Atrial Fibroblasts Research Articles

High hydrostatic pressure participates in atrial fibrosis through the p300/p53/Smad3 pathway.

As an independent risk factor of atrial fibrillation (AF), hypertension (HTN) can induce atrial fibrosis through cyclic stretch and hydrostatic pressure. The mechanism by which high hydrostatic pressure promotes atrial fibrosis is unclear yet. p300 and p53/Smad3 play important roles in the process of atrial fibrosis. This study investigated whether high hydrostatic pressure promotes atrial fibrosis by activating the p300/p53/Smad3 pathway. Biochemical experiments were used to study the expression of p300/p53/Smad3 pathway in left atrial appendage (LAA) tissues of patients with sinus rhythm (SR), AF, AF + HTN, and C57/BL6 mice, hypertensive C57/BL6 mice and atrial fibroblasts of mice. To investigate the roles of p300 and p53 in the process of atrial fibrosis, p300 and p53 in mice atrial fibroblasts were knocked in or knocked down, respectively. The expression of p300/p53/Smad3 and fibrotic factors was higher in patients with AF and AF + HTN than those with SR only. The expressions of p300/p53/Smad3 and fibrotic factors increased in hypertensive mice. Curcumin (Cur) and knocking down of p300 reversed the expressions of these factors. 40 mmHg hydrostatic pressure/overexpression of p300 upregulated the expressions of p300/p53/Smad3 and fibrotic factors in mice LAA fibroblasts. While Cur or knocking down p300 reversed these changes. Knocking down/overexpression of p53, the expressions of p53/Smad3 and fibrotic factors also decreased/increased, correspondingly. High hydrostatic pressure promotes atrial fibrosis by activating the p300/p53/Smad3 pathway, which further increases the susceptibility to AF.

In Vitro Mechanical Stimulation to Reproduce the Pathological Hallmarks of Human Cardiac Fibrosis on a Beating Chip and Predict The Efficacy of Drugs and Advanced Therapies.

Cardiac fibrosis is one of the main causes of heart failure, significantly contributing to mortality. The discovery and development of effective therapies able to heal fibrotic pathological symptoms thus remain of paramount importance. Micro-physiological systems (MPS)are recently introduced as promising platforms able to accelerate this finding. Here a 3D in vitro model of human cardiac fibrosis, named uScar, is developed by imposing a cyclic mechanical stimulation to human atrial cardiac fibroblasts (AHCFs) cultured in a 3D beating heart-on-chip and exploited to screen drugs and advanced therapeutics. The sole provision of a cyclic 10% uniaxial strain at 1Hz to the microtissues is sufficient to trigger fibrotic traits, inducing a consistent fibroblast-to-myofibroblast transition and an enhanced expression and production of extracellular matrix (ECM) proteins. Standard of care anti-fibrotic drugs (i.e., Pirfenidone and Tranilast) are confirmed to be efficient in preventing the onset of fibrotic traits in uScar. Conversely, the mechanical stimulation applied to the microtissues limit the ability of a miRNA therapy to directly reprogram fibroblasts into cardiomyocytes (CMs), despite its proved efficacy in 2D models. Such results demonstrate the importance of incorporating in vivo-like stimulations to generate more representative 3D in vitro models able to predict the efficacy of therapies in patients.

LncRNA LENGA sponges miR-378 to promote myocardial fibrosis in atrial fibrillation.

miR-378 is known to suppress myocardial fibrosis, while its upstream regulators have not been identified. lncRNA LENGA is a recently identified lncRNA in cancer biology. We observed the altered expression of LENGA in atrial fibrillation (AF) patients and predicted its interaction with miR-378. We then explored the interaction between LENGA and miR-378 in AF. Angiotensin-II (Ang-II)-induced human atrial cardiac fibroblasts and human atrial muscle tissues were collected and the expression of LENGA and miR-378 was determined by RT-qPCR. The interaction between LENGA and miR-378 was analyzed through bioinformatics analysis and confirmed by RNA pulldown assay. Cell proliferation and collagen production were analyzed through in vitro assay to analyze the role of LENGA and miR-378 in MF. AF patients showed increased expression of LENGA and deceased expression of miR-378 compared to the sinus rhythm group. LENGA and miR-378 interacted with each other, while they are not closely correlated with each other. Overexpression assay showed that LENGA and miR-378 overexpression failed to affect each other's expression. LENGA promoted collagen production and proliferation of Ang-II-induced atrial fibroblasts, while miR-378 played opposite roles. Moreover, LENGA suppressed the function of miR-378. Therefore, LENGA may sponge miR-378 to promote MF in AF.

