LV dyssynchrony and fibroblast activation in PVC-induced Cardiomyopathy

背景室性早搏（PVC）是最常见的室性心律失常， 在老年人中非常普遍。已知持续性PVC会导致LV收缩功能障碍，称为 PVC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 心脏频繁发生室性心动过速的原因很难确定，但最近我们已经取得了重大进展， 了解PVC-CM动物模型左心室（LV）发生的结构和功能变化 (see初步结果）。我们假设左心室不同步是激活驻留在左心室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）可以命令细胞外基质的重塑 （弥漫性纤维化），释放细胞因子和神经体液因子，可影响心肌细胞结构 （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促进成纤维细胞 活化涉及RhoA/HIPPO途径的活化。活化的成纤维细胞（肌成纤维细胞）释放 促进组织重塑（纤维化）的细胞因子，这些因子对心肌细胞具有旁分泌作用 导致改变对Ca 2+循环起关键作用的蛋白质的功能/表达，最终损害收缩。 目的1：确定整个组织中RhoA/HIPPO通路的活化水平，特别是在 使用杂交链反应（HCR）免疫组织化学（IHC）和HCR-RNA-FISH检测成纤维细胞 技术（原位技术）以及基于人工智能的图像处理量化， 高通量分析。 目的2：使用qPCR和RT-PCR方法测定成纤维细胞活化的细胞因子标志物（TGFβ 1、2、3、IL 6和IL-1β）， WB用于全局变化，HCR-RNA-FISH技术用于原位确定特定细胞类型 其中这些标志物的表达被上调。 目的3：参与兴奋收缩偶联的蛋白质，如RyR 2和磷酸化RyR 2，L-型Ca ~（2+） 通道、Rad和磷酸化Rad、NCX、SERCA 2a、受磷蛋白（PLB）和磷酸化PLB以及钙螯合蛋白 将通过WB确定，并从从母基金获得的Ca 2+动力学结果，数学 模拟将评估Ca 2+处理对收缩性的作用和复极异常的可能性 在PVC-CM中。 方法.猪将植入改良的起搏器，并随机分为3个队列：1）假手术组 （正常心律，无心律失常），2）房性期前收缩（PAC）组 （3）PVC组同时存在PESP和LV失同步。使用 在12周期间，将对超声心动图、PESP和LV不同步以及LVEF的变化进行沿着量化 对于每一个动物。因此，这些变化将与目标1、2和3中观察到的生化变化相关 对于每一个动物。这些相关性将提供PESP和LV不同步性在以下方面的相关性的证据： 成纤维细胞活化及其下游有害作用。蛋白质的组合测量 表达/调控与“在硅片”测定，我们将确定收缩性的变化，并评估风险 与PVC-CM中观察到的Ca 2+动力学相关的复极异常。 意义该提案将通过以下方式确定成纤维细胞活化的作用和分子机制： 与PVC期间LV不同步相关的机械应力，并使用数学模型评估 Ca 2+动力学和复极在PVC-CM中的作用。在这个项目结束时，潜在的分子靶点将 确定，指导未来的临床前研究的治疗干预。

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.