TRPV4 blockade suppresses atrial fibrillation in sterile pericarditis rats

Abstract

Atrial fibrillation (AF) commonly occurs after surgery and is associated with atrial remodeling. TRPV4 is functionally expressed in the heart, and its activation affects cardiac structure and functions. We hypothesized that TRPV4 blockade alleviates atrial remodeling and reduces AF induction in sterile pericarditis (SP) rats. TRPV4 antagonist GSK2193874 or vehicle was orally administered 1 day before pericardiotomy. AF susceptibility and atrial function were assessed using in vivo electrophysiology, ex vivo optical mapping, patch clamp, and molecular biology on day 3 after surgery. TRPV4 expression increased in the atria of SP rats and patients with AF. GSK2193874 significantly reduced AF vulnerability in vivo and the frequency of atrial ectopy and AF with a reentrant pattern ex vivo. Mechanistically, GSK2193874 reversed the abnormal action potential duration (APD) prolongation in atrial myocytes through the regulation of voltage-gated K+ currents (IK); reduced the activation of atrial fibroblasts by inhibiting P38, AKT, and STAT3 pathways; and alleviated the infiltration of immune cells. Our results reveal that TRPV4 blockade prevented abnormal changes in atrial myocyte electrophysiology and ameliorated atrial fibrosis and inflammation in SP rats; therefore, it might be a promising strategy to treat AF, particularly postoperative AF.

Keywords: Arrhythmias; Calcium; Cardiology; Fibrosis.

Figure 1. Upregulated expression of TRPV4 in the atria of SP rats and AF patients.

(A and B) Representative Western blot (A) and quantification (B) of TRPV4 in atrial tissue of sham (n = 14) and SP rats 1 day (d) (n = 5), 2 d (n = 7), 3 d (n = 6), 4 d (n = 6), 5 d (n = 6), 7 d (n = 6), and 14 d (n = 5) after surgery. (C) The expression of TRPV4 in hearts was measured at day 3 after surgery using IHC. The negative control shown was treated using the same immunohistochemical procedure, but the primary antibody step was omitted. Scale bar: 50 μm. (D and E) Representative Western blot (D) and quantification (E) of TRPV4 in atrial tissue of patients with in sinus rhythm (SNR, n = 16) and AF (n = 12). Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test (B) and 2-tailed unpaired Student’s t test (E); *P < 0.05, ***P < 0.001. Data are expressed as the mean ± SEM.

Figure 2. Blockage of TRPV4 suppresses atrial fibrillation induction and duration in sterile pericarditis rats.

(A) Typical ECG recording results from the sham, vehicle, and GSK2193874 groups. (B and C) Statistical results of atrial fibrillation duration and probability of induced atrial fibrillation (AF) before and 3 d after operation among 3 groups. n = 6 each group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test (B and C); **P < 0.01, ***P < 0.001 versus sham; ^#P < 0.05, ###P < 0.001 versus vehicle. Data are expressed as the mean ± SEM.

Figure 3. Blockage of TRPV4 suppresses atrial arrhythmia propensity in isolated hearts.

(A–C) Representative optical action potential (AP) and ECG recorded in a vehicle rat, showing atrial ectopy (B), fibrillation (C), and none of both (A) induced by using an extrastimulus (S1S2; S2 intervals ranging from 50 to 30 ms) method. (C) Activation maps of pacing, ectopy, reentry, and sinus rhythm corresponding to the AP traces. (D) Incidence of atrial ectopy or fibrillation for each S2 interval in the 3 groups; sham, n = 10; vehicle, n = 11; GSK2193874, n = 7. Statistical analyses: χ² test; *P < 0.05, **P < 0.01 versus sham. (E) Quantification of atrial effective refractory period in the 3 groups; sham n = 8; vehicle n = 8; GSK2193874 n = 7. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test. Data are expressed as the mean ± SEM.

Figure 4. Blockage of TRPV4 prevents atrial electrical remodeling in sterile pericarditis rats.

