Amelioration of myocardial global ischemia/reperfusion injury with volume-regulatory chloride channel inhibitors in vivo1

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Abstract
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. We performed global ischemia for 8 min at 37°C or 4°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. The transplanted hearts showed TUNEL-positivity and DNA laddering with a peak at 24 hr during reperfusion after ischemia at 37°C, but not at 4°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. 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.

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