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.

IP3-mediated Ca2+ release regulates atrial Ca2+ transients and pacemaker function by stimulation of adenylyl cyclases.

Inositol trisphosphate (IP3) is a Ca2+-mobilizing second messenger shown to modulate atrial muscle contraction and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP3-mediated Ca2+ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca2+-stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sinoatrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified colocalization of type 2 IP3 receptors with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP3 by UV light significantly enhanced the amplitude of the Ca2+ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6% increase 60 s after photorelease, n = 16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP3. In mouse, spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP3-mediated Ca2+ release, increased the maximum beating rate by 14.7 ± 0.5%, n = 10. This effect was substantially reduced by 2.5 µmol/L 2-aminoethyl diphenylborinate and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP3 and cAMP signaling involving Ca2+ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP3 receptor pathways and Ca2+-stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias.NEW & NOTEWORTHY This study provides evidence supporting the proposal that IP3 signaling in cardiac atria and sinoatrial node involves stimulation of Ca2+-activated adenylyl cyclases (AC1 and AC8) by IP3-evoked Ca2+ release from junctional sarcoplasmic reticulum. AC8 and IP3 receptors are shown to be located close together, while AC1 is nearby. Greater understanding of these novel aspects of the IP3 signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly atrial fibrillation.

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.

Regulation of left atrial fibrosis induced by mitral regurgitation by SIRT1.

SIRT1 (silent information regulator 1) is a histone deacetylase. It can sense the energy level in cells and delay cell senescence, leading to resistance to external stress and improving metabolism. Mitral regurgitation (MR) is a common disease in cardiac surgery. However, there are no previous studies on SIRT1 and left atrial fibrosis caused by MR. In this study, we aimed to explore the regulatory effect of SIRT1 on left atrial fibrosis induced by MR. We used Guizhou miniature pigs to establish an MR model and a sham operation model after anaesthesia induction and respiratory intubation, and these model animals were followed for 30 months after the surgery. The differential distribution and expression of SIRT1 and collagen I in the left atrium was determined by immunofluorescence and Western blotting. Furthermore, we treated NIH3T3 fibroblasts (CFs) with resveratrol and Angiotensin II (Ang II) to analyse the specific mechanism involved in the development of myocardial fibrosis. The results showed that the MR model was successfully constructed. There were 8 pigs in the MR group and 6 pigs in the control group. In both the animal experiments and the cell experiments, the expression of collagen I in the MR group was increased significantly compared to that in the control group, while the expression of SIRT1 was decreased.

Puerarin Decreases Collagen Secretion in AngII-Induced Atrial Fibroblasts Through Inhibiting Autophagy Via the JNK-Akt-mTOR Signaling Pathway.

Puerarin is used to treat cardiovascular diseases due to its anti-inflammatory and antifibrotic effects. However, its mechanism of action in atrial fibroblasts is unknown. In this study, we investigated the autophagy pathway and molecular changes in angiotensin II (AngII)-stimulated atrial fibroblasts in response to puerarin treatment. Atrial fibroblasts were cultured and then subjected to stimulation with AngII and puerarin or other chemical drugs (3-MA, CQ, and SP600125). Quantitative real-time polymerase chain reaction and Western blot experiments were used to quantify the expression levels of mRNA and protein. mCherry-GFP-LC3 adenovirus was applied to reflect the autophagic flux. The results showed aggravating levels of autophagy and collagen deposit in the presence of AngII. Puerarin inhibited autophagy and decreased collagen secretion in a dose-dependent manner in atrial fibroblasts. Furthermore, phosphorylation of JNK was down-regulated in response to puerarin, whereas phosphorylation of Akt and mammalian target of rapamycin (mTOR) was upregulated. Interestingly, reduced autophagy and collagen secretion were observed when the JNK signaling pathway was blocked using SP600125. We also observed upregulation of Akt and mTOR phosphorylation in the presence of SP600125. These results suggest that puerarin exerts its antifibrotic effect in atrial fibroblasts partly through the inhibition of autophagy. Furthermore, the mechanism of action of puerarin in fibroblast autophagy seems to be mediated partly through JNK-Akt-mTOR signaling.

