Differentially expressed platelet activation-related genes in dogs with stage B2 myxomatous mitral valve disease.

BACKGROUND: Peripheral blood carries a reservoir of mRNAs that regulate cardiac structure and function potential. Although it is well recognized that the typical symptoms of Myxomatous Mitral Valve Disease (MMVD) stage B2 are long-standing hemodynamic disorder and cardiac structure remodeling caused by mitral regurgitation, the transcriptomic alterations in blood from such dogs are not understood. RESULTS: In the present study, comparative high-throughput transcriptomic profiling of blood was performed from normal control (NC) and naturally-occurring MMVD stage B2 (MMVD) dogs. Using Weighted Gene Co-expression Network Analyses (WGCNA), Gene Ontology (GO), and Kyoto Encyclopedia of Gene and Genomes (KEGG), we identified that the turquoise module was the most highly correlated with echocardiographic features and found 64 differentially expressed genes (DEGs) that were significantly enriched in platelet activation related pathways. Therefore, from the turquoise module, we selected five DEGs (MDM2, ROCK1, RIPK1, SNAP23, and ARHGAP35) that, according to real-time qPCR, exhibited significant enrichment in platelet activation related pathways for validation. The results showed that the blood transcriptional abundance of MDM2, ROCK1, RIPK1, and SNAP23 differed significantly (P < 0.01) between NC and MMVD dogs. On the other hand, Correlation Analysis revealed that MDM2, ROCK1, RIPK1, and SNAP23 genes negatively regulated the heart structure parameters, and followed the same trend as observed in WGCNA. CONCLUSION: We screened four platelet activation related genes, MDM2, ROCK1, RIPK1, and SNAP23, which may be considered as the candidate biomarkers for the diagnosis of MMVD stage B2. These findings provided new insights into MMVD pathogenesis.

Elevated cardiac hemoglobin expression is associated with a pro-oxidative and inflammatory environment in primary mitral regurgitation.

BACKGROUND: Primary mitral regurgitation (PMR) is associated with oxidative and inflammatory myocardial damage. We reported greater exosome hemoglobin (Hb) in pericardial fluid (PCF) versus plasma, suggesting a cardiac source of Hb. OBJECTIVE: Test the hypothesis that Hb is produced in the PMR heart and is associated with increased inflammation. METHODS AND RESULTS: Hb gene expression for subunits alpha (HBA) and beta (HBB) was assessed in right atria (RA), left atria (LA) and left ventricular (LV) tissue from donor hearts (n = 10) and PMR patient biopsies at surgery (n = 11). PMR patients (n = 22) had PCF and blood collected for macrophage markers, pro-inflammatory cytokines, and matrix metalloproteinases (MMPs). In-situ hybridization for HBA mRNA and immunohistochemistry for Hb-alpha (Hbα) and Hb-beta (Hbß) protein was performed on PMR tissue. RESULTS: HBA and HBB genes are significantly increased (>4-fold) in RA, LA, and LV in PMR vs. normal hearts. In PMR tissue, HBA mRNA is expressed in both LV cardiomyocytes and interstitial cells by in-situ hybridization; however, Hbα and Hbß protein is only expressed in interstitial cells by immunohistochemistry. PCF oxyHb is significantly increased over plasma along with low ratios (<1.0) of haptoglobin:oxyHb and hemopexin:heme supporting a highly oxidative environment. Macrophage chemotactic protein-1, tumor necrosis factor-α, interleukin-6, and MMPs are significantly higher in PCF vs. plasma. CONCLUSION: There is increased Hb production in the PMR heart coupled with the inflammatory state of the heart, suggests a myocardial vulnerability of further Hb delivery and/or production during cardiac surgery that could adversely affect LV functional recovery.

Recruited macrophages elicit atrial fibrillation.

Atrial fibrillation disrupts contraction of the atria, leading to stroke and heart failure. We deciphered how immune and stromal cells contribute to atrial fibrillation. Single-cell transcriptomes from human atria documented inflammatory monocyte and SPP1+ macrophage expansion in atrial fibrillation. Combining hypertension, obesity, and mitral valve regurgitation (HOMER) in mice elicited enlarged, fibrosed, and fibrillation-prone atria. Single-cell transcriptomes from HOMER mouse atria recapitulated cell composition and transcriptome changes observed in patients. Inhibiting monocyte migration reduced arrhythmia in Ccr2-∕- HOMER mice. Cell-cell interaction analysis identified SPP1 as a pleiotropic signal that promotes atrial fibrillation through cross-talk with local immune and stromal cells. Deleting Spp1 reduced atrial fibrillation in HOMER mice. These results identify SPP1+ macrophages as targets for immunotherapy in atrial fibrillation.

