We examine the effects of nitrite repletion on mitochondrial function, reperfusion ROS generation, organ function and survival in a 12-minute mouse cardiac arrest model. Cardiac arrest results in systemic nitrite depletion and low dose nitrite replacement (therapy with 50 nmol) at CPR initiation repletes these levels to near baseline and increases cardiac S-nitrothiols. Therapeutic nitrite repletion and S-nitrosation in heart is associated with transient, reversible inhibition of complex I reducing mitochondrial reperfusion ROS generation and oxidative injury. Nitrite improved pulmonary gas exchange, cardiac contractility and survival with a suggestion of neuroprotection.
Moderate NO reperfusion therapy is known to be protective29
but NO formation is limited by NO synthase’s dependence on oxygen and reduced substrates30
. Nitrite acts as a reservoir for NO during ischemia and nitrite reduction generates NO through NOS-independent pathways 6, 31, 32
. We demonstrate that global ischemia depletes nitrite systemically reducing its availability to act as a reperfusion NO source. This profound depletion with brief global ischemia was surprising but not unprecedented13
and explains why our nitrite dose did not achieve the “optimal” plasma levels (11.9 μM) noted after focal ischemia8
. Nitrite repletion early in reperfusion provides a NOS-independent source of NO to ischemic tissues.
Mitochondrial complexes I and III are major sources of pathological reperfusion ROS 33
and transient, reversible inhibition of complex I has been proposed as a mechanism to achieve cardioprotection34–36
. The protective effects of complex I inhibition have been described for nitrite23
, S-nitrosothiol donors28
and observed during classical ischemic preconditioning25
. Complex I has numerous cysteine residues available for S-nitrosation with resultant inhibition of electron flow37, 38
. Nadtochiy and colleagues S-nitrosated complex I in cardiomyocytes and isolated heart using S-nitroso-2-mercaptopropionyl glycine with associated reduction in ROS production and improved cardiac contractility after ischemia-reperfusion28
. Similar to our findings, these authors noted reversal of S-nitrosation and complex I inhibition 30 minutes after ischemia. Shiva and colleagues provided the first evidence that nitrite S-nitrosates complex I and reduces ROS production in liver mitochondria after in vitro
though inhibition persisted for 5 hours and was bypassed via complex II. Sun and colleagues have shown complex I to be one of several proteins S-nitrosated in cardioprotective ischemic preconditioning 25
Nitrite therapy is complex I specific based on the lack of effects using succinate. Complex I efficiency is uneffected therefore this isn’t due to complex I damage. Complex I inhibition is reversible based on restored oxygen and NADH consumption and ATP generation by 60 minutes. The increase in complex I oxygen consumption with placebo in the absence of increased NADH oxidation implies pathological oxygen consumption to form ROS rather than ATP which is prevented with nitrite. Based on our ROS and aconitase data, nitrite is an antioxidant. This mechanism complements prior observations of reduced tissue nitrotyrosine staining9, 39
, lipid peroxidation9
and superoxide production9
with nitrite therapy.
Cardiac arrest’s poor prognosis is driven primarily by brain and heart injury15
. Excepting hypothermia, no beneficial post-resuscitation therapies exist since CPR’s description ~50 years ago. Present post-resuscitation care is largely supportive15
. Nitrite’s role as a novel therapeutic would be of great importance in this setting.
Human myocardial dysfunction (stunning) is common cardiac arrest40, 41
, ultimately reversible42
and strongly associated with mortality40, 41
The molecular mechanisms of myocardial stunning after cardiac arrest remain unknown but loss of excitation-contraction coupling is believed to result from ROS injury and calcium-mediated proteolysis43
. Nitrite, by reducing ROS, may mitigate stunning similar to other antioxidants44
. The reduction in myocardial dysfunction likely explains the 50% relative survival advantage we noted. Further work is needed to characterize nitrite’s effects on brain injury but our results are encouraging.
We designed a mouse model of cardiac arrest with prolonged asystole to study the effects of nitrite on heart and brain injury after resuscitation. Our model utilizes hyperkalemia to induce arrest, limiting its clinical relevance and potentially causing artifacts which may be organ protective (eg cardioplegia) or injurious (endothelial damage perhaps causing RV dysfunction). In the context of these limitations, we demonstrate improvements in gas exchange, heart and brain function and survival. We demonstrate that nitrite transiently inhibits complex I resulting in an antioxidant effect. The ease in delivering IV nitrite,, its established human safety14, 31
, its reproducible cytoprotective effects in multiple organs and species6
all suggest that nitrite represents a promising post-resuscitation therapy after cardiac arrest.