Alterations in Cardiac Structure and Function in a Modified Rat Model of Myocardial Hypertrophy

This study was aimed to establish a stable animal model of left ventricular hypertrophy （LVH） to provide theoretical and experimental basis for understanding the development of LVH. The abdominal aorta of male Wistar rats （80-100 g） was constricted to a diameter of 0.55 mm between the branches of the celiac and anterior mesenteric arteries. Echocardiography using a linear phased array probe was performed as well as pathological examination and plasma B-type natriuretic peptide （BNP） measurement at 3, 4 and 6 weeks after abdominal aortic constriction （AAC）. The results showed that the acute mortality rate （within 24 h） of this modified rat model was 8%. Animals who underwent AAC demonstrated significantly increased interventricular septal （IVS）, LV posterior wall （LVPWd）, LV mass index （LVMI）, cross-sectional area （CSA） of myocytes, and perivascular fibrosis; the ejection fraction （EF）, fractional shortening （FS）, and cardiac output （CO） were consistently lower at each time point after AAC. Notably, differences in these parameters between AAC group and sham group were significant by 3 weeks and reached peaks at 4th week. Following AAC, the plasma BNP was gradually elevated compared with the sham group at 3rd and 6th week. It was concluded that this modified AAC model can develop LVH, both stably and safely, by week four post-surgery; echocardiography is able to assess changes in chamber dimensions and systolic properties accurately in rats with LVH.

Icariin ameliorate diabetic myocardial hypertrophy by inhibiting autophagy via the AMPK/mTOR pathway

Objective:To observe the effect of Icariin on diabetic myocardial hypertrophy and explore its molecular mechanism.Methods:C57BL/6 mice were randomly divided into Ctrl group(normal control group),DM group(STZ intraperitoneal injection model),and DM+ICA group(diabetic C57BL/6 mice by intragastric Icariin solution 80mg/kg/d,for 3 consecutive weeks).Real-time quantitative PCR was used to detect myocardial hypertrophy markers BNP andβ-MHC.Western blotting was used to detect myocardial AMPK,p-AMPK,mTOR,p-mTOR,LC3B and Beclin1 protein expression.Echocardiogram was used to detect left ventricular mass and ejection fraction.Results:Compared with the normal control group,the expression of myocardial hypertrophy markers BNP andβ-MHC mRNA in diabetic mice were significantly increased;the expression of phosphorylated AMPK protein,autophagy-related protein LC3B and Beclin1 were significantly increased,and the expression of phosphorylated mTOR protein is significantly reduced;the left ventricular mass is significantly increased.The above changes can be reversed after treatment with Icariin,but the effect of Icariin is blocked by the autophagy inhibitor rapamycin.Conclusion:Icariin may inhibit autophagy and reduce diabetic myocardial hypertrophy through AMPK-mTOR signaling pathway.

Effects of TanshinoneⅡA on the Myocardial Hypertrophy Signal Transduction System Protein Kinase B in Rats

Objective： To study the effect of tanshinone ⅡA on the cell signal transduction system protein kinase B （Akt） in rats with hypertrophy of the myocardium induced by partial constriction of the thoracic aorta. Methods： Rat models of myocardial hypertrophy were established by the thoracic aorta partial constriction method. Forty-eight rats were randomly divided into the sham-operative group, the model group, the valsartan treatment group, and the low-, medium-, and high-dose tanshinone treatment groups. The heart mass index （HMI）, left ventricular mass index （LVMI）, ejection fraction （EF）, left ventricular posterior wall （LVPW）, and interventricular septal thickness （IVS） were detected by high-frequency ultrasonography. The myocardial fiber diameter （MFD） was detected by HE staining, and the contents of p-Akt and p-Gsk3β in the myocardium were detected by Western blot. Results： Compared with the sham-operative group, the levels of HMI, LVMI, LVPW, IVS, and MFD were increased respectively in the other groups （P〈0.05）; the contents of p-Akt and p-Gsk3β were also increased in the other groups. Compared with the model group, the levels of HMI, LVMI, LVPW, IVS, and MFD were decreased respectively in all treatment groups （P〈0.05）; the contents of p-Akt and p-Gsk3β were decreased in all treatment groups as well. There was no significant difference, however, among the low-, medium-, and high-dose tanshinone treatment groups and the valsartan treatment group （P〉0.05）. Conclusion： Tanshinone HE A can prevent myocardial hypertrophy by its action on the protein kinase B （Akt） signaling pathway.

