Xanthine oxidase inhibitor febuxostat reduces atrial fibrillation susceptibility by inhibition of oxidized CaMKII in Dahl salt-sensitive rats.

Oxidative stress could be a possible mechanism and a therapeutic target of atrial fibrillation (AF). However, the effects of the xanthine oxidase (XO) inhibition for AF remain to be fully elucidated. We investigated the effects of a novel XO inhibitor febuxostat on AF compared with allopurinol in hypertension rat model. Five-week-old Dahl salt-sensitive rats were fed either low-salt (LS) (0.3% NaCl) or high-salt (HS) (8% NaCl) diet. After 4 weeks of diet, HS diet rats were divided into three groups: orally administered to vehicle (HS-C), febuxostat (5 mg/kg/day) (HS-F), or allopurinol (50 mg/kg/day) (HS-A). After 4 weeks of treatment, systolic blood pressure (SBP) was significantly higher in HS-C than LS, and it was slightly but significantly decreased by treatment with each XO inhibitor. AF duration was significantly prolonged in HS-C compared with LS, and significantly suppressed in both HS-F and HS-A (LS; 5.8 ± 3.5 s, HS-C; 33.9 ± 23.7 s, HS-F; 15.0 ± 14.1 s, HS-A; 20.1 ± 11.9 s: P<0.05). Ca2+ spark frequency was obviously increased in HS-C rats and reduced in the XO inhibitor-treated rats, especially in HS-F group. Western blotting revealed that the atrial expression levels of Met281/282-oxidized Ca2+/Calmodulin-dependent kinase II (CaMKII) and Ser2814-phosphorylated ryanodine receptor 2 were significantly increased in HS-C, and those were suppressed in HS-F and HS-A. Decreased expression of gap junction protein connexin 40 in HS-C was partially restored by treatment with each XO inhibitor. In conclusion, XO inhibitor febuxostat, as well as allopurinol, could reduce hypertension-related increase in AF perpetuation by restoring Ca2+ handling and gap junction.

Inducibility, but not stability, of atrial fibrillation is increased by NOX2 overexpression in mice.

AIMS: Gp91-containing NADPH oxidases (NOX2) are a significant source of myocardial superoxide production. An increase in NOX2 activity accompanies atrial fibrillation (AF) induction and electrical remodelling in animal models and predicts incident AF in humans; however, a direct causal role for NOX2 in AF has not been demonstrated. Accordingly, we investigated whether myocardial NOX2 overexpression in mice (NOX2-Tg) is sufficient to generate a favourable substrate for AF and further assessed the effects of atorvastatin, an inhibitor of NOX2, on atrial superoxide production and AF susceptibility. METHODS AND RESULTS: NOX2-Tg mice showed a 2- to 2.5-fold higher atrial protein content of NOX2 compared with wild-type (WT) controls, which was associated with a significant (twofold) increase in NADPH-stimulated superoxide production (2-hydroxyethidium by HPLC) in left and right atrial tissue homogenates (P = 0.004 and P = 0.019, respectively). AF susceptibility assessed in vivo by transoesophageal atrial burst stimulation was modestly increased in NOX2-Tg compared with WT (probability of AF induction: 88% vs. 69%, respectively; P = 0.037), in the absence of significant alterations in AF duration, surface ECG parameters, and LV mass or function. Mechanistic studies did not support a role for NOX2 in promoting electrical or structural remodelling, as high-resolution optical mapping of atrial tissues showed no differences in action potential duration and conduction velocity between genotypes. In addition, we did not observe any genotype difference in markers of fibrosis and inflammation, including atrial collagen content and Col1a1, Il-1ß, Il-6, and Mcp-1 mRNA. Similarly, NOX2 overexpression did not have consistent effects on RyR2 Ca2+ leak nor did it affect PKA or CaMKII-mediated RyR2 phosphorylation. Finally, treatment with atorvastatin significantly inhibited atrial superoxide production in NOX2-Tg but had no effect on AF induction in either genotype. CONCLUSION: Together, these data indicate that while atrial NOX2 overexpression may contribute to atrial arrhythmogenesis, NOX2-derived superoxide production does not affect the electrical and structural properties of the atrial myocardium.

Ibrutinib-Mediated Atrial Fibrillation Attributable to Inhibition of C-Terminal Src Kinase.

