Myocardial Infarction In The Experimental Study Of Gene Therapy

rAAV-mediated angiogenin gene transfer induces angiogenesis and modifies left ventricular remodeling in rats with myocardial infarctionBackgroundCardiac dysfunction following myocardial infarction (MI) results from two major mechanisms: loss of cardiomyocyte contractility in the area of infarction, and remodeling of the spared myocardium in the left ventricle. Inadequate structural adaptation of the vascular bed to the hypertrophy of the surrounding cardiomyocytes may play a role in the progression from left ventricular dysfunction to heart failure. It has been reported that vascular growth is inadequate to maintain normal capillary density in the hypertrophied myocardium of infarcted hearts. Moreover, the lower capillary density as a consequence of cardiomyocyte hypertrophy may limit the oxygen supply and lead to ischemia, which may result in ongoing loss of viable tissue, infarct extension, and replacement fibrosis. Induction of neovascularization is recognized to be a valid approach to modify the pathophysiological changes of ventricular remodeling. Transfer of angiogenic genes and some drug administration are known to induce angiogenesis and eliminate or attenuate cardiac dysfunctionafter MI in animal models. Angiogenesis is a multi-step process that involves many angiogenic factors, and it has been reported that angiogenin (ANG) is essential in the course of neovessel formation. In vitro studies have confirmed that bovine ANG stimulates both the migration of endothelial cells and the formation of tube-like structures. In vivo studies have demonstrated that ANG is beneficial in bind limb ischemic models, and ANG-expressing bone marrow stem cell transplantation is effective in repairing damaged myocardium and enhancing angiogenesis in ischemic myocardium. However, as yet the effects of direct ANG gene transfer into myocardium of animals with MI remain unclear.ObjectiveIn this study, we performed experiments to test the hypothesis that intramyocardial injection of a recombinant adeno-associated virus vector encoding the ANG gene (rAAV-ANG) can augment therapeutic neovascularization, leading to restoration of left ventricular remodeling, and prevention of cardiac dysfunction in a rat model of MI.MethodsUsing an AAV helper-free system, pAAV-ANG (or pAAV-lacZ), pAAV-RC, and pHelper were cotransfected into 293 cells to obtain rAAV-ANG and rAAV-lacZ vectors. MI model was generated in male Wistar rats by ligating the left anterior descending coronary artery. Then MI rats were randomly divided into 3 groups, designated Mi-saline, MI-rAAV-lacZ and MI-rAAV-ANG Immediately after the coronary artery ligation, saline or rAAV-lacZ or rAAV-ANG was injected into 4 sites of myocardium around the infarction area. In the normal-rAAV-ANG group, normal rats were anesthetized and thorax opened, and administered intramyocardial injection of same dose of rAAV-ANG with the same procedure. Normal rats without any intervention and sham-operation rats served as control groups. Four weeks later, left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD) and left ventricular ejection fraction (LVEF) were detected byechocardiography; left ventricular hemodynamic parameters were detected to evaluate left ventricular function; western blot and immunohistochemistry were performed to detect myocardium ANG protein expression; immunohistochemistry to vWF was performed to detect myocardium capillary density; left ventricular weight to body weight ratio, cardiomyocyte diameter and fibrosis infiltration in noninfarction area were measured to evaluate left ventricular remodeling.Results1. rAAV-ANG and rAAV-lacZ vectors were constructed successfully. The infection titer of the vectors was 3×10¹⁰ IU /mL.2. Evaluated by echocardiography and histological detection, MI rat model was generated successfully. There was no significant difference of MI area among the groups of MI-saline, MI-rAAV-lacZ and MI-rAAV-ANG3. X-gal staining showed that, in MI-rAAV-lacZ group, cytoplasm of a large number of cardiomyocytes around the infarction area was stained blue, indicating effective transfection. X-gal staining of the brain, kidney, liver and lung tissue slices of the MI-rAAV-lacZ group showed no positive staining.4. Low-level expression of myocardium ANG protein was found in the normal, sham-operation, MI-saline, and MI-rAAV-lacZ groups, while rAAV-ANG transfection resulted in a significantly higher myocardium ANG protein levels in both the normal-rAAV-ANG group and MI-rAAV-ANG group. Immunohistochemical detection showed that the ANG protein diffusely distributed in the cytoplasm of cardiomyocytes. We did not score the positive staining in each experimental group, but observed an obvious tendency that was concordant with the results of western blot.5. Quantitative assessment at 4 weeks after the induction of MI revealed reduced capillary density in Mi-saline and MI-rAAV-lacZ rats compared with the normal and sham-operation rats. Myocardium capillary density in normal-rAAV-ANG rats was relatively higher than that in the normal and sham-operation groups (both P<0.05). Also a significantly higher capillary count was observed in the MI-rAAV-ANG group than in the Mi-saline group (P<0.05).6. Compared with the normal and sham-operation rats, rAAV-ANG transfection had no effects on left ventricular diameter, cardiac