LV dyssynchrony and fibroblast activation in PVC-induced Cardiomyopathy

背景资料。室性早搏是最常见的室性心律失常， 在老年人中非常普遍。众所周知，持续性的静脉曲张会导致左心室收缩功能障碍，称为 聚氯乙烯诱导的心肌病(PVC-CM)。适应不良反应背后的触发因素和分子机制 频发的室上性心动过速患者的心脏部位难以确定，但最近我们取得了重大进展。 了解PVC-CM动物模型左心室结构和功能的变化 (见初步结果)。我们推测，左心室不同步是激活驻留在心脏的成纤维细胞的关键触发因素。 心脏。活化的成纤维细胞(也称为肌成纤维细胞)可以控制细胞外基质的重塑。 (弥漫性纤维化)，释放可影响心肌细胞结构的细胞因子和神经体液因子 (肥大)和功能(射血分数降低)。 假设：我们的工作假设是机械应力通过左心室不同步性促进成纤维细胞 激活涉及RhoA/Hippo途径的激活。活化的成纤维细胞(肌成纤维细胞)释放 细胞因子促进组织重构(纤维化)，这些因子对心肌细胞有旁分泌作用 导致钙循环关键蛋白的功能/表达改变，最终损害收缩。 目的1：测定RhoA/Hippo通路在整个组织中的激活水平，特别是在 成纤维细胞的杂交链反应免疫组织化学和HCR-RNA-FISH 技术(原位技术)与基于人工智能的图像处理量化 高通量分析。 目的：应用定量聚合酶链式反应和免疫印迹技术检测成纤维细胞活化的细胞因子标志物(转化生长因子β1、2、3、IL 6和IL 1β)。 WB用于全球变化，而HCR-RNA-FISH技术将用于特定细胞类型的原位测定 其中这些标记物的表达上调。 目的3：参与兴奋收缩偶联的蛋白质，如RyR2和磷酸化RyR2，L-型钙离子 通道、Rad和磷酸-Rad、NCX、SERCA2a、磷蛋白(PLB)和磷酸-PLB以及钙调素 将通过WB和从父母拨款获得的钙动力学结果来确定，数学 模拟将评估钙离子处理对收缩和复极异常的可能性的作用 在PVC-CM中。 方法：研究方法。将猪植入改良的起搏器，并随机分成3组：1)假手术组 (正常节律，无心律失常)，2)早搏(PAC)组有后收缩 增强(PESP)，但无左室不同步；3)PVC组同时存在PESP和LV不同步。vbl.使用 超声心动图、PESP和左心室不同步将在12周内与左心室射血分数的变化一起量化 对于每种动物。因此，这些变化将与在目标1、2和3中观察到的生化变化相关。 对于每种动物。这些相关性将为PESP和LV不同步的相关性提供证据 成纤维细胞的激活及其下游的有害影响。蛋白质的综合测量 表达/调节通过“电子计算机”分析，我们将识别收缩能力的变化并评估风险。 用于与在PVC-CM中观察到的钙动态有关的复极异常。 意义重大。这项建议将确定成纤维细胞激活的作用和分子机制，通过 与静脉曲张期间左室不同步相关的机械应力，并使用数学模型来评估 钙离子动力学和复极在PVC-CM中的作用。在这个项目结束时，潜在的分子目标将 被确定，指导未来的治疗干预的临床前研究。

Background. Premature ventricular contractions (PVCs) are the most frequent ventricular arrhythmia and are highly prevalent in the elderly. Persistent PVCs are known to cause an LV systolic dysfunction, referred as PVC-induced cardiomyopathy (PVC-CM). Triggers and molecular mechanisms behind maladaptive responses of the heart subjected to frequent PVCs are elusive to identify, but recently we have done significant progress understanding structural and functional changes happening in the left ventricle (LV) of PVC-CM animal models (see preliminary results). We postulate that LV dyssynchrony is the key trigger to activate fibroblast resident in the heart. Activated fibroblasts (also called myofibroblasts) can command remodeling of the extracellular matrix (diffuse fibrosis), release cytokines and neurohumoral factors that can affect cardiac myocyte structure (hypertrophy) and function (decreased ejection fraction). Hypothesis: Our working hypothesis is that mechanical stress though LV dyssynchrony promotes fibroblast activation involving the activation of the RhoA/HIPPO pathway. Activated fibroblasts (myofibroblasts) release cytokines promoting tissue remodeling (fibrosis), and these factors have a paracrine effect on cardiac myocytes leading to altering function/expression of proteins key for Ca2+ cycling, ultimately impairing contraction. Aim 1: Determine the activation level of the RhoA/HIPPO pathway in the whole tissue, and specifically in fibroblasts using hybridization chain reaction (HCR) immunohistochemistry (IHC) and HCR-RNA-FISH technologies (in situ techniques) together with artificial intelligence based-image processing quantification for high throughput analysis. AIM 2: Cytokine markers of fibroblast activation (TGFβ1,2,3, IL6 and IL-1β) will be determined using qPCR and WB for global changes, and HCR-RNA-FISH technology will used for in situ determination of specific cell type where expression of these markers is upregulated. AIM 3: Proteins involved in excitation contraction (EC) coupling such as RyR2 and phospho-RyR2, L-type Ca2+ channel, Rad and phospho-Rad, NCX, SERCA2a, phospholamban (PLB) and phospho-PLB, and calsequestrin will be determined via WB, and from Ca2+ dynamics results obtained from the parent grant, mathematical simulations will assess the role of Ca2+ handling on contractility and the likelihood of repolarization abnormalities in PVC-CM. Methods. Swine will be implanted with modified pacemakers and will be randomized in 3 cohorts: 1) Sham group (normal rhythm, no arrhythmias), 2) premature atrial contractions (PAC) group having post-extrasystolic potentiation (PESP) but no LV dyssynchrony, 3) PVC group having both PESP and LV dyssynchrony. Using echocardiography, PESP and LV dyssynchrony will be quantified along with changes in LVEF during 12 weeks for each animal. Thus, these changes will be correlated with biochemical changes observed in Aims 1, 2, and 3 for each animal. These correlations will provide evidence of the relevance of PESP and LV dyssynchrony in fibroblast activation and its downstream deleterious effects. Combining measurements of protein expression/regulation with “in silico” assays, we will identify changes in contractility and assess risk for repolarization abnormalities linked to Ca2+ dynamics observed in PVC-CM. Significance. This proposal will identify the role and molecular mechanisms of fibroblast activation by mechanical stress associated with LV dyssynchrony during PVCs, and use mathematical models to assess the role of Ca2+ dynamics and repolarization in PVC-CM. At the end of this project, potential molecular targets will be identified, directing future preclinical studies for therapeutic intervention.