Xinshuaikang inhibits myocardial autophagy and MAPK/ERK1/2 signaling pathway in rats with chronic heart failure

AIM:To explore the effect of Xinshuaikang on myocardial autophagy in the rats with chronic heart failure and its relationship with the MAPK/ERK1/2 signaling pathway. METHODS:The rats were divided into sham group, model group (rat model of chronic heart failure was established by ligation of anterior descending branch of left coronary artery), low-, middle-, and high-dose Xinshuaikang treatment (TL, TM and TH) groups and captopril group (treated with captopril as positive control), with 12 in each group. Doppler echocardiography was used to evaluate the cardiac function. The morphological changes of the myocardium were observed by HE staining. TUNEL staining was used to detect cardiomyocyte apoptosis. The expression of microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) in the myocardium was detected by immunofluorescence labeling. The protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ, beclin-1 and p62 in the myocardium were determined by Western blot. RESULTS:Compared with sham group, left ventricular end-diastolic dia-meter (LVEDD) and left ventricular end-systolic diameter (LVESD) in model group were increased, while left ventricular posterior wall thickness at end-diastole (LVPWTd), left ventricular posterior wall thickness at end-systole (LVPWTs), left ventricular ejection fraction (LVEF), cardiac output (CO), left ventricular diastolic pressure (LVDP), left ventricular systolic pressure (LVSP) and maximum rate of rise/decrease of left ventricular pressure (+dp/dt_max/-dp/dt_max) were decreased (P<0.05). The myocardial cells were deformed and necrotic, and the myocardial fibers were broken, with inflammatory cell infiltration. The apoptotic rate, the positive rate of LC3-Ⅱ, and the protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ/LC3-I and beclin-1 were increased, and the protein expression of p62 was decreased (P<0.05). Compared with model group, the levels of LVEDD and LVESD were decreased, LVPWTd, LVPWTs, LVEF, CO, LVSP, LVDP, +dp/dt_max and -dp/dt_max were increased in Xinshuaikang groups and captopril group (P<0.05). The morphological changes of myocardial cells were gradually returned to normal, and inflammatory cell infiltration, the apoptotic rate and the positive rate of LC3-Ⅱ were decreased. The protein levels of p-ERK, p-p38 MAPK, LC3-Ⅱ/LC3-I and beclin-1 were decreased, and the protein expression of p62 was increased (P<0.05). CONCLUSION:Xinshuaikang inhibits myocardial auto-phagy to play a role of cardiac protection in the rats with chronic heart failure, and its mechanism may be related to inhibition of MAPK/ERK1/2 signaling pathway.     

Bone marrow-derived mesenchymal stem cells protect against hypoxia-induced injury and apoptosis of PC12 cells via up-regulation of erythropoietin expression

AIM： To investigate the effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) on cobalt chloride (CoCl2)-induced injury and apoptosis of PC12 cells. METHODS：PC12 cells were divided into control group, CoCl2 group, BM-MSCs-siCTL+CoCl2 group and BM-MSCs-siEPO+CoCl2 group. The viability of the PC12 cells was measured by MTT assay. Flow cytometry and Hoechst 33258 staining were used to determine the apoptotic rate and the changes of chromatin distribution in PC12 cells. The expression of erythropoietin (EPO) in BM-MSCs was measured by RT-PCR and Western blotting. The mRNA expression of Bcl-2 and Bax in PC12 cells was detected by RT-PCR. Caspase-9 and caspase-3 assay kits were used to detect the activity of caspase-9 and caspase-3. RESULTS：The viability of PC12 cells treated with CoCl2 for 24 h and 48 h decreased to （43.0±6.4）% and （33.8±5.7）%, respectively, while 1∶15 ratio of BM-MSCs co-culture increased the cell viability to （77.9±3.8）% and （75.2±9.7）%,respectively. The expression of EPO in BM-MSCs was up-regulated after treated with 0.6 mmol/L CoCl2 for 24 h and 48 h, while EPO siRNA significantly abrogated the EPO expression in BM-MSCs. BM-MSCs-siCTL co-culture significantly inhibited the apoptosis of PC12 cells induced by CoCl2. However, EPO siRNA the protective effect of BM-MSCs. Compared with CoCl2 treatment group, BM-MSCs co-culture induced remarkable increase in the expression of Bcl-2 and decrease in the expression of Bax in PC12 cells, which was reversed by EPO siRNA. BM-MSCs-siCTL co-culture remarkably abrogated the CoCl2 induced up-regulation of caspase-9 and -3, while BM-MSCs-siEPO co-culture significantly reversed the down-regulation of caspase-9 and -3 induced by BM-MSCs-siCTL co-culture. CONCLUSION：BM-MSCs protect PC12 cells from apoptosis induced by CoCl2. The protective effect of BM-MSCs might be executed by up-regulating the expression of EPO.     

