The Role Of Mitochondrial Nitric Oxide Synthase In Myocardial Depression Of Septic Shock

BACKGROUNDSeptic shock is the most common cause of death in medical and surgical intensive-care units. Although care of critically ill patients and knowledge about pathophysiological mechanisms have progressed significantly in the past years, mortality still ranges from 16% in patients with sepsis to 40-60% in patients with septic shock. The yearly incidence of sepsis is 50-95 cases per 100 000, and has been increasing by 9% each year. This disease accounts for 2% of hospital admissions;roughly 9% of patients with sepsis progress to severe sepsis, and 3% of those with severe sepsis experience septic shock, which accounts for 10% of admissions to intensive care units.The septic shock patients have their specific changes of hemodynamic parameters. In the early stage of septic shock, the clinical presentations are described as a high cardiac output, increasing in tissue perfusion flow and the transport of oxygen, this stage is also called hyperdynamic phase;while in the late stage of septic shock, the clinical presentations aredescribed as a low cardiac output, decreasing in tissue perfusion flow and the transport of oxygen, this stage is also called hypodynamic phase, in this phase, the hypocardiovascular response is manifested as systemic hypotension. The systemic hypotension which is the symbol of septic shock will lead to the progressive, irreversible failure and death.During the late phase of sepsis, a large amount of cytokines are produced by multiple activated cells in response to septic shock, including tumour necrosis factor (TNF), interleukins, etc. These variety of cytokines induce the expression of inducible nitric oxide synthase (iNOS) in the vascular endothelial cells, resulting in the excessive production of nitric oxide (NO). Previous studies have demonstrated that excessive amounts of NO produced by the vascular tissue play an important role in the hyporesponsiveness in septic shock.The cellular NO is mainly synthesized by nitric oxide synthase (NOS) accomplished by oxidation of L-arginine and NADPH, yielding L-citrulline and NADP as coproduct. At least three NOS are known, they are referred as inducible NOS (iNOS or NOS2), endothelial NOS (eNOS or NOS3) and neuronal NOS (nNOS or NOS1). eNOS and nNOS are also called constitutive NOS (cNOS). A fourth type, mitochondrial NOS (mtNOS), this time differentiated by its subcellulart localization in the mitochondrial inner membrane, was simultaneously reported in rat liver mitochondria by Ghafourifar and Richter in 1997. Since mitochondria is one of the most important organelles in the body, many people focused on the finding and several paper reported that mtNOS may involved in the hyperproduction of NO in septic shock.In order to test the hypothsis, two methods that were lipopolysaccharide (LPS) method and cecal ligation and puncture (CLP) method to mimic septic shock were used to examine the role of mtNOS in myocardial depression of septic shock.OBJECTIVES1. To compare the difference between LPS and CLP induced septic shock model onhemodynamic parameters and NOS activity.2. To examine the role of mtNOS in myocardial depression of septic shock.3. To observe the relationship of mtNOS and mitochondrial permeability transition pore (MPTP) in septic shock.METHODS1. Experimental septic shock model(1) LPS model: Male Sprague-Dawley rats were injected with lmg/g body weight LPS intraperitoneally. Saline group injected with saline was used as control.(2) CLP model: Male Sprague-Dawley rats were randomised into two groups, CLP group and SHAM group. All the animals were anaesthetized by 4% chloral hydrate injected with lml/lOOg rat body weight intraperitoneally followed by an abdominal incision. The cecum was then ligated and punctured twice with an 18-gauge needle. Sham operated animals underwent the same surgical procedure except that the cecum was neither ligated nor punctured.2. The measurement of hemodynamic parametersAll the animals were anaesthetized by 4% chloral hydrate injected with lml/lOOg rat body weight intraperitoneally. The carotid artery was subsequently cannulated and connected to a blood pressure transducer to determine mean arteral blood pressure (MABP). Ventricular-dynamic parameters were determined following intraventricular cannulation via the carotid artery, including heart rate (HR), left ventricular developed pressure (LVDP), maximal rise/fall velocity of ventricular pressure (+/-dP/dtmax). All the parameters were recored via a data acquisition system (MedLab).3. The measurement of NOS activityHeart tissues were homogenized in ice-cold buffer. The homogenate was centrifuged at lOOOxg for 10 min at 2°C, and the supernatant was centrifuged at 8000xg for 10 min at 2°C. The supernatant was the cyroplasm. The pellets were re-suspended in buffer, and centrifuged at 8000xg for 10 min at 2°C. The pellets were the mitochondria. The NOS activities in isolated mitochondria and cytosol were determined respectively using the NOS activity assay kit. Protein concentration was determined by the Lowry assay with BSA as standard.4. The measurement of nitrite/nitrate (NOX")The cytoplasm was staining with Griess agent and measure the absorbance at 530nm.5. The measurement of NOThe NO fluorescent