Abstract 13116: Profiling the Role of Gut Microbiota-Derived Trimethylamine- N -Oxide in Cardiac Fibrosis — Evidence From a Primary Human in vitro Model

Introduction: The gut derived trimethylamine- N -oxide (TMAO) is correlated with increased atrial inflammation and fibrosis and associated with atrial fibrillation, heart failure, and stroke. However, the mechanisms mediating these links remain unresolved. Hypothesis: TMAO and its precursor metabolite L-carnitine (LCART) induce the transformation of atrial fibroblasts into pro-fibrotic phenotypes. Aim: To characterise the association between TMAO and cardiac fibrosis. Methods: Primary human cardiac (atrial) fibroblasts (hCFs) were sourced from healthy donors. hCFs were starved for 24 h and treated with PBS (control), 20 ng/mL transforming growth factor beta 1 (TGFβ1), 10 mM TMAO, or 1.0 mM LCART for 72 h (n=6 in each), then analysed by flow cytometry for α-smooth muscle actin (αSMA) expression. Unbiased proteomics was performed using LC-MS/MS to determine protein expression profile. Results: Analysis of αSMA expression revealed 3 hCF states: quiescent (Fbs), quiescent-to-myofibroblast (intermediate [Fbs-myoFbs]), and fully activated myofibroblast (myoFbs), (Fig-A). Compared to controls, there was no difference in the intermediate state after TGFβ1, TMAO, or LCART treatment (p=0.30). TMAO and LCART resulted in a significant induction of myofibroblast states compared to controls and TGFβ1 group. Unbiased proteomics identified 92, 65, and 43 proteins to be overexpressed and 84, 46, and 78 proteins downregulated following TGFβ1, TMAO, and LCART treatments (Fig-B). Both TGFβ1 and TMAO demonstrated shared enrichment for oxidative stress-regulated ferroptosis pathway (fold enrichment [FE] 31.8 and 22.2, p <.01 and 0.03); LCART showed enrichment for cell motility (FE 3.8, p=0.03). Conclusions: TMAO and its precursor L-carnitine are associated with activation of pro-fibrotic states, which may be due to oxidative stress and abnormal cell migration related mechanisms.

Abstract 15845: Unraveling Postoperative Atrial Fibrillation at the Single-Cell Level

Background: Postoperative atrial fibrillation (poAF) is one of the most common complications after cardiac surgery. It increases the length of stay in the ICU and hospital, increases the risk of stroke, and leads to death. Several clinical factors, including age, obesity, and co-occurring conditions such as prior atrial fibrillation, hypertension, kidney disease, etc., are associated with poAF. We hypothesize that the atrial transcriptome differs between patients who remain in sinus rhythm following surgery and those who develop poAF, allowing the identification of genes, pathways, and cell types necessary for the understanding and treatment of poAF. Methods: Single-cell RNA sequencing (scRNA-seq) was performed to gain insights into the heterogeneous cell populations of the left atrium. It provided an in-depth view of the variability in the cellular response to poAF through the molecular changes within cells, allowing for identifying biomarkers and therapeutic targets. Changes in gene expression associated with poAF were modeled, including batch, sex, age, atrial size, and history of atrial fibrillation and covariates. Results: sc-RNA-seq data of thirty-one human left atrium samples were analyzed. The samples included 14 females and 17 males aged 35-86 years. Cell clusters were identified after the quality check, preprocessing, and normalization. Nine distinct cell types were identified, including atrial cardiomyocytes, fibroblasts, endothelial cells, smooth muscle cells, adipocytes, pericytes, myeloid, lymphoid, and neuronal cells. The atrial cardiomyocytes and fibroblasts were the most abundant identified cell types. The enrichment of these cell types and significant changes in many marker genes in the disease condition suggest their crucial role in their response to the disease. Conclusion: The comprehensive analysis revealed distinct gene expression patterns and cellular heterogeneity. It provided valuable insights into the cellular landscape and transcriptional alterations in poAF. By shedding light on the molecular mechanisms, the findings pave the way for developing targeted interventions to mitigate the impact of poAF.

Abstract 15697: Exposure to Serum From Exclusive Heated Tobacco Product Smokers Induces mTOR Activation and Fibrotic Features in Human Cardiac Fibroblasts

