Mechanisms of Adenylyl Cyclase Effects in the Heart

腺苷酸环化酶对心脏的作用机制

基本信息

批准号：
8426173
负责人：
H. Kirk Hammond
金额：
$ 33.36万
依托单位：
VETERANS MEDICAL RESEARCH FDN/SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-09-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8426173
关键词：
Action Potentials Acute Adenosine Triphosphate Adenovirus Vector Adenylate Cyclase Adrenergic Receptor Adverse effects Alanine Amino Acid Substitution Animal Model Apoptosis Aspartic Acid Award Biodistribution Biological Biological Assay Cardiac Cardiac Myocytes Cardiotoxicity Catalytic Domain Cell membrane Cells Chimeric Proteins Clinical Coronary Occlusions Coupling Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytoplasm Cytoplasmic Tail DNA Data Disease Effector Cell Engineering Event Family suidae Foundations Funding G-Protein-Coupled Receptors Gene Transfer Generations Heart Heart failure Hypertrophy In Vitro Laboratories Left Left ventricular structure Length Logic Measures Methods Modeling Myocardial National Heart, Lung, and Blood Institute Nuclear Envelope Nucleic Acid Regulatory Sequences Organ Outcome Parents Patients Pattern Pharmaceutical Preparations Phosphorylation Plasmids Positioning Attribute Pre-Clinical Model Production Property Protein Isoforms Proteins Protocols documentation Recombinants Research Design Reticulum Route SERCA2a Safety Series Signal Transduction Structure Testing Therapeutic Time Transgenes Transgenic Mice Transgenic Organisms Translating Translations Transmembrane Domain Troponin I Ventricular Virus adeno-associated viral vector alanylaspartic acid base clinical application clinically relevant design efficacy testing expression vector gene transfer vector heart function improved improved functioning in vivo inhibitor/antagonist lipofection mortality mutant phospholamban pre-clinical protein protein interaction public health relevance research study safety testing therapeutic transgene transgene expression vector

项目摘要

DESCRIPTION (provided by applicant): Data from the initial funding period of the current award indicate that increased cardiac AC type 6 (AC6), a dominant AC isoform expressed in mammalian cardiac myocytes, has protean beneficial effects on the failing left ventricle (LV). These effects, so consistent in a variety of species and pathophysiological models, must be reconciled with the dire consequences on the heart of 2-adrenergic receptor stimulation and elevations in intracellular cAMP. Logic would dictate that either: a) cAMP is not bad for the heart after all-that it is something else which leads to poor outcomes when cAMP levels are increased in the failing heart; or b) increased AC6 has beneficial cardiac effects independent of cAMP, which counterbalance its expected deleterious effects. Using pharmacological inhibitors and other approaches, our data suggest that many of the beneficial effects of increased cardiac AC6 expression do not require increased cAMP generation. Because of the inherent limitations of studies using pharmacological inhibition, we generated a catalytically inactive AC6 mutant (AC6mut) molecule by substitution of Ala for Asp at position 426 in the catalytic core. This AC6mut does not generate cAMP, but retains the cellular distribution pattern and favorable signaling effects associated with AC6. These data indicate that the beneficial effects of AC6, at least in part, are independent of cAMP. We propose now to conduct a series of experiments, both in vitro and in vivo, to establish unassailable mechanisms for the beneficial effects. The ideal inotrope would increase cardiac contractile function in the absence of 2AR stimulation or cAMP generation, improve Ca2+ handling, reduce adverse remodeling and apoptosis, and have favorable electrophysiological properties. With the exception of its effects on cAMP generation, increased cardiac AC6 expression accomplishes many of these features, and the FDA has approved an NHLBI-funded trial of AC6 gene transfer in patients with severe CHF (ClinicalTrials.gov NCT00787059). However, there are four reasons to conduct studies of the cAMP-incompetent AC6 mutant: 1) to obtain definitive mechanistic data on whether AC6's beneficial cardiac effects are unrelated to cAMP generation; 2) to conduct structure-activity studies of the AC6mut molecule by generating a variety of AC6mut fragment molecules and to determine the effects of subcellular targeting of the AC6mut fragments on Ca2+ handling and Akt activation; 3) to determine the cardiac effects of AC6mut in vivo and 4) to generate a suitable long term regulated expression vector encoding the optimal AC6mut fragment, and conduct preclinical gene transfer studies to determine its efficacy and safety in improving function of the failing heart. The studies are designed to uncover mechanisms, but also to have clinical relevance. For example, in clinical heart failure, agents that increase cardiac contractile function by increasing cAMP levels have failed to prolong survival. The AC6mut, based on preliminary data, preserves the beneficial effects seen with AC6, but does so in the absence of increased cAMP production, thus fulfilling features of a potentially ideal therapeutic inotrope.

描述（由申请人提供）：当前奖励的初始资金期间的数据表明，在哺乳动物心肌细胞中表达的主要AC同工型增加了心脏AC型6（AC6），对失败的左心室（LV）具有蛋白质的有益作用。这些作用在多种物种和病理生理模型中如此一致，必须与对2-肾上腺素能受体刺激和细胞内cAMP升高的心脏的可怕后果进行调和。逻辑要么决定：a）在全部情况下，训练营对心脏不利 - 当营地级别增加心脏时，这是其他事情会导致不良的结果；或b）增加的AC6具有独立于CARP的有益心脏影响，这抵消了其预期的有害影响。使用药理抑制剂和其他方法，我们的数据表明，增加心AC6表达的许多有益作用不需要增加营地产生。由于使用药理抑制的研究固有局限性，我们通过将ALA取代ALA代替ASP在催化核心中产生了催化无效的AC6突变体（AC6MUT）分子。该AC6MUT不会产生cAMP，而是保留与AC6相关的细胞分布模式和有利的信号效应。这些数据表明，至少部分地，AC6的有益效应与CAMP无关。我们现在建议在体外和体内进行一系列实验，以建立对有益作用的无懈可击的机制。理想的唯一性将在没有2AR刺激或营地产生的情况下提高心脏收缩功能，改善CA2+处理，减少不良重塑和凋亡，并具有有利的电生理特性。除了其对营地生成的影响外，心脏AC6表达的增加还可以实现许多特征，而FDA批准了严重CHF患者的AC6基因转移的NHLBI资助的试验（ClinicalTrials.gov NCT00787059）。但是，有四个原因是对cAMP不足的AC6突变体进行研究：1）获得有关AC6的有益心脏影响是否与cAMP的产生无关的确定机械数据； 2）通过产生各种AC6MUT片段分子，并确定AC6MUT片段对Ca2+处理和Akt激活的亚细胞靶向的影响，对AC6MUT分子进行结构活性研究； 3）确定体内AC6MUT的心脏效应和4）产生编码最佳AC6MUT片段的合适的长期调节表达矢量，并进行临床前基因转移研究，以确定其在改善失败心脏功能方面的功效和安全性。这些研究旨在发现机制，但也具有临床相关性。例如，在临床心力衰竭中，通过提高营地水平增加心脏收缩功能的药物无法延长生存率。基于初步数据的AC6MUT保留了AC6看到的有益效果，但在没有增加的营地生产的情况下，因此可以实现潜在理想的治疗性障碍的特征。