BACKGROUND: Ibrutinib is a Bruton tyrosine kinase inhibitor with remarkable efficacy against B-cell cancers. Ibrutinib also increases the risk of atrial fibrillation (AF), which remains poorly understood. METHODS: We performed electrophysiology studies on mice treated with ibrutinib to assess inducibility of AF. Chemoproteomic analysis of cardiac lysates identified candidate ibrutinib targets, which were further evaluated in genetic mouse models and additional pharmacological experiments. The pharmacovigilance database, VigiBase, was queried to determine whether drug inhibition of an identified candidate kinase was associated with increased reporting of AF. RESULTS: We demonstrate that treatment of mice with ibrutinib for 4 weeks results in inducible AF, left atrial enlargement, myocardial fibrosis, and inflammation. This effect was reproduced in mice lacking Bruton tyrosine kinase, but not in mice treated with 4 weeks of acalabrutinib, a more specific Bruton tyrosine kinase inhibitor, demonstrating that AF is an off-target side effect. Chemoproteomic profiling identified a short list of candidate kinases that was narrowed by additional experimentation leaving CSK (C-terminal Src kinase) as the strongest candidate for ibrutinib-induced AF. Cardiac-specific Csk knockout in mice led to increased AF, left atrial enlargement, fibrosis, and inflammation, phenocopying ibrutinib treatment. Disproportionality analyses in VigiBase confirmed increased reporting of AF associated with kinase inhibitors blocking Csk versus non-Csk inhibitors, with a reporting odds ratio of 8.0 (95% CI, 7.3-8.7; P<0.0001). CONCLUSIONS: These data identify Csk inhibition as the mechanism through which ibrutinib leads to AF. Registration: URL: https://ww.clinicaltrials.gov; Unique identifier: NCT03530215.

Atrial Myocyte NLRP3/CaMKII Nexus Forms a Substrate for Postoperative Atrial Fibrillation.

RATIONALE: Postoperative atrial fibrillation (POAF) is a common and troublesome complication of cardiac surgery. POAF is generally believed to occur when postoperative triggers act on a preexisting vulnerable substrate, but the underlying cellular and molecular mechanisms are largely unknown. OBJECTIVE: To identify cellular POAF mechanisms in right atrial samples from patients without a history of atrial fibrillation undergoing open-heart surgery. METHODS AND RESULTS: Multicellular action potentials, membrane ion-currents (perforated patch-clamp), or simultaneous membrane-current (ruptured patch-clamp) and [Ca2+]i-recordings in atrial cardiomyocytes, along with protein-expression levels in tissue homogenates or cardiomyocytes, were assessed in 265 atrial samples from patients without or with POAF. No indices of electrical, profibrotic, or connexin remodeling were noted in POAF, but Ca2+-transient amplitude was smaller, although spontaneous sarcoplasmic reticulum (SR) Ca2+-release events and L-type Ca2+-current alternans occurred more frequently. CaMKII (Ca2+/calmodulin-dependent protein kinase-II) protein-expression, CaMKII-dependent phosphorylation of the cardiac RyR2 (ryanodine-receptor channel type-2), and RyR2 single-channel open-probability were significantly increased in POAF. SR Ca2+-content was unchanged in POAF despite greater SR Ca2+-leak, with a trend towards increased SR Ca2+-ATPase activity. Patients with POAF also showed stronger expression of activated components of the NLRP3 (NACHT, LRR, and PYD domains-containing protein-3)-inflammasome system in atrial whole-tissue homogenates and cardiomyocytes. Acute application of interleukin-1ß caused NLRP3-signaling activation and CaMKII-dependent RyR2/phospholamban hyperphosphorylation in an immortalized mouse atrial cardiomyocyte cell-line (HL-1-cardiomyocytes) and enhanced spontaneous SR Ca2+-release events in both POAF cardiomyocytes and HL-1-cardiomyocytes. Computational modeling showed that RyR2 dysfunction and increased SR Ca2+-uptake are sufficient to reproduce the Ca2+-handling phenotype and indicated an increased risk of proarrhythmic delayed afterdepolarizations in POAF subjects in response to interleukin-1ß. CONCLUSIONS: Preexisting Ca2+-handling abnormalities and activation of NLRP3-inflammasome/CaMKII signaling are evident in atrial cardiomyocytes from patients who subsequently develop POAF. These molecular substrates sensitize cardiomyocytes to spontaneous Ca2+-releases and arrhythmogenic afterdepolarizations, particularly upon exposure to inflammatory mediators. Our data reveal a potential cellular and molecular substrate for this important clinical problem.