Introduction: Smokers are at higher risk of fibrosis-related atrial fibrillation. The use of heated tobacco products (HTPs) is rising worldwide with unclear effects on health. The SUR-VAPES chronic trial showed that exclusive HTP smokers have increased serum markers of oxidative/endothelial stress versus non-smokers (NS), comparable to cigarette smokers. Hypothesis: We hypothesized that the serological profile of HTP smokers can activate atrial fibroblasts towards a pro-fibrotic phenotype. Methods: CFs were isolated from discarded atrial tissue from elective cardiac surgery procedures, and exposed to serum lots (20 healthy young subjects each) from HTP smokers (mean exclusive use=1.5 years) or clinically matched NSs from the SUR-VAPES Chronic trial. ANOVA testing was applied. Results: CFs treated with HTP serum increased expression of fibrotic genes, including PDGFA (1.18-fold, p<0.01) and THY1 (1.22-fold, p<0.01), and increased aSMA protein levels (1.87-fold, p<0.01), compared to NS serum. Connexin 43 was downregulated at both mRNA (1.17-fold, p<0.05) and protein levels (1.56-fold, p<0.01) in CFs after exposure to HTP serum versus NS. COL1A1 expression increased (1.46-fold, p<0.01), with higher soluble collagen release (1.56-fold, p<0.05). CFs pre-treated with HTP serum released lower levels of cardioprotective proteins (e.g. Adiponectin, Chi3L1, VEGF) by protein arrays/ELISA (p<0.05 vs NS). Paracrine support of CF-conditioned media to endothelial tube formation (63.3±4.7 vs 37.3±5.7 mesh #/well, p<0.05) and to cardiomyocyte viability under starvation (0.60±0.04 vs 0.47±0.01 normOD vs t0, p<0.001) were reduced when pre-treated with HTP versus NS serum. Array screening of 37 phospho-proteins in CFs exposed to HTP serum for 1h found several beneficial pathways blunted, with many inhibiting mTOR, including decreased P-PRAS40 (-2.43-fold) and P-p53 (-2.32-fold, p<0.05). Western blot analysis on CFs after 48h of HTP serum treatment confirmed increased P-mTOR (1.40-fold), and its targets P-S6K (1.39-fold) and P-4EBP1 (1.83-fold, all p<0.05). Conclusions: The serological profile of chronic exclusive HTP smokers induces CF activation and fibrotic features pointing to a potential risk for atrial fibrosis.

MicroRNA-499-5p inhibits transforming growth factor-β1-induced Smad2 signaling pathway and suppresses fibroblast proliferation and collagen synthesis in rat by targeting TGFβ-R1

BackgroundAtrial fibrosis has been recognized as a typical pathological change in atrial fibrillation. Although present evidence suggests that microRNA-499-5p (miR-499-5p) plays an important role in the development of atrial fibrosis, the specific mechanism is not fully understood. Therefore, this study attempted to assess the influence of miR-499-5p on atrial fibroblasts and explore the potential molecular mechanism.MethodsAtrial fibroblasts from sprague dawley rat were respectively transfected with miR-499-5p mimic, miR-499-5p negative control and miR-499-5p inhibitor, atrial fibroblasts without any treatment were also established. Cell counting kit-8 assay and transwell assay were used to detect the proliferation and migration of atrial fibroblasts in each group. Expressions of miR-499-5p, TGF-β1, smad2, α-SMA, collagen-I and TGFβ-R1 in mRNA and protein level were subsequently detected via quantitative real-time polymerase chain reaction and western blot. Furthermore, the prediction of the binding sites of miR-499-5p and TGFβ-R1 was performed via the bioinformatics online software TargetScan and verified by dual luciferase reporter.ResultsBy utilizing miR-499-5p-transfected atrial fibroblasts model, expression of miR-499-5p in the miR-499-5p mimic group was upregulated, while it was downregulated in the miR-499-5p inhibitor group. Upregulated miR-499-5p expression led to a significant decrease in the proliferative and migratory ability of cultured atrial fibroblasts, while downregulated miR-499-5p expression led to a significant increase in the proliferative and migratory ability of cultured atrial fibroblasts. Additionally, upregulated miR-499-5p expression made a significant rise in TGF-β1-induced mRNA and protein expression of TGF-β1, TGFβ-R1, smad2, α-SMA and collagen-I in atrial fibroblasts. Furthermore, results from the dual luciferase reporter conformed that miR-499-5p may repress TGFβ-R1 by binding the 3′UTR of TGFβ-R1 directly.ConclusionsmiR-499-5p is able to inhibit the activation of transforming growth factor β-induced Smad2 signaling and eventually suppressed the proliferation, migration and invasion of atrial fibroblasts and collagen synthesis by targeting TGFβ-R1.

Cfa-circ002203 was upregulated in rapidly paced atria of dogs and involved in the mechanisms of atrial fibrosis.

The role of circular RNAs (circRNAs) in the pathophysiology of cardiovascular disease is gradually being elucidated; however, their roles in atrial fibrillation (AF)-related fibrosis are largely unknown. This study aimed to characterize the different circRNA profiles in the rapid-pacing atria of dogs and explore the mechanisms involved in atrial fibrosis. A rapid right atrial-pacing model was established using electrical stimulation from a pacemaker. After 14 days, atrial tissue was collected for circRNA sequencing analysis. In vitro fibrosis was established by stimulating canine atrial fibroblasts with angiotensin II (Ang II). The fibroblasts were transfected with siRNA and overexpressing plasmids to explore the effects of cfa-circ002203 on fibroblast proliferation, migration, differentiation, and the expression of fibrosis-related proteins. In total, 146 differentially expressed circRNAs were screened, of which 106 were upregulated and 40 were downregulated. qRT-PCR analysis showed that cfa-circ002203 was upregulated in both in vivo and in vitro fibroblast fibrosis models. The upregulation of cfa-circ002203 enhanced proliferation and migration while weakening the apoptosis of fibroblasts. Western blotting showed that cfa-circ002203 overexpression increased the protein expression levels of fibrosis-related indicators (Col I, Col III, MMP2, MMP9, and α-SMA) and decreased the protein expression levels of pro-apoptotic factors (Bax and Caspase 3) in Ang II-induced fibroblast fibrosis. Cfa-circ002203 might serve as an active promoter of the proliferation, migration, and fibrosis of atrial fibroblasts and is involved in AF-induced fibroblast fibrosis.

