Epigenetic Regulation on Atrial Regional Difference of Fibroblast Activity and Calcium Homeostasis in Atrial Myopathy and Fibrogenesis

Atrial fibrosis frequently occurs in cardiovascular diseases and plays a pivotal pathological role in the genesis of atrial arrhythmia. Our previous study found that left atrium (LA) fibroblasts have distinctive biological characteristic with easier myofibroblasts transformation and a higher collagen production activity than right atrium (RA) fibroblasts, which is considered to be caused by its higher MAPK signaling and oxidative stress. Ca2+ signaling and homeostasis are pivotal characters in fibrogenesis. However, the role of Ca2+ homeostasis in distinctive LA fibroblasts activity is not clear. Angiotensin II induced cardiac fibroblasts proliferation and myofibroblast differentiation through modulating Ca2+ signalling. Intermediate conductance calcium-activated potassium channel (KCa3.1) can be activated by an increase in intracellular Ca2+ leading to an hyperpolarization membrane potential thereby amplifying the driving force for Ca2+ entry. KCa3.1 can up-regulate the collagen production, and myofibroblast differentiation through an MAPK signaling pathway. Transient receptor potential (TRP) channels induced Ca2+ influx make a significant contribution to the pathogenesis of cardiac fibrosis. Moreover, heart failure (HF) increases atrial fibrosis with enhancing fibroblast activity. HF atrial fibroblasts have lower voltage-dependent potassium current, but higher inward-rectifier potassium currents than control fibroblasts, which suggest that increasing store operated Ca2+ entry may contribute to HF-induced fibrogenesis. Similarly, uremic cardiomyopathy is characterized by diffuse cardiac fibrosis. Our previous study revealed that chronic kidney disease (CKD) significantly increased LA fibrosis, but not in RA tissues, which suggests that LA has a high vulnerability to atrial myopathy. Nevertheless, it is not clear whether the atrial regional differences of Ca2+ homeostasis through current modulation may contribute to the LA disinvite fibrosis activity and atrial myopathy-related fibrogenesis. MicroRNA (miR), and long non-coding RNAs (LncRNA) critically regulates cardiac fibrosis. Up-regulated miR24, miR101, miR133 decrease cardiac fibrosis and attenuating cardiac fibroblasts profibrotic abilities. Up-regulated miR21 enhance collagen production of cardiac fibroblasts. Moreover, LncRNAs also contribute to cardiac fibrogenesis. Upregulated LncRNA H19, n379599, n379519, n384648, n380433, and n410105 increased cardiac myofibroblast differentiation ability. Recently, new generation sequencing (NGS) and analysis through bioinformatics approaches has been used to evaluate MicroRNA differences in different tissues or managements Nevertheless, distinct microRNA and LncRNA characteristics between different atrial region and different disease (HF and CKD) fibroblasts and the connection with fibroblast activity have not been fully elucidated. In this study, we will investigate the Ca2+ homeostasis in LA and RA fibroblasts and evaluate whether HF or CKD will change fibroblast electrophysiological characteristics and result in high atrial fibrogenesis and atrial myopathy. Additionally, we will study whether microRNAs and LncRNAs will contribute to distinctive LA fibroblasts activity and atrial myopathy. In the first year experiment, we will evaluate the different electrophysiological characteristics and calcium homeostasis of LA and RA fibroblasts from healthy and HF rats and study the background mechanisms. In the second year experiment, we will evaluate the different microRNA and LncRNA expressions of LA and RA fibroblasts from healthy and HF rats by NGS and analysis the distinct epigenetic regulation on fibroblast activity and calcium homeostasis by bioinformatic methods. In the third year experiment, we will evaluate the different microRNA and LncRNA expressions of LA and RA fibroblasts from healthy and CKD rats by NGS and analysis the distinct epigenetic regulation on fibroblast activity and calcium homeostasis by bioinformatic methods. Methods: In the first year, KCa3.1, TRPC3, TRPC6, Calcium imaging will be used in evaluating the activity of primary isolated LA and RA fibroblasts from healthy and isoproterenol induced HF rats and analysis the electrophysiologic regulation of fibroblast activites. In the second year, microRNA and LncRNA analysis of LA and RA fibroblasts from healthy and isoproterenol induced HF rats will be done with new generation sequencing. Distinct epigenetic regulation on fibroblast activity and calcium homeostasis will be evaluated by bio-informatic methods. Further microRNA validation and functional assay will be done. In the third year, microRNA and LncRNA analysis of LA and RA fibroblasts from healthy and neomycin induced CKD rats will be done with new generation sequencing. Distinct epigenetic regulation on fibroblast activity and calcium homeostasis will be evaluated by bio-informatic methods. Further microRNA validation and functional assay will be done. Preliminary results: (1) Both in LA and RA fibroblasts, TRP channel mediated non-selective cation (NSC) current can be suppressed by gadolinium. (2) Ca2+ current in LA fibroblasts measured by Fura2-Ca2+ imaging (ratio of Fluorescent wavelength 340nm, 380nm) were 0.13±0.03 (3) Compared to isolated HF RA fibroblasts, isolated HF LA fibroblasts have higher calcium-activated potassium current density