Early and severe tricuspid valve dysplasia in a fetus with cardiospondylocarpofacial syndrome due to a variant c.616T>G p.(Tyr206Asp) in MAP3K7.

Cardiospondylocarpofacial syndrome (CSCF; MIM#157800) is a rare condition caused by monoallelic variants in the MAP3K7 gene. The characteristic features of CSCF include growth retardation, facial dysmorphism, carpal-tarsal fusion, dorsal spine synostosis, deafness, inner ear malformation, cardiac septal defect and valve dysplasia. We present here a 20-week-old fetus with cardiospondylocarpofacial syndrome arising from a de novo variant c.616T>G p.(Tyr206Asp) in the MAP3K7 (NM_145331.3) gene with early and severe tricuspid valve dysplasia as a prenatal manifestation. Fetal echocardiography revealed tricuspid regurgitation with valve prolapse. Fetus had facial dysmorphism and dilated right atrium and right ventricle with tricuspid valve dysplasia on perinatal evaluation. To the best of our knowledge, this is the first report mentioning the prenatal manifestation of cardiospondylocarpofacial syndrome.

A novel MAP3K7 mutation in a child with cardiospondylocarpofacial syndrome and orofacial clefting.

Here we present the case of a patient with a novel de novo, likely pathogenic, heterozygous MAP3K7 variant (c.528dupT, p.G177WfsX5) causing cardiospondylocarpofacial syndrome (CSCFS). The variant, which falls in exon 6, is the first frameshift or non-sense mutation to be connected to CSCFS and presents with a phenotype that shares features with other MAP3K7-linked pathologies, including frontometaphyseal dysplasia 2 (FMD2) and the syndrome arising from an interstitial 6q15 deletions which envelop the gene. Other known mutations associated with CSCFS are plotted in black text (1,2,3).

Mitral regurgitation severity at left ventricular assist device implantation is associated with distinct myocardial transcriptomic signatures.

OBJECTIVES: We examined for differences in pre-left ventricular assist device (LVAD) implantation myocardial transcriptome signatures among patients with different degrees of mitral regurgitation (MR). METHODS: Between January 2018 and October 2019, we collected left ventricular (LV) cores during durable LVAD implantation (n = 72). A retrospective chart review was performed. Total RNA was isolated from LV cores and used to construct cDNA sequence libraries. The libraries were sequenced with the NovaSeq system, and data were quantified using Kallisto. Gene Set Enrichment Analysis (GSEA) and Gene Ontology analyses were performed, with a false discovery rate <0.05 considered significant. RESULTS: Comparing patients with preoperative mild or less MR (n = 30) and those with moderate-severe MR (n = 42), the moderate-severe MR group weighted less (P = .004) and had more tricuspid valve repairs (P = .043), without differences in demographics or comorbidities. We then compared both groups with a group of human donor hearts without heart failure (n = 8). Compared with the donor hearts, there were 3985 differentially expressed genes (DEGs) for mild or less MR and 4587 DEGs for moderate-severe MR. Specifically altered genes included 448 DEGs for specific for mild or less MR and 1050 DEGs for moderate-severe MR. On GSEA, common regulated genes showed increased immune gene expression and reduced expression of contraction and energetic genes. Of the 1050 genes specific for moderate-severe MR, there were additional up-regulated genes related to inflammation and reduced expression of genes related to cellular proliferation. CONCLUSIONS: Patients undergoing durable LVAD implantation with moderate-severe MR had increased activation of genes related to inflammation and reduction of cellular proliferation genes. This may have important implications for myocardial recovery.

Prospective pilot study on the predictive significance of plasma miR-30b-5p through the study of echocardiographic modifications in Cavalier King Charles Spaniels affected by different stages of myxomatous mitral valve disease: The PRIME study.

