Understanding the cell fate and behavior of progenitors at the origin of the mouse cardiac mitral valve.

Congenital heart malformations include mitral valve defects, which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal undergoes an endothelial-to-mesenchymal transition to give rise to mitral valvular cells. However, the identity and fate decisions of these progenitors as well as the behavior and distribution of their derivatives in valve leaflets remain unknown. We used single-cell RNA sequencing (scRNA-seq) of genetically labeled endocardial cells and microdissected mouse embryonic and postnatal mitral valves to characterize the developmental road. We defined the metabolic processes underlying the specification of the progenitors and their contributions to subtypes of valvular cells. Using retrospective multicolor clonal analysis, we describe specific modes of growth and behavior of endocardial cell-derived clones, which build up, in a proper manner, functional valve leaflets. Our data identify how both genetic and metabolic mechanisms specifically drive the fate of a subset of endocardial cells toward their distinct clonal contribution to the formation of the valve.

Dynamic changes in mitral valve extracellular matrix, tissue mechanics and function in a mouse model of Marfan syndrome.

OBJECTIVE: Mouse models of Marfan syndrome (MFS) with Fibrillin 1 (Fbn1) variant C1041G exhibit cardiovascular abnormalities, including myxomatous valve disease (MVD) and aortic aneurism, with structural extracellular matrix (ECM) dysregulation. In this study, we examine the structure-function-mechanics relations of the mitral valve related to specific transitions in ECM composition and organization in progressive MVD in MFS mice from Postnatal day (P)7 to 1 year-of-age. APPROACH AND RESULTS: Mechanistic links between mechanical forces and biological changes in MVD progression were examined in Fbn1C1041G/+ MFS mice. By echocardiography, mitral valve dysfunction is prevalent at 2 months with a decrease in cardiac function at 6 months, followed by a preserved cardiac function at 12 months. Mitral valve (MV) regurgitation occurs in a subset of mice at 2-6 months, while progressive dilatation of the aorta occurs from 2 to 12 months. Mitral valve tissue mechanical assessments using a uniaxial Permeabilizable Fiber System demonstrate decreased stiffness of MFS MVs at all stages. Histological and microscopic analysis of ECM content, structure, and fiber orientation demonstrate that alterations in ECM mechanics, composition, and organization precede functional abnormalities in Fbn1C1041G/+MFS MVs. At 2 months, ECM abnormalities are detected with an increase in proteoglycans and decreased stiffness of the mitral valve. By 6-12 months, collagen fiber remodeling is increased with abnormal fiber organization in MFS mitral valve leaflets. At the same time, matrifibrocyte gene expression characteristic of collagen-rich connective tissue is increased, as detected by RNA in situ hybridization and qPCR. Together, these studies demonstrate early prevalence of proteoglycans at 2 months followed by upregulation of collagen structure and organization with age in MVs of MFS mice. CONCLUSIONS: Altogether, our data indicate dynamic regulation of mitral valve structure, tissue mechanics, and function that reflect changes in ECM composition, organization, and gene expression in progressive MVD. Notably, increased collagen fiber organization and orientation, potentially dependent on increased matrifibrocyte cell activity, is apparent with altered mitral valve mechanics and function in aging MFS mice.

Epicardial deletion of Sox9 leads to myxomatous valve degeneration and identifies Cd109 as a novel gene associated with valve development.

Epicardial-derived cells (EPDCs) are involved in the regulation of myocardial growth and coronary vascularization and are critically important for proper development of the atrioventricular (AV) valves. SOX9 is a transcription factor expressed in a variety of epithelial and mesenchymal cells in the developing heart, including EPDCs. To determine the role of SOX9 in epicardial development, an epicardial-specific Sox9 knockout mouse model was generated. Deleting Sox9 from the epicardial cell lineage impairs the ability of EPDCs to invade both the ventricular myocardium and the developing AV valves. After birth, the mitral valves of these mice become myxomatous with associated abnormalities in extracellular matrix organization. This phenotype is reminiscent of that seen in humans with myxomatous mitral valve disease (MVD). An RNA-seq analysis was conducted in an effort to identify genes associated with this myxomatous degeneration. From this experiment, Cd109 was identified as a gene associated with myxomatous valve pathogenesis in this model. Cd109 has never been described in the context of heart development or valve disease. This study highlights the importance of SOX9 in the regulation of epicardial cell invasion-emphasizing the importance of EPDCs in regulating AV valve development and homeostasis-and reports a novel expression profile of Cd109, a gene with previously unknown relevance in heart development.

