Activation of extracellular-signal regulated kinase and protein phosphatases in human atria during atrial fibrillation

AIM: The purpose of this study was to determine whether the signal transduction systems were activated at the molecular atrial tissue level in patients with atrial fibrillation (AF) and whether atrial expression of extracellular-signal regulated kinase (ERK) and protein phosphatases is altered. METHODS: Atrial tissue sample of 30 patients undergoing cardiac surgery were examined. 20 patients had AF, 10 patients had no history of AF. The mRNA expression of calcineurin B and MKP-1 were detected by semi-quantitative RT-PCR. ERK1 and phospho-ERK1 were analyzed at the protein level by Western blot. RESULTS: Western blot analysis showed that atrial fibrillation did not induce significant change in ERK1 expression level in the left atrium. In contrast , phospho-ERK1 content was increased in the patients with AF in comparison with those who had sinus rhythm (SR). The mRNA expression of calcineurin B and MKP-1 in the patients with AF were significantly higher than that in patients with SR. CONCLUSION: The activation of extracellular-signal regulated kinase and protein phosphatases may have correlation with the initiation or maintenance of atrial fibrillation.     

Effects of the calpain system expression on atrial structural remodeling in canine with atrial fibrillation

AIM: To evaluate the influence of the calpain system mRNA and protein expression on the progress of atrial structural remodeling in fibrillating canine.METHODS: 17 dogs were randomly divided into 2 groups: normal control group (SR,n=6) and atrial fibrillation (AF,n=11) group.AF was induced by rapid pacing for 8 weeks and all dogs underwent transthoratic echocardiography before and after rapid pacing.The mRNA and protein expression of calpainⅠ,calpainⅡand calpastatin were assessed by real-time quantitative PCR and Western blotting,respectively.RESULTS: Compared with SR group,the left atrial diameters and the content of calcium in atrial myocardium increased significantly in AF group (P<0.05).There was no significant difference in mRNA expression of calpainⅠand calpainⅡ (P>0.05) between two groups.The expression of calpastatin mRNA was upregulated significantly in AF group (P<0.05).The levels of calpainⅠand calpainⅡprotein were significantly increased in AF group compared with SR group (P<0.05).The expression of calpastatin protein was significantly decreased in AF group (P<0.05).The calpainⅠand calpainⅡprotein levels were positively correlated with the left atrial diameter.The calpastatin protein level was negatively correlated with the left atrial diameter (P<0.05).CONCLUSION: The changes of the calpain system protein expression in AF result in the disturbance of calpain/calpastatin system and degradation of many proteins,which may play an important role in mechanism of atrial remodeling.     

Protein kinase A regulates small-conductance calcium-activated potassium type 2 channel in chronic atrial fibrillation patients

AIM：To investigate the alteration of small-conductance calcium-activated potassium type 2 (SK2) channel currents in atrial myocytes from atrial fibrillation (AF) patients, and the relationship between protein kinase A (PKA) and SK2 channel. METHODS：Right auricular tissues were obtained from the patients undergoing open-heart surgery with extracorporeal circulation. Single atrial myocytes were isolated by modified enzymatic dissociation method. The SK2 channel currents in the isolated human atrial myocytes were recorded using whole-cell patch-clamp technique. The alteration of SK2 channel currents and the regulation of SK2 channel by PKA were compared between sinus rhythm (SR) group and AF group. The total protein and PKA levels in human atrial tissues were detected by BCA assay and ELISA, respectively. RESULTS：The SK2 channel current densities and the proportion of SK2 channel currents in the integrated inward currents were significantly increased in AF group (all P<0.05 vs SR group). PKA-selective inhibitor H-89 reduced SK2 channel current densities and the proportion of SK2 channel currents in the integrated inward currents in both SR and AF groups, with larger reduction in AF group (all P<0.05 vs SR group). The PKA level was significantly decreased in AF atrial tissues (P<0.05 vs SR group). CONCLUSION: The increase in SK2 channel currents underlies the occurrence and maintenance of AF. PKA-dependent regulation may be involved in the remodeling of SK2 channel in both SR and AF human atrial myocytes, with a more powerful effect in AF.     