Xanthine Oxidase Inhibitor Allopurinol Prevents Oxidative Stress-Mediated Atrial Remodeling in Alloxan-Induced Diabetes Mellitus Rabbits.

BACKGROUND: There are several mechanisms, including inflammation, oxidative stress and abnormal calcium homeostasis, involved in the pathogenesis of atrial fibrillation. In diabetes mellitus (DM), increased oxidative stress may be attributable to higher xanthine oxidase activity. In this study, we examined the relationship between oxidative stress and atrial electrical and structural remodeling, and calcium handling abnormalities, and the potential beneficial effects of the xanthine oxidase inhibitor allopurinol upon these pathological changes. METHODS AND RESULTS: Ninety rabbits were randomly and equally divided into 3 groups: control, DM, and allopurinol-treated DM group. Echocardiographic and hemodynamic assessments were performed in vivo. Serum and tissue markers of oxidative stress and atrial fibrosis, including the protein expression were examined. Atrial interstitial fibrosis was evaluated by Masson trichrome staining. ICaL was measured from isolated left atrial cardiomyocytes using voltage-clamp techniques. Confocal microscopy was used to detect intracellular calcium transients. The Ca2+ handling protein expression was analyzed by Western blotting. Mitochondrial-related proteins were analyzed as markers of mitochondrial function. Compared with the control group, rabbits with DM showed left ventricular hypertrophy, increased atrial interstitial fibrosis, oxidative stress and fibrosis markers, ICaL and intracellular calcium transient, and atrial fibrillation inducibility. These abnormalities were alleviated by allopurinol treatment. CONCLUSIONS: Allopurinol, via its antioxidant effects, reduces atrial mechanical, structural, ion channel remodeling and mitochondrial synthesis abnormalities induced by DM-related increases in oxidative stress.

Transcriptional regulation of stress kinase JNK2 in pro-arrhythmic CaMKIIδ expression in the aged atrium.

Aims: c-jun N-terminal kinase (JNK) is a critical stress response kinase that activates in a wide range of physiological and pathological cellular processes. We recently discovered a pivotal role of JNK in the development of atrial arrhythmias in the aged heart, while cardiac CaMKIIδ, another pro-arrhythmic molecule, was also known to enhance atrial arrhythmogenicity. Here, we aimed to reveal a regulatory role of the stress kinase JNK2 isoform on CaMKIIδ expression. Methods and results: Activated JNK2 leads to increased CaMKIIδ protein expression in aged human and mouse atria, evidenced from the reversal of CaMKIIδ up-regulation in JNK2 inhibitor treated wild-type aged mice. This JNK2 action in CaMKIIδ expression was further confirmed in HL-1 myocytes co-infected with AdMKK7D-JNK2, but not when co-infected with AdMKK7D-JNK1. JNK2-specific inhibition (either by a JNK2 inhibitor or overexpression of inactivated dominant-negative JNK2 (JNK2dn) completely attenuated JNK activator anisomycin-induced CaMKIIδ up-regulation in HL-1 myocytes, whereas overexpression of JNK1dn did not. Moreover, up-regulated CaMKIIδ mRNA along with substantially increased phosphorylation of JNK downstream transcription factor c-jun [but not activating transcription factor2 (ATF2)] were exhibited in both aged atria (humans and mice) and transiently JNK activated HL-1 myocytes. Cross-linked chromatin-immunoprecipitation assays (XChIP) revealed that both c-jun and ATF2 were bound to the CaMKIIδ promoter, but significantly increased binding of c-jun only occurred in the presence of anisomycin and JNK inhibition alleviated this anisomycin-elevated c-jun binding. Mutated CaMKII consensus c-jun binding sites impaired its promoter activity. Enhanced transcriptional activity of CaMKIIδ by anisomycin was also completely reversed to the baseline by either JNK2 siRNA or c-jun siRNA knockdown. Conclusion: JNK2 activation up-regulates CaMKIIδ expression in the aged atrium. This JNK2 regulation in CaMKIIδ expression occurs at the transcription level through the JNK downstream transcription factor c-jun. The discovery of this novel molecular mechanism of JNK2-regulated CaMKII expression sheds new light on possible anti-arrhythmia drug development.

