Meglumine cyclic adenylate induces differentiation of bone marrow mesenchymal stem cells into cardiomyocytes in vitro

AIM: To study the effect of meglumine cyclic adenylate (MCA) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes in vitro. METHODS: The whole bone marrow adherent culture method was used to isolate, culture and amplify the BMSCs. The surface markers of BMSCs were determined by flow cytometry analysis. MCA at concentrations of 10^-2 mol/L, 10^-3 mol/L, 10^-4 mol/L, 10^-5 mol/L, 10^-6 mol/L and 10^-7 mol/L was added to the culture medium containing the second generation of BMSCs.5-Azacytidine(5-Aza) was used as a positive control. The cell viability was measured by MTT method.The cAMP content in BMSCs was detected by ELISA. The mRNA expression of GATA-4, Cx43 and β-MHC in MCA group and MCA+H89 (a PKA inhibitor) group was measured by SYBR-RT-PCR. The differentiation effects of MCA and 5-Aza were compared by flow cytometry. RESULTS: Most of the BMSCs expressed CD44 and CD71, and did not express CD45. MCA inhibited the viability of BMSCs in a time-and dose-dependent manner, and MCA atthe concentration of 10^-2 mol/L showed particularly remarkable effect. MCA significantly increased intracellular cAMP level in BMSCs in a concentration-dependent manner. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group were significantly higher than that in blank group (P<0.05), and the highest effect was under the condition of MCA induction at the concentration of 10^-3 mol/L for 3 days. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group was higher than that in 5-Aza group and H89+MCA group (both P<0.05). Differentiation rate in MCA group was slightly higher than that in 5-Aza group (20.24%±1.02% vs 18.39%±0.58%, P<0.05). CONCLUSION: MCA stimulates BMSCs to increase intracellular cAMP production and inhibits the viability of BMSCs, thus promoting the mRNA expression of GATA-4, β-MHC and Cx43 through the cAMP/PKA signaling pathway.     

Effects of Tongxinluo on connexin 43 remodeling and ventricular arrhythmia after myocardial infarction in rats

AIM: To determine the effects of Tongxinluo(TXL) on connexin 43(Cx43) remodeling and ventricular arrhythmia(VA) after myocardial infarction(MI) in rats. METHODS: Male SD rats were randomly divided into sham-operated(sham) group(n=25) and operation group(n=75). The left anterior descending(LAD) was ligated in operated group, while the rats in sham group only underwent pericardiotomy. The rats in operation group which survived for 3 d after operation were randomly assigned to TXL group and MI group. The rats in TXL group was administrated with TXL(2 g·kg^-1·d^-1, intragastric administration) for 4 weeks, while normal saline was applied to the rats in sham group and MI group. The levels of interleukin-1β(IL-1β) and endothelin-1(ET-1) in the tissue from the border zone were measured by ELISA after treatment. The distribution and the mRNA and protein expression of Cx43 were detected by immunohistochemical staining, RT-PCR and Western blotting, respectively. The burst pacing was used to induce ventricular arrhythmia(VA). RESULTS: Compared with sham group, the levels of IL-1β and ET-1 and the incidence of VA were significantly increased, while the mRNA and protein expression of Cx43 was markedly reduced with irregular distribution in MI group(P<0.05). Compared with MI group, the levels of IL-1β and ET-1 and the incidence of VA were significantly reduced, while the expression of Cx43 at mRNA and protein levels was markedly increased with augmented linear distribution in the myocardial cell intercalated disc in TXL group(P<0.05). CONCLUSION: TXL reduces the incidence of VA after MI via inhibiting the Cx43 remodeling.     

Inhibitory effect of aerobic exercise on left ventricular remodeling and sympathetic neural remodeling in rats with heart failure after myocardial infarction

