LV dyssynchrony and fibroblast activation in PVC-induced Cardiomyopathy

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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.