Attenuation of Oxidative Injury With Targeted Expression of NADPH Oxidase 2 Short Hairpin RNA Prevents Onset and Maintenance of Electrical Remodeling in the Canine Atrium: A Novel Gene Therapy Approach to Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common heart rhythm disorder in adults and a major cause of stroke. Unfortunately, current treatments of AF are suboptimal because they are not targeted to the molecular mechanisms underlying AF. Using a highly novel gene therapy approach in a canine, rapid atrial pacing model of AF, we demonstrate that NADPH oxidase 2 (NOX2) generated oxidative injury causes upregulation of a constitutively active form of acetylcholine-dependent K+ current (IKACh), called IKH; this is an important mechanism underlying not only the genesis, but also the perpetuation of electric remodeling in the intact, fibrillating atrium. METHODS: To understand the mechanism by which oxidative injury promotes the genesis and maintenance of AF, we performed targeted injection of NOX2 short hairpin RNA (followed by electroporation to facilitate gene delivery) in atria of healthy dogs followed by rapid atrial pacing. We used in vivo high-density electric mapping, isolation of atrial myocytes, whole-cell patch clamping, in vitro tachypacing of atrial myocytes, lucigenin chemiluminescence assay, immunoblotting, real-time polymerase chain reaction, immunohistochemistry, and Masson trichrome staining. RESULTS: First, we demonstrate that generation of oxidative injury in atrial myocytes is a frequency-dependent process, with rapid pacing in canine atrial myocytes inducing oxidative injury through the induction of NOX2 and the generation of mitochondrial reactive oxygen species. We show that oxidative injury likely contributes to electric remodeling in AF by upregulating IKACh by a mechanism involving frequency-dependent activation of PKCε (protein kinase C epsilon). The time to onset of nonsustained AF increased by >5-fold in NOX2 short hairpin RNA-treated dogs. Furthermore, animals treated with NOX2 short hairpin RNA did not develop sustained AF for up to 12 weeks. The electrophysiological mechanism underlying AF prevention was prolongation of atrial effective refractory periods, at least in part attributable to the attenuation of IKACh. Attenuated membrane translocation of PKCε appeared to be a likely molecular mechanism underlying this beneficial electrophysiological remodeling. CONCLUSIONS: NOX2 oxidative injury (1) underlies the onset, and the maintenance of electric remodeling in AF, as well, and (2) can be successfully prevented with a novel, gene-based approach. Future optimization of this approach may lead to a novel, mechanism-guided therapy for AF.

Metformin improves lipid metabolism and reverses the Warburg effect in a canine model of chronic atrial fibrillation.

BACKGROUND: Previous studies demonstrated impaired lipid metabolism and augmented aerobic glycolysis in AF. The authors aimed to investigate whether the use of metformin, an AMPK activator, could reverse this metabolic remodeling in chronic AF and to explore the underlying mechanisms. METHODS: We conducted chronic AF animal models with 18 beagle dogs and divided them into SR (pacemaker implanted without pacing), AF (pacemaker implanted with sustained pacing at a frequency of 400 beats/min for 6 weeks), and metformin+AF group (daily oral administration of metformin was initiated 1 week before surgery and continued throughout the study period). After electrophysiological measurements, the left atrial appendage tissue samples were taken from the beating heart for further analysis. Protein expression, histological analysis, and biochemical measurements were conducted. RESULTS: The AF groups showed decreased expression of FAT/CD36, CPT-1, VLCAD, increased concentration of free fatty acid and triglyceride, and increased lipid deposition. The activation of AMPK/PGC-1α/PPARα pathway was decreased. The key factors of the Warburg effect, including HIF-1α, GLUT-1, PDK1, HK, and LDH, increased in AF group compared to SR group. The expression of PDH decreased significantly, accompanied by increased atrial lactate production. The extent of fibrosis increased significantly in the left atrial appendage of AF group. dERP, ∑WOV, and AF inducibility increased while ERP decreased in AF group compared to SR group. The use of metformin attenuated all these changes effectively. CONCLUSIONS: Metformin improves lipid metabolism and reverses the Warburg effect in chronic AF via AMPK activation. It attenuates atrial electrical and structural remodeling.

Expression of Rho Kinase and Its Mechanism in the Left Atrial Appendage in Patients with Atrial Fibrillation.

AIM: To study the expression of Rho kinase (Rho associated coil forming protein kinase-1, ROCK-1) and its substrate myosin phosphatase target subunit 1 (myosin phosphatase target subunit-1, MYPT-1), connexin 40 (Cx40) and connexin 43 (Cx43) in the left atrial appendage of patients with atrial fibrillation, and explore the role of ROCK signaling pathway in patients with atrial fibrillation and its underlying mechanism. Methods: 40 patients undergoing open heart surgery were divided into two groups; atrial fibrillation group (AF group) and sinus rhythm group (SR group). About 100 mg of left atrial appendage tissue was taken during surgery and quickly frozen in liquid nitrogen. Immunohistochemistry and western blot were performed to evaluate the expression and location of ROCK-1, MYPT-1, Cx40 and Cx43 in the left atrial appendage tissue. Results: The results indicated that the expression of ROCK-1, MYPT-1, and Cx40 in the left atrial appendage in patients with atrial fibrillation was significantly upregulated (P < .01), the difference in the two groups was statistically significant, and ROCK-1, Cx40, and MYPT-1 expression in the AF group were higher than those in sinus rhythm group; there was a weakly positive expression of Cx43 protein in the AF group and sinus rhythm group, the difference was not statistically significant, and ROCK-1 and MYPT-1 expression showed a significant positive correlation (r = 0.968, P < .05), MYPT 1 and Cx40 protein expression was also positively correlated (r = 0.983, P < .05). Evidence in the left atrial appendage tissue of patients with atrial fibrillation showed that some proteins in Rho/ROCK pathway were upregulated, and MYPT-1 and Cx40 protein expression in AF group were significantly higher than that of SR group, which was also positively correlated; Cx43 showed a weak positive expression in both the SR group and AF group, which indicates that Rho kinase may induce expression of Cx40 by phosphorylation of MYPT-1; Cx43 may not be involved, suggesting that Rho kinase signaling pathway may activate and play an important role in the pathogenesis of atrial fibrillation lesions.