Effects of non-excitatory electric stimulation on cardiac function in normal and infarcted heart rabbits and it's regional effect on myocardium

AIM: To investigate the influences of electric stimulation applied during the absolute refractory period (ARP) on the cardiac function of normal rabbits and rabbits after myocardial infarction (MI) and to observe the regional effects of this electric stimulation. METHODS: 64 rabbits were randomly assigned to normal and MI groups and each group was then divided into the anterior and posterior groups. A thoracotomy was performed 4 weeks after MI in rabbits. One set of electrodes was inserted into the anterior and posterior wall of left ventricle of the anterior and posterior groups, respectively. Current pulses were delivered during the ARP (called CCM) during sinus rhythm in rabbits. The left ventricular systolic pressure (LVSP) and the left ventricular end diastolic pressure(LVEDP) as well as maximum positive and negative left ventricular pressure change (±dp/dt_max) were observed. RESULTS: In the normal and MI groups, LVSP, +dp/dt_max significantly increased, and LVEDP, -dp/dt_max were reduced during CCM stimulation compared with the baseline (P<0.05). In the normal rabbits, electric stimulation in the anterior group improved the cardiac function more significantly than that in the posterior group (P<0.05). In the MI rabbits, there was no difference between the anterior and the posterior groups (P>0.05). CONCLUSION: Electric stimulation delivered during the ARP significantly enhances the contractility and the relaxation of myocardium in normal rabbits and rabbits after MI, and the effects of CCM stimulation on heart are regional.     

Relationship between the intracellular calcium concentration changes and left ventricular hypertrophy and function in the spontaneously hypertensive rats

AIM:To elucidate the relationship between the intracellular calcium concentration changes and left ventricular hypertrophy and function in the spontaneously hypertensive rats (SHR).METHODS:Intracellular free calcium concentrations were measured by Fura 2 methodology and left ventricular function quantitated by cardiac catheterization in 20 SHR aged 10, 22, and 34 weeks and 20 age-matched Wistar-kyoto (WKY) rats.RESULTS:(1) The systolic blood pressure(SBP), intracellular calcium concentrations and left ventricular mass / body weight index (LVM/BW) were significantly higher in all three age groups of SHR than the corresponding groups of WKY; (2) Compared with age-matched WKY groups, the peak left ventricular pressure descending rate(-dp／dt_max) decreased while left ventricular relaxation time constant (τ)increased significantly in SHR aged 22 and 34 weeks. The peak left ventricular pressure ascending rate(dp／dt_max) and the left ventricular contractility index were significantly increased only in the 34 weeks SHR; (3) Intracellular calcium concentrations showed a positive correlation with LVM/BW,SBP,-dp／dt_max and τ(r=0.47-0.83,P<0.01)and a negative correlation with dp／dt_max and the left ventricular contractility index (r=-0.46,P<0.05 and r=-0.81, P<0.01).CONCLUSION:Intracellular calcium overload is one of the potential mechanisms in the induction of left ventricular hypertrophy as well as of systolic and diastolic dysfunction.     

Short-term cardioprotective effects of trimetazidine on acute myocardial infarction in rats

AIM: To observe the myocardial protective effects of trimetazidine on myocardial infarction (MI) in Sprague-Dawley (SD) rats. METHODS: Ninety SD rats were randomly assigned to 3 groups (n=30 each): myocardial infarction group (MI group), MI+trimetazidine group (MT group) and sham group (S group). By permanently ligating the left anterior descending artery, the MI model was set up in the rats in MI group and MT group. Before and after setting up the MI model, normal saline was given to the rats in MI and S group by gavage. On the other hand, trimetazidine (3 mg/kg,twice per day) was given to the rats in MT group by gavage. At 8 h, 24 h and 48 h after applying trimetazidine, the serum level of cardiac troponin I (cTnI) was measured. At the 1st week, 2nd week and 4th week after treated with trimetazidine, the size of myocardial infarction, the maximum rising rate of the left ventricular systolic pressure (+dp/dt_max) and the maximum descending rate of the left ventricular diastolic pressure (-dp/dt_max) were measured. Also at the 1st week after applying trimetazidine, the cardiomyocyte apoptotic index was detected. RESULTS: Compared with MI group 2 weeks after applying trimetazidine, +dp/dt_max significantly increased in MT group , and -dp/dt_max also significantly increased in MT group . Four weeks after applying trimetazidine, +dp/dt_max significantly increased in MT group , and -dp/dt_max also significantly increased in MT group . At 8 h and 48 h after applying trimetazidine, no statistically significant difference (P＞0.05) of serum cTnI between MI group and MT group was observed. However, at 24 h after applying trimetazidine, the serum level of cTnI decreased in MT group as compared with MI group . Aditionally, trimetazidine significantly decreased the infarction size of myocardium in MT group (0.248±0.052) as compared with MI group (0.362±0.082, P<0.01). CONCLUSION: Trimetazidine has short-term cardioprotective effects on the rats with acute MI by improving myocardial systolic and diastolic functions, reducing infarct size and inhibiting apoptosis.     