Mitral valve transcriptome analysis in thirty-four age-matched Cavalier King Charles Spaniels with or without congestive heart failure caused by myxomatous mitral valve disease.

We here report the results of a mitral valve transcriptome study designed to identify genes and molecular pathways involved in development of congestive heart failure (CHF) following myxomatous mitral valve disease (MMVD) in dogs. The study is focused on a cohort of elderly age-matched dogs (n = 34, age ~ 10 years) from a single breed-Cavalier King Charles Spaniels (CKCS)-with a high incidence of MMVD. The cohort comprises 19 dogs (10♀, 9♂) without MMVD-associated CHF, and 15 dogs (6♀, 9♂) with CHF caused by MMVD; i.e., we compare gene expression in breed and age-matched groups of dogs, which only differ with respect to CHF status. We identify 56 genes, which are differentially expressed between the two groups. In this list of genes, we confirm an enrichment of genes related to the TNFß-signaling pathway, extracellular matrix organization, vascular development, and endothelium damage, which also have been identified in previous studies. However, the genes with the greatest difference in expression between the two groups are CNTN3 and MYH1. Both genes encode proteins, which are predicted to have an effect on the contractile activity of myocardial cells, which in turn may have an effect on valvular performance and hemodynamics across the mitral valve. This may result in shear forces with impact on MMVD progression.

PROX1 Inhibits PDGF-B Expression to Prevent Myxomatous Degeneration of Heart Valves.

BACKGROUND: Cardiac valve disease is observed in 2.5% of the general population and 10% of the elderly people. Effective pharmacological treatments are currently not available, and patients with severe cardiac valve disease require surgery. PROX1 (prospero-related homeobox transcription factor 1) and FOXC2 (Forkhead box C2 transcription factor) are transcription factors that are required for the development of lymphatic and venous valves. We found that PROX1 and FOXC2 are expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 and FOXC2 regulate cardiac valve development and disease is not known. METHODS: We used histology, electron microscopy, and echocardiography to investigate the structure and functioning of heart valves from Prox1ΔVEC mice in which Prox1 was conditionally deleted from VECs. Isolated valve endothelial cells and valve interstitial cells were used to identify the molecular mechanisms in vitro, which were tested in vivo by RNAScope, additional mouse models, and pharmacological approaches. The significance of our findings was tested by evaluation of human samples of mitral valve prolapse and aortic valve insufficiency. RESULTS: Histological analysis revealed that the aortic and mitral valves of Prox1ΔVEC mice become progressively thick and myxomatous. Echocardiography revealed that the aortic valves of Prox1ΔVEC mice are stenotic. FOXC2 was downregulated and PDGF-B (platelet-derived growth factor-B) was upregulated in the VECs of Prox1ΔVEC mice. Conditional knockdown of FOXC2 and conditional overexpression of PDGF-B in VECs recapitulated the phenotype of Prox1ΔVEC mice. PDGF-B was also increased in mice lacking FOXC2 and in human mitral valve prolapse and insufficient aortic valve samples. Pharmacological inhibition of PDGF-B signaling with imatinib partially ameliorated the valve defects of Prox1ΔVEC mice. CONCLUSIONS: PROX1 antagonizes PDGF-B signaling partially via FOXC2 to maintain the extracellular matrix composition and prevent myxomatous degeneration of cardiac valves.

Serum Proteomic Profiles Reflect the Stages of Myxomatous Mitral Valve Disease in Dogs.

