Knockdown of Ift88 in fibroblasts causes extracellular matrix remodeling and decreases conduction velocity in cardiomyocyte monolayers

Abstract

Background: Atrial fibrosis plays an important role in the development and persistence of atrial fibrillation by promoting reentry. Primary cilia have been identified as a regulator of fibroblasts (FB) activation and extracellular matrix (ECM) deposition. We hypothesized that selective reduction of primary cilia causes increased fibrosis and facilitates reentry. Aim: The aim of this study was to disrupt the formation of primary cilia in FB and examine its consequences on ECM and conduction in a co-culture system of cardiomyocytes (CM) and FB. Materials: Using short interfering RNA (siRNA), we removed primary cilia in neonatal rat ventricular FB by reducing the expression of Ift88 gene required for ciliary assembly. We co-cultured neonatal rat ventricular cardiomyocytes (CM) with FB previously transfected with Ift88 siRNA (siIft88) or negative control siRNA (siNC) for 48 h. We examined the consequences of ciliated fibroblasts reduction on conduction and tissue remodeling by performing electrical mapping, microelectrode, and gene expression measurements. Results: Transfection of FB with siIft88 resulted in a significant 60% and 30% reduction of relative Ift88 expression in FB and CM-FB co-cultures, respectively, compared to siNC. Knockdown of Ift88 significantly increased the expression of ECM genes Fn1, Col1a1 and Ctgf by 38%, 30% and 18%, respectively, in comparison to transfection with siNC. Conduction velocity (CV) was significantly decreased in the siIft88 group in comparison to siNC [11.12 ± 4.27 cm/s (n = 10) vs. 17.00 ± 6.20 (n = 10) respectively, p < 0.05]. The fraction of sites with interelectrode activation block was larger in the siIft88 group than in the siNC group (6.59 × 10^-2 ± 8.01 × 10^-2 vs. 1.18 × 10^-2 ± 3.72 × 10^-2 respectively, p < 0.05). We documented spontaneous reentrant arrhythmias in two cultures in the siIft88 group and in none of the siNC group. Action potentials were not significantly different between siNC and siIft88 groups. Conclusion: Disruption of cilia formation by siIft88 causes ECM remodeling and conduction abnormalities. Prevention of cilia loss could be a target for prevention of arrhythmias.

Keywords: atrial fibrillation; cardiac; fibroblasts; fibrosis; primary cilia.

FIGURE 1

Reduction of Ift88 expression in FB leads to increased ECM genes expression in CM and FB co-culture. (A) Relative expression of (mRNA) Ift88 in FB previously transfected with siNC or siIft88. Data are mean ± SD. n = 6 from tree independent CM/FB isolation, Student’s t-test. (B,C) Relative expression of (protein) IFT88 in FB previously transfected with siNC or siIft88. Data are mean ± SD. n = 4 from two independent CM/FB isolation, Student’s t-test. Full unedited blots are provided in Supplementary Figure S1. (D) Relative expression of (mRNA) Ift88 in CM-FB co-cultures after FB transfection with siNC or siIft88. Data are mean ± SD. n = 12 from four independent CM/FB isolation, Student’s t-test. (E) Relative expression of ECM genes Fn1, Col1a1 and Ctfg after 48 h of co-culture of CM-FB. Data are mean ± SD. n = 12 from four independent CM/FB isolation, Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

FIGURE 2

Reduced conduction velocity in CM monolayers co-cultured with decreased Ift88 FB. (A,B) Representative activation maps obtained after electrical mapping of spontaneously active CM co-cultured for 48 h with FB previously transfected with siNC or siIft88. Colors indicate activation times, according to the scale at right. Isochrones, 5 ms. White arrows indicate where conduction velocity was measured. Black line indicates 30 ms conduction block. Interelectrode distance of 700 μM. (C) Conduction velocity (CV) and number of block lines in spontaneously active CM monolayers co-cultured for 48 h with FB transfected with siNC or siIft88. Data are mean ± SD for CV and median for number of block lines, n = 10 monolayers from three independent CM/FB isolation. Student’s t-test for CV, Mann-Whitney test for number of block lines. Factor correction was carried out on CV to avoid inter-isolations differences.

FIGURE 3

Reentry in CM co-cultured with siIft88 FB. (A) Activation maps of two following beats showing reentrant circuit and obtained from one CM monolayer co-cultured for 48 h with FB previously transfected with siIft88. Colors indicate activation times, according to the scale at right. Dashed black line indicated reentrant conduction pattern. Letters indicate electrodes from which selected unipolar electrograms are depicted in (B). (B) Electrograms selected along the reentry circuit. Red arrow shows that the diastolic interval is spanned within local activation (electrogram from electrode A is repeated on top and at the bottom) as expected during reentry. The letters next to the electrograms indicate electrodes from which selected unipolar electrograms are shown (A). (C) Proportion of CM-FB co-cultures showing reentrant arrhythmia or no arrhythmia in the siNC and siIft88 groups.

FIGURE 4

Co-culture between FB with reduced (mRNA) Ift88 expression and CM does not lead to changes of the action potential characteristics. (A) Representative spontaneous APs recorded in CM monolayers co-cultured for 48 h with FB previously transfected with siNC or siIft88. (B–E) Effect of co-culture on spontaneous APs duration at 40%, 60%, and 80% (APD₄₀, APD₆₀, and APD₈₀) of repolarization (B), resting membrane potential (RMP), AP amplitude (APA) (C), maximal upstroke velocity (V_max) (D) and cycle length (E). n ≥ 9 microelectrode measurements from two independent CM/FB isolation. Data are mean ± SD.