| Infarct size after acute myocardial infarction is highly related with post-infarct arrhythmia, heart failure and mortality. The most important therapeutic goal in myocardial infarction management is to decrease infarct size. Early reperfusion therapy is an effective method to save myocardium and reduce infarct size. But unfortunately, reperfusion itself will cause death of cardiomyocytes and decrease myocardial contractility. It is clear that inhibit or diminish reperfusion injury will improve the effects of reperfusion therapy, reduce complication and further decrease myocardial infarction mortality.Ischemia conditioning is defined as repetitive intermittent ischemia myocardium or other unlethal organs again before, after or during events may decrease the reperfusion injury. This means ischemia pre-conditioning, post-conditioning, per-conditioning and remote conditioning may protect myocardium from reperfusion injury. Recently, a Netherland group reported that pacing induced pre-conditioning and post-conditioning might protect reperfusion injuries in rabbit and pig models.First, we established right ventricular apex pacing models in vivo in New Zealand rabbits and SD rats by using Metronic Select Secure3830active lead and rapid atrial stimulation pacing mode. Second, we tested left ventricular dyssynchrony in these two pacing models by electrophysiology and echocardiogram. We verified20-cylce pacing induced pre-conditioning,10,20-cycle per-conditioning and20-cycle post-conditioning decreased the infarct sizes stained by Evans blue and TTC in rabbit and rat models. Among the pacing induced condition groups, the positive effects of20-cycle pre-condition were the best. The effects of20-cycle per-conditioning is better than those of10-cycle. And we also found that the decrease of infarct size was accompanied with decrease of cardiomyocyte apoptosis. And we verified pacing induced conditioning might protect reperfused myocardium through activating PKC signal pathway, inhibit expression of inflammatory factors, decrease apoptosis of cardiomyocytes and finally decrease reperfusion injury through ELISA, Western blot and qPCR. Last not the least, pacing induced pre-conditioning, per-conditioning and post-conditioning decreased8-week ischemic reperfusion injury by inhibiting myocardial fibrosis, decreasing left ventricle wall thickness and left ventricular enlargement and increasing left ventricular systolic function in SD rats. Pacing induced conditioning protects long-tern ischemic reperfusion injury through down-regulating AT1R expression, activating PKC signal pathway and decreasing phosphorylation of ERK, following inhibition of myocardial fibrosis and myocardial remodeling and improvement of heart function.Right ventricular apex pacing induced pre-conditioning, per-conditioning and post-conditioning protect post acute myocardial infarction reperfusion injury in New Zealand rabbit and SD rat models. The molecular mechanism of this protection is that stretch down-regulating PKC signal pathway, inhibiting expression of inflammatory factors, decreasing apoptosis of cardiomyocytes and finally decreasing reperfusion injury. On the other hand, pacing induced conditioning down-regulates AT1R expression, activates PKC signal pathway and decreases phosphorylation of ERK, following inhibits of myocardial fibrosis and myocardial remodeling and improves of heart function. PKC signal pathway plays a key role not only in decreasing cardiomyocyte apoptosis but also in myocardium fibrosis.Part ⅠSetup of acute myocardial infarction and reperfusion injury and in vivo right ventricular apex pacing in rabbits and SD ratsObjective:To setup acute myocardial infarction and reperfusion injury and in vivo right ventricular apex pacing in animal models of rabbits and SD rats.Methods:Left ventricular myocardial infarction models were setup by ligation coronary artery in an open chest manner in New Zealand rabbits or Sprague Dawley rats. Electronic activities of rabbits and rats were detected by using12leads EKG. Rat coronary angiography was taken in an isolated rat heart by30%Barium Sulfate retro perfusion aortic artery and exposed under Carestream live image system. To setup live pacing model, a Select Secure3830active pace leads was screwed into right ventricle apex portion of rabbits or rats