Investigations On The Therapeutic Cardioprotective Effect Of Noninvasive Remote Ischemic Conditioning Against Acute Phase Of Myocardial Infarction

Objective:To investigate the effect of noninvasive remote ischemic conditioning（nRIC）on cardiac function,myocardial apoptosis,mitochondrial respiratory function and myocardial morphology against acute phase of myocardial infarction（MI）.Methods:1.Effect of nRIC on myocardial injury against acute phase of MI in ratsThe male Wistar rats were randomly divided into three groups:（1）MI group:MI model was established by permanent ligation of the left anterior descending artery（LAD）;（2）nRIC group:After the ligation of the LAD,the rats were treated with 4 cycles of 5 min ischemia/5 min reperfusion on the left hind limb once a day.（3）Sham group:the rats were taken heart LAD threading but no ligation,and nRIC was not performed.Animals in each group were fed under normal condition for 1,7,14days.After the experiment,hemodynamic parameters and heart rate were measured.Myocardial infarction size was determined by TTC staining.Changes of myocardial morphology were observed by hematoxylin-eosin（HE）staining.2.Effect of nRIC on myocardial apoptosis against acute phase of MI in ratsExperimental groups were same with the method 1.After the experiments,the blood samples were withdrawn from the abdominal aorta,and serum levels of Bcl-2and Bax were measured by ELISA.Left ventricular anterior wall was homogenized.Caspase-3 levels in the myocardial tissue were detected by ELISA.Apoptosis of cardiomyocytes were assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling（TUNEL）assay.3.Effect of nRIC on mitochondrial respiratory function against acute phase of MI in ratsExperimental groups were same with the method 1.After the experiment,left ventricular anterior wall was taken for lysis and homogenization.The activity of mitochondrial chain complexesⅠ,Ⅱ,Ⅲ,Ⅳand the content of ATP in myocardium were determined by colorimetry.Mitochondria was isolated from myocardium.Mitochondrial membrane potential and the opening of mPTP were measured.4.Effect of nRIC on oxidation stress and myocardial fibrosis against acute phase of MI in ratsExperimental groups were same with the method 1.After the experiment,heart weight/body weight（HW/BW）was determined.Left ventricle anterior wall was lysed and homogenized.The content of iNOS in myocardium was measured by ELISA.The extent of myocardial fibrosis was determined by MASSON staining.Results:1.Effect of nRIC on myocardial injury against acute phase of MI in ratsCompared with MI groups,at 1,7 and 14 day,hemodynamic indexs in the nRIC group were improved.In 1,7,14 day nRIC group,left ventricular end diastolic pressure（LVEDP）was decreased（11.38±1.15,12.48±2.36,9.85±2.01 mmHg vs17.68±1.41,17.40±2.66,18.40±2.39 mmHg,P<0.05）.The maximal rate of rise of left ventricular pressure(+dp/dt_max)was significantly increased（3.81±0.44,3.65±0.32,3.66±0.35 mmHg/ms vs 3.00±0.38,3.07±0.34,2.98±0.24 mmHg/ms,P<0.05,P<0.01）;In 7,14 day nRIC group,left ventricular systolic pressure（LVSP）was significantly elevated（112.65±5.95,112.28±5.57 mmHg vs 103.27±3.42,103.25±4.28 mmHg,P<0.05）and the maximal rate of decrease of left ventricular pressure(-dp/dt_max)were elevated（3.49±0.28,3.57±0.55 mmHg/ms vs 2.60±0.49,2.75±0.37 mmHg/ms,P<0.05,P<0.01）.Compared with MI group,heart rate was significantly reduced in nRIC group（433±13,437±13,420±16 beats/min vs 462±18,461±14,462±20 