Atrial epicardial adipose tissue abundantly secretes myeloperoxidase and activates atrial fibroblasts in patients with atrial fibrillation

BackgroundEpicardial 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.MethodsAtrial 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).ResultsThe 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).ConclusionIn 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.

SHP-1 alleviates atrial fibrosis in atrial fibrillation by modulating STAT3 activation.

Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) has a well-established role in myocardial infarction, yet its involvement in atrial fibrosis and atrial fibrillation (AF) has not been elucidated. As cardiac arrhythmias caused by AF are a major global health concern, we investigated whether SHP-1 modulates AF development. The degree of atrial fibrosis was examined using Masson's trichrome staining, and SHP-1 expression in the human atrium was assessed using quantitative polymerase chain reaction (qPCR), immunohistochemistry (IHC), and western blotting (WB). We also examined SHP-1 expression in cardiac tissue from an AF mouse model, as well as in angiotensin II (Ang II)-treated mouse atrial myocytes and fibroblasts. We found that SHP-1 expression was reduced with the aggravation of atrial fibrosis in clinical samples of patients with AF. SHP-1 was also downregulated in the heart tissue of AF mice and Ang II-treated myocytes and fibroblasts, compared with that in the control groups. Next, we demonstrated that SHP-1 overexpression alleviated AF severity in mice by injecting a lentiviral vector into the pericardial space. In Ang II-treated myocytes and fibroblasts, we observed excessive extracellular matrix (ECM) deposition, reactive oxygen species (ROS) generation, and transforming growth factor beta 1 (TGF-β1)/mothers against decapentaplegic homolog 2 (SMAD2) pathway activation, all of which were counteracted by the overexpression of SHP-1. Our WB data showed that STAT3 activation was inversely correlated with SHP-1 expression in samples from patients with AF, AF mice, and Ang II-treated cells. Furthermore, administration of colivelin, a STAT3 agonist, in SHP-1-overexpressing, Ang II-treated myocytes and fibroblasts resulted in higher levels of ECM deposition, ROS generation, and TGF-β1/SMAD2 activation. These findings indicate that SHP-1 regulates AF fibrosis progression by modulating STAT3 activation and is thus a potential treatment target for atrial fibrosis and AF.

MiR-135a Regulates Atrial Fibrillation by Targeting Smad3.

Atrial fibrillation (AF) is the most common arrhythmia in clinical. Atrial fibrosis is a hallmark feature of atrial structural remodeling in AF, which is regulated by the TGF-β1/Smad3 pathway. Recent studies have implicated that miRNAs are involved in the process of AF. However, the regulatory mechanisms of miRNAs remain largely unknown. This study is aimed at investigating the function and regulatory network of miR-135a in AF. In vivo, the plasma was collected from patients with AF and non-AF subjects. Adult SD rats were induced by acetylcholine (ACh) (66 μg/ml)-CaCl2 (10 mg/ml) to establish an AF rat model. In vitro, atrial fibroblasts (AFs), isolated from adult SD rats, were treated with high-frequency electrical stimulation (HES) (12 h) and hypoxia (24 h) to mimic the AF and atrial fibrosis, respectively. miR-135a expression was detected through quantitative real-time polymerase chain reaction (qRT-PCR). The association between miR-135a and Smad3 was speculated by the TargetScan database and confirmed by the luciferase reporter assay. Fibrosis-related genes, Smad3, and TRPM7 were all assessed. The expression of miR-135a was markedly decreased in the plasma of AF patients and AF rats, which was consistent with that in HES-treated and hypoxia-treated AFs. Smad3 was identified as a target of miR-135a. the downregulation of miR-135a was associated with the enhancement of Smad3/TRPM7 expressions in AFs. Additionally, the knockdown of Smad3 significantly reduced the expression of TRPM7 and further inhibited atrial fibrosis. Our study demonstrates that miR-135a regulates AF via Smad3/TRPM7, which is a potential therapeutic target for AF.

