| At present, coronary heart disease is still a major disease to threaten the human life and health. Acute myocardial infarction(AMI) is one of the important clinical types with the clinical characteristics of high morbidity and high mortality. To open infarction related artery(IRA) rapidly and restore myocardial effective reperfusion is a basic principle for the treatment of AMI. Today, thrombolysis therapy or primary percutaneous coronary intervention(PCI) has become the important treatment strategies for patients with ST elevation myocardial infarction(STEMI). However, after restoring reperfusion of ischemic myocardium, myocardial ultrastructure, function, metabolism and electrophysiology were further damaged. This secondary injury on basis of ischemic injury was called myocardial ischemia/reperfusion(I/R) injury. Although rapid development of interventional treatment technology and therapeutic drug has been made, there is still no exactly effective method for the prevention and treatment of myocardial I/R injury. Therefore, reducing myocardial I/R injury in order to restore effective myocardial reperfusion is still popular in current research field.Anisodamine(Ani) is a belladonna alkaloid extracted from the Anisodus tangutica Maxim of the Solanaceae family. As a muscarinic(M) cholinergic receptor antagonist in the original classification of pharmacology, Ani is less toxic than atropine to the peripheral nervous system and also less toxic than scopolamine to the central nervous system. Ani has multiple beneficial effects of pharmacology on myocardium and coronary microcirculation which could improve average intracoronary perfusion pressure at the same time of raising heart rate. Ani also has high security and low price. Studies in recent decades showed that Ani also has many pharmacological effect of non-cholinergic antagonist, including regulating microvascular tension, increasing cell membrane lipid fluidity and blood cell deformation ability, inhibiting platelet aggregation and thrombosis, antioxidation effect and scavenging superoxide, inhibiting apoptosis and intracellular calcium overload, reducing the myocardial infarction area and so on, which are associated with the cardiovascular protective effect of Ani. And our previous clinical studies showed that intracoronary administration of 500 μg, 1000 μg, 1500μg or 3000μg Ani could improve myocardial reperfusion of STEMI patients undergoing primary PCI as well as prevent and improve no reflow phenomenon. However, it is not clear whether the protective effects against I/R injury will be further enhanced when the dose of Ani is increased, whether different doses of anisodamine have different myocardial protective effects and whether the relationship between the dosage and efficacy exists. Further research showed that Ani could reduce damage to structure and function of myocardial mitochondria in rat model of cardiac arrest resuscitation, but specific mechanism has not yet been elucidated.Based on the early clinical and basic research, this study was designed to observe the protective effects of intracoronary administration of different doses of Ani and possible relationship between the dosage and efficacy, and mitochondrial ATP-sensitive K+ channel(mito KATP)-mediated mitochondrial protection mechanism was further investigated. This study was divided into three parts. In the first part, different doses of Ani were intracoronarily administrated in patients with STEMI undergoing primary PCI before stenting. Effects of different doses of Ani on myocardial reperfusion index and cardiac function parameters were observed and compared. And the relationship between the dosage and efficacy was analyzed. In the second part, from the level of isolated rat heart, the influence of neurohumoral factors was excluded, and then we investigated whether mito KATP participated in the protective effect of Ani against myocardial I/R injury. In the third part, neonatal rat cardiomyocytes were used to investigate the protective mechanism of Ani against cardiomyocyte hypoxia/reoxygenation(H/R) injury medicated by mito KATP.Part one Protective effect of preventively intracoronary administration of different doses of anisodamine on myocardial reperfusion in patients with ST-segment elevation myocardial infarction undergoing primary percutaneous coronary interventionObjectives: To investigate the protective effect of preventively intracoronary administration of Ani on myocardial reperfusion in patients with STEMI undergoing primary percutaneous coronary intervention(PCI), and possible relationship between the dosage and