Cardioprotective Effects of Alpha-Lipoic Acid on Myocardial Reperfusion Injury: Suppression of Reactive Oxygen Species Generation and Activation of Mitogen-Activated Protein Kinase

오석규,윤경호,유남진,김남호,김민선,박병림,정진원 대한심장학회 2009 Korean Circulation Journal Vol.39 No.9

Background and Objectives: Reactive oxygen species (ROS) and mitogen-activated protein (MAP) kinase play an important role in the development of myocardial reperfusion injury. In this study, we examined whether treatment with alpha-lipoic acid (ALA) before reperfusion could prevent myocardial reperfusion injury in vivo. Materials and Methods: Sprague-Dawley rats were subjected to a 45-minute left anterior descending coronary artery ligation followed by 45- or 10-minute reperfusion. ALA was administered 10 minutes prior to reperfusion. The infarct size ratio of the infarct area to the ischemic area at risk, was measured based on 10, 25, 50, and 100 mg/kg of ALA, with propidium iodide (PI) fluorescence. Apoptosis was evaluated by TdT-mediated dUDP nick end labeling (TUNEL) staining. The generation of intracellular ROS was evaluated using the fluorogenic probe, dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Western blot analysis was performed for MAP kinase (pERK 1/2 and pJNK 1/2) activity. Results: The infarct size, according to ALA dose, was significantly suppressed 29.1% with ALA 25 mg/kg (p<0.0001), 41.5% with 50 mg/kg (p<0.05), and 41.4% with 100 mg/kg (p<0.05) compared to the controls (54.3%). However, the results were not significantly different with 47.2% of the ALA 10 mg/kg (p=0.192). A few apoptotic nucleoli were detected in the ALA 25 mg/kg group, but were frequently detected in the control group. The ROS generation was significantly suppressed (p<0.0001), the activity of pERK 1/2 was significantly increased (p<0.05) and the activity of pJNK 1/2 was significantly decreased (p<0.05) in the ALA 25 mg/kg group compared to the controls. Conclusion: The results of this study suggested that adequate doses of ALA before reperfusion was effective for the prevention of myocardial reperfusion injury in vivo. This cardioprotective activity of ALA might be associated with an anti-apoptotic effect of ALA via suppression of ROS generation, increase of pERK 1/2 and decrease of pJNK 1/2 activity. Background and Objectives: Reactive oxygen species (ROS) and mitogen-activated protein (MAP) kinase play an important role in the development of myocardial reperfusion injury. In this study, we examined whether treatment with alpha-lipoic acid (ALA) before reperfusion could prevent myocardial reperfusion injury in vivo. Materials and Methods: Sprague-Dawley rats were subjected to a 45-minute left anterior descending coronary artery ligation followed by 45- or 10-minute reperfusion. ALA was administered 10 minutes prior to reperfusion. The infarct size ratio of the infarct area to the ischemic area at risk, was measured based on 10, 25, 50, and 100 mg/kg of ALA, with propidium iodide (PI) fluorescence. Apoptosis was evaluated by TdT-mediated dUDP nick end labeling (TUNEL) staining. The generation of intracellular ROS was evaluated using the fluorogenic probe, dichlorodihydrofluorescein diacetate (CM-H2DCFDA). Western blot analysis was performed for MAP kinase (pERK 1/2 and pJNK 1/2) activity. Results: The infarct size, according to ALA dose, was significantly suppressed 29.1% with ALA 25 mg/kg (p<0.0001), 41.5% with 50 mg/kg (p<0.05), and 41.4% with 100 mg/kg (p<0.05) compared to the controls (54.3%). However, the results were not significantly different with 47.2% of the ALA 10 mg/kg (p=0.192). A few apoptotic nucleoli were detected in the ALA 25 mg/kg group, but were frequently detected in the control group. The ROS generation was significantly suppressed (p<0.0001), the activity of pERK 1/2 was significantly increased (p<0.05) and the activity of pJNK 1/2 was significantly decreased (p<0.05) in the ALA 25 mg/kg group compared to the controls. Conclusion: The results of this study suggested that adequate doses of ALA before reperfusion was effective for the prevention of myocardial reperfusion injury in vivo. This cardioprotective activity of ALA might be associated with an anti-apoptotic effect of ALA via suppression of ROS generation, increase of pERK 1/2 and decrease of pJNK 1/2 activity.

