Characterization of Cardiac Anoctamin1 Ca²⁺-Activated Chloride Channels and Functional Role in Ischemia-Induced Arrhythmias

Abstract

Anoctamin1 (ANO1) encodes a Ca(2+)-activated chloride (Cl(-)) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1-encoded CaCCs in ischemia-induced arrhythmias in the heart. Quantitative real-time RT-PCR, immunofluorescence staining assays, and immunohistochemistry identified the molecular expression, location, and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch-clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca(2+)-activated Cl(-) current (I(Cl.Ca)) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of I(Cl.Ca), which was inhibited by a specific ANO1 inhibitor, T16A(inh)-A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia-induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper "spike and dome" compared to control cardiomyocytes from non-ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia-induced early phase 1 repolarization acceleration and caused a much shallower "spike and dome". We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia-induced increase in ANO1 expression may be responsible for the increased density of I(Cl.Ca) in the ischemic heart and may contribute, at least in part, to ischemia-induced arrhythmias.

Fig. 1

(A) qRT-PCR revealed that ANO1, but not ANO2 expressed in LV tissues (n = 3); (B) Representative immunohistochemistry images demonstrate that ANO1 distributes from epi- (the white arrow head), mid-myocardial and endocardial (the yellow arrow head) layers of the mouse LV. (C) Representative immunofluorescence staining images suggest that ANO1 is localized on the plasma membrane of the ventricular myocytes. Ventricular myocytes labeled with antibody against ANO1 (C,a; red), or co-labeled with antibodies raised against ANO1 (red) and against α-actinin (green) (C,b). Scale bars by white solid lines represent 20 µm. (C,c) The constructed histograms of fluorescence intensity across the cell (where indicated by the yellow solid line in C,b) for ANO1 (the red solid line) and α-actinin (the green solid line) suggest that ANO1 is mainly localized at the plasma membrane of mVMs (C,c).

Fig. 2

A. Representative whole-cell currents were recorded from control (A,a) and in the presence of 150 µmol L⁻¹ DIDS (A,b); average I–V relationships (A,c) were constructed as a function of voltages from records as shown in A,a and A,b (n = 6). B. Representative whole-cell currents were recorded under control condition (B,a) and in the presence of 30 µmol L⁻¹ specific ANO1 blocker T16A_inh-A01 (B,b); I–V relationships (B,c) were constructed against voltages from records as shown in B,a and B,b. (n = 5). C. Representative whole-cell currents were recorded under control condition (C,a) and in the presence of the specific pore-targeting anti-ANO1 antibody in the same ventricular myocyte (C,b); I–V relationships (C,c) were constructed as a function of voltages from records as shown in C,a and C,b (n = 5). D. Representative whole-cell currents were respectively recorded under control condition (D,a), in the presence of the boiled specific pore-targeting anti-ANO1 antibody (D,b) and in the presence of a non-pore-targeting anti-ANO1 antibody (D,c) ; I–V relationships (D,d) were constructed as a function of voltages from records as shown in D,a–c (n = 5). Anti-pore-ANO1, B-anti-pore-ANO1 and Anti-ANO1 respectively represent specific pore-targeting anti-ANO1 antibody, boiled specific pore-targeting anti-ANO1 antibody and non-pore-targeting anti-ANO1 antibody.

Fig. 3

A. Representative whole-cell currents were recorded from control (A,a) and extracellular Cl⁻ substituted by equimolar gluconate⁻ (A, b). I–V relationships demonstrated that replacement of Cl⁻ with gluconate⁻ led to a significantly decreased density of the outward currents and the E_rev shifted toward more positive potentials (A,c) (n = 5). B. Representative whole-cell currents were recorded from control (B,a) and extracellular Cl⁻ replaced with equimolar SCN⁻ (B,b). I–V relationships showed that replacement of Cl⁻ with SCN⁻ resulted in a significantly increased density of the outward currents and the E_rev shifted toward more negative potentials (B,c) (n = 5).

Fig. 4

A. The representative macroscopic currents were recorded in the absence (A,a) or in the presence of 5 µmol L⁻¹ nifedipine (A,b). I–V relationships from the records as shown in A,a and A,b are shown in panel A,c (n = 7). B. The representative whole-cell currents were recorded in the absence of (B,a) or in the presence of 10 µmol L⁻¹ extracellular caffeine plus 10 mmol L⁻¹ pipette BAPTA (B,b). Panel B,c shows the average I–V curves from the recordings (n = 6) as shown in B,a and B,b.

Fig. 5

Ischemia-induced increase in I_Cl.Ca in mVMs was respectively inhibited by a specific pore-targeting anti-ANO1 antibody and T16A_inh-A01. I_Cl.Ca was recorded in mVMs respectively isolated from sham (A,a and B,a) and myocardial ischemic groups before (A,b and B,b) or after application of specific pore-targeting anti-ANO1 antibody and 30 µmol/L T16A_inh-A01 (A,c and B,c). (A,d and B,d) Summarized densities of I_Cl.Ca were plotted as a function of membrane potentials under given conditions; I_Cl.Ca was significantly up-regulated by myocardial ischemia and was dramatically inhibited by either specific pore-targeting anti-ANO1 antibody (n = 5) or T16A_inh-A01 (n = 5).

Fig. 6

Representative I_Cl.Ca was respectively recorded in the mVMs under the normoxic (A,a and B,a), hypoxic (A,b and B,b) conditions and in the presence of inhibitors (A,c and B,c). (A,d and B,d) Summarized densities of I_Cl.Ca were plotted against membrane potentials; I_Cl.Ca was significantly up-regulated by hypoxia and was dramatically inhibited by a specific pore-targeting anti-ANO1 antibody (n = 6) and T16A_inh-A01 (n = 5).

Fig. 7

(A) Relative mRNA abundance of ANO1 examined by qRT-PCR in LV tissues from control (Ctrl), sham and myocardial ischemia (Isch) groups (n = 3). (B) Representative western blot (B,a) and averaged ANO1 protein densities in the isolated mVMs from control, sham, and myocardial ischemia group (B,b) (n = 5). * Indicates P < 0.05 vs. control or sham.

Fig. 8

A. Representative ECG recordings in mice during surgeries. Isch-a: an example of ischemia with ventricular premature beats (VE); Isch-b: an example of ischemia with ventricular fibrillation (VF); Isch-c: an example of ischemia with ventricular tachycardia (VT). B. Representative APs were recorded in the mVMs isolated from sham (the black line) and myocardial ischemia (the red line) groups, respectively (B,a). Amplified portion of AP (square boxes in B,a) is shown in panel B,b. C. Representative APs were recorded in the same ventricular myocytes isolated from control mice, before (the black line) or after application of specific pore-targeting anti-ANO1 antibody (the red line) (C,a). C,b. The enlarged portion indicated by square boxes in C,a.