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

Abstract

BackgroundHeart ischemia can rapidly induce apoptosis and mitochondrial dysfunction via mitochondrial permeability transition-induced cytochrome c release. We tested whether nitric oxide (NO) can block this damage in isolated rat heart, and, if so, by what mechanisms.MethodsHearts were perfused with 50 μM DETA/NO (NO donor), then subjected to 30 min stop-flow ischemia or ischemia/reperfusion. Isolated heart mitochondria were used to measure the rate of mitochondrial oxygen consumption and membrane potential using oxygen and tetraphenylphosphonium-selective electrodes. Mitochondrial and cytosolic cytochrome c levels were measured spectrophotometrically and by ELISA. The calcium 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 activity of lactate dehydrogenase in perfusate, respectively.Results30 min ischemia caused release of mitochondrial cytochrome c to the cytoplasm, inhibition of the mitochondrial respiratory chain, and stimulation of mitochondrial proton permeability. 3 min perfusion with 50 μM DETA/NO of hearts prior to ischemia decreased this mitochondrial damage. The DETA/NO-induced blockage of mitochondrial cytochrome c release was reversed by a protein kinase G (PKG) inhibitor KT5823, or soluble guanylate cyclase inhibitor ODQ or protein kinase C inhibitors (Ro 32-0432 and Ro 31-8220). Ischemia also 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. Incubation of isolated heart mitochondria with activated PKG blocked calcium-induced mitochondrial permeability transition and cytochrome c release. Perfusion of non-ischemic heart with DETA/NO also made the subsequently isolated mitochondria resistant to calcium-induced permeabilisation, and this protection was blocked by the PKG inhibitor.ConclusionThe results indicate that NO rapidly protects the ischemic heart from apoptosis and mitochondrial dysfunction via PKG-mediated blockage of mitochondrial permeability transition and cytochrome c release.

Levosimendan Modulates Programmed Forms of Cell Death Through KATP Channels and Nitric Oxide

“…41,50 In particular, low levels (in nanomolars) of NO were shown to reversibly inhibit MPTP opening, whereas at higher than physiological release rates (.2 mM/s), NO would accelerate MPTP opening and Cytochrome c release. 26 It is noteworthy in this context that H 2 O 2 treatment used in the present study significantly reduced NO release; moreover, this response was counteracted by levosimendan, which increased in a dose-dependent manner NO production by an amount which was kept in the physiological rate.…”

Cytosolic Ca2+–Induced Apoptosis in Rat Cardiomyocytes via Mitochondrial NO-cGMP-Protein Kinase G Pathway

“…swelling, was significantly reduced by the external addition of PKG in isolated mitochondria (Borutaite et al, 2009). These phenomena reflect the sudden influx of excess Ca 21 into the mitochondria mimicked ischemia-reperfusion injury.…”

“…In cardiomyocytes, cytosolic PKG activators such as diazoxide and sildenafil, a typical inhibitor of phosphodiesterase type 5, reportedly inhibit MPTP opening and apoptosis by stimulating the mitochondrial K ATP channel (Takimoto et al, 2005;Das et al, 2005;Costa et al, 2006;Xi et al, 2009;Borutaite et al, 2009). On the other hand, Taimor et al (2000) reported that PKG inhibitor inhibited NO-induced cardiomyocyte apoptosis.…”

Effect of Nesfatin-1 on Ischaemia-Reperfusion Injury in Isolated Heart of Albino Rats

“…It is important to distinguish between the effects of ischemia and the effects of reperfusion on the heart. Necrosis does not occur during ischemia, but rather during the subsequent reperfusion, and this has been attributed to production of excessive reactive oxygen radicals [60] , which are an important mechanism of reperfusion injury, when molecular oxygen is reintroduced into a previously ischemic myocardium, undergoes a sequential reduction leading to the formation of oxygen free radicals and the influx of the inflammatory cells, leading to rupture of the cell membrane [61] .Those deleterious effects were counteracted by giving Nesfatin-1 during reperfusion as a post-conditioning factor (group III), but this was not the case if given before ischemia (group II). The resulting decrease in CRP in case of Nesfatin-1 preconditioning (group II) could be attributed to its intrinsic biological properties as an acute-phase reactant in case of ischaemic heart disease.…”