Cytokine-Mediated Alterations of Human Cardiac Fibroblast's Secretome.

Fibroblasts contribute to approximately 20% of the non-cardiomyocytic cells in the heart. They play important roles in the myocardial adaption to stretch, inflammation, and other pathophysiological conditions. Fibroblasts are a major source of extracellular matrix (ECM) proteins whose production is regulated by cytokines, such as TNF-α or TGF-ß. The resulting myocardial fibrosis is a hallmark of pathological remodeling in dilated cardiomyopathy (DCM). Therefore, in the present study, the secretome and corresponding transcriptome of human cardiac fibroblasts from patients with DCM was investigated under normal conditions and after TNF-α or TGF-ß stimulation. Secreted proteins were quantified via mass spectrometry and expression of genes coding for secreted proteins was analyzed via Affymetrix Transcriptome Profiling. Thus, we provide comprehensive proteome and transcriptome data on the human cardiac fibroblast's secretome. In the secretome of quiescent fibroblasts, 58% of the protein amount belonged to the ECM fraction. Interestingly, cytokines were responsible for 5% of the total protein amount in the secretome and up to 10% in the corresponding transcriptome. Furthermore, cytokine gene expression and secretion were upregulated upon TNF-α stimulation, while collagen secretion levels were elevated after TGF-ß treatment. These results suggest that myocardial fibroblasts contribute to pro-fibrotic and to inflammatory processes in response to extracellular stimuli.

The E3 ubiquitin ligase Asb2ß is downregulated in a mouse model of hypertrophic cardiomyopathy and targets desmin for proteasomal degradation.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease with mutations in genes encoding sarcomeric proteins. Previous findings suggest deregulation of the ubiquitin proteasome system (UPS) in HCM in humans and in a mouse model of HCM (Mybpc3-targeted knock-in (KI) mice). In this study we investigated transcript levels of several muscle-specific E3 ubiquitin ligases in KI mice and aimed at identifying novel protein targets. METHODS AND RESULTS: Out of 9 muscle-specific E3 ligases, Asb2ß was found with the lowest mRNA level in KI compared to wild-type (WT) mice. After adenoviral-mediated Asb2ß transduction of WT neonatal mouse cardiomyocytes with either a WT or inactive Asb2ß mutant, desmin was identified as a new target of Asb2ß by mass spectrometry, co-immunoprecipitation and immunoblotting. Immunofluorescence analysis revealed a co-localization of desmin with Asb2ß at the Z-disk of the sarcomere. Knock-down of Asb2ß in cardiomyocytes resulted in higher desmin protein levels. Furthermore, desmin levels were higher in ventricular samples of HCM mice and patients than controls. CONCLUSIONS: This study identifies desmin as a new Asb2ß target for proteasomal degradation in cardiomyocytes and suggests that accumulation of desmin could contribute to UPS impairment in HCM mice and patients.

Single-nucleus RNA/ATAC-seq in early-stage HCM models predicts SWI/SNF-activation in mutant-myocytes, and allele-specific differences in fibroblasts.

Hypertrophic cardiomyopathy (HCM) is associated with phenotypic variability. To gain insights into transcriptional regulation of cardiac phenotype, single-nucleus linked RNA-/ATAC-seq was performed in 5-week-old control mouse-hearts (WT) and two HCM-models (R92W-TnT, R403Q-MyHC) that exhibit differences in heart size/function and fibrosis; mutant data was compared to WT. Analysis of 23,304 nuclei from mutant hearts, and 17,669 nuclei from WT, revealed similar dysregulation of gene expression, activation of AP-1 TFs (FOS, JUN) and the SWI/SNF complex in both mutant ventricular-myocytes. In contrast, marked differences were observed between mutants, for gene expression/TF enrichment, in fibroblasts, macrophages, endothelial cells. Cellchat predicted activation of pro-hypertrophic IGF-signaling in both mutant ventricular-myocytes, and profibrotic TGFß-signaling only in mutant-TnT fibroblasts. In summary, our bioinformatics analyses suggest that activation of IGF-signaling, AP-1 TFs and the SWI/SNF chromatin remodeler complex promotes myocyte hypertrophy in early-stage HCM. Selective activation of TGFß-signaling in mutant-TnT fibroblasts contributes to genotype-specific differences in cardiac fibrosis.

