Amelioration of myocardial global ischemia/reperfusion injury with volume-regulatory chloride channel inhibitors in vivo.
BACKGROUND
Recently, the apoptotic volume decrease was suggested to be regulated by volume regulatory Cl- channels in cultured cell lines. We thus examined whether inhibition of volume-regulatory Cl- channels is cardioprotective, like caspase inhibition, by hindering the apoptosis of cardiomyocytes induced by global ischemia/reperfusion (I/R) in vivo.


METHODS
We performed global ischemia for 8 min at 37 degrees C or 4 degrees C in isolated rat hearts, followed by 24-hr reperfusion via heterotopic heart transplantation. The heart tissue was examined by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, genomic DNA electrophoresis, and caspase-3 activity. Two blockers of volume-regulatory Cl- channels, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), and a broad-spectrum caspase inhibitor, benzoyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-Asp-DCB), were administered intravenously. Triphenyltetrazolium chloride (TTC) staining and ultrasound cardiography were performed to examine myocardial viability. The TTC-unstained region was assessed by means of horseradish peroxidase (HRP) infiltration and the TUNEL method.


RESULTS
The transplanted hearts showed TUNEL-positivity and DNA laddering with a peak at 24 hr during reperfusion after ischemia at 37 degrees C, but not at 4 degrees C. NPPB and DIDS were as potent as Z-Asp-DCB for recovery of cardiac function and for blocking the appearance of TUNEL-positivity, DNA laddering, caspase 3 activity, and a TTC-unstained area. TTC-unstained areas were composed of either TUNEL- and slightly HRP-positive or TUNEL-negative and strongly HRP-positive cardiomyocytes.


CONCLUSIONS
The present results demonstrated that myocardial DNA fragmentation, caspase activation, and loss of cardiac function after global I/R were blocked by NPPB and DIDS, similar to in the case of Z-Asp-DCB. These results suggest that inhibition of volume-regulatory Cl- channels is also effective for preventing cardiac I/R injury.
Mizoguchi
Kazuhiro
K
First Department of Surgery and Second Department of Physiology, Kagawa Medical University, Kagawa 761-0793, Japan.
Maeta
Hajime
H
Yamamoto
Akira
A
Oe
Masahiro
M
Kosaka
Hiroaki
H
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001224:Aspartic Acid / Q000031:analogs & derivatives*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D018118:Chloride Channels / Q000037:antagonists & inhibitors*; D053938:DNA Fragmentation; D016027:Heart Transplantation / Q000502:physiology*; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D008297:Male; D017202:Myocardial Ischemia / Q000517:prevention & control; D015428:Myocardial Reperfusion Injury / Q000517:prevention & control*; D009579:Nitrobenzoates / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D061945:Caspase Inhibitors; D018118:Chloride Channels; D009579:Nitrobenzoates; D011480:Protease Inhibitors; C093260:benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene; D001224:Aspartic Acid; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid