Human Atrial Samples Research Articles

Building a Career in Cardiovascular Research: A Conversation With Barbara Casadei, MD, DPhil.

Building a Career in Cardiovascular Research: A Conversation With Barbara Casadei, MD, DPhil.

A Novel Loss-of-Function Variant in the Chloride Ion Channel Gene Clcn2 Associates with Atrial Fibrillation

Atrial Fibrillation (AF) is the most common cardiac arrhythmia. Its pathogenesis is complex and poorly understood. Whole exome sequencing of Danish families with AF revealed a novel four nucleotide deletion c.1041_1044del in CLCN2 shared by affected individuals. We aimed to investigate the role of genetic variation of CLCN2 encoding the inwardly rectifying chloride channel ClC-2 as a risk factor for the development of familiar AF. The effect of the CLCN2 variant was evaluated by electrophysiological recordings on transiently transfected cells. We used quantitative PCR to assess CLCN2 mRNA expression levels in human atrial and ventricular tissue samples. The nucleotide deletion CLCN2 c.1041_1044del results in a frame-shift and premature stop codon. The truncated ClC-2 p.V347fs channel does not conduct current. Co-expression with wild-type ClC-2, imitating the heterozygote state of the patients, resulted in a 50% reduction in macroscopic current, suggesting an inability of truncated ClC-2 protein to form channel complexes with wild type channel subunits. Quantitative PCR experiments using human heart tissue from healthy donors demonstrated that CLCN2 is expressed across all four heart chambers. Our genetic and functional data points to a possible link between loss of ClC-2 function and an increased risk of developing AF.

Investigation of the underlying hub genes and mechanisms of reperfusion injury in patients undergoing coronary artery bypass graft surgery by integrated bioinformatic analyses.

Although coronary artery bypass graft (CABG) surgery is the main method to revascularize the occluded coronary vessels in coronary artery diseases, the full benefits of the operation are mitigated by ischemia-reperfusion (IR) injury. Although many studies have been devoted to reducing IR injury in animal models, the translation of this research into the clinical field has been disappointing. Our study aimed to explore the underlying hub genes and mechanisms of IR injury. A weighted gene co-expression network analysis (WGCNA) was executed based on the expression profiles in patients undergoing CABG surgery (GSE29396). Functional annotation and protein-protein interaction (PPI) network construction were executed within the modules of interest. Potential hub genes were predicted, combining both intramodular connectivity (IC) and degrees. Meanwhile, potential transcription factors (TFs) and microRNAs (miRNAs) were predicted by corresponding bioinformatics tools. A total of 336 differentially expressed genes (DEGs) were identified. DEGs were mainly enriched in neutrophil activity and immune response. Within the modules of interest, 5 upregulated hub genes (IL-6, CXCL8, IL-1β, MYC, PTGS-2) and 6 downregulated hub genes (C3, TIMP1, VSIG4, SERPING1, CD163, and HP) were predicted. Predicted miRNAs (hsa-miR-333-5p, hsa-miR-26b-5p, hsa-miR-124-3p, hsa-miR-16-5p, hsa-miR-98-5p, hsa-miR-17-5p, hsa-miR-93-5p) and TF (STAT1) might have regulated gene expression in the most positively related module, while hsa-miR-333-5p and HSF-1 were predicted to regulate the genes within the most negatively related module. Our study illustrates an overview of gene expression changes in human atrial samples from patients undergoing CABG surgery and might help translate future research into clinical work.