Canine myxomatous mitral valve disease (MMVD) is similar to Barlow's form of MMVD in humans. These valvulopathies are complex, with varying speeds of progression. We hypothesized that the relative abundances of serum proteins would help identify the consecutive MMVD stages and discover new disease pathways on a systemic level. To identify distinction-contributing protein panels for disease onset and progression, we compared the proteomic profiles of serum from healthy dogs and dogs with different stages of naturally occurring MMVD. Dogs were divided into experimental groups on the basis of the left-atrium-to-aorta ratio and normalized left ventricular internal dimension in diastole values. Serum was collected from healthy (N = 12) dogs, dogs diagnosed with MMVD in stages B1 (N = 13) and B2 (N = 12) (asymptomatic), and dogs diagnosed with MMVD in chronic stage C (N = 13) (symptomatic). Serum biochemistry and selected ELISAs (galectin-3, suppression of tumorigenicity, and asymmetric dimethylarginine) were performed. Liquid chromatography-mass spectrometry (LC-MS), tandem mass tag (TMT) quantitative proteomics, and statistical and bioinformatics analysis were employed. Most of the 21 serum proteins with significantly different abundances between experimental groups (p < 0.05, FDR ˂ 0.05) were classified as matrix metalloproteinases, protease inhibitors, scaffold/adaptor proteins, complement components, anticoagulants, cytokine, and chaperone. LC-MS TMT proteomics results obtained for haptoglobin, clusterin, and peptidase D were further validated analytically. Canine MMVD stages, including, for the first time, asymptomatic B1 and B2 stages, were successfully distinguished in dogs with the disease and healthy dogs on the basis of the relative abundances of a panel of specific serum proteins. Most proteins with significantly different abundances were involved in immune and inflammatory pathways. Their role in structural remodeling and progression of canine MMVD must be further investigated. Further research is needed to confirm the resemblance/difference with human MMVD. Proteomics data are available via ProteomeXchange with the unique dataset identifier PXD038475.

TGF-ß-induced PI3K/AKT/mTOR pathway controls myofibroblast differentiation and secretory phenotype of valvular interstitial cells through the modulation of cellular senescence in a naturally occurring in vitro canine model of myxomatous mitral valve disease.

PI3K/AKT/mTOR signalling contributes to several cardiovascular disorders. The aim of this study was to examine the PI3K/AKT/mTOR pathway in myxomatous mitral valve disease (MMVD). Double-immunofluorescence examined expression of PI3K and TGF-ß1 in canine valves. Valve interstitial cells (VICs) from healthy or MMVD dogs were isolated and characterized. Healthy quiescent VICs (qVICs) were treated with TGF-ß1 and SC-79 to induce activated myofibroblast phenotypes (aVICs). Diseased valve-derived aVICs were treated with PI3K antagonists and expression of RPS6KB1 (encoding p70 S6K) was modulated using siRNA and gene overexpression. SA-ß-gal and TUNEL staining were used to identify cell senescence and apoptosis, and qPCR and ELISA to examine for senescence-associated secretory phenotype. Protein immunoblotting was used to examine expression of phosphorylated and total proteins. TGF-ß1 and PI3K are highly expressed in mitral valve tissues. Activation of PI3K/AKT/mTOR and increased expression of TGF-ß are found in aVICs. TGF-ß transitions qVICs to aVICs by upregulation of PI3K/AKT/mTOR. Antagonism of PI3K/AKT/mTOR reverses aVIC myofibroblast transition by inhibiting senescence and promoting autophagy. Upregulation of mTOR/S6K induces transformation of senescent aVICs, with reduced capacity for apoptosis and autophagy. Selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy. TGF-ß-induced PI3K/AKT/mTOR signalling contributes to MMVD pathogenesis and plays crucial roles in the regulation of myofibroblast differentiation, apoptosis, autophagy and senescence in MMVD.

Decreased serotonin transporter activity in the mitral valve contributes to progression of degenerative mitral regurgitation.