Effect of Yiqi Huoxue Recipe(益气活血方)on Cardiac Function and Ultrastructure in Regression of Pressure Overload-induced Myocardial Hypertrophy in Rats

Objective： To investigate the effect of Yiqi Huoxue Recipe （YHR, 益气活血方） on the cardiac function and ultrastructure during the regression of myocardial hypertrophy induced by pressure overload in rats. Methods： The model of myocardial hypertrophy was established by abdominal aortic banding. Eighty male Wistar rats were divided into six groups, the normal control group Ⅰ （n=20）, the normal control group Ⅱ（n=12）, the hypertension model group Ⅰ （n=12）, the hypertension model group Ⅱ （n=12）, the YHR group （n=12） and the Captopril group （n=12）. The observation was carried out in the normal control group Ⅰ and the hypertension model groupⅠ after 4 weeks of modeling, and the other four groups were observed after 16 weeks of modeling （12 weeks of administration）. The cardiac function was measured with a multichannel biological signal analysis system, and the myocardium ultrastructure was observed by a transmission electron microscope. Results： （1） Compared with the normal control group Ⅰ, the systolic blood pressure and cardiac coefficient （left ventricular weight/body weight） in the model Ⅰ group was higher （P〈0.05, P〈0.01）. （2） In the YHR group, cardiac coefficient and -dp/dtmax were lower, left ventricular systolic pressure and ＋dp/dtmin were higher when compared with the model group Ⅱ and the Captopril group （P〈0.05 or P〈0.01）. In the Captopril group, only cardiac coefficient was lower when compared with the mode group Ⅱ （P〈0.05）. （3） Compared with the normal control group Ⅱ, ＋dp/dtmax was higher （P〈0.01）, -dp/dtmax and isovolumetric contraction time （ICT） was lower （P〈0.05, P〈0.01） in both the YHR group and the Captopril group. （4） Results of the myocardium ultrastructure showed edema under myocardium plasmalemma, enlarged sarcoplasmic reticulum and T tube, and significantly enlarged intercalated disc of the cardiac muscle in the model groups. In the Captopril group, the extension of sarcoplasmic reticulum and T tube as well as the pathological changes of intercalated disc were lighter, with slight edema under the myocardium plasmalemma. In the YHR group, the expansion of the sarcoplasmic reticulum was less than in the Captopril group, part of the pathological changes of intercalated discs was slightly more severe than that in the Captopril group, the dissolution of nuclear chromatin was not found, which was similar to that of the Captopril group, and no injury of the nucleus was found, either. Conclusion： YHR could reverse myocardial hypertrophy in rats with abdominal aortic banding and improve the systolic and diastolic function of the left ventricle. The ultrastructure of the myocardium such as arcoplasmic reticulum, intercalated disc, and cell nucleus in abdominal aortic banding rats could be partly reversed by the recipe.

Protective effect of tanshinone Ⅱ A on signal transduction system protein kinase B in rats with myocardial hypertrophy

The effects of tanshinone II A on cell signal transduction system protein kinase B in rats with myocardial hypertrophy induced by the abdominal aorta partial coarctation were investigated.Rat models of myocardial hypertrophy were established by using abdom-inal aorta partial coarctation method.Forty-eight rats were randomly divided into sham group(S group),model group(M group),valsartan treatment group(X group),low-dose tanshinone treatment group(LD group),medium-dose tanshinone treatment group(MD group),and high-dose tanshinone treatment group(HD group)(n=8 in each group).Left ventricular mass index(LVMI),left ventri-cular posterior wall(LVPW),and septal thickness(IVS)were detected by high frequency ultrasonography.Myocardialfiber diameter(MFD)was examined by Hematoxylin-Eosin(HE)staining,and the contents of phosphorylated protein kinase B(p-Akt)and p-Gsk3βin myocardium were assayed by Western blot.The results showed that compared with S group,the values of LVMI,LVPW,IVS and MFD were increased in other groups(P<0.05),and the contents of p-Akt,and p-Gsk3βwere also increased in other groups.As compared with MD group,the values of LVMI,LVPW,IVS and MFD were decreased in all treatment groups(P<0.05),and the contents of p-Akt,and p-Gsk3βwere also decreased in all treatment groups.However,there were no significant differences among LD,MD,and HD groups(P>0.05),and there were no significant differences between X group and tanshinone treatment groups(P>0.05).It was suggested that tanshinone II A could prevent myocardial hypertrophy by its action on the Akt signaling pathway.

