Beta-adrenergic Response in Cardiac Hypertrophy/Failure

描述(申请人提供)：信号转导的改变 调节心脏内钙依赖性力量的途径有助于 心力衰竭时收缩功能受损。这些功能变化是 可能是通过改变cAMP依赖蛋白的磷酸化来调节的 激酶(PKA)和蛋白激酶C(PKC)底物。主要的PKA/PKC之一 心肌细胞中的底物是细丝调节蛋白， 肌钙蛋白I(TnI)。由于TnI分子的构象变化 在不同的PKA和PKC位点TnI被磷酸化时，相互作用 在TnI与细丝的其他蛋白质之间-因此是收缩的 功能-被更改。也就是说，TnI及其磷酸化指纹图谱 代表调节收缩状态的通路中的一个关键控制点 作为内源性钙信号的函数。我们已经证明了PKA 在人类心力衰竭患者中，TnI的磷酸化水平降低了25%。这将导致 增加肌钙蛋白C(TNC)的钙亲和力，并可能有助于增强 肌丝对钙离子的敏感性，以及衰竭心脏的延长松弛。在……里面 相比之下，据报道，心力衰竭时PKC增加；PKC增加 TnI上一个或多个位点的磷酸化降低最大肌动球蛋白(AM) ATPase活性，因此也可能导致心脏收缩受损 失败了。然而，关于PKC活性升高对TNI的影响的报道 磷酸化和心脏功能是相互矛盾的。最后，活动 蛋白磷酸酶-蛋白磷酸酶1(PP1)和/或PP2A-也将 测定TnI的磷酸化状态。在具体目标1中，我们将确定 TnI在心脏衰竭中的完整磷酸化谱 扩张型心脏病(DCM)，并将其与非心力衰竭心脏进行比较。 将使用电喷雾电离质谱仪(ESI/MS)进行定量 TnI胰酶消化液中磷酸化残基的化学计量 从失败和不失败的心脏，通过快速的一步隔离来绊倒 体内的磷酸化状态。在具体目标2中，我们将(A)审查 PKC和PKA联合变化引起的构象变化 衰竭心脏与非衰竭心脏中TnI的磷酸化。这将会实现的 通过测定cTnI中的荧光猝灭色氨酸残基， 选定的丝氨酸和苏氨酸突变为天冬氨酸或丙氨酸，然后 用人心脏TnT和TnC重组。(B)职能后果 将通过测量钙离子来评估改变的TnI磷酸化 心力衰竭与非心力衰竭时皮肤小梁的依赖性 红心。具体目标3将检验如下假设：TnI的活动具有靶向性 在衰竭的心脏中，磷酸酶会发生变化。这些研究应该提供新的 关于PKA和依赖PKC的TNI变化的完整补充信息 人类心力衰竭中的磷酸化。的结构和功能结果 这些变化加上改变的磷酸酶活性的鉴定将会减少 阐明导致心力衰竭功能衰退的机制。

DESCRIPTION (provided by applicant): Alterations in the signal transduction pathways which regulate Ca2+ dependent force in the heart contribute to the impaired contractile function in heart failure. These functional changes are likely to be mediated by altered phosphorylation of cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) substrates. One of the major PKA/PKC substrates in the cardiac muscle cell is the thin filament regulatory protein, troponin I (TnI). As a result of conformational changes in the TnI molecular upon phosphorylation of the different PKA and PKC sites TnI, interactions between TnI with other proteins of the thin filament - and thus contractile function - are altered. In other words, TnI and its phosphorylation fingerprint represent a critical control point in the pathway regulating contractile state as a function of the incominb Ca2+ signal. We have shown that PKA phosphorylation of TnI is decreased by 25% in human heart failure. This results in increased Ca2+ affinity of troponin C (TnC), and may contribute to enhanced myofilament Ca2+ sensitivity, and prolonged relaxation of failing hearts. In contrast, PKC is reportedly increased in failing hearts; increased PKC phosphorylation of one or more sites on TnI decreases maximal actomyosin (AM) ATPase activity and thus could also contribute to impaired contraction in heart failure. However, reports on the effect of elevated PKC activity on TnI phosphorylation and cardiac function are conflicting. Finally, activity of protein phosphatases - protein phosphatase 1 (PP1) and/or PP2A - will also determine the phosphorylation state of TnI. In Specific Aim 1, we will identify the complete phosphorylation profile of TnI in failing human hearts with dilated cardiomopathy (DCM) and compare this with non-failing hearts. Electrospray ionization mass spectrometry (ESI/MS) will be used to quantify stoichiometry of the phosphorylated residues in tryptic digests of TnI obtained from failing and non-failing hearts, by a rapid one-step isolation to trop the in vivo phosphorylation state. In Specific Aim 2, we will (a) examine conformational changes that result from the combined changes of PKC and PKA phosphorylation of TnI in failing vs non-failing hearts. This will be achieved by measurement of fluorescence quenching tryptophan residues in cTnI, with selected serines and threonine mutated to aspartates or alanines, then reconstituted with human cardiac TnT and TnC. (b) The functional consequences of altered TnI phosphorylation will be assessed by measurement of Ca2+ dependent force in skinned cardiac trabeculae from failing and non-failing hearts. Specific Aim 3 will test the hypothesis that activity of TnI targeted phosphatases is altered in failing hearts. These studies should provide new information on the complete complement of changes in PKA and PKC-dependent TnI phosphorylation in human heart failure. Structural and functional outcomes of these changes plus identification of the altered phosphatase activity will shed light on mechanisms responsible for the functional decline in heart failure.