Enhanced cell-volume regulation in cyclosporin A cardioprotection

Cyclosporin A (CsA) has been shown to protect against ischaemia/reperfusion injury presumably by its inhibition of mitochondrial permeability transition pore opening through cyclophilin D inhibition. We examine if CsA cardioprotection involves a cell-volume regulatory mechanism. To address this issue, cultured rabbit cardiomyocytes were subjected to the following protocols: (i) cardiomyocytes were treated with 200 nM CsA either given for 10 min followed by 10 min of washout prior to 30 min hypo-osmotic stress (200 mOsm) or administered throughout 75 min simulated ischaemia/60 min simulated reperfusion. Cell necrosis and cell swelling were determined by trypan blue staining and cell-volume measurements, respectively; (ii) SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium) dye loaded cardiomyocytes were treated with 200 nM CsA for 10 min followed by 10 min washout and intracellular Cl concentration measured (Cl efflux); (iii) 5,5,6,6-tetrachloro-1,1,3,3- tetraethylbenzimi-dazolylcarbocyanine iodide(JC-1) loaded cardiomyocytes were treated with 200 nM CsA to inhibit mitochondrial membrane potential (m) dissipation (an index of mitochondria permeability transition pore opening) by either valinomycin (2 M) or ischaemia/reperfusion injury. Cl channels were blocked by indanyloxyacetic acid 94 (IAA-94, 50 M). CsA not only significantly (P 0.001) reduced the of dead cells following simulated ischaemia/reperfusion but it also triggered an efflux of Cl, hence enhancing cardiomyocyte cell-volume regulatory response. CsA protection against cell necrosis and its effect on Cl transport/volume regulation were all blocked by IAA-94. IAA-94 had no effect on m. These data indicate that CsA protects against cell necrosis at least in part by enhancing cardiomyocyte volume regulation, and not simply by inhibiting MPTP opening.