Identification of atrial-enriched lncRNA Walras linked to cardiomyocyte cytoarchitecture and atrial fibrillation.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in humans. Genetic and genomic analyses have recently demonstrated that the homeobox transcription factor Pitx2 plays a fundamental role regulating expression of distinct growth factors, microRNAs and ion channels leading to morphological and molecular alterations that promote the onset of AF. Here we address the plausible contribution of long non-coding (lnc)RNAs within the Pitx2>Wnt>miRNA signaling pathway. In silico analyses of annotated lncRNAs in the vicinity of the Pitx2, Wnt8 and Wnt11 chromosomal loci identified five novel lncRNAs with differential expression during cardiac development. Importantly, three of them, Walaa, Walras, and Wallrd, are evolutionarily conserved in humans and displayed preferential atrial expression during embryogenesis. In addition, Walrad displayed moderate expression during embryogenesis but was more abundant in the right atrium. Walaa, Walras and Wallrd were distinctly regulated by Pitx2, Wnt8, and Wnt11, and Wallrd was severely elevated in conditional atrium-specific Pitx2-deficient mice. Furthermore, pro-arrhythmogenic and pro-hypertrophic substrate administration to primary cardiomyocyte cell cultures consistently modulate expression of these lncRNAs, supporting distinct modulatory roles of the AF cardiovascular risk factors in the regulation of these lncRNAs. Walras affinity pulldown assays revealed its association with distinct cytoplasmic and nuclear proteins previously involved in cardiac pathophysiology, while loss-of-function assays further support a pivotal role of this lncRNA in cytoskeletal organization. We propose that lncRNAs Walaa, Walras and Wallrd, distinctly regulated by Pitx2>Wnt>miRNA signaling and pro-arrhythmogenic and pro-hypertrophic factors, are implicated in atrial arrhythmogenesis, and Walras additionally in cardiomyocyte cytoarchitecture.

Increased Density of Endogenous Adenosine A2A Receptors in Atrial Fibrillation: From Cellular and Porcine Models to Human Patients.

Adenosine, an endogenous nucleoside, plays a critical role in maintaining homeostasis during stressful situations, such as energy deprivation or cellular damage. Therefore, extracellular adenosine is generated locally in tissues under conditions such as hypoxia, ischemia, or inflammation. In fact, plasma levels of adenosine in patients with atrial fibrillation (AF) are elevated, which also correlates with an increased density of adenosine A2A receptors (A2ARs) both in the right atrium and in peripheral blood mononuclear cells (PBMCs). The complexity of adenosine-mediated effects in health and disease requires simple and reproducible experimental models of AF. Here, we generate two AF models, namely the cardiomyocyte cell line HL-1 submitted to Anemonia toxin II (ATX-II) and a large animal model of AF, the right atrium tachypaced pig (A-TP). We evaluated the density of endogenous A2AR in those AF models. Treatment of HL-1 cells with ATX-II reduced cell viability, while the density of A2AR increased significantly, as previously observed in cardiomyocytes with AF. Next, we generated the animal model of AF based on tachypacing pigs. In particular, the density of the key calcium regulatory protein calsequestrin-2 was reduced in A-TP animals, which is consistent with the atrial remodelling shown in humans suffering from AF. Likewise, the density of A2AR in the atrium of the AF pig model increased significantly, as also shown in the biopsies of the right atrium of subjects with AF. Overall, our findings revealed that these two experimental models of AF mimicked the alterations in A2AR density observed in patients with AF, making them attractive models for studying the adenosinergic system in AF.

Apolipoprotein J protects cardiomyocytes from lipid-mediated inflammation and cytotoxicity induced by the epicardial adipose tissue of diabetic patients.