AIM: To investigate the effects of long-term aerobic exercise on the heart and sympathetic neural remodeling (structure and function remodeling) in heart failure rats induced by myocardial infarction. METHODS: Heart failure model after myocardial infarction was performed by ligating anterior descending coronary artery in the Wistar rats. Four weeks after operation, the rats were randomly divided into sham operation sedentary (S) group, heart failure sedentary (H) group and heart failure exercise (HE) group. The animals in HE group underwent 10-week treadmill running, while those in S group and H group were sustained in a resting state. The cardiac structure and function including left ventricular internal diameter at diastole (LVIDd), left ventricular internal diameter at systole (LVIDs), left ventricular anterior wall diameter at diastole (LVAWDd), left ventricular anterior wall diameter at systole (LVAWDs), left ventricular posterior wall diameter at diastole (LVPWDd) and left ventricular posterior wall diameter at systole (LVPWDs), and cardiac function parameters including fractional shortening (FS) and left ventricular ejection fraction (LVEF) were measured by echocardiography. The myocardium was collected for histopathological observation with Masson staining, and the collagen volume fraction (CVF) was determined. The concentrations of norepinephrine (NE) in the myocardium and plasma were measured by high-pressure liquid chromatography. The frequency domain analysis was applied for determining the heart rate variability (HRV) via subcutaneous recording electrode involving total power (TP), normalized low power (LFn), normalized high power (HFn) and LF/HF ratio. The mRNA expression of collagen type I (Col-I), collagen type III (Col-III), atrial natriuretic factor (ANF), α-myosin heavy chain (α-MHC), β-myosin heavy chain (β-MHC), sarcoplasmic endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) was detected by real-time PCR. The protein levels of nerve growth factor (NGF) and its receptor (TrkA), and tyrosine hydroxylase (TH) were measured by Western blotting. RESULTS: (1) Compared with S group, body weight (BW), LVIDd, FS, LVEF, TP, HFn, the mRNA expression of α-MHC and SERCA2a, and the protein levels of NGF, TrkA and TH decreased (P<0.05). Left ventricular weight (LVW), left ventricular mass index (LVMI), LVAWDd, LVAWDs, LVPWDd, LVPWDs, CVF, plasma and myocardial NE content, LFn, LF/HF, and the mRNA expression of ANF, β-MHC, Col-I and Col-III increased (P<0.05) in H group. (2) Compared with H group, LVW, LVMI, LVIDd, FS, LVEF, TP, HFn, the mRNA expression of α-MHC and SERCA2a, and the protein levels of NGF, TrkA and TH were raised (P<0.05), while CVF, plasma and myocardial NE content, LFn, LF/HF, and the mRNA expression of ANF, β-MHC, Col-I and Col-III decreased (P<0.05) in HE group. CONCLUSION: Long-term aerobic exercise training leads to inhibition of heart and sympathetic neural remodeling and improvement of cardiac function and autonomic modulation in the rats after myocardial infarction.     

Roles of gap junction in TGF-β1-induced proliferation of vascular smooth muscle cells from spontaneous hypertensive rats

AIM: To investigate whether gap junction participates in transforming growth factor β₁(TGF-β₁)-induced proliferation of spontaneous hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). METHODS: The thoracic aorta of the rats were sampled. The primary SHR VSMCs were isolated and cultured in vitro. The cells were divided into 4 groups: control group, TGF-β₁ group,18α-glycyrrhetinic acid(18α-GA) group and TGF-β₁+18α-GA group. The proliferation of SHR VSMCs was observed by the methods of MTT and flow cytometry. The protein expression and co-localization of connexin(Cx)43 and Cx40 in SHR VSMCs were detected by immunofluorescence staining. The protein levels of Cx43 and Cx40 in the cells were also measured by Western blotting. The method of molecular dye transfer (scrape dye transfer method) was applied to detect the function of gap junction in SHR VSMCs. RESULTS: The protein expression of Cx43 and Cx40 in SHR VSMCs was positive and co-localized in the cytoplasm. Compared with control group, the percentage of S-phase detected by cell cycle and A value detected by MTT in TGF-β₁ group were obviously increased (P<0.05), indicating that the proliferation of the cells was enhanced. However, the proliferation of the cells decreased in 18α-GA group (P<0.05). Compared with TGF-β₁ group, the percentage of S-phase and A value in TGF-β₁+18α-GA group were both significantly decreased (P<0.05), indicating that the proliferation of the cells decreased. Compared with control group, the protein expression of Cx43 in TGF-β₁ group was increased (P<0.05), whereas the protein expression of Cx40 was not changed (P>0.05), and the protein expression of Cx43 and Cx40 in 18α-GA group were decreased (P<0.05). Compared with TGF-β₁ group, the expression of Cx43 in TGF-β₁+18α-GA group was significantly decreased (P<0.05),but no difference of the Cx40 protein levels between the two groups was observed. Compared with control group, the function of gap junction detected by scrape dye transfer method in TGF-β₁ group was enhanced (P<0.05), and weakened in 18α-GA group (P<0.05). Compared with the TGF-β₁ group, the function of gap junction in TGF-β₁+18α-GA group was significantly attenuated (P<0.05). CONCLUSION: TGF-β₁ enhances the function of gap junction to stimulate the proliferation of SHR VSMCs through the expression of Cx43 protein. The expression of Cx40 protein may not play a major role in this process.     