Relationship between fibrosis and remodeling of gap junction in atrium of patients with atrial fibrillation

AIM: To examine fibrosis and remodeling of gap junction in atrial myocardium of patients with or without atrial fibrillation and to investigate the relationship between them. METHODS: Right atrial appendage (RAA) samples were collected from 44 patients with rheumatic heart disease during heart operation, 28 of which were clinically diagnosed as atrial fibrillation (AF), the left remained sinus rhythm (SR). Fibrosis and remodeling of connexin 43 were examined by polarization microscope and microscopy respectively, and analyzed with an image analyzer. Meanwhile, intercalated disc was counted under transmission electron microscope. The collagen volume fraction of type I (CVF-I) and the volume fraction of Cx43 (Cx43VF) were studied between groups of atrial fibrillation and sinus rhythm. The relationship between CVF-I and fraction of remodeled intercalated disc was studied as well. RESULTS: (1) Polarization microscope demonstrated that CVF-I collagen increased (P<0.01) in atrial fibrillation group. (2) The ratio of remodeled of intercalated disc in patients with AF was higher (P<0.01) than that in SR group whereas the number of intercalated disc was not different (P>0.05) between the two groups. (3) Cx43VF decreased (P<0.01) in the AF patients compared to those with SR. (4) A positive correlation between fibrosis and the remodeling of intercalated disc (r=0.96, P<0.01) was observed. The CVF-I was negatively correlated with the Cx43VF (r=-0.98, P<0.01). CONCLUSION: These results suggest that both fibrosis of atrial muscle and remodeling of intercalated disc are involved in the pathogenesis of human atrial fibrillation. Fibrosis of atrial muscle may play an important role in the process of atrial fibrillation by interfering with remodeling of intercalated disc and thereby involves in the remodeling of connexins.     

Effects of atorvastatin on atrial electrical remodeling in a rabbit model of chronic atrial fibrillation

AIM: To evaluate the effects of atorvastatin (ATO) on atrial electrical remodeling in a rabbit mo-del of chronic atrial fibrillation (AF) produced by 3 weeks of rapid atrial pacing (RAP). METHODS: The sternotomy was performed and the pacing and testing electrodes were fixed to the left atria of 24 New Zealand white rabbits. The animals were randomly divided into 3 groups. The rabbits in model group and ATO group were subjected to RAP for 3 weeks, and then were treated with placebo and ATO (2.5 mg·kg^-1·d^-1), respectively. The rabbits in sham group did not receive RAP and drugs. Electrophysiological examination was performed to test heart rate, P-wave duration, atrial effective refractory period (AERP) and AF inducibility. The protein expression levels of Cav1.2, Kv4.3 and myeloperoxidase (MPO) were detected by Western blot. RESULTS: Sustained AF was induced in 5 and 4 rabbilts in model group and atorvastatin group and no rabbits in sham group was found. After 3 weeks of RAP, compared with sham group, heart rate and P-wave duration were increased and AERP was shortened in model group and ATO group (P<0.05). Compared with model group, AERP was increased in ATO group (P<0.05), while heart rate and P-wave duration had no difference between these 2 groups. Compared with sham group, the protein levels of Cav1.2 and Kv4.3 were decreased, and protein level of MPO was increased in model group and ATO group (P<0.05). Compared with model group, Cav1.2 was increased and MPO was decreased in ATO group (P<0.05), while Kv4.3 had no difference between these 2 groups. CONCLUSION: Atorvastatin suppresses the down-regulation of atrial Cav1.2 protein level and the shortening of AERP, thus preventing atrial electrical remodeling in a rabbit model of chronic AF. The effect of atrovastatin on reducing atrial MPO level may be the potential mechanism.     