PO-01-158 MODELING AN ATRIAL FIBRILLATION-CAUSING TITIN MUTATION USING HUMAN IPSC-DERIVED ATRIAL CARDIOMYOCYTES

Atrial fibrillation (AF) affects more than 33 million people worldwide and, if left untreated, can lead to increased morbidity and mortality. Although AF has been classified as an ‘ion channelopathy’, next-generation sequencing has identified mutations in sarcomeric proteins like TTN, the gene encoding titin, that are associated with AF. Despite progress in understanding the genetic basis of AF, the translation of these discoveries to the bedside care of patients has been limited due to challenges in understanding the myocardial substrate for AF, especially that develops from the mutation in the structural protein. As human atrial tissue is rarely available and animal models are mechanistically limited, our goal is to model AF using mature human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) by a CRISPR-Cas9-mediated single amino acid substitution of a rare missense ethnic-specific mutation (TTN_T32756I) in an immunoglobulin (Ig)-like domain of the A-band of titin that is enriched in AF-causing pathogenic variants. Isogenic TTN_T32756I hiPSC-aCM mutants were generated using the CRISPR-Cas9 technique. Cells were matured either with metabolic conditioning or co-culturing with atrial fibroblasts in a micropatterned platform. We used confocal microscopy to assess sarcomeric integrity, MuscleMotion to assess contractility, and optical voltage mapping to assess the electrophysiological properties of TTN_T32756I hiPSC-aCMs. T32756I results in a non-conservative amino acid change, which in-silico tools have previously predicted results in an impairment in titin function. Here, we show that TTN_T32756I hiPSC-aCM mutants display aberrant contractility (Fig. 1A-C) and Ca2+ homeostasis (Fig. 1D) leading to abnormal electrophysiology (Fig. 1E-F) at the cellular level without compromising the sarcomeric integrity of the atrial cardiomyocytes (Fig. 1G-H). Our results suggest that a single amino acid change could have a profound effect on titin function, further establishing titin as a major signaling hub for contractility. Ongoing studies will not only identify the key pathways and transcription factors involved in the pathogenesis of TTN_ T32756I mutant but also uncover the impact of the mutation on atrial action potential and cardiac ion channel activity. Our study will shed new light on the mechanisms by which TTN mutation cause AF and identify novel therapeutic targets for patients carrying AF-associated rare variants in the structural genes.

High-phosphate diet causes atrial remodeling and increases atrial fibrillation vulnerability via STAT3/NF-κB signaling and oxidative stress.

Hyperphosphatemia is associated with adverse cardiovascular outcomes in both the general population and patients with end-stage renal disease. We evaluated whether high inorganic phosphate (Pi) intake causes atrial remodeling and increased atrial fibrillation (AF) risk. The 5/6 nephrectomized chronic kidney disease (CKD) mice were fed a high-Pi (2%) diet for 10 weeks. AF vulnerability was evaluated through transesophageal burst atrial pacing. Phosphoproteomic, Western blotting, and immunohistochemistry were used to evaluate the effects of high Pi in atrial fibroblasts, atrial myocytes, and HL-1 myocytes. CKD and sham mice fed a high-Pi diet exhibited increased AF vulnerability, atrial fibrosis, and oxidative stress compared with mice fed a normal diet. Compared with normal (1 mM) Pi, high (2 mM) Pi significantly increased the activity of atrial fibroblasts and mitochondrial oxidative stress. Phosphoproteomic analysis revealed that compared with normal Pi, high Pi considerably increased the phosphorylation of intracellular proteins in atrial fibroblasts, including proteins related to NF-κB signaling and STAT3. Inhibition of NF-κB, STAT3, and Nox4 by small interfering RNA reduced the high-Pi-induced expression of collagen. In HL-1 myocytes, the high Pi induced the degradation of myofibril proteins and hyperphosphorylation of RyR2, which was abolished by Nox4 and CaMKII inhibition. Switching back to a normal-Pi diet improved the atrial abnormalities induced by high-Pi diet. High-Pi intake causes atrial structural and electrical remodeling and increases AF vulnerability, which is mediated through STAT3/NF-κB signaling and oxidative stress. High dietary Pi intake can exert detrimental effects on atria and may increase AF risk.

Correction to: Myocardial Cytoskeletal Adaptations in Advanced Kidney Disease.

