Phospholemman and Na-Pump Function in Cardiac Myocytes

心肌细胞中的磷酸化和钠泵功能

• 批准号: 7088862
• 负责人: Donald M Bers
• 金额: $ 36.25万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088862
• 关键词: cardiac myocytes; enzyme activity; fluorescence resonance energy transfer; glycosides; immunocytochemistry; laboratory mouse; laboratory rabbit; laboratory rat; phosphorylation; protein kinase A; protein kinase C; protein structure function; sarcolemma; sodium potassium exchanging ATPase

DESCRIPTION (provided by applicant): Na/K-ATPase (NKA) is the main Na extrusion pathway in cardiac myocytes and therefore is essential in [Na]i regulation. Na and Ca transport are tightly linked in heart via Na/Ca exchange, and the importance of Ca in heart makes understanding [Na]i regulation extremely important. Phospholemman (PLM), a member of FXYD gene family of proteins that associate with and modulate Na/K-ATPase activity. It is also a major target substrate for protein kinase A (PKA) and C (PKC) in heart during sympathetic nervous system activation (and b- and a-adrenergic activation). Here we will test the hypothesis that PLM regulates NKA similar to the way phospholamban regulates SR Ca-ATPase. Aim 1 will examine the roles of phosphorylation of PLM at Ser63 and Ser68 in mediating a- and b-adrenergic effects on NKA activity in intact myocytes (more broadly assessing PKA and PKC effects on PLM-NKA). For these studies we will measure [Na]i and NKA pump current in isolated myocytes from rabbits and mice (including PLM-knockout mice), and complementary measures of PLM phosphorylation. It has been suggested that different NKA isoforms may be localized differently in the sarcolemma and may also interact differently with PLM. Thus, Aim 2 will address the interaction between PLM and NKA and how it is affected by subcellular localization of NKA isoforms and PLM phosphorylation. This will include immunohistochemistry of NKA isoforms, separation of isoforms function by glycoside selectivity, assessment of fluorescence resonance energy transfer (FRET) between CFP-NKA and YFP-PLM and how this is modified by PKA and PKC. Cardiac myocyte Na regulation is perturbed in heart failure and this contributes to altered Ca regulation. In Aim 3 we will address how the adrenergic (or PKA/PKC) dependent modulation of NKA via PLM is altered in heart disease and how it contributes to altered myocyte function. This includes studies in our non-ischemic rabbit heart failure model and complementary work in a rat post myocardial infarction model. The measurements here will focus mainly on isolated myocyte NKA function (modulation by PKA/PKC) and PLM complementary biochemical/molecular measurements. This work will provide comprehensive new information regarding the roles of PLM in cardiac myocytes in normal and failing hearts.

描述（由申请方提供）：Na/K-ATP酶（NKA）是心肌细胞中的主要Na排出途径，因此在[Na]i调节中至关重要。Na和Ca的转运在心脏中通过Na/Ca交换紧密相连，Ca在心脏中的重要性使得理解[Na]i调节极其重要。Phospholemman（PLM）是与Na/K-ATP酶活性相关并调节Na/K-ATP酶活性的蛋白质FXYD基因家族的成员。在交感神经系统激活（以及B-和a-肾上腺素能激活）期间，它也是心脏中蛋白激酶A（PKA）和蛋白激酶C（PKC）的主要靶底物。在这里，我们将测试PLM调节NKA类似于受磷蛋白调节SR Ca-ATP酶的方式的假设。目的1将检查PLM在Ser 63和Ser 68处的磷酸化在介导a-和b-肾上腺素能对完整肌细胞中NKA活性的作用中的作用（更广泛地评估PKA和PKC对PLM-NKA的作用）。对于这些研究，我们将测量来自兔和小鼠（包括PLM敲除小鼠）的分离的肌细胞中的[Na]i和NKA泵电流，以及PLM磷酸化的补充测量。有人认为，不同的NKA亚型可能是本地化不同的肌膜，也可能与PLM不同的相互作用。因此，目标2将解决PLM和NKA之间的相互作用，以及它如何受到NKA亚型和PLM磷酸化的亚细胞定位的影响。这将包括NKA亚型的免疫组织化学，通过糖苷选择性分离亚型功能，评估CFP-NKA和YFP-PLM之间的荧光共振能量转移（FRET）以及PKA和PKC如何修饰。心力衰竭时心肌细胞Na调节受到干扰，这有助于改变Ca调节。在目标3中，我们将讨论如何肾上腺素能（或PKA/PKC）通过PLM的NKA依赖性调节在心脏病中改变，以及它如何有助于改变心肌细胞功能。这包括在我们的非缺血性兔心力衰竭模型中的研究和在大鼠心肌梗死后模型中的补充工作。这里的测量将主要集中在分离的肌细胞NKA功能（PKA/PKC调节）和PLM补充生化/分子测量。这项工作将提供有关PLM在正常和衰竭心脏的心肌细胞中的作用的全面的新信息。