Role of microRNA-582 in cardiac signal transduction, hypertrophy and heart failure

microRNA-582 在心脏信号转导、肥大和心力衰竭中的作用

• 批准号: 406416108
• 负责人: Professor Dr. Norbert Frey
• 金额: --
• 依托单位: Klinik für Kardiologie, Angiologie und Pulmologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/406416108?language=en
• 关键词: Role; microRNA; 582; cardiac; signal

Heart failure still a leading cause of death worldwide. Cardiac muscle diseases (cardiomyopathies) are a major underlying cause for the development of heart failure and a substantial fraction is due to genetic causes. Mutations in human Muscle LIM protein (MLP) can lead to cardiomyopathy. Likewise, in mice, loss of MLP also results in dilated cardiomyopathy and progressive heart failure. However, despite of multiple efforts so far, the exact molecular mechanism of MLP function is still elusive.In a microRNA microarray screen performed with MLP knockout mice versus wildtype mice we identified miR-582 to be highly upregulated in MLP knockout mice. For this microRNA no cardiac function is known so far. We also observed differential regulation of miR-582 in several mouse models of cardiac hypertrophy as well as in vitro in stretched cardiomyocytes. These findings lead to the hypothesis that this microRNA might play an important role during the development or progression of cardiac muscle diseases. Interestingly, microRNA-582 is localized in an intron of the cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D) gene. PDE’s control cAMP-signaling in various types of cells, including cardiomyocytes, and ablation of PDE4D leads to the development of cardiac hypertrophy in mice. We could show that the PDE4D-isoform PDE4D7 is upregulated along with microRNA-582 in MLP knockout mice.To further elucidate the function of miR-582 in the heart, we generated both transgenic mice that overexpress microRNA-582 specifically in the heart as well as a miR-582 knockout mouse line. As no cardiac phenotype for any of these mouse lines was observed at baseline, we now plan to subject MiR-582-KO mice to biomechanical stress with TAC (transverse aortic constriction) surgery, a widely used model to induce cardiac hypertrophy that eventually progresses to heart failure. Additionally we aim to crossbreed these lines to the MLP KO mice to assess if overexpression or knockout of miR-582 modulates the MLP knockout phenotype. Another important goal will be to identify relevant cardiac target genes for miR-582, which we plan to achieve by applying several methods, including Ago2-HITS-CLIP. Finally, we plan to further analyze the host gene of miR-582, PDE4D, and especially the isoform PDE4D7, which we found to be coregulated with miR-582. It is known that PDE4D plays an important role in the regulation of cardiac calcium homeostasis which is also dysregulated in MLP knockout mice. Therefore we plan to perform calcium transient measurements, to analyze the expression of calcium cycling genes and to identify the subcellular localization of PDE4D7. Taken together, we hope that our proposal will assist in the identification of new molecular mechanisms in MLP-mediated cardiomyopathy as well as general heart failure pathogenesis.

心力衰竭仍然是全球死亡的主要原因。心肌疾病（心肌病）是心力衰竭发展的主要潜在原因，很大一部分是由于遗传原因。人类肌肉LIM蛋白（MLP）的突变可导致心肌病。同样，在小鼠中，MLP的丧失也会导致扩张型心肌病和进行性心力衰竭。然而，尽管迄今为止做出了多种努力，MLP功能的确切分子机制仍然难以捉摸。在对MLP敲除小鼠与野生型小鼠进行的microRNA微阵列筛选中，我们发现miR-582在MLP敲除小鼠中高度上调。到目前为止，还不知道这种微小RNA的心脏功能。我们还观察到miR-582在几种心肌肥大小鼠模型中以及在体外拉伸心肌细胞中的差异调节。这些发现导致了这样的假设，即这种microRNA可能在心肌疾病的发展或进展过程中发挥重要作用。有趣的是，microRNA-582位于cAMP特异性3 '，5'-环磷酸二酯酶4D（PDE 4D）基因的内含子中。PDE控制包括心肌细胞在内的各种类型细胞中的cAMP信号传导，并且PDE 4D的消融导致小鼠心脏肥大的发展。我们可以证明，PDE 4 D亚型PDE 4 D 7在MLP敲除小鼠中与microRNA-582一起沿着上调。为了进一步阐明miR-582在心脏中的功能，我们产生了在心脏中特异性过表达microRNA-582的转基因小鼠以及miR-582敲除小鼠系。由于在基线时没有观察到任何这些小鼠系的心脏表型，我们现在计划用TAC（横向主动脉缩窄）手术使MiR-582-KO小鼠经受生物力学应力，TAC是一种广泛使用的诱导心脏肥大的模型，最终进展为心力衰竭。此外，我们的目标是将这些品系与MLP KO小鼠杂交，以评估miR-582的过表达或敲除是否调节MLP敲除表型。另一个重要的目标是确定miR-582的相关心脏靶基因，我们计划通过应用几种方法来实现，包括Ago 2-HITS-CLIP。最后，我们计划进一步分析miR-582、PDE 4D的宿主基因，特别是同工型PDE 4D 7，我们发现它与miR-582共调节。已知PDE 4D在调节心脏钙稳态中起重要作用，这在MLP敲除小鼠中也失调。因此，我们计划进行钙瞬变测量，分析钙循环基因的表达，并确定亚细胞定位的PDE 4D 7。总之，我们希望我们的建议将有助于识别新的分子机制，在MLP介导的心肌病以及一般的心力衰竭发病机制。