Pathway analysis with genome-wide association study (GWAS) data detected the association of atrial fibrillation with the mTOR signaling pathway

BackgroundGenome-wide association studies (GWAS) have identified numerous loci associated with diseases and traits. However, the elucidation of disease mechanisms followed by drug development has remained a challenge owing to complex gene interactions. We performed pathway analysis with MAGENTA (Meta-Analysis Geneset Enrichment of variaNT Associations) to clarify the pathways in genetic background of AF. MethodsThe existing GWAS data were analyzed using MAGENTA. A microarray analysis was then performed for the identified pathways with human atrial tissues, followed by Gene-Set Enrichment Analysis (GSEA). ResultsMAGENTA identified two novel candidate pathways for AF pathogenesis, the CTCF (CCCTC-binding factor, p = 1.00 × 10−4, FDR q = 1.64 × 10−2) and mTOR pathways (mammalian target of rapamycin, p = 3.00 × 10−4, FDR q = 3.13 × 10−2). The microarray analysis with human atrial tissue using the GSEA indicated that the mTOR pathway was suppressed in AF cases compared with non-AF cases, validating the MAGENTA results, but not CTCF pathway. ConclusionsMAGENTA identified a novel pathway, mTOR, followed by GSEA with human atrial tissue samples. mTOR pathway is a key interface that adapts the change of environments by pressure overload and metabolic perturbation. Our results indicate that the MTOR pathway is involved in the pathogenesis of AF, although the details of the basic mechanism remain unknown and further analysis for causal-relationship of mTOR pathway to AF is required. CTCF pathway is essential for construction of chromatin structure and transcriptional process. The gene-set components of CTCF overlap with those of mTOR in Biocarta.

DNA methylation profiling allows for characterization of atrial and ventricular cardiac tissues and hiPSC-CMs

BackgroundCardiac disease modelling using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) requires thorough insight into cardiac cell type differentiation processes. However, current methods to discriminate different cardiac cell types are mostly time-consuming, are costly and often provide imprecise phenotypic evaluation. DNA methylation plays a critical role during early heart development and cardiac cellular specification. We therefore investigated the DNA methylation pattern in different cardiac tissues to identify CpG loci for further cardiac cell type characterization.ResultsAn array-based genome-wide DNA methylation analysis using Illumina Infinium HumanMethylation450 BeadChips led to the identification of 168 differentially methylated CpG loci in atrial and ventricular human heart tissue samples (n = 49) from different patients with congenital heart defects (CHD). Systematic evaluation of atrial-ventricular DNA methylation pattern in cardiac tissues in an independent sample cohort of non-failing donor hearts and cardiac patients using bisulfite pyrosequencing helped us to define a subset of 16 differentially methylated CpG loci enabling precise characterization of human atrial and ventricular cardiac tissue samples. This defined set of reproducible cardiac tissue-specific DNA methylation sites allowed us to consistently detect the cellular identity of hiPSC-CM subtypes.ConclusionTesting DNA methylation of only a small set of defined CpG sites thus makes it possible to distinguish atrial and ventricular cardiac tissues and cardiac atrial and ventricular subtypes of hiPSC-CMs. This method represents a rapid and reliable system for phenotypic characterization of in vitro-generated cardiomyocytes and opens new opportunities for cardiovascular research and patient-specific therapy.

Abstract 16786: ADP Ribosylation Factor 6 Modulates the Cell Surface Expression of the Acetylcholine Activated Inward Rectifier Potassium Current

Introduction: The ADP ribosylation factor 6 (Arf6), a GTP binding protein, is suggested to play a role in the trafficking of potassium inward rectifiers. We investigated the effects of Arf6 on the cell surface expression of Kir3.1 and Kir3.4, the molecular correlates of the acetylcholine activated inward rectifier potassium current (I KACh ). Hypothesis: The GDP and GTP bound forms of Arf6 modulate the surface expression of I KACh proteins. Methods: We used human embryonic kidney cells stably expressing Kir3.1 and Kir3.4 (GIRK cells). Cell surface I KACh proteins were labeled with TertiapinQ ATTO488, visualized with confocal microscopy, or quantified using flow cytometry in the GIRK cells transiently transfected with Arf6WT (wild type), dominant negative Arf6T44N (GDP bound), or constitutively active Arf6Q67L(GTP bound). Patch clamp was used to quantify the effects of Arf6 on the barium sensitive I KACh current, and coimmunoprecipitation in human atrial tissue was used to probe the interaction between Kir3.1, Kir3.4 and Arf6. Results: In confocal microscopy, Arf6WT, and Arf6T44N localized mainly to the cell membrane, while Arf6Q67L localized to intracellular vesicles. In flow cytometry, Arf6T44N significantly decreased channels expression at the cell surface compared to Arf6WT. Arf6Q67L did not affect cell surface expression compared to Arf6WT. Patch clamp showed that I KACh current density was significantly smaller in Arf6T44N compared to WT and Q67L, consistent with the flow cytometry experiment. Kir3.1, Kir3.4 and Arf6 coimmunoprecipitated in whole cell lysates form human atrial tissue samples. Conclusion: Expression of the I KACh proteins at the cellular membrane is in part modulated by an Arf6-dependent pathway. Constitutive interference with Arf6 cycling between GDP- and GTP-bound forms leads to changes in the cell surface expression of the I KACh proteins.