Therapeutic Potential of a Novel Necrosis Inhibitor, 7-Amino-Indole, in Myocardial Ischemia–Reperfusion Injury

Hwang, In-Chang,Kim, Ju-Young,Kim, Ji-Hyun,Lee, Joo-Eun,Seo, Ji-Yun,Lee, Jae-Won,Park, Jonghanne,Yang, Han-Mo,Kim, Soon-Ha,Cho, Hyun-Jai,Kim, Hyo-Soo Lippincott, WilliamsWilkins 2018 Hypertension Vol.71 No.6

<▼1><P>Supplemental Digital Content is available in the text.</P></▼1><▼2><P>Opening of mitochondrial permeability transition pore and Ca<SUP>2+</SUP> overload are main contributors to myocardial ischemia–reperfusion injury, which paradoxically causes a wide variety of myocardial damage. We investigated the protective role of a novel necrosis inhibitor (NecroX-7; NecX) against myocardial ischemia–reperfusion injury using in vitro and in vivo models. H9C2 rat cardiomyoblasts and neonatal cardiomyocytes were exposed to hypoxia–reoxygenation stress after pre-treatment with NecX, vitamin C, a combination of vitamin C and E, N-acetylcysteine, an apoptosis inhibitor (Z-VAD-fmk), or cyclosporine A. The main mechanism of cell death after hypoxia–reoxygenation stress was not apoptosis but necrosis, which was prevented by NecX. Protective effect of NecX was based on its potent reactive oxygen species scavenging activity, especially on mitochondrial reactive oxygen species. NecX preserved mitochondrial membrane potential through prevention of Ca<SUP>2+</SUP> influx and inhibition of mitochondrial permeability transition pore opening, which was more potent than that by cyclosporine A. Using Sprague-Dawley rats exposed to myocardial ischemia for 45 minutes followed by reperfusion, we compared therapeutic efficacies of NecX with cyclosporine A, vitamin C, a combination of vitamin C and E, and 5% dextrose, each administered 5 minutes before reperfusion. NecX markedly inhibited myocardial necrosis and reduced fibrotic area to a greater extent than did cyclosporine A and other treated groups. In addition, NecX preserved systolic function and prevented pathological dilatory remodeling of left ventricle. The novel necrosis inhibitor has a significant protective effect against myocardial ischemia–reperfusion injury through inhibition of mitochondrial permeability transition pore opening, indicating that it is a promising candidate for cardioprotective adjunctive measure on top of reperfusion therapy.</P></▼2>

The Effect of High Dose Melatonin on Cardiac Ischemia- reperfusion Injury

Hakan Ceyran,Figen Narin,Nazmi Narin,Hülya Akgün,A. Bahar Ceyran,Figen Öztürk,Yiğit Akçah 연세대학교의과대학 2008 Yonsei medical journal Vol.49 No.5