probe DAF-FM diacetate was used. Freshly isolated mitochondria were dispersed in K+-buffer for 30 min at 4°C. Mitochondria were washed with the K+-buffer twice. Fluorescence measurements were obtained with an fluorescence spectrophotometer using excitation and emission wavelength of 485 nm and 520 nm, respectively. Autofluorescence was corrected for by the inclusion of parallel blanks and it did not exceed 10% of the total fluorescence.6. Western blot analysisThe proteins of mitochondrial membranes and the postmitochondrial supernatant were separated by SDS-PAGE, blotted into nitrocellulose films, and probed with 1/500 diluted rabbit polyclonal anti-NOS3 and with a secondary anti-rabbit antibody conjugated with horseradish peroxidase and revealed by chemiluminescence.7. Measurement of mitochondrial permeability transition pore opening in isolated cardiac mitochondriaOpening of the MPTP causes mitochondrial swelling, which results in a reduction ofabsorbance at 520 run (A52o).RESULTSPART 1. The comparison of LPS model and CLP model in hemodynamic parameters and cardiomyocyte NOS activity1. Hemodynamic parameters(1) MABP: In the CLP model, it showed little increase in the early septic shock stage while significant decrease in the middle and late septic shock stage (PO.01). The LPS model also showed significant decrease but less than late septic shock stage (P<0.01).(2) Ventricular-dynamic parameters: In the CLP model, LVDP or +dP/dtmax showed significant increase and -dP/dtmax showed significant decrease in the early stage;in middle stage, ±dP/dtmax decreased obviously (PO.01). Both CLP late stage and LPS model showed significant decrease in all parameters (PO.01).(3) After using NOS non-selective inhibitor L-NAME in CLP-late stage or LPS model, the hemodynamic parameters recovered or partly recovered, but there were also several significant changes (PO.01)..2. The NOS activity in cardiac myocyte cytoplasmIn the CLP model, the NOS activity reached its peak at middle stage, in this stage, the total NOS activity was 5 times higher than the early stage while cNOS and iNOS were 3 times and 9 times seperately. The LPS model showed the same trend as the CLP late stage.3. The mtNOS activity in cardiac myocyteIn the CLP model, the mtNOS activity increased significantly in the middle stage andreached its peak in the late stage in which the mtNOS activity increased 5 times. The LPS model also showed the significant increase but much lower than CLP late stage and only 58.0% of the activity in CLP.late stage.4 The effect of L-NAME on the nitrite/nitrate (NOx') production in septic shock cardiacmyocyteAfter using L-NAME, the NOx* production drcreased obviously in both CLP late stage model and LPS model (PO.01). There were no differences in the production by iNOS but less production by total NOS or cNOS in CLP late stage group after this injection.PART 2. The role of mtNOS in myocardial depression of septic shock1. Hemodynamic parameters(1) MABP: After using the NOS inhibitors, the MABP which were 86.2%, 74.3% and 79.2% as the SHAM separately partly recovered.(2) Ventricular-dynamic parameters: All the Ventricular-dynamic parameters partly recovered after using different NOS inhibitors, including HR, LVDP and ±dP/dtmax, AND there were few differences between injection of AMG and 7-NI.3. Western blot of mtNOSThere was almost no expression in normal rat cardiac mitochondria. Its expression increased significantly about 51.8% in the CLP late stage and 7-NI could inhibite the overexpression of mtNOS (PO.01).4. The comparison of NO produced by mtNOS and iNOSMtNOS and iNOS showed less effect of the NO production in the SHAM group, but in theCLP late stage, both of the two NOS paly the most important role in the NO production and they produced 50.99% and 37.04% of the NO in the cell. And determined the NO produced by iNOS in the cytosol, it produced 86.62% of the total NO in the cytosol. iNOS may play more responsility in the NO production.PART 3. The relationship between mtNOS and MPTP1. The effect of NOS inhibitors on the cardiac MPTP of septic shockAfter adding 200 uM CaCh, only L-NAME group and 7-NI group showed the significant decrease of absorbance (PO.01) while there were no changes in the other groups..2. The effevt of CsA and Atr on the cardiac MPTP of septic shockThere were no obvious changes on the absorbance by adding 200 uM CaCl2.3. Western blot of mtNOS in the septic shock pretreated with CsA or AtrAtr could increase the mtNOS expression (PO.01) but it had no effect on the 7-NI treated rats;CsA could inhibite the mtNOS expression completely.CONCLUSIONS1. In the LPS model, iNOS may be the only reason to induce the hyperproduction of NO in septic shock while both cNOS and iNOS involved the NO overproduction. And in the comparison of mtNOS activity in the two models, we found that mtNOS also involved in the NO production and it may play the more important role in the CLP model.2. The overexpression of mtNOS is the one of the important reasons to induce myocardial depression in septic shock, but iNOS may play more crucial role than mtNOS.3. The opening of MPTP may take part in the mtNOS overexpression, the NO production by mtNOS can also influence the MPTP function, but it also need further reaserch on the mechanism of the interaction between the two parts.