HomeJournal of the American Heart AssociationVol. 12, No. 6Correction to: Myocardial Cytoskeletal Adaptations in Advanced Kidney Disease Open AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citations ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toOpen AccessCorrectionPDF/EPUBCorrection to: Myocardial Cytoskeletal Adaptations in Advanced Kidney Disease Originally published9 Mar 2023https://doi.org/10.1161/JAHA.121.027645Journal of the American Heart Association. 2023;12:e027645This article corrects the followingMyocardial Cytoskeletal Adaptations in Advanced Kidney DiseaseOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: March 9, 2023: Ahead of Print In the article by Arvin Halim et al, “Myocardial Cytoskeletal Adaptations in Advanced Kidney Disease” which published online February 18, 2022 (J Am Heart Assoc. 2022;11:e022991. DOI: 10.1161/JAHA.121.022991) and was included in the March 1, 2022 issue of the journal, corrections were needed.Figure 1 indicated that samples in all 3 cohorts (50 total) underwent RNA sequencing. The figure has been revised to show that that only a subset of samples from 2 cohorts underwent the final transcriptomic analysis.On page 3 of the Methods section, in the first paragraph under the heading Next Generation RNA Sequencing, the following sentence described the removal of samples from the final analysis: “Samples with low total reads (<1×106) were removed from further analyses.”This sentence has been revised to clarify the number of samples that were removed from the final analysis and the reasons for their removal: “Several samples were removed from the final analysis because of poor sample quality (ie, low total reads of <1×106) and technical batch effect that could not be corrected. The final analysis includes 13 hemodialysis and 5 healthy control hearts.”On page 4 of the Methods section, in the last paragraph under the heading Next Generation RNA Sequencing, the authors stated, “All RNA‐seq data have been deposited into the NCBI Gene Expression Omnibus (GEO) repository (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE160145).”The following text has been added to provide additional detail about the samples deposited and to indicate which samples were included in the final transcriptomic analyses: “Ex vivo human heart samples are designated as N (Control), CAD (HTN), RF (HD), and PD (peritoneal dialysis group) followed by sample number. Heart samples that were included in the final RNA‐seq analysis were: N10, N12, N13, N14, N15, RF2, RF3, RF4, RF8, RF9, RF11, RF12, RF13, RF15_2, RF16, RF17, RF18, and RF19. In vitro human cell samples are designated as A (atrial fibroblasts), V (ventricular fibroblasts), M (mixed atrial and ventricular fibroblasts), ‐N (Control), ‐P (elevated phosphate treatment), ‐C (elevated calcium treatment), and ‐PC (combined elevated phosphate and elevated calcium treatment) followed by technical repeat number (1‐3). Cell samples that were included in the supplemental final RNA‐seq analysis were: VN1, VN2, VN3, VPC1_2, VPC2, and VPC3.”On page 5 of the Results section, in the first paragraph under the heading Transcriptomic Profiling of Human Hearts, the RNA sequencing analysis was described as follows: “Among the top 1000 differentially expressed genes between hemodialysis and healthy control hearts, 5 were significantly upregulated and 21 were significantly downregulated in hearts from the hemodialysis group compared with control (P<0.05) (Figure 3A).”This sentence has been revised to provide additional information about the samples included in the analysis: “Among the top 1000 differentially expressed genes between hemodialysis (n=13) and healthy control (n=5) hearts, 5 were significantly upregulated and 21 were significantly downregulated in hearts from the hemodialysis group compared with control (P<0.05) (Figure 3A).”The authors regret the errors.The online version of the article has been updated and is available here: https://www.ahajournals.org/doi/10.1161/JAHA.121.022991. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMyocardial Cytoskeletal Adaptations in Advanced Kidney DiseaseKenneth Lim, et al. Journal of the American Heart Association. 2022;11 March 21, 2023Vol 12, Issue 6Article InformationMetrics Copyright © 2023 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley BlackwellThis is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.https://doi.org/10.1161/JAHA.121.027645PMID: 36892070 Originally publishedMarch 9, 2023 PDF download

Comparative analysis of the cardiomyocyte differentiation potential of induced pluripotent stem cells reprogrammed from human atrial or ventricular fibroblasts.

Background: We had shown that cardiomyocytes (CMs) were more efficiently differentiated from human induced pluripotent stem cells (hiPSCs) when the hiPSCs were reprogrammed from cardiac fibroblasts rather than dermal fibroblasts or blood mononuclear cells. Here, we continued to investigate the relationship between somatic-cell lineage and hiPSC-CM production by comparing the yield and functional properties of CMs differentiated from iPSCs reprogrammed from human atrial or ventricular cardiac fibroblasts (AiPSC or ViPSC, respectively). Methods: Atrial and ventricular heart tissues were obtained from the same patient, reprogrammed into AiPSCs or ViPSCs, and then differentiated into CMs (AiPSC-CMs or ViPSC-CMs, respectively) via established protocols. Results: The time-course of expression for pluripotency genes (OCT4, NANOG, and SOX2), the early mesodermal marker Brachyury, the cardiac mesodermal markers MESP1 and Gata4, and the cardiovascular progenitor-cell transcription factor NKX2.5 were broadly similar in AiPSC-CMs and ViPSC-CMs during the differentiation protocol. Flow-cytometry analyses of cardiac troponin T expression also indicated that purity of the two differentiated hiPSC-CM populations (AiPSC-CMs: 88.23% ± 4.69%, ViPSC-CMs: 90.25% ± 4.99%) was equivalent. While the field-potential durations were significantly longer in ViPSC-CMs than in AiPSC-CMs, measurements of action potential duration, beat period, spike amplitude, conduction velocity, and peak calcium-transient amplitude did not differ significantly between the two hiPSC-CM populations. Yet, our cardiac-origin iPSC-CM showed higher ADP and conduction velocity than previously reported iPSC-CM derived from non-cardiac tissues. Transcriptomic data comparing iPSC and iPSC-CMs showed similar gene expression profiles between AiPSC-CMs and ViPSC-CMs with significant differences when compared to iPSC-CM derived from other tissues. This analysis also pointed to several genes involved in electrophysiology processes responsible for the physiological differences observed between cardiac and non-cardiac-derived cardiomyocytes. Conclusion: AiPSC and ViPSC were differentiated into CMs with equal efficiency. Detected differences in electrophysiological properties, calcium handling activity, and transcription profiles between cardiac and non-cardiac derived cardiomyocytes demonstrated that 1) tissue of origin matters to generate a better-featured iPSC-CMs, 2) the sublocation within the cardiac tissue has marginal effects on the differentiation process.