Specific microRNAs expressions may accurately characterize different stages of canine myxomatous mitral valve disease. This preliminary pilot study aimed to (1) describe the clinical and echocardiographic parameters of Cavalier King Charles Spaniels affected by myxomatous mitral valve disease at different American College of Veterinary Internal Medicine (ACVIM) stages (B1, B2 and C) and healthy control group (ACVIM A), comparing the parameters collected during the first examination (T0) and the end of the follow-up (T1); (2) assess the association between the values of echocardiographic parameters at T1 and the expression profile of miR-30b-5p at T0. Thirty-five Cavalier King Charles Spaniels (median age 4.29 years and median weight 9 Kg) in different ACVIM stages were included (7 A, 19 B1, 6 B2 and 3 C). Inverse probability weighting analysis was performed to estimate the association of the exposure variable (miR-30b-5p) with the outcome variables (clinical and echocardiographic variables). Time was included as variable. The results pointed out that high levels of plasma miR-30b-5p corresponded to lower values of left ventricular end-diastolic diameter normalized for body weight, end-diastolic and end-systolic volumes indexed for body weight, and left atrium-to aortic root ratio. Hence, higher miR-30b-5p expressions were associated with milder forms of mitral valve disease in our study population. In contrast, the results obtained for the intensity of heart murmur, the mitral regurgitation severity, and the Mitral INsufficiency Echocardiographic score) were not statistically significant. A relationship between high abundance of miR-30b-5p and myxomatous mitral valve disease that appear echocardiographically more stable over time has been demonstrated. In conclusion, Cavalier King Charles Spaniels affected by myxomatous mitral valve disease that at the first cardiologic evaluation showed an upregulation of miR-30b-5p are expected to experience lesser variations on their echocardiographic examination between T0 and T1.

Molecular Signatures of Human Chronic Atrial Fibrillation in Primary Mitral Regurgitation.

OBJECTIVES: Transcriptomics of atrial fibrillation (AFib) in the setting of chronic primary mitral regurgitation (MR) remains to be characterized. We aimed to compare the gene expression profiles of patients with degenerative MR in AFib and sinus rhythm (SR) for a clearer picture of AFib pathophysiology. METHODS: After transcriptomic analysis and bioinformatics (n = 59), differentially expressed genes were defined using 1.5-fold change as the threshold. Additionally, independent datasets from GEO were included as meta-analyses. RESULTS: QRT-PCR analysis confirmed that AFib persistence was associated with increased expression molecular changes underlying a transition to heart failure (NPPB, P = 0.002; ANGPTL2, P = 0.002; IGFBP2, P = 0.010), structural remodeling including changes in the extracellular matrix and cellular stress response (COLQ, P = 0.003; COMP, P = 0.028; DHRS9, P = 0.038; CHGB, P = 0.038), and cellular stress response (DNAJA4, P = 0.038). Furthermore, AFib persistence was associated with decreased expression of the targets of structural remodeling (BMP7, P = 0.021) and electrical remodeling (CACNB2, P = 0.035; MCOLN3, P = 0.035) in both left and right atrial samples. The transmission electron microscopic analysis confirmed ultrastructural atrial remodeling and autophagy in human AFib atrial samples. CONCLUSIONS: Atrial cardiomyocyte remodeling in persistent AFib is closely linked to alterations in gene expression profiles compared to SR in patients with primary MR. Study findings may lead to novel therapeutic targets. This trial is registered with ClinicalTrials.gov identifier: NCT00970034.

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

[Figure: see text].

Putative Circulating MicroRNAs Are Able to Identify Patients with Mitral Valve Prolapse and Severe Regurgitation.