Effects of electroacupuncture at Neiquan (PC6) on protein expressions of Raf-1,ERK 1/2,and p-ERK 1/2 of Rats with myocardial hypertrophy

Objective To observe the effects of electroacupuncture(EA)at Neiguan(PC6)on protein expressions of proto-oncogene serine/threonine-protein kinase-1(Raf-1),phospho-extracellular signal-regulated kinase 1/2(p-ERK 1/2),and extracellular signal-regulated kinase1/2(ERK 1/2)in rats with myocardial hypertrophy.Methods Totally 40 healthy Sprague-Dawley(SD)rats of clean grade were divided into 4 groups according to random digit table,i.e.,the normal group,the

Magnolol attenuates right ventricular hypertrophy and fibrosis in hypoxia-induced pulmonary arterial hypertensive rats through inhibition of the JAK2/STAT3 signaling pathway

OBJECTIVE Right ventricular(RV)remodeling is one of the essential pathological features in pulmonary arterial hypertension(PAH).RV hypertrophy or fibrosis are the leading causes of RV remodeling.Magnolol is a compound isolated from Magnolia officinalis.It possesses multiple pharmacological activities,such as anti-oxidation and anti-inflammation.This study aims to evaluate the effects and underlying mechanisms of magnolol on RV remodeling in hypoxia-induced PAH.METHODS①Male SD rats(220 g)were randomly divided into 5 groups(n=10):the normoxia group,the hypoxia group,the hypoxia plus Magnolol(10 and 20 mg·kg^(-1)·d-1)group,and the vehicle group.Rats in the normoxia group were kept in a normoxia environment for 4 weeks,while rats in the hypoxia group were kept in a hypoxic chamber(10%O2).The rats in the hypoxia plus magnolol groups were administered with magnolol at 10 or 20 mg·kg^(-1)(ip)once a day for 4 weeks.At the end of 4 weeks,the heart function was assessed by Doppler echocardiography,and then the rats were anesthetized with sodium pentobarbital(30 mg·kg^(-1),ip).The RVSP was measured by the right heart catheterization method.The heart tissues were collected and dissected to calculate the index of RV remodeling(RV/LV+IVS,RV/tibial length,or RV/body weight).Part of the RV samples was fixed with 4%paraformaldehyde for morphological analysis,while other samples were frozen at-80℃for molecular studies(measurements of ANP,BNP,α-SMA,and collagenⅠ/ⅢmRNA expression as well as p-JAK2/JAK2 and p-STAT3/STAT3 protein levels).②To evaluate the effect of magnolol on hypoxia-induced myocardial hypertrophy and fibrosis,H9c2 or cardiac fibroblasts were divided into 7 groups:the control group,cells were cultured under normal conditions;the hypoxia group,cells were cultured under hypoxic condition(3%O2);the hypoxia plus magnolol 10 mg·kg^(-1) group,magnolol10μmol·L^(-1) was added to the culture medium before the hypoxia treatment;the hypoxia plus magnolol 30 mg·kg^(-1) group,magnolol 20μmol·L^(-1) was added to the culture medium before the hypoxia treatment;the hypoxia plus TG-101348 group,TG-101348(a specific inhibitor of JAK2)1μmol·L^(-1) was added to the culture medium before the hypoxia treatment;the hypoxia plus JSI-124 group,JSI-124(a specific inhibitor of JAK2)1μmol·L^(-1) was added to the culture medium before the hypoxia treatment;and the hypoxia plus vehicle group,an equal volume of vehicle(DMSO)was added to the culture medium before the hypoxia treatment.At the end of the experiments,the cells were collected for morphological and molecular analysis.RESULTS In vivo,male Sprang-Daley rats were exposed to 10%O2 for 4 weeks to establish an RV remodeling model,which showed hypertrophic and fibrotic features(increases of RV remodeling index,cellular size,hypertrophic and fibrotic marker expression),accompanied by an elevation in phosphorylation levels of JAK2 and STAT3;these changes were attenuated by treating rats with magnolol.In vitro,the cultured H9c2 cells or cardiac fibroblasts were exposed to 3%O2 for 48 h to induce hypertrophy or fibrosis,which showed hypertrophic(increases in cellular size as well as the expression of ANP and BNP)or fibrotic features(increases in the expression of collagenⅠ,collagenⅢandα-SMA).Administration of magnolol and TG-101348 or JSI-124 (JAK2 selective inhibitors) could prevent the process of myocardial hypertrophy and fibrosis, accompanied by the decrease in the phosphorylation level of JAK2 and STAT3. CONCLUSION Magnolol can attenuate RV hypertrophy and fibrosis in hypoxia-induced PAH rats through a mechanism involving inhibition of the JAK2/STAT3 signaling pathway.