function, left ventricular weight, left ventricular weight to body weight ratio, cardiomyocyte diameter and interstitial fibrosis in normal rats.7. As indicated by LVEDD and LVESD, the left ventricle was dilated in all the MI rats 4 weeks after coronary artery ligation, but rAAV-ANG treatment partly alleviated this. MI induced an increase of ventricular weight, which was reflected by the data of MI-saline and MI-rAAV-lacZ groups, while rAAV-ANG transfection normalized the ventricular weight to the level of sham-operation group. Moreover, ventricular weight to body weight ratio, which increased in MI-saline and MI-rAAV-lacZ groups, was reduced to the level of sham-operation group in the MI-rAAV-ANG group. MI-induced hypertrophy, which was microscopically confirmed by the increase of cardiomyocyte diameter, was restored by rAAV-ANG transfer. Significantly higher extent of interstitial fibrosis in the noninfarcted region in both the MI-saline group and MI-rAAV-lacZ group was significantly alleviated by rAAV-ANG transfection.8. Four weeks after coronary artery ligation, compared with the normal and sham-operation groups, LVEF, LVSP and ±dp/dt_max were significantly decreased and LVEDP increased in all MI rats, indicating marked cardiac dysfunction, but these parameters were partly restored in MI-rAAV-ANG rats.Conclusions1. Four weeks after the induction of MI, rats showed significant left ventricular dysfunction and left ventricular remodeling, including increased left ventricular weight, dilation of left ventricle, and increase of myocardiocyte diameter and fibrosis infiltration in the noninfarction area, accompanied with decreased capillary density in the noninfarction area.2. Compared with normal and sham-operation rats, rats in norml-rAAV-ANG group showed increased myocardium ANG protein expression, increase of capillary density. But rAAV-ANG treatment had no effect on the cardiac function and left ventricular remodeling in normal rats.3. rAAV-ANG treatment in MI rats increased myocardium ANG protein expression and capillary density, alleviated left ventricular remodeling and attenuated left ventricular dysfunction.rAAV-asPLB transfer attenuates abnormal sarcoplasmic reticulum Ca²⁺-ATPase activity and cardiac dysfunction in rats with myocardial infarctionBackgroundAbnormality of myocardium sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) activity and phospholamban (PLB) level during the cardiac dysfunction have been observed by many studies. Overexpression of SERCA by gene transfer in failing human cardiomyocytes significantly restored calcium handling and contractility. On the other hand, inhibition of PLB expression had positive results in heart failure therapies. Decreased PLB expression by antisense PLB (asPLB) resulted in increased velocity of both contraction and relaxation in cardiomyocytes isolated from failing human hearts. PLB-null mutation was reported to prevent systolic dysfunction and improve exercise intolerance in a murine model of hypertrophic cardiomyopathy.ObjectiveCurrent study was designed to examine the effects of rAAV-antisense phospholamban (asPLB) gene transfer on cardiac function, myocardium SERCA expression and activity, as well as PLB expression and its phosphorylation (Pser16-PLB) in a rat myocardial infarction (MI) model.MethodsrAAV-asPLB was constructed using an AAV Helper-Free System. MI was induced in adult male Wistar rats by ligating the left anterior descending coronary artery, and then MI rats were randomly divided into 3 groups: Mi-saline group, MI-rAAV-lacZ group and MI-rAAV-asPLB group. Immediately after the coronary artery ligation in the three groups, 120 μL saline or 120 μL rAAV-lacZ (1.2×10⁹ IU) or 120 μL rAAV-asPLB (1.2×10⁹ IU) was injected into 4 sites of the myocardium around the infarction area with a 30-gauge needle. Additionally, in the normal-rAAV-asPLB group, normal rats were anesthetized and thorax opened, and administered intramyocardial injection of same dose of rAAV-asPLB into the same region with the same procedure. One week after the operation, echocardiography was performed to estimate left ventricular infarct size with the centerline method. Four weeks after the operation, left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD) and left ventricular ejection fraction (LVEF) were detected using echocardiography. Left ventricular systolic pressure (LVSP), the maximum velocity of ascending and descending in intraventricular pressure (±dp/dt_max) and left ventricular end diastolic pressure (LVEDP) were detected with hemodynamic measurements. Myocardial SERCA activity, protein expression levels of PLB, Pser16-PLB and SERCA were measured.Results1. Four weeks after MI, compared with sham-operation group, rats in MI-saline group and MI-rAAV-lacZ group showed significant left ventricular dilation and left ventricular dysfunction, as was indicated by increased LVEDD and LVESD, markedly depressed LVEF, LVSP and ±dp/dt_max, and significantly increased LVEDP. Compared with sham-operation group, depressed myocardial SERCA activity, markedly increased myocardial PLB level and decreased Pser16-PLB level were observed in rats of MI-saline and MI-rAAV-lacZ groups.2. Compared with sham-operation group, left ventricular diameter and function were not significantly altered in normal-rAAV-asPLB group. rAAV-asPLBtransfer slightly inhibited myocardial PLB protein