The stress kinase JNK regulates gap junction Cx43 gene expression and promotes atrial fibrillation in the aged heart.

BACKGROUND: The stress kinase c-jun N-terminal kinase (JNK) is critical in the pathogenesis of cardiac diseases associated with an increased incidence of atrial fibrillation (AF), the most common arrhythmia in the elderly. We recently discovered that JNK activation is linked to the loss of gap junction connexin43 (Cx43) and enhanced atrial arrhythmogenicity. However, direct evidence for JNK-mediated impairment of intercellular coupling (cell-cell communication) in the intact aged atrium is lacking, as is evidence for whether and how JNK suppresses Cx43 in the aged human atrium. METHODS AND RESULTS: JNK activity in human atrial samples is correlated with both reduced Cx43 expression and increasing age. Using a unique technique of optical mapping space constant measurement, we found that impaired intercellular coupling and reduced Cx43 were linked to enhanced activation of JNK in intact aged rabbit atria. These JNK-associated alterations were further confirmed in naturally JNK activated aged mice and in cardiac-specific inducible MKK7D (JNK upstream activator) young mice. Moreover, JNK inhibition, using either JNK specific inhibitors in aged wild-type (WT) mice and JNK activator anisomycin-treated young WT mice or JNK1/2 dominant-negative mice with genetically inhibited cardiac JNK activity, completely eliminated these functional abnormalities. Furthermore, we discovered for the first time that long-term JNK activation downregulates Cx43 expression via c-jun suppressed transcriptional activity of the Cx43 gene promoter. CONCLUSION: Our results demonstrate that JNK is a critical regulator of Cx43 expression, and that augmented JNK activation in aged atria downregulates Cx43 to impair cell-cell communication and promote the development of AF. JNK inhibition may represent a promising therapeutic approach to prevent or treat AF in the elderly.

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.

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.

Differential expression of the angiotensin-(1-12)/chymase axis in human atrial tissue.

OBJECTIVE: Heart chymase rather than angiotensin converting enzyme has higher specificity for angiotensin (Ang) I conversion into Ang II in humans. A new pathway for direct cardiac Ang II generation has been revealed through the demonstration that Ang-(1-12) is cleaved by chymase to generate Ang II directly. We address here whether Ang-(1-12) and chymase gene expression and activity are detected in the atrial appendages of 44 patients (10 females) undergoing heart surgery for the correction of valvular heart disease, resistant atrial fibrillation or ischemic heart disease. METHODS AND RESULTS: Immunoreactive Ang-(1-12) expression was 54% higher in left atrial compared with right atrial appendages. This was associated with higher abundance of left atrial appendage chymase gene transcripts and chymase activity, but no differences in angiotensinogen mRNA. Atrial chymase enzymatic activity was highly correlated with left atrial but not right atrial enlargement as determined by echocardiography, while both tyrosine hydroxylase and neuropeptide Y atrial appendage mRNAs correlated with atrial angiotensinogen mRNAs. CONCLUSIONS: Higher Ang-(1-12) expression and upregulation of chymase gene transcripts and enzymatic activity from the atrial appendages connected to the enlarged left versus right atrial chambers of subjects with left heart disease defines a role of this alternate Ang II forming pathway in the processes accompanying adverse atrial and ventricular remodeling.

Enhanced expression of ROCK in left atrial myocytes of mitral regurgitation: a potential mechanism of myolysis.