Degenerative mitral valve (MV) regurgitation (MR) is a highly prevalent heart disease that requires surgery in severe cases. Here, we show that a decrease in the activity of the serotonin transporter (SERT) accelerates MV remodeling and progression to MR. Through studies of a population of patients with MR, we show that selective serotonin reuptake inhibitor (SSRI) use and SERT promoter polymorphism 5-HTTLPR LL genotype were associated with MV surgery at younger age. Functional characterization of 122 human MV samples, in conjunction with in vivo studies in SERT-/- mice and wild-type mice treated with the SSRI fluoxetine, showed that diminished SERT activity in MV interstitial cells (MVICs) contributed to the pathophysiology of MR through enhanced serotonin receptor (HTR) signaling. SERT activity was decreased in LL MVICs partially because of diminished membrane localization of SERT. In mice, fluoxetine treatment or SERT knockdown resulted in thickened MV leaflets. Similarly, silencing of SERT in normal human MVICs led to up-regulation of transforming growth factor ß1 (TGFß1) and collagen (COL1A1) in the presence of serotonin. In addition, treatment of MVICs with fluoxetine not only directly inhibited SERT activity but also decreased SERT expression and increased HTR2B expression. Fluoxetine treatment and LL genotype were also associated with increased COL1A1 expression in the presence of serotonin in MVICs, and these effects were attenuated by HTR2B inhibition. These results suggest that assessment of both 5-HTTLPR genotype and SERT-inhibiting treatments may be useful tools to risk-stratify patients with MV disease to estimate the likelihood of rapid disease progression.

Multimodality imaging and transcriptomics to phenotype mitral valve dystrophy in a unique knock-in Filamin-A rat model.

AIMS: Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD. METHODS AND RESULTS: Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-ß and inflammation in the disease. CONCLUSION: The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.

Cardiac valve calcification in patients on maintenance dialysis. The role of malnutrition-inflammation syndrome, adiposity andcomponents of sarcopenia. A cross-sectional study.

BACKGROUND & AIMS: Cardiac valve calcification (CVC) is a predictor of cardiovascular disease and all-cause mortality in end stage kidney disease (ESKD) patients. Several risk factors are related to CVC in patients with ESKD including traditional ones as well as inflammation, bone mineral disease and malnutrition. Adiposity is associated with dyslipidemia and proinflammatory activity which could predispose for CVC. Sarcopenia or dynapenia is a state common in patients with ESKD. This study aimed to investigate the relationship of adiposity, sarcopenia and malnutrition-inflammation markers with CVC in patients on maintenance hemodialysis. METHODS: CVC in aortic (AVC), mitral valves (MVC) and systolic and diastolic dysfunction (DD) were assessed by using two-dimensional echocardiography. Nutritional, adiposity and anthropometric assessments were made using several indices respectively. Creatinine index and muscle strength measurements were also performed. Biochemical parameters such as total proteins, albumin, calcium, phosphate, plasma lipoproteins, C-Reactive Protein and parathyroid hormone were also measured. RESULTS: Adiposity, nutritional, and sarcopenia parameters did not show any difference between patients with or without CVC. Age ≥ 65 years [PR: 1.47 p = 0.012], DD [PR: 2.31, p = 0.005], high CRP/albumin ratio [PR: 1.46, p = 0.01], mid arm circumference (MAC) < 26 cm [PR: 1.37, p = 0.03] were associated with increased prevalence of AVC, while DD [PR: 1.97 p = 0.02], high CRP/albumin ratio [PR: 1.56, p = 0.02], and MAC < 26 cm [PR: 1.52, p = 0.01], showed positive correlation with MVC. Age ≥ 65 years [PR: 1.33, p = 0.028], DD [PR: 1.72, p = 0.01], high CRP/albumin ratio [PR: 1.53, p = 0.003], and MAC < 26 cm [PR: 1.4, p = 0.006], related to greater prevalence of calcification at any valve. CONCLUSIONS: Ageing, diastolic dysfunction, MAC and increased CRP/albumin ratio were powerful predictors of CVC in patients on hemodialysis.