Combinational effects of trimetazidin and parecoxib on acute myocardial infarction in rats

AIM: To investigate the protective effects and mechanisms of combinational use of trimetazidine(TMZ) and parecoxib sodium on acute myocardial infarction (AMI) in rats. METHODS: Sixty-six Sprague-Dawley rats were randomly divided into 5 groups: sham group; AMI group; AMI+TMZ group; AMI+parecoxib group; AMI+TMZ+parecoxib group. All rats were sacrificed and cardiac functions (HR, LVSP, LVEDP, +dp/dt_max,-dp/dt_max) were measured with a Pclab-3804 biological signal processing system on the 8th day. The infarct size in each group was checked up by TTC staining method. RT-PCR was employed to detect the bax mRNA and bcl-2 mRNA. The protein levels of COX-2, Bax, Bcl-2 and cleaved caspase-3 in myocardium were determined by Western blotting. The activity of caspase-3 in each group was measured by colorimetric assay kit, and the apoptotic rates were detected with DNA ladder kit.RESULTS: Compared with sham group, increased expression of COX-2 protein (P<0.01) was observed in AMI group. The expression of COX-2 protein in parecoxib group was lower than that in AMI group (P<0.01). Compared with AMI group, the combinational use of trimetazidin and parecoxib improved contractile functions (LVSP and +dp/dt_max), reduced the infarct size and lowered the apoptotic rates remarkably. Specifically, the combinational use of trimetazidin and parecoxib showed better effects than use of trimetazidin or parecoxib alone. Reduced expression of Bax/Bcl-2 mRNA and protein, the reduced caspase-3 activity and cleaved caspase-3 expression were also found in combinational group as compared with other groups (P<0.05).CONCLUSION: The combinational use of trimetazidin and parecoxib effectively improves cardiac functions and reduces infarct size. The mechanism of the protective effect is probably associated with inhibiting apoptosis of cardiac myocytes.     

Effect of transplantation of hRAMP1 gene-modified bone marrow MSCs on postangioplasty neointima formation in rabbits

AIM: To explore the effect of transplantation of human receptor activity-modifying protein 1 ( hRAMP1 ) gene-modified bone marrow mesenchymal stem cells (MSCs) on neointima formation after carotid balloon angioplasty in carotid atherosclerosis rabbits. METHODS: MSCs were collected through density gradient centrifugation and adherent culture. MSCs were transfected with adenovirus vector carrying hRAMP1 gene to generate hRAMP1 gene-modified MSCs (hRAMP1-MSCs). All animals with carotid atherosclerosis and balloon angioplasty were randomly divided into hRAMP-MSCs group, MSCs group and control group. After the model was established, MSCs transfected with pAd2-EGFP-hRAMP1 or pAd2-EGFP and PBS were injected to the ear vein,respectively. The injured carotid arteries were harvested to detect the homing of MSCs,reendothelialization and neointima thickness 7 d, 14 d and 28 d after cell transplantation. The plasma samples were collected for detecting vascular endothelial growth factor (VEGF) by ELISA. The expression of endothelial nitric oxide synthase (eNOS) in injured carotid arteries was measured by Western blotting. RESULTS: The expression of CD31 and EGFP was observed in the neointima at different time points in hRAMP1-MSCs group and MSCs group. Compared to control group, the reendothelialization of carotid significantly increased in both hRAMP1-MSCs group and MSCs group at different time points (P<0.05), and that in hRAMP1-MSCs group showed better than that in MSCs group (P<0.05). The area of neointima and the rate of restenosis were lower in hRAMP1-MSCs group and MSCs group than those in control group, and those in hRAMP1-MSCs group were significantly lower than those in MSCs group. The plasma level of VEGF and the expression of eNOS in the injured carotid arteries were significantly higher in both hRAMP1-MSCs group and MSCs group than those in control group at different time points (P<0.05), and those in hRAMP1-MSCs group were better than those in MSCs group (P<0.05). In the injured carotid arteries, the expression level of proliferating cell nuclear antigen (PCNA) in hRAMP1-MSCs group was the lowest,with the middle level in MSCs group and the highest level in control group. CONCLUSION: The hRAMP1 gene-modified MSCs are better in promoting reendothelialization and attenuating neointima than natural MSCs. The recombinant hRAMP1 adenovirus vectors dont affect the differentiation potential of MSCs into endothelial cells.These findings indicate that the modified stem cells have the potency of more effective reendothelialization to decrease restenosis after angioplasty.     