BACKGROUND: Severe mitral regurgitation (MR) may cause myolysis in the left atrial myocytes. Myolysis may contribute to atrial enlargement. However, the relationship between Rho-associated kinase (ROCK) and myolysis in the left atrial myocytes of MR patients remain unclear. METHODS: This study comprised 22 patients with severe MR [12 with atrial fibrillation (AF) and ten in sinus rhythm]. Left atrial appendage tissues were obtained during surgery. Normal left atrial tissues were purchased. Immunofluorescence histochemical and immunoblotting studies were performed. RESULTS: The expression of ROCK2 in the myolytic left atrial myocytes of MR AF patients (p = 0.009) and MR sinus patients (p = 0.011) were significantly higher than that of the normal subjects. Similarly, the expression of ROCK1 in the myolytic left atrial myocytes of MR AF patients was significantly higher than that of the normal subjects (p = 0.010), and the expression of ROCK1 in the myolytic left atrial myocytes of MR sinus patients was higher than that of the normal subjects (p = 0.091). Immunofluorescence study revealed significant co-localization and juxtaposition of ROCK2 and cleaved caspase-3 in the left atrial myocytes both in the MR AF group (Pearson's coefficient = 0.74 ± 0.03) and the MR sinus group (Pearson's coefficient = 0.73 ± 0.02). Similarly, immunofluorescence study revealed significant co-localization and juxtaposition of ROCK1 and cleaved caspase-3 in the left atrial myocytes both in the MR AF group (Pearson's coefficient = 0.65 ± 0.03) and the MR sinus group (Pearson's coefficient = 0.65 ± 0.03). Correlation analysis demonstrated that there was a significant direct relationship between the expression of ROCK2 in the myolytic left atrial myocytes and left atrial diameter in the MR patients (p = 0.041; r = 0.440). Moreover, the ratio of phosphorylated myosin-binding subunit of myosin light chain phosphatase (pMBS)/total MBS of left atrial tissues was significantly higher in the MR AF group (p < 0.04) and the MR sinus group (p < 0.04) compared with the normal control group. CONCLUSIONS: The enhanced expression of ROCKs might be involved in the myolysis of the left atrial myocytes of MR patients.

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.

Left atrial myxoma presenting as pulmonary embolism: potential role of heme oxygenase-1.

We present the case of a patient with left atrial myxoma that presented with pulmonary embolism. The patient did not have any intracardiac communication between right and left sides of the heart. Using thrombelastography, the patient was determined to have an abnormally large velocity of plasma thrombus growth and strength with reduced vulnerability to lysis. Critically, increased carboxyhemoglobin concentrations were present, likely secondary to hemolysis from the tumor and engagement of systemic heme oxygenase-1. It was determined that the patient's plasmatic hypercoagulability was in part due to carboxyhemefibrinogen formation via a thrombelastographic method. In addition to circulating hypercoagulability, the patient also had an area of chronic venous stasis in his left ankle that had not changed for over a decade prior to this thrombophilic episode. In conclusion, we present the first case of paradoxical pulmonary embolism in the presence of a left atrial myxoma, potentially secondary to a combination of hemolysis, heme oxygenase-1 up-regulation, systemic hypercoagulability/hypofibrinolysis, and regional venous stasis.

Mechanisms with clinical implications for atrial fibrillation-associated remodeling: cathepsin K expression, regulation, and therapeutic target and biomarker.