Protective role of carnosine on cardiac dysfunction in type 1 diabetes mellitus rat model

AIM:To explore the effects of carnosine (CAR) on cardiac dysfunction in type 1 diabetic mellitus rats and the underlying mechanism. METHODS:The SD rats were randomly divided into 4 groups:control (C) group, control+carnosine (C+CAR) group, diabetes mellitus (DM) group and diabetes mellitus+carnosine (DM+CAR) group (n=10). The rats were sacrificed after 12 weeks. The cardiac function was assessed by ventricular cannulation. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were assessed by ELISA. The mRNA levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β) and IL-6 were measured by real-time PCR. The distribution of connexin 43 (Cx43) was examined by immunofluorescence. The protein levels of Cx43 and protein kinase C (PKC) were determined by Western blot. RESULTS:Compared with the C group, the left ventricular end diastolic pressure (LVEDP) was increased whereas the left ventricular pressure maximum rise/fall velocity (±dp/dt_max) was decreased in the DM group (P<0.01). The activity of SOD decreased while the MDA increased in the left ventricular tissues (P<0.01). The mRNA levels of TNF-α, IL-1β and IL-6 were increased (P<0.01). The Cx43 distribution was irregular. The protein levels of phosphorylated Cx43 and PKCε were elevated (P<0.01). Compared with the DM group, the cardiac function of LVEDP and ±dp/dt_max in DM+CAR group was ameliorated (P<0.01), with increased SOD activity and decreased MDA content (P<0.05). The mRNA levels of TNF-α, IL-1β and IL-6 were reduced (P<0.01). The Cx43 distribution was improved and the protein levels of phosphorylated Cx43 and PKCε were decreased (P<0.01). CONCLUSION:CAR treatment can improve the cardiac function by its anti-oxidative and anti-inflammation effects and suppression of Cx43 abnormalities through PKCε in DM rats.     

Changes of cardiac function,RAGE expression and calcium dysregulation in type 2 diabetic rats

AIM: To investigate the changes of cardiac structure and function in rats with type 2 diabetic mellitus (T2DM), and to explore the mechanisms underlying diabetic cardiomyopathy. METHODS: The cardiac structure and function were measured by echocardiography in Zucker diabetic fatty (ZDF) rats and their control Zucker lean (ZL) rats. The size of the cardiomyocytes was determined by wheat germ agglutinin staining. The protein expression of atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), receptor for advanced glycation end products (RAGE), L-type cal-cium channel α1C subunit (Ca_V1.2) and Orai1 was assessed by Western blot. RESULTS: Compared with the ZL control rats, the thickness of left ventricular wall, ejection fraction (EF), fractional shortening (FS) and the sizes of cardiomyocytes were significantly increased, and diastolic function was decreased in the ZDF rats (P<0.05). The protein expression of β-MHC, ANP, RAGE and Orai1 was increased, while the expression of Ca_V1.2 was decreased in ZDF rats (P<0.05). CONCLUSION: T2DM rats show the prominent features including cardiomyocyte hypertrophy, ventricular hypertrophy and compensatory enhancement of cardiac function, and the Ca²⁺ handling and increase in RAGE expression may play important roles in the processes.     

Mechanisms of histone acetylation modification in paternal alcohol consumption-induced heart development abnormalities of offspring