efficacy.Methods: This research was a randomized-controlled study. Patients with STEMI undergoing primary PCI who were hospitalized in Cardiology Department of the second hospital of Hebei Medical University beteween Jan 2014 and Jun 2015 were enrolled in. Eligible patients were divided randomly into four groups: Group A(normal saline), Group B(1000 μg Ani), Group C(2000 μg Ani), and Group D(4000 μg Ani). As soon as the lesion of the IRA was crossed by the guidewire or dilated by the balloon(before stenting) and thrombolysis myocardial infarction trial(TIMI) flow was above grade zero, drug was administered intracoronarily through a guiding catheter. Patients in Group A were administered intracoronarily 4 ml normal saline. Patients in Group B, C and D were administered intracoronarily 1000 μg, 2000 μg and 4000 μg Ani, respectively. Coronary angiography and primary PCI were performed through an artery of forearm(a radial or an ulnar artery) access point. The parameters of coronary angiography and PCI were determined and analyzed by two or more interventional cardiologists who were unaware of patients ’ conditionand study protocol, including TIMI flow grade, TIMI myocardial perfusion grade(TMPG), the corrected TIMI frame count(c TFC), the increased value of TMPG and the decreased value of c TFC at the baseline and after stenting. Thrombosis burden integral was evaluated. The invasive hemodynamic parameters were monitored through a guiding catheter, and then heart rates, SBP(systolic blood pressure), DBP(diastolic blood pressure) and MBP(mean blood pressure) before drug administration as well as 5min and 10 min after administration were recorded. The incidences of reperfusion arrhythmia were also observed. The intracoronary use of tirofiban was decided by the operator according to patients’ clinical and angiographic characteristics. Electrocardiograph(ECG) was recorded on admission to the Emergency Department and 90 min after operation, and the ratio of complete ST-segment resolution(STR) on the ECG was calculated. CK-MB and c Tn I levels were measured on admission to the Emergency Department and every 6 h after PCI, and the peak values were recorded. High sensitivity C-reactive protein(hs-CRP) was measured on admission, 24, and 72 h after PCI. Echocardiogram at baseline, at 1 week after primary PCI and at 3-month follow-up was conducted to assess left ventricular ejection fraction(LVEF). The primary endpoint was the combined outcome of c TFC and TMPG after primary PCI. The secondary endpoint was major adverse cardiac events(MACEs) during hospitalization and at the 3-month follow-up.Results: A total of 140 patients were finally enrolled in this study: Group A(n=36), Group B(n=35), group C(n=35), and group D(n=34). There were no significant differences with respect to patients’ baseline characteristics, mean onset-to-balloon time and door-to-balloon time, and coronary angiography parameters among the four groups. The rates of complete STR in Groups C and D(77.14% and 79.41%) were both significantly higher than those in Group A(52.78%)(P=0.032 and P=0.019, respectively). With the increase of Ani doses, the rates of complete STR in Groups B, C and D also increased(71.43%, 77.14% and 79.41%), but have not reached statistical differences among the three groups(all P > 0.05).There was no significant difference in rates of TIMI flow 3 and c TFC at baseline. Compared with Group A, a tendency towards higher rates of TIMI flow 3 after PCI was seen in Groups B, C and D, but the difference is not significant(P=0.062). Rates of TMPG 3 after PCI and the increasing ranges of TMPG in Groups B, C and D were significantly higher than those in Group A(all P<0.05). The c TFCs after PCI in Groups B, C and D were significantly reduced as compared with Group A(all P<0.01), and the decreasing ranges of c TFCs in Groups B, C and D were significantly higher than those in Group A(B vs. A: P<0.05; C vs. A: P<0.05; D vs. A: P<0.01). With the increase of Ani doses, the c TFCs after PCI in Groups B, C and D were decreased(22.7±6.17, 21.82±6.88 and 21.30±5.49), and the decreasing ranges of c TFCs were increased(12.58±8.61, 13.14±8.19 and 14.19±7.83), but there were no significant differences among the three groups.SBP, DBP, MBP and heart rates in coronary artery before drug administration(baseline values) were similar among the four groups. After five minutes of administration, there were increasing trends of SBP, DBP and MBP in Group B as compared with baseline values, but the differeces were not significant. But heart rates in Group B and SBP, DBP, MBP and heart rates in Groups C and D were all