Beta-adrenergic Response in Cardiac Hypertrophy/Failure

心脏肥大/衰竭中的β-肾上腺素能反应

• 批准号: 6823706
• 负责人: Meredith Bond
• 金额: $ 14.06万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6823706
• 关键词: beta adrenergic receptor; calcium flux; conformation; electrospray ionization mass spectrometry; enzyme activity; heart contraction; heart enlargement; heart failure; heart innervation; heart metabolism; human tissue; idiopathic dilated cardiomyopathy; laboratory rat; microfilaments; molecular pathology; phosphoprotein phosphatase; phosphorylation; protein kinase A; protein kinase C; protein protein interaction; spontaneous hypertensive rat; troponin

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.

描述（由申请人提供）：信号转导的改变 调节心脏中 Ca2+ 依赖性力的途径有助于 心力衰竭时收缩功能受损。这些功能变化是 可能是由 cAMP 依赖性蛋白磷酸化改变介导的 激酶 (PKA) 和蛋白激酶 C (PKC) 底物。主要PKA/PKC之一 心肌细胞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 不同 PKA 和 PKC 位点 TnI 磷酸化后，相互作用 TnI 与细丝的其他蛋白质之间 - 因此具有收缩性 功能 - 被改变。换句话说，TnI 及其磷酸化指纹 代表调节收缩状态途径中的关键控制点 作为传入 Ca2+ 信号的函数。我们已经证明 PKA 人类心力衰竭时 TnI 磷酸化降低 25%。这个结果 肌钙蛋白 C (TnC) 的 Ca2+ 亲和力增加，并可能有助于增强 肌丝 Ca2+ 敏感性，以及衰竭心脏的长时间松弛。在 相比之下，据报道，在衰竭的心脏中 PKC 会增加； PKC增加 TnI 上一个或多个位点的磷酸化会降低最大肌动球蛋白 (AM) ATP 酶活性，因此也可能导致心脏收缩受损 失败。然而，有关 PKC 活性升高对 TnI 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 确定 TnI 的磷酸化状态。在具体目标 1 中，我们将确定 衰竭人类心脏中 TnI 的完整磷酸化谱 扩张型心肌病 (DCM) 并将其与非衰竭心脏进行比较。 电喷雾电离质谱 (ESI/MS) 将用于定量 获得的 TnI 胰蛋白酶消化物中磷酸化残基的化学计量 从失败的心和未失败的心中，通过快速的一步隔离来摆脱困境 体内磷酸化状态。在具体目标 2 中，我们将 (a) 检查 PKC 和 PKA 联合变化导致的构象变化 衰竭心脏与非衰竭心脏中 TnI 的磷酸化。这将实现 通过测量 cTnI 中的荧光猝灭色氨酸残基， 选择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 重建。 (b) 功能性后果 通过测量 Ca2+ 来评估 TnI 磷酸化的改变 因衰竭和非衰竭而导致的带皮心脏小梁的依赖性力 心。具体目标 3 将检验以下假设：TnI 的活性靶向 磷酸酶在衰竭的心脏中发生改变。这些研究应该提供新的 有关 PKA 和 PKC 依赖性 TnI 变化的完整信息 人类心力衰竭中的磷酸化。结构和功能结果 这些变化加上磷酸酶活性改变的鉴定将导致 阐明导致心力衰竭功能下降的机制。