AIM:To investigate the role of histone acetylation modification in paternal alcohol consumption-induced heart development abnormalities of offspring. METHODS:Male C57 mice were exposed to 40% alcohol for 6 weeks at a dose of 10 mL·kg^-1·d^-1. Normal saline was used as negative control and non-treatment was for blank control. Heart samples of the newborn offspring mice were collected. Birth weight was measured. Cardiac ultrastructure was observed by transmission electron microscope (TEM). The apoptosis of cardiomyocytes was measured by TUNEL method. The mRNA levels of caspase-3 and Bcl-2 were detected by qPCR. The protein levels of active caspase-3 and Bcl-2 were determined by Western blot. ChIP-qPCR was employed to analyze the levels of acH3K9 and acH3K27 on the promoters of caspase-3 and Bcl-2. RESULTS:Low birth weight was found in the mice of alcohol exposure group. TEM observation showed broad existence of sarcomere dissolution, enlargement of sarcoplasmic reticulum and disorder of sarcomere arrangement. The results of TUNEL displayed increased cardiac apoptosis in alcohol group compared with the controls. The mRNA levels of caspase-3 and the protein levels of active caspase-3 were increased compared with control group (P<0.05). The protein level of active caspase-3 was decreased compared with the controls (P<0.05). Both mRNA and protein levels of Bcl-2 were decreased compared with the controls (P<0.05). The acH3K9 on the promoter of caspase-3 went up significantly (P<0.05), and acH3K27 on the promoter of Bcl-2 was decreased (P<0.05). CONCLUSION:Paternal alcohol exposure causes heart development abnormalities in offspring mice. Histone acetylation may play an important role in the apoptosis of cardiomyocytes.     

Role of Cx43 gene in the process of myocardialization of proximal outflow tract septum in the mouse heart

AIM:To investigate the role of Cx43 in the myocardialization of the proximal outflow tract (OFT) septum in the mouse heart. METHODS:C57/BL6 mice of ED11.5 to 1 day after birth were used in this study, which included Cx43 knockout homozygotes (Cx43-/-)， heterozygotes (Cx43+/-) and wildtypes (Cx43+/+). Pathohistological analysis was used to examine the structure of the hearts. The expression of alpha-sarcomeric actin (α-SCA), active caspase-3 and activator protein-2 (AP-2) were detected by immunohistochemistry. RESULTS:Most Cx43-/- mice died within 24 h after birth with a swelling and blockage of the conotruncal region, which led to the obstruction of OFT and enlargement of right ventricle. HE staining showed plenty of abnormal tissues in this region forming many pouches. No apparent malformations were observed in Cx43+/- and Cx43+/+ mice. The expression of α-SCA in the proximal OFT septum was delayed obviously in Cx43-/-. The apoptotic cells existed in the proximal OFT septum of Cx43+/+ mostly during ED12.5 to ED15.5. However, there were less apoptotic cells observed in Cx43+/-, and few in Cx43-/-. The expression of AP-2, marker of neural crest cells, was increased in Cx43-/- and abnormally located in the proximal OFT septum. CONCLUSIONS:Cx43 KO mice are characterized by hyperplasia in conotruncal region, which may be associated with the delayed myocardialization of OFT septum. The decreased apoptosis and the abnormal distribution of cardiac neural crest cells are likely to contribute to the abnormal myocardialization in mice with Cx43 defects.     

Effects of adiponectin on expression of connexin 43 in rat myocardium during ischemia-reperfusion

AIM: To observe the effects of adiponectin(APN) on the expression of connexin 43 (Cx43) in rat myocardium during ischemia-induced arrhythmias. METHODS: The SD rats were randomly divided into 4 groups (n=12): sham operation group (SM group), ischemia and reperfusion group (I/R group), I/R+adiponectin(APN1) group: pre-ischemia with 3.5 μg/kg of APN; I/R+APN2 group: post-ischemia with 3.5 μg/kg of APN. The incidence of ventricular arrhythmias and ventricular arrhythmia score (VAS) were determined. The expression of Cx43 in the ischemic myocardium was studied by the techniques of immunohistochemistry and RT-PCR. The levels of malondialdehyde(MDA) and superoxide dismutase(SOD) were measured by the methods of xanthine oxidase and thiobarbituric acid. The expression of endothelial nitric oxide synthase (eNOS) at mRNA and protein levels was determined by RT-PCR and Western blotting,respectively.The morphological changes of the myocardial tissues were observed under electronic microscope. RESULTS: The VAS and concentration of MDA increased obviously and the activity of SOD was decreased in I/R group as compared with SM group (P<0.01). The expression of Cx43 was evidently decreased and the distribution of Cx43 in the myocardium was disturbed. The expression of eNOS at mRNA and protein levels was decreased in I/R group (P<0.05). The ultrastructure of ventricular myocardium was abnormal in I/R group. Compared with I/R group, APN obviously decreased the VAS caused by ischemia and reperfusion (P<0.01) no matter the drug was given before or after ischemia. APN increased the activity of SOD, inhibited the MDA content in serum, and resulted in normal distribution of Cx43 and increased the expression of Cx43 and eNOS. Compared with I/R group, the changes of heart ultrastructure attenuated greatly in APN group, but didn't recover to normal state. CONCLUSION: Adiponectin antagonizes the arrhythmias during myocardial ischemia and reperfusion via inhibiting oxidative stress and regulating Cx43.     