Purpose: Melatonin, the most potent scavenger of toxic free radicals, has been found to be effective in protecting against pathological states due to the release of reactive oxygen species. This study was performed to establish the effect of high dose melatonin on protection against ischemia- reperfusion (I/R) injury in rat hearts. Materials and Methods: Forty male Sprague-Dawley rats were used in this study. They were separated into four groups of ten rats each. A left coronary artery occlusion was induced in the rats by ligating the artery for 20 minutes and then releasing the ligation (reperfusion) afterwards. The control group was Group A. Group B was subjected to myocardial ischemia-reperfusion without any treatment, while Group C underwent myocardial ischemia-reperfusion with a melatonin treatment before the ischemia. Group D was subjected to myocardial ischemia- reperfusion with a melatonin treatment before the reperfusion. After 20 minutes of reperfusion, blood samples were obtained from each group for biochemical studies, and the animals were sacrificed for histological and, immunohistochemical examinations of the myocardial tissue. Results: We found that the cardiac troponin T(cTn-T) levels were significantly increased in Group B when all groups were compared. In the Group C rats treated with melatonin, the cTn-T values were significantly lower than those in Groups B and D. In addition, malondialdehyde (MDA) and antioxidant enzymes including, superoxide dismutase (SOD) and myeloperoxidase (MPO) were lower than those in Group B in the melatonin treated groups. The differences were statistically significant (p<0.05). Histopathologic and immunohistopathologic studies also supported the effectiveness of melatonin. Conclusion: Our study suggests that high dose melatonin, appears to offer protection against cardiac ischemia-reperfusion injuries in rats by scavenging the free radicals and could have a potential clinical use in the management of myocardial ischemia.

Cryptotanshinone, a lipophilic compound of Salvia miltiorrriza root, inhibits TNF-α-induced expression of adhesion molecules in HUVEC and attenuates rat myocardial ischemia/reperfusion injury in vivo

Jin, Y.C.,Kim, C.W.,Kim, Y.M.,Nizamutdinova, I.T.,Ha, Y.M.,Kim, H.J.,Seo, H.G.,Son, K.H.,Jeon, S.J.,Kang, S.S.,Kim, Y.S.,Kam, S.C.,Lee, J.H.,Chang, K.C. North-Holland ; Elsevier Science Ltd 2009 european journal of pharmacology Vol.614 No.1

The aim of the present study was to evaluate the protective effect of cryptotanshinone (CTS), one of active ingredients of Salvia miltiorrhiza root, on myocardial ischemia-reperfusion injury in rat due to inhibition of some inflammatory events that occur by NF-kB-activation during ischemia and reperfusion. Myocardial ischemia and reperfusion injury was induced by occluding the left anterior descending coronary artery for 30 min followed by either 2 h (biochemical analysis) or 24 h (myocardial function and infarct size measurement) reperfusion. CTS injected (i.v.) 10 min before ischemia and reperfusion insult. CTS significantly reduced the infarct size and improved ischemia and reperfusion-induced myocardial contractile dysfunction. Furthermore, CTS inhibited NF-kB translocation, expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), neutrophil infiltration and MPO activity in ischemic myocardial tissues. CTS also significantly reduced plasma levels of TNF-α, IL-1β due to ischemia and reperfusion. Interestingly, H<SUB>2</SUB>O<SUB>2</SUB>-stimulated NF-kB-luciferase activity and TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) expressions in human umbilical vein endothelial cells (HUVEC) were significantly inhibited by CTS. Taken together, it is concluded that CTS may attenuate ischemia and reperfusion-induced microcirculatory disturbances by inhibition of proinflammatory cytokine production, reduction of neutrophil infiltration and possibly inhibition of adhesion molecules through inhibition of NF-kB-activation during ischemia and reperfusion.