significantly higher than baseline values(all P<0.01). Moreover, SBP in Group D and DBP, MBP and heart rates in Groups B, C and D were all higher than those in Group A(all P<0.05). With the increase of Ani doses, SBP, DBP, MBP and heart rates in Groups B, C and D also increased, but there were no significant differences among the three groups except that heart rates in Group D were faster than Groups B and C(both P<0.001). After ten minutes of administration, SBP, DBP and MBP all declined to near the baseline levels, while heart rates in Groups C and D decreased slowly and were still faster than baseline, and heart rates in Group D were still faster than Groups A and C(P<0.001 and P<0.01, respectively).With respect to side effects during PCI, results showed that four patients in Group A(11.11%) and one in Group B(2.86%) devolped hypotension, and none in Groups C and D. There were significant differences between C or D and A(both P < 0.05). Three patients in Group A(8.33%) developed symptomatic bradycardia and none in Groups B, C and D(B or C or D vs. A: all P<0.05). Five patients in Group A(13.89%) and two in Group B(5.71%) developed rapid reperfusion arrhythmias, and none in Groups C and D(C or D vs. A: both P<0.05). No severe sinus tachycardia(defined as heart rate was more than 130 beats/min or heart rate increased by more than 40 beats/min) was observed after 10 min of administration of anisodamine at three doses.The peak values of CK-MB and c Tn I in Groups B, C, and D were all lower than those in group A(CK-MB: B or C vs. A: both P<0.05; D vs. A: P<0.01; c Tn I: all P<0.05), which showed a dose-dependent manner. There were no significant differences in the levels of hs-CRP on admission among the four groups. The hs-CRP levels at 24 and 72 h after PCI in patients of Groups B, C, and D were both decreased compared with Group A(24h: B vs. A: P<0.01, C or D vs. A: P<0.001; 72h: all P<0.05). With the increase of Ani doses, the peak values of CK-MB and c Tn I as well as hs-CRP levels at 24 and 72 h after PCI in Groups B, C and D all decreased, but there were no significant differences among the three groups.The LVEF at 1 week after PCI in Groups B, C, and D were both higher than Group A(both P<0.05), and at 3-month follow-up the LVEF in Groups B, C, and D was significantly increased as compared with Group A(all P<0.05). With the increase of Ani doses, the LVEF at 1 week after PCI and at 3-month follow-up in Groups B, C and D also increased, but have not reached statistical differences among the three groups. The incidence of total MACEs in Group D was significantly lower than Group A during hospitalization(P=0.025) and at the 3-month follow-up(P=0.012). With the increase of Ani doses, the incidences of total MACEs during hospitalization and at the 3-month follow-up in Groups B, C and D both decreased, but there were also no significant differences among the three groups. Kaplan–Meier analysis showed that the MACE-free survival rates of the 3-month follow-up in Groups B, C and D were significantly higher than those in Group A(P=0.019).Conclusions: All the three doses of anisodamine(1000 μgã€2000 μgã€4000 μg) by preventively intracoronary administration in patients with STEMI undergoing primary PCI could improve myocardial reperfusion in different extent by reducing myocardial injury and myocardial infarction area as well as inflammation, and also improve left ventricular function and the prognosis of patients, which showed a trend of dose-dependent relationship with no obvious side effects.Part two Protective effect of anisodamine against ischemia/reperfusion injury in isolated rat hearts and possible protection mechanism of mitochondriaObjectives: To observe the protective effect of Ani against myocardial I/R injury in isolated rat heart during early reperfusion, and preliminarily investigate whether mito KATP participated in this effect.Methods: Langendorff isolated heart perfusion equipment was used to perfuse isolated rat hearts. Isolated heart I/R model was established by stopping perfusion globally for 30 min and then reperfusion for 60 min. Firstly, isolated hearts from healthy and male Sprague-Dawley(SD) were divided randomly into six drug concentration groups: Control, I/R, I/R+Ani 0.03 m M, I/R+Ani 0.10 m M, I/R+Ani 0.30 m M and I/R+Ani 1.00 m M groups, in order to determine the optimal drug concentration. All hearts were perfused for 30 min initially. BIOPAC 16 channels physiological signal collection and analysis system were applied to monitor hemodynamic parameters, including heart rate(HR), the peak value of left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), left