Neuronal nitric oxide synthase in heart mitochondria: a matter of life or death

Abstract

Due to its high energy demand, cardiac muscle has the highest density of mitochondria of all mammalian organs. Mitochondria supply the cells with ATP generated from the electron transport chain by oxidative phosphorylation (0.5 μmol ATP (g wet wt)−1 s−1 at rest). Oxygen is the terminal electron acceptor in this chain. A side-effect of this high metabolic rate is an important production of oxygen radicals, which are an inevitable side product of the electron transport chain due to ‘electron leakage’ generated in the sequential donation of electrons to molecular oxygen, which normally ends in H2O production. Mitochondria also generate nitric oxide (NO), another radical species. NO is generated by nitric oxide synthases (NOS). The specific isoform that is expressed in this organelle is still not clear. Nevertheless, in the heart, there is consistent evidence suggesting that neuronal NOS (nNOS) is the isoform that is found in mitochondria, based on pharmacological evidence, and on the fact that genetic deletion of nNOS abolishes NO production in mouse heart mitochondria (Kanai et al. 2001). The actual role of mitochondrial nitric oxide synthase (NOS) has remained elusive. The importance of NOS in heart mitochondria is high, given the critical role of this organelle in energy production in such a metabolically active organ. Beside their role in energy production, mitochondria are deeply connected to the processes that lead to cell death. In the case of the heart, the impact of apoptosis and necrosis is clearly evidenced in myocardial infarction or after an episode of ischaemia–reperfusion. For instance, an episode of ischemia/reperfusion is followed by a burst of reactive oxygen species (ROS). In this phenomenon, mitochondria also play in important role generating these species. On the other hand, the protective effects of nitric oxide on cardiac disease are also established in the literature. In a recent issue of The Journal of Physiology, Dedkova and Blatter explored the role of mitochondrial NO production and its relationship with ROS production and the mitochondrial permeability transition (Dedkova & Blatter, 2009). Using permeabilized cat cardiomyocytes as a model, the authors used pharmacologic probes, together with confocal microscopy that imaged a battery of pathway-specific fluorophores, to investigate some of the mechanisms by which NO modulates mitochondrial permeability. NO production was assessed using DAF-2, in the absence of functional caveolae and sarcoplasmic reticulum (disrupted by cyclodextrin and thapsigargin, respectively), to rule out the possible influence of eNOS and nNOS activity from those compartments. Although these manoeuvres may appear questionable, these results were also confirmed using specific inhibitors for NOS isoforms. More importantly, mitochondria-derived NO was abolished when mitochondria were uncoupled (blocking the respiratory chain or dissipating the mitochondrial membrane potential, Δψ), or when the mitochondrial Ca2+ uniporter was blocked. Since nNOS activity is Ca2+ dependent, dissipating the membrane potential that creates the driving force for Ca2+ influx or blocking the uniporter directly abolished NO production. These results confirmed the observations of Kanai et al. (2001) on isolated heart mitochondria using a NO-sensitive electrode. Interestingly, cytosolic [Ca2+] above 1 μm (a concentration observed during adrenergic stimulation or reperfusion, for instance) was necessary to activate mtNOS, and this Ca2+ requirement also included calmodulin. Since the cardiomyocytes were permeabilized, supplementation with l-arginine was necessary for mitochondrial NO synthesis. Importantly, part of the urea cycle in which l-arginine is produced and consumed takes place in the mitochondria. Arginase II, an enzyme that catabolizes arginine, is located in mitochondria and competes with mtNOS for substrate. In absence of l-arginine, ROS production was observed upon Ca2+ rise. The addition of arginine almost abolished ROS production and arginase inhibition decreased ROS production by 50% (without arginine supplementation). Another target for NO assessed by the authors (and probably the most critical experiment) was the mitochondrial permeability transition pore (PTP). The permeability transition pore is a large conductance channel (about 1 nS) in the inner mitochondrial membrane that opens in response to high [Ca2+], low [ATP] and ROS. Opening of this channel causes a dramatic depolarization of the mitochondria followed by ATP depletion and cell death. The PTP opening (induced by high [Ca2+] and monitored by using calcein-loaded mitochondria) was prevented when ROS production was neutralized using a superoxide dismutase mimetic, or when l-arginine or tetrahydrobiopterin (BH4), a co-factor for NOS, was added as a supplement. Notably, supplementation with l-arginine nearly abolishes the pore opening, with an effect similar to cyclosporine A, a PTP inhibitor. These results suggest that mtNOS-derived NO inhibits the PTP opening when cytosolic [Ca2+] is high. It is not clear whether this effect is mediated directly by NO (for instance direct S-nitrosylation of reactive thiols) in the pore or is a result of neutralizing ROS production. On the contrary, in the presence of oxidative stress (like after reperfusion or during congestive heart failure) or in the absence of enough l-arginine, an increase in cytosolic [Ca2+] triggers opening of the permeability transition pore and, inevitably, leads to cell death. These results strongly support a novel cardioprotective role for mtNOS. It seems likely that mtNOS is nNOS or a spliced variant of it (likely nNOSα or nNOSμ), as nNOS-deficient mice exhibit increased ROS production (Kinugawa et al. 2005) and after a myocardial infarction, they show increased myocardial damage and lower survival rates than wild-type animals (Saraiva et al. 2005). Consistent with this idea, heart-specific nNOS overexpression has been shown to be cardioprotective in a model of volume overload-hypertrophy conductive to heart failure (Loyer et al. 2008). These results have been attributed to nNOS located in the sarcolemma or in the sarcoplasmic reticulum, but not to nNOS in mitochondria. An exciting question that arises from Dedkova's observations is whether the effects of NO are direct on the permeability transition pore, or indirect, based on modulation of another target that may prevent its opening, like the mitochondrial K+ channel (mitoKATP) or protein kinase Cɛ, both known as cardioprotective mediators that prevent PTP induction. Indeed, the PTP may be an interesting target for preventing myocardial damage. Recently, a pilot clinical study showed that treatment with cyclosporine A, a PTP inhibitor, decreased myocardial damage in patients who underwent percutaneous coronary intervention (reperfusion) after a myocardial infarction (Piot et al. 2008). This is encouraging for the search of other compounds that inhibit PTP. In summary, the work by Dedkova and Blatter suggests that mtNOS, likely to be an nNOS, plays an important role in cardioprotection, especially under circumstances that produce ROS generation or high calcium load into the mitochondria such as reperfusion after a myocardial infarction.