Ca-Calmodulin Activated Phosphorylation Signaling Pathways in the Heart

项目总结/摘要 Ca[2+]是将电信号（即动作电位）转化为心脏机械过程（即收缩）的主要第二信使。这种高度协调的兴奋-收缩偶联（ECC）过程是通过肌膜上以及肌浆网（SR）中的许多转运蛋白、泵和离子通道实现的，从而导致在心跳到心跳周期期间观察到的Ca[2+]波。普遍表达的Ca[2+]/钙调蛋白依赖性蛋白激酶II（CaMKII）和钙调神经磷酸酶（CaN）对这些Ca[2+]处理蛋白的调节已被发现在心脏中发挥越来越重要的作用。ECC期间的心肌细胞信号传导通过细胞内[Ca[2+]]i浓度的流动来描述。在[Ca[2+]]i的增加期间，Ca[2+]结合的钙调蛋白（Ca-CaM）结合并激活CaMK II和CaN;从而使几种重要的Ca[2+]处理蛋白磷酸化或去磷酸化，在细胞内具有多种功能后果。CaMKII和CaN信号传导彼此独立，但在磷酸化信号传导级联中也可以是拮抗的。CaMKII活性和表达在人心脏肥大、心力衰竭和心力衰竭动物模型中显示增加。在心力衰竭动物模型中抑制CaMKII活性可减少或预防病理。在心力衰竭动物模型中，抑制CaN已显示出有益的效果;然而，其信号传导机制尚不清楚，需要进一步研究。因此，这些Ca-CaM依赖性途径是心力衰竭和心律失常的发生和治疗中的关键节点和潜在有价值的治疗靶点。具体的目的是集中在Ca-CaM CaMK Ⅱ- CaN信号通路的定位和易位（A）和激活动力学（B），其假设如下：（1A）Ca-CaM定位和易位由不同的刺激诱导。(1B)Ca-CaM信号动力学在关键肌细胞位置上不同。(2A)CaMK Ⅱ δ {B}与δ {C}在定位和易位方面不同。(2B)心肌细胞中CaMKII的激活局部不同，并整合以引起记忆。(3A)CaN定位于Z线，但与局部Ca[2+]信号易位。(3B)在成年心肌细胞中，CaN激活的动力学和位置与CaMKII不同。Bers实验室已经产生了一组荧光标记的蛋白质来监测CaM，CaMKII和CaN的定位和易位。也有基于FRET的Ca-CaM、CaMKII和CaN活性报告基因。将这些构建体转染到分离的成年心肌细胞中进行活细胞成像，并使用FRAP和TIRF来研究信号通路的动力学。在表征健康心脏细胞中的Ca-CaM-CaMKIICaN途径后，来自心力衰竭模型的心肌细胞将用于研究病理变化。这将极大地增强我们对CaMKII和CaN活性如何通过病理和生理信号通路在心脏中调节的理解。这将有助于确定新的信号级联，从而确定新的治疗心力衰竭的治疗靶点。

Project Summary/Abstract Ca[2+] is the principal second messenger in translating electrical signals (i.e. action potentials) into mechanical process in the heart (i.e. contractions). This highly orchestrated process of excitation-contraction coupling (ECC) is achieved through a number of transporters, pumps, and ion channels on the sarcolemmal membrane as well as in the sarcoplasmic reticulum (SR), resulting in the Ca[2+] waves observed during a beat-to-beat cycle. Modulation of these Ca[2+] handling proteins by the ubiquitously expressed Ca[2+]/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) have been found to play an increasingly important role in the heart. Cardiomyocytes signaling during ECC is described by the flux in intracellular [Ca[2+]]i concentration. During the increase in [Ca[2+]]i Ca[2+] bound calmodulin (Ca-CaM) binds to and activates CaMKII and CaN; thereby phosphorylating or dephosphorylating several important Ca[2+] handling proteins with multiple functional consequences within the cell. CaMKII and CaN signaling are independent of each other, but can also be antagonistic in the phosphorylation signaling cascade. CaMKII activity and expression were shown to be increased in human cardiac hypertrophy, heart failure, and animal models of heart failure. Inhibiting CaMKII activity in animal models of heart failure reduces or prevents pathology. The inhibition of CaN has demonstrated beneficial effect in animal models of heart failure; however the signaling mechanisms are not understood and need further investigation. Thus, these Ca-CaM-dependent pathways are critical nodal points and potentially valuable therapeutic targets in the genesis and treatment of heart failure and arrhythmias. The specific aims are focus on localization and translocation (A) and activation dynamics (B) of the Ca-CaMCaMKII- CaN signaling pathways with the following hypothesizes: (1A) Ca-CaM localization and translocation are induced by different stimuli. (1B) Ca-CaM signaling dynamics differ in key myocyte locations. (2A) CaMKIIdelta{B} vs. delta{C} differ in localization and translocation. (2B) CaMKII activation in myocytes differs locally and integrates to cause ¿memory¿. (3A) CaN localizes at the Z-line, but translocates with local Ca[2+] signals. (3B) Kinetics and location of CaN activation differ from CaMKII in adult cardiomyocytes. The Bers Lab has generated a set of fluorescently tagged proteins to monitor localization and translocation of CaM, CaMKII and CaN. There are also FRET based activity reporters for Ca-CaM, CaMKII and CaN. These constructs will be transfected into isolated adult cardiomyocytes for live cell imaging and the use of FRAP and TIRF will be employed to investigate the dynamics of the signaling pathways. After characterization of the Ca-CaM-CaMKIICaN pathway in heathly heart cells, cardiomyocytes from heart failure models will be used to investigate pathological changes. This will greatly enhance our understanding of how CaMKII and CaN activity is regulated in the heart by both pathological and physiological signaling pathways. This will help to identify novel signaling cascades and thereby identify new therapeutic targets for the treatment of heart failure.