Effects of spironolactone on atrial structural remodeling in rabbit model of chronic atrial fibrillation

AIM: To evaluate the effects and potential mechanism of spironolactone (SP) on atrial structural remodeling in rabbit model of chronic atrial fibrillation (AF). METHODS: The sternotomy was performed and the pacing electrodes were fixed to the left atria of New Zealand white rabbits. The animals were randomly divided into 3 groups. The rabbits were subjected to rapid atrial pacing (RAP) for 3 weeks in RAP group (intragastric administration with placebo) and RAP+SP group (intragastric administration with spironolactone at 20 mg·kg^-1·d^-1), respectively. The rabbits in sham group did not receive RAP and drugs. Before and after RAP, the structure and function of the atria were evaluated and AF inducibility was tested. After RAP, the atrial fibrosis was evaluated, and the expression levels of collagen I, collagen Ⅲ, matrix metalloproteinase (MMP)-2 and MMP-9 were determined. RESULTS: After 3 weeks of RAP, compared with sham group, obvious left atrial enlargement and dysfunction were observed in RAP group and RAP+SP group, but those had no significant differences in these 2 groups. Sustained AF was induced in 7, 5, and 0 rabbits in RAP group, RAP+SP group, and sham group, respectively. Compared with sham group, atrial interstitial fibrosis and the protein expression levels of collagen Ⅰ, collagen Ⅲ, MMP-2 and MMP-9 were all significantly increased in RAP group and RAP+SP group(P<0.05). Compared with RAP group, the the above indexes were all decreased in RAP+SP group(P<0.05). CONCLUSION: Spironolactone suppresses the atrial interstitial fibrosis and collagen expression, thus preventing atrial structural remodeling in rabbit model of chronic AF. The effect of spironolactone on reducing atrial MMP-2 and MMP-9 levels may be the potential mechanism.     

Effect of spironolactone on hyperthyroxine-induced atrial fibrillation and atrial remodeling in rabbits

AIM: To detect the effect of spironolactone on hyperthyroxine-induced atrial remodeling. METHODS: New Zealand rabbits were divided into control group (C), hyperthyroxine group (H) and spironolactone group (S). Thyroxin was given to the rabbits in group H and group S by intraperitoneal injection for 4 weeks, and then spironolactone was given in group S by gavage for 2 weeks. Atrial fibrillation (AF) was induced by "burst" stimulation after administration. The inducing rate of AF and atrial effective refractory period (AERP) were tested by intra-cardiac electrophysiologic instrument. The expression of AF-related Ca²⁺ channel (Cav1.2), K⁺ channels (Kv1.5 and Kv4.3) and connexins (Cx40 and Cx43) at mRNA and protein levels was detected by real-time PCR, immunohistochemistry and Western blot. RESULTS: Spironolactone reduced the inducing rate of AF. No significant difference of AERP between group H and group S was observed (CONCLUSION: Spironolactone attenuates the hyperthyroxine-induced atrial remodeling in rabbits, and reduces the susceptibility of the myocardium to AF.     

Cx43 is involved in electrical remodeling of atrial myocytes through regulating L-type calcium current

AIM: To investigate whether the association of connexin 43(Cx43) and L-type calcium channel involved in the pathogenesis of atrial fibrillation (AF). METHODS: The biochemical assays and whole-cell patch-clamp technique were used to study the expression of Cx43 in human atrial tissue. The co-localization of Cx43 and L -type calcium channel, and the regulation of L-type calcium current in atrial myocytes were investigated. RESULTS: The expression of Cx43 at mRNA and protein levels was decreased in human atrial tissues of AF patients. In cultured atrium-derived myocytes (HL-1 cells), knockdown of Cx43 significantly inhibited the mRNA expression of L-type calcium channel α1c subunit, as well as L-type calcium current. Co-localization of Cx43 with L-type calcium channel α1c subunit in mouse atrial myocytes was observed. CONCLUSION: The decrease in Cx43 is involved in the pathogenesis of AF, probably through reducing the L-type calcium current in atrial myoctyes by co-localization with L-type calcium channel, thus representing the potential pathogenesis in atrial fibrillation.     