Effect of endoplasmic reticulum stress on cardiac myocyte apoptosis in mouse congestive heart failure induced by myocardial infraction

AIM: To explore the effect of endoplasmic reticulum stress on cardiac myocyte apoptosis in mouse congestive heart failure induced by myocardial infraction.METHODS: The mouse model of heart failure was established by ligating the left anterior descending coronary to produce acute myocardial infarction. Thirty-two mice were divided into 4 groups: sham group and groups of post-operation at time points of 2, 4 or 6 weeks, respectively. The ventricular dilatation and left ventricular functions were assessed by echocardiography. The expression of GRP78, CHOP, caspase-12, cleaved caspase-12, JNK and phosphorylated-JNK was detected by Western blotting. The cardiac myocyte apoptosis was determined by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL).RESULTS: The cardiac expression of endoplasmic reticulum chaperones GRP78 was significantly increased in the hearts with functional failure. The upregulated expression of CHOP, phosphorylated-JNK and cleaved caspase-12 illuminated that the CHOP-JNK- caspase-12 dependent pathways for endoplasmic reticulum-initiated apoptosis were activated in the heart with functional failure by myocardial infraction. CONCLUSION: These findings suggest that the congestive heart failure induced by myocardial infraction is associated with endoplasmic reticulum stress and activation of CHOP, JNK, caspase-12 dependent pathways for endoplasmic reticulum-initiated apoptosis.     

Effects of different β-adrenergic receptors on cardiac systolic and diastolic functions in hypoxic stress rats

AIM:To explore the effects of different β-adrenergic receptors (β-AR) on the left and right ventricular systolic and diastolic functions in rats under acute hypoxic stress. METHODS:The healthy male SD rats were randomly divided into 4 groups (n=7):control group, non-selected β-AR blocker propranolol group, selected β₁-AR blocker atenolol group and selected β₂-AR blocker ICI 118,551 group, and then the rats were exposed to normoxia (20.9% O₂, 79.1% N₂) and hypoxia (15.0% O₂, 85.0% N₂) condition respectively at the altitude of 2 260 m (Xining, China). The heart rate (HR), the left ventricular systolic pressure (LVSP), the right ventricular systolic pressure (RVSP), and the maximum raise/decline rate of left and right ventricular pressure (±dp/dt_max) were monitored, and the arterial blood gas in normoxia and hypoxia condition were compared to explore the effect of β-AR on the left and right ventricular systolic and diastolic functions in acute hypoxic stress rats. RESULTS:Under normoxia condition, the LVSP, ±dp/dt_max of left ventricular were decreased in propranolol group, atenolol group and ICI 118,551 group, the RVSP and ±dp/dt_max of right ventricle were decreased in propranolol group and atenolol group (P<0.05). Under hypoxia condition, the PaO₂, LVSP, ±dp/dt_max of left ventricle were decreased in all groups compared with the normoxia group, and the ±dp/dt_max of right ventricle was increased in all groups (P<0.05), also the degree of index change in control group was more obvious than that in propranolol group and atenolol group. CONCLUSION:The activation of β₁-AR is an important compensatory regulation for heart function during hypoxic stress. However, the compensatory enhancement of right heart function under acute hypoxia condition which through tonogenic dilation is more significant for maintaining the normal circulating blood flow.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

Effect of human β-NGF gene-modified bone marrow-derived mesenchymal stem cells on rotational behavior of rats with Parkinson disease

AIM：To investigate the effect of human β-nerve growth factor (β-NGF) gene-modified bone marrow-derived mesenchymal stem cells (MSCs) transplantation on the rotational behavior improvement in a rat model of Parkinson disease (PD). METHODS：The rat model of PD was established successfully and the animals were divided into 4 groups:β-NGF-MSCs group (transplanted with 5×105 β-NGF-engineered MSCs), MSCs group (transplanted with 5×105 MSCs), DMEM/F12 group (5 μL transplantation medium was injected in the right striatum of the rats) and PD model group (without transplantation). The rotational scores were assessed 2 weeks, 4 weeks and 6 weeks after transplantation. At different time points after transplantation, the rats were tested for apomorphine (APO)-induced rotational behavior and the brains of the PD model rats were examined by fluorescence microscopy and immunohistochemical staining. RESULTS：Transplantation of human β-NGF gene-modified MSCs effectively improved the behavioral performance in the rats. At the 2nd, 4th and 6th weeks after cell transplantation, the rotational frequencies after injection of APO decreased significantly in β-NGF-MSCs group compared with MSCs group and PD group (P<0.05). Both β-NGF gene-modified MSCs and MSCs survived in the brains of PD model rats, had good compatibility with the host cells, and showed no signs of destroying the host and the glial cicatrisation. The β-NGF gene-modified MSCs expressed β-NGF stablely in the brains of PD model rats, and showed obvious improvement of the rotational behavior in the PD model rats induced by APO compared with MSCs group. CONCLUSION:The behavior of the rats with PD is significantly improved by transplanting β-NGF-modified MSCs in right striatum, and β-NGF gene therapy has potential clinical value．     

