Ca-Calmodulin Activated Phosphorylation Signaling Pathways in the Heart

描述（由申请人提供）：Ca2+是将电信号（即动作电位）转换为心脏机械过程（即收缩）的主要第二信使。这种高度协调的兴奋-收缩耦合（ECC）过程是通过肌层膜和肌浆网（SR）上的许多转运蛋白、泵和离子通道实现的，导致在搏动周期中观察到Ca2+波。这些Ca2+处理蛋白通过无处不在表达的Ca2+/钙调素依赖性蛋白激酶II （CaMKII）和钙调神经磷酸酶（CaN）的调节已经被发现在心脏中起着越来越重要的作用。心肌细胞在ECC期间的信号传导是通过细胞内[Ca2+]i浓度的通量来描述的。在[Ca2+]i增加的过程中，Ca2+结合钙调蛋白（Ca-CaM）结合并激活CaMKII和CaN；从而磷酸化或去磷酸化几种重要的Ca2+处理蛋白，在细胞内产生多种功能后果。CaMKII和CaN信号相互独立，但在磷酸化信号级联中也可能是拮抗的。CaMKII的活性和表达在人类心脏肥厚、心力衰竭和心力衰竭动物模型中均有所增加。在心力衰竭动物模型中抑制CaMKII活性可减少或预防病理。抑制CaN在心力衰竭动物模型中显示出有益的效果；然而，信号机制尚不清楚，需要进一步研究。因此，这些ca - cam依赖性通路是心衰和心律失常发生和治疗的关键节点和潜在的有价值的治疗靶点。具体目的是关注Ca-CaM- CaMKII-CaN信号通路的定位和易位(A)和激活动力学(B)，并提出以下假设：（1A） Ca-CaM的定位和易位是由不同的刺激诱导的。（1B） Ca-CaM信号动力学在关键的肌细胞位置不同。（2A） CaMKII4B与4C在定位和易位上存在差异。（2B） CaMKII在肌细胞中的激活局部不同，并整合形成“记忆”。（3A） CaN定位于z线，但随局部Ca2+信号易位。（3B）成人心肌细胞中CaN活化的动力学和位置与CaMKII不同。Bers实验室已经生成了一组荧光标记蛋白来监测CaM、CaMKII和CaN的定位和易位。Ca-CaM、CaMKII和CaN也有基于FRET的活动报告。这些构建体将被转染到分离的成人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。在对健康心脏细胞中的Ca-CaM-CaMKII- CaN通路进行表征后，将使用心力衰竭模型中的心肌细胞来研究病理变化。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。这将有助于识别新的信号级联，从而确定治疗心力衰竭的新靶点。

DESCRIPTION (provided by applicant): Ca2+ 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 Ca2+ waves observed during a beat-to-beat cycle. Modulation of these Ca2+ handling proteins by the ubiquitously expressed Ca2+/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 [Ca2+]i concentration. During the increase in [Ca2+]i Ca2+ bound calmodulin (Ca-CaM) binds to and activates CaMKII and CaN; thereby phosphorylating or dephosphorylating several important Ca2+ 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-CaM- CaMKII-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) CaMKII4B vs. 4C 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 Ca2+ 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-CaMKII- CaN 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.