Impact of Renal Denervation on Atrial Arrhythmogenic Substrate in Ischemic Model of Heart Failure.

BACKGROUND: Myocardial infarction increases the risk of heart failure (HF) and atrial fibrillation. Renal denervation (RDN) might suppress the development of atrial remodeling. This study aimed to elucidate the molecular mechanism of RDN in the suppression of atrial fibrillation in a HF model after myocardial infarction. METHODS AND RESULTS: HF rabbits were created 4 weeks after coronary ligation. Rabbits were classified into 3 groups: normal control (n=10), HF (n=10), and HF-RDN (n=6). Surgical and chemical RDN were approached through midabdominal incisions in HF-RDN. Left anterior descending coronary artery in HF and HF-RDN was ligated to create myocardial infarction. After electrophysiological study, the rabbits were euthanized and the left atrial appendage was harvested for real-time polymerase chain reaction analysis and Trichrome stain. Left atrial dimension and left ventricular mass were smaller in HF-RDN by echocardiography compared with HF. Attenuated atrial fibrosis and tyrosine hydroxylase levels were observed in HF-RDN compared with HF. The mRNA expressions of Cav1.2, Nav1.5, Kir2.1, KvLQT1, phosphoinositide 3-kinase, AKT, and endothelial nitric oxide synthase in HF-RDN were significantly higher compared with HF. The effective refractory period and action potential duration of HF-RDN were significantly shorter compared with HF. Decreased atrial fibrillation inducibility was noted in HF-RDN compared with HF (50% versus 100%, P<0.05). CONCLUSIONS: RDN reversed atrial electrical and structural remodeling, and suppressed the atrial fibrillation inducibility in an ischemic HF model. The beneficial effect of RDN may be related to prevention of the downregulation of the phosphoinositide 3-kinase/AKT/endothelial nitric oxide synthase signaling pathway.

DNA methylation dysregulations in valvular atrial fibrillation.

BACKGROUND: The epigenetic changes underlying the development of atrial fibrillation (AF) remain incompletely understood. Limited evidence suggests that abnormal DNA methylation might be involved in the pathogenesis of AF. In the present study, we evaluated the methylation status of genomic DNA from myocardial tissue in AF patients and sinus rhythm (SR) patients systematically. HYPOTHESIS: DNA methylation dysregulations will be associated with valvular AF. METHODS: Right atrial myocardial tissue was obtained from rheumatic valvular patients who had undergone valve replacement surgery (SR group, n = 10; AF group, n = 10). The global DNA methylation level, the promoter methylation level of the natriuretic peptide receptor-A gene (NPRA), and its correlation with the mRNA expression level of DNA methyltransferase genes were detected. RESULTS: The global DNA methylation level was significantly higher in the AF group than in the SR group (P < 0.05). The NPRA mRNA expression was decreased and the NPRA gene was hypermethylated in the AF group (P < 0.05). Meanwhile, the NPRA mRNA expression level has a negative correlation with the mean methylation level in the promoter region of the NPRA gene. CONCLUSIONS: DNA methylation dysregulations may be relevant in the pathogenesis of AF. DNA methyltransferase 3B likely plays an essential role in the DNA methylation dysregulations in AF.

Prevention of Atrial Fibrillation by Using Sarcoplasmic Reticulum Calcium ATPase Pump Overexpression in a Rabbit Model of Rapid Atrial Pacing.