Cardiomyocyte apoptosis and related gene expression after acute myocardial infarction in rats

AIM: To investigate cardiomyocyte apoptosis and the expression of caspase-3, Bcl-2 and Bax after acute myocardial infarction (AMI) in rats.METHODS: AMI model was established with the ligation of left coronary artery in 78 randomly selected female SD rats.Twenty-four hours after operation, 43 survivors were randomly divided into 48-hour and 4-week two groups according to the time points: MI 48 h (n=11) and MI4 weeks (n=13) groups, sham-operated rats (S, n=27) were also randomly selected and reassigned to S48 h (n=10) and S4 weeks (n=10) groups.Cardiomyocyte apoptosis was detected with in situ terminal deoxynucleotidyl transferase (TdT)-dUTP nick-end labeling (TUNEL staining) and DNA gel electrophoresis.Caspase-3, Bcl-2 expression and Bax expression were detected with immunohistochemistry and Western blotting analysis.RESULTS: Compared with sham-operated group, after AMI, systolic, diastolic, and mean arterial blood pressures (SBP, DBP, MAP), left ventricular systolic pressure (LVSP) and the maximum change rate of left ventricular pressure rise and fall (±dp/dt) were significantly decreased (P<0.05, P＜0.01), while left ventricular end diastolic pressure (LVEDP) was significantly increased (P<0.05) in MI 48 h group.All the above indices in MI 4 weeks group had the same change as that in MI48h group, with the LVEDP significantly higher (P<0.01), except for a non-significantly change in SBP, DBP and MAP (all P>0.05).In both MI 48 h and MI 4 weeks groups, myocyte apoptotic index was significantly increased in the infracted/scar, border and non-infarcted areas (P<0.05,P＜0.01) with caspase-3 and Bax expressions increased significantly (P<0.05, P＜0.01) in myocytes of the above three areas and Bcl-2 expression increased only in myocytes of the infracted area in MI 48 h group.Western blotting indicated that Bcl-2/Bax ratio was also decreased in MI 48 h subgroup.CONCLUSIONS: After AMI in rats, cardiomyocyte apoptosis happened in the infarction/scar, border and non-infarcted areas, with caspase-3 and Bax expression in myocytes increased, and with Bcl-2 expression increased in myocytes of infracted area and Bcl-2/Bax ratio decreased only early after AMI.     

Role of bradykinin in the therapeutic effect of enalapril on left ventricular hypertrophy and cardiac function after myocardial infarction in rats

AIM:To investigate the influences of bradykinin(BK)on left ventricular hypertrophy and cardiac function in angiotensin-converting enzyme inhibitor(ACEI) therapy in rats after myocardial infarction.METHODS:The effects of enalapril (500 μg·kg^-1·d^-1), enalapril (500 μg·kg^-1·d^-1)with BKB₂ receptor antagonist (Hoe-140 500 μg·kg^-1·d^-1), losartan(3 mg·kg^-1·d^-1) on left ventricular end-diastolic pressure (LVEDP), maximum positive left ventricular pressure change (+dp/dt_max) and LVW/BW as well as V(m)n of noinfarcted area were examined after 4 weeks treatment in rats after myocardial infarction.RESULTS:The values of LVEDP, LVW/BW and V(m)n of three treatment groups were higher than that of untreated MI group (P＜0.05),but the +dp/dt_max of three treatment groups were not significantly different compared with the untreated MI group. In addition, no significant difference in MAP was observed among the three treatment groups, but the LVW/BW and V(m)n of enalapril+Hoe-140-treated group were higher than that of enalapril-treated group (P＜0.05) .CONCLUSION:Enalapril can prevent left ventricular hypertrophy and improve cardiac function independent of blood pressure after myocardial infarction, which is partly due to the inhibition of BK degradation.     