Effect of high-rate left atrial pacing on atrial remodeling and functions of sinoatrial node and atrioventricular node in dogs

AIM: To determine whether sinoatrial node (SAN) and atrioventricular node (AVN) undergo functional remodeling during atrial fibrillation (AF). METHODS: The Beagle dogs were randomized into pacing group (n=9) and control group (n=6). In open-chest dogs, the electrode catheters were sutured at left atria for pacing and data recording. SAN and AVN conductive properties were studied. The dogs in pacing group underwent 4 weeks of high-rate left atrial pacing (400 min-1). The dogs in control group were not subject to pacing. RESULTS: The animal model of chronic AF was successfully established by pacing the left atrium at 400 min-1 in the dogs. Two of those animals were recorded with spontaneous AF at the end of 2 weeks, and the induction rate of AF reached 100% following 4 weeks of pacing. The incidences of paroxysmal AF and permanent AF were significantly increased by pacing compared to control group. After 4 weeks of pacing, atrial effective refractory periods (AERP) at various at pacing cycle lengths (PCL; 250 ms, 300 ms and 350 ms) were all statistically shorter than those in control group. Compared with control group, a longer AVN Wenckebach point ［(294.44±26.06)min-1 vs (328.33±24.01)min-1,P<0.05］ and longer atrioventricular node effective refractory period (AVERP) (P<0.01) in pacing group were observed. The sinus node recovery time (SNRT) and corrected SNRT both showed significant increases. No significant change of P-wave and PA interval between the two groups was found. The left atrial dimensions (anteroposterior, superoinferior and left-right diameters) and the right atrial superoinferior diameter were measured to be significantly increased after 2 weeks of pacing. CONCLUSION: The animal model of left atrium pacing can induce AF occurrence with a higher incidence. The characteristic electrophysiological indexes about atria, AVN and SAN were observed during AF in the canine model, indicating that electrical and structural remodeling accompanies with AVN and SAN remodeling during AF.     

Effects of serum from patients with atrial fibrillation on chemotaxis of cardiac fibroblasts

AIM: To investigate the effects of the serum from the patients with atrial fibrillation (AF) on the chemotaxis of rat cardiac fibroblasts.METHODS: Cardiac fibroblasts were isolated from the ventricles of neonatal Sprague-Dawley rats and primarily cultured with digestion and differential adhesion. The cells in 3 to 4 passages were used for Transwell chamber assay to determine the chemotatic effects of the sera.RESULTS: Compared with control group, the cells that migrated through the polycarbonate membrane were obviously increased in AF group. The strongest chemotaxis was induced by the serum from the patients with persistent atrial fibrillation(pers-AF).The number of migrated cells in non-AF atrial arrhythmia(AA) group was higher than that in paroxysmal atrial fibrillation(paro-AF) group, and that in control group was the lowest. The results of multiple Logistic regression analysis showed that the migrated cells were related to AF and left atrial diameter.CONCLUSION: The chemotactic effect of AF serum is obviously higher than that of control serum. The differences of AF sera among groups show that myocardial fibrosis is a chronic, insidious and delayed process. The migration and infiltration of cardiac fibroblasts indirectly reflect the presence, severity and extent of the myocardial damage. The changes of migrated cells precede the changes of left atrial diameter, indicating that the cause of fibrosis is more important, and the positive correlation between AF and left atrial diameter may not be the direct causality.     