Key nutrients important in the management of canine myxomatous mitral valve disease and heart failure.

The most common cause of heart failure in dogs is myxomatous mitral valve disease (MMVD), which accounts for approximately 75% of canine heart disease cases and is especially common in smaller dogs. Although low-sodium diets have been recommended for humans with heart diseases for decades, there is little evidence to support this practice in dogs. In recent years, however, it has become clear that other nutrients are important to heart health. Dogs with heart disease secondary to MMVD experience patterns of metabolic changes that include decreased mitochondrial energy metabolism and ATP availability, with increased oxidative stress and inflammation. These changes occur early in disease and progress with worsening heart disease. Key nutrients that may support normal function and address these changes include omega-3 fatty acids, medium-chain triglycerides, magnesium, antioxidants including vitamin E and taurine, and the amino acids methionine and lysine. The long-chain omega-3 fatty acids provide anti-inflammatory, antithrombotic, and other benefits. Medium-chain fatty acids and ketones derived from medium-chain triglycerides provide an alternative energy source for cardiac mitochondria and help reduce free radical production. Magnesium supports mitochondrial function, normal cardiac rhythm, and provides other benefits. Both vitamin E and taurine counter oxidative stress, and taurine also has direct cardiac benefits. Dogs with MMVD have reduced plasma methionine. Methionine and lysine are important for carnitine production as well as other functions. This article reviews the evidence supporting the functions and benefits of these and other nutrients in MMVD and other cardiac conditions.

Whole blood and plasma taurine reference intervals in adult Cavalier King Charles Spaniels and correlations between taurine concentration, diet and mitral valve disease.

OBJECTIVE: To determine breed-specific reference intervals for whole blood (WB) and plasma taurine concentrations in adult, overtly healthy Cavalier King Charles Spaniels (CKCSs) and determine whether taurine concentrations differ across preclinical myxomatous mitral valve disease (MMVD) stages or between CKCSs eating diets that meet World Small Animal Veterinary Association (WSAVA) nutritional guidelines versus other diets. ANIMALS: 200 privately owned CKCSs. PROCEDURES: Clinically healthy adult CKCSs were recruited prospectively. Diet and supplement history was collected. Dogs were staged by echocardiography using MMVD consensus guidelines. Taurine concentrations were measured in deproteinized lithium heparin WB and plasma samples with the postcolumn ninhydrin derivatization method on a dedicated amino acid analyzer. RESULTS: There were 12 stage A (6%), 150 stage B1 (75%), and 38 stage B2 (19%) CKCSs. Seventy-eight dogs (39%) were reported by their owners to be eating diets meeting WSAVA nutritional guidelines; 116 (58%) were not. Taurine concentrations in plasma (P = .444) and WB (P = .073) were not significantly different across MMVD stages or between CKCSs eating diets meeting WSAVA nutritional guidelines versus other diets (P = .345 and P = .527, respectively). Reference intervals for WB taurine (152 to 373 µM) and plasma taurine (51 to 217 µM) concentrations in CKCSs were generated. CLINICAL RELEVANCE: In CKCSs, taurine concentrations do not differ significantly based on preclinical MMVD stage, nor do they differ significantly based on consumption of a diet that does or does not meet WSAVA nutritional guidelines.

Circulating MiR-30b-5p is upregulated in Cavalier King Charles Spaniels affected by early myxomatous mitral valve disease.