17β-Estradiol의 심근 보호작용에 대한 연구 ; 재관류 부정맥을 유발한 동물실험

홍정석,김원,조규종,이미우,장성은,임경수 대한응급의학회 2001 대한응급의학회지 Vol.12 No.4

Background: Although reperfusion certainly prevents tissue ischemia from possible cardiac death, several lines of evidence suggest that reperfusion may paradoxically aggravate the frequency of serious reperfusion-induced lethal arrhythmias. It has been reported that acute administration of estrogen at physiological concentrations reduced with myocardial ischemic injury in women with coronary heart disease. In studies with canines, acute administration by either the intra-muscular or the intra-coronary route similarly prevented ischemia and reperfusion dysrhythmias and also reduced the infarct size because the estrogen increased the distal coronary perfusion pressure, scavenged free radicals and had other effects during both ischemia and reperfusion. However, the canine heart is notoriously well collateralized. 17β-estradiol induces very little vasorelaxation in cat coronary rings, suggesting that increased ischemic myocardial blood flow dose not contribute to the protective effect. In the present study, employing a cat model of regional cardiac ischemia, we examined whether reperfusion rendered after acute administration of 17β-estradiol could lower the incidence of reperfusion-induced lethal arrhythmia and the death rate. Method: Adult mongrel male cats(n=31, 2.7∼4.5 kg) were anesthetized under positive-pressure artificial ventilation with room air. Electrocardiograms were recorded. The animals of the control group(n=15) were subjected to 20-minute left anterior descending coronary artery(LAD) occlusion followed by abrupt reperfusion. The animals in the experimental 17β-estradiol(2 or 20 ㎍/kg) group were subjected to ischemia/reperfusion insult following drug treatment: 17 β -estradiol was applied intravenously within the 60 seconds just before LAD ligation followed by abrupt reperfusion. The Fisher's exact test was used to compare the data from different animal groups(p<0.05). Results: The number of arrhythmias(ventricular premature beat, ventricular tachycardia and ventricular fibrillation) emerging during the reperfusion phase were not statistically different from that in the control group. The death rate in the 17β-estradiol 20㎍/kg group was lower from that in the control group(P value = 0.039). Conclusion: Acute administration of 17β -estradiol at a supraphysiological concentration might produce cardioprotective effects, not by modificating the coronary blood flow into the threatened myocardial region, but by other mechanisms that directly or indirectly increase the intrinsic myocardial ischemic tolerance in the cat during the reperfusion phase.

가토 허혈-재관류 심근에서의 Bcl-2 단백의 발현

류재욱,김삼현,서필원,박성식,최창휴,류경민,김영권,박이태,김성숙 대한흉부심장혈관외과학회 1998 Journal of Chest Surgery (J Chest Surg) Vol.31 No.10

연구배경 : 심근의 허혈 또는 재관류에 의한 세포사에는 괴사 이외에 세포고사가 존재함이 알려져 있다. Bcl-2 단백은 세포질에 존재하는 단백으로 세포고사를 억제하는 기능을 하며 정상심근에서는 발현되지 않으나 심근경색의 급성기에서 발현됨이 보고되어 있다. 본 연구는 가토 허혈-재관류 심근에서 Bcl-2 단백의 발현 여부와 재관류의 시간에 따른 발현의 변화를 알아보고자 하였다. 방법: 평균 무게가 2.9Kg(1.5-4.8Kg)인 가토 39마리를 이용하였다. 허혈-재관류 모델의 각 실험동물에서 좌전하행지를 30분간 결찰한 다음 1, 4, 8, 12, 24시간, 3, 7일 동안 재관류시켰다. 이후 즉시 실험동물을 희생시킨 다음 심장을 적출하여 심근조직을 얻고 10% buffered formalin에 고정하였다. Bcl-2 단백의 발현은 파라핀에 포매된 조직에서 단일클론항체를 이용한 면역조직화학적 염색으로 확인하였다. 결과: 허혈-재관류 심근 중 12, 24시간, 3일 재관류군에서 Bcl-2 단백의 발현을 관찰할 수 있었으며, 특히 24시간 재관류 심근에서 잘 관찰되었다. Bcl-2 양성염색의 심근세포는 위험부위의 구제심근에서 관찰되었다. 결론: Bcl-2 단백은 심근의 허혈-재관류에서 급성기의 비교적 후기에 발현되며, 이는 재관류 초기에서 보다는 후기에서 세포고사를 억제하는데 일부 역할을 할 것으로 사료된다. Background: Myocardial cell death after myocardial infarction or reperfusion is classified into necrosis and apoptosis. Bcl-2 protein is a cytoplasmic protein, which inhibits apoptosis and is expressed in acute stage of myocardial infarction but not in normal heart. This study was performed to investigate whether Bcl-2 protein was expressed respectively to the reperfusion time. Materials and methods: Thirty nine New Zealand white rabbits weighing 1.5-4.8 kg (mean, 2.9kg) were alloted into 7 groups (n=5 in each group) which underwent left anterior descending coronary artery(LAD) occlusion for 30 minutes, followed by reperfusion. The animals were sacrificed at 1, 4, 8, 12, 24 hours, and 3, 7 days after occlusion. Ventricle was excised immediately after intervention. Tissues were fixed in 10% buffured formalin and embedded in paraffin. Bcl-2 protein was detected by immunohistochemical stain with using monoclonal antibody against Bcl-2 protein. Results: The positive immunohistochemical reactivity for Bcl-2 protein was observed in 12, 24 hours, and 3 days reperfusion groups. Bcl-2 protein was detected in salvaged myocytes surrounding the infarcted area. Conclusions: Bcl-2 protein is expressed at the late acute stage of infarct. Therefore, the expression of Bcl-2 protein may not protect acute cell death, but may play a role in the prevention of late cell death after myocardial is chemia-reperfusion.

