Inhibition Of Poly (ADP-ribose) Polymerase And Inducible Nitric Oxide Synthase Protects Against Ischemic Myocardial Damage By Reduction Of Apoptosis

BackgroundMyocardial infarction (MI) is a common cardiovascular disease and one of the leading causes of death in the world. Despite the importance of treatment, but about 6-12% patients of myocardial infarction can not be timely coronary revascularization (such as PTCA, CABG etc.) to save dying myocardium.Therefore, clinical research on new methods of treatment not only has important scientific significance, but also has significant social benefits.MI is caused by deprivation of oxygen and nutrition and induces inflammatory response, resulting in apoptosis of cardiomyocytes. A variety of factors have involved in the process, includingPARPl.PARP1 is a nuclear enzyme which functions as a DNA damage sensor and can be activated by DNA breaks. Activation of PARP1 leads to synthesis of poly(ADP-ribose) (PAR) from nicotinamide adenine dinucleotide (NAD) and ATP, which is important for DNA repair. However, excessive activation of PARP1 can cause depletion of NAD and ATP, which results in cellular dysfunction and eventually cell death. Moreover, PARP1 is required for specific NF-κB-dependent gene activation and acts as a transcriptional co-activator of NF-κB. It can regulate a variety of key inflammatory cytokines including monocyte chemotactic protein-1(MCP-1), inducible nitric oxide synthase (iNOS), and adhesion molecules, all of which are regulated by NF-κB.iNOS is a critical member among these inflammatory cytokines regulated by PARP1 via NF-κB pathway. Induction of iNOS results in excessive production of nitric oxide (NO), which reacts with superoxide anion to form peroxynitrite with resultant tyrosine nitration, DNA damage, and activation of PARP1, which lead to changes in inflammatory responses and promotion of cell death via apoptosis and necrosis.In the present study, we hypothesized that the oxidative DNA damage that results from the generation of reactive species during the onset of myocardial infarction causes excessive activation of PARP1, which results in an increased expression of iNOS and imbalance of cell survival mechanisms that contribute to the death of cardiomyocytes and aggravating cardiac functions. Pharmacologically inhibition of PARP1 or iNOS protects the cardiomyocytes from death and improves heart functions. We used a rat model of myocardial infarction and investigated a potential role for PARP1 and iNOS in the process of MI and examined the protective effects of their inhibition.Objectives1. To investigate the effects of PARP1, iNOS on cardiac structure and heart function after myocardial infarction in rats2. To investigate the effects of inhibitors of PARP1, iNOS on cardiac function after myocardial infarction in rats.3. To further explore the possible molecular mechanism of inhibitor PARP1, iNOS protective effect on heart function after myocardial infarction.Methods1. Animal model and inhibtor treatmentRat were divided into 2 groups:control and MI. MI group rats were anesthetized and given descending coronary artery ligation. After the induction of MI, rats were divided into 3 groups for treatment:MI group, PARP-1 inhibitor (DPQ), iNOS inhibitor (1400W).2. Measurement of body basic indicatorsWe measuring the heart rate, body weight and blood pressure in rat of each groups before and after ligation of coronary artery.3. Cardiac function measurementThe cardiac diameter and function were measured by the use of echocardiography on 3 days, two weeks and four weeks after operation. The indicators includes:left ventricular systolic diameter (ESD), left ventricular end diastolic diameter (EDD), fractional shortening (FS)=((EDD-ESD)/EDD) × 100, take multiple measurements multiple sets of data for statistical statistics.4. Assessment of caspase-3 activityWe selected caspase-3 for further evaluation the apoptosis level in regional myocardial infarction. The activity of caspase-3 were determined by kit instructions.5. Assessment of active oxygen and its productsBuy DHE staining kit, follow the steps of the experimental animals stained paraffin sections drawn DHE probe labeled cells in the generation of superoxide amon.6. ImmunohistochemistryTissue was paraffin-embedded and sectioned for staining with PAR, 3-nitro-tyrosine (3-NT) and vWF in myocardium, then comprehensive analysis.7. Real-time RT-PCRTotal RNA was extracted from frozen myocardium in -80℃. In the experiment, the expression of mRNA was analyzed.8. Western blotProteins were extracted from left ventricular myocardium stored in -80 ℃. Caspase-3, PAR and vWF were analyzed in our experiment.9. Statistical analysisAll statistical analyses involved use of SPSS 16.0. Data are reported as mean ± standard deviation. Comparisons of 2 groups involved unpaired t test and multiple groups were performed by one-way ANOVA followed by post-hoc individual comparisons. Differences were considered statistically significant at p<0.05.Results1 The general conditions of rats.The whole experiment died in 5, finally 35 rats completed the final experiment. At the end, each group compared with the control group, the MI group increased heart rate (P<0.05), and no difference in MI+DPQ group and MI+1400W group, heart rate, blood pressure, body weight (both P> 0.05).2. Inhibitor of PARP-1 or iNOS could improve