The Effects of Singal Protein SMADs on Rat Cardiocyte Hypertrophy

Objectives To investigate the role of signal protein SMADs in rat cardiac hypertrophy. Methods The rat models of cardiac hypertrophy were produced by constriction of the abdominal aorta. The left vertricular mass index （LVMI） was investigated. The expression of transforming growth factor-β1 mRNA （TGF-β1） and Smad 2,3,7 mRNA were assessed by RT-PCR. Reslutes The LVMI and the expression of TGF-β1 and Smad 2,3,7mRNA in hypertrophic left ventricule were increased on day 3 after the operation and continued to 4th weeks. The peak expression of TGF-β1 and Smad 2,3 mRNA were in 2 weeks after operation. The expression of Smad 7 was increased in 3 day after operation, but the peak was in 1 week after operation, then decreased. Conclusions The TGF-β1 and signal protein Smad 2,3,7 were included in the progress of rat cardiac hypertrophy produced by constriction of abdominal aorta.

The Effect of TanshinoneⅡ A upon the TGF-beta1/Smads Signaling Pathway in Hypertrophic Myocardium of Hypertensive Rats

To investigate the molecular mechanism by which Tanshinone Ⅱ A （TSN Ⅱ A） prevents left ventricular hypertrophy （LVH）, we examined the expression of AT1R, TGF-β1 and Smads gene in the hypertrophic myocardium of hypertensive rats with abdominal aorta constriction. LVH model was established by creating abdominal aorta constriction. Four weeks later, animals were randomly divided into 4 groups with 8 animals in each. One group was used as model control, the other three groups were treated with TSN ⅡA （20 mg/kg）, TSN ⅡA （10 mg/kg） and valsartan （10 mg/kg）, respectively. Another 8 SD rats were subjected to sham surgery and served as blank control. After 8- week treatment, the caudal artery pressure of the animals was measured. The tissues of left ventricle were taken for the measurement of the left ventricular mass index （LVMI） and pathological sectioning and HE-staining were used for determining the myocardial fiber dimension （MFD）. The mRNA expression of AT1R, protein expression of TGF-betal and activity of Smad-2, 4, 7 were detected by RT-PCR and Western blotting, respectively. Our results showed that （1） the blood pressure of rats treated with TSN Ⅱ A, either at high or low dose, was significantly higher than those in the control and valsartan-treated group （P〈0.01, P〈0.05）; （2） LVMI and MFD in TSN Ⅱ A and valsartan-treated rats were higher than those in the control group （P〈0.05） but significantly lower than those in the model control （P〈0.01）; （3） the high doses of TSN Ⅱ A and valsartan significantly down-regulated the mRNA expression of AT 1R and protein expression of TGF-beta l and Smad-3 in the hypertrophic myocardium （P〈0.01）, and TGF-betal in valsartan-treated animals was more significantly lower than that in rats treated with TSN Ⅱ A; （4） the two doses of TSN Ⅱ A and valsartan significantly up-regulated the protein expression of Smad-7 in the hypertrophic myocardium （P〈0.01）, and Smad-7 in the animals treated with high-dose TSN Ⅱ A was significantly higher than that in rats treated with valsartan. It is concluded that inhibition of myocardial hypertrophy induced by TSN ⅡA independent of blood pressure. The underlying mechanism might be the down-regulated expression of AT1R mRNA and Smad-3, increased production of Smad-7, and blocking effect of TSN Ⅱ A on TGF betal/Smads signal pathway in local myocardium.