There is a growing interest in developing new molecular markers of heart disease in young dogs affected by myxomatous mitral valve disease. The study aimed to measure 3 circulating microRNAs and their application as potential biomarkers in the plasma of Cavalier King Charles Spaniels with early asymptomatic myxomatous mitral valve disease. The hypothesis is that healthy Cavalier King Charles Spaniels have different microRNA expression profiles than affected dogs in American College of Veterinary Internal Medicine (ACVIM) stage B1. The profiles can differ within the same class among subjects of different ages. This is a prospective cross-sectional study. Thirty-three Cavalier King Charles Spaniels in ACVIM stage B1 were divided into three groups (11 younger than 3 years, 11 older than 3 years and younger than 7 years, and 11 older than 7 years), and 11 healthy (ACVIM stage A) dogs of the same breed were included as the control group. Three circulating microRNAs (miR-1-3p, miR30b-5p, and miR-128-3p) were measured by quantitative real-time PCR using TaqMan® probes. Diagnostic performance was evaluated by calculating the area under the receiver operating curve (AUC). MiR-30b-5p was significantly higher in ACVIM B1 dogs than in ACVIM A subjects, and the area under the receiver operating curve was 0.79. According to the age of dogs, the amount of miR-30b-5p was statistically significantly higher in group B1<3y (2.3 folds, P = 0.034), B1 3-7y (2.2 folds, P = 0.028), and B1>7y (2.7 folds, P = 0.018) than in group A. The area under the receiver operating curves were fair in discriminating between group B1<3y and group A (AUC 0.780), between B1 3-7y and A (AUC 0.78), and good in discriminating between group B1>7y and A (AUC 0.822). Identifying dogs with early asymptomatic myxomatous mitral valve disease through the evaluation of miR-30b-5p represents an intriguing possibility that certainly merits further research. Studies enrolling a larger number of dogs with preclinical stages of myxomatous mitral valve disease are needed to expand further and validate conclusively the preliminary findings from this report.

Coordination of PRKCA/PRKCA-AS1 interplay facilitates DNA methyltransferase 1 recruitment on DNA methylation to affect protein kinase C alpha transcription in mitral valve of rheumatic heart disease.

In the present study, mitral valve tissues from three mitral stenosis patients with RHD by valve replacement and two healthy donors were harvested and conducted DNA methylation signature on PRKCA by MeDIP-qPCR. The presence of hypomethylated CpG islands at promoter and 5' terminal of PRKCA was observed in RHD accompanied with highly expressed PRKCA and down-regulated antisense long non-coding RNA (lncRNA) PRKCA-AS1 compared to health control. Furthermore, the enrichments of DNMT1/3A/3B on PRKCA were detected by ChIP-qPCR assay in vivo and in human cardiomyocyte AC16 and RL-14 cells exposed to TNF-α in vitro, and both demonstrated that DNMT1 substantially contributed to DNA methylation. Additionally, PRKCA-AS1 was further determined to bind with promoter of PRKCA via 5' terminal and interact with DNMT1 via 3' terminal. Taken together, our results illuminated a novel regulatory mechanism of DNA methylation on regulating PRKCA transcription through lncRNA PRKCA-AS1, and shed light on the molecular pathogenesis of RHD occurrence.

Impact of Mitral Annular Calcium and Mitral Stenosis on Outcomes After Transcatheter Aortic Valve Implantation.

Mitral annular calcium (MAC) is a common finding in patients undergoing transcatheter aortic valve implantation (TAVI) and may be associated with mitral stenosis (MAC-MS). Their impact on post-TAVI outcomes remains controversial. We sought to assess the impact of MAC and MAC-MS on clinical outcomes following TAVI. We included 1,177 patients who consecutively underwent TAVI in our institution between January 2008 and May 2018. MAC diagnosis reposed on echocardiogram and computed tomography. The combination of MAC and a mean transmitral gradient ≥ 5 mmHg defined MAC-MS. The study included 1,177 patients, of whom 504 (42.8%) had MAC and 85 (7.2%) had MAC-MS. Patients with and without MAC had similar outcomes except for a higher rate of pacemaker implantation in MAC patients (adjusted HR: 1.32, 95% CI: 1.03-1.69, p = 0.03). The subgroup of patients with severe MAC had similar outcomes. However, MAC-MS was an independent predictor of all-cause mortality at 30 days (adjusted HR: 2.30, 95% CI: 1.08-4.86, p = 0.03) and 1 year (adjusted HR: 1.73, 95% CI: 1.04-2.89, p = 0.04). In conclusion, MAC is present in nearly half of the patients treated with TAVI but MAC-MS is far less frequent. In itself, even severe, MAC does not influence outcomes while MAC-MS is an independent predictor of all-cause 1-year mortality. Measurement of mean transmitral gradient identifies patients with MAC at high risk after TAVI.