Diabetic patients present increased volume and functional alterations in epicardial adipose tissue (EAT). We aimed to analyze EAT from type 2 diabetic patients and the inflammatory and cytotoxic effects induced on cardiomyocytes. Furthermore, we analyzed the cardioprotective role of apolipoprotein J (apoJ). EAT explants were obtained from nondiabetic patients (ND), diabetic patients without coronary disease (DM), and DM patients with coronary disease (DM-C) after heart surgery. Morphological characteristics and gene expression were evaluated. Explants were cultured for 24 h and the content of nonesterified fatty acids (NEFA) and sphingolipid species in secretomes was evaluated by lipidomic analysis. Afterwards, secretomes were added to AC16 human cardiomyocytes for 24 h in the presence or absence of cardioprotective molecules (apoJ and HDL). Cytokine release and apoptosis/necrosis were assessed by ELISA and flow cytometry. The EAT from the diabetic samples showed altered expression of genes related to lipid accumulation, insulin resistance, and inflammation. The secretomes from the DM samples presented an increased ratio of pro/antiatherogenic ceramide (Cer) species, while those from DM-C contained the highest concentration of saturated NEFA. DM and DM-C secretomes promoted inflammation and cytotoxicity on AC16 cardiomyocytes. Exogenous Cer16:0, Cer24:1, and palmitic acid reproduced deleterious effects in AC16 cells. These effects were attenuated by exogenous apoJ. Diabetic secretomes promoted inflammation and cytotoxicity in cardiomyocytes. This effect was exacerbated in the secretomes of the DM-C samples. The increased content of specific NEFA and ceramide species seems to play a key role in inducing such deleterious effects, which are attenuated by apoJ.

Spatial Distribution of Calcium Sparks Determines Their Ability to Induce Afterdepolarizations in Human Atrial Myocytes.

Analysis of the spatio-temporal distribution of calcium sparks showed a preferential increase in sparks near the sarcolemma in atrial myocytes from patients with atrial fibrillation (AF), linked to higher ryanodine receptor (RyR2) phosphorylation at s2808 and lower calsequestrin-2 levels. Mathematical modeling, incorporating modulation of RyR2 gating, showed that only the observed combinations of RyR2 phosphorylation and calsequestrin-2 levels can account for the spatio-temporal distribution of sparks in patients with and without AF. Furthermore, we demonstrate that preferential calcium release near the sarcolemma is key to a higher incidence and amplitude of afterdepolarizations in atrial myocytes from patients with AF.

Beta-blocker treatment of patients with atrial fibrillation attenuates spontaneous calcium release-induced electrical activity.

AIMS: Atrial fibrillation (AF) has been associated with excessive spontaneous calcium release, linked to cyclic AMP (cAMP)-dependent phosphorylation of calcium regulatory proteins. Because ß-blockers are expected to attenuate cAMP-dependent signaling, we aimed to examine whether the treatment of patients with ß-blockers affected the incidence of spontaneous calcium release events or transient inward currents (ITI). METHODS: The impact of treatment with commonly used ß-blockers was analyzed in human atrial myocytes from 371 patients using patch-clamp technique, confocal calcium imaging or immunofluorescent labeling. Data were analyzed using multivariate regression analysis taking into account potentially confounding effects of relevant clinical factors RESULTS: The L-type calcium current (ICa) density was diminished significantly in patients with chronic but not paroxysmal AF and the treatment of patients with ß-blockers did not affect ICa density in any group. By contrast, the ITI frequency was elevated in patients with either paroxysmal or chronic AF that did not receive treatment, and ß-blocker treatment reduced the frequency to levels observed in patients without AF. Confocal calcium imaging showed that ß-blocker treatment also reduced the calcium spark frequency in patients with AF to levels observed in those without AF. Furthermore, phosphorylation of the ryanodine receptor (RyR2) at Ser-2808 and phospholamban at Ser-16 was significantly lower in patients with AF that received ß-blockers. CONCLUSION: Together, our findings demonstrate that ß-blocker treatment may be of therapeutic utility to prevent spontaneous calcium release-induced atrial electrical activity; especially in patients with a history of paroxysmal AF displaying preserved ICa density.

Impact of R-Carvedilol on ß2-Adrenergic Receptor-Mediated Spontaneous Calcium Release in Human Atrial Myocytes.