Expression of MPO,MMP-2 and MMP-9 in atrial myocardium of rabbit atrial fibrillation model

ATM: To investigate the expression of myeloperoxidase (MPO), matrix metalloproteinase (MMP)-2 and MMP-9 in the atrial myocardium, and their potential effects on atrial structural remodeling in a rabbit atrial fibrillation (AF) model. METHODS: The sternotomy was performed and the pacing electrode was fixed to the left atria of 20 New Zealand white rabbits. The animals were randomly divided into 2 groups:rapid atrial pacing (RAP) group and sham group. The rabbits in RAP group were subjected to RAP for 3 weeks. The structure and function of the atria and ventricle were analyzed by echocardiography. Atrial burst stimulation was performed to test AF inducibility. The atrial fibrosis was evaluated by Masson trichrome-staining. The mRNA and protein levels of MPO, MMP-2 and MMP-9 were detected by RT-qPCR and Western blot. RESULTS: After 3 weeks of RAP, obvious left atrial enlargement and dysfunction were observed, but almost no change of left ventricular diameter and function was found in RAP group compared with sham group. AF inducibility, atrial interstitial fibrosis and the mRNA and protein levels of MPO, MMP-2 and MMP-9 were all significantly increased in RAP group compared with sham group. CONCLUSION: Obvious atrial structural remodeling is found in the rabbit AF model induced by sustained RAP, and the up-regulation of MPO, MMP-2 and MMP-9 may be the potential molecular mechanism of atrial structural remodeling.     

Relationship of microRNA-133a and TGF-β1 protein in myocardial tissues of spontaneously hypertensive rats

AIM：To observe the changes of microRNA-133a and transforming growth factor β1 (TGF-β1) protein in the myocardium of spontaneously hypertensive rats (SHR). METHODS：Male SHR (18 weeks old, n=12) and male Wistar-Kyoto rats (WKY, 18 weeks old, n=12) served as SHR group and control group, respectively. Caudal arterial blood pressure was detected by a noninvasive blood pressure measurement and analysis system. Myocardial collagen volume fraction (CVF) and perivascular collagen area ratio (PVCA) were determined by Masson staining. The level of miR-133a in the heart was detected by real-time quantitative PCR. The protein level of TGF-β1 in the heart was also analyzed by the methods of immunohistochemisty and Western blotting. RESULTS：Compared with control group, systolic and diastolic blood pressure, CVF and PVCA significantly increased, the expression of TGF-β1 protein was significantly up-regulated, and the level of miR-133a was significantly reduced in SHR group. In SHR group, the expression of miR-133a was decreased to (23.9±4.6)% in control group. A negative correlation between the levels of miR-133a and TGF-β1 protein in SHR group was observed (r=-0.791, P<0.01). CONCLUSION：The level of miR-133a is down-regulated along with the up-regulation of TGF-β1 protein expression and collagen synthesis in the myocardial tissues of SHR. miR-133a and TGF-β1 may be involved in myocardial fibrosis in SHR.     

Role of hepatitis B virus in intrahepatic TGF-β1/Smads signaling pathway

AIM:To explore the effects of hepatitis B virus (HBV) on intrahepatic expression of transforming growth factor β1（TGF-β1） and Smads. METHODS:The expression of intrahepatic TGF-β1, HBsAg and HBcAg in control group and chronic hepatitis B (CHB) group was detected by immunohistochemical method.The serum HBV DNA content was determined by real-time PCR. The role of HBV in the expression of TGF-β1, Smad3 and Smad7 in human hepatic stellate cell line LX-2 in vitro was observed by cell culture and Western blotting. RESULTS:The average score of intrahepatic TGF-β1 expression in CHB group was higher than that in control group. With the increase in serum HBV DNA content, intrahepatic TGF-β1 expression was also enhanced. In the HBcAg positive hepatic tissue, there was higher TGF-β1 expression than that in the liver tissue of HBcAg negative. Compared with control group and HBV+anti-TGF-β1 group, HBV caused increased expression of TGF-β1 and Smad3 in HBV group in vitro. No difference of Smad7 protein among control group, HBV group and HBV+anti-TGF-β1 group was observed. CONCLUSION: The expression of intrahepatic TGF-β1 is related to serum HBV DNA and hepatocellular HBcAg in the patients with CHB. HBV-induced liver fibrosis mainly relies on positive regulatory mechanisms of Smad3,and the negative regulation by Smad7 almost does not function.     

