Nitric oxide protects the heart from ischemia-induced apoptosisand mitochondrial damage via protein kinase G mediated blockageof permeability transition and cytochrome c release

摘要

Background: Heart ischemia can rapidly induce apoptosis and mitochondrial dysfunction via mitochondrialpermeability transition-induced cytochrome c release. We tested whether nitric oxide (NO) can block thisdamage in isolated rat heart, and, if so, by what mechanisms.Methods: Hearts were perfused with 50 μM DETA/NO (NO donor), then subjected to 30 min stop-flowischemia or ischemia/reperfusion. Isolated heart mitochondria were used to measure the rate of mitochondrialoxygen consumption and membrane potential using oxygen and tetraphenylphosphonium-selective electrodes.Mitochondrial and cytosolic cytochrome c levels were measured spectrophotometrically and by ELISA. Thecalcium retention capacity of isolated mitochondria was measured using the fluorescent dye Calcium Green-5N.Apoptosis and necrosis were evaluated by measuring the activity of caspase-3 in cytosolic extracts and the activityof lactate dehydrogenase in perfusate, respectively.Results: 30 min ischemia caused release of mitochondrial cytochrome c to the cytoplasm, inhibition of themitochondrial respiratory chain, and stimulation of mitochondrial proton permeability. 3 min perfusion with 50μM DETA/NO of hearts prior to ischemia decreased this mitochondrial damage. The DETA/NO-inducedblockage of mitochondrial cytochrome c release was reversed by a protein kinase G (PKG) inhibitor KT5823, orsoluble guanylate cyclase inhibitor ODQ or protein kinase C inhibitors (Ro 32-0432 and Ro 31-8220). Ischemiaalso stimulated caspase-3-like activity, and this was substantially reduced by pre-perfusion with DETA/NO.Reperfusion after 30 min of ischemia caused no further caspase activation, but was accompanied by necrosis,which was completely prevented by DETA/NO, and this protection was blocked by the PKG inhibitor. Incubationof isolated heart mitochondria with activated PKG blocked calcium-induced mitochondrial permeability transitionand cytochrome c release. Perfusion of non-ischemic heart with DETA/NO also made the subsequ