Abstract 252: Downregulated Striated Muscle Preferentially Expressed Protein Kinase Enhances Susceptibility to Post-Operative Atrial Fibrillation

Post-operative atrial fibrillation (poAF) is the most common perioperative arrhythmia associated with increased hospital morbidity and mortality. It is known that atrial myocyte Ca 2+ handling changes can play a crucial role in AF pathogenesis, although the specific mechanisms involved in poAF have not been elucidated. Our lab has previously identified Striated Muscle Preferentially Expressed Protein Kinase (SPEG) as a binding partner of the major cardiac Ca 2+ release channel RyR2 and its negative regulator JPH2, which are critical for cardiac Ca 2+ homeostasis. We hypothesized that decreased SPEG levels may create an arrhythmogenic substrate promoting poAF development. SPEG protein and mRNA levels were assessed using Western blotting and qRT-PCR, respectively. To establish a mouse model of poAF, we performed thoracotomy in C57Bl/6J mice followed by rapid atrial pacing three days post-operatively to assess for poAF susceptibility. To test for a cause-effect relationship between decreased SPEG and AF susceptibility, we used a cardiac specific tamoxifen-inducible SPEG conditional knockout (cKO) mouse. Rapid atrial pacing was performed in SPEG cKO mice two weeks post-tamoxifen injection prior to onset of heart failure as assessed by echocardiography. In human atrial samples from patients with poAF we found 41±9% lower SPEGβ mRNA expression (n=7, p=0.04) compared to patients with normal sinus rhythm (NSR, n=8), while SPEGα expression was unchanged. Similarly, SPEGβ protein was 45±7% lower in poAF vs NSR patients (n=6, p=0.04). In contrast, SPEGβ mRNA expression was not decreased in patients with persistent or paroxysmal AF. Similar to poAF patients, our novel mouse model exhibited 90±4% lower SPEGβ protein levels (n=4, p=0.03) compared to mice with NSR (n=4) after rapid atrial pacing three days post-thoracotomy. Furthermore, SPEG cKO mice had increased AF duration after rapid atrial pacing (19.43 ± 15.16 s, n=3) compared to controls (0.27 ± 0.27 s, n=6, p=0.01), suggesting that loss of SPEG is sufficient to drive AF. In conclusion, we discovered reduced SPEGβ mRNA and protein in both humans and mice with poAF and established that a loss of SPEG might causally contribute to poAF pathogenesis.

Combined Analysis of Human and Experimental Murine Samples Identified Novel Circulating MicroRNAs as Biomarkers for Atrial Fibrillation.

