A role for cardiomyocyte pannexin 1 in non-ischemic heart failure

心肌细胞pannexin 1在非缺血性心力衰竭中的作用

• 批准号: 10680109
• 负责人: Caitlin Marie Pavelec
• 金额: $ 3.67万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680109
• 关键词: Address; Adrenergic Agents; Adrenergic Receptor; Adrenergic beta-Agonists; Affect; American; Autopsy; Binding; Binding Sites; Biological Assay; Blood Pressure; Cardiac; Cardiac Myocytes; Cardiometabolic Disease; Caspase; Cell Line; Cells; Creatine; Creatinine; Data; Deposition; Disease; Disease Progression; Dyes; Echocardiography; Electrophysiology (science); Environment; Fibroblasts; Fibrosis; Functional disorder; G-protein Beta gamma; GTP-Binding Proteins; Gene Deletion; Heart; Heart Diseases; Heart Hypertrophy; Heart Rate; Heart failure; Histologic; Hypertension; Immune; Immune response; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Inflammation; Injections; Ion Channel; Isoproterenol; Lead; Left ventricular structure; Leucocytic infiltrate; Link; Magnetic Resonance Imaging; Measurement; Measures; Mediating; Modeling; Mus; Mutation; Myoblasts; Myocardial; Myocardial Infarction; Myocardial dysfunction; Myocardium; Myofibroblast; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Onset of illness; Organ; Osmosis; Pathologic; Patients; Perfusion; Plasma; Play; Population; Protein Isoforms; Protein Subunits; Pump; Radio; Rattus; Regulation; Role; Signal Transduction; Small Interfering RNA; Spironolactone; Sterility; Survival Rate; Tamoxifen; Telemetry; Testing; Therapeutic; Universities; Virginia; Weight; Work; beta-2 Adrenergic Receptors; beta-adrenergic receptor; blood pressure regulation; comparison control; coronary fibrosis; extracellular; gallein; heart function; immune activation; immune cell infiltrate; implantation; in vivo; inhibitor; knock-down; male; mouse model; mutant; new therapeutic target; novel; osmotic minipump; pressure; promoter; recruit; response; uptake

Project Summary Cardiac fibrosis is currently untargeted in many cardiac disease settings, yet this is a hallmark of decreased myocardial compliance. Current therapy for patients with heart failure, which has a hallmark of cardiac fibrosis, includes spironolactone, a pannexin 1 (Panx1) channel inhibitor. Despite this, the role for Panx1 in cardiomyocytes in non-ischemic heart failure has not been studied. Additionally, Panx1 plays a role in fibrosis in other diseases, and it has been shown to play roles in inflammation, blood pressure regulation, and myocardial infarction. Furthermore, it can be activated by G-protein subunits during alpha1D and β3-adrenergic stimulation. This evidence provides reason to hypothesize that protection from cardiac dysfunction and decreases pathophysiology including immune cell recruitment and cardiac fibrosis are Panx1 dependent. Preliminary data in a model of non-ischemic heart failure, 14 days of isoproterenol (ISO) injections, shows that male mice of a novel mouse with cardiomyocyte-specific pannexin 1 deletion (Panx1MyHC6) are protected from cardiac hypertrophy and increases in left ventricle volume compared to their Panx1fl/fl counterparts. Additionally, H9c2 cells, a rat myoblast cell line, with a siRNA-mediated knock-down of Panx1 have blunted release of ATP compared to control after β-adrenergic stimulation with ISO. This data and other preliminary studies lead me to address the hypothesis in two aims. In subaim 1A, I will investigate the mechanism of Panx1 activation by β-adrenergic stimulation in cardiomyocytes. I will first determine the mechanism by which ISO activates Panx1 using established Panx1 mutants in Panx1-null H9c2 cells. I hypothesize that the regulation occurs via binding of the G-protein βγ-subunits to Panx1 and have a proposed binding site identified from previous work. I will evaluate this binding site using BioID. In subaim 1B, I will investigate the role of Panx1 channel activation by β-adrenergic stimulation in whole hearts. In subaim 1B, I will perform pressurized contractile studies ex vivo on Langendorff perfused murine hearts to evaluate Panx1’s effect on cardiac contraction. For subaim 1B, I will use our novel Panx1MyHC6 mouse. I will test the role of Panx1 in the induction of cardiac dysfunction in non-ischemic heart failure, using a model of ISO administration for 14 days. Finally, in subaim 2B I will investigate the role of Panx1 in the progression of non-ischemic heart failure. For this aim, I will use another novel mouse model which has inducible deletion of Panx1 under the Tnnt2 promoter. I will use ISO in an osmotic pump for 28 days to induce heart failure and intervene by deleting Panx1 to delineate the role of Panx1 in the onset versus the progression of non-ischemic heart failure. Both aims will evaluate cardiac function using echocardiography with Dr. Wolf. I will use histological analysis for fibrotic content and immune cell infiltration of the myocardium, measure plasma lactate and creatinine as markers of secondary organ damage, and radiotelemetry for blood pressure changes. Together these studies will identify Panx1 as a novel therapeutic target and elucidate whether Panx1 channel activation plays a differential role in heart failure disease states.

