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A novel miRNA screen identifies miRNA-4454 as a candidate biomarker for ventricular fibrosis in patients with hypertrophic cardiomyopathy

ABSTRACT: Left ventricular hypertrophy, myocardial disarray and interstitital fibrosis are the hallmarks of hypertrophic cardiomyopathy (HCM). Access to the myocardium for diagnostic purposes is limited. Circulating biomolecules reflecting the myocardial disease processes could improve early detection of HCM. Circulating miRNAs have been found to reflect disease processes in several cardiovascular diseases. Circulating miR-1, miR-495-3p and miR-4454 levels are elevated in plasma of HCM patients. miR-4454 is suggested as a potential biomarker for fibrosis in these patients.

ORGANISM(S): Homo sapiens

PROVIDER: GSE188324 | GEO | 2021/11/20

REPOSITORIES: GEO

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Project description:Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant genetic disorder, characterized by cardiomyocyte hypertrophy, cardiomyocyte disarray and fibrosis, which has a prevalence of ~1:200-500 and predisposes individuals to sudden death and heart failure. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs have emerged as critical regulators of gene expression and disease phenotype in various pathologies. We explored whether miRNAs could play a role in HCM pathogenesis and offer potential therapeutic targets. Methods and Results: Using high-throughput miRNA expression profiling and qPCR analysis in two distinct mouse models of HCM, we found that miR-199a-3p expression levels are upregulated in mutant mice compared to age- and treatment-matched wild-type mice. We also found that miR-199a-3p expression is enriched in cardiac non-myocytes compared to cardiomyocytes. When we expressed miR-199a-3p mimic in cultured primary cardiac non-myocytes and analyzed the conditioned media by proteomics, we found that several ECM proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially expressed. We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac non-myocytes drives upregulation of ECM genes including Tsp2, Fbln3, Pcoc1, Col1a1 and Col3a1. To examine the role of miR-199a-3p in vivo, we inhibited its function using lock-nucleic acid (LNA)-based inhibitors (antimiR-199a-3p) in an HCM mouse model. Our results revealed that progression of cardiac fibrosis is attenuated when miR-199a-3p function is inhibited in mild-to-moderate HCM. Finally, guided by computational target prediction algorithms, we identified mRNAs Cd151 and Itga3 as direct targets of miR-199a-3p and have shown that miR-199a-3p mimic expression negatively regulates AKT activation in cardiac non-myocytes. Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac non-myocytes. Thus, inhibition of miR-199a-3p in mild-to-moderate HCM may offer therapeutic benefit in combination with complementary approaches that target the primary defect in cardiac myocytes.

Project description:Background – Hypertrophic cardiomyopathy (HCM) is a common genetic cardiac disorder that is characterized by the presence of left ventricular hypertrophy with histological evidence of myocyte disarray and fibrosis. Pathologic DNA variants in sarcomeric genes are seen in 50–60% of families, but the mechanistic origins of hypertrophy and disarray remain poorly understood. More molecular insights could contribute to a better understanding of these origins and improve therapeutic strategies for HCM. Methods – To identify gene expression patterns relevant to HCM pathology, we quantified intercellular heterogeneity in cardiomyocyte gene expression by performing single-cell RNA sequencing (scRNA-seq) on myectomy tissue from HCM patients. Results – Septal myectomy samples provide a unique opportunity to access diseased human heart tissue for scRNA-seq. We analysed gene expression of individual cardiomyocytes for three HCM patients, who each showed a similar presence of cardiomyocyte subpopulations. This similarity indicated these subpopulations emerge from biological processes, and not from experimental variability. Moreover, our data revealed functional links between genes important to HCM. A subpopulation characterized by elevated titin (TTN) expression showed increased expression of genes involved in muscle contraction and development. A subpopulation of Natriuretic Peptide A (NPPA)-enriched cells showed elevated expression of Natriuretic Peptide B (NPPB). Further analysis of gene co-expression revealed five groups of genes with tightly correlated expression (regulons), one consisting of mostly sarcomeric genes and another containing some well-known stress markers. These correlations were confirmed in 97 additional HCM myectomy samples and allowed us to de novo identify transcription factors related to sarcomeric gene expression and stress. Finally, using forward scatter, we charted functional implications of regulon expression and showed a link between the stress-marker regulon and cell size. Histology samples independently confirmed that regulon member skeletal alpha actin (ACTA1) correlates with cell size. Conclusion – We have shown that single-cell sequencing on myectomy samples from HCM patients identified new functional links between genes, transcription factors, and a functional link between genes and cell size. This highlights the relevance of cardiomyocyte heterogeneity, and additionally provides a wealth of novel insights into molecular events at the base of HCM, which potentially contribute to understanding disease progression.