beats/min P<0.05,P<0.01）;nRIC decreased myocardial infarct size significantly（44.84±2.44%,35.78±4.41%,36.61±4.19%vs 56.25±2.64%,47.88±2.16%,48.45±1.81%,P<0.01）.Compared with the MI group,at 1,7 and 14day,HE staining displayed that nRIC can reduce inflammatory cell infiltration and alleviate the degree of myocardial damage.2.Effect of nRIC on myocardium apoptosis against acute phase of MI in ratsCompared with the MI group,at 1,7 and 14 day,the serum level of Bcl-2 in the nRIC group was elevated（490.29±60.31,470.44±62.75,450.79±44.45 pg/ml vs347.32±12.20,350.14±10.58,352.86±12.23 pg/ml,P<0.01）.The level of Bax in the nRIC group was decreased（116.00±18.21,114.89±25.74,151.58±44.57 pg/ml vs392.27±56.44,614.43±33.52,568.72±43.67 pg/ml,P<0.05）.Compared with the MI group,at 1,7 and 14 day,TUNEL staining displayed that the apoptotic index of myocardium was significantly reduced in the nRIC group（26.45±2.88%,25.00±1.57%,28.43±2.43%vs 30.32±1.34%,35.83±2.43%,38.45±1.95%,P<0.05）.3.Effect of nRIC on mitochondrial respiratory function against acute phase of MI in ratsCompared with MI group,at 1,7 and 14 days,nRIC improved mitochondrial respiratory,and mitochondrial respiratory chain complex activity was significantly increased.The activity of mitochondrial respiratory chain complexⅠin nRIC group was increased（0.37±0.04,0.43±0.09,0.38±0.07μmol/mg/min vs 0.23±0.06,0.27±0.03,0.25±0.08μmol/mg/min,P<0.05,P<0.01）;the activity of mitochondrial respiratory chain complexⅡwas elevated（0.043±0.005,0.058±0.006,0.055±0.007μmol/mg/min vs 0.038±0.005,0.040±0.006,0.039±0.009μmol/mg/min,P<0.01）;the activity of mitochondrial respiratory chain complexⅢwas increased（0.33±0.04,0.37±0.05,0.39±0.07μmol/mg/min vs 0.26±0.06,0.27±0.04,0.25±0.08μmol/mg/min,P<0.01）;the activity of mitochondrial respiratory chain complexⅣwas elevated（0.48±0.09,0.52±0.06,0.46±0.11μmol/mg/min vs 0.38±0.11,0.30±0.06,0.27±0.09μmol/mg/min,P<0.01）.ATP content was significantly elevated（17.34±5.09,20.75±8.57,22.98±7.63μmol/g vs 11.56±0.69,10.76±0.58,11.76±1.36μmol/g,P<0.05）.Compared with MI group,1,7,14 day nRIC could restrain the opening of mPTP（0.16±0.01,0.19±0.05,0.16±0.01 vs 0.22±0.01,0.21±0.01,0.25±0.04,P<0.05,P<0.01）and prevent the fall of mitochondrial membrane potential（7.51±0.43,9.79±0.74,10.95±0.99 vs 4.62±0.35,7.53±0.25,7.67±0.33,P<0.01）.4.Effect of nRIC on myocardial fibrosis against acute phase of MI in ratsCompared with MI group,at 1,7 and 14 day,HW/BW was decreased significantly in nRIC group（3.47±0.13,3.41±0.10,3.22±0.16 mg/g vs 4.09±0.15,3.58±0.16,3.48±0.12 mg/g,P<0.05,P<0.01）.iNOS expression was significantly decreased in nRIC group（54.56±6.22,49.50±7.23,52.72±6.38 ng/mg vs 68.03±3.75,68.39±7.24,66.97±7.15 ng/mg,P<0.05）;and nRIC can reduce myocardial fibrosis level and reduce collagen deposition.Conclusion:1.nRIC improves hemodynamics index,reduces myocardial infarct size,alleviates myocardial damage,thus exerting myocardial protective effect.2.nRIC increases the expression of Bcl-2,reduces the expression of Bax and Caspase-3,and inhibiting cardiomyocyte apoptosis in acute stage of myocardial infarction.3.nRIC can restrain the opening of mPTP,elevate mitochondrial respiratory chain complex activity and increase ATP level in myocardial tissue,thus improving the mitochondrial function.4.nRIC reduces the expression of iNOS protein and alleviates the degree of myocardial fibrosis,which might be involved in the cardioprotective mechanisms.