A hallmark of atrial fibrillation is an excess of spontaneous calcium release events, which can be mimicked by ß1- or ß2-adrenergic stimulation. Because ß1-adrenergic receptor blockers (ß1-blockers) are primarily used in clinical practice, we here examined the impact of ß2-adrenergic stimulation on spontaneous calcium release and assessed whether the R- and S-enantiomers of the non-selective ß- blocker carvedilol could reverse these effects. For this purpose, human atrial myocytes were isolated from patients undergoing cardiovascular surgery and subjected to confocal calcium imaging or immunofluorescent labeling of the ryanodine receptor (RyR2). Interestingly, the ß2-adrenergic agonist fenoterol increased the incidence of calcium sparks and waves to levels observed with the non-specific ß-adrenergic agonist isoproterenol. Moreover, fenoterol increased both the amplitude and duration of the sparks, facilitating their fusion into calcium waves. Subsequent application of the non ß-blocking R-Carvedilol enantiomer reversed these effects of fenoterol in a dose-dependent manner. R-Carvedilol also reversed the fenoterol-induced phosphorylation of the RyR2 at Ser-2808 dose-dependently, and 1 µM of either R- or S-Carvedilol fully reversed the effect of fenoterol. Together, these findings demonstrate that ß2-adrenergic stimulation alone stimulates RyR2 phosphorylation at Ser-2808 and spontaneous calcium release maximally, and points to carvedilol as a tool to attenuate the pathological activation of ß2-receptors.

Mini-extracorporeal circulation surgery produces less inflammation than off-pump coronary surgery.

OBJECTIVES: Both off-pump coronary artery bypass grafting surgery (OPCABG) and mini-extracorporeal circulation (MECC) have been associated with lower morbidity and mortality and less inflammation than conventional cardiopulmonary bypass. However, studies comparing the 2 techniques are scarce and the results are controversial. We compared the clinical outcomes and inflammatory response of low-risk patients undergoing coronary bypass grafting with MECC versus OPCABG. METHODS: We conducted a prospective, randomized study in patients undergoing coronary heart surgery. Two hundred and thirty consecutive low-risk patients were randomly assigned to either receive OPCABG (n = 117) or MECC (n = 113). Clinical outcomes and postoperative biochemical results were analysed in both groups. We also analysed 19 circulating inflammatory markers in a subgroup of 40 patients at 4 perioperative time points. The area under the curve for each marker was calculated to monitor differences in the inflammatory response. RESULTS: No significant differences were found between groups regarding perioperative clinical complications and no deaths occurred during the trial. Plasma levels in 9 of the 19 inflammatory markers were undetectable or showed no temporal variation, 3 were higher in the MECC group [interleukin (IL)-10, macrophage inflammatory protein-1ß and epidermal growth factor] and 7 were higher in the OPCABG group (growth regulator oncogene, IL-6, IL-8, soluble CD40 ligand, monocyte chemoattractant protein-1, monocyte chemoattractant protein-3 and tumour necrosis factor-α). Differences in 2 proinflammatory cytokines, IL-6 and monocyte chemoattractant protein 1, between the 2 surgical procedures were statistically significant. CONCLUSIONS: No clinical differences were observed between in low-risk patients undergoing MECC or OPCABG surgery, but OPCABG was associated with an increased release of proinflammatory cytokines compared with MECC. Studies in larger cohorts and in patients at higher risk are needed to confirm these findings. CLINICAL TRIAL REGISTRATION NUMBER: NCT02118025.

Orthotopic heart transplantation in a patient with situs invs, transposition of the great arteries and Mustard operation.

Orthotopic heart transplantation has become standard treatment for end-stage cardiomyopathy, but experience with this technique for complex congenital heart diseases is limited. We report a patient with visceroatrial situs invs, transposition of the great arteries and previous Mustard correction, who successfully underwent orthotopic heart transplantation.

The Sorin Freedom SOLO stentless aortic valve: easier implantation technique with potentially less risk of complications.

