Chronic kidney disease (CKD) promotes development of cardiac abnormalities and is highly prevalent in patients with heart failure, particularly in those with preserved ejection fraction. CKD is associated with endothelial dysfunction, however, whether CKD can induce impairment of endothelium-to-cardiomyocyte crosstalk leading to impairment of cardiomyocyte function is not known. The sodium-glucose co-transporter 2 inhibitor, empagliflozin, reduced cardiovascular events in diabetic patients with or without CKD, suggesting its potential as a new treatment for heart failure with preserved ejection fraction. We hypothesized that uremic serum from patients with CKD would impair endothelial control of cardiomyocyte relaxation and contraction, and that empagliflozin would protect against this effect. Using a co-culture system of human cardiac microvascular endothelial cells with adult rat ventricular cardiomyocytes to measure cardiomyocyte relaxation and contraction, we showed that serum from patients with CKD impaired endothelial enhancement of cardiomyocyte function which was rescued by empagliflozin. Exposure to uremic serum reduced human cardiac microvascular endothelial cell nitric oxide bioavailability, and increased mitochondrial reactive oxygen species and 3-nitrotyrosine levels, indicating nitric oxide scavenging by reactive oxygen species. Empagliflozin attenuated uremic serum-induced generation of endothelial mitochondrial reactive oxygen species, leading to restoration of nitric oxide production and endotheliummediated enhancement of nitric oxide levels in cardiomyocytes, an effect largely independent of sodiumhydrogen exchanger-1. Thus, empagliflozin restores the beneficial effect of cardiac microvascular endothelial cells on cardiomyocyte function by reducing mitochondrial oxidative damage, leading to reduced reactive oxygen species accumulation and increased endothelial nitric oxide bioavailability.
BackgroundImproved understanding of the interconnectedness of structural remodeling processes in atrial fibrillation (AF) in patients could identify targets for future therapies.MethodsWe present transcriptome sequencing of atrial tissues of patients without AF, with paroxysmal AF, and persistent AF (total n = 64). RNA expression levels were validated in the same and an independent cohort with qPCR. Biological processes were assessed with histological and immunohistochemical analyses.ResultsIn AF patients, epicardial cell gene expression decreased, contrasting with an upregulation of epithelial‐to‐mesenchymal transition (EMT) and mesenchymal cell gene expression. Immunohistochemistry demonstrated thickening of the epicardium and an increased proportion of (myo)fibroblast‐like cells in the myocardium, supporting enhanced EMT in AF. We furthermore report an upregulation of endothelial cell proliferation, angiogenesis, and endothelial signaling. EMT and endothelial cell proliferation concurred with increased interstitial (myo)fibroblast‐like cells and extracellular matrix gene expression including enhanced tenascin‐C, thrombospondins, biglycan, and versican. Morphological analyses discovered increased and redistributed glycosaminoglycans and collagens in the atria of AF patients. Signaling pathways, including cell–matrix interactions, PI3K‐AKT, and Notch signaling that could regulate mesenchymal cell activation, were upregulated.ConclusionOur results suggest that EMT and endothelial cell proliferation work in concert and characterize the (myo)fibroblast recruitment and ECM remodeling of AF. These processes could guide future research toward the discovery of targets for AF therapy.