Effects of Calmodulin-dependent Protein Kinase Ⅱ Inhibitor,KN-93,on Electrophysiological Features of Rabbit Hypertrophic Cardiac Myocytes

Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to determine the effects of the calmodulin-dependent protein kinase(CaMK) Ⅱ inhibitor,KN-93,on L-type calcium current(I Ca,L) and early after-depolarizations(EADs) in hypertrophic cardiomyocytes.A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation(LVH group).The control group(sham group) received a sham operation,in which the abdominal aortic was dissected but not coarcted.Eight weeks later,the degree of left ventricular hypertrophy(LVH) was evaluated using echocardiography.Individual cardiomyocyte was isolated through collagenase digestion.Action potentials(APs) and I Ca,L were recorded using the perforated patch clamp technique.APs were recorded under current clamp conditions and I Ca,L was recorded under voltage clamp conditions.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were observed under the conditions of low potassium(2 mmol/L),low magnesium(0.25 mmol/L) Tyrode’s solution perfusion,and slow frequency(0.25-0.5 Hz) electrical stimulation.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92(KN-92 group) and KN-93(KN-93 group).Eight weeks later,the model was successfully established.Under the conditions of low potassium,low magnesium Tyrode’s solution perfusion,and slow frequency electrical stimulation,the incidence of EADs was 0/12,11/12,10/12,and 5/12 in sham group,LVH group,KN-92 group(0.5 μmol/L),and KN-93 group(0.5 μmol/L),respectively.When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group,the incidence of EADs was 10/12 and 2/12,respectively.At 0 mV,the current density was 6.7±1.0 and 6.3±0.7 PA·PF-1 in LVH group and sham group,respectively(P>0.05,n=12).When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups,the peak I Ca,L at 0 mV was decreased by(9.4±2.8)% and(10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups,respectively(P>0.05,n=12).When the drug concentration was increased to 1 μmol/L,the peak I Ca,L values were lowered by(13.4±3.7)% and(40±4.9)%,respectively(P<0.01,n=12).KN-93,a specific inhibitor of CaMKII,can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I Ca,L,which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.

EXPERIMENTAL STUDY ON THE BIOCHEMICAL REMODELING OF VENTRICULAR COLLAGEN MAT

The present study is to characterize the biochemicai remodeling of ventricular collagen matrix and its natural history following myocardial infarction(MI)in rats.Collagen concentration,the total collagen content and the ratio of types Ⅰ,Ⅲ collagen (Ⅰ/Ⅲ) were measured at 3,15 and 42 days after MI.The results showed:1)The total collagen content of non-infarcted area (NIA) and right ventricle (RV) foliowing the development of ventricular hypertrophy increased progressively. Although the collagen concentration had no difference in RV, it showed dynamic changes in NIA. Both the collagen concentration and the total collagen content in infarcted area (IA) increased rapidly following reparative fibrosis.2)At the early stage or MI,type Ⅲ collagen in NIA increased significantly;later,type collagen was remarkedly higher in NIA and RV.Ⅰ/Ⅲ of IA showed difrerent patterns than that of NIA.The results suggest:(1) the blochemical remodeling or collagen matrix in NIA, IA and RV occurred following MI and its time courses were different;(2)the mechanism of the biochemlcal remodeling of collagen matrix in ventricles may be different.

