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）：当前奖项初始资助期间的数据表明，增加的心脏 AC 6 型 (AC6)（一种在哺乳动物心肌细胞中表达的主要 AC 亚型）对衰竭的左心室 (LV) 具有千变万化的有益作用。这些效应在多种物种和病理生理学模型中如此一致，必须与 2-肾上腺素受体刺激和细胞内 cAMP 升高对心脏造成的可怕后果相协调。从逻辑上讲，要么： a) cAMP 毕竟对心脏来说并不是坏事——当衰竭心脏中 cAMP 水平升高时，它是其他东西，会导致不良结果； b) AC6 增加具有独立于 cAMP 的有益心脏作用，这抵消了其预期的有害作用。使用药物抑制剂和其他方法，我们的数据表明，增加心脏 AC6 表达的许多有益作用不需要增加 cAMP 的生成。由于药理抑制研究的固有局限性，我们通过在催化核心的 426 位用 Ala 取代 Asp 生成了催化失活的 AC6 突变体 (AC6mut) 分子。该 AC6mut 不产生 cAMP，但保留了与 AC6 相关的细胞分布模式和有利的信号传导效应。这些数据表明 AC6 的有益作用至少部分独立于 cAMP。我们现在建议进行一系列体外和体内实验，以建立无懈可击的有益作用机制。理想的正性肌力药将在没有 2AR 刺激或 cAMP 生成的情况下增强心脏收缩功能，改善 Ca2+ 处理，减少不良重塑和细胞凋亡，并具有良好的电生理特性。除了对 cAMP 生成的影响之外，心脏 AC6 表达的增加可以实现许多这些特征，FDA 已批准了一项由 NHLBI 资助的针对严重 CHF 患者进行 AC6 基因转移的试验 (ClinicalTrials.gov NCT00787059)。然而，对 cAMP 无能力的 AC6 突变体进行研究有四个原因：1）获得关于 AC6 的有益心脏作用是否与 cAMP 生成无关的明确机制数据； 2) 通过生成多种AC6mut片段分子来进行AC6mut分子的结构-活性研究，并确定AC6mut片段的亚细胞靶向对Ca2+处理和Akt激活的影响； 3) 确定AC6mut在体内的心脏作用，4) 生成编码最佳AC6mut片段的合适的长期调节表达载体，并进行临床前基因转移研究以确定其在改善衰竭心脏功能方面的功效和安全性。这些研究旨在揭示机制，但也具有临床意义。例如，在临床心力衰竭中，通过增加 cAMP 水平来增强心脏收缩功能的药物未能延长生存期。根据初步数据，AC6mut 保留了 AC6 所见的有益效果，但在没有增加 cAMP 产生的情况下实现了这一效果，从而实现了潜在理想的治疗性正性肌力药物的特征。