Phospholemman and Na-Pump Function in Cardiac Myocytes

描述(申请人提供)：Na/K-ATPase(NKA)是心肌细胞中主要的钠排出途径，因此在[Na]i调节中是必不可少的。钠和钙的转运在心脏中通过钠/钙交换紧密相连，而钙在心脏中的重要性使得了解[Na]i的调节非常重要。磷脂酶原(PLM)是FXYD基因家族的一员，与Na/K-ATPase活性相关并参与调节。在交感神经系统激活(以及b-和a-肾上腺素能激活)过程中，它也是心脏中蛋白激酶A(PKA)和C(PKC)的主要靶底物。在这里，我们将检验PLM调节NKA的假设，类似于磷蛋白调节SR Ca-ATPase的方式。目的1研究PLM Ser63和Ser68的磷酸化在介导a-和b-肾上腺素能对完整心肌细胞NKA活性的影响中的作用(更广泛地评估PKA和PKC对PLM-NKA的影响)。在这些研究中，我们将测量兔和小鼠(包括PLM基因敲除小鼠)分离的心肌细胞的[Na]i和NKA泵电流，以及PLM磷酸化的补充措施。有人认为，不同的NKA亚型在肌膜中的定位不同，与PLM的相互作用也可能不同。因此，目标2将解决PLM和NKA之间的相互作用，以及NKA亚细胞定位和PLM磷酸化如何影响它。这将包括NKA异构体的免疫组织化学，通过糖苷选择性分离异构体的功能，评估CFP-NKA和YFP-PLM之间的荧光共振能量转移(FRET)，以及PKA和PKC如何改变这一点。心力衰竭时，心肌细胞钠调节受到干扰，从而导致钙调节改变。在目标3中，我们将阐述依赖肾上腺素(或PKA/PKC)的NKA通过PLM的调制在心脏病中是如何改变的，以及它是如何促进心肌细胞功能改变的。这包括对我们的非缺血性兔心力衰竭模型的研究，以及对大鼠心肌梗死后模型的补充工作。这里的测量将主要集中在分离的心肌细胞NKA功能(由PKA/PKC调节)和PLM互补的生化/分子测量。这项工作将提供关于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。

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.