Effect of Sodium Tanshinone ⅡA Sulfonate on Phosphorylation of Extracellular Signal-regulated Kinasel/2 in Angiotensin Ⅱ-induced Hypertrophy of Myocardial Cells

Objective： To observe the effects of sodium tanshinone ⅡA sulfonate （STS） on angiotensin Ⅱ （Ang Ⅱ）-induced hypertrophy of myocardial cells through the expression of phosphorylated extracellular signal-regulated kinase （p-ERK1/2）. Methods： In the primary culture of neonatal rat myocardial cells, the total protein content in myocardial cells was determined by coomassie brilliant blue and the protein synthesis rate was measured by [3H]-Leucine incorporation as indexes for hypertrophy of myocardial cells. The expression of p-ERK1/2 was determined using Western blot and immunofluorescence labeling. Results： （1） The total protein and protein synthesis rate increased significantly in contrast to the control group after the myocardial cells were stimulated by Ang Ⅱ （1 μ mol/L） for 24 h; STS markedly inhibited the increment of the total protein level induced by Ang Ⅱ and the syntheses of protein. （2） After pretreatment of myocardial cells with Ang Ⅱ （1 μmol/L） for 5 min, the p-ERK1/2 protein expression was increased, with the most obvious effect shown at about 10 min; pretreatment of myocardial cells with STS at different doses （2, 10, 50μmol/L） for 30 min resulted in obvious inhibition of the expression of p-ERK1/2 stimulated by Ang Ⅱ in a dose-dependent manner. （3） After the myocardial cells were stimulated by AngⅡ （1 μ mol/L）, the immunofluorescence of ERK1/2 rapidly appeared in the nucleus. The activation and translocation process of ERK1/2 induced by Ang Ⅱ was blocked distinctly by STS. （Conclusion： STS inhibited the myocardial cell hypertrophy induced by Ang Ⅱ, and the mechanism may be associated with the inhibition of p-ERK1/2 expression.

Effects of transforming growth factor-β_1 and signal protein Smad3 on rat cardiomyocyte hypertrophy

Background SMAD proteins have recently been identified as the first family of putative transforming growth factor-β_1(TGF-β_1) signal transducers. This study was to investigate the effects of TGF-β_1 and signal protein Smad3 on rat cardiac hypertrophy.Methods The incorporation of [3H]-leucine was measured to determine the hypertrophy of cardiomyocyte incubated with different doses of TGF-β_1 in cultured neonatal cardiomyocytes. The model of rat cardiac hypertrophy was produced with constriction of the abdominal aorta. At different times after the operation, rats were killed, and their left ventricular mass index (LVMI) determined. The mRNA expression of TGF-β_1 and Smad3 of cultured cells and hypertrophic left ventricles were assessed by RT-PCR. The protein expression of Smad3 was assessed by Western blot.Results In cultured neonatal cardiomyocytes, TGF-β_1 significantly promoted incorporation of [3H]-leucine. With the concentration of 3 pg/L, it increased the expression of Smad3 in mRNA and protein levels after 15 minutes, and continued for up to 8 hours of cultured cardiomyocytes. The LVMI and the expression of TGF-β_1 (mRNA) and Smad3 (mRNA and protein) of hypertrophic left ventricle were increased by day 3 after the operation and continued to the 4th week. The peak expression of these was in the second week after operation.Conclusion TGF-β_1 has positive effects on rat cardiomyocyte hypertrophy. Signal protein Smad3 could be related to the pathologic progression of rat cardiac hypertrophy.

Depletion of Kindlin-2 induces cardiac dysfunction in mice

Kindlin-2, a member of the Kindlin family focal adhesion proteins, plays an important role in cardiac development. It is known that defects in the Z-disc proteins lead to hypertrophic cardiomyopathy(HCM) or dilated cardiomyopathy(DCM). Our previous investigation showed that Kindlin-2 is mainly localized at the Z-disc and depletion of Kindlin-2 disrupts the structure of the Z-Disc. Here, we reported that depletion of Kindlin-2 leads to the disordered myocardial fibers, fractured and vacuolar degeneration in myocardial fibers. Interestingly, depletion of Kindlin-2 in mice induced cardiac myocyte hypertrophy and increased the heart weight. Furthermore, decreased expression of Kindlin-2 led to cardiac dysfunction and also markedly impairs systolic function. Our data indicated that Kindlin-2 not only maintains the cardiac structure but also is required for cardiac function.