and connected with Metronic2090programmer plus2290analyzer. A VOO pace model, pacing rate20beats per minute faster than self heart rate, was proceeded to capture self heart rhythm. QRS widths were measured and compared before and after pacing. Septal to posterior wall motion delay (SPWMD) and pre ejection period (PEP) were measured and calculated by transthoracic echocardiogram after closing the chest and compared before and after pacing in rabbits. After inserting a catheter into left ventricle through carotid arteries, the changing of left ventricular pressures and dp/dt were measured and compared before and after pacing in rabbits too.Results:After ligation30minutes and releasing of left ventricular branch in rabbit or left descending anterior artery in SD rat, the typical ST-T dynamic changes and reperfusion arrhythmia were recorded in12-lead EKG Normal coronary and blockage of left anterior descending artery angiogram were taken successfully. After VOO pacing, the QRS widths were wider significantly compared with that of before in right ventricle apex pacing models of rabbits or rats separately. A total5rabbits were measured by echocardiogram. SPWMD was (20.02±8.32)ms before pacing and (78.80±26.57) ms (P=0.007) after pacing. PEP was (14.40±5.86)ms before pacing and (-26.80±7.33) ms (P<0.001) after pacing. The maximum dp/dt was (1072.82±154.85)mmHg/ms and (648.75±135.09)mmHg/ms (P<0.001) before and after pacing, the minimum dp/dt was (-989.49±100.51)mmHg/ms and (-547.57±148.11)mmHg/ms (P=0.002) before and after pacing.Conclusion:It is reliable to establish acute myocardial infarction and reperfusion injury models by ligation coronary arteries. And it is also reliable to setup right ventricle apex pacing models by Select Secure3830active pace lead in rabbits or rats. And right ventricle apex pacing may induce inter and intra ventricles dyssynchronization verified by electrophysiology and echocardiogram and decrease left ventricular systolic function. Part ⅡShort term effects of right ventricular apex pacing induced dyssynchronization protects ischemic reperfusion injury in New Zealand rabbits Objective:To explore whether right ventricular apex pacing induced dyssynchronization might protect ischemic reperfusion injury in New Zealand rabbits.Methods:Acute myocardial infarction reperfusion injury models were setup by ligation left ventricle branch (LVB)30minutes and reperfusion210minutes in an open chest manner in New Zealand rabbits. Right ventricle apex pacing was established by Metronic Select Secure3830active pacing lead. Sixty rabbits were divided into sham group, without pacing; per-conditioning group (Per10), pacing at self heart rate plus20bpm frequency for ten cycle of30seconds pacing and30seconds interval after ligation LVB20minutes, following release ligation for reperfusion; post-conditioning group (Post20),20cycle pacing immediately after release ligation for reperfusion; per-conditioning group (Per20),20cycle pacing before release ligation for reperfusion; and pre-conditioning group (Pre20),20cycle pacing before ligation. Venous blood were taken before opening chest and sacrifice for test of CK-MB, TNT, LDH, CRP, TNF-α, IFN-γ and IL-10levels. Infarct ratio and ischemia ratio were measured and calculated by Evans blue and TTC staining in6to9rabbits each group. The hearts were fixed in10%formaldehyde instantly after sacrifice. Haematoxylin-eosin stained sections, immunohistochemistry connexin43expression and TUNEL for apoptosis ratio were performed.Results:1. There were no statistical differences of heart/body ratio and ischemia ratio, but statistical differences of infarct ratio among5groups. The infarct ratio of sham group (n=9) was61.23%, higher than42.46%of Pre20group (n=7) and41.03%of Per20group (n=6). The infarct ratio of Post20group (n=7) was47.87%, higher than those of Pre20group and Per20group but lower than that of Per10group (n=6,50.90%) with statistic significant difference.2. Comparing with those of baseline, the serum myocardium enzymes including creatine kinase isoform MB (CKMB), troponin T (TNT) and lactate dehydrogenase (LDH) detected after ligation LVB30minutes and reperfusion210minutes were increased significantly. Pacing induced conditionings decreased the serum enzyme levels comparing those of sham pacing group and more effective