(A) Representative action potentials (APs) recorded from isolated atrial myocytes of indicated groups. (B–D) Mean rest membrane potential (RMP) and AP amplitude (APA) (B), AP slope (C), and action potential duration (APD) (D) until 20%, 50%, and 90% of repolarization (APD₂₀, APD₅₀, and APD₇₀, respectively) in atrial myocytes. (E) Voltage clamp protocol. (F) Representative the outward voltage-gated K⁺ currents (I_K) recorded from isolated atrial myocytes of indicated groups.(G–I) Mean current-voltage (I–V) curves for the peak (I_peak, G), sustained (I_ss, H), and transient (I_to, I). Sham, n = 19 myocytes/6 rats; vehicle, n = 18 myocytes/7 rats; GSK2193874, n = 11 myocytes/7 rats. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test (B, C, D, G, H, and I); *P < 0.05, **P < 0.01, ***P < 0.001 versus sham; ^#P < 0.05 versus vehicle. Data are expressed as the mean ± SEM.

Figure 5. Blockage of TRPV4 attenuates atrial fibrosis and related gene expression in sterile pericarditis rats.

(A) Representative histological sections stained with Masson trichrome and percentage of left atrial interstitial fibrosis. n = 10/group. Scale bars: 50 μm.(B) Examples of α-SMA immunohistochemical staining and quantification. Sham, n = 10; vehicle, n = 10; GSK2193874, n = 8. Scale bars: 50 μm. (C) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time PCR. n = 6/group, each in triplicate. (D) The mRNA expression of IL-6, TNF-α, and TGF-β by real-time PCR. n = 6/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test (A–D); *P < 0.05, ***P < 0.001 versus sham; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus vehicle. Data are expressed as the mean ± SEM.

Figure 6. Effect of TRPV4 blockage on atrial fibrosis-related signaling pathways in sterile pericarditis rats.

(A and B) Representative Western blot (A) and quantification (B) of SMAD3, p-SMAD3, ERK, p-ERK, P38, p-P38, JNK, p-JNK, AKT, p-AKT, STAT3, and p-STAT3 in atrial tissue of indicated group. n = 6–7/group. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test (B); *P < 0.05, ***P < 0.001 versus sham; ^##P < 0.01, ^###P < 0.001 versus vehicle. Data are expressed as the mean ± SEM.

Figure 7. The function of TRPV4 enhances in atrial fibroblasts from sterile pericarditis rats.

(A) Time course of whole-cell current at +90 and –90 mV evoked by 300 nM GSK1016790A (left panel) and current-voltage (I–V) relations taken at time points a and b (right panel) in atrial fibroblasts from sham and SP rats. A ramp protocol elicited by a voltage ramp from –100 mV to +100 mV. Horizontal bars denote the time courses for applications of GSK1016790A. (B) Mean current-voltage (I–V) curves for GSK1016790A-induced TRPV4 current. Sham, n = 8 cells/5 rats; SP, n = 9 cells/5 rats. (C and D) Representative time course (C) of the changes in [Ca²⁺]_i and quantification (D) induced by GSK1016790A in atrial fibroblasts from sham and cells from SP rats with/without pretreatment with a selective TRPV4 antagonist, GSK2193874 (300 nM). n = 6/group. Statistical analyses: 2-tailed unpaired Student’s t test (B) and 1-way ANOVA with Bonferroni’s post hoc test (D); **P < 0.01, ***P < 0.001 versus sham; ^###P < 0.001 versus SP; **P < 0.01 versus sham; ^###P < 0.001 versus SP. Data are expressed as the mean ± SEM.

Figure 8. TRPV4 contributes to the differentiation and proliferation of atrial fibroblasts from sterile pericarditis rats via the activation of P38, AKT, and STAT3.

(A) The mRNA expression of α-SMA, collagen-1, and collagen-3 by real-time polymerase chain reaction (PCR). n = 4/group. (B) Proliferation of CFs by BrdU assay. Cells were treated with DMSO or GSK1016790A, or with GSK2193874 or GSK2193874 + multiple signaling pathway inhibitors. LY294002, an AKT inhibitor; S3I-201, a STAT3 specific inhibitor; SB 203580, P38 inhibitor; or SIS3, a SMAD3 inhibitor. n = 4/group, each in triplicate. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test (D); *P < 0.05, **P < 0.01, ***P < 0.001 versus DMSO; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus GSK1016790A. Data are expressed as the mean ± SEM.