ventricular developed pressure(LVDP=LVSP-LVEDP), the maximum rate of left ventricular pressure rise(dp/dtmax) and the maximum rate of left ventricular pressure rise(-dp/dtmax) and coronary flow(CF). Myocardial infarction size was measured by 2, 3, 5-trihenylterzolium chloride(TTC) staining. Cardiac troponin I(c Tn I) levels in coronary effluent were detected by enzyme-linked immunosorbent assay(ELISA). Secondly, Ani of the optimal concentration was perfused in different time. The isolated hearts were divided randomly into six time groups: Control, I/R, I/R+ Ani 15 min, I/R+ Ani 30 min, I/R+ Ani 45 min and I/R+ Ani 60 min groups. The levels of c Tn I in coronary effluent were detected by ELISA in order to determine the optimal duration of Ani treatment. Thirdly, influence of mito KATP blocker 5-hydroxy decanoic acid(5-HD) on Ani protecting I/R injured isolated hearts was observed. The isolated hearts were divided randomly into five groups: Control, I/R, I/R+Ani, I/R+Ani+5-HD and I/R+5-HD groups. Hemodynamic parameters in each group were monitored,the number and duration of any kind of ventricular reperfusion arrhythmia were observed, and arrhythmia score was used to quantify the arrhythmias. Myocardial infarction size was measured by TTC staining. The levels of c Tn I in coronary effluent were detected by ELISA. Contents of ATP and malondialdehyde(MDA) and superoxide dismutase(SOD) activity in myocardial tissue were detected with colorimetric method. Transmission electron microscope was used to observe the change of myocardial and mitochondrial ultrastructure.Results: A total of 30 isolated hearts were finally included in the six drug concentration groups in the first step, with 5 hearts in each group. There were no significant difference in hemodynamic parameters during equilibrium among the six groups. Compared with Control group, all the hemodynamic parameters in I/R group declined significantly(all P<0.05). When the concentration of Ani was at 0.30 m M, effect of improvement in these parameters was the most obvious, that is, LVDP, dp/dtmax,-dp/dtmax and CF after 15, 30, 45 and 60 min reperfusion were all significantly increased as compared with I/R group. However, the effect of Ani on HR was not significant. Furthermore, compared with I/R group, the decrease of myocardial infarction size in I/R+0.30 m M Ani group was the greatest(P<0.001), and the inhibitory effect of 0.30 m M Ani on c Tn I levels was the strongest(P<0.001). Therefore, the optimal drug concentration of Ani was 0.30 m M, which was used in the following experiment. A total of 24 isolated hearts were finally included in the six time groups in the second step, with 4 hearts in each group. Compared with I/R group, the decline of c Tn I levels was the most obvious when heart was perfused with 0.30 m M Ani(P<0.001). So the optimal duration of Ani treatment was 30 min, which was used in the following experiment.In the third step, a total of 40 isolated hearts were finally included in the five groups, with 8 hearts in each group. There were no significant difference in hemodynamic parameters at baseline among the five groups. Compared with I/R group, Ani improved all hemodynamic parameters except HR, including increase of LVDP, dp/dtmax,-dp/dtmax and CF(all P<0.05). At each time point of reperfusion, all the parameters apart from HR in I/R+Ani+5-HD group declined(all P<0.05). When heart was reperfused with 5-HD alone, all the hemodynamic parameters were not affected as compared with I/R group. The myocardial infarction size and c Tn I levels in I/R+Ani group were both significantly smaller than I/R group(P<0.001 and P<0.01, respectively). Both values in I/R+Ani+5-HD were all higher than those in I/R+Ani group(P<0.001 and P<0.01, respectively). There were no significant differences in infarction size and c Tn I levels between I/R+5-HD and I/R groups.The number of all kinds of ventricular reperfusion arrhythmia including PVC, VT and VF and the duration of VT and VF were significantly lower than I/R group(P<0.05). Compared with I/R+Ani group, the number of PVC and VT, and the duration of VT were increased(P<0.05). Reperfusion arrhythmia score in I/R+Ani group was lower than that in I/R group, while simultaneous use of Ani and 5-HD partially inhibited the beneficial effect of Ani. Reperfusion of 5-HD only did not affect the incidence of reperfusion arrhythmia.The ATP content and SOD activity in I/R group were both significantly lower than Control group, while MDA level was higher all(P<0.001). Compared with I/R group, the ATPcontent and SOD activity in I/R+Ani group