Saikosaponin a inhibits PTZ-induced activation of hippocampal astrocytes in mice

AIM: To investigate the inhibitory effect of saikosaponin a (SSa) on pentylenetetrazole (PTZ)-induced activation of hippocampal astrocytes in mice. METHODS: Hippocampal astrocytes were isolated and cultured. The cells were randomly divided into control group, PTZ group, PTZ+0.625 mg/L SSa group and PTZ+1.25 mg/L SSa group. The cells were identified by detection of glial fibrillary acidic protein (GFAP). The cell viability was measured by MTT assay. The cell cycle was analyzed by flow cytometry. The expression of GFAP and connexin 43 (Cx43) was mea-sured by ELISA. The level of apoptosis was determined by flow cytometry and Hoechst 33258 staining. RESULTS: The primary hippocampal astrocytes grew by adherent culture, and the processes of the astrocytes were obvious. Immunofluorescence showed positive GFAP expression in the astrocytes. Compared with control group, the viability of the cells and the percentage of the cells in G₂/M phase in PTZ group were significantly increased (P<0.05), and the expression of GFAP and Cx43 was also markedly increased (P<0.05). Compared with PTZ group, the viability of the cells and the percentage of the cells in G₂/M phase were obviously decreased in PTZ+0.625 mg/L SSa group and PTZ+1.25 mg/L SSa group, and the expression of GFAP and Cx43 was also reduced, whereas the percentage of apoptotic cells was significantly increased (P<0.05).CONCLUSION: SSa significantly suppresses PTZ-induced activation of hippocampal astrocytes, inhibits the cell proliferation and induced apoptosis.     

Effect of ischemic preconditioning on myoelectricity and the structure of myocardial gap junction during heart valve replacement surgery

AIM: To investigate the cardio-protective mechanism of ischemic preconditioning (IP) during heart valve replacement of the perspective of architectural changes of myocardial gap junction. METHODS: Fifty-four patients were prospectively randomized to receive or not ischemic preconditioning before cold cardioplegic arrest. The IP protocol in IP patients (n=22) consisted of a single 2-minute ischemia followed by 3-minute reperfusion just before aortic clamping and cold crystalloid cardioplegia for myocardial protection. The control group (n=32) received no ischemic preconditioning prior to cold cardioplegic arrest. The parameters including arrhythmias occurrence, Cx43 expression (immunohistochemistry SABC method) and myocardial structure and intercalated discs observed under electronic microscope were recorded before and after surgery in each group. RESULTS: In IP group, one case (4.55%) of ventricular arrhythmia (sporadic ventricular premature beat), 11 cases (50.00%) of supraventricular arrhythmia (atrial fibrillation, atrial flutter, supraventricular tachycardia, atrioventricular block) and 10 cases (45.50%) of ischemic ST-T changes were observed. In control group, there were 14 cases (43.75%) of ventricular arrhythmia (ventricular premature beat, tachycardia), 18 cases (56.25%) of supraventricular tachycardia and 28 cases (87.50%) of ischemic ST-T changes. No statistical difference in preoperative positive unit of Cx43 expression between the two groups was found (P>0.05). Postoperatively, the positive unit of Cx43 expression in IP group was 16.15±4.40, but the difference was not significant compared to the preoperative value (P>0.05). In control group, Cx43 expression was 11.92±1.26, significantly lower than that of the preoperative value (P<0.05). Cx43 expression between the two postoperative groups showed a significant difference (P<0.05). In control group, electronic microscopic observation revealed disrupted intercalated discs, with some partially or even totally ruptured and disintegrated. Enormous necrotic structural changes of myocardial fibers were also observed, including swelling, dissolution and disorganization of myofilaments and fibers, widening of the Z striae and disorganization. However in IP group, the intercalated discs appeared intact, continuous with normal myocardial structure. CONCLUSION: IP maintains normal expression of the myocardial junctional gap protein Cx43, which preserves a seamless intercellular gap junction and a normal myocardial electric conduction activity.     

