A novel Cl − inward rectifier channel (Cl,ir) encoded by ClC-2, a member of the ClC voltage-gated Cl − channel gene superfamily, has been recently discovered in cardiac myocytes of several species. However, the physiological role of Cl,ir channels in the heart remains unknown. In this study we tested the hypothesis that Cl,ir channels may play an important role in cardiac pacemaker activity. In isolated guinea-pig sinoatrial node (SAN) cells, Cl,ir current was activated by hyperpolarization and hypotonic cell swelling. RT-PCR and immunohistological analyses confirmed the molecular expression of ClC-2 in guinea-pig SAN cells. Hypotonic stress increased the diastolic depolarization slope and decreased the maximum diastolic potential, action potential amplitude, APD 50 , APD 90 , and the cycle-length of the SAN cells. These effects were largely reversed by intracellular dialysis of anti-ClC-2 antibody, which significantly inhibited Cl,ir current but not other pacemaker currents, including the hyperpolarization-activated non-selective cationic "funny" current (I f ), the L-type Ca 2+ currents (I Ca,L ), the slowly-activating delayed rectifier I Ks and the volume-regulated outwardlyrectifying Cl − current (I Cl,vol ). Telemetry electrocardiograph studies in conscious ClC-2 knockout (Clcn2 −/− ) mice revealed a decreased chronotropic response to acute exercise stress when compared to their age-matched Clcn2 +/+ and Clcn2 +/− littermates. Targeted inactivation of ClC-2 does not alter intrinsic heart rate but prevented the positive chronotropic effect of acute exercise stress through a sympathetic regulation of ClC-2 channels. These results provide compelling evidence that ClC-2-encoded endogenous Cl,ir channels may play an important role in the regulation of cardiac pacemaker activity, which may become more prominent under stressed or pathological conditions.

Background— Recent evidence suggests that chloride channels may be involved in ischemic preconditioning (IPC). In this study, we tested whether the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels, which are expressed in the heart and activated by protein kinase A and protein kinase C, are important for IPC in isolated heart preparations from wild-type (WT) and CFTR knockout (CFTR −/− ) mice. Methods and Results— Hearts were isolated from age-matched WT or CFTR −/− (B6.129P2-Cftr tm1Unc and STOCKCftr tm1Unc -TgN 1Jaw) mice and perfused in the Langendorff or working-heart mode. All hearts were allowed to stabilize for 10 minutes before they were subjected to 30 or 45 minutes of global ischemia followed by 40 minutes of reperfusion (control group) or 3 cycles of 5 minutes of ischemia and reperfusion (IPC group) before 30 or 45 minutes of global ischemia and 40 minutes of reperfusion. Hemodynamic indices were recorded to evaluate cardiac functions. Release of creatine phosphate kinase (CPK) in the samples of coronary effluent and infarct size of the ventricles were used to estimate myocardial tissue injury. In WT adult hearts, IPC protected cardiac function during reperfusion and significantly decreased ischemia-induced CPK release and infarct size. A selective CFTR channel blocker, gemfibrozil, abrogated the protective effect of IPC. Furthermore, targeted inactivation of the CFTR gene in 2 different strains of CFTR −/− mice also prevented IPC’s protection of cardiac function and myocardial injury against sustained ischemia. Conclusions— CFTR Cl − channels may serve as novel and crucial mediators in mouse heart IPC.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the ATP-binding cassette transporter superfamily and encodes a PKC- and PKA-activated chloride (Cl−) channel in the heart. Previous study in isolated mouse heart supports a potential role of CFTR in acute ischemic preconditioning (IPC). This study was designed to further investigate the functional role of CFTR in the early and late (second window) IPC- and postconditioning (POC)-mediated cardioprotection against ischemia/reperfusion (I/R) injury. In the in vivo I/R models, early IPC significantly reduced the myocardial infarct size in the wild-type (CFTR+/+) (from 40.4±5.3% to 10.4±2.0%, n=8, p<0.001) and the heterozygous (CFTR+/−) littermates (from 39.4±2.4% to 15.4±5.1%, n=6, p<0.001) but failed to protect the CFTR knockout (CFTR−/−) mice (46.9±6.2% vs 55.5±7.8%, n=6, p>0.5). Similar results were observed in the in vivo late IPC experiments. Furthermore, both in vivo and ex vivo POC significantly reduced myocardial infarction in the CFTR+/+ mice but not in the CFTR−/− mice. Targeted inactivation of CFTR abolished the protective effects of IPC on I/R-induced apoptosis, suggesting that inhibition of apoptosis by activation of CFTR channels may be a novel mechanism of IPC- and POC-mediated cardioprotection against I/R injury. These results provide compelling evidence for a critical role of CFTR Cl− channels in the IPC- and POC-mediated cardioprotection against myocardial injury. Therefore, CFTR Cl− channels may represent novel therapeutic targets for the treatment of ischemic cardiac diseases.

Activation of volume regulated chloride channels (VRCCs) has been shown to be cardioprotective in ischemic preconditioning (IPC) of isolated hearts but the underlying molecular mechanisms remain unclear. Recent independent studies support that ClC-3, a ClC voltage-gated chloride channel, may function as a key component of the VRCCs. Thus, ClC-3 knockout (Clcn3^-/-) mice and their age-matched heterozygous (Clcn3^+/-) and wild-type (Clcn3^+/+) littermates were used to test whether activation of VRCCs contributes to cardioprotection in early and/or second-window IPC. Targeted disruption of ClC-3 gene caused a decrease in the body weight but no changes in heart/body weight ratio. Telemetry ECG and echocardiography revealed no differences in ECG and cardiac function under resting conditions among all groups. Under treadmill stress (10 m/min for 10 min), the Clcn3^-/- mice had significant slower heart rate (648±12 bpm) than Clcn3^+/+ littermates (737±19 bpm, n=6, P<0.05). Ex vivo IPC in the isolated working-heart preparations protected cardiac function during reperfusion and significantly decreased apoptosis and infarct size in all groups. In vivo early IPC significantly reduced infarct size in all groups including Clcn3^-/- mice (22.7±3.7% vs control 40.1±4.3%, n=22, P=0.004). Second-window IPC significantly reduced apoptosis and infarction in Clcn3^+/+ (22.9±3.2% vs 45.7±5.4%, n=22, P<0.001) and Clcn3^+/- mice (27.5±4.1% vs 42.2±5.7%, n=15, P<0.05) but not in Clcn3^-/- littermates (39.8±4.9% vs 41.5±8.2%, n=13, P>0.05). Impaired cell volume regulation of the Clcn3^-/- myocytes may contribute to the failure of cardioprotection by second-window IPC. These results strongly support that activation of VRCCs may play an important cardioprotective role in second-window IPC.