Imaging Myocardial Ischemia and Reperfusion Injury via Cy5.5-Annexin V

Tian, Rong,Pan, DongFeng The Korea Society of Nuclear Medicine 2012 핵의학 분자영상 Vol.46 No.3

Aim The aim of this article is to present the results of an imaging study of myocardial apoptosis induced by ischemia/reperfusion injury. Methods Twenty nude mice were randomly divided into an experimental group (10 mice) and control group (10 mice). In the experimental group, myocardial apoptosis was induced by ligation of the left anterior descending coronary artery (LAD) for 30 min. This was followed by reperfusion for 90 min. In the control group, the heart was exposed for the same length of time as in the experimental group. Cy5. 5-annexin V (25 ${\mu}g$) was injected into both sets of mice after the onset of reperfusion. At 90 min post-injection, the mice were imaged. The region of interest (ROI) was obtained, and the fluorescence intensity of the ROI was quantified. The animals were sacrificed, and myocardial apoptosis was assayed by TUNEL assay. Results Fluorescence intensity in the ischemia/reperfusion hearts was significantly higher than that in the control group (P<0.05). In the TUNEL assay, more apoptotic cells were observed in the experimental group than in the control group, correlating with imaging results. Conclusion Fluorescence imaging of Cy5.5-annexin V in a mouse model of myocardial ischemia/reperfusion can be used in vivo as a noninvasive means of detecting ischemia/reperfusion-induced apoptotic cells in the heart.

Myocardial Protective Effect of Tezosentan, an Endothelin Receptor Antagonist, for Ischemia-Reperfusion Injury in Experimental Heart Failure Models

류세민,김학제,조규란,조원민 대한의학회 2009 Journal of Korean medical science Vol.24 No.5

The myocardial protective effects of endothelin antagonist in ischemic cardiomyopathy (ICMP), doxorubicin-induced cardiomyopathy (DOX) and pressure-overload hypertrophy by transverse aortic constriction (TAC) models have been predicted to be different. The objective of this experiment, therefore, is to evaluate the myocardial protective effect of tezosentan, an endothelin receptor antagonist, in various experimental heart failure models. Sprague-Dawley rats (6-8 weeks old, 200-300 g) were randomized to three experimental groups (n=30 each): ICMP; DOX; and TAC group. Each of these groups was randomly assigned further to the following subgroups (n=10 each): sham-operated ischemia-reperfusion subgroup (SHAM); tezosentan treated ischemia-reperfusion subgroup (Tezo); and tezosentan non-treated ischemia-reperfusion subgroup (N-Tezo). Total circulatory arrest was induced for 1 hr, followed by 2 hr of reperfusion. The left ventricular developed pressure, peak positive and negative first derivatives, and coronary blood flow were significantly different (P<0.05) among the SHAM, Tezo, and N-Tezo subgroups of the ICMP group at 30 min of reperfusion, but there were no statistically significant differences among the subgroups of the DOX and TAC groups. In conclusion, tezosentan, an endothelin receptor antagonist, showed myocardial protection effects only on the ischemic cardiomyopathy rat model, but not in the non-ischemic heart failure rat models.