Recent experimental studies have demonstrated that several microRNAs (miRNAs) expressed in atrial tissue promote a substrate of atrial fibrillation (AF). However, because it has not been fully elucidated whether these experimental data contribute to identifying circulating miRNAs as biomarkers for AF, we used a combined analysis of human serum and murine atrial samples with the aim of identifying these biomarkers for predicting AF.Methods and Results:Comprehensive analyses were performed to screen 733 miRNAs in serum from 10 AF patients and 5 controls, and 672 miRNAs in atrial tissue from 6 inducible atrial tachycardia model mice and 3 controls. We selected miRNAs for which expression was detected in both analyses, and their expression levels were changed in the human analyses, the murine analyses, or both. This screening identified 11 candidate miRNAs. Next, we quantified the selected miRNAs using a quantitative RT-PCR in 50 AF and 50 non-AF subjects. The individual assessment revealed that 4 miRNAs (miR-99a-5p, miR-192-5p, miR-214-3p, and miR-342-5p) were significantly upregulated in AF patients. A receiver-operating characteristics curve indicated that miR-214-3p and miR-342-5p had the highest accuracy. The combination of the 4 miRNAs modestly improved the predictive accuracy for AF (76% sensitivity, 80% specificity). Novel circulating miRNAs were upregulated in the serum of AF patients and might be potential biomarkers of AF.

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

BackgroundThe 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 resultsJNK 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. ConclusionOur 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.

0118 : Fatty infiltration of the subepicardium of the atrial myocardium is replaced by fibrosis during atrial fibrillation in human and sheep

Accumulation of atrial adipose tissue is associated with atrial fibrillation (AF). However the underlying mechanisms remain poorly understood. We examined the relationship between fatty infiltrations in the atrial myocardium and the development of AF structural/fibrotic substrate. Atrial samples, collected in 92 patients during cardiac surgery and in a sheep model of persistent AF, were subjected to a detailed histological analysis. A transition from fatty to fibro-fatty infiltrates with persistence of AF was tested in the AF sheep model using a cardiac magnetic resonance imaging. In sections of human right atrial samples, subepicardial fatty infiltrations were commonly observed in the majority of patients. A clear difference in the appearance and fibrotic content of these fatty infiltrations was observed. Fibro-fatty infiltrates predominated in patients with permanent AF (no AF 37±24% vs paroxysmal AF 50±21% vs permanent AF 64±23%, P <0.001). An inverse correlation between fibrotic remodeling and the amount of subepicardial adipose tissue is suggested. A shift from pure fatty to dense fibro-fatty infiltrations was observed in the left atria of AF sheep. Secondly, cellular inflammation, mainly consisting of functional cytotoxic T lymphocytes, was observed together with adipocyte cell death in human atria. AF is associated with a transformation of fatty into fibro-fatty infiltrations, a process in which cytotoxic lymphocytes might be involved. The replacement of fatty infiltrates by fibrosis could be an early step in the remodelling of the subepicardium and the formation of the AF substrate. The author hereby declares no conflict of interest

0113 : Epicardial progenitors are source of adipocyte in human atria

and aims The accumulation of the adipose tissue (AT) in the sup- and subepicardium of the atrial myocardium is associated with a high risk of atrial fibrillation. Here we addressed the question of the cellular origin of atrial AT. Human right atrial specimen obtained during cardiac surgery were used for histological, biochemical studies and to harvest epicardial progenitors. Cells were characterized using flow cytometry, proteomic and genic expression assays and maintained in culture conditions, in some experiments they were co cultured with human atrial myocytes using a Transwell system. In atrial section, epicardial progenitors expressing the pre-adipocyte factor 1 (Pref-1) were detected in the epicardial and sub-epicardial layer which contained a number of progenitor cells referred as epicardic progenitorderived cells (EPDCs). Some EPDCs expressed Pref-1 suggesting that they could engaged in the adipocyte pathways. This was tested in vitro using EPDCs harvested from human right atrial samples (n=20). From 1 st to 5-6 cells passages, EPDCs underwent an epithelial-to-mesenchymal transition (EMT), acquired mesenchymal phenotypes and could differentiate into osteocyte or chondrocyte. When cultured using an adipogenic medium, around 40±6% of EPDCs cells showed lipid droplet colored with oil red and expressed mature adipocyte perilipin marker. Culture medium enriched with the secretome of human atria too induced the differentiation of EPDCs in adipocytes (n=6). A shift from pre- to mature adipocytes was indicated by the detection of Pref-1 at 7 but not 30 days of culture, it was the opposite for perilipin. Human atrial myocyte could be the source of pro-adipogenic factor. In fact, after 7 days in coculture, EPDCs are differentiated into adipocytes (n=6). Human EPDCs from atria can differentiate into adipocyte; factors secreted by atrial myocytes regulate this differentiation process. The author hereby declares no conflict of interest

