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


RESULTS
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 microM 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 microM 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.


CONCLUSIONS
These findings provide the first direct evidence of activation of sarcolemmal Cl(-) channels by ischaemic and pharmacological preconditioning in cardiomyocytes.
Diaz
Roberto J
RJ
Division of Cell Biology, The Hospital for Sick Children, McMaster Building, Room 7019C, 555 University Avenue, Toronto, ON, Canada M5G 1X8.
Hinek
Alina
A
Wilson
Gregory J
GJ
D000241:Adenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D058907:Adenosine A1 Receptor Agonists; D058909:Adenosine A3 Receptor Agonists; D000818:Animals; D002317:Cardiovascular Agents / Q000494:pharmacology*; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D005456:Fluorescent Dyes; D006016:Glycolates / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects*; D019157:Ischemic Preconditioning, Myocardial; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D011806:Quinolinium Compounds; D011817:Rabbits; D043682:Receptor, Adenosine A1 / Q000378:metabolism; D043684:Receptor, Adenosine A3 / Q000378:metabolism; D012508:Sarcolemma / Q000187:drug effects* / Q000378:metabolism; D013997:Time Factors
D058907:Adenosine A1 Receptor Agonists; D058909:Adenosine A3 Receptor Agonists; D002317:Cardiovascular Agents; D018118:Chloride Channels; D002712:Chlorides; D005456:Fluorescent Dyes; D006016:Glycolates; C086112:N(6)-2-(4-aminophenyl)ethyladenosine; D011806:Quinolinium Compounds; D043682:Receptor, Adenosine A1; D043684:Receptor, Adenosine A3; C015765:MK 473; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium; D000241:Adenosine