Anisodamine Protecting Cardiac Mitochondrial Function In Post-cardiopulmonary Resuscitation Rat Model

Objective To reveal the mitochondrial dysfunctions in post-cardiac arrest resuscitation rat model, and the effect of intervention by anisodamine on mitochondrial dysfunctions. The protecting mechanisms of anisodamine on cardiac mitochondrial function in post-cardiac arrest resuscitation rat model were studied too.Methods 1.Eighty wistar rats were divided into 2 groups randomly, and the cardiac arrest was induced by alternating current via pacing electrode placed in esophagus.PCR was performed 5 minutes later, and epinephrine was administrated at 0.1mg/kg as soon as PCR was performed in Epinephrine group. Epinephrine at 0.1mg/kg and anisodamine at 10mg/kg were administrated in epinephrine and anisodamine group. Incidence of cardiac arrest and ratio of ROSC were calculated, and the types of cardiac arrest electrocardiogram were analyzed.2. One hundred and twenty wistar rats were divided into 3 groups randomly, A group was control group, B group was epinephrine group and C group was epinephrine plus anisodamine group. Every group was divided into five subgroups according to the time after ROSC. A1,B1 and C1 were instant group after ROSC. A2, B2 and C2 were 3 hours group after ROSC. A3, B3 and C3 were 6 hours group after ROSC. A4, B4 and C4 were 12 hours group after ROSC. A5, B5 and C5 were 24 hours group after ROSC. The cardiac arrest wasn't induced in group A, and the cardiac arrest was induced in B and C groups. Physiological saline at 1 ml/kg was consecutively administrated to A and B groups after ROSC, and anisodamine at 10mg/kg was consecutively administrated to C groups after ROSC.The rat model were executed at the setting time, and heart tissues were obtained for centrifugating mitochondrion. The ATP contents of heart tissues were assayed with high performance liquid chromatography. The mitochondrial ROC contents and the mitochondrial membrane potentials of heart tissues were assayed with ELIASA. The mitochondrial Ca2+ contents and membrane permeability of heart tissues were assayed with Laser Scanning Confocal Microscopy and ELIASA.The mitochondrial structures of heart tissues were observed by transmission electron microscope.The ANT2mRNA and ANT contents of heart tissues of instant,3 hours and 24 hours after ROSC groups were assayed with Real-Time PCR and Western-Blot.Results 1. Incidence of the cardiac arrest induced by modified alternating current via pacing electrode placed in esophagus was 85%. The percentage of PEA was 59%, ventricular rhythm 16% and electrocardiac silence 25% in all lectrocardiogram of cardiac arrest rat model. Epinephrine plus anisodamine group had higher ratio of ROSC,67%vs33% P<0.05, comparing with epinephrine group.The ATP contents of heart tissues in both epinephrine plus anisodamine group and epinephrine group were less than control group in every assaying time point, P<0.05. The differences of ATP contents between epinephrine plus anisodamine group and epinephrine group had no statistical significance at instant, 3 hours and 6 hours after ROSC, but they were obvious, P<0.05, at 12 hours and 24 hours after ROSC. The ATP contents in epinephrine plus anisodamine group had a rapider recovery comparing with epinephrine group. The results of ROS contents revealed that the mitochondrial ROS contents increased at every measuring point in both intervention groups comparing with control group, P<0.05, but more ROS could be found in epinephrine group comparing with epinephrine plus anisodamine group, P<0.05, at every measuring point. Calcium overload was showed at every measuring point in epinephrine group, and it was more serious with 22 times content of control group at 3 hours after ROSC. Comparing with epinephrine group, the mitochondrial Ca2+ contents of heart tissues was lower at every measuring point in epinephrine plus anisodamine group, P<0.05,and had come to normal level at 12 hours after ROSC, P>0.05. The results of red fluorescence intensity seen with Laser Scanning Confocal Microscopy, depending on the mitochondrial Ca2+ contents, was coincident with the results of ELIASA. The results demonstrated that membrane potentials decreased at every measuring point in epinephrine group, comparing with control group, P<0.05,and that decreased more obviously at instant,3 hours and 6 hours after ROSC than 12 and 24 hours after ROSC. Although the membrane potentials in epinephrine plus anisodamine group also decreased before 6 hours of ROSC, the decreasing range was smaller comparing with epinephrine group, P>0.05, and the level of membrane potentials in epinephrine plus anisodamine group were nearly the same level of control group at 12 hours after ROSC, P>0.05. The mitochondrial membrane permeability at instant time point after ROSC increased in both epinephrine group and epinephrine plus anisodamine group, P>0.05. The peak of mitochondrial membrane permeability increasing was at 3 hours after ROSC, subsequently, the mitochondrial membrane permeability recovered gradually. Comparing with epinephrine group, smaller mitochondrial membrane permeability and more rapid recovery could be seen in epinephrine plus anisodamine group, P>0.05. The mitochondrial membrane permeability in epinephrine plus anisodamine group had resumed at 24 hours after ROSC, P>0.05. Electron microscope results of myocardium showed mitochondria cristae disappearance, vacuolization et al in epinephrine group and mitochondrial swelling and blurred mitochondria cristae could be seen in epinephrine plus anisodamine group. The mitochondrial damaging degree in epinephrine plus anisodamine group was between control group and epinephrine group.The difference of ANT2mRNA contents had no statistics significance between epinephrine group and control group at instant point after ROSC. The results of ANT2mRNA contents at 3 hours and 24 hours point after ROSC showed higher level in both epinephrine group and epinephrine plus ansodamine group than that in control group, P>0.05,but the difference between epinephrine group and epinephrine plus ansodamine group had no statistic significance, P>0.05.The results of ANT expression in myocardium revealed ANT/B-actin in both epinephrine group and epinephrine plus ansodamine group was higher than that in control group at any meaturing point, P>0.05,but the difference of ANT expression between epinephrine group and epinephrine plus ansodamine group had no statistic significance, P>0.05.Conclusion 1. Incidence of the cardiac arrest rat model induced by modified alternating current via pacing electrode placed in esophagus was high, and this cardiac arrest model suits CPR research more.2. This study confirmed that administration of anisodamine and epinephrine can rise the ratio of ROSC again.3.The myocardium mitochondrial dysfunction was obvious after cardiac arrest and resuscitation. The mitochondrial dysfunction was more obvious at 3 hours after ROSC.4. Administration of anisodamine can release mitochondrial calcium overload during and after resuscitation,and protect mitochondrial function. 5. The protecting mechanisms of anisodamine on mitochondrial function don't include increasing ANT expression on mitochondrial membrane.