Effects of long-term cigarette smoke exposure on pulmonary vascular remodeling and TGF-β1 expression in rat pulmonary vessels

AIM: To observe the effects of long-term cigarette smoke exposure on pulmonary vascular remodeling and the protein expression of transforming growth factor-β1(TGF-β1) in the rats, and to explore the mechanism.METHODS: SD rats(n=36) were randomly divided into control group, 2-week smoke exposure(S-2W) group and 12-week smoke exposure(S-12W) groups. HE staining and α-smooth muscle actin staining were performed to observe the pulmonary vascular remodeling.The protein expression of proliferating cell nuclear antigen(PCNA) and TGF-β1 in the pulmonary arteries was determined by the method of immunohistochemistry. The mRNA expression of TGF-β1 in the pulmonary arteries was evaluated by RT-qPCR.RESULTS: Compared with control group, ratio of pulmonary vessel wall thickness to vessel diameter(WT%) and percentage of muscularized vessels were significantly increased in S-2W group and S-12W group(P<0.01). Significant increases in the protein expression of PCNA and TGF-β1 in smoke exposure groups were observed compared with control group. There was significant difference between 2 model groups(P<0.01). Compared with control group, the mRNA expression of TGF-β1 in pulmonary artery walls obviously increased in smoke exposure groups. There was significantly difference between S-2W and S-12W groups(P<0.05). The mRNA expression of TGF-β1 was positively correlated with pulmonary vascular muscularization, WT% and the protein expression of PCNA. CONCLUSION: Long-term cigarette smoke exposure results in pulmonary artery remodeling in rats. The possible mechanism is that cigarette smoking exposure induces the over-expression of TGF-β1 at mRNA level in pulmonary vessels and promotes the proliferation of pulmonary vascular smooth muscle cells in rats.     

Effect of c-SKI on coronary endothelial cell proliferation and endothelial-mesenchymal transition

AIM: To investigate the effect of cellular Sloan-Kettering Institute (c-SKI) on the proliferation and endothelial-mesenchymal transition of human coronary artery endothelial cells (HCAECs). METHODS: HCAECs were treated with transforming growth factor-β1 (TGF-β1) at varying concentrations for different time points. Western blot was used to test the expression of c-SKI and mesenchymal markers such as α-smooth muscle actin (α-SMA) and vimentin. Meanwhile, the endothelial marker E-cadherin was also detected. HCAECs were transfected with c-ski gene mediated by lentivirus (LV), the efficiency of LV-SKI transfection was detected by RT-qPCR. The HCAECs were divided into 4 groups:control group, TGF-β1 (5 μg/L) group, LV-SKI+ TGF-β1 group, LV-NC+ TGF-β1 group. The cell viability and colony formation were measured by MTT assay and colony formation assay. The protein levels of vimentin, α-SMA, E-cadherin, Smad2, Smad3, p-Smad2 and p-Smad3 were determined by Western blot. RESULTS: The expression of c-SKI was down-regulated in the HCAECs treated with TGF-β1 (P<0.01). Over-expression of c-SKI inhibited the proliferation of HCAECs (P<0.01). Compared with LV-NC group, over-expression of c-SKI down-regulated the expression of α-SMA and vimentin (P<0.01), up-regulated the expression of E-cadherin (P<0.01), and inhibited the protein phosphorylation of Smad2 and Smad3 (P<0.01), reversed the endothelial-mesenchymal transition induced by TGF-β1. CONCLUSION: The expression of c-SKI in the HCAECs is down-regulated in the process of endothelial-mesenchymal transition. Over-expression of c-SKI inhibits proliferation and endothelial-mesenchymal transition of HCAECs, the mechanism may be related to regulation of the TGF-β1/Smad signaling pathway.     