Empagliflozin suppressed cardiac fibrogenesis through sodium-hydrogen exchanger inhibition and modulation of the calcium homeostasis

BackgroundThe novel sodium-glucose co-transporter 2 inhibitor (SGLT2i) potentially ameliorates heart failure and reduces cardiac arrhythmia. Cardiac fibrosis plays a pivotal role in the pathophysiology of HF and atrial myopathy, but the effect of SGLT2i on fibrogenesis remains to be elucidated. This study investigated whether SGLT2i directly modulates fibroblast activities and its underlying mechanisms.Methods and resultsMigration, proliferation analyses, intracellular pH assay, intracellular inositol triphosphate (IP3) assay, Ca2+ fluorescence imaging, and Western blotting were applied to human atrial fibroblasts. Empagliflozin (an SGLT2i, 1, or 5 μmol/L) reduced migration capability and collagen type I, and III production. Compared with control cells, empagliflozin (1 μmol/L)- treated atrial fibroblasts exhibited lower endoplasmic reticulum (ER) Ca2+ leakage, Ca2+ entry, inositol trisphosphate (IP3), lower expression of phosphorylated phospholipase C (PLC), and lower intracellular pH. In the presence of cariporide (an Na+-H+ exchanger (NHE) inhibitor, 10 μmol/L), control and empagliflozin (1 μmol/L)-treated atrial fibroblasts revealed similar intracellular pH, ER Ca2+ leakage, Ca2+ entry, phosphorylated PLC, pro-collagen type I, type III protein expression, and migration capability. Moreover, empagliflozin (10 mg/kg/day orally for 28 consecutive days) significantly increased left ventricle systolic function, ß-hydroxybutyrate and decreased atrial fibrosis, in isoproterenol (100 mg/kg, subcutaneous injection)-induced HF rats.ConclusionsBy inhibiting NHE, empagliflozin decreases the expression of phosphorylated PLC and IP3 production, thereby reducing ER Ca2+ release, extracellular Ca2+ entry and the profibrotic activities of atrial fibroblasts.

Increased β1-adrenergic receptor antibody confers a vulnerable substrate for atrial fibrillation via mediating Ca2+ mishandling and atrial fibrosis in active immunization rabbit models.

Autoimmune disorder is the emerging mechanism of atrial fibrillation (AF). The β1-adrenergic receptor antibody (β1-AAb) is associated with AF progress. Our study aims to investigate whether β1-AAbs involves in atrial vulnerable substrate by mediating Ca2+ mishandling and atrial fibrosis in autoimmune associated AF. Active immunization models were established via subcutaneous injection of the second extracellular loop (ECL2) peptide for β1 adrenergic receptor (β1AR). Invasive electrophysiologic study and ex vivo optical mapping were used to evaluate the changed electrophysiology parameters and calcium handling properties. Phospho-proteomics combined with molecular biology assay were performed to identify the potential mechanisms of remodeled atrial substrate elicited by β1-AAbs. Exogenous β1-AAbs were used to induce the cellular phenotypes of HL-1 cells and atrial fibroblasts to AF propensity. β1-AAbs aggravated the atrial electrical instability and atrial fibrosis. Bisoprolol alleviated the alterations of action potential duration (APD), Ca2+ transient duration (CaD), and conduction heterogeneity challenged by β1-AAbs. β1-AAbs prolonged calcium transient refractoriness and promoted arrhythmogenic atrial alternans and spatially discordant alternans, which were partly counteracted through blocking β1AR. Its underlying mechanisms are related to β1AR-drived CaMKII/RyR2 activation of atrial cardiomyocytes and the myofibroblasts phenotype formation of fibroblasts. Suppressing β1-AAbs effectively protects the atrial vulnerable substrate by ameliorating intracellular Ca2+ mishandling and atrial fibrosis, preventing the process of the autoimmune associated AF.