Effect of fluvastatin on left ventricular remodeling and caspase-3 expression after myocardial infarction in rats

AIM: To observe the effect of fluvastatin (FV) on left ventricular remodeling and expression of caspase-3 after myocardial infarction (MI) in rats. METHODS: Rats were divided into 4 groups: group Ⅰ (sham), group Ⅱ (sham+FV), group Ⅲ (MI) and group Ⅳ (MI+FV). group Ⅱ and Ⅳ were treated with FV (10 mg·kg^-1·d^-1) for 4 weeks. The left ventricular structure, echocardiography and hydroxyproline were observed. The expression of caspase-3 was measured by immunohistochemistry and RT-PCR. RESULTS: Compared with MI group, there was a improvement of ultrastructure and index of left ventricular remodeling, and decrease in hydroxyproline in MI+FV group (all P<0.05). The number of caspase-3 positive cells also decreased in MI+FV group, and RT-PCR showed the level of caspase-3 mRNA expression was lower than that in MI group (P<0.05). CONCLUSION: Fluvastatin improves left ventricular remodeling after myocardial infarction, decreases the expression of caspase-3 and inhibits apoptosis.     

Effects of rAAV9-mediated siRNA for p65 knockdown on myocardial fibrosis and apoptosis in rats with pressure overload

AIM To investigate the effect of p65 gene knock-down mediated by recombinant adeno-associated virus serotype 9 （rAAV9） on the cardiac function of pressure overload rat and its possible mechanism. METHODS The rat model of left ventricular hypertrophy was established by abdominal aortic coarctation（AAC）. SD rats were randomly divided into sham operation group, AAC group, AAC+rAAV9-eGFP group and AAC+rAAV9-eGFP-P65-siRNA group. The abdominal cavity was closed directly after laparotomy in the rats of sham operation group, the abdominal cavity was closed after ligation of the abdominal aorta in the rats of AAC group, and normal saline, rAAV9-eGFP and rAAV9-eGFP-P65-siRNA were injected into the tail vein 3 d after operation. After 4 weeks, the hemodynamic indexes were measured, the heart mass parameters were calculated, the degree of myocardial fibrosis was detected by Masson staining, the expression level of myocardial P65 was detected by Western blot, the degree of apoptosis was detected by TUNEL staining, and the serum contents of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） of the rats in each group were measured by ELISA. RESULTS The expression of P65 in AAC group and AAC+rAAV9-eGFP group was higher than that in sham operation group, while the expression of P65 in AAC+rAAV9-eGFP-P65-siRNA group was significantly lower than that in AAC group. The levels of systolic blood prossure （SBP）, diastolic blood pressure （DBP）, left ventricular weight/body weight （LVW/BW）, cardiomyocyte apoptotic rate and TNF- α and IL-6 in AAC group and AAC+rAAV9-eGFP group were higher than those in sham operation group, while SBP, DBP, LVW/BW, cardiomyocyte apoptosis rate and TNF-α in AAC+rAAV9-eGFP-P65-siRNA group were significantly lower than those in AAC group. The results of Masson staining showed that the deposition of collagen in cardiac tissue in AAC group and AAC+rAAV9-eGFP group was higher than that in sham operation group, and treatment with rAAV9-eGFP-P65-siRNA alleviated hypertension-induced fibrosis. CONCLUSION Knockdown of p65 gene reduces the degree of left ventricular fibrosis and apoptosis in rats with stress overload, and its mechanism is related to the regulation of NF-κB pathway and the reduction of inflammatory response.     

Effect of arachidonylethanolamide in paraventricular nucleus on cardiac function and sympathetic nerve activity in rats with heart failure

AIM:To determine the roles of the arachidonylethanolamide (AEA) in the paraventricular nucleus (PVN) in cardiac function and sympathetic activity in the rats with chronic heart failure (CHF). METHODS:Chronic heart failure was induced by left coronary ligation in Wistar rats and was confirmed using echocardiography. The rats with CHF and the sham-operated controls (sham group) were treated for 4 weeks with a continuous PVN infusion of AEA, cal-cium-calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) selective inhibitor KN-93, transient receptor potential vanilloid type 1 (TRPV1) channel blocker capsazepine (CPZ), intracellular calcium chelator BAPTA-AM, small-conductance calcium-activated potassium channel (SK channel) blocker apamin and artificial cerebrospinal fluid (vehicle). Sympathetic drive indexes and cardiac function were detected. NG108 cells were incubated with AEA, and then the intracellular cal-cium concentration was measured by fluorometry. The protein expression levels of CaMKⅡ, SK₂ and phosphorylated TRPV1 were determined by Western blot. RESULTS:Compared with sham group, the left ventricular end-diastolic pressure (LVEDP) increased significantly, while peak rate of rise/decline of left ventricular pressure (±dp/dt_max) and ejection fraction (EF) decreased significantly in the CHF group. The concentrations of AEA and intracellular calcium, and the protein levels of CaMKⅡ, SK₂ and phosphorylated TRPV1 in PVN were significantly lower in CHF rats. Compared with the vehicle group, the mortality and sympathetic drive were decreased significantly and cardiac function was improved after treatment with AEA in CHF group. However, PVN perfusion of KN-93, CPZ, BAPTA-AM or apamin contributed to the sympathetic drive and deteriorated the cardiac function. AEA dose-dependently increased intracellular calcium ion concentration, and the protein levels of CaMKⅡ, SK₂ and phosphorylated TRPV1 in NG108 cells. CONCLUSION:AEA in the PVN may be involved in the improvement of cardiac function and sympathetic overdrive via CaMKⅡ/TRPV1/Ca²⁺/SK₂ pathway in rats with CHF.     