Effect of captopril and betaloc on the changes in expression of MHC of left ventricle in hypertensive rats

AIM:To investigate myosin heavy chain(MHC)gene expression and the effects of captopril and betaloc at an early stage of hypertension. METHODS:Model of hypertension was made by partly narrowing two bilateral renal arteries(2K2C). The rats were divided into four groups at random. (1) control group; (2)2K2C group;(3)captopril group;(4)betaloc group.Levels of α-MHC and β-MHC mRNA were determined by dot-blot. RESULTS:α-MHC mRNA expression were gradual1y reduced in 2K2C group, while β-MHC mRNA expression were increased, and the marked changes were observed at 72h postoperation. Captopril could inhibit the changes in MHC gene expression; but betaloc could not. CONCLUSION: The expression of MHC gene has changed at an early stage of renal hypertensive rat, and renin-angiotensin system may play an important role in this change.     

High glucose induces H9c2 cardiomyocyte hypertrophy through Ca2+-CaN-NFAT3 signaling pathway

AIM: To study the morphological changes of cardiac H9c2 cells during the developmental process of fetal rat. METHODS: Embryonic rat heart-derived H9c2 cells were maintained in DMEM supplemented with 10% fetal bovine serum. The H9c2 cells were plated at a density of 6000 cells/cm and divided into 5 groups: H9c2 cells were treated with 5 mmol/L glucose, 25 mmol/L glucose, 50 mmol/L glucose, Norvasc (25 nmol/L)+25 mmol/L glucose, or Norvasc (25 nmol/L)+50 mmol/L glucose for 48 h. The morphology of H9c2 cells was observed. The cell surface area was measured by Image-Pro Plus 6.1 software. Fluorescence spectrophotometry was used to detect the concentration of intracellular calcium ion ([Ca²⁺]_i)in the cardiomyocytes. The concentration of CaN in the cell was measured by ELISA. The mRNA expression of CaNAβ, NFAT3 and β-MHC in the cells was detected by real-time PCR. The protein levels of CaNAβ, NFAT3 and β-MHC in cultural H9c2 cells were detected by Western blot. RESULTS: The mean area of the cells, the mean fluorescence value of [Ca²⁺]_i and the concentration of CaN in 25 mmol/L glucose group were higher than those in 5 mmol/L glucose group, and those were lower than those in 50 mmol/L glucose group. After treated with Norvasc, those results decreased significantly. The expression of CaNAβ, NFAT3 and β-MHC at mRNA and protein levels in 25 mmol/L glucose group was higher than those in 5 mmol/L glucose group, but was lower than those in 50 mmol/L glucose group. The expression of CaNAβ, NFAT3 and β-MHC at mRNA and protein levels decreased significantly in Norvasc treatment group. CONCLUSION: Ca²⁺-CaN-NFAT3 signaling pathway is perhaps involved in high glucose-induced H9c2 cardiomyocyte hypertrophy.     

Effects of cardiac contractility modulation on ventricular electrical remodeling in rabbits with chronic heart failure

AIM: To investigate the effects of cardiac contractility modulation (CCM) on ventricular electrical remodeling in a rabbit model of chronic heart failure. METHODS: The rabbits were divided into sham group, heart failure(HF) group and HF+ CCM group. The rabbit model of chronic heart failure was established by ligating the ascending aortic root. Then electrical stimulations during the absolute refractory period were delivered lasting 6 h everyday for 4 weeks in rabbits of HF+ CCM group. The QTc and ventricular effective refrective period (VERP) were recorded. The protein and mRNA levels of Kv1.4, Kv4.3 and connexin 43 (Cx43) were determined by Western blot and RT-qPCR. RESULTS: Compared with sham group, QTc were significantly prolonged in HF rabbits at week 12 (P<0.05). CCM therapy shortened QTc of rabbits with heart failure at week 16 (P<0.05). Compared with sham group, VERP significantly increased in HF group and HF+ CCM group, while CCM therapy shortened VERP of rabbits with heart failure at week 16 (P<0.05). Compared with sham group, the mRNA and protein levels of Kv1.4, Kv4.3 and Cx43 were decreased in HF group and HF+ CCM group (P<0.05). However, CCM therapy restored the mRNA and protein levels of Kv1.4, Kv4.3 and Cx43 of rabbits with heart failure (P<0.05). CONCLUSION: CCM suppresses ventricular electrical remodeling in heart failure and the underlying mechanism may be associated with increasing Kv1.4, Kv4.3 and Cx43 expression.     

