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

A calcium (Ca2+)-sensitive voltage-gated chloride (Cl−) current (ICl.Ca) with distinct biophysical properties to classic Ca2+-activated Cl− channels (CaCCs) has been described in atrial and ventricular cardiomyocytes of several species (Duan et al., 2005; Duan, 2009), including swine (Li et al., 2003, 2004), canine (Tseng and Hoffman, 1989; Yue et al., 1997), rabbit (Zygmunt and Gibbons, 1991, 1992; Sipido et al., 1993; Wang et al., 1995), mouse (Xu et al., 2002), and human (Wang et al., 1995). It was reported that Cl− channel blockers niflumic acid and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) (Li et al., 2003), or replacement of external Cl− with methanesulfonate (Wang et al., 1995) resulted in an increase in APD. It was also suggested that ICl.Ca may likely contribute to the rate- and rhythm-dependent repolarization of the cardiac action potential (AP) depending on intracellular Ca2+ activities (Zygmunt, 1994). In normal heart, however, ICl.Ca may play an insignificant role in regulation of the diastolic membrane potential and action potential duration (APD) since the resting intracellular Ca2+ concentration ([Ca2+]i) is low under physiological conditions. But, when [Ca2+]i is substantially increased above the physiological resting level, ICl.Ca may carry a significant amount of transient outward current. It was proposed that under Ca2+ overload conditions ICl.Ca may contribute to the arrhythmogenic transient inward current (IT) and cause delayed after depolarization (DAD). However, it remains unknown about the molecular identity and the exact physiological and pathophysiological role of CaCCs in the heart (Duan et al., 2005; Duan, 2009). Recently, two members of the anoctamin (ANO) transmembrane protein family, ANO1 and ANO2, were determined to function as CaCCs in mouse over-expressed in Axolotl oocytes and HEK293 cells (Schroeder et al., 2008). Mouse ANO1 (mANO1) is broadly expressed in tissues known to contain native CaCCs. The human ANO1 mRNA is present in multiple human tissues including heart, lung, placenta, liver, skeletal muscle, and small intestine (Huang et al., 2006). The ANO1 (or TMEM16A)-encoded CaCC may participate in the control of cellular excitability, and regulation of smooth muscle contraction, slow wave activity in the gut, and fluid and salt transport by epithelia (Caputo et al., 2008; Schroeder et al., 2008; Yang et al., 2008; Duran and Hartzell, 2011). In this study, we investigated whether ANO1 underlies ICl.Ca in mouse ventricular myocytes (mVMs) and whether it plays a functional role in ischemia-induced alteration of APD and arrhythmias in the heart.