BACKGROUND Recent research suggests that abnormal Ca2+ handling plays a role in the occurrence and maintenance of atrial fibrillation (AF). Therefore, Ca2+ release and ingestion depend on properties of the ryanodine receptor (RyR) and sarcoplasmic reticulum Ca2+ATPase2a (SERCA2a). This study aimed to detect whether SERCA2a gene overexpression has a preventive effect on atrial fibrillation caused by rapid pacing right atrium. MATERIAL AND METHODS Forty-eight New Zealand white rabbits were randomly divided into a control group, AF group, AAV9/GFP group, and AAV9/SERCA2a group. The right atrium was rapidly paced at 600 beats/min for 30 days after an intraperitoneal injection of an adeno-associated virus expressing the SERCA2a gene and GFP. The AF induction rate and the effective refraction period (ERP) were measured after 0, 4, 8, 12, and 24 h of pacing. Western blot analysis was used to test for the expression of SERCA2a. Changes in atrial tissue structure were observed by H&E staining and electron microscopy. RESULTS The AF induction rate was higher in the AF groups than in the AAV9/SERCA2a group at different time points of pacing. After 12 h of pacing, ERP was significantly prolonged in the AAV9/SERCA2a group compared to the AF and AAV9/GFP groups (p<0.05). SERCA2a protein expression was significantly lower in the AF and AAV9/GFP groups compared to the control group (p<0.05), while expression was significantly higher in the AAV9/SERCA2a group than in the AF and AAV9/GFP groups (p<0.05). The myocardial structure of the AAV9/SERCA2a group was significantly improved compared with the AF group, indicating that SERCA2a overexpression relieved the structural remodeling of atrial fibrillation. CONCLUSIONS SERCA2a overexpression is capable of suppressing ERP shortening and AF induced by rapid pacing atrium. SERCA2a gene therapy is expected to be a new anti-atrial fibrillation strategy.

Impact of citrate pretreatment on ventricular arrhythmia and myocardial capase-3 expression in ischemia/reperfusion injury.

Ischemia/reperfusion (I/R) injury often triggers ventricular arrhythmia. Citrate binds calcium ions, forming a soluble calcium citrate complex that may reduce I/R injury by affecting calcium ion concentration. We tested the effects of citrate pretreatment on ventricular heart rate and related factors in a rat I/R model. Fifty male Sprague Dawley rats weighing 350-400 g were randomly divided into equally sized control (A), model (B), and 0.1 M (C), 0.05 M (D), and 0.025 M (E) citrate groups. An I/R model was established by ligating the left anterior descending coronary artery. Serum calcium ion concentration was measured before and after citrate treatment. Triphenyltetrazolium chloride staining and spectrophotometry were used to determine infarction area and caspase-3 protein levels in myocardial tissue, respectively. Polymerase chain reaction was performed to test myocardial calmodulin (CAM) expression. The frequency of ventricular arrhythmia in group B was significantly higher than in the sham surgery group (P < 0.05). Citrate pretreatment resulted in lower and higher frequencies than those observed in the model and control groups, respectively, in a dose-independent manner. The most obvious reduction in ventricular arrhythmia was seen in Group D. Serum calcium ion concentration decreased markedly after citrate treatment (P < 0.05), with a specific pattern emerging over time. Infarction area and caspase-3 and CAM levels were significantly lower in the citrate groups compared with the model group (P < 0.05). Citrate can reduce myocardial cell apoptosis, alleviating ventricular arrhythmia and protecting the myocardium by reducing serum calcium ion concentration and downregulating caspase-3 and CAM expression.

Association of smoking with the risk of incident atrial fibrillation: A meta-analysis of prospective studies.

BACKGROUND: Several studies have investigated the role of smoking in incident atrial fibrillation (AF) but have reported contradictory results. Identifying the quantitative association between smoking and AF risk is important for AF management and prevention; therefore, we aimed to estimate the association of smoking with incident AF. METHODS: We systematically retrieved relevant studies reporting on the association between smoking and AF using the Cochrane Library, PubMed, and Embase databases. The data were extracted from applicable articles, and we used a random effects model to pool the effect estimates. RESULTS: Sixteen prospective studies with an overall number of 286,217 participants and 11,878 AF cases met the inclusion criteria. A higher prevalence of AF was confirmed among smokers (risk ratio [RR]=1.23, 95% confidence interval [CI] 1.08-1.39; P=0.001). These results were stable in the sensitivity analysis. The pooled RRs showed consistent positive associations in most subgroups. Specifically, 8 articles compared both current smokers (RR=1.39, 95% CI 1.11-1.75) and former smokers (RR=1.16, 95% CI: 1.00-1.36) with never smokers. Four articles compared ever smokers (pooled RR=1.21, 95% CI 0.93-1.57) with never smokers, and 7 articles compared current smokers (pooled RR=1.21, 95% CI 1.03-1.42) with non-current smokers. Additionally, we estimated that 6.7% of the total risk of AF in men and 1.4% of the risk in women were attributable to smoking worldwide. CONCLUSIONS: Based on the published literature, smoking is associated with a modest increased risk of incident AF. Smoking cessation seemed to reduce but not entirely eliminate the excess risk of AF.

Selective downregulation of mitochondrial electron transport chain activity and increased oxidative stress in human atrial fibrillation.

