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

心力衰竭仍然是世界范围内死亡的主要原因。心肌疾病（心肌病）是心力衰竭发展的主要潜在原因，其中很大一部分是由于遗传原因。人类肌肉LIM蛋白（MLP）突变可导致心肌病。同样，在小鼠中，MLP的缺失也会导致扩张型心肌病和进行性心力衰竭。然而，尽管迄今为止多方努力，MLP功能的确切分子机制仍不明确。在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在几种心脏肥大小鼠模型以及体外拉伸心肌细胞中的差异调控。这些发现导致假设该microRNA可能在心肌疾病的发生或进展中发挥重要作用。有趣的是，microRNA-582定位于camp特异性3',5'-环磷酸二酯酶4D （PDE4D）基因的内含子中。PDE在包括心肌细胞在内的各种类型的细胞中控制camp信号，PDE4D的消融导致小鼠心脏肥厚的发生。我们可以证明pde4d -异构体PDE4D7在MLP敲除小鼠中与microRNA-582一起上调。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的转基因小鼠和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，我们现在计划对MiR-582-KO小鼠施加TAC（横断面主动脉收缩）手术的生物力学应力，TAC是一种广泛使用的模型，可诱导心脏肥厚，最终进展为心力衰竭。此外，我们的目标是将这些系与MLP KO小鼠杂交，以评估miR-582的过表达或敲除是否会调节MLP敲除表型。另一个重要的目标将是鉴定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Ago2-HITS-CLIP。最后，我们计划进一步分析miR-582的宿主基因PDE4D，特别是我们发现与miR-582共调控的PDE4D7亚型。已知PDE4D在MLP敲除小鼠心脏钙稳态的调节中起重要作用，而心肌钙稳态也在MLP敲除小鼠中失调。因此，我们计划进行钙瞬态测量，分析钙循环基因的表达，并确定PDE4D7的亚细胞定位。综上所述，我们希望我们的建议将有助于确定mlp介导的心肌病以及一般心力衰竭发病机制的新分子机制。

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.