Background: Atrial fibrosis plays an important role in the development and persistence of atrial fibrillation by promoting reentry. Primary cilia have been identified as a regulator of fibroblasts (FB) activation and extracellular matrix (ECM) deposition. We hypothesized that selective reduction of primary cilia causes increased fibrosis and facilitates reentry.Aim: The aim of this study was to disrupt the formation of primary cilia in FB and examine its consequences on ECM and conduction in a co-culture system of cardiomyocytes (CM) and FB.Materials: Using short interfering RNA (siRNA), we removed primary cilia in neonatal rat ventricular FB by reducing the expression of Ift88 gene required for ciliary assembly. We co-cultured neonatal rat ventricular cardiomyocytes (CM) with FB previously transfected with Ift88 siRNA (siIft88) or negative control siRNA (siNC) for 48 h. We examined the consequences of ciliated fibroblasts reduction on conduction and tissue remodeling by performing electrical mapping, microelectrode, and gene expression measurements.Results: Transfection of FB with siIft88 resulted in a significant 60% and 30% reduction of relative Ift88 expression in FB and CM-FB co-cultures, respectively, compared to siNC. Knockdown of Ift88 significantly increased the expression of ECM genes Fn1, Col1a1 and Ctgf by 38%, 30% and 18%, respectively, in comparison to transfection with siNC. Conduction velocity (CV) was significantly decreased in the siIft88 group in comparison to siNC [11.12 ± 4.27 cm/s (n = 10) vs. 17.00 ± 6.20 (n = 10) respectively, p < 0.05]. The fraction of sites with interelectrode activation block was larger in the siIft88 group than in the siNC group (6.59 × 10−2 ± 8.01 × 10−2 vs. 1.18 × 10−2 ± 3.72 × 10−2 respectively, p < 0.05). We documented spontaneous reentrant arrhythmias in two cultures in the siIft88 group and in none of the siNC group. Action potentials were not significantly different between siNC and siIft88 groups.Conclusion: Disruption of cilia formation by siIft88 causes ECM remodeling and conduction abnormalities. Prevention of cilia loss could be a target for prevention of arrhythmias.
Background Epicardial adipose tissue (EAT) secretome induces fibrosis. Fibrosis, primarily extracellular matrix (ECM) produced by fibroblasts, creates a substrate for atrial fibrillation (AF). Whether the EAT secretome from patients with AF activates human atrial fibroblasts and through which components, remains unexplored. Research aims (a) To investigate if the EAT secretome from patients with versus without AF increases ECM production in atrial fibroblasts. (b) To identify profibrotic proteins and processes in the EAT secretome and EAT from patients with, who will develop (future onset), and without AF. Methods Atrial EAT was obtainded during thoracoscopic ablation (AF, n = 20), or open-heart surgery (future onset and non-AF, n = 35). ECM gene expression of human atrial fibroblasts exposed to the EAT secretome and the proteomes of EAT secretome and EAT were assessed in patients with and without AF. Myeloperoxidase and neutrophil extracellular traps (NETs) were assessed immunohistochemically in patients with paroxysmal, persistent, future onset, and those who remain free of AF (non-AF). Results The expression of COL1A1 and FN1 in fibroblasts exposed to secretome from patients with AF was 3.7 and 4.7 times higher than in patients without AF (p < 0.05). Myeloperoxidase was the most increased protein in the EAT secretome and EAT from patients with versus without AF (FC 18.07 and 21.57, p < 0.005), as was the gene-set neutrophil degranulation. Immunohistochemically, myeloperoxidase was highest in persistent (FC 13.3, p < 0.0001) and increased in future onset AF (FC 2.4, p = 0.02) versus non-AF. Myeloperoxidase aggregated subepicardially and around fibrofatty infiltrates. NETs were increased in patients with persistent versus non-AF (p = 0.03). Conclusion In AF, the EAT secretome induces ECM gene expression in atrial fibroblasts and contains abundant myeloperoxidase. EAT myeloperoxidase was increased prior to AF onset, and both myeloperoxidase and NETs were highest in persistent AF, highlighting the role of EAT neutrophils in the pathophysiology of AF.
Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This study was supported by Netherlands Organization for Health Research and Development Background Myeloperoxidase (MPO), an enzyme most abundantly expressed by neutrophils, is increased in the atrial tissue of AF patients and is associated with treatment failure. Moreover, MPO increases fibrosis and AF susceptibility in mouse atria. However, how MPO promotes AF has yet to be fully established. Purpose To investigate the role of MPO in electrophysiological and structural remodelling. Methods We incubated cultured neonatal rat ventricular myocyte (NRVM) monolayers with 1 μg/mL MPO or vehicle for 24 hours. We then performed electrophysiological mapping using an 8x8 multielectrode array (MEA) setup with 100 μm-sized electrodes and 700 μm inter-electrode distance (n=8 MPO, n=7 control) and recorded action potentials (AP) using a microelectrode during pacing. Additionally, we quantified the number of fibroblasts using vimentin staining (n=5/group) and the expression of extracellular matrix (ECM) genes (COL1A1, COL8A2, FN1, CTGF, TNC, THBS2, MMP2, MMP12) using qPCR (n=4/group). Multielectrode data were analyzed with custom-made software in MATLAB. Statistical analysis was performed in R. Data presented as median [interquartile range]. Results NRVM monolayers incubated with MPO were unresponsive to pacing (5/8 vs 0/7 control) or had a higher pacing threshold (1300 [1000-2500] vs 500 [400-600] μA) (p=0.02). The percent of excitable tissue was lower in the MPO than in the control group, and the propagation of spontaneous activations was slower and more heterogeneous (index: 5 [3.6-9.5] vs 2 [1.5-3], p=0.004). More fractionated electrograms were present in MPO than in the control (29 [25-40] vs 11 [4-23] electrodes per grid, p=0.02). Re-entry was observed in 3/8 MPO-incubated monolayers vs 0/7 controls. Intracellular recordings revealed a less negative resting membrane potential (-50 [-24- -52] vs -69 [-56- -79] mV, p=0.04) and lower AP upstroke velocity (4 [2.5-14] vs 26 [22-28] V/s, p=0.01) in MPO-incubated cardiomyocytes than in the control. Moreover, relative to the control, MPO significantly increased the number of fibroblasts (126 [120-128] vs 87 [70-106] cells/0.26 mm^2, p=0.02) and the expression of MMP12 (1.17 (fold-change), p=0.04), whereas a trend toward increase was observed for other ECM genes, such as COL8A2 (1.5 (fold change), p=0.1). Conclusion Incubation of NVRM with MPO results in depolarization of cardiomyocytes and an increase in the number of fibroblasts and ECM gene expression. Thereby, MPO increases conduction heterogeneity and susceptibility to re-entry.
Introduction. Atrial fibrillation (AF) is more prevalent in men than in women. However, women with AF are more symptomatic, have a worse quality of life, a higher stroke risk and may therefore benefit most from ablation. In this study we aim to identify the risk of recurrent AF after thoracoscopic ablation, and assess the differential impact of the risk factors for recurrence between women and men. Method. This is a single center cohort study, including patients undergoing thoracoscopic ablation for advanced AF between 2008 and 2019. All patients were clinically followed up for two years with quarterly 24 h Holter monitoring and ECGs for the detection of recurrent AF. Left atrial appendage (LAA) tissue was collected for collagen analysis. Results. We included 571 patients, of whom 143 (25%) were women. Women were older than men (63 ± 8.3 y vs. 59 ± 8.5, p < 0.001), but had fewer cardiovascular risk factors, myocardial infarctions (1.4% vs. 6.5%, p = 0.03) and, in particular, vascular disease (7.0% vs. 16.1%, p = 0.01). Women suffered more from AF recurrence, driven by more atrial tachycardias, and sex was an independent risk factor for recurrence (HR1.41 [1.04–1.91], p = 0.028]). The presence of vascular disease was associated with an increased risk for AF recurrence in women, but not in men. In LAA histology, women had more collagen than men, as had patients with persistent compared to paroxysmal AF. Conclusion. Women had 15% more recurrences, driven by more atrial tachycardias, which may be explained by a more fibrotic atrial substrate. What’s new? Women undergoing thoracoscopic AF ablation have a higher risk of recurrent AF, driven by more atrial tachycardias. Among patients with left atrial enlargement or persistent AF, women have worse outcomes than men. Vascular disease was a risk factor for recurrence in women, but not in men. In a histopathologic analysis of the left atrial appendage, women had more collagen than men, as had patients with persistent compared to paroxysmal AF.
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