Mitochondria are critical for maintaining normal cardiac function, and a deficit in mitochondrial energetics can lead to the development of the substrate that promotes atrial fibrillation (AF) and its progression. However, the link between mitochondrial dysfunction and AF in humans is still not fully defined. The aim of this study was to elucidate differences in the functional activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes and oxidative stress in right atrial tissue from patients without (non-AF) and with AF (AF) who were undergoing open-heart surgery and were not significantly different for age, sex, major comorbidities, and medications. The overall functional activity of the electron transport chain (ETC), NADH:O2 oxidoreductase activity, was reduced by 30% in atrial tissue from AF compared with non-AF patients. This was predominantly due to a selective reduction in complex I (0.06 ± 0.007 vs. 0.09 ± 0.006 nmol·min(-1)·citrate synthase activity(-1), P = 0.02) and II (0.11 ± 0.012 vs. 0.16 ± 0.012 nmol·min(-1)·citrate synthase activity(-1), P = 0.003) functional activity in AF patients. Conversely, complex V activity was significantly increased in AF patients (0.21 ± 0.027 vs. 0.12 ± 0.01 nmol·min(-1)·citrate synthase activity(-1), P = 0.005). In addition, AF patients exhibited a higher oxidative stress with increased production of mitochondrial superoxide (73 ± 17 vs. 11 ± 2 arbitrary units, P = 0.03) and 4-hydroxynonenal level (77.64 ± 30.2 vs. 9.83 ± 2.83 ng·mg(-1) protein, P = 0.048). Our findings suggest that AF is associated with selective downregulation of ETC activity and increased oxidative stress that can contribute to the progression of the substrate for AF.

The serum matrix metalloproteinase-9 level is an independent predictor of recurrence after ablation of persistent atrial fibrillation

OBJECTIVES: This study investigated whether the serum matrix metalloproteinase-9 level is an independent predictor of recurrence after catheter ablation for persistent atrial fibrillation. METHODS: Fifty-eight consecutive patients with persistent atrial fibrillation were enrolled and underwent catheter ablation. The serum matrix metalloproteinase-9 level was detected before ablation and its relationship with recurrent arrhythmia was analyzed at the end of the follow-up. RESULTS: After a mean follow-up of 12.1±7.2 months, 21 (36.2%) patients had a recurrence of their arrhythmia after catheter ablation. At baseline, the matrix metalloproteinase-9 level was higher in the patients with recurrence than in the non-recurrent group (305.77±88.90 vs 234.41±93.36 ng/ml, respectively, p=0.006). A multivariate analysis showed that the matrix metalloproteinase-9 level was an independent predictor of arrhythmia recurrence, as was a history of atrial fibrillation and the diameter of the left atrium. CONCLUSION: The serum matrix metalloproteinase-9 level is an independent predictor of recurrent arrhythmia after catheter ablation in patients with persistent atrial fibrillation.

Atrial Fibrillation Activates AMP-Dependent Protein Kinase and its Regulation of Cellular Calcium Handling: Potential Role in Metabolic Adaptation and Prevention of Progression.

BACKGROUND: Atrial fibrillation (AF) is associated with metabolic stress, which activates adenosine monophosphate-regulated protein kinase (AMPK). OBJECTIVES: This study sought to examine AMPK response to AF and associated metabolic stress, along with consequences for atrial cardiomyocyte Ca(2+) handling. METHODS: Calcium ion (Ca(2+)) transients (CaTs) and cell shortening (CS) were measured in dog and human atrial cardiomyocytes. AMPK phosphorylation and AMPK association with Ca(2+)-handling proteins were evaluated by immunoblotting and immunoprecipitation. RESULTS: CaT amplitude and CS decreased at 4-min glycolysis inhibition (GI) but returned to baseline at 8 min, suggesting cellular adaptation to metabolic stress, potentially due to AMPK activation. GI increased AMPK-activating phosphorylation, and an AMPK inhibitor, compound C (CompC), abolished the adaptation of CaT and CS to GI. The AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) increased CaT amplitude and CS, restoring CompC-induced CaT and CS decreases. CompC decreased L-type calcium channel current (ICa,L), along with ICa,L-triggered CaT amplitude and sarcoplasmic reticulum (SR) Ca(2+) content under voltage clamp conditions in dog cells and suppressed CaT and ICa,L in human cardiomyocytes. Small interfering ribonucleic acid-based AMPK knockdown decreased CaT amplitude in neonatal rat cardiomyocytes. L-type Ca(2+) channel α subunits coimmunoprecipitated with AMPKα. Atrial AMPK-activating phosphorylation was enhanced by 1 week of electrically maintained AF in dogs; fractional AMPK phosphorylation was increased in paroxysmal AF and reduced in longstanding persistent AF patients. CONCLUSIONS: AMPK is activated by metabolic stress and AF, and helps maintain the intactness of atrial ICa,L, Ca(2+) handling, and cell contractility. AMPK contributes to the atrial compensatory response to AF-related metabolic stress; AF-related metabolic responses may be an interesting new therapeutic target.

