Direct evidence of chloride ion efflux in ischaemic and pharmacological preconditioning of cultured cardiomyocytes

We have previously shown that reduction of ischaemic cell swelling via enhanced cell volume regulation is a key mechanism of ischaemic preconditioning (IPC) in cardiomyocytes. We have also shown that pharmacological blockade of Cl- channels abolishes cardioprotection achieved by IPC in Langendorff-perfused hearts and freshly isolated cardiomyocytes, thus suggesting that Cl- plays a key role in IPC cardioprotection. However, direct evidence of Cl- channel activation resulting in transsarcolemmal Cl- efflux by IPC had been lacking. To address this issue, 24 h cultured rabbit cardiomyocytes were loaded with 5 mM 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), a specific fluorescence indicator that is quenched by Cl- so that cellular efflux of Cl- results in an increase in SPQ fluorescence. After stabilization for 10 min, cardiomyocytes were preconditioned either with 10 min simulated ischaemia/10 min simulated reperfusion or with 10 min treatment with 1 mu M N-6-2-(4-aminophenyl)ethyladenosine (APNEA). IPC and APNEA significantly (P < 0.001) reduced the intracellular Cl- concentration ([Cl-](i)) to 31.9 +/- 3.2 mM (mean +/- SEM) and 32.5 +/- 2.8 mM, respectively, from an initial [Cl-](i) (pooled stabilization 61.5 +/- 7.1 mM). [Cl-](i) did not change in control (non-preconditioned) cardiomyocytes (control 58.1 +/- 1.9 mM and control + vehicle 62.6 +/- 4.9 mM, P = 0.98 and 0.99 vs. pooled pre-treatment baseline, respectively). Inhibition of Cl- channels with 50 mu M indanyloxyacetic acid 94 completely blocked preconditioning-induced Cl- efflux. Thus, a net Cl- efflux of 29.6 and 29.0 mM was triggered by IPC and APNEA. These findings provide the first direct evidence of activation of sarcolemmal Cl- channels by ischaemic and pharmacological preconditioning in cardiomyocytes.