expression without statistical significance, and had no effects on myocardial Pser16-PLB level, SERCA acitivity and SERCA protein level in normal rats.3. Compared with sham-operation group, left ventricular function was not completely restored in MI-rAAV-asPLB group, but compared with MI-saline group, LVEF, LVSP and ±dp/dt_max were markedly increased and LVEDP decreased in MI-rAAV-asPLB group. Although rAAV-asPLB treatment normalized LVESD to the level of sham-operation group, no significant improvement of LVEDD was observed in rat of MI-rAAV-asPLB group.4. Compared with MI-saline group, myocardial PLB expression was significantly inhibited; PLB phosphorylation in Ser16 site and myocardial SERCA activity were restored in MI-rAAV-asPLB group.5. No significant difference was observed in myocardium SERCA protein level among all the experimental groups.Conclusions1. Four weeks after ligation of left anterior descending coronary artery, significant left ventricular dilation and cardiac dysfunction were observed in rats, and myocardial PLB protein expression was increased, Pser16-PLB decreased and SERCA activity decreased.2. In normal rats, rAAV-asPLB gene transfer by intramyocardial injection inhibited myocardial PLB protein expression slightly without statistical significance, and had no effects on left ventricular diameter and cardiac function, myocardial SERCA activity, Pser16-PLB level, and SERCA protein level.3. In MI rats, left ventricular function was significantly improved by rAAV-asPLB transfer, which inhibited myocardial PLB protein expression, restored phosphorylation of PLB on Ser16 site, and restored myocardial SERCA activity, and had no effects on myocardium SERCA protein level.Alterations of myocardium phospholamban expression and cardiac sarcoplasmic reticulum Ca²⁺ -ATPase activity in diabetic ratBackgroundDiabetic cardiomyopathy is one of the most important complications of diabetes mellitus (DM). The gene alterations which involved in the occurrence and progression of diabetic cardiomyopathy remain unclear to date. Contractility and dilation of myocardium is related to sarcoplasmic reticulum calcium ATPase (SERCA) activity, which is regulated by phospholamban (PLB). Previous studies found that, in idiopathic dilated cardiomyopathy, chronic phospholamban-SERCA interaction is the critical calcium cycling defect, and expression of additional phospholamban molecules resulted in inhibition of sarcoplasmic reticulum Ca²⁺ transport, decreases in systolic Ca²⁺ levels and contractile parameters in ventricular myocytes, and depression of basal left ventricular systolic function in vivo. As to diabetic cardiomyopathy, it was found that altered sarcoplasmic reticulum protein expression was associated with contractile dysfunction in diabetic rat hearts, andinsulin treatment normalized sarcoplasmic reticulum protein expression and function. In vitro study showed that in cardiomyocytes isolated from diabetic rats, systolic and diastolic function were markedly depressed, which were related with altered sarcoplasmic reticulum functions of uptake and/or release of Ca²⁺. Some in vivo studies have observed the altered expression of Ca²⁺-handling proteins in diabetic rats and obtained conflicting results.ObjectiveThis study was designed to investigate the alterations of SERCA protein and activity, the expression of PLB mRNA and protein in diabetic myocardium and their relationship with cardiac function in a DM rat model.MethodsDM was induced in male adult Wistar rats (8 weeks old) by intraperitoneal injection of streptozotocin (65mg/kg), and the development of DM was confirmed by urine analysis and serum glucose level (≥16.7mmol/L) on the 7th day of streptozotocin injection. DM rats were then randomly subdivided into 3 groups according to the time they were detected and sacrificed: 4 weeks, 6 weeks and 8 weeks after streptozotocin-injection (4-wk-DM, 6-wk-DM, 8-wk-DM), respectively. At each time point, hemodynamic measurement was performed to evaluate left ventricular function, the protein levels of PLB and SERCA in rat myocardium were detected with Western blot, myocardium PLB mRNA level was semi-quantitatively detected with RT-PCR. Myocardium SERCA activity was evaluated.Results1. As was evaluated by Western blot analysis, no significant difference of myocardium SERCA protein level was found between 4-week DM rats andnormal control rats; myocardium SERCA protein level showed a trend of decrease without statistical significance in 6-wk-DM rats and 8-wk-DM rats.2. Compared with normal control rats, myocardium SERCA activity decreased significantly in 6-wk-DM rats and 8-wk-DM rats.3. Compared with normal rats, myocardium PLB mRNA and protein levels were not significantly changed in 4-wk-DM rats and were significantly increased in 6-wk-DM rats and 8-wk-DM rats. There were no significant differences between the latter two groups.4. In 4-wk-DM rats, LVSP, LVEDP and ±dp/dt_max were at the same level as the control group. Compared with normal control rats, LVSP and ±dp/dt_max were markedly decreased and LVEDP increased in 6-wk-DM rats and 8-wk-DM rats.ConclusionsIn streptozotocin-induced diabetic rats, myocardium PLB protein expression was upregulated, SERCA activity was inhibited, which was accompanied with left ventricular dysfunction. These alterations were observed 6 weeks and 8 weeks after the induction of diabetes mellitus.