Angiotensin-converting enzyme-2 overexpression improves atrial remodeling and function in a canine model of atrial fibrillation.

BACKGROUND: Atrial fibrosis is an important factor in initiating and maintaining atrial fibrillation. The purpose of this study was to test the hypothesis that atrial angiotensin-converting enzyme-2 (ACE2) overexpression might inhibit atrial collagen accumulation and improve atrial remodeling in a canine atrial pacing model. METHODS AND RESULTS: Thirty-two mongrel dogs of both genders were divided randomly into 4 groups: sham-operated, control, gene therapy with adenovirus-enhanced green fluorescent protein (Ad-EGFP), and gene therapy with Ad-ACE2. All of the dogs in the control, Ad-EGFP, and Ad-ACE2 groups were paced at 450 bpm for a period of 14 days. The dogs in the sham group were instrumented without pacing. After 2 weeks, all of the dogs underwent a thoracotomy operation and received epicardial gene painting. On post-gene transfer day 21, the animals underwent electrophysiology, histology, and molecular studies. The percentage of fibrosis in the Ad-ACE2 group was markedly lower than the percentage in the control and Ad-EGFP groups. Compared with the other groups, ACE2 expression was increased significantly in the Ad-ACE2 group. Compared with the sham and Ad-ACE2 groups, the expression levels of transforming growth factor-ß1 and Smad3 were significantly higher in the Ad-EGFP and control groups; however, the expression levels of Smad7 were lower in the atrial tissue as detected by Western blot and reverse transcription polymerase chain reaction. CONCLUSIONS: Our results demonstrate that the overexpression of ACE2 inhibits atrial collagen accumulation and improves left atrial remodeling and function in a canine model of atrial fibrillation. Thus, targeted gene ACE2 therapy provides a promising approach for the treatment of atrial fibrillation.

LKB1 knockout mouse develops spontaneous atrial fibrillation and provides mechanistic insights into human disease process.

BACKGROUND: Atrial fibrillation (AF) is a complex disease process, and the molecular mechanisms underlying initiation and progression of the disease are unclear. Consequently, AF has been difficult to model. In this study, we have presented a novel transgenic mouse model of AF that mimics human disease and characterized the mechanisms of atrial electroanatomical remodeling in the genesis of AF. METHODS AND RESULTS: Cardiac-specific liver kinase B1 (LKB1) knockout (KO) mice were generated, and 47% aged 4 weeks and 95% aged 12 weeks developed spontaneous AF from sinus rhythm by demonstrating paroxysmal and persistent stages of the disease. Electrocardiographic characteristics of sinus rhythm were similar in KO and wild-type mice. Atrioventricular block and atrial flutter were common in KO mice. Heart rate was slower with persistent AF. In parallel with AF, KO mice developed progressive biatrial enlargement with inflammation, heterogeneous fibrosis, and loss of cardiomyocyte population with apoptosis and necrosis. Atrial tissue was infiltrated with inflammatory cells. C-reactive protein, interleukin 6, and tumor necrosis factor α were significantly elevated in serum. KO atria demonstrated elevated reactive oxygen species and decreased AMP-activated protein kinase activity. Cardiomyocyte and myofibrillar ultrastructure were disrupted. Intercellular matrix and gap junction were interrupted. Connexins 40 and 43 were reduced. Persistent AF caused left ventricular dysfunction and heart failure. Survival and exercise capacity were worse in KO mice. CONCLUSIONS: LKB1 KO mice develop spontaneous AF from sinus rhythm and progress into persistent AF by replicating the human AF disease process. Progressive inflammatory atrial cardiomyopathy is the genesis of AF, through mechanistic electrical and structural remodeling.

Serum NOX2 and urinary isoprostanes predict vascular events in patients with atrial fibrillation.