Inhibition of desmoplakin expression induces connexin 43 remodeling in mouse HL-1 myocardial cells

AIM: To evaluate the content, distribution, and function of connexin 43 (Cx43) gap junctions in mouse HL-1 myocardial cells under the condition of desmoplakin (DSP) silencing.METHODS: The technique of RNA interference was used to inhibit the protein expression of DSP in HL-1 cells. The protein expression of DSP and Cx43 was analyzed by Western blotting and flow cytometry. The immunofluorescence staining was used to detect the distribution and co-localization of DSP and Cx43. The techniques of scrape loading and dye transfer were also used to determine the function of Cx43 gap junctions.RESULTS: Compared with non-treatment group and negative control group, the protein expression of DSP and Cx43 was reduced (P<0.05). Co-localization to the site of cell-cell contact was apparent in untreated and control conditions, but loss of DSP expression induced by siRNA-DSP correlated with a drastic redistribution of Cx43. Instead, Cx43 was found mostly within the intracellular space. The results of dye transfer assay indicated a significant decrease in the function of Cx43 gap junctions of the cells treated with siRNA-DSP.CONCLUSION: Inhibition of DSP expression induces redistribution of Cx43 and decrease in dye coupling between cells.     

Effect of leonurine on expression of miR-1 in rats with myocardial fibrosis induced by isoproterenol

AIM: To investigate effect of leonurine on the expression of microRNA-1 (miR-1) in rats with myocardial fibrosis induced by isoproterenol (ISO). METHODS: SD rats (n=10) were used as normal control group, and 80 rats were given ISO by intraperitoneal injection daily for 2 weeks to establish the model of myocardial fibrosis. The model rats were randomly divided into 5 groups:model group, low-dose (7.5 mg·kg^-1·d^-1) leonurine group, middle-dose (15 mg·kg^-1·d^-1) leonurine group, high-dose (30 mg·kg^-1·d^-1) leonurine group and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor (0.3 mg·kg^-1·d^-1) group. After the treatment for 2 weeks, the ultrastructure of left ventricular myocardial tissues was observed under electron microscope. Masson staining was used to detect collagen fibrosis, and the expression of collagen I and collagen Ⅲ was determined by the method of immunohistochemistry. The contents of endothelin-1 (ET-1) and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. The expression of miR-1 and ET-1 mRNA was detected by real-time PCR, and the protein expression of p38 MAPK, β-myosin heavy chain (MHC) and α-MHC was determined by Western blot. RESULTS: Compared with model group, the ultrastructure of left ventricular myocardial tissues in high-dose leonurine group was attenuated, and the expression of miR-1 and the protein expression of α-MHC in left ventricular myocardial tissues of high-dose leonurine group were increased (P<0.05). Collagen volume fraction, collagen I, collagen Ⅲ, the ratio of collagen Ⅰ/collagen Ⅲ, the contents of ET-1 and Ang Ⅱ, the mRNA expression of ET-1, and the protein expression of p38 MAPK and β-MHC in high-dose leonurine group were lower than those in model group (P<0.05). CONCLUSION: Leonurine attenuates myocardial fibrosis in the rats induced by ISO, and it is potentially associated with affecting the expression of miR-1, and inhibiting ET-1/p38 MAPK signaling pathway.     

Effect of rosuvastatin combined with irbesartan on remodeling of myocardial hypertrophy in rats

AIM: To evaluate the effect of rosuvastatin combined with irbesartan on the remodeling of myocardial hypertrophy in the rats. METHODS: Male SD rats(n=50) were randomly divided into control group, model group, rosuvastatin group, irbesartan group and combination group. The model of myocardial hypertrophy was established by subcutaneous injection of isoproterenol at dose of 2.5 mg/kg for 14 d. From the first day of modeling, the rats in control group and model group received intragastrical saline, and the rats in rosuvastatin group, irbesartan group and combination group were treated with rosuvastatin(4 mg·kg^-1·d^-1), irbesartan(15 mg·kg^-1·d^-1) and rosuvastatin(4 mg·kg^-1·d^-1)+ irbesartan(15 mg·kg^-1·d^-1), respectively. The interventions continued for 4 weeks. After the interventions, the cardiac mass index and left ventricular mass index of the SD rats were measured. Besides, the degree of myocardial hypertrophy was observed with HE staining. The mRNA expression of hypertrophy-related factors, such as ANF, β-MHC and AT1R was determined by RT-PCR, and the protein expression of AT1R was determined by Western blot. RESULTS: Compared with control group, the cardiac mass index, left ventricular mass index, as well as the mRNA expression of ANF and β-MHC in model group were significantly increased(P<0.05). Compared with model group, the above factors in rosuvastatin group and irbesartan group were decreased(P<0.05), and the factors in combination group were lower than those in rosuvastatin group and irbesartan group(P<0.05). In addition, the expression of AT1R at mRNA and protein levels in rosuvastatin group and irbesartan group was lower than that in model group(P<0.05), while the expression AT1R at mRNA and protein levels in combination group was lower than that in rosuvastatin group and irbesartan group(P<0.05). CONCLUSION: Rosuvastatin and irbesartan are equally effective drugs to resist the formation of myocardial hypertrophy by decreasing the expression of AT1R. Moreover, combination of the 2 drugs is more effective to improve the degree of myocardial hypertrophy than the 2 drugs alone.     