Stiff substrates inhibit collagen accumulation via integrin α2β1, FAK and Src kinases in human atrial fibroblast and myofibroblast cultures derived from patients with aortal stenosis

The aim of the study was to confirm whether cell substrate stiffness may participate in the regulation of fibrosis. The involvement of integrin α2β1, focal adhesion kinase (FAK) and Src kinase in signal transmission was investigated. Human atrial fibroblasts and myofibroblasts were cultured in both soft (2.23 ± 0.8 kPa) and stiff (8.28 ± 1.06 kPa) polyacrylamide gels. The cells were derived from the right atrium of patients with aortal stenosis undergoing surgery. The isolated cells, identified as fibroblasts or myofibroblasts, were stained positively with α smooth muscle actin, vimentin and desmin. The cultures settled on stiff gel demonstrated lower intracellular collagen and collagen type I telopeptide (PICP) levels; however, no changes in α1 chain of procollagen type I and III expression were noted. Inhibition of α2β1 integrin by TC-I 15 (10−7 and 10−8 M) or α2 integrin subunit silencing augmented intracellular collagen level. Moreover, FAK or Src kinase inhibitors increased collagen content within the culture. Lower TIMP4 secretion was reported within the stiff gel cultures but neither MMP 2 nor TIMP-1, 2 or 3 release was altered. The stiff substrate cultures also demonstrated lower interleukin-6 release. Substrate stiffness modified collagen deposition within the atrial fibroblast and myofibroblast cultures. The elasticity of the cellular environment exerts a regulatory influence on both synthesis and breakdown of collagen. Integrin α2β1, FAK and Src kinase activity participates in signal transmission, which may influence fibrosis in the atria of the human heart.

Intermittent fasting attenuates obesity-related atrial fibrillation via SIRT3-mediated insulin resistance mitigation

ObjectiveAtrial fibrillation (AF) is the most common tachyarrhythmia in urgent need of therapeutic optimization. Obesity engenders AF, and its pathogenesis is closely intertwined with insulin resistance (IR), but mechanism-based management is still underinvestigated. Intermittent fasting (IF) is a novel lifestyle intervention that mitigates IR, a potential AF driver, yet whether IF can prevent obesity-related AF remains elusive. Here, we aimed to evaluate the impacts of short-term IF on AF and to uncover the underlying mechanism. MethodsWe subjected obese mice (high-fat diet for 8-week) to IF (alternative-day fasting for another 5-week) for AF vulnerability and substrate formation assessment, and similarly treated neonatal atrial cardiomyocytes (NRCMs) and fibroblasts (NRCFs) (palmitate, 200 μM) with IF (alternative-day short-term starvation for 8-day) for mechanism investigation. ResultsObese mice were prone to AF and atrial remodeling. IF reduced AF inducibility, duration, and reversed atrial remodeling including channel disturbance, left atrial dilation, cardiac hypertrophy and fibrosis in obese mice independent of weight loss. Mechanistically, IF up-regulated the SIRT3 protein level both in vivo and in vitro, and pharmacologic inhibition (3-(1H-1,2,3-Triazol-4-yl) pyridine, 50 μM) and genetic suppression of SIRT3 could attenuate the IF-mediated benefits against hypertrophy and fibrosis. Furthermore, IF activated AMPK and Akt signaling, two positive downstream targets of SIRT3, and inactivated HIF1α signaling, a negative downstream target of SIRT3 in both obese mice atria and palmitate-treated cells, while inhibition of SIRT3 reversed these effects. ConclusionIF prevents obesity-related AF via SIRT3-mediated IR mitigation, thus representing a feasible lifestyle intervention to improve AF management.

ANGPTL4 suppresses the profibrogenic functions of atrial fibroblasts induced by angiotensin II by up-regulating PPARγ.

The present study's objective was to investigate the association between angiopoietin-like 4 (ANGPTL4) levels and the prognosis of Atrial fibrillation (AF), the causative effect in angiotensin II- (Ang II) induced AF, and its underlying mechanisms. Baseline serum ANGPTL-4 concentrations were measured in 130 patients with AF. Rat atrial fibroblasts were isolated from 14-day-old SD rats and transfected with Ang II treatment. Transfected cells were divided into: The control group, ANGPTL4-OE group, Ang II group, and Ang II+ANGPTL4-OE group. Transfected cells were used to analyze fibroblasts' proliferation, migration, and collagen production at the cellular level. RT-qPCR and western blotting evaluated the ANGPTL4-targeted gene and PPARγ-Akt pathway. In patients with AF, serum ANGPTL4 concentrations decreased significantly compared with the healthy group. ANGPTL4 mRNA and protein expressions were significantly down-regulated in Ang II-induced cardiac fibroblasts. ANGPTL4 overexpression potentially attenuated Ang IIinduced fibroblast proliferation, migration, and collagen production in atrial tissue. ANGPTL4 inhibited the signaling proteins, such as PPARγ, α-SMA, and Akt. Our experimental data speculate that ANGPTL4 is a key factor in regulating AF progression. Therefore, increasing ANGPTL4 expression could be an effective strategy for AF treatment.