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.     

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.     

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.     

An approach to calcium signal transmission between L-type calcium channel and sarcoplasmic reticulum of atrial myocytes during atrial fibrillation

AIM: To inquire into the Ca2+ signal transmission from L-type calcium channel to the sarcoplasma reticulum in atrial fibrillation. METHODS: Ten adult cross-bred dogs were used in the experiment. Five dogs underwent continuous rapid atrial pacing (500±20 beats/min) for twenty-four weeks to create persistent atrial fibrillation. Another group of size-matched dogs (n=5) without pacemaker implantation was used as a control group. Canine atrial myocytes were isolated by enzymatic dissociation. The Ca2+ cytosolic transient in atrial myocytes was analyzed by confocal imaging after loading myocytes with the acetoxymethyl ester of fluo-3 (Fluo-3/AM). Ca2+ signal transmission from L-type Ca2+ channels in the plasma membrane to the sarcoplasma reticular IP3R and RyR in atrial myocytes during atria fibrillation were measured. RESULTS: (1) Ca2+ signal transmission from L-type calcium channel to IP3R in the sarcoplasma reticulum:intracellular Ca2+concentration was slightly increased in two groups after blocking T-type calcium channel and RyR, but showed no statistic significance (P＞0.05) between them. (2) Ca2+ signal transmission from L-type calcium channel to RyR in the sarcoplasma reticulum: intracellular Ca2+concentration was risen (1.5576±0.1989) in control groups after blocking T-type calcium channel and IP3R, and no significance was observed (P＞0.05) compared with that in atrial fibrillation group (1.5372±0.2952). CONCLUSIONS: Ca2+ signal transmission possibly exists from L-type calcium channel to RyR and IP3R in the sarcoplasma reticulum, but it does not play an important role in intracellular Ca2+-overload and abnormal Ca2+ signal transmission during atrial fibrillation.     

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.     

Resveratrol reduces electrical remodeling in atrial fibrillation by down-regulating microRNA-21 in neonatal rat atrial myocytes

AIM: To detect the effects of resveratrol (RSV) on the expression of microRNA-21 (miR-21) in primarily cultured neonatal rat atrial myocytes with electric remodeling induced by rapid electrical stimulation (RES). Furthermore, to find out the possible mechanism of miR-21 regulating electrical remodeling. METHODS: The neonatal rat atrial myocytes were isolated by double-enzyme (trypsin and collagenase I) digestion and differential adhesion method. The atrial fibrillation (AF) model was induced by RES. Atrial myocytes were randomly divided into 4 groups:control group, RSV group, RES group, and RSV+RES group. To further detect whether RSV regulated electric remodeling by miR-21, except the 4 groups, we add miR-21 over-expression group and miR-21 inhibitor group:RES+negative control (NC) group, RES+miR-21 mimics group, RES+miR-21 mimics+RSV group, RES+miR-21 inhibitor group, and RES+miR-21 inhibitor+RSV group. The optimal concentration and pretreatment time of resveratrol were determined by CCK-8 assay. The expression of miR-21 and the mRNA expression of L-type calcium channels CACNA1C and CACNB2 in atrial myocytes were detected by qPCR. The protein expression of L-type calcium channels Cav1.2 and Cavβ₂ in the atrial myocytes was analyzed by Western blot. RESULTS: The expression of miR-21 in RES group was significantly increased compared with control group, while preconditioning with RSV decreased the expression of miR-21. Compared with RES+miR-21 mimics group, the expression of miR-21 in RES+miR-21 mimics+RSV group was significantly decreased. Meanwhile, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ₂ were increased (P<0.05). Compared with RES group, the expression of miR-21 in RES+miR-21 inhibitor group and RES+miR-21 inhibitor+RSV group was decreased, while the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ₂ were increased. However, no difference of the expression of miR-21, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ₂ among RSV+RES, RES+miR-21 inhibitor and RES+miR-21 inhibitor+RSV groups was observed (P<0.05).CONCLUSION: In AF model induced by RES, RSV may reduce electric remodeling by inhibiting the expression of miR-21 and regulating the downstream target genes.     

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.     

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.     

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.     

Over-expressed calreticulin interacts with integrin-α5 and calcineurin system to induce atrial remodeling in patients with atrial fibrillation and valvular disease

AIM: To determine whether calreticulin over-expression contributes to atrial fibrosis in the patients with atrial fibrillation (AF) and valvular heart disease (VHD).METHODS: Right and left atrial specimens were obtained from 78 patients undergoing valve replacement surgery. The patients were divided into sinus rhythm (SR) group, paroxysmal AF (PaAF) group and persistent AF (PeAF, AF lasting >6 months) group. The protein expression of calreticulin, integrin-α5, and transforming growth factor-β1 (TGF-β1) was measured. Immunoprecipitation was also performed to determine whether calreticulin interacted with either calcineurin B or integrin-α5. RESULTS: The protein expression of calreticulin, integrin-α5 and TGF-β1 was increased in AF groups, especially in the left atrium of the patients with mitral valve disease as compared with SR group. Calreticulin interacted with both calcineurin B and integrin-α5. The expression level of integrin-α5 was significantly correlated with the expression level of TGF-β1, while the expression level of calreticulin was correlated with that of integrin-α5 and TGF-β1. Under similar classification of the cardiac function, the expression level of calreticulin in PeAF group was higher than that in SR group. CONCLUSION: The expression of calreticulin, integrin-α5, and TGF-β1 is increased in the atrial tissues of the AF patients and is related to the AF type, suggesting that calreticulin is involved in atrial remodeling in AF and VHD patients.     

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.     