Sevoflurane exerts a more marked influence compared with propofol on gene expression in patients undergoing coronary artery bypass graft surgery

The aim of the present study was to elucidate the influence of the anesthetics propofol and sevoflurane on gene expression in patients undergoing coronary artery bypass graft surgery (CABG) and to provide a basis for the selection of the appropriate anesthetic. The gene expression profiles of patients receiving one of the two anesthetics were analyzed prior to and following the induction of anesthesia. GSE4386 microarray data obtained from the Gene Expression Omnibus database was used to identify the differentially expressed genes (DEGs) by significance analysis of the microarray. The data set contained data regarding atrial tissue samples from 40 patients that underwent CABG, and that received either propofol (n=10) or sevoflurane (n=10) or were control subjects (n=20). The 20 control samples comprised the same patients prior to undergoing CABG. The Kyoto Encyclopedia of Genes and Genomes and Gene Ontology (GO) Enrichment Analysis was applied to the DEGs using the Database for Annotation, Visualization and Integration Discovery functional annotation bioinformatics microarray tool. A total of 242 and 560 DEGs were identified in the human atrial samples treated with propofol and sevoflurane, respectively. Among these, 116 upregulated DEGs and no downregulated DEGs were found to be unique to sevoflurane, while 10 upregulated and 212 downregulated DEGs were unique to propofol. The majority of the pathways that were significantly over-represented among the upregulated DEGs were associated with the immune response, such as Toll- and NOD-like receptors and Jak-STAT signaling pathways. GO enrichment analysis revealed that the downregulated DEGs unique to sevoflurane treatment were involved in the immune response and glucose metabolism, while the upregulated DEGs were associated with cellular ion homeostasis and epithelial cell development. Compared with propofol, sevoflurane appeared to exert a more marked effect on biological pathways, such as drug metabolism, glycolysis, cellular ion homeostasis and epithelial cell development.

Atrial fibrillation is associated with the fibrotic remodelling of adipose tissue in the subepicardium of human and sheep atria.

Accumulation of atrial adipose tissue is associated with atrial fibrillation (AF). However, the underlying mechanisms remain poorly understood. We examined the relationship between the characteristics of fatty infiltrates of the atrial myocardium and the history of AF. Atrial samples, collected in 92 patients during cardiac surgery and in a sheep model of persistent AF, were subjected to a detailed histological analysis. In sections of human right atrial samples, subepicardial fatty infiltrations were commonly observed in the majority of patients. A clear difference in the appearance and fibrotic content of these fatty infiltrations was observed. Fibro-fatty infiltrates predominated in patients with permanent AF (no AF: 37 ± 24% vs. paroxysmal AF: 50 ± 21% vs. permanent AF: 64 ± 23%, P < 0.001). An inverse correlation between fibrotic remodelling and the amount of subepicardial adipose tissue suggested the progressive fibrosis of fatty infiltrates with permanent AF. This hypothesis was tested in a sheep model of AF. In AF sheep, an increased accumulation of peri-atrial fat depot was observed on cardiac magnetic resonance imaging and dense fibro-fatty infiltrations predominated in the left atria of AF sheep. Cellular inflammation, mainly consisting of functional cytotoxic T lymphocytes, was observed together with adipocyte cell death in human atria. Atrial fibrillation is associated with the fibrosis of subepicardial fatty infiltrates, a process in which cytotoxic lymphocytes might be involved. This remodelling of the atrial subepicardium could contribute to structural remodelling forming a substrate for AF.