BACKGROUND: The cysteine protease cathepsin K (CatK) has been implicated in the pathogenesis of cardiovascular disease. We sought to determine the link between atrial fibrillation (AF) and plasma CatK levels and to investigate the expression of and therapeutic target for CatK in vivo and in vitro. METHODS AND RESULTS: Plasma CatK and extracellular matrix protein peptides (intact procollagen type I of N-terminal propeptide; carboxyl-terminal telopeptide of type I collagen [ICTP]) were measured in 209 consecutive patients with AF (paroxysmal AF, 146; persistent AF, 63) and 112 control subjects. In addition, the regulation of CatK expression was investigated in vivo and vitro. Patients with AF had higher plasma CatK and ICTP levels than did control subjects. Patients with persistent AF had higher levels of plasma CatK and ICTP than did patients with paroxysmal AF. CatK was correlated with ICTP concentration and left atrial diameter in all subjects. In rabbits, superoxide production, CatK activity, fibrosis, and the levels of atrial tissue angiotensin II, angiotensin type 1 receptor, gp91phox, phospho-p38 mitogen-activated protein kinase, and CatK were greater in those with tachypacing-induced AF than in controls, and these changes were reversed with angiotensin type 1 receptor antagonist. Olmesartan and mitogen-activated protein kinase inhibitor decreased the CatK expression induced by angiotensin II in rat neonatal myocytes. CONCLUSIONS: These data indicated that increased plasma CatK levels are linked with the presence of AF. Angiotensin type 1 receptor antagonist appears to be effective in alleviating atrial fibrosis in a rabbit AF model, partly reducing angiotensin type 1 receptor-p38mitogen-activated protein kinase-dependent and -independent CatK activation, thus preventing AF.

Age and obesity-associated changes in the expression and activation of components of the AMPK signaling pathway in human right atrial tissue.

BACKGROUND: Obesity is associated with an increased incidence of left ventricular hypertrophy, diastolic dysfunction, heart failure, and premature cardiac aging. In the heart, intrinsic activation of the AMP-dependent protein kinase (AMPK) plays a pivotal role in the stress response to ischemia and hypertrophy. Furthermore, AMPK is an important regulator of cardiac mitochondrial biogenesis. The purpose of the present study was to investigate the influence of obesity and aging on the AMPK signaling pathway in human cardiac tissue. METHODS: 60 male cardiac surgery patients were included in the study and divided into 4 groups (old normal weight: ON; old obese: OO; young normal weight: YN, young obese: YO) according to their body mass index (18.5-25: normal weight or 30-35: obese) and age (<55 years: young or >70: old) with 15 patients each. Right atrial tissue (RA) was analyzed for the expression of the AMPK upstream kinases CAMKK and LKB1, activation of AMPK as well as phosphorylation of the AMPK downstream targets ACC, eEF2, mTOR and eNOS. Epicardial adipose tissue was analyzed for the expression of the endogenous AMPK activator adiponectin. The metabolic state of all patients was further characterized in fasting blood samples. RESULTS: Old patients (ON, OO) and young obese (YO) subjects displayed higher fasting glucose, insulin and leptin serum levels compared to the young, normal weight group, although HbA1c was below the threshold required for the diagnosis of type 2 diabetes. Serum adiponectin as well as total adiponectin protein expression in epicardial adipose tissue was decreased in these three groups. Analyses of adiponectin isoforms by native gel electrophoresis revealed significant differences in the high molecular weight (HMW) isoforms between the groups. Despite the low total serum adiponectin and HMW adiponectin, AMPK activation was high in the RA of obese patients (YO, OO). Among the AMPK upstream kinases, LKB1 expression showed a strong positive correlation with AMPK activation. While the phosphorylation of the AMPK downstream targets mTOR, eEF-2 and ACC was not altered, phospho-eNOS was significantly lower in old patients (ON, OO). Despite strong AMPK activation, mitochondrial biogenesis and respiration were impaired in old (ON, OO) and young obese (YO) subjects. CONCLUSION: These data indicate that obesity and aging result in significant changes although many direct parameters in the AMPK signaling pathway are not changed in the same direction. LKB1 may represent a stronger activator of the AMPK pathway than adiponectin or the CAMKKs in human right atrial tissue.

Xanthine oxidase inhibition prevents atrial fibrillation in a canine model of atrial pacing-induced left ventricular dysfunction.