Recent advance in cardiac ion channel remodeling in diabetes

Occurrence of lethal cardiac arrhythmia, which is associated with prolongation of the QT interval in electrocardiogram(ECG), is related to increased mortality in the patients with diabetes mellitus (DM). Increased QT interval reflects the prolonged cardiac action potential duration (APD). APD abnormality is based on by cardiac ion channel remodeling induced by DM-related pathological changes. As DM is becoming an important risk factor for atrial fibrillation, the most clinically common arrhythmia, this review mainly summarizes the alterations of ion channel currents and relevant protein expression in diabetic ventricular myocytes to explore the underlying mechanisms of arrhythmia, and also discusses some research achievements from investigating the atrial myocytes in DM.     

The altered transient outward (Ito1) and ultra-rapid delayed rectifier (IKur) K+ currents in right atrial myocytes of human atrial fibrillation

AIM: To study and evaluate the changes of two main kinds of voltage-gated K+ currents in human atrial fibrillation (AF) and to discuss the role of these changes in the atrial electrical remodeling (AER) caused by AF. METHODS: Specimens of human atrial appendage were obtained from 36 RHD patients (18 with chronic AF and 18 without AF). Single atrial myocytes were acutely dissociated by tissue chunk enzymatic digestion. Ito1 and IKur in the two groups were measured respectively with the patch-clamp technique in a whole-cell configuration and the I-V curves were compared. RESULTS: Ito1 and IKur amplitudes in AF groups were significantly reduced and the current densities of both Ito1 and IKur in AF patients were lower than those in NAF patients. CONCLUSION: The reduction of Ito1 and IKur may be related to changes in atrial conduction, refractory period and may constitute two main parts of the major mechanisms in the AER of chronic AF. Whether exists a relation between changes of the above K+ currents and that of other ionic currents and the AF initiation and perpetuation deserves further investigation.     

Mitochondrial reactive oxygen species and atrial fibrillation

Atrial fibrillation (AF) is the most common arrhythmia in clinical practice. Mitochondrial oxidative stress is supposed to contribute to development, progression and self-perpetuation of AF. Reactive oxygen species (ROS) is the major molecule mediating mitochondrial oxidative stress damage. ROS can alter the redox status of various molecular targets, which quite specifically leads to functional alterations of ion channel activity or activation of a variety of redox sensitive signal transduction pathways. Eventually, it leads to atrial electrical remodeling and promotes the development of AF. Therefore, mitochondrial oxidative stress pathways may be a new target for the therapy of atrial fibrillation.     

Effect of autophagy on L-type calcium channel current induced by gp120V3 in hippocampus neurons

AIM: To observe the influence of autophagy on L-type calcium channel current induced by gp120V3 loop in hippocampal neurons.METHODS: Hippocampal neurons were exteriorized from the newborn rats within 1 d and primarily cultured for 7 d. The neurons were divided into 2 parallel groups, namely control group, gp120V3 group, autophagy inhibitor 3-MA group, gp120V3 + 3-MA group; control group, gp120V3 group, autophagy activator rapamycin group, gp120V3 + rapamycin group. Whole-cell pathch-clamp was used to record L-type calcium channel current. RESULTS: Compared with control group, the density of L-type calcium channel current increased in gp120V3 group and 3-MA group(P<0.05). Compared with gp120V3 group, the density of L-type calcium channel current increased in gp120V3 + 3-MA group(P<0.05). Compared with control group, the density of L-type calcium channel current decreased in rapamycin group(P<0.05). Compared with gp120V3 group, the density of L-type calcium channel current increased in gp120V3 + rapamycin group(P<0.05).CONCLUSION: Autophagy may be involved in the gp120V3 loop-mediated L-type calcium channels in hippocampus neurons.     

Effects of adipose tissue-derived mesenchymal stem cells on calcium channels of pulmonary artery in rats with pulmonary arterial hypertension induced by monocrotaline

AIM: To investigate the effects of adipose tissue-derived mesenchymal stem cells (ADMSCs) on calcium channels of pulmonary artery in monocrotaline (MCT)-induced pulmonary hypertensive rats.METHODS: ADMSCs were isolated from adipose tissue by collagenase digestion. Twenty-four Sprague-Dawley rats were randomly divided into 3 groups: normal control (Ctr) group, pulmonary arterial hypertension (PAH) group and ADMSCs transplantation group. Mean pulmonary arterial pressure (MPAP) was measured by catheterization, and right ventricular hypertrophy index (RVHI) was calculated. The expression of voltage-gated calcium channel α1c subunit (CaVα1c), sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA-2a), inositol 1,4,5-triphosphate receptor 1(IP3R-1), transient receptor potential channel 1 (TRPC1) and TRPC6 at mRNA and protein levels in the pulmonary trunks was determined by RT-PCR and Western blotting, respectively.RESULTS: MPAP and RVHI were higher in PAH group than those in Ctr group, while those in ADMSCs group were significantly decreased as compared with PAH group. The expression of CaVα1c, TRPC1 and TRPC6 at mRNA and protein levels was obviously increased in PAH group as compared with Ctr group, while that in ADMSCs group was significantly decreased as compared with PAH group. Compared with Ctr group, the expression of SERCA-2a and IP3R-1 at mRNA and protein levels was obviously decreased in PAH group, while that in ADMSCs group was significantly increased as compared with PAH group.CONCLUSION: MPAP and RVHI are attenuated by ADMSCs in MCT-induced pulmonary hypertensive rats. The reduction of pulmonary arterial pressure by ADMSCs transplantation in MCT-induced pulmonary hypertensive rats may be related to the changes of calcium channels.     

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.