Regulation of the novel mTOR suppressor DDIT4 in the failing heart

描述（由申请人提供）：amp活化蛋白激酶（AMPK）负调控哺乳动物雷帕霉素靶蛋白（mTOR），雷帕霉素靶蛋白是翻译机制和细胞生长的主要调节剂。最近，我们证明了在AMPK12基因缺失（AMPK12-/-）的小鼠中，由横切主动脉收缩（TAC）引起的进行性左心室（LV）肥厚和心力衰竭（HF）会加剧，这与mTOR复合物1 （mTORC1）的下游靶点p- p70S6KThr389的增加有关。我们还发现，用AICAR或二甲双胍激活AMPK，或过表达组成型活性AMPK12，都能显著减轻体外心肌细胞肥大和p-p70S6KThr389。然而，AMPK12缺失导致TAC后mTORC1/p-p70S6k Thr389信号通路激活增加的机制尚不清楚。dna损伤诱导转录本4 （DDIT4）是一种新的应激反应基因，在几种肿瘤来源的细胞系中负调控mTORC1通路。虽然没有发表的研究检查心脏中的DDIT4，但使用全局微阵列分析，我们发现AMPK12-/-小鼠心肌中DDIT4的表达降低。这些发现表明，DDIT4可能是AMPK12-/-引起mTORC1激活的重要环节，从而在应激条件下加剧左室肥大。因此，研究人员提出确定新的mTORC1抑制因子DDIT4是否有助于减轻TAC诱导的小鼠TAC后左室肥大和HF，以及AMPK12促进心脏适应血流动力学超载的分子机制。我们的中心假设是，DDIT4通过减弱mTORC1信号通路来减弱慢性tac诱导的心肌细胞肥大。我们还假设AMPK至少部分通过DDIT4调节mTORC1信号。我们计划通过以下两个具体目标来检验我们的中心假设：i)确定DDIT4对心肌mTORC1信号传导、左室肥厚和心衰的总体影响。我们的工作假设是，DDIT4基因缺陷（DDIT4-/-）将放大mTOR信号通路的激活和左室肥大，当心脏暴露于收缩负荷时发生；ii)确定AMPK抑制mTORC1/p70s6k活化和心肌细胞肥厚的分子机制。我们的工作假设是DDIT4在AMPK减轻病理性左室肥大中起重要作用。该项目具有创新意义，因为之前没有研究检测过DDIT4对左室肥厚和心衰的影响。使用DDIT4-/-小鼠联合TAC将使我们能够在临床相关条件下破译DDIT4在减轻心肌细胞肥大中的作用。该项目具有重要意义，因为所获得的知识将使我们更好地了解心室肥厚和心衰的分子机制，这可能为开发治疗这些疾病的特异性干预措施提供基础。

DESCRIPTION (provided by applicant): AMP-activated protein kinase (AMPK) negatively regulates mammalian target of rapamycin (mTOR), the master regulator of translational machinery and cell growth. Recently, we demonstrated that the progressive left ventricular (LV) hypertrophy and heart failure (HF) caused by transverse aortic constriction (TAC) is exacerbated in mice with AMPK12 gene deficiency (AMPK12-/-), and this was associated with increased p- p70S6KThr389, a downstream target of mTOR complex 1 (mTORC1). We also found that activation of AMPK with AICAR or metformin, or overexpression of constitutively active AMPK12, all significantly attenuated cardiac myocyte hypertrophy and p-p70S6KThr389 in vitro. However, the mechanism by which loss of AMPK12 causes increased activation of the mTORC1/p-p70S6k Thr389 signaling pathway after TAC is not clear. DNA-damage-inducible transcript 4 (DDIT4) is a novel stress-responsive gene that negatively regulates the mTORC1 pathway in several tumor-derived cell lines. Although no published studies examining DDIT4 in the heart are available, using global microarray profiling we found that AMPK12-/- mice have decreased expression of myocardial DDIT4. These findings suggest that DDIT4 could provide the essential link by which AMPK12-/- causes activation of the mTORC1 and thereby exacerbates LV hypertrophy during stress conditions. Consequently, studies are proposed to determine whether the novel mTORC1 suppressor DDIT4 is instrumental in attenuating TAC-induced LV hypertrophy and HF in mice after TAC, and the molecular mechanisms by which AMPK12 facilitates adaptation of the heart to hemodynamic overload. Our central hypothesis is that DDIT4 attenuates chronic TAC-induced cardiac myocyte hypertrophy by attenuating the mTORC1 signaling pathway. We also hypothesize that AMPK regulates mTORC1 signaling at least partially through DDIT4. We plan to test our central hypothesis by pursuing the following two Specific Aims: i) Identify the overall impact of DDIT4 on myocardial mTORC1 signaling, LV hypertrophy and HF. Our working hypothesis is that DDIT4 gene deficiency (DDIT4-/-) will amplify activation of the mTOR signaling pathway and LV hypertrophy that occurs when hearts are exposed to systolic overload; ii) Determine the molecular mechanism by which AMPK attenuates mTORC1/p70s6k activation and cardiac myocyte hypertrophy. Our working hypothesis is that DDIT4 plays an essential role for AMPK to attenuate pathological LV hypertrophy. The project is innovative as no previous studies have examined the influence of DDIT4 on LV hypertrophy and HF. Using DDIT4-/- mice combined with TAC will allow us to decipher the role of DDIT4 in attenuating cardiac myocyte hypertrophy under clinically relevant conditions. This project is significant as the knowledge obtained will lead to a better understanding of the molecular mechanisms mediating ventricular hypertrophy and HF, which may provide the basis for developing specific interventions to treat these diseases. PUBLIC HEALTH RELEVANCE: DNA-damage-inducible transcript 4 (DDIT4) is a novel stress-responsive gene that negatively regulate the mTOR pathway in tumor cell lines. However, the effect of DDIT4 on ventricular hypertrophy and dysfunction has not been studied. Using global microarray profiling we recently found that AMPK12 KO mice have decreased expression of myocardial DDIT4, a change may explain the enhanced activation of myocardial mTOR signaling and ventricular hypertrophy in AMPK12 mice in response to chronic stress overload. Studies are proposed to determine whether the novel mTOR suppressor DDIT4 is instrumental in attenuating the development of ventricular hypertrophy and heart failure.