Mitral valve prolapse (MVP) associated with severe mitral regurgitation is a debilitating disease with no pharmacological therapies available. MicroRNAs (miRNA) represent an emerging class of circulating biomarkers that have never been evaluated in MVP human plasma. Our aim was to identify a possible miRNA signature that is able to discriminate MVP patients from healthy subjects (CTRL) and to shed light on the putative altered molecular pathways in MVP. We evaluated a plasma miRNA profile using Human MicroRNA Card A followed by real-time PCR validations. In addition, to assess the discriminative power of selected miRNAs, we implemented a machine learning analysis. MiRNA profiling and validations revealed that miR-140-3p, 150-5p, 210-3p, 451a, and 487a-3p were significantly upregulated in MVP, while miR-223-3p, 323a-3p, 340-5p, and 361-5p were significantly downregulated in MVP compared to CTRL (p ≤ 0.01). Functional analysis identified several biological processes possible linked to MVP. In addition, machine learning analysis correctly classified MVP patients from CTRL with high accuracy (0.93) and an area under the receiving operator characteristic curve (AUC) of 0.97. To the best of our knowledge, this is the first study performed on human plasma, showing a strong association between miRNAs and MVP. Thus, a circulating molecular signature could be used as a first-line, fast, and cheap screening tool for MVP identification.

Identification of Biomarkers Related to Atrial Fibrillation With Mitral Regurgitation.

BACKGROUND: We aimed to explore the biomarkers associated with atrial fibrillation (AF) with mitral regurgitation (MR). METHODS: The gene expression profile data GSE115574 were downloaded from Gene Expression Omnibus database, which were obtained from patients with degenerative MR with AF and sinus rhythm (SR). The differentially expressed genes (DEGs) in samples of AF with MR compared with those of SR with MR were selected, followed by functional enrichment analysis, protein-protein interaction (PPI) network analysis, transcription factor (TF) prediction, and drug-gene interaction prediction. RESULTS: By comparing the genes' expression profiles between AF with MR and SR with MR, 379 DEGs were obtained. The upregulated genes, such as NMNAT2, LDHB, and hexosaminidase subunit beta (HEXB), were significantly enriched in metabolic pathways. Hub genes, such as amyloid beta precursor protein (APP), CDH2, SPP1, and STC2, were significantly associated with functions related to extracellular matrix organization and vitamin D response. Additionally, two TFs, PRDM3 and LSM6, were predicted for the key module genes. APP predicted the most drug molecules, that is, 22 molecules, and SPP1 predicted 10 drug molecules. CONCLUSIONS: Dysregulation of the metabolic pathway may play a critical role in AF with MR. Changes in functions related to the extracellular matrix and vitamin D response may also be associated with AF progression in patients with MR. Furthermore, APP, STC2, and SPP1 may serve as potential therapeutic targets of AF.

Secondary mitral regurgitation-Insights from microRNA assessment.

BACKGROUND: While secondary mitral regurgitation (sMR) is associated with adverse outcome in heart failure with reduced ejection fraction (HFrEF), key pathophysiologic mechanisms remain poorly understood and might be elucidated by microRNAs (miRNA/miR), that were recently related to cardiac remodelling. This study sought to assess (i) the differences of miRNA profiles in patients with severe sMR compared to matched disease controls, (ii) the correlation between circulating miRNAs and surrogates of sMR severity as well as (iii) the prognostic implications of miRNA levels in severe sMR. MATERIALS AND METHODS: Sixty-six HFrEF patients were included, of these 44 patients with severe sMR 2:1 matched to HFrEF controls with no/mild sMR. A comprehensive set of miRNAs (miR-21, miR-29a, miR-122, miR-132, miR-133a, miR-let7i) were measured and correlated to echocardiographic sMR severity. RESULTS: miRNA patterns differed distinctly between patients with severe sMR and HFrEF controls (P < .05). Among the panel of assessed miRNAs, miR-133a correlated most strongly with surrogates of sMR severity (r = -0.41, P = .001 with sMR vena contracta width). Interestingly, elevated levels of miR-133 were associated with an increased risk for cardiovascular death and/or HF hospitalizations with and adjusted HR of 1.85 (95% CI 1.24-2.76, P = .003). CONCLUSIONS: This study unveils distinct pathophysiologic maladaptions at a cellular level in patients with severe sMR compared to no/mild sMR by showing significant differences in miRNA profiles and correlations with sMR severity, supporting the concept that sMR drives cardiac remodelling in heart failure. Moreover, the increased risk for adverse outcome in HFrEF patients with severe sMR conveyed by miR-133a might indicate irreversible myocardial damage.

Clinical presentation and evolution of Xia-Gibbs syndrome due to p.Gly375ArgfsTer3 variant in a patient from DR Congo (Central Africa).