MI-induced cardiac remodeling and function.LVIDs and LVIDd were significantly expanded in MI group compared with control group (P<0.05, while drugs intervention could significantly reduce MI-increased LVIDs and LVIDd (Both P<0.05). FS and LVEF were reduced in MI group compared with control group (Both P<0.05), however the FS and LVEF were improved in both two drug intervention groups at different degree (P<0.05).3. The effect of PARP inhibitor and iNOS inhibitor on cardiomyocyte apoptosis in infarcted myocardium.The apoptosis of cardiomyocyte significantly increased in MI group compared with control group. While the PARP and iNOS inhibitor could reduced MI-induced apoptosis. PARP inhibitor group and iNOS inhibitor group was not significantly different (P>0.05).4. The effect of PARP inhibitor and iNOS inhibitor on expression and activity of caspase-3.The activity and expression of caspase-3 protein in MI group were significantly higher than that in sham operation group, while inhibition of PARP-1 and iNOS could decrease the activity and expression of caspase-3 (Both P<0.05). But there is no significant difference between the two groups (P>0.05).5. PARP inhibitor and iNOS inhibitor could decrease the expression of PARP-1 and iNOS, respectively.In MI rat myocardium, the inhibitive efficient of DPQ and iNOS was 51.14% and 51.84%, respectively. The expression pyrolysis PARP fragment significantly increased in MI group, while the PARP inhibitor and iNOS inhibitor could reduced the activity of PAR in infarcted myocardium (P<0.05). The expression of iNOS was increased in MI group, while inhibition of PARP-1 could reduced iNOS in infarcted myocardium.6. Inhibition of PARP or iNOS could reduce the level of ROS and 3-NT in infarcted myocardium.The level of ROS and 3-NT in MI group were significantly increased in MI group (P<0.05), while Inhibition of PARP or iNOS could reduce the level of ROS and 3-NT in infarcted myocardium (P<0.05).7. Inhibitor PARP or iNOS could both increased the expression of vWF in infarcted myocardium.The expression of vWF was decreased in MI group, while inhibition of PARP or iNOS could increased the vWF level in infarcted myocardium (P<0.05), suggested that angiogenesis was enhanced.Conclusion1 PARP1 and the excessive activation of iNOS apoptosis, and myocardial cells is closely related to inflammatory reaction, DPQ and 1400W can effectively suppress the activation of myocardial ischemia and necrosis induced by PARP1 and iNOS.2 PARP1 and iNOS inhibitors can reduce the expression of apoptosis related factors, can reduce the peroxynitrite ischemia site production, inhibition of peroxidation, thereby reducing the cell apoptosis, inflammatory reaction, the protection of heart function.3 PARP1 inhibitors and iNOS inhibitors reduced left ventricular myocardial infarction rat systolic diameter (ESD) and left ventricular end diastolic diameter (EDD), increased fractional shortening (FS), showed that PARP1 inhibitors and iNOS inhibitors improve ventricular remodeling in rats after myocardial infarction, improve heart function.BackgroundWith the aging of the population, the incidence rate of heart failure is increasing day by day. As one of the main causes of death in heart diseases, heart failure is the final stage of various heart diseases. And it is a group of syndrome which is due to various causes of myocardial injury, leading to changes of cardiac structure and function, eventually leading to low ventricular pump function. The pathophysiological mechanism of heart failure is very complicated, including the renal hypoperfusion and sodium and water retention, increased tension of ventricular wall and disturbance of hemodynamics, left ventricular remodeling, hypertrophy and addition of preload and afterload caused by myocardial damage. Although the resesarches about heart failure have been deepened, the prevention, assessment and treatment of heart failure is still a medical problem now. Poly two ADP ribose transferase 1 (PARP-1) is involved in many physiological and pathological processes of cell injury and repair. It is also closely related with the occurrence and development of many cardiovascular diseases. Accompanied by vascular induced nitric oxide synthase’s (iNOS) activity enhanced, endothelial nitric oxide synthase’s (eNOS) activity decreased and decrease of the NO generation, the injury to vascular endothelial cell function occur with the stimulation of inflammation or oxidative. There are some consensuses and evaluation standards on clinical heart failure diagnosis, risk stratification, and treatment, such as echocardiography, biochemical detection of BNP and its precursors. These play important role in the diagnosis and treatment of heart failure. Based on the research result of the generation and biological function of PARP-1 and iNOS, we focus on the observation of theri correlation between clinical assessments standards of the severity of heart failure patients, furtherly estimate its role in the progression of heart failure.Methods1. Experimental patients and groupsFrom 2011 February to 2013 August, the cardiac insufficiency subjects were selected from the patients in the cardiac department of the Second Hospital of Shandong University. The clinical evaluation of cardiac function was about NYHA grade Ⅱ-Ⅳ, based on echocardiography measurement of left ventricular ejection fraction<50%, or