Project description:Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure-function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes. In this work, we test the hypothesis that changes in cardiac energetics represent a common pathophysiologic pathway in HCM. We performed a comprehensive multi-omics profile of the molecular (transcripts, metabolites, and complex lipids), ultrastructural, and functional components of HCM energetics using myocardial samples from 27 HCM patients and 13 normal controls (donor hearts). Integrated omics analysis revealed alterations in a wide array of biochemical pathways with major dysregulation in fatty acid metabolism, reduction of acylcarnitines, and accumulation of free fatty acids. HCM hearts showed evidence of global energetic decompensation manifested by a decrease in high energy phosphate metabolites (ATP, ADP, and phosphocreatine) and a reduction in mitochondrial genes involved in the creatine kinase and ATP synthesis. Accompanying these metabolic derangements, quantitative electron microscopy showed an increased fraction of severely damaged mitochondria with reduced cristae density, coinciding with reduced citrate synthase (CS) activity and mitochondrial oxidative respiration. These mitochondrial abnormalities were associated with elevated reactive oxygen species (ROS) and reduced antioxidant defenses. However, despite significant mitochondrial injury, HCM hearts failed to upregulate mitophagic clearance. Overall, our findings suggest that perturbed metabolic signaling and mitochondrial dysfunction are common pathogenic mechanisms in patients with HCM. These results highlight potential new drug targets for attenuation of the clinical disease through improving metabolic function and reducing mitochondrial injury.

Project description:Rationale There is a need for new and better biomarkers for hypertrophic cardiomyopathy (HCM) which correlate more closely with disease progression as determined by clinical imaging and biohumoral information. We have used a combination of heart tissue and plasma proteomics to identify potential biomarkers for HCM and developed them into an exploratory targeted proteomic assay. Objective To identify informative staging biomarkers for HCM and develop them into a blood test. The test using 10 µl of plasma, was developed into a 10 min liquid chromatography-tandem/mass spectrometry (LC-MS/MS) assay to analyze multiple candidate biomarkers and evaluate their association with clinical phenotypes in patients with HCM. Methods and Results Myocardial tissue and plasma samples from patients with HCM and healthy volunteers (controls) were screened using a combined gel- and nano-LC quadrupole time of flight MS approach. Twenty-six potential biomarkers were identified from the proteomics screens and developed into a multiplexed targeted proteomic assay. Their association with clinical phenotypes was tested in plasma samples collected from 207 prospectively recruited participants: 110 patients with HCM (50.1 ± 15.0 years, 70% male; 48 [44%] with identified genetic mutations) and 97 controls (49.6 ± 13.4 years, 58% male), randomly split into training (80 HCM, 67 controls) and validation datasets (30 HCM, 30 controls). Six markers (Aldolase Fructose-Bisphosphate A, Complement C3, Glutathione S-Transferase Omega 1, Ras Suppressor Protein 1, Talin 1, and Thrombospondin 1) were significantly increased (P<0.006) in the plasma of HCM patients compared to controls in the training dataset. These markers correlated with left ventricular (LV) wall thickness, LV mass and % myocardial scar on cardiovascular magnetic resonance imaging. Using supervized machine learning (ML) this panel differentiated HCM from controls (area under the curve: 0.89 in the training dataset, sensitivity 96%, 95% confidence interval [CI] 77–93; specificity 87%, 95%CI 77–94; and 0.87 in the validation dataset, sensitivity 97%, 95%CI 83–100; specificity 77%, 95%CI 58–90). Four of the biomarkers as well as the composite ML score of the plasma proteome correlated with the presence of nonsustained ventricular tachycardia and the estimated 5-year risk of sudden cardiac death. Conclusion By developing a high-throughput, multiplex, and targeted proteomic plasma assay we identified 6 biomarkers that correlate with the presence of disease and with clinical risk score for sudden cardiac death.

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A novel miRNA screen identifies miRNA-4454 as a candidate biomarker for ventricular fibrosis in patients with hypertrophic cardiomyopathy

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