Mitochondrial apoptotic pathway activation in the atria of heart failure patients due to mitral and tricuspid regurgitation

Apoptosis occurs in atrial cardiomyocytes in mitral and tricuspid valve disease. The purpose of this study was to examine the respective roles of the mitochondrial and tumor necrosis factor-α receptor associated death domain (TRADD)-mediated death receptor pathways for apoptosis in the atrial cardiomyocytes of heart failure patients due to severe mitral and moderate-to-severe tricuspid regurgitation. This study comprised eighteen patients (7 patients with persistent atrial fibrillation and 11 in sinus rhythm). Atrial appendage tissues were obtained during surgery. Three purchased normal human left atrial tissues served as normal controls. Moderately-to-severely myolytic cardiomyocytes comprised 59.7±22.1% of the cardiomyocytes in the right atria and 52.4±12.9% of the cardiomyocytes in the left atria of mitral and tricuspid regurgitation patients with atrial fibrillation group and comprised 58.4±24.8% of the cardiomyocytes in the right atria of mitral and tricuspid regurgitation patients with sinus rhythm. In contrast, no myolysis was observed in the normal human adult left atrial tissue samples. Immunohistochemical analysis showed expression of cleaved caspase-9, an effector of the mitochondrial pathways, in the majority of right atrial cardiomyocytes (87.3±10.0%) of mitral and tricuspid regurgitation patients with sinus rhythm, and right atrial cardiomyocytes (90.6±31.4%) and left atrial cardiomyocytes (70.7±22.0%) of mitral and tricuspid regurgitation patients with atrial fibrillation. In contrast, only 5.7% of cardiomyocytes of the normal left atrial tissues showed strongly positive expression of cleaved caspase-9. Of note, none of the atrial cardiomyocytes in right atrial tissue in sinus rhythm and in the fibrillating right and left atria of mitral and tricuspid regurgitation patients, and in the normal human adult left atrial tissue samples showed cleaved caspase-8 expression, which is a downstream effector of TRADD of the death receptor pathway. Immunoblotting of atrial extracts showed that there was enhanced expression of cytosolic cytochrome c, an effector of the mitochondrial pathways, but no expression of membrane TRADD and cytosolic caspase-8 in the right atrial tissue of mitral and tricuspid regurgitation patients with sinus rhythm, and right atrial and left atrial tissues of mitral and tricuspid regurgitation patients with atrial fibrillation. Taken together, this study showed that mitochondrial pathway for apoptosis was activated in the right atria in sinus rhythm and in the left and right atria in atrial fibrillation of heart failure patients due to mitral and tricuspid regurgitation, and this mitochondrial pathway activation may contribute to atrial contractile dysfunction and enlargement in this clinical setting.

Human pluripotent stem cell-derived cardiomyocytes: Genome-wide expression profiling of long-term in vitro maturation in comparison to human heart tissue.

Cardiomyocyte-like cells (CMs) derived from human pluripotent stem cells (hPSCs) present a valuable model for human disease modeling, studying early human development and, potentially, developing cell therapeutic approaches. However, the specification of early hPSC-derived CMs into defined cardiac subtypes such as atrial and ventricular cells is not well understood and, thus, poorly controlled. Moreover, the maturation status of hPSC-CMs is not well defined, yet it is known that these cells undergo at least some degree of maturation upon longer term in vitro culture. To gain insight into this process, and to assess their developmental status, we have recently generated a data set of hPSC-CMs monitoring global changes in gene expression upon long term maintenance in vitro, in comparison to human atrial and ventricular heart samples (GEO accession number GEO: GSE64189). These data present a rich resource for evaluating the maturation status of hPSC-CMs, for identifying suitable markers for subtype-specific gene expression, as well as for the generation of functional hypotheses. Here, we provide additional details and quality checks of this data set, and exemplify how it can be used to identify maturation-associated as well as cardiac subtype-specific markers.