There are limited prospective data evaluating the role of urinary F2-IsoP and NOX2 as predictive markers in atrial fibrillation (AF). The aim of this study was to analyse the role of urinary prostaglandin PGF2alpha (8-iso-PGF2α) and NOX2, markers of systemic oxidative stress, in predicting cardiovascular (CV) events and mortality in anticoagulated non-valvular AF patients. This was a prospective study including 1,002 anticoagulated AF patients, followed for a median time of 25.7 months (interquartile range: 14.8-50.9). All major CV events, CV deaths and all-cause deaths were considered as primary outcomes of the study. CV events included fatal/nonfatal ischaemic stroke, fatal/nonfatal myocardial infarction (MI), cardiac revascularisation and transient ischaemic attack (TIA). Oxidative stress biomarkers, such as urinary 8-iso-PGF2α and serum sNOX2-dp, a marker of NOX2 activation, were measured. A CV event occurred in 125 patients (12.5 %); 78 CV deaths and 31 non-CV deaths were registered. 8-iso-PGF2α and sNOX2-dp were correlated (Rs=0.765 p< 0.001). A significant increased cumulative incidence of CV events and CV deaths was observed across tertiles for 8-iso-PGF2α and sNOX2-dp. An increased rate of all-cause death was observed across tertiles of urinary 8-iso-PGF2α. In Cox or Fine and Gray models, 8-iso-PGF2α predicted CV events and CV and non-CV deaths. The addition of tertiles of 8-iso-PGF2α to CHA2DS2-VASc score improved ROC curves for each outcome and NRI for CV events (0.24 [0.06-0.53] p=0.0067). The study shows that in AF patients 8-iso-PGF2α and NOX2 levels are predictive of CV events and total mortality. F2-IsoP may complement conventional risk factors in prediction of CV events.

Monoamine oxidase is a major determinant of redox balance in human atrial myocardium and is associated with postoperative atrial fibrillation.

BACKGROUND: Onset of postoperative atrial fibrillation (POAF) is a common and costly complication of heart surgery despite major improvements in surgical technique and quality of patient care. The etiology of POAF, and the ability of clinicians to identify and therapeutically target high-risk patients, remains elusive. METHODS AND RESULTS: Myocardial tissue dissected from right atrial appendage (RAA) was obtained from 244 patients undergoing cardiac surgery. Reactive oxygen species (ROS) generation from multiple sources was assessed in this tissue, along with total glutathione (GSHt) and its related enzymes GSH-peroxidase (GPx) and GSH-reductase (GR). Monoamine oxidase (MAO) and NADPH oxidase were observed to generate ROS at rates 10-fold greater than intact, coupled mitochondria. POAF risk was significantly associated with MAO activity (Quartile 1 [Q1]: adjusted relative risk [ARR]=1.0; Q2: ARR=1.8, 95% confidence interval [CI]=0.84 to 4.0; Q3: ARR=2.1, 95% CI=0.99 to 4.3; Q4: ARR=3.8, 95% CI=1.9 to 7.5; adjusted Ptrend=0.009). In contrast, myocardial GSHt was inversely associated with POAF (Quartile 1 [Q1]: adjusted relative risk [ARR]=1.0; Q2: ARR=0.93, 95% confidence interval [CI]=0.60 to 1.4; Q3: ARR=0.62, 95% CI=0.36 to 1.1; Q4: ARR=0.56, 95% CI=0.34 to 0.93; adjusted Ptrend=0.014). GPx also was significantly associated with POAF; however, a linear trend for risk was not observed across increasing levels of the enzyme. GR was not associated with POAF risk. CONCLUSIONS: Our results show that MAO is an important determinant of redox balance in human atrial myocardium, and that this enzyme, in addition to GSHt and GPx, is associated with an increased risk for POAF. Further investigation is needed to validate MAO as a predictive biomarker for POAF, and to explore this enzyme's potential role in arrhythmogenesis.

Targeting inflammation and oxidative stress in atrial fibrillation: role of 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibition with statins.

SIGNIFICANCE: Atrial fibrillation (AF) is a burgeoning health-care problem, and the currently available therapeutic armamentarium is barely efficient. Experimental and clinical evidence implicates inflammation and myocardial oxidative stress in the pathogenesis of AF. RECENT ADVANCES: Local and systemic inflammation has been found to both precede and follow the new onset of AF, and NOX2-dependent generation of reactive oxygen species in human right atrial samples has been independently associated with the occurrence of AF in the postoperative period in patients undergoing cardiac surgery. Anti-inflammatory and antioxidant agents can prevent atrial electrical remodeling in animal models of atrial tachypacing and the new onset of AF after cardiac surgery, suggesting a causal relationship between inflammation/oxidative stress and the atrial substrate that supports AF. CRITICAL ISSUES: Statin therapy, by redressing the myocardial nitroso-redox balance and reducing inflammation, has emerged as a potentially effective strategy for the prevention of AF. Evidence indicates that statins prevent AF-induced electrical remodeling in animal models of atrial tachypacing and may reduce the new onset of AF after cardiac surgery. However, whether statins have antiarrhythmic properties in humans has yet to be conclusively demonstrated, as data from randomized controlled trials specifically addressing the relevance of statin therapy for the primary and secondary prevention of AF remain scanty. FUTURE DIRECTIONS: A better understanding of the mechanisms underpinning the putative antiarrhythmic effects of statins may afford tailoring AF treatment to specific clinical settings and patient's subgroups. Large-scale randomized clinical trials are needed to support the indication of statin therapy solely on the basis of AF prevention.