Xia-Gibbs syndrome (XGS) is a very rare genetic condition. The clinical spectrum is very broad and variable. The phenotype and evolution in a Congolese boy with XGS have been reported. At 6 years he had speech delay, drooling, marked hyperactivity, attention deficit, aggressive behavior, and intellectual disability. Dysmorphological evaluation revealed strabismus, mild unilateral ptosis, uplifted ear lobes, flat philtrum, thin upper lip vermillion, high arched palate, and flat feet. Patient-only whole exome sequencing identified a known pathogenic frameshift variant in the AHDC1 gene [NM_001029882.3(AHDC1):c.1122dupC;(p.Gly375ArgfsTer3)]. The clinical follow-up revealed the deterioration of his fine motor skills and significant cerebellar phenotype including tremor, pes cavus, and gait instability at the age of 12 years. This patient was compared with three previously reported patients with the same variant but did not identify a consistent pattern in the evolution of symptoms with age.

Hemodynamic and transcriptomic studies suggest early left ventricular dysfunction in a preclinical model of severe mitral regurgitation.

OBJECTIVE: Primary mitral regurgitation is a valvular lesion in which the left ventricular ejection fraction remains preserved for long periods, delaying a clinical trigger for mitral valve intervention. In this study, we sought to investigate whether adverse left ventricular remodeling occurs before a significant fall in ejection fraction and characterize these changes. METHODS: Sixty-five rats were induced with severe mitral regurgitation by puncturing the mitral valve leaflet with a 23-G needle using ultrasound guidance. Rats underwent longitudinal cardiac echocardiography at biweekly intervals and hearts explanted at 2 weeks (n = 15), 10 weeks (n = 15), 20 weeks (n = 15), and 40 weeks (n = 15). Sixty age- and weight-matched healthy rats were used as controls. Unbiased RNA-sequencing was performed at each terminal point. RESULTS: Regurgitant fraction was 40.99 ± 9.40%, with pulmonary flow reversal in the experimental group, and none in the control group. Significant fall in ejection fraction occurred at 14 weeks after mitral regurgitation induction. However, before 14 weeks, end-diastolic volume increased by 93.69 ± 52.38% (P < .0001 compared with baseline), end-systolic volume increased by 118.33 ± 47.54% (P < .0001 compared with baseline), and several load-independent pump function indices were reduced. Transcriptomic data at 2 and 10 weeks before fall in ejection fraction indicated up-regulation of myocyte remodeling and oxidative stress pathways, whereas those at 20 and 40 weeks indicated extracellular matrix remodeling. CONCLUSIONS: In this rodent model of mitral regurgitation, left ventricular ejection fraction was preserved for a long duration, yet rapid and severe left ventricular dilatation, and biological remodeling occurred before a clinically significant fall in ejection fraction.

A nonsense variant in FBN1 caused autosomal dominant Marfan syndrome in a Chinese family: a case report.

BACKGROUND: Marfan syndrome (MFS) is a common autosomal dominant inherited disease, and the occurrence rate is around 0.1-0.2‰. The causative variant of FNB1 gene accounts for approximately 70-80% of all MFS cases. In this study, we found a heterozygous c.3217G > T (p.Glu1073*) nonsense variant in the FBN1 gene. This finding extended the variant spectrum of the FBN1 gene and will provide a solution for patients to bear healthy offspring by preimplantation genetic testing or prenatal diagnosis. CASE PRESENTATION: The patient was treated due to tachycardia during excitement in a hospital. Echocardiography showed dilatation of the ascending aorta and main pulmonary artery, mitral regurgitation (mild), tricuspid regurgitation (mild), and abnormal left ventricular filling. Electrocardiograph showed sinus rhythm. In addition, flutters of shadows in front of his eyes and vitreous opacity were present in the patient. Genomic DNA was extracted from peripheral blood samples from members of the family and 100 unrelated controls. Potential variants were screened out by next-generation sequencing and confirmed by MLPA & Sanger sequencing. Real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the relative mRNA quantitation in the patient. A heterozygous nonsense variant c.3217G > T of the FBN1 gene, which resulted in p. Glu1073Term, was identified in both patients. Only wild type bases were found in the cDNA sequence of the patient. Real-time fluorogenic quantitative PCR results showed that the relative expression level of FBN1 cDNA in the patient was only about 21% compared to that of normal individuals. This variant c.3217G > T of the FBN1 gene introduces a Stop codon in the cb-EGF12 domain. We speculated that a premature translational-termination codon (PTC) was located in the mRNA and the target mRNA was disintegrated through a process known as nonsense-mediated mRNA decay (NMD), which led to a significant decrease of the fibrillin-1 protein, eventually causing clinical symptoms in the patient. CONCLUSIONS: In this study, we found a heterozygous c.3217G > T (p.Glu1073*) nonsense variant in the FBN1 gene, which eventually led to Marfan syndrome in a Chinese family.