Altered Responsiveness to TGFß and BMP and Increased CD45+ Cell Presence in Mitral Valves Are Unique Features of Ischemic Mitral Regurgitation.

[Figure: see text].

Activation of the Interleukin-33/ST2 Pathway Exerts Deleterious Effects in Myxomatous Mitral Valve Disease.

Mitral valve disease (MVD) is a frequent cause of heart failure and death worldwide, but its etiopathogenesis is not fully understood. Interleukin (IL)-33 regulates inflammation and thrombosis in the vascular endothelium and may play a role in the atherosclerotic process, but its role in mitral valve has not been investigated. We aim to explore IL-33 as a possible inductor of myxomatous degeneration in human mitral valves. We enrolled 103 patients suffering from severe mitral regurgitation due to myxomatous degeneration undergoing mitral valve replacement. Immunohistochemistry of the resected leaflets showed IL-33 and ST2 expression in both valve interstitial cells (VICs) and valve endothelial cells (VECs). Positive correlations were found between the levels of IL-33 and molecules implicated in the development of myxomatous MVD, such as proteoglycans, extracellular matrix remodeling enzymes (matrix metalloproteinases and their tissue inhibitors), inflammatory and fibrotic markers. Stimulation of single cell cultures of VICs and VECs with recombinant human IL-33 induced the expression of activated VIC markers, endothelial-mesenchymal transition of VECs, proteoglycan synthesis, inflammatory molecules and extracellular matrix turnover. Our findings suggest that the IL-33/ST2 system may be involved in the development of myxomatous MVD by enhancing extracellular matrix remodeling.

Chromatin Accessibility of Human Mitral Valves and Functional Assessment of MVP Risk Loci.

RATIONALE: Mitral valve prolapse (MVP) is a common valvopathy that leads to mitral insufficiency, heart failure, and sudden death. Functional genomic studies in mitral valves are needed to better characterize MVP-associated variants and target genes. OBJECTIVE: To establish the chromatin accessibility profiles and assess functionality of variants and narrow down target genes at MVP loci. METHODS AND RESULTS: We mapped the open chromatin regions in nuclei from 11 human pathogenic and 7 nonpathogenic mitral valves by an assay for transposase-accessible chromatin with high-throughput sequencing. Open chromatin peaks were globally similar between pathogenic and nonpathogenic valves. Compared with the heart tissue and cardiac fibroblasts, we found that MV-specific assay for transposase-accessible chromatin with high-throughput sequencing peaks are enriched near genes involved in extracellular matrix organization, chondrocyte differentiation, and connective tissue development. One of the most enriched motifs in MV-specific open chromatin peaks was for the nuclear factor of activated T cells family of TFs (transcription factors) involved in valve endocardial and interstitial cell formation. We also found that MVP-associated variants were significantly enriched (P<0.05) in mitral valve open chromatin peaks. Integration of the assay for transposase-accessible chromatin with high-throughput sequencing data with risk loci, extensive functional annotation, and gene reporter assay suggest plausible causal variants for rs2641440 at the SMG6/SRR locus and rs6723013 at the IGFBP2/IGFBP5/TNS1 locus. CRISPR-Cas9 deletion of the sequence including rs6723013 in human fibroblasts correlated with increased expression only for TNS1. Circular chromatin conformation capture followed by high-throughput sequencing experiments provided evidence for several target genes, including SRR, HIC1, and DPH1 at the SMG6/SRR locus and further supported TNS1 as the most likely target gene on chromosome 2. CONCLUSIONS: Here, we describe unprecedented genome-wide open chromatin profiles from human pathogenic and nonpathogenic MVs and report specific gene regulation profiles, compared with the heart. We also report in vitro functional evidence for potential causal variants and target genes at MVP risk loci involving established and new biological mechanisms. Graphic Abstract: A graphic abstract is available for this article.