Adrenomedullin gene transfection enhances the therapeutic effects of transplantation of bone marrow mesenchymal stem cells on cardiac function and ventricle remodeling in acute myocardial infarction rats

AIM: To investigate adrenomedullin gene transfection enhances the therapeutic effects of homogeneous transplantation of bone marrow mesenchymal stem cells (MSCs) on cardiac function and ventricle remodeling in acute myocardial infarction rats. METHODS: MSCs were isolated and expanded using the preplating method. The infection efficiency of adenovirus vector to MSCs was tested by X-gal staining. Ad-ADM expression in MSCs and its secretion in culture medium were measured by ELISA. The left anterior descending branch of rats was ligated to establish a myocardial infarction model. The MSCs were labeled by DAPI, and were directly implanted into the acute infarct site via focal injection. Four weeks later, cardiac function was evaluated using physiological recorder. Hearts were harvested and sliced to be analyzed by immunohistochemistry (factor Ⅷ and ADM) and the DAPI-labeled cells were identified. Sirius red staining was used to identify interstitial collagen on slides. Analysis of collagen type I and III was performed using a polarized filter on sections stained for collagen with Sirius red, and the ratio of collagen type I and III were detected. RESULTS: With X-gal staining, MSCs were effectively transfected by adenovirus in vitro. The transfection efficiency showed the dose-effect relationship with multiplicities of infection (MOI). When MOI was 150, the infection efficiency was 95.4%. The expression of ADM was traced in culture medium and expressed in the time-dependent manner. A maximum production of ADM was observed at 7 d after infection ［(26.53±1.42) ng/L vs (1.34±0.08) ng/L, P<0.05］, and ADM secretion reduced to normal level at 15 d ［(2.20±1.44) ng/L vs (1.52±0.33) ng/L, P>0.05］. DAPI-labeled MSCs transplantation was found in the hearts of the recipients. Immunohistochemical studies demonstrated that intense immunostaining for ADM was higher in Ad-ADM plus MSCs group, compared to other groups. Compared with control, MSCs transplantation significantly increased capillary density in infarct area (P<0.01). A combination of Ad-ADM trensfection and MSCs transplantation demonstrated a further increase in capillary density compared with Ad-ADM or MSCs alone. MSCs transplantation decreased the ratio of collagen type I and III, obviously improved the left ventricular functions. Furthermore the combination treatment resulted in further decrease in the ratio of collagen type I and III, and significantly improved the left ventricular functions. CONCLUSION: Ad-ADM transfection enhances the angiogenic potency of MSCs transplantation and decreases the ratio of collagen type I and III through increasing ADM expression in infarct area, thus contributes to reverse the ventricular remodeling and improves the cardiac function.     

Transfection of rAAV1-SERCA2a improves cardiac function of beagles with heart failure

AIM:To study the effects of recombinant adeno-associated virus type 1 (rAAV1)-sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) transfection on the cardiac function of beagles with heart failure (HF). METHODS:The beagles were used to make an animal model with heart failure after rapid right ventricular pacing (230 beats/min) for 30 d. A reduced rate (180 beats/min) was continuously applied for another 30 d. The beagles were divided into 4 groups (n=4): control group, HF group, HF+EGFP group and HF+SERCA2a group. rAAV1-EGFP and rAAV1-SERCA2a (both 1×1012 viral genomes) were intramyocardially injected into the animals in the latter 2 groups, respectively. RESULTS:After transfection for 30 d, the left ventricular systolic function in HF+SERCA2a group was similar to that in control group, and significantly higher than that in HF group (P<0.05). The ratio of SERCA2a mRNA/GAPDH mRNA was significantly higher in HF+SERCA2a group than that in HF group (P<0.05). The expression level of SERCA2a in the myocardial tissues was higher in HF+SERCA2a group than that in HF group (P<0.05). The apoptotic index of the cardiomyocytes and the protein expression of MMP-9 were much lower in HF+ SERCA2a group than those in HF group (P<0.05). No significant difference of all parameters was observed between HF group and HF+EGFP group. The mRNA level of phospholamban was unchanged. CONCLUSION:Transfection of SERCA2a improves the expression of SERCA2a, restores the cardiac function and inhibits left ventricular remodeling by reducing the cardiac cell apoptosis and the MMP-9 expression in the heart failure beagles.     