Differential gene expression in patients with primary mitral valve disease: identifying potential therapeutic targets in the era of precision medicine.

Primary (degenerative) mitral valve (MV) disease is a result of structural remodeling due to degenerative and adaptive changes of MV tissue. We hypothesized that in patients with primary MV disease undergoing surgery for severe mitral regurgitation (MR), a distinct genetic expression profile within the MV leaflet tissue could be identified as compared with patients without MV disease. Tissue samples from the MV leaflets of 65 patients undergoing MV surgery for MR due to primary MV disease and 4 control cadavers without MV disease were collected and analyzed. MicroRNA transcripts were hybridized to Illumina HumanHT-12 v4 Beadchips. Ingenuity pathway analyses (IPAs) were conducted to provide biological interpretation. Of the approximately 20 000 genes examined, 4092 (20%) were differentially expressed between patients with primary MV disease and normal controls (false discovery rate<0.05). The differentially expressed genes could be clustered into five regulator effect networks from the Ingenuity Knowledge IPA database with a consistency score of >6. These five networks have been previously implicated in pathophysiological cardiac abnormalities, including inhibited contractility of the heart and fatty acid oxidation as well as activation of apoptosis of smooth muscle cells, cardiac degeneration, and hypertrophy of cardiac cells. MV tissue in patients with primary MV disease demonstrated distinct genetic expression patterns as compared with normal controls. Further studies are necessary to determine whether the molecular pathways identified in this experiment may represent potential therapeutic targets to prevent degeneration of MV tissue leading to severe MR.

Ubiquitin Pathway Is Associated with Worsening Left Ventricle Function after Mitral Valve Repair: A Global Gene Expression Study.

The molecular mechanism for worsening left ventricular (LV) function after mitral valve (MV) repair for chronic mitral regurgitation remains unknown. We wished to assess the LV transcriptome and identify determinants associated with worsening LV function post-MV repair. A total of 13 patients who underwent MV repair for chronic primary mitral regurgitation were divided into two groups, preserved LV function (N = 8) and worsening LV function (N = 5), for the study. Specimens of LV from the patients taken during surgery were used for the gene microarray study. Cardiomyocyte cell line HL-1 cells were transfected with gene-containing plasmids and further evaluated for mRNA and protein expression, apoptosis, and contractile protein degradation. Of 67,258 expressed sequence tags, microarrays identified 718 genes to be differentially expressed between preserved-LVF and worsening-LVF, including genes related to the protein ubiquitination pathway, bone morphogenetic protein (BMP) receptors, and regulation of eIF4 and p70S6K signaling. In addition, worsening-LVF was associated with altered expressions of genes pathologically relevant to heart failure, such asdownregulated apelin receptors and upregulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A). HL-1 cardiomyocyte cells transfected with ubiquitination-related genes demonstrated activation of the protein ubiquitination pathwaywith an increase in the ubiquitin activating enzyme E1 (UAE-E1). It also led to increased apoptosis, downregulated and ubiquitinated X-linked inhibitor of apoptosis protein (XIAP), and reduced cell viability. Overexpression of ubiquitination-related genes also resulted in degradation and increased ubiquitination of α-smooth muscle actin (SMA). In conclusion, worsening-LVF presented differential gene expression profiles from preserved-LVF after MV repair. Upregulation of protein ubiquitination-related genes associated with worsening-LVF after MV repair may exert adverse effects on LV through increased apoptosis and contractile protein degradation.

A rare association of divided left atrium with mitral valve prolapse.

We hereby present a case of divided left atrium with mitral valve prolapse and discuss the hemodynamic consequences that ensue. This case demonstrates the utility of computed tomography angiography in identifying such associations.