We have shown that atrial natriuretic peptide (ANP) modulates the development of pressure overload-?induced cardiac hypertrophy; and ANP， via the cGMP-protein kinase G (PKG) signaling， has direct anti-fibrogenic actions on heart by modulating expression of extracellular matrix (ECM) molecules in cardiac fibroblasts (CFs). Transforming growth factor (TGF)-?1， via the Smad signaling， stimulates ECM expression and fibrosis in heart under stress conditions.  The current study tests the specific hypothesis that activation of ANP-cGMP-PKG signaling inhibits TGF-?1-induced ECM expression in CFs and defined the specific site(s) at which the molecular merging of signaling pathways occurs.  Left ventricular (LV) hypertrophy and fibrosis， collagen deposition and myofibroblast (MF) transformation of CFs in response to pressure overload by transverse aortic constriction (TAC) were exaggerated in ANP null mice compared to wild type control mice.  Pretreatment with ANP and 8-?Br-cGMP (a cGMP analog) for 30 min inhibited TGF-?1-?induced MF transformation， proliferation， collagen synthesis and plasminogen activator inhibitor 1 (PAI-1) expression in CFs isolated from wild type mice.  Following pretreatment with cGMP， TGF-?1 induced phosphorylation of Smad3， but the resultant pSmad3 could not be translocated to the nucleus.  The effects of ANP and cGMP were blocked by pretreating CFs with KT5823 (a PKG inhibitor)， indicating that the effects of ANP and cGMP were mediated via PKG activation.  pSmad3 that had been phosphorylated with recombinant PKG-1? was analyzed by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ion trap tandem mass spectrometry (MS/MS).  The analysis revealed phosphorylation of Ser309 and Thr388 residues， sites distinct from the C-terminal Ser423/425 residues that are phosphorylated by TGF-? receptor kinase and are critical for the nuclear translocation and down-stream signaling of pSmad3.  These results suggest that phosphorylation of Smad3 by PKG is a potential molecular mechanism by which activation of ANP-cGMP-PKG signaling disrupts TGF-?1-induced nuclear translocation of pSmad3 and downstream events， including MF transformation， proliferation and expression of ECM molecules in CFs.  We postulate that this process contributes to the anti-fibrogenic effects of the natriuretic peptide in the pathogenesis of cardiac fibrosis and remodeling under stress conditions. 


This study tested the hypothesis that activation of atrial natriuretic peptide (ANP)/cGMP/protein kinase G (PKG) signaling inhibits transforming growth factor (TGF)-β1-induced extracellular matrix (ECM) expression in cardiac fibroblasts (CFs) and defined the specific site(s) at which this molecular merging of signaling pathways occurs.  Left ventricular (LV) hypertrophy and fibrosis， collagen deposition and myofibroblast (MF) transformation of CFs in response to pressure overload by transverse aortic constriction (TAC) were exaggerated in ANP null mice compared to wild type controls.  ANP and cGMP inhibited TGF-?1-?induced MF transformation， proliferation， collagen synthesis and plasminogen activator inhibitor 1 (PAI-1) expression in CFs isolated from wild type mice.  Following pretreatment with cGMP， TGF-?1 induced phosphorylation of Smad3， but the resultant pSmad3 could not be translocated to the nucleus.  pSmad3 that had been phosphorylated with recombinant PKG-1? was analyzed by use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and ion trap tandem mass spectrometry (MS/MS).  The analysis revealed phosphorylation of Ser309 and Thr388 residues， sites distinct from the C-terminal Ser423/425 residues that are phosphorylated by TGF-? receptor kinase and are critical for the nuclear translocation and down-stream signaling of pSmad3.  These results suggest that phosphorylation of Smad3 by PKG is a potential molecular mechanism by which activation of ANP/cGMP/PKG signaling disrupts TGF-?1-induced nuclear translocation of pSmad3 and downstream events， including MF transformation， proliferation and expression of ECM molecules in CFs.  We postulate that this process contributes to the anti-fibrogenic effects of the natriuretic peptide in the heart.

（中文对照）
压力超负荷性心肌肥厚与重构的机制研究
心房利钠肽（ANP）在压力超负荷性心肌肥厚的发生中起着调节作用，且通过cGMP- PKG信号途径调节心肌成纤维细胞（CFs）ECM分子的表达，对心脏有直接的抗纤维生成作用。在压力条件下，TGF-?1通过Smad信号传导途径刺激ECM表达和纤维化。本研究旨在检验下述假说：ANP-cGMP-PKG信号途径的激活抑制CFs中TGF-?1诱导的ECM表达，并定位信号传导途径中分子合并发生位点。在ANP敲除鼠中，主动脉缩窄（TAC）引起的压力超负荷性左室肥厚和纤维化、胶原沉着和肌纤维母细胞（MF）转化均较对照鼠严重。以ANP和cGMP类似物预治疗30分钟抑制了野生型小鼠CFs TGF-?1诱导的MF转化、增殖、胶原合成和纤溶酶原激活剂抑制因子1（PAI-1）的表达。cGMP预治疗后，TGF-?1诱导了Smad3磷酸化，但pSmad3不能转位至细胞核。ANP和cGMP的效应被CFs的KT5823（PKG抑制剂）预处理阻断，提示ANP和cGMP的效应是通过PKG激活介导的。对pSmad3进行质谱分析显示，磷酸化Ser309和Thr388残基位点不同于被TGF-?受体激酶磷酸化的C端ser423/425残基，这对pSmad3的细胞核转运和下游信号转导至关重要。结果提示，在ANP-cGMP-PKG信号途径激活从而中断TGF-?1诱导的pSmad3核转位及下游事件（包括CFs中MF转化、增殖、核ECM分子表达）中，PKG磷酸化Smad3是一种可能的分子机制。该过程可能是利钠肽在压力所致心脏纤维化和重构发病中发挥抗纤维化作用的机制。