Transplantation of bcl-2 gene-modified bone marrow mesenchymal stem cells improves cardiac function and angiogenesis in rabbit ischemic car-diac insufficiency model

AIM: To investigate the effects of transplantation of bone marrow mesenchymal stem cells (BMSCs) modified by bcl-2 gene on myocardial cell apoptosis, angiogenesis and cardiac function in the rabbit after acute myocardial infarction (MI).METHODS: The rabbit BMSCs were isolated, cultured and purified in vitro. The BMSCs were transfected with adenovirus or adenovirus-Bcl-2. The rabbit model of MI was established by ligation of left anterior descending branch. The rabbits were injected with Ad-Bcl-2-BMSCs (MI+Bcl-2-BMSCs group), Ad-BMSCs (MI+BMSCs group) and DMEM (MI group) in infarction marginal zone 2 weeks after ligation. The cardiac function was evaluated by echocardiography.The apoptosis of myocardial cells was measured by TUNEL. The mRNA expression of VEGF was detected by real-time PCR. The expression of CD31 was examined by immunohistochemical staining, and new blood capillaries were counted at 4 weeks after BMSCs transplantation. The correlation of the above values with cardiac function was analyzed.RESULTS: The cardiac function was better, the apoptotic rate was lower, the mRNA expression of VEGF and the capillary density were higher in both MI+Bcl-2-BMSCs group and the MI+BMSCs group than those in MI group, and those in MI+Bcl-2-BMSCs group increased more obviously.The left ventricular ejection fraction (LVEF) had a negative correlation with the myocardial cell apoptosis rate. A positive correlation with the mRNA expression level of VEGF and the capillary density was also observed.CONCLUSION: The transplantation of BMSCs modified by bcl-2 gene significantly reduces the myocardial cell apoptosis, promotes angiogenesis, improves heart function of the rabbits with MI.     

Therapeutic effect of neuregulin-1β on pressure overload-induced myocardial hypertrophy in rats

AIM：To investigate the therapeutic effect and the mechanism of neuregulin-1β (NRG-1β) on the rat model of myocardial hypertrophy induced by pressure overload．METHODS：Eight weeks after coarctation of abdominal aorta, the Wistar rats were randomly divided into 4 groups: myocardial hypertrophy (model) group, sham operation (sham) group, NRG-1β treatment group (intravenous injection of NRG-1β at dose of 10 μg/kg daily for 7 d) and NRG-1β+Herceptin (HERCE) treatment group ［intravenous injection of NRG-1β (10 μg/kg) plus HERCE (10 μg/kg) daily for 7 d］. The characteristics of heart functions were evaluated by the methods of hemodynamics and echocardiography. Masson staining was employed to observe the pathological changes of myocardial tissues. The concentration of angiotensin II (Ang II) in myocardial tissues was measured by radioimmunoassay. The level of tumor necrosis factor α (TNF-α) in myocardial tissues was detected by ELISA. The mRNA expression of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2-associated X protein (bax) in the myocardium was determined by RT-PCR. RESULTS：The left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were higher, while the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were smaller in NRG-1β group than those in model group. The left ventricular end-systolic pressure (LVESP) and maximal rate of increase/decrease in left ventricular pressure (±dp/dtmax) were higher, and left ventricular end-diastolic pressure (LVEDP) was significantly lower in NRG-1β group than those in model group. Compared with model group, treatment with NRG-1β decreased collagen volume fraction (CVF), reduced the Ang II and TNF-α, increased bcl-2 mRNA expression, and decreased bax mRNA expression in myocardial tissues. No difference of the above parameters between model group and NRG-1β+HERCE treatment group was observed. CONCLUSION：NRG-1 reduces the expression of Ang II and TNF-α in myocardial tissues in pressure-overload rats, thus reducing Ang II and TNF-α mediated myocardial interstitial remodeling. Increase in the mRNA expression of bcl-2 and decrease in the mRNA expression of bax by NRG-1 inhibit myocardial cell apoptosis, which is responsible for its role of improving cardiac function of myocardial hypertrophy induced by pressure overload.