ANP Modulates Cardiac Remodeling via Cardiac Fibroblasts

描述(由申请人提供)：心钠素(ANP)是胎儿基因程序的一个众所周知的组成部分，在应激条件下在成人心脏中过度表达。然而，心钠素在应激/肥厚心脏中的作用却鲜有人关注。目前的应用将测试这一假设，即ANP具有直接的抗生长和抗纤维化作用，通过阻断它们的信号级联，对抗转化生长因子(TGF)-β和血管紧张素II(AngII)的促纤维化作用，这两种物质已知在应激条件下在心脏中过度表达。我们已经证明，与非转基因(NTG)对照组相比，ANP基因纯合子突变(NPPA-/-)的小鼠在基础状态下表现出心脏增大和过度的心脏重构/纤维化，以及对横动脉缩窄(TAC)诱导的压力超负荷应激的早期过渡到衰竭。初步研究显示，服用TAG的NPPA-/-小鼠心脏肌成纤维细胞转化和细胞外基质(ECM)分子的表达显著增加。我们还证明，在表达可诱导的转化生长因子-β受体II型基因(DriTGFbRIl)的显性负突变(DriTGFbRIl)的小鼠中，TAC诱导的心脏重构和纤维化被消除，因此对转化生长因子-β信号没有反应。最近，我们在小鼠心脏成纤维细胞(CFs)中进行了令人兴奋的初步观察，即ANP信号抑制了转化生长因子-β诱导的磷酸化Smads的核移位，首次明确了ANP信号可能通过其保护心脏重构/纤维化和血流动力学应激反应失败的精确分子机制。本研究的目的是：1)建立静息状态和压力超负荷应激后不同阶段(急性应激、早期代偿纤维化和重塑、晚期失代偿/向衰竭过渡阶段)NTG小鼠心脏NPPA-/-、DnTGFbRII的表型，并控制NTG小鼠心脏的表型。这一目标将提供第一个严格的体内测试，以了解转化生长因子-β信号在介导压力超负荷诱导的左室纤维化/重塑中的作用，以及心钠素在调节这些过程中的作用。2)以体外培养的CFs为模型，探讨促纤维化/生长因子刺激的CFs增殖转化和细胞外基质表达的细胞内信号机制。这一目的将为ANP信号具有负性，而转化生长因子-β(和/或AngII)信号对这些过程具有正刺激作用这一假说提供严格的检验。ANP和/或其信号通路的组成部分将被用来定义ANP级联系统截获促纤维化/生长因子信号的特定部位(S)，从而抑制CFs的表型转化和细胞外基质的产生。这些机制研究将为心功能不全/衰竭患者的治疗干预提供更合理的基础，他们中的许多人都降低了ANP水平。

DESCRIPTION (provided by applicant): Atrial natriuretic peptide (ANP) is a well known component of the fetal gene program that is overexpressed in adult heart under stress conditions. Yet little attention has been devoted to the function of ANP in the stressed/hypertrophic heart. The current application will test the hypothesis that ANP has direct anti-growth and anti-fibrogenic effects that oppose the pro-fibrogenic actions of transforming growth factor (TGF)-beta and angiotensin II (ANGII), which are known to be overexpressed in heart under stress conditions, by interrupting their signaling cascades. We have shown that, compared to nontransgenic (NTG) controls, mice with homozygous disruption of the ANP gene (Nppa-/-) exhibit cardiac enlargement at baseline and exaggerated cardiac remodeling/fibrosis, as well as an early transition to failure, in response to transverse aortic constriction (TAC)-induced pressure overload stress. Preliminary studies revealed remarkable increases in myofibroblast transformation and expression of extracellular matrix (ECM) molecules in hearts of Nppa-/- mice subjected to TAG. We have also demonstrated that TAC-induced cardiac remodeling and fibrosis are abolished in mice that express an inducible dominant negative mutation of the TGF-beta receptor type II gene (DriTGFbRIl), and thus do not respond to TGF-beta signaling. Most recently, we have made the exciting preliminary observation that ANP signaling inhibits TGF-beta-induced nuclear translocation of phosphorylated-SmadS in mouse cardiac fibroblasts (CFs), defining for the first time a precise molecular mechanism by which ANP signaling may protect against cardiac remodeling/fibrosis and failure in response to hemodynamic stress. The Aims of this proposal are: 1) To establish the phenotypes of Nppa-/-, DnTGFbRII and control NTG mouse hearts under resting conditions and at various phases (acute stress, early compensatory stage of fibrosis and remodeling, and late decompensate/transition stage to failure) during pressure overload stress following TAG. This Aim will provide the first rigorous in vivo test of the role of TGF-beta signaling in mediating pressure overload-induced LV fibrosis/remodeling and of ANP in modulating these processes. 2) Using isolated CFs as an in vitro model, to define the intracellular signaling mechanisms leading to pro-fibrogenic/growth factor-stimulated CF proliferation/transformation and ECM expression. This Aim will provide a rigorous test of the hypothesis that ANP signaling has negative, and TGF-beta (and/or ANGII) signaling has positive stimulatory effects on these processes. ANP and/or components of its signaling pathway will be used to define the specific site(s) at which the ANP cascade intercepts pro-fibrogenic/growth factor signaling, thus inhibiting phenotypic transformation of CFs and ECM production. These mechanistic studies will provide a more rational basis for therapeutic intervention in patients with cardiac dysfunction/failure, many of whom have decreased levels of ANP.