Cardiodynamics and Infarct Size in Regional and Global Ischemic Isolated Heart Model: Comparison of 1 Hour and 2 Hours Reperfusion

김준홍,김준,박용현,전국진,김정수,Young Ho Jang,이미영,Zhelong Xu 대한심장학회 2012 Korean Circulation Journal Vol.42 No.9

Background and Objectives: We investigated whether 1 hour reperfusion is enough to assess cardiodynamics and infarct size in both regional ischemia (RI) and global ischemia (GI) in isolated rat heart models. Materials and Methods: Hearts were randomly assigned to one of the following groups (each n=14): 1) Sham hearts for 1 hour; 2)Sham hearts for 2 hours; 3) 30 minutes RI followed by 1 hour reperfusion; 4) 30 minutes of RI followed by 2 hours reperfusion; 5) 30minutes GI followed by 1 hour reperfusion; and 6) 30 minutes GI followed by 2 hours reperfusion. Results: There were no significant differences in infarct size between 1 hour and 2 hours reperfusion in both RI and GI. Left ventricular developed pressure was significantly decreased at both 1 hour and 2 hours reperfusion in groups of RI and GI compared to baseline (p<0.01). Rate-pressure product and +dP/dt max also significantly decreased compared to baseline level at both 1 hour and 2 hours reper-fusion in groups of RI and GI (p<0.05). Conclusion: There was no significant difference in infarct size between 1 hour and 2 hours reperfusion in groups of RI and GI. Cardiody-namic variables measured at 1 hour and 2 hours reperfusion significantly decreased compared to baseline level. Our data suggests that reperfusion of 1 hour is sufficient to assess cardiodynamics in both regional and global ischemic isolated hearts model.

급성 심근 경색 환자에서 재관류 후 조기에 시행한 휴식 / 24시간 지연 T1-201 심근 SPECT 의 심근벽 운동 호전 예측능

현인영,권준 ( In Young Hyun,June Kwan ) 대한핵의학회 1998 핵의학 분자영상 Vol.32 No.3

Purpose: We studied early rest/24 hour delay Tl-201 perfusion SPECT for prediction of wall motion improvement after reperfusion in patients with acute myocardial infarction. Materials and Methods: Among 17 patients (male/female ll/6, age: 59+13) with acute myocardial infarction, 15 patients were treated with percutaneous transcoronary angioplasty (direct:2, delay:11) and intravenous urokinase (2). Spontaneous resolution occurred in infarct-related arteries of 2 patients. We confirmed TIMI 3 flow of infarct-related artery after reperfusion in all patients with coronary angiography. We performed rest Tl-201 perfusion SPECT less then 6 hours after reperfusion and delay Tl-201 perfusion SPECT next day. Tl-201 uptake was visually graded as 4 point score from norrnal (0) to severe defect (3). Rest Tl-201 uptake <2 or combination of rest Tl-201 uptake<2 or late reversibility were considered to be viable. Myocardial wall motion was graded as 5 point score from normal (1) to dyskinesia (5). Myocardial wall motion was considered to be improved when a segment showed an improvement > 1 grade in follow up echo compared with the baseline values. Results: Among 98 segments with wall rnotion abnormality, the severity of myocardial wall motion decrease was as follow: mild hypokinesia: 18/98 (18%), severe hypokinesia: 28/98 (29%), akinesia: 5l/98 (52%), dyskinesia: 1/98 (1%), The wall rnotion improved in 85%. Redistribution (13%), and reverse redistribution (4%) were observed in 24 hour delay SPECT. Positive predictive value (PPV) and negative predictive value (NPV) of combination of late reversibility and rest Tl-201 uptake were 99%, and 54%. PPV and NPV of rest T1-201 uptake were 100% and 52% respectively. Predictive values of combination of rest Tl-201 uptake and late reversibility were not significantly different compared with predictive values of rest Tl-201 uptake only. Conclusion: We conclude that early Tl-201 perfusion SPECT predict myocardial wall motion improvement with excellent positive but relatively low negative predictive values in patients with acute myocardial infarction after reperfusion. (Korean J Nucl Med 1998;32:259-65)