Effects of meglumine cycle adenylate phosphate combined with transplantation of mesenchymal stem cells on heart functions in rat model of heart failure

AIM: To investigate the therapeutic effects of recombinant meglumine cycle adenylate phosphate (MCA) and bone marrow mesenchymal stem cells (BMSCs) on the enhancement of the cell survival and improvement of the cardiac functions in the rat model of adriamycin-induced cardiomyopathic heart failure. METHODS: BMSCs were isolated and expanded using the pre-plating method. Doxorubicin was used by intraperitoneal injection into the Wistar rats to establish the model of cardiomyopathic heart failure. The model animals randomly received the injection of PBS, MCA, BMSCs or MCA+BMSCs respectively, and normal controls were without any treatment. Four weeks after injection, the cardiac functions were determined by echocardiography and multichannel physiological recorder. The levels of brain natriuretic peptide (BNP) were measured by ELISA. The positive rate of BrdU-labeled BMSCs in the myocardium was analyzed by the method of immunohistochemistry. The expression of myocardium-specific protein, GATA-4, connexin 43(Cx43) and cardiac troponin 1(cTNI), was detected by Western blotting. Myocardial fibrosis was observed with Masson's staining. RESULTS: Compared with other groups, the results of echocardiography and hemodynamic showed that the left ventricular functions in BMSCs+MCA group improved significantly (P<0.05). The BMSCs numbers in the myocardium in BMSCs+MCA group were significantly higher than those in BMSCs group (P<0.05). The level of BNP was significantly lower in BMSCs+MCA group than that in BMSCs group (P<0.05). Compared with other groups, the expression of GATA-4, Cx43 and cTNI was significantly increased in BMSCs+MCA group. CONCLUSION: Combination of MCA with BMSCs transplantation improves the cardiac functions, possibly due to the enhancement of BMSCs survival and the increase in the protein expression of GATA-4.     

Regulatory effect of miR-199a-5p on cardiomyocyte hypertrophy in rat

AIM：To investigate the expression of miR-199a-5p in rat cardiomyocyte hypertrophy models. METHODS：The in vivo cardiomyocyte hypertrophy model was established by transverse abdominal aortic constriction (TAAC) and the in vitro model was induced by angiotensin II. The content of miR-199a-5p was detected by qRT-PCR in the plasma of the TAAC rats and in the cardiomyocytes (CM) of the newborn rats. The CM was isolated and transfected with miR-199a-5p mimic or inhibitor at concentration of 100 nmol/L by Lipofectamine RNAiMAX. The mRNA levels of atrial natriuretic factor (ANF) and β-myosin heavy chain (β-MHC) were detected by qRT-PCR. Tritium-labeled leucine incorporation was employed to determine the protein synthesis rate in the CM. The method of cyto-fluorescent staining was applied to measure the changes of the CM surface area. RESULTS：Compared with control group, the content of miR-199a-5p significantly increased in the TAAC rats and in the CM induced by angiotensin II. In addition, over-expression of miR-199a-5p in the CM up-regulated the mRNA expression of ANF and β-MHC, accelerated the protein synthesis rate and enlarged the CM surface area. In the CM transfected with miR-199a-5p inhibitor following induced by angiotensin II, the hypertrophy effect receded inversely (P<0.05). CONCLUSION:Over-expression of miR-199a-5p may promote cardiomyocyte hypertrophy, and repression of miR-199a-5p may inhibit cardiomyocyte hypertrophy in the CM.