Abstract 17106: Transition From Adipose to Fibrotic Tissue in the Atrial Subepicardium in Humans and in a Sheep Model of Atrial Fibrillation

Epicardial adipose tissue (EAT) is recognized as potentially involved in the pathophysiology of atrial fibrillation (AF). In addition to EAT fatty infiltration commonly observed in myocardium might be also associated with the substrate for AF. We conducted a histological study in 93 human right atrial samples obtained during cardiac surgery and in a sheep model of long-term persistent AF (PAF) induced by atrial tachypacing (24±8 weeks) (15 PAF, 11 SR). Upon Sirius Red and Harris Haematoxylin staining, human atria showed various degree of fatty infiltration starting from the epicardium (rarely from vessels), which could be associated with various degree of subepicardial fibrosis realizing in some cases a true fibro-fatty infiltration. The extent of remodeled epicardium was assessed as % of infiltrating and fibrotic epicardium±adipose tissue to total epicardial length; 44±26 % of the epicardium was remodeled. A multiple regression model (including history of AF, % adipose tissue, age, BodyMassIndex, coronary artery bypass, ejection fraction) significantly predicted the percentage of epicardial remodeling (R=0.501, p=0.003). Only AF and the % adipose tissue were significant predictors (respectively β=0.27;p=0.016 and β=-0.267;p=0.016). To further analyze the relationship between fibro-fatty infiltration and AF, a histological study was performed in LA specimen of a sheep PAF model which revealed 4 grades of subepicardial infiltrates from pure fatty to dense fibro-fatty infiltration (fig A). EAT infiltrates (316) were graded, demonstrating a shift towards more severe grades in the AF group p&lt;0.0001) (fig B). Inflammatory cells were detected in 14 fatty infiltrations (grade 2 and 3) of 6 AF sheep (and only in 1 fatty infiltration of SR sheep). Conclusion: , AF is associated with the transformation of fat into fibro-fatty infiltrations suggesting that the subepicardial adipose tissue plays a role in the atrial fibrotic remodeling.

THREE DIMENSIONAL BLOOD AND CARDIAC STEM CELL SPHERES ENHANCE THE REGENERATIVE PERFORMANCE OF TRADITIONAL MONOLAYER CULTURE CELLS

Combination therapy with blood and cardiac stem cells after myocardial injury provide a complimentary paracrine profile that promotes angiogenesis and the generation of new myocardium. Given evidence that culture of cardiac stem cells (CSCs) as cardiospheres enhances regenerative performance, we test the hypothesis that growing both blood and cardiac stem cells as three dimensional spheres will recapitulate a stem cell niche environment to promote cell survival and cell-mediated repair. Human left atrial appendages and blood samples were obtained from patients undergoing clinically-indicated heart surgery. After seven days of monolayer culture, equivalent numbers of circulating angiogenic cells (CACs) and CSCs were combined in suspension culture which, over 7 days of culture, spontaneously formed spherical aggregates. Immunofluorescent lineage tracking demonstrated that both CACs and CSCs were heterogeneously distributed throughout the 50-80 uM spheres. CACs and CSCs grown together as spheres proliferated slowly as compared to monolayer culture of CSCs alone (2.0±0.4 fold less, p=0.01) or monolayer culture of CACs and CSCs together (1.8±0.18 fold less, p=0.02). Media conditioned under hypoxic, low serum conditions demonstrated that spherical culture did not inhibit cytokine production of angiogenin and VEGF while providing a 1.3±0.3 fold (n=4) increase in the production of the pro-survival cytokine SDF-1α. Consistent with these findings, media conditioned by CACs and CSCs after spherical culture promoted comparable degrees of HUVEC tubule formation when compared to media conditioned by monolayer culture of CACs and CSCs together (41.1±10.6 vs. 53.4±6.5 um respectively; p=0.36) while providing markedly greater recruitment of circulating stem cells (76.6±9.3 vs. 65.7±7.8 respectively; p=0.02). Implantation of sphered CACs and CSCs one week into immunodeficient mice after LAD ligation provided greater myocardial function relative to monolayer cultured CSCs (5±4 vs. 11±1% delta LVEF, p=0.04) with hints of superior regenerative performance when compared to traditional cardiospheres (9±1% delta LVEF, p=0.09). Spherical culture of CACs and CSCs together provides an incremental benefit towards cytokine production, recruitment of circulating stem cells and cell-mediated cardiac repair. This innovative culture technique may provide a means to directly apply stem cell therapy to the clinical setting when cell product yields are severely constrained.