immune detecting blood BNP>100ng/ml. The subjects with normal heart function were selected as control group (n=32), which has no dysfunction symptoms, left ventricular ejection fraction >50% and clinical detection of blood BNP<100ng/ml.2. Measurement criteriaWe kept an account of the gender, age, weight, height, heart rate, blood pressure; drugs as digoxin, ACEI/ARB, beta blockers and diuretics and others before admission of every selected patient. After admission, blood routine, liver and kidney function, blood glucose, TC, TG, LDL-C, HDL-C, BNP, CRP, ESR, myocardial enzyme, ECG, echocardiography, chest x-ray and other related inspection were all need detected.Method for blood pressure:The left upper arm blood pressure were measured after waking up at early morning without any form of physical activity once a day, and taking the average of SBP as the indexes, with calculated BMI according to kg/m2.The measurement of echocardiography is completed by experienced ultrasound physicians, which is inspected in strict accordance with the America Echocardiography Guide. Measure the left ventricular end diastolic diameter (EDD), left ventricular end systolic diameter (ESD), left ventricular ejection fraction (LVEF), left atrial diameter (LA), mitral regurgitation area (MRA) at the view of parasternal long axis of the left heart. Select the average of three consecutive cardiac cycles of the measured results as the study data.3. Others(1) The peripheral vein blood plasma separation and preservation:Collect elbow venous blood 3ml when limosis at early morning of the research objects, inject into blood collectors of sodium citrate (EDTA) anticoagulation, centrifuging for 20 min in a low temperature, supernatant were packeted in different labeled EP tubes and frozen at-80 degree refrigerator rapidly.(2) The separation of white blood cells and attentions:Collect the elbow venous blood 5ml of objects, transfer human peripheral white blood cells separated liquid will the same volume into 15ml centrifuge tube. The second layer was leukocyte enriched layer after centrifugation at room temperature, and after washing it was frozen at -80 degree refrigerator as soon as possible.(3) The detection step of plasma BNP (enzyme-linked immunosorbent assay):Plasma BNP was measured with a double antibody sandwich ELISA method.(4) The expression of PARP-1 and iNOS proteins in the cell by Weston-blot method.(5) The expression of PARP-1 and iNOS gene in the cells by RT-PCR.4 StatisticsStatistical analysis was performed by SPSS 17.0 software, the measurement data using x±s to express; using t test to express the comparison between measurement data, using chi square test to express the comparison between count data; using linear correlation analysis (Person’s related) to express correlation among variables; there were statistically significant differences if P<0.05.Results1. Compared with the control group, there was no significant difference in the indexes such as body mass index (BMI), hemoglobin, blood glucose, blood lipid (heart rate, TG, TC, LDL-C, HDL-C) (P>0.05), blood pressure (SBP) levels in heart failure (IV) group decreased than in the control group, the difference was statistically significant (P<0.05).2. Compared with the control group, the EDD, ESD and LVEF were all statistically significant difference in cardiac dysfunction patients, LVEF which could prompt cardiac systolic function decreased significantly (P<0.05), conversely BNP concentration in serum increased obviously (P<0.05). The correlation coefficient between LVEF and heart dysfunction was 0.9676 (P<0.01), and the correlation coefficient between concentration of BNP and heart dysfunction was 0.8796 (P<0.01).3. The relative gene expression of different groups of PARP1 and iNOS, compared with the control group, were significantly increased in the heart dysfunction group (II, III, IV group) (P<0.05); PARP1 gene relative expression is positively correlated with the grade of cardiac function (R2=0.7919, P<0.05); iNOS gene relative expression is also positively correlated with the grade of cardiac function (R2=0.6737, P<0.05).4. The relative protein levels of PARP1 and iNOS in the heart dysfunction group (II, Ⅲ, IV group) were significantly increased (P<0.05), compared with the control group. PARP1 and iNOS protein levels were positively correlated with the grade of cardiac function (R2=0.973, R2=0.7696, P<0.05)5. Correlation analysis between the expression of PARP1/iNOS and LVEF showed that both the relative gene expression and protein level of PARP1/iNOS were negatively correlated with the level of LVEF (P<0.05).6. Both the relative gene and protein expression quantity of PARP1/iNOS were positively correlated with BNP (P<0.05), and the severity is consistent with heart function.Conclusion1. Both the gene and protein level results showed that the expression of PARP-1 and iNOS were significantly higher in cardiac dysfunction patients than control group.2. Expression of PARP-1 and iNOS kept a significant positive correlation with the grade of cardiac dysfunction, and the level were positively correlated with BNP and negatively correlated with the heart function detected by echocardiography.3. Increased expression levels of PARP-1 and iNOS participate in heart failure played an important role in the process of heart failure, might prompt the severity of heart failure.