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）负调节哺乳动物雷帕霉素靶蛋白（mTOR），mTOR是翻译机制和细胞生长的主要调节因子。最近，我们证明了由横向主动脉收缩（TAC）引起的进行性左心室（LV）肥大和心力衰竭（HF）在具有AMPK 12基因缺陷（AMPK 12-/-）的小鼠中加剧，并且这与增加的p-p70 S6 KThr 389（mTOR复合物1（mTORC 1）的下游靶点）相关。我们还发现，用AICAR或二甲双胍激活AMPK，或过表达组成型活性AMPK 12，都能显著减弱体外心肌细胞肥大和p-p70 S6 KThr 389。然而，TAC后AMPK 12缺失导致mTORC 1/p-p70 S6 k Thr 389信号通路激活增加的机制尚不清楚。DNA损伤诱导转录本4（DDIT 4）是一种新的应激反应基因，在几种肿瘤细胞系中负调控mTORC 1通路。虽然没有发表的研究检查DDIT 4在心脏是可用的，使用全球微阵列分析，我们发现AMPK 12-/-小鼠心肌DDIT 4的表达减少。这些发现表明，DDIT 4可以提供AMPK 12-/-引起mTORC 1激活的重要联系，从而在应激条件下加剧LV肥大。因此，提出研究以确定新型mTORC 1抑制剂DDIT 4是否有助于减轻TAC诱导的小鼠TAC后LV肥大和HF，以及AMPK 12促进心脏适应血流动力学过载的分子机制。我们的中心假设是DDIT 4通过减弱mTORC 1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DDIT 4调节mTORC 1信号传导。我们计划通过追求以下两个特定目标来测试我们的中心假设：i）确定DDIT 4对心肌mTORC 1信号传导、LV肥大和HF的总体影响。我们的工作假设是，DDIT 4基因缺陷（DDIT 4-/-）将放大激活的mTOR信号通路和左心室肥大时，心脏暴露于收缩超负荷; ii）确定AMPK减弱mTORC 1/p70 s6 k激活和心肌细胞肥大的分子机制。我们的工作假设是DDIT 4在AMPK减弱病理性LV肥大中起重要作用。该项目具有创新性，因为之前没有研究检查过DDIT 4对LV肥大和HF的影响。使用DDIT 4-/-小鼠与TAC组合将使我们能够在临床相关条件下破译DDIT 4在减弱心肌细胞肥大中的作用。该项目意义重大，因为获得的知识将导致更好地了解介导心室肥大和HF的分子机制，这可能为开发治疗这些疾病的特定干预措施提供基础。 公共卫生相关性：DNA损伤诱导转录本4（DDIT 4）是一种新的应激反应基因，在肿瘤细胞系中负调控mTOR通路。然而，DDIT 4对心室肥大和功能障碍的影响尚未研究。使用全局微阵列分析，我们最近发现AMPK 12 KO小鼠心肌DDIT 4表达减少，这一变化可能解释AMPK 12小鼠对慢性应激超负荷的反应中心肌mTOR信号转导和心室肥大的激活增强。提出研究以确定新型mTOR抑制剂DDIT 4是否有助于减轻心室肥大和心力衰竭的发展。

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.