Knockdown of HMGA2 gene inhibits TGF-β1-induced human embryonic lung fibroblast growth and collagen synthesis

AIM: To investigate the effects of high mobility group A2(HMGA2) gene knockdown on the cell viability, apoptosis, collagen synthesis and oxidative stress of human embryonic lung fibroblast (HELF) induced by transforming growth factor-β1 (TGF-β1). METHODS: The HELF were divided into blank group, TGF-β1 group,negative control (NC) group and HMGA2 siRNA(si-HMGA2) group. The protein levels of HMGA2, AKT and p-AKT were determined by Western blot. The cell viability and apoptotic rate was analyzed by MTT assay and flow cytometry,respectively. The mRNA expression of collagen I (COL-Ⅰ) and COL-Ⅲ was detected by RT-qPCR. DCFH-DA was used to detect the content of reactive oxygen species (ROS). RESULTS: Compared with blank group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in TGF-β1 group were significantly increased, but the apoptotic rate and ROS level were significantly decreased (P<0.05). Compared with TGF-β1 group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in si-HMGA2 group were significantly decreased, but the apoptotic rate and ROS level were significantly increased (P<0.05). CONCLUSION: Knockdown of HMGA2 gene expression decreases the viability and collagen synthesis, and promotes apoptosis and ROS production of human embryonic lung fibroblasts induced by TGF-β1. The mechanism may be related to down-regulation of PI3K/AKT signaling pathway.     

Expression of Sonic hedgehog pathway-related genes in kidney tissues of rats with unilateral ureteral obstruction

AIM: To investigate the role of Sonic hedgehog (Shh) signaling pathway in renal interstitial fibrosis in the rats with unilateral ureteral obstruction (UUO). METHODS: Forty-eight male Sprague-Dawley rats were divided randomly into sham operation group and UUO model group with 24 rats each. The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fiber in the kidneys was detected with HE and Masson staining. Immunohistochemical analysis was performed to evaluate the expression of Shh signaling pathway-related proteins, including Shh, Smo,Ptch1 and Gli1. The contents of TGF-β₁ and Shh in the kidney tissues were determined by ELISA. Real-time RT-PCR was used to detect the mRNA expression of TGF-β₁, Col I, Col III and Shh signaling-related genes.RESULTS: Fibrosis observed with HE and Masson staining was obviously increased in UUO kidneys, and aggravated as time prolonged. The contents of TGF-β₁, Col I and Col III were also increased. In addition, the expression of Shh, Smo and Gli1 was markedly increased in obstructive kidneys, and the expression of Ptch1 was decreased (P<0.01), suggesting that Shh signaling was activated. The level of Shh in UUO rats was associated with the content of TGF-β₁. CONCLUSION: Shh signaling is activated in the progress of renal interstitial fibrosis in UUO rats, and the possible mechanism triggering the fibrogenic response is that Shh signaling promotes the expression of TGF-β₁.     

Effect of chronic atrial fibrillation on Ca2+/calmodulin dependent protein kinase Ⅱ expression in human atrial myocytes

AIM: To investigate the effect of chronic atrial fibrillation (AF) on free calcium concentration and expression of Ca2+/calmodulin dependent protein kinase Ⅱ (CaMKⅡ) in human atrial myocytes. METHODS: The intracellular free calcium concentration in acute isolated atrial myocytes and the expression of CaMKⅡ in atrial tissue of rheumatic heart disease patients with atrial fibrillation (AF) and with normal sinus rhythm were measured by laser scanning cofocal microscopy technique and Western blotting, respectively. RESULTS: The intracellular Ca2+ concentration in patients with atrial fibrillation was significantly higher than that in patients with normal sinus rhythm ［(276.38±38.12） nmol/L vs （122.28±45.63) nmol/L, P<0.05］. Western blotting analysis of atrial samples showed that CaMKⅡ expression was enhanced during chronic atrial fibrillation (10.14±0.31 vs 6.86±0.89，P<0.05). CONCLUSION: Chronic AF leads to intracellular calcium overload in human atrial myocytes. Ca2+/calmodulin dependent protein kinase signaling cascades may play an important role in maintenance of chronic AF.