Quantitative Imaging of Preamyloid Oligomers, a Novel Structural Abnormality, in Human Atrial Samples.

Abnormalities in atrial myocardium increase the likelihood of arrhythmias, including atrial fibrillation (AF). The deposition of misfolded protein, or amyloidosis, plays an important role in the pathophysiology of many diseases, including human cardiomyopathies. We have shown that genes implicated in amyloidosis are activated in a cellular model of AF, with the development of preamyloid oligomers (PAOs). PAOs are intermediates in the formation of amyloid fibrils, and they are now recognized to be the cytotoxic species during amyloidosis. To investigate the presence of PAOs in human atrium, we developed a microscopic imaging-based protocol to enable robust and reproducible quantitative analysis of PAO burden in atrial samples harvested at the time of elective cardiac surgery. Using PAO- and myocardial-specific antibodies, we found that PAO distribution was typically heterogeneous within a myocardial sample. Rigorous imaging and analysis protocols were developed to quantify the relative area of myocardium containing PAOs, termed the Green/Red ratio (G/R), for a given sample. Using these methods, reproducible G/R values were obtained when different sections of a sample were independently processed, imaged, and analyzed by different investigators. This robust technique will enable studies to investigate the role of this novel structural abnormality in the pathophysiology of and arrhythmia generation in human atrial tissue.

Characterization of a novel multifunctional resveratrol derivative for the treatment of atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with an increased risk for stroke, heart failure and cardiovascular-related mortality. Candidate targets for anti-AF drugs include a potassium channel K(v)1.5, and the ionic currents I(KACh) and late I(Na), along with increased oxidative stress and activation of NFAT-mediated gene transcription. As pharmacological management of AF is currently suboptimal, we have designed and characterized a multifunctional small molecule, compound 1 (C1), to target these ion channels and pathways. We made whole-cell patch-clamp recordings of recombinant ion channels, human atrial I(Kur), rat atrial I(KACh), cellular recordings of contractility and calcium transient measurements in tsA201 cells, human atrial samples and rat myocytes. We also used a model of inducible AF in dogs. C1 inhibited human peak and late K(v)1.5 currents, frequency-dependently, with IC₅₀ of 0.36 and 0.11 μmol·L(-1) respectively. C1 inhibited I(KACh)(IC₅₀ of 1.9 μmol·L(-1)) and the Na(v)1.5 sodium channel current (IC₅₀s of 3 and 1 μmol·L(-1) for peak and late components respectively). C1 (1 μmol·L(-1)) significantly delayed contractile and calcium dysfunction in rat ventricular myocytes treated with 3 nmol·L(-1) sea anemone toxin (ATX-II). C1 weakly inhibited the hERG channel and maintained antioxidant and NFAT-inhibitory properties comparable to the parent molecule, resveratrol. In a model of inducible AF in conscious dogs, C1 (1 mg·kg(-1)) reduced the average and total AF duration. C1 behaved as a promising multifunctional small molecule targeting a number of key pathways involved in AF.

Activation of prosurvival signaling pathways during the memory phase of volatile anesthetic preconditioning in human myocardium: a pilot study

According to a compelling body of evidence anesthetic preconditioning (APC) attenuates the deleterious consequences of ischemia-reperfusion and protects the heart through a mechanism similar to ischemic preconditioning. The present study was purported to investigate the intracellular signaling pathways activated in human myocardium in response to a preconditioning protocol with two different volatile anesthetics, namely isoflurane and sevoflurane. To this aim, phosphorylation of PKCα and -δ, ERK1/2, Akt, and GSK3β was determined at the end of the APC protocol, in human atrial samples harvested from patients undergoing open-heart surgery. The results demonstrate that preconditioning with volatile anesthetics triggers the activation of PKCδ and -α isoforms and of prosurvival kinases, ERK1/2, and Akt, while inhibiting their downstream target GSK3β during the memory phase.