in Per20and Pre20group.3. There were significant differences of cardiomyocyte apoptosis rate among five groups (P<0.001). The highest rate was82.42%in sham pacing group, then68.93% and69.04%in Per10and Post20group individually (no statistic difference between Per10and Post20). The lowest rates were62.85%and58.88%in Per20and Pre20group individually, with no statistic difference between two groups but with difference comparing other three groups.4. Comparing with those of baseline, the serum inflammatory factors including C reacting protein (CRP), tumor necrosis factor-a (TNF-a), interleukin-10(IL-10) and interferon-y (IFN-y) detected after ligation LVB30minutes and reperfusion210minutes were increased significantly. There was no statistic difference of inflammatory factors level between sham pacing group with those of Per10group. The serum levels of TNF-a and IFN-y were decreased in Post20group and CRP> TNF-α、IL-10and IFN-y levels decreased in Per20and Pre20group.Conclusion:1. Right ventricular apex pacing induced conditioning, including precondition, percondition and postcondition, can decrease ischemic reperfusion injury in rabbits. Among all the pacing induced conditioning modes, preconditioning decrease more ischemic reperfusion injury comparing with those of perconditioning and postconditioning. The protective effects of20-cycle perconditioning are better than that of10-cycle perconditioning, similar to preconditioning.2. Pacing induced conditioning can decrease the apoptosis rate of cardiomyocytes after onset of acute myocardial infarction in rabbits, and preconditioning and20-cycle perconditioning are the most effective ones. In the meantime, pacing induced conditioning can also decrease the serum levels of inflammatory factors, including CRP, TNF-a, IL-10and IFN-y significantly when comparing with that of without pacing.3. Pacing induced conditioning may protect reperfused myocardium through inhibiting inflammatory factors which cause apoptosis of cardiomyocytes. Part ⅢShort term effects of right ventricular apex pacing induced dyssynchronization protects ischemic reperfusion injury in ratsObjective:To explore right ventricular apex pacing induced myocardial conditioning inhibits ischemia reperfusion injury in rats.Methods:Acute myocardial infarction reperfusion injury models were setup by ligation left anterior descending artery (LAD)30minutes and reperfusion90minutes in an open chest manner in SD rats. Right ventricle apex pacing was established by Metronic Select Secure3830active pacing lead. The rats were divided into sham group, without pacing; per-conditioning group (Per10), pacing at self heart rate plus20bpm frequency for ten cycle of30seconds pacing and30seconds interval after ligation LAD20minutes, following release ligation for reperfusion; post-conditioning group (Post20),20cycle pacing immediately after release ligation for reperfusion; per-conditioning group (Per20),20cycle pacing before release ligation for reperfusion; and pre-conditioning group (Pre20),20cycle pacing before ligation. Venous blood were taken before opening chest and sacrifice for test of CK-MB, TNT, LDH, CRP, TNF-α, IFN-γ and IL-10levels. Infarct ratio and ischemia ratio were measured and calculated by Evans blue and TTC staining in each group. Haematoxylin-eosin stained sections, immunohistochemistry connexin43expressions and TUNEL for apoptosis ratio were performed. Myocardial RNA was extracted to detect inflammation related genes (IL12a、IL6R、IL8Ra and IL8Rb) and transcriptional activating related genes (HSP ala and TGF betal) expressions. The expression of phosphorylated (phos-) protein kinase C (PKC) α, phos-PKCβ1, phos-PKCβ2, phos-PKC y, phos-extracellular signal-regulated kinase (ERK), pIκB, pNFκB, IκB, NFκB, Akt family, Bcl-2family, surviving and active caspase-3were measured by western blot and normalized to the protein level of GAPDH or b-actin.Results:1. There were no statistical differences of heart/body weight ratio and ischemia ratio, but statistical differences of infarct ratio among5groups (Χ2value was52.40, P<0.001). The infarct ratio of sham group (n=9) was the highest,48.21%. The infarct ratio of Per10group (n=6), Post20group(n=7), Per20group (n=6) and Pre20group (n=7) were37.07%,43.53%,27.71%and22.66%with statistic significant differences. The infarct ratio of Pre20group was the lowest. 