Structural Valve Deterioration Is Linked to Increased Immune Infiltrate and Chemokine Expression.

We aim to investigate whether structural valve deterioration (SVD) of bioprosthetic xenogenic tissue heart valves (XTHVs) is associated with increased immune cell infiltration and whether co-expression of several chemokines correlates with this increase in immune infiltrate. Explanted XTHVs from patients undergoing redo valve replacement for SVD were obtained. Immunohistochemical, microscopic, and gene expression analysis approaches were used. XTHVs (n = 37) were obtained from 32 patients (mean 67.7 years) after a mean time of 11.6 years post-implantation. Significantly increased immune cellular infiltration was observed in the explanted SVD valves for all immune cell types examined, including T cells, macrophages, B cells, neutrophils, and plasma cells, compared to non-SVD controls. Furthermore, a significantly increased chemokine gradient in explanted SVD valves accompanied immune cell infiltration. These data suggest the development of SVD is associated with a significantly increased burden of immune cellular infiltrate correlated to the induction of a chemokine gradient around the XHTV, representing chronic immune rejection.Graphical abstract Proposed interaction between innate and adaptive immunity leading to the development of structural valve deterioration in xenogenic tissue heart valves.

Activation of activin/Smad2 and 3 signaling pathway and the potential involvement of endothelial­mesenchymal transition in the valvular damage due to rheumatic heart disease.

Rheumatic heart disease (RHD) is an autoimmune disease caused by rheumatic fever following group A hemolytic streptococcal infection and primarily affects the mitral valve. RHD is currently a major global health problem. However, the exact pathological mechanisms associated with RHD­induced cardiac valve damage remain to be elucidated. The endothelial­mesenchymal transition (EndMT) serves a key role in a number of diseases with an important role in cardiac fibrosis and the activin/Smad2 and 3 signaling pathway is involved in regulating the EndMT. Nevertheless, there are no studies to date, to the best of the authors' knowledge, investigating the association between RHD and EndMT. Thus, the aim of the current study was to investigate the potential role of EndMT in cardiac valve damage and assess whether activin/Smad2 and 3 signaling was activated during RHD­induced valvular injury in a rat model of RHD induced by inactivated Group A streptococci and complete Freund's adjuvant. Inflammation and fibrosis were assessed by hematoxylin and eosin and Sirius red staining. Serum cytokine and rheumatoid factor levels were measured using ELISA kits. Expression levels of activin/Smad2 and 3 signaling pathway­related factors [activin A, Smad2, Smad3, phosphorylated (p­)Smad2 and p­Smad3], EndMT­related factors [lymphoid enhancer factor­1 (LEF­1), Snail1, TWIST, zinc finger E­box­binding homeobox (ZEB)1, ZEB2, α smooth muscle actin (α­SMA) and type I collagen α 1 (COL1A1)], apoptosis­related markers (BAX and cleaved caspase­3) and valvular inflammation markers (NF­κB and p­NF­κB) were detected using reverse transcription­quantitative PCR and western blot analyses. Compared with the control group, the degree of valvular inflammation and fibrosis, serum levels of IL­6, IL­17, TNF­α and expression of apoptosis­related markers (BAX and cleaved caspase­3) and valvular inflammation marker (p­NF­κB), activin/Smad2 and 3 signaling pathway­related factors (activin A, p­Smad2 and p­Smad3), EndMT­related factors (LEF­1, Snail1, TWIST, ZEB 1, ZEB2, α­SMA and COL1A1) were significantly increased in the RHD group. These results suggested that the activin/Smad2 and 3 signaling pathway was activated during the development of valvular damage caused by RHD and that the EndMT is involved in RHD­induced cardiac valve damage.