were both significantly increased, while MDA level was decreased(P<0.01, P<0.001 and P<0.01, respectively). Simultaneous reperfusion of Ani and 5-HD partially weakened the beneficial effect of Ani, making ATP content and SOD activity decline and MDA level increase(P<0.001).Transmission electron microscope was used to observe myocardial ultrastructure. Results showed that in I/R group myocardial and mitochondria structure were damaged obviously, sarcomere disappeared, myofibril fractured and was dissolved, nuclear substance was highly swollen, most of the mitochondria crest fractured and was dissolved, and mitochondrial membrane ruptured. The damage in Ani+I/R group was lighter than that in I/R group. Myofibril arranged neatly, nuclear substance was not obviously swollen, and mitochondria crest and mitochondrial membrane were not dissolved.Compared with Ani+I/R group, the damage in Ani+I/R+5-HD group was heavier. Myofibril was swollen, nuclear substance was mildly swollen, and mitochondria crest and mitochondrial membrane were partially fused. The damage between I/R+5-HD group and I/R group was similar.Conclusions:1 Ani could improve hemodynamic parameters of isolated rat heart after I/R injury, and reduce the incidence of ventricular reperfusion arrhythmia.2 Ani could improve myocardial energy metabolism after I/R injury, reduce myocardial infarction area and oxidative stress reaction, and reduce myocardial and mitochondrial ultrastructure damage.3 The mito KATP inhibitor 5-HD partially suppressed the protective effects of Ani above, indicating that the protective effect of Ani against myocardial I/R injury was possibly associated with opening of mito KATP..Part three Protective effect of anisodamine against neonatal rat cardiomyocyte hypoxia/reoxygenation injury and mechanism mediated by mitochondrial ATP-sensitive potassium channelObjectives: To observe protective effect of anisodamine against neonatal rat cardiomyocyte H/R injury and mitochondrial function at the beginning of reoxygenation, and investigate the mechanism mediated by mito KATP.Methods: Cardiomyocytes were isolated from 1- to 3- day- old healthy SD neonatal rat hearts and were primarily cultured. After cell culture for 72 h, the isolated neonatal rat cardiomyocyte developed a woven mesh and a synchronization pulse appeared. Immunocytochemistry method for the striated muscle actin(α- sarcomeric actin) was used to identify purity of isolated neonatal rat cardiomyocytes, which was above 99%. Cardiomyocyte H/R injury model was established by hypoxia culture in serum- and glucose-free medium for 3h and then by normal culture in complete medium for 12 h. Firstly, neonatal rat cardiomyocytes after 72 h culture were randomly divided into seven drug concentration groups: Control, H/R, H/R+Ani 10-8m M, H/R+Ani 10-7m M, H/R+Ani 10-6m M, H/R+Ani 10-5m M and H/R+Ani10-4m M group. Neonatal rat cardiomyocyte viability after H/R injury and different concentration of Ani treatment was detected by MTT staining in order to determine the optimal drug concentration. Secondly, influence of mito KATP inhibitor 5-HD on protection of Ani against neonatal rat cardiomyocyte H/R injury was observed. Neonatal rat cardiomyocytes after 72 h culture were randomly divided into five groups: Control, H/R, H/R+Ani, H/R+Ani+5-HD and H/R+5-HD group. At the end of experimental treatments, cardiomyocyte viability was detected by MTT staining, and c Tn I levels in cell supernatant were measured by ELISA. Contents of ATP and MDA as well as activity of SOD and mitochondrial respiratory chain complex V were quantitatively detected by colorimetric method. And total protein concentration and mitochondrial protein concentration were determined by BCA(bicinchonininc acid) method. Myocardiocyte was incubated with calcium fluorescent probe Fluo-3/AM, and the fluorescence intensity change was observed by fluorescence microscope for the determination of cytoplasm calcium concentration. Similarly, calcium fluorescent probe Rhod-2/AM was used to determine mitochondrial calcium concentration, and fluorescent probe JC-1 was used to detect the change of mitochondrial membrane potential.Results: The OD values of seven drug concentratin groups detected by MTT assay showed that cell viability in H/R group significantly declined as compared with the Control group(P<0.001). Compared with the H/R group and other Ani concentration groups, when the concentration of Ani was 10-6m M, the OD value of myocardiocyte achieved the maximum, namely, cell viability was the best. So 10-6m M was the optimal drug concentration of Ani, which was adopted in the following