Control of Cardiac Growth by Ca2+Dependent Phosphorylation of Histones

描述（由申请人提供）： 钙在许多生理过程的调节中起着核心作用。在心脏中，钙信号由钙/钙调蛋白依赖性蛋白激酶（CaMK）解码，所述钙/钙调蛋白依赖性蛋白激酶调节肌肉收缩、基因表达、细胞生长和凋亡。了解钙信号如何传递到细胞核来调节各种细胞活动仍然是人类生物学中的一个重要问题。我们最近有了一个新的发现，表明CaMKII偶联钙信号染色质重塑心肌细胞。初步结果表明，一个特定的核异构体的CaMKII选择性磷酸化组蛋白H3在体外和原代心肌细胞。通过激动剂处理激活CaMK活性，诱导肥大性生长，增加组蛋白H3磷酸化。相反，心肌细胞中细胞核CaMKII的特异性消除显著降低组蛋白H3磷酸化并抑制心脏生长。这些令人兴奋的新结果首次揭示了CaMK酶与染色质相关，并导致我们目前的假设，即CaMK依赖的组蛋白磷酸化控制心脏生长，并可能在心脏发病机制中发挥重要作用。本申请的目的是证明CaMK在心脏中的这种新功能。为此，我们建议测试以下假设来自我们的初步结果：1）证明CaMK介导的染色质重塑在心脏疾病中的功能作用。为此，我们将测试组蛋白H3磷酸化的变化是否在胎儿心脏基因中检测到，以及体外转录是否从突变组蛋白重建的染色质中改变。2)检测组蛋白H3磷酸化是否与其他心脏疾病有关，并通过研究组蛋白H3中被核CaMK II磷酸化的各种氨基酸，开始阐明CaMK介导的染色质重塑的生化机制。全面的实验方法将包括染色质免疫沉淀，操纵基因表达，使用重组腺病毒和RNA干扰原代心脏细胞，共聚焦显微镜，质谱和体外转录测定。这项研究可能会导致一个新的领域的研究在心脏领域，并可能在其他领域的CaMK发挥其功能的作用。最重要的是，它将导致潜在的新的治疗策略，以治疗心脏疾病，如心力衰竭。 公共卫生相关性： 钙/钙调蛋白依赖性蛋白激酶（CaMK）是存在于所有主要器官中的酶，并参与各种心脏疾病。我们有了一个新的发现，表明在心肌细胞核中富集的一种特定的CaMK亚型，将钙信号与染色质重塑偶联。本申请的目的是证明CaMK的这种新功能是导致心脏肥大和其他心脏疾病的病理事件的核心。因此，拟议的研究有可能在CaMK领域开辟新的途径，并可能为心脏病的治疗带来新的治疗干预。

DESCRIPTION (provided by applicant): Calcium is central in the regulation of many physiological processes. In the heart, calcium signals are decoded by calcium/calmodulin-dependent protein kinases (CaMKs) that regulate muscle contraction, gene expression, cell growth and apoptosis. Understanding how calcium signals are transmitted to the nucleus to modulate various cellular activities remains an important question in human biology. We have recently made a novel discovery showing that CaMKII couples calcium signals to chromatin remodeling in cardiac cells. Preliminary results show that a specific nuclear isoform of CaMKII selectively phosphorylates histone H3 in vitro and in primary cardiomyocytes. Activation of CaMK activity by agonist treatment, which induces hypertrophic growth, increases histone H3 phosphorylation. Conversely specific elimination of nuclear CaMKII in cardiac cells significantly reduces histone H3 phosphorylation and inhibits cardiac growth. These exciting new results reveal for the first time that CaMK enzymes are linked to chromatin and lead to our current hypothesis that CaMK- dependent phosphorylation of histones controls cardiac growth and may play an important role in heart pathogenesis. The goal of this application is to demonstrate this novel function of CaMK in the heart. For this, we propose to test the following hypotheses derived from our preliminary results: 1) To demonstrate the functional role of CaMK-mediated chromatin remodeling in cardiac diseases. For this we will test whether changes in histone H3 phosphorylation is detected at fetal cardiac genes and whether in vitro transcription is altered from chromatin reconstituted with mutant histones. 2) To test whether histone H3 phosphorylation is implicated in other cardiac disorders, and to begin to elucidate the biochemical mechanisms of CaMK- mediated chromatin remodeling by investigating the various amino acids that are phosphorylated by nuclear CaMKII in histone H3. The comprehensive experimental approaches will include chromatin immunoprecipitation, manipulation of gene expression using recombinant adenovirus and RNA interference in primary cardiac cells, confocal microscopy, mass spectrometry and in vitro transcription assays. This study may lead to a new area of investigation on CaMKs in the cardiac field and possibly in other fields where CaMKs exert their functional role. Most importantly, it will lead to potential new therapautic strategies to treate cardiac diseases such as heart failure. PUBLIC HEALTH RELEVANCE: Calcium/calmodulin-dependent protein kinases (CaMKs) are enzymes that are present in all major organs and are involved in a variety of cardiac disorders. We made a new discovery showing that a specific isoform of CaMK enriched in cardiac nuclei, couples calcium signals to chromatin remodeling. The goal of this application is to demonstrate that this new function of CaMK is central to pathological events leading to cardiac hypertrophy and to other cardiac diseases. Thus, the proposed study has potential to open new avenues in the CaMK field and may bring novel therapeutic intervention for the treatment of heart disease.