2. Comparing with those of baseline, the serum myocardium enzymes including creatine kinase isoform MB (CKMB), troponin T (TNT) and lactate dehydrogenase (LDH) detected after ligation LAD30minutes and reperfusion90minutes were increased significantly. Pacing induced conditionings decreased the serum enzyme levels comparing those of sham pacing group and the most effective in Pre20group.3. There were significant differences of cardiomyocyte apoptosis rate among five groups (P<0.001). The highest rate was58.92%in sham pacing group, then48.83%and49.57%in Per10and Post20group individually (no statistic difference between Per10and Post20). The lowest rates were40.73%and40.11%in Per20and Pre20group individually, with no statistic difference between two groups but with difference comparing other three groups.4. Comparing with those of baseline, the serum inflammatory factors including C reacting protein (CRP), tumor necrosis factor-a (TNF-a), interleukin-10(IL-10) and interferon-y (IFN-y) detected after ligation LAD30minutes and reperfusion90minutes were increased significantly. The serum levels of inflammatory factors were decreased in conditioning group with statistic differences. The serum levels of inflammatory factors of Pre20group decreased the most.5. The expression of connexin43showed obvious disorder and mass like aggression in sham group but milder disorder in pacing condition group; In Pre20group, the expression of connexin43showed normal density with short brand like staining at intercalated disc position of cardiomyocytes.6. The pacing induced condition decreased the activating expression of subgroups PKC and downloaded the phosphorylation of ERK, especially in Pre20group (P<0.05).7. The pacing induced condition downloaded the expression of inflammatory factors (IL12a、IL6R、IL8Ra and IL8Rb) and of transcriptional activating related genes (HSP ala and TGF betal) significantly. In the meantime, the expressions of inflammatory related protein pIκB and pNFκB were downloaded and total IκB and NFκB were maintained.8. The pacing induced condition uploaded the expression of apoptosis related protein, pAKT/AKT、Bcl-2/Bax and Survivin and downloaded the expression of Caspase3, the most in Pre20group.Conclusion:1. Right ventricular apex pacing induced conditioning, including pre-conditioning, per-conditioning and post-conditioning, can decrease ischemic reperfusion injury in SD rats. Among all the pacing induced conditioning modes, pre-conditioning decrease more ischemic reperfusion injury comparing with those of per-conditioning and post-conditioning. The protective effects of20-cycle per-conditioning are better than that of10-cycle per-conditioning, similar to pre-conditioning.2. Pacing induced conditioning may protect reperfused myocardium through activating PKC signal pathway, inhibit expression of inflammatory factors, decrease apoptosis of cardiomyocytes and finally decrease reperfusion injury. On the other hand, pacing induced dyssynchronization may regulate expression of connexin43, which also decrease the apoptosis of cardiomyocytes, to protect injured myocardium. Part ⅣLong-term effects of pacing induced conditioning of ischemic reperfusion injury in ratsObjective:To explore the long-term effects of pacing induced conditioning of ischemic reperfusion injury in acute myocardial infarction SD rats.Methods:Acute myocardial infarction reperfusion injury models were setup by ligation left anterior descending artery (LAD)30minutes and reperfusion90minutes in an open chest manner in SD rats. Right ventricle apex pacing was established by Metronic Select Secure3830active pacing lead. The rats were divided into sham group, without pacing; per-conditioning group (Per20), pacing at self heart rate plus20bpm frequency for20cycle of30seconds pacing and30seconds interval after ligation LAD20minutes, following release ligation for reperfusion; post-conditioning group (Post20),20cycle pacing immediately after release ligation for reperfusion and pre-conditioning group (Pre20),20cycle pacing before ligation. Transthoracic echocardiogram was performed before opening chest and8-week after the surgery. Left ventricular anterior wall (diastole)(LVAWd), left ventricular posterior wall (diastole)(LVPWd), Left ventricular internal diameter (diastole)(LVIDd), LV