Effect Of Cardiac Deacetylase SIRT3 On Ischemia Reperfusion Arrhythmias In Mice Heart And The Underlying Mechanism

Introduction:The coronary heart disease has become the first major type illnese to threaten Chinese people’s health with the remarkable improvement of people’s material life. With the extensive application of drug thrombolysis, coronary stent implantation and bypass operation of coronary artery, myocardial ischemia/reperfusion injury(MIRI) is increasingly becoming a hot spot in cardiovascular research field. One of the main manifestation of MIRI is arrhythmia and it would be a life-threatening condition if it is serious and leads to cardiogenic shock.Therefore,to clarify the etiology of the arrhythmia caused by MIRI and to find therapeutic measures are important subjects for clinical medicine.The etiology of the ischemia-reperfusion arrhythmias is complex. Current studies suggests that energy metabolism dysfunction and too much reactive oxygen species(ROS) produced by reperfusion are main physiopathologic basis of MIRI. Organisms have ROS clean systems.The key enzymes represented by manganese superoxide dismutase(Mn SOD) and Catalase(CAT) can effectively clean overproduced ROS.However, when cardiac muscle cells are under anoxia and ischemia, the function of ROS clean systems decreases or loses.That ROS damage mitochondriaaggravates disorder of energy metabolism, and spurs mitochondria to produce more ROS.We speculate that protecting mitochondria function in cardiac muscle cells and antioxidasactivity in ROS clean systems is the feasible approach to prevent and treat the arrhythmia caused by MIRI.Sirtuin protein family is a NAD+-dependent histone deacetylaseclass.Researches have found that Sirtuin3( Sirt3) takes part in regulation of many crucial enzyme in mitochondriaenergy metabolism with its deacylation function and then increases adenosine triphosphate(ATP) amount.Another research has found that Sirt 3 could increase gene expression of Mn SOD and CAT and rise Mn SOD gene activity.These studies hint Sirt 3afunction possibly play an important role in the emergence of reperfusion arrhythmias.Our research is to observe Sirt3’s impact on the reperfusion arrhythmias with Sirt3 gene knockout( KO) mice and further analysis whether the decrease of Sirt3 expression would aggravate MIRI-inducedcardiac oxidative stress and then aggravate reperfusion arrhythmias.The research will affords clinical data and theory for prevention and cure of reperfusion arrhythmias. Objective1.To be clear the key function of Sirt3 in triggering reperfusion arrhythmias. The emphasis to be discussed is the biological function of myocardial antioxidase system regulated by Sirt3 during this process.2.To investigate whether Sirt3 KO can reduce gene expression of Mn SOD and CAT and lower Mn SOD activity.3.To discuss NAD+ treatment whether can increase Sirt 3 activity in myocardium,raise Mn SOD activity,and surpress excessive production of ROS and at last inhibit I/R-induced arrhythmia. Methods1.Twenty-four grown(3 months) Sirt3 KO mice and twenty-fourgrown wild-type(WT) mice were randomized into control group(Control), sham group(Sham), ischemia reperfusion group(I/R) and NAD+ treated I/R group(I/R+NR). NAD+ treatment of I/R+NR group was performed by intraperitoneal injection 7 days(1 mg/kg/day),then acute MI/R mice model was built.. Silk thread cut across the left descending anterior coronary artery of mice of sham group,while their coronary artery not tied.2.2nd- standard lead ECG was monitored in the whole process of making MI/R mice model to record types of reperfusion arrhythmias, electrodes inserted into skin underneath on their limbs. Objective judgement criteria is in accordance with Lambeth standards and grading according to the papers by Fryer et al.Arrhythmia scores in 30 minutes were calculated and then we further analysis the data.3.After ROS sensitive fluorescent probe CM2-H2 DCFDA labeled myocardial cells, fluorescence intensity variation of H2 DCFDA was detected using fluorescence microscope,to observe ROS content variation.ROS positive cells were dyed red in cell nuclear region.Image analysis software Image-Pro Plus 6.0(Media-Cybernetics Inc) was used to calculate average optical density.4.BCA method was used to measure concentration ofcardiac protein and Western Blot was performed to measure Sirt3, Mn SOD and CAT expression level.5.Sirt3 activity in myocardium was detected by colorimetry detection kit and Mn SOD activity by Mn SOD activity detection kit. Results1.Western Blot showed that Sirt3 was expressed in normal myocardium.Compared with WT mice group,Sirt3 expression was significantly decreased in SIRT3 KO mice.Thus showed mice models were successfully built.2.Analysing the calculated arrhythmia scores in 30 minutes suggested arrhythmia was hardly detected in WT Sham mice group,while myocardial ischemiareperfusion injury could induce arrhythmia in WT mice group. Arrhythmia could be observed in SIRT3 KO Sham mice group,and the arrhythmia scores in SIRT3 KO mice group were significantly higher than in WT mice group.Thus indicated that SIRT3 KO facilitated arrhythmia.3.After myocardial ischemia reperfusion,ROS production in WT mice group were significantly higher than in WT Sham mice group.ROS production augment could be observed in SIRT3 KO Sham mice group and after myocardial ischemiareperfusion increment in SIRT3 KO mice group was greater than that in WT I/R mice group.The trend was similar to that of arrhythmia scores.4.Compared with those in WT mice, cardiac Mn SOD and CAT expression decreased in SIRT3 KO mice.5.NAD+ treatment significantly increased cardiac Sirt3 and Mn SOD activity, inhibited ROS production and consequently suppressed I/R-induced arrhythmia in WT mice. However, NAD+ induced cardioprotctive effects were blunted in SIRT3 KO mice.Conclusions1.Sirt3 is an important endogenous myocardial protection factor,and its reduction or shortage will induce the inhibition of ROS scavenge system,further aggravate reperfusion arrhythmias.2.NAD+ treatment can significantly increase Sirt3 activity,and then increase Mn SOD activity, inhibite ROS production and consequently suppresse reperfusion arrhythmias.3.Decrease of Sirt3 expression or activity with subsequent ROS production plays an important role during reperfusion arrhythmias.