项目摘要 心脏纤维化目前在许多心脏疾病环境中没有靶向，但这是 心肌顺应性降低。心力衰竭患者的当前治疗方法，这是心脏疾病的标志 纤维化，包括螺内酯，一种pAnnexin 1(Panx1)通道抑制剂。尽管如此，Panx1在 心肌细胞在非缺血性心力衰竭中的作用还没有研究。此外，Panx1在肝纤维化中发挥作用。 其他疾病，已被证明在炎症、血压调节和心肌梗死中起作用。 脑梗塞。此外，在α1D和β3肾上腺素能刺激过程中，G蛋白亚基也可以激活它。 这一证据提供了假设的理由，即对心脏功能障碍的保护作用 包括免疫细胞募集和心肌纤维化在内的病理生理机制依赖于Panx1。 非缺血性心力衰竭模型的初步数据显示，注射异丙肾上腺素(ISO)14天 一种新的心肌细胞特异性pAnnexin 1缺失小鼠(Panx1MyHC6)的雄性小鼠受到保护 与Panx1fl/fl对应的心脏肥厚和左心室容量增加有关。 此外，具有siRNA介导的Panx1基因敲除的大鼠成肌细胞系H9c2细胞已经钝化 β肾上腺素能刺激后三磷酸腺苷释放与对照组比较。这一数据和其他初步数据 研究让我从两个方面阐述了这一假说。在子目标1A中，我将研究Panx1的机制 心肌细胞受β-肾上腺素能刺激激活。我将首先确定ISO所依据的机制 在Panx1缺失的H9c2细胞中使用已建立的Panx1突变体激活Panx1。我假设这项规定 通过将G蛋白βγ亚基与Panx1结合而发生，并具有如下建议的结合位点 以前的工作。我会用BioID来评估这个结合位点。在子目标1B中，我将调查Panx1的作用 β-肾上腺素能刺激激活整个心脏的通道。在子目标1B中，我将执行加压 朗宁多夫灌流小鼠心脏的体外收缩实验评价潘生丁对S心脏的影响 收缩。对子目标1B，我将使用我们的新型Panx1MyHC6小鼠。我将测试Panx1在诱导中的作用 使用ISO给药14天的模型，研究非缺血性心力衰竭患者的心功能障碍。最后，在 亚目标2B I将研究Panx1在非缺血性心力衰竭进展中的作用。为此，我 将使用另一种新的小鼠模型，该模型在Tnnt2启动子下具有可诱导的Panx1缺失。我会用 ISO在渗透泵中28天诱导心力衰竭，并通过删除Panx1进行干预以描述其作用 Panx1在发病时与非缺血性心力衰竭进展之间的关系。这两个目标都将评估心脏 与沃尔夫医生一起使用超声心动图检查心脏功能。我将使用组织学分析纤维化内容物和免疫细胞 心肌的浸润，测量血浆乳酸和肌酐作为继发性器官损害的标志， 以及用于血压变化的无线电遥测。总之，这些研究将确定Panx1是一种新的治疗方法 目的并阐明Panx1通道激活在心力衰竭疾病状态中是否起着不同的作用。