NADPH Oxidase4in Cardiomyocytes Hypoxia/Reoxygenation Injury: Key Role Of Mitochondrial NAD~+/NADH-Sirt3Signaling

Background and objectivesMyocardial ischemia caused by coronary occlusion and reperfusion injury followingthrombolytic therapy or stent placement severely affect the prognosis of coronary heartdisease. How to reduce the myocardial ischemia/reperfusion (MI/R) injury at the time ofrestoration of blood flow remains to be a hot spot in medical research which needed to beresolved. Reactive oxygen species (ROS) and their associated oxidative stress are one ofthe most important mechanisms of the MI/R injury. The mitochondrial electron transportchain enzymes and NADPH oxidases(Noxs) are the two major donors of ROS in MI/Rinjury. Moreover, the two donors have mutual influence and connect with each other.Some researches suggest that Nox2and Nox4are able to induce a large number of ROSgeneration, which cause great injuries in a variety of heart diseases. However, more andmore studies show that Nox4plays an important role in protecting heart from chronicstress. Previous studies of Nox4mainly focused on vascular injury, myocardialhypertrophy and heart failure, while the role of Nox4in the MI/R injury is not completely understood.Therefore, we established the hypoxia/reoxygenation (H/R) model with H9C2myocytesto investigate the role of endogenous Nox4in the H/R injury and its underlyingmechanisms, which could provide a new direction for clinical prevention and treatment ofMI/R injury.MethodsSection I: To establish H/R model with H9C2myocytes to investigate the role ofendogenous Nox4in the H/R injury.1. H9C2myocytes were exposed to H/R and Nox4was downregulated by RNAinterference. H9C2myocytes were randomly divided into control group, NC-siRNA group,Nox4-siRNA group, H/R group, H/R+NC-siRNA group and H/R+Nox4-siRNA group.2. The expression of Nox4protein was analyzed by Western blot. Cellular survival ratewas detected by MTT colorimetry. The apoptotic index of cells were determined byTUNEL assay. The activity of caspase-3was measured by spectrophotometry.CellularROS production was determined by DCFH-DA fluorescent probe and MDA level wasanalyzed by colorimetry.Section II: To investigate the molecular mechanism that endogenous Nox4protectsH9C2myocytes from H/R injury.1. H9C2myocytes were exposed to H/R and Nox4was downregulated by RNAinterference. H9C2myocytes were randomly divided into control组group, NC-siRNAgroup, Nox4-siRNA group, H/R group, H/R+NC-siRNA group and H/R+Nox4-siRNAgroup.2. Mitochondrial ROS production was determined by MitoSOX fluorescent probe. Themitochondrial membrane potential was detected by JC-1fluorescent probe and cellularATP level was measured by luciferase chemiluminescence assay. NAD+/NADH ratio wasassessed by colorimetry. The expression of Sirt3protein was analyzed by western blot.Results1. Compared with control group, H/R increased Nox4protein expression (P<0.01) andsignificantly reduced cellular survival rate. Moreover, H/R increased the AI index,theactivity of caspase-3and cellular ROS production as well as lipid peroxidation remarkably(P<0.01). H/R+Nox4-siRNA group decreased Nox4protein expression compared with H/R group (P<0.01). Furthermore, H/R+Nox4-siRNA group exhibited aggravated declinein cellular survival rate and significant increase of AI index and caspase-3activity(P<0.01). Meanwhile, H/R+Nox4-siRNA group enhanced cellular ROS production andlipid peroxidation further dramatically (P<0.05).2. Compared with control group, H/R resulted in significant increase in mitochondrialROS production measured by MitoSOX fluorescence (P<0.01). Meanwhile, mitochondrialmembrane potential and ATP content significantly reduced. More importantly,NAD+/NADH ratio increased after H/R (P<0.01), whereas Sirt3protein expressiondecreased (P<0.05). In H/R+Nox4-siRNA group, mitochondrial ROS production furtherenhanced, and mitochondrial membrane potential and ATP level further declinedcompared with H/R group (P<0.05). Nox4knockdown also led to a robust decline inNAD+/NADH ratio (P<0.01), while Sirt3protein expression was further suppressed(P<0.05).ConclusionDownregulation of Nox4significantly increased the susceptibility of cardiomyocytes toH/R injury as evidenced by compromised cell survival, increased cell apoptosis,intracellular ROS production and lipid peroxidation suggesting that endogenous Nox4may play an important role in myocardial protection against H/R injury. The protectiveeffects of endogenous Nox4in H/R injury may attributive to upregulatedNAD+/NADH-Sirt3signaling which plays a key role in reducing mitochondrial oxidativestress and dysfunction of energy synthesis.