experiment.As soon as 5-HD was applied, MTT result showed that cell viability in H/R+Ani+5-HD group was significantly decreased as compared with H/R+Ani group(P<0.01), while using 5-HD only did not affect the viability of myocardiocytes. The c Tn I level in H/R group was significantly higher than that in Control group(P<0.01), while the c Tn I level in H/R+Ani group was signicantly reduced as compared with H/R group(P<0.01). Compared with the H/R+Ani group, simultaneously adding Ani and 5-HD into the medium could inhibit the decrease of c Tn I level(P<0.05). Application of 5-HD alone did not influnce the c Tn I level after H/R injury.Compared with Control group, ATP content of cardiomyocyte in H/R group was siginificantly decreased(P<0.001). The ATP content in H/R+Ani group was significantly increased as compared with the H/R group(P<0.001), while the content in H/R+Ani+5-HD group was signicantly lower than that in the H/R+Ani group(P<0.01). The ATP content in H/R+5-HD group was similar to the H/R+Ani group. Compared with Control group, the activity of myocardiocyte mitochondrial respiratory chain complexes V in H/R group decreased significantly(P<0.001). The activity of the complexes in H/R+Ani group was significanly higher than that in the H/R group(P<0.05). At the same time of using Ani, adding 5-HD could reduce its activity obviously(P<0.05). Application of 5-HD alone had no effect on the activity of the complexes.The MDA content of myocardiocyte in H/R group was significantly higher than that in Control group(P<0.001). The content of MDA in H/R+Ani group was significantly reduced as compared with that in the H/R group(P<0.01), while the content in H/R+Ani+5-HD group was significantly higher than that in the H/R+Ani group(P<0.05). No effect on the MDA content of myocardiocyte occurred with the application of 5-HD alone. Significantly decreased activity of SOD was observed in H/R group as compared with Control group(P<0.001), while the activity in H/R+Ani group was significantly increased as compared with the H/R group(P<0.001). The SOD activity in H/R+Ani+5-HD group was significantly lower than that in the H/R+Ani group(P<0.001). No impact of application of 5-HD alone on the SOD activity was demonstrated.The intensity of fluorescence marked with fluorescent probe Fluo-3/AM in H/R group observed with fluorescent microscope was obviously stronger than Control group(P<0.001). Compared with the H/R group, the fluorescence intensity of H/R+Ani group obviously weakened(P<0.01). At the same time of using Ani, adding 5-HD could make the relative fluorescence intensity value in myocardiocytes obviously higher than that of the H/R+Ani group(P<0.05). Treating myocardiocytes with 5-HD alone did not affect the intracellular calcium concentration.The intensity of fluorescence marked with fluorescent probe Rhod-2/AM in H/R group was observed obviously stronger than Control group(P<0.001). Compared with the H/R group, the fluorescence intensity of H/R+Ani group was obviously attenuated(P<0.05), while the intensity of H/R+Ani+5-HD group was significantly higher than that of the H/R+Ani group(P<0.05). Treating myocardiocytes with 5-HD alone did not affect the mitochondrial calcium concentration.After myocardiocytes were marked with fluorescent probe JC-1, the intensity of JC-1 aggregates and JC-1 monomers was corresponding to high and low level of mitochondrial membrane potential, respectively. Compared with Control group, the relative fluorescence intensity value of JC-1 aggregates in H/R group was significantly reduced(P<0.001), while the value of JC-1 monomers was obviously increased(P<0.01). In H/R+Ani group, the increased relative fluorescence intensity value of JC-1 aggregates and the decreased value of JC-1 monomers were observed as compared with the H/R group(both P<0.01). The relative fluorescence intensity value of JC-1 aggregates in H/R+Ani+5-HD group was significantly lower than that in H/R+Ani group(P<0.05), while the value of JC-1 monomers was higher(P<0.05). Treating myocardiocytes after H/R injury with 5-HD alone did not affect the mitochondrial membrane potential.Conclusions:1 At the beginning of neonatal rat cardiomyocyte reoxygenation, Ani could significantly enhance cell viability, reduce the degree of myocardial injury and oxidative stress, and improve energy metabolism and mitochondrial function.2 The protective effects of Ani above were partially suppressed by mito KATP inhibitor 5-HD, indicating that Ani played an important role in reducing myocardiocyte H/R injury by opening mito KATP. |
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