adult myocardium. The investigator will now apply these in vivo gene transfer approaches to examine the molecular mechanisms regulating TnI isoform switching in the heart and assess the consequences of TnI isoform switching to calcium-based myofilament function. (i) New lines of transgenic mice will be analyzed to assess the role of individual transcriptional elements to temporal and spatial gene regulation in the heart; (ii) Transient transfection of primary myocytes, gel mobility shift assays, and DNA footprinting studies will be used to identify factors regulating the TnIs gene in the neonatal versus adult heart; (iii) Null mutations will be introduced into the mouse cardiac and slow skeletal muscle TnI isoform genes to assess the consequences of these knock out mutations on the differentiation and Ca2+ activation of myocytes derived from embryonic stem cells; (iv) To address the functional importance of previously identified TnI interaction and phosphorylation domains, expression vectors containing chimerical cDNA constructs between the TnIc and TnIs isoforms will be expressed in adult rat hearts along with a marker plasmid by direct DNA injection. Myocytes will be prepared from the injected hearts, and the effect of the hybrid TnI proteins on calcium based myofilament regulation will be evaluated. This is an integrative study using in vitro and in vivo approaches to examine the molecular mechanisms controlling thin filament protein gene expression and the physiological consequences of TnI isoform switching in the maturing heart.