AIMS: Oxidative stress could be a possible mechanism and a therapeutic target of atrial fibrillation (AF). Xanthine oxidase (XO) inhibition reduces oxidative stress, but the effects of XO inhibitor on AF have not been evaluated. Hence, we assessed the effects of XO inhibitor, allopurinol, on progression of atrial vulnerability in dogs associated with tachycardia-induced cardiomyopathy. METHODS AND RESULTS: The dogs were subjected to atrial tachypacing (ATP, 400 bpm) without atrioventricular block for 4 weeks. The dynamics of atrial-tachycardia remodeling were evaluated in allopurinol-treated dogs (ALO, n = 5), placebo-treated controls (CTL, n = 6), and sham-operated dogs (n = 6). In CTL dogs, 4 weeks of ATP significantly increased AF duration (DAF; from 0.2 ± 0.2 seconds to 173 ± 67 seconds, P < 0.05) and decreased atrial effective refractory period (ERP; from 152 ± 9 milliseconds to 80 ± 4 milliseconds at a cycle length of 350 milliseconds, P < 0.01). Allopurinol attenuated the ATP effects on ERP (118 ± 6 milliseconds, P < 0.01) or DAF (0.6 ± 0.3 seconds, P < 0.05). In CTL dogs, ATP-induced rapid ventricular responses decreased left ventricular ejection fraction (LVEF; from 58.6 ± 0.1 to 23.5 ± 2.4%, P < 0.01), and increased left atrial diameter (LAD; from 17 ± 1 mm to 24 ± 1 mm, P < 0.01). ATP increased atrial fibrosis when compared with sham-operated dogs (CTL 10.7 ± 0.8% vs Sham 1.1 ± 0.3%, P < 0.01). Allopurinol suppressed atrial fibrosis (2.3 ± 0.6%, P < 0.01 vs CTL) and eNOS reduction without affecting LVEF (20.6 ± 2.2%, ns) and LAD (23 ± 1 mm, ns). CONCLUSION: Allopurinol suppresses AF promotion by preventing both electrical and structural remodeling. These results suggest that XO may play an important role in enhancement of atrial vulnerability, and might be a novel target of AF therapy.

Short-term magnesium deficiency upregulates ceramide synthase in cardiovascular tissues and cells: cross-talk among cytokines, Mg2+, NF-κB, and de novo ceramide.

The present study tested the hypotheses that 1) short-term dietary deficiency (MgD) of magnesium (21 days) would result in the upregulation of ceramide synthase (CS) in left ventricular (LV), right ventricular, atrial, and aortic smooth muscle, as well as induce a synthesis/release of select cytokines and chemokines into the LV and aortic smooth muscle and serum; 2) exposure of primary cultured vascular smooth muscle cells (VSMCs) to low extracellular Mg concentration would lead to the synthesis/release of select cytokines/chemokines, activation of N-SMase, and the de novo synthesis of ceramide; and 3) inhibition of CS by fumonisin B1 (FB1) or inhibition of neutral sphingomyelinase (N-SMase) by scyphostatin (SCY) in VSMCs exposed to low Mg would result in reductions in the levels of the cytokines/chemokines and lowered levels of ceramide concomitant with inhibition of NF-κB activation. The data indicated that short-term MgD (10% normal dietary intake) resulted in the upregulation of CS in ventricular, atrial, and aortic smooth muscles coupled to the synthesis/release of 12 different cytokines/chemokines, as well as activation of NF-κB in the LV and aortic smooth muscle and sera; even very low levels of water-borne Mg (e.g., 15 mg·l(-1)·day(-1)) either prevented or ameliorated the upregulation and synthesis of the cytokines/chemokines. Our experiments also showed that VSMCs exposed to low extracellular Mg resulted in the synthesis of 5 different cytokines and chemokines concomitant with synthesis/release of ceramide. However, inhibition of the synthesis and release of ceramide by either FB1 or SCY attenuated, markedly , the generation of ceramide, release of the cytokines/chemokines, and activation of NF-κB (as measured by activated p65 and cRel).