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: 6747571
• 负责人: Meredith Bond
• 金额: $ 27.37万
• 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 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6747571
• 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Ca 2+依赖力的途径有助于 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PKA和PKC位点TnI磷酸化后， 在TnI与细丝的其他蛋白质之间， 功能-改变。换句话说，TnI及其磷酸化指纹 代表调节收缩状态的途径中的关键控制点 作为incominb Ca 2+信号的函数。我们已经证明PKA 在人心力衰竭中，TnI的磷酸化降低了25%。这导致 肌钙蛋白C（TnC）的Ca 2+亲和力增加，并可能有助于增强 肌丝Ca 2+敏感性和衰竭心脏的延长舒张。在 相反，据报道，PKC在衰竭的心脏中增加;增加的PKC TnI上一个或多个位点的磷酸化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，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（PP 1）和/或PP 2A-也将 确定TnI的磷酸化状态。在具体目标1中，我们将确定 在衰竭的人类心脏中， 扩张性心脏病（DCM），并将其与非衰竭心脏进行比较。 将使用电喷雾电离质谱法（ESI/MS）定量 获得的TnI胰蛋白酶中磷酸化残基的化学计量 从失败和非失败的心脏，通过快速一步隔离， 体内磷酸化状态。在具体目标2中，我们将（a）检查 由PKC和PKA的组合变化引起的构象变化 衰竭与非衰竭心脏中TnI的磷酸化。完成这项工作的方法是 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成丙氨酸或丙氨酸，然后 用人心脏TnT和TnC重建。(b)功能性后果 将通过测量Ca 2+来评估TnI磷酸化的改变 剥离的心脏小梁在失效和非失效时的依赖力 心中具体目标3将检验以下假设： 磷酸酶在衰竭的心脏中改变。这些研究将提供新的 PKA和PKC依赖性TnI变化的完整补充信息 磷酸化在人类心力衰竭中的作用。结构和功能成果 这些变化加上磷酸酶活性改变的鉴定， 阐明心力衰竭功能下降的机制。

项目成果

Sustained apoptosis in human cardiac allografts despite histologic resolution of rejection.

尽管组织学上消除了排斥反应，但人心脏同种异体移植物仍持续凋亡。

• DOI: 10.1097/01.tp.0000084824.70320.da
• 发表时间: 2003
• 期刊: Transplantation.
• 作者: Masri,SofiaC;Yamani,MohamadH;Russell,MaryA;Ratliff,NormanB;Yang,Jiacheng;Almasan,Alex;Apperson-Hansen,Carolyn;Li,Jianbo;Starling,RandallC;McCarthy,Patrick;Young,JamesB;Bond,Meredith
• 通讯作者: Bond,Meredith

The intermediate filament protein, synemin, is an AKAP in the heart.

中间丝蛋白 Synemin 是心脏中的 AKAP。

• DOI: 10.1016/j.abb.2006.06.010
• 发表时间: 2006
• 期刊: Archives of biochemistry and biophysics
• 影响因子: 3.9
• 作者: Russell,MaryA;Lund,LindaM;Haber,Roy;McKeegan,Kathleen;Cianciola,Nicholas;Bond,Meredith
• 通讯作者: Bond,Meredith

Selectivity and regulation of A-kinase anchoring proteins in the heart. The role of autophosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase.

心脏中 A 激酶锚定蛋白的选择性和调节。

• DOI: 10.1074/jbc.m004212200
• 发表时间: 2000
• 期刊: The Journal of biological chemistry
• 作者: Zakhary,DR;Fink,MA;Ruehr,ML;Bond,M
• 通讯作者: Bond,M

Targeting of protein kinase A by muscle A kinase-anchoring protein (mAKAP) regulates phosphorylation and function of the skeletal muscle ryanodine receptor.

肌肉 A 激酶锚定蛋白 (mAKAP) 以蛋白激酶 A 为目标，调节骨骼肌兰尼碱受体的磷酸化和功能。

• DOI: 10.1074/jbc.m213279200
• 发表时间: 2003
• 期刊: The Journal of biological chemistry
• 作者: Ruehr,MaryL;Russell,MaryA;Ferguson,DonaldG;Bhat,Manju;Ma,Jianjie;Damron,DerekS;Scott,JohnD;Bond,Meredith
• 通讯作者: Bond,Meredith