ejection fraction(LVEF) and LV Fractional Shortening(LV%FS) were detected and calculated through left parasternal long axis view at M-mode. Nucleo plasmic relation ratio was measured and calculated under HE staining and myocardial fibrosis ratio under Masson staining. The expression of phosphorylated (phos-) protein kinase C (PKC)a, phos-PKCβ1, phos-PKCβ2, phos-PKC y, phos-extracellular signal-regulated kinase (ERK) and ATI Receptor (AT1R) were measured by western blot and normalized to the protein level of GAPDH.Results:Pacing induced conditioning improved the left ventricular systolic function (LVEF and FS%) significantly under echocardiogram after8-week ischemic reperfusion injury. Comparing with those of sham pacing group, LVAWd and LVPWd were shortened and LVIDd decreased in3pacing induced conditioning groups. And myocardial fibrosis ratio and nucleo plasmic relation ratio of condition groups were decreased significantly comparing with those of sham group. The pacing induced condition decreased the expression of AT1R (P<0.01) which mediated myocardial hypertrophy, the activating expression of downstream subgroups PKC (P<0.001) and downloaded the phosphorylation of ERK (P<0.001).Conclusion:Twenty-cycle right ventricular apex pacing induced conditioning, including pre-conditioning, per-conditioning and post-conditioning, can decrease8-week ischemic reperfusion injury by inhibiting myocardial fibrosis, decreasing left ventricle wall thickening and left ventricular enlargement and increasing left ventricular systolic function in SD rats. Pacing induced conditioning protects long-tern ischemic reperfusion injury through downloading AT1R expression, activating PKC signal pathway and decreasing phosphorylation of ERK, following inhibition of myocardial fibrosis and myocardial remodeling and improvement of heart function. Conclusion1. Acute anterior myocardial infarction and right ventricular apex pacing models are successfully established in vivo in New Zealand rabbits and Sprague Dawley rats.2. Right ventricular apex pacing induced pre-conditioning, per-conditioning and post-conditioning decrease ischemic reperfusion injury.3. The molecular mechanism of pacing induced condition is that stretch down-regulating PKC signal pathway, decreasing the expression of inflammatory factors, inhibiting cardiomyocyte apoptosis and reducing reperfusion injury.4. Pacing induced pre-conditioning, per-conditioning and post-conditioning inhibit myocardial fibrosis, left ventricle enlargement and deterioration of left ventricular systolic function after myocardial infarction in a long term manner.5. Pacing induced conditioning protects ischemia reperfusion injury via down-regulating AT1R-PKC-ERK signal pathway, inhibiting myocardial remodeling and improving heart function.The potential application and novelty of this project1. We discover the existence of right ventricular apex pacing induced per-condition protecting ischemic reperfusion injury for the first time ever.2. We establish right ventricular apex pacing models in vivo in Sprague Dawley rats for the first time and verify pacing induced pre-conditioning, per-conditioning and post-conditioning decreasing ischemic reperfusion injury.3. We discover the molecular mechanism of pacing induced condition is that stretch down-regulating PKC signal pathway, decreasing the expression of inflammatory factors, inhibiting cardiomyocyte apoptosis and reducing reperfusion injury. This provides the new theoretic evidence of different forms of conditioning.4. We also find pacing induced conditioning inhibits myocardial fibrosis, left ventricle enlargement and deterioration of left ventricular systolic function after myocardial infarction in long term manner via down-regulating AT1R-PKC-ERK signal pathway. This provides the new evidence that PKC signal pathway plays a role in fibrosis formation.5. Our study is as a supplement of conditioning, a unique interfere form of myocardial infarction, in different form and mechanism. Under the circumstances of translational medicine, pacing induced conditioning with its clinical convenience may decrease the mortality of acute myocardial infarction in short-term and long-term manner and may change the therapeutic model of acute myocardial infarction. |
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