Aortic valve replacement by bioprosthesis is increasingly being used. Among these, stentless bioprosthesis is becoming a valid choice due to its potential hemodynamic improvement. The Sorin Freedom SOLO stentless aortic valve (FS) (Sorin Biomedica Cardio, Saluggia, Italy) is widely used, with initial and midterm promising results. It is designed to be deployed in a straightforward manner in the supraannular position with a single running suture due to its reproducible implantation technique. Few specific valve-related complications, such as transient thrombocytopenia or rare thrombotic events, are known. Our experience, after the implantation of 33 Freedom SOLO, suggests that using the classical implantation technique, the noncoronary sinus adopts a conformation that increases the difficulty of the implant. Besides, it could also be distorted increasing the risk of periprosthetic leakage and hypothetically becoming the origin of thrombus. We present an easy and safe variation in the implantation technique of the FS that decreases the technical difficulty of the implantation, reduces the risk of periprosthetic leakage, and may additionally reduce the risk of thrombosis.

Long term outcome of mechanical valve prosthesis in the pulmonary position.

OBJECTIVES: Assessment of the long term outcome of mechanical valve prosthesis at pulmonary position in a population of grown-up congenital heart disease patients from a tertiary referral center. METHODS: From 1977 to 2007, 22 consecutive patients underwent a total of 25 pulmonary valve replacements with mechanical prosthesis. The most frequent underlying cardiac condition was tetralogy of Fallot (n=16, 64%) and the mean age at the time of pulmonary valve replacement was 32 ± 11 years (range 14-50 years). RESULTS: The postoperative mortality rate was 4% (n=1) with no late deaths documented after a mean follow-up of 7.6 ± 7.6 years (range 0.29-24 years). No major bleeding episodes occurred. Three patients presented with valve thrombosis in the setting of long term anticoagulation withdrawal and required valve re-replacement. Two of these patients, both with poor right ventricular function and overt clinical signs of right heart failure at the time of valve re-replacement, experienced further episodes of thrombosis despite correct anticoagulation. All episodes resolved with thrombolysis. After addition of antiplatelet treatment in one case and anticoagulation self-control, in the other, no further thrombosis has been documented. CONCLUSIONS: Mechanical valve prosthesis may be an alternative to tissue valve prosthesis in patients with congenital heart disease requiring pulmonary valve replacement. Optimal anticoagulation is crucial and additional antiplatelet treatment should be considered. Our data also suggest that patients with severe right ventricular dysfunction and congestive heart failure might be at particular risk for valve thrombosis.

Right atrium traumatic rupture presenting as chronic tamponade.

Non-penetrating cardiac traumatisms can cause cardiac rupture, myocardial contusion or, rarely, commotio cordis. In cases of rupture of a cardiac cavity, acute and severe cardiac tamponade almost invariably occurs. This paper presents an exceptionally unusual case of non-penetrating cardiac trauma resulting in right atrium rupture contained by the pericardial cavity. A situation of exceptional hemodynamic balance was established with subacute, progressive cardiac tamponade that evolved during three months, presenting gradual right-heart failure instead of the expected acute and severe cardiac tamponade. The rupture of the atrium was successfully repaired.

Orthotopic heart transplantation in patients with transposition of the great arteries.

Thanks to progress in cardiac surgery and cardiology, pediatric patients with complex congenital heart conditions who would previously have died are now reaching adulthood. Patients with transposition of the great arteries who have undergone atrial repair can present during follow-up with progression towards heart failure as a result of progressive systemic right ventricular failure. In this situation, heart transplantation is a possible therapeutic option. Anatomic abnormalities and the presence of intraatrial conduits ensure that transplantation must involve a number of technical modifications. Here, we present our experience during 1992-2004 with heart transplantations in four patients with transposition of the great arteries and atrial repair. There was no operative mortality. During follow-up (mean period, 75 months), there was one death due to chronic rejection. The other patients remain in New York Heart Association class I, with normally functioning grafts.