Lipid metabolism drives allele-specific early-stage hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) results from pathogenic variants in sarcomeric protein genes, that increase myocyte energy demand and lead to cardiac hypertrophy. But it is unknown whether a common metabolic trait underlies the cardiac phenotype at early disease stage. This study characterized two HCM mouse models (R92W-TnT, R403Q-MyHC) that demonstrate differences in mitochondrial function at early disease stage. Using a combination of cardiac phenotyping, transcriptomics, mass spectrometry-based metabolomics and computational modeling, we discovered allele-specific differences in cardiac structure/function and metabolic changes. TnT-mutant hearts had impaired energy substrate metabolism and increased phospholipid remodeling compared to MyHC-mutants. TnT-mutants showed increased incorporation of saturated fatty acid residues into ceramides, cardiolipin, and increased lipid peroxidation, that could underlie allele-specific differences in mitochondrial function and cardiomyopathy.

A Novel miRNA Screen Identifies miRNA-4454 as a Candidate Biomarker for Ventricular Fibrosis in Patients with Hypertrophic Cardiomyopathy.

(1) Background: Left ventricular hypertrophy, myocardial disarray and interstitial 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 the early detection of HCM. Circulating miRNAs have been found to reflect disease processes in several cardiovascular diseases. (2) Methods: We quantified circulating miRNA molecules in the plasma of 24 HCM and 11 healthy controls using the Human v3 miRNA Expression Assay Kit Code set (Nanostring Tech., Seattle, WA, USA) and validated differentially expressed miRNAs using RT-PCR. (3) Results: In comparison to healthy controls, the levels of six miRNAs (miR-1, miR-3144, miR-4454, miR-495-3p, miR-499a-5p and miR-627-3p) were higher in the plasma of HCM patients than healthy individuals (p < 0.05). Of these, higher levels of miR-1, miR-495 and miR-4454 could be validated by real-time PCR. In addition, elevated miR-4454 levels were significantly correlated with cardiac fibrosis, detected by magnetic resonance imaging in HCM patients. (4) Conclusions: Circulating miR-1, miR-495-3p and miR-4454 levels are elevated in the plasma of HCM patients. To the best of our knowledge, this is the first report showing a correlation between miR-4454 levels and cardiac fibrosis in HCM. This suggests miR-4454 as a potential biomarker for fibrosis in these patients.

Increased myocardial mass and attenuation of myocardial strain in professional male soccer players and competitive male triathletes.

The purpose of this prospective study was to analyze the relationship between ventricular morphology and parameters of cardiac function in two different athletic groups and controls, using feature tracking cardiac magnetic resonance (FT-CMR). Twenty-three professional soccer players (22 ± 4 years), 19 competitive triathletes (28 ± 6 years) and 16 controls (26 ± 3 years) were included in the study. CMR was performed using a 1.5 T scanner. Cardiac chamber volumes, mass and biventricular global myocardial strain were obtained and compared. In comparison to the control subjects, athletes were characterized by a higher cardiac volume (p < 0.0001), higher cardiac mass (p < 0.001), reduced longitudinal strain of the left and right ventricle (p < 0.05 and p < 0.01 respectively) and reduced left ventricular radial strain (p < 0.05). Soccer players revealed higher amounts of left ventricular mass (87 ± 15 vs. 75 ± 13 g/m2, p < 0.05) than triathletes. Moreover, they showed a greater decrease in left and right ventricular longitudinal strain (p < 0.05 and p < 0.05) as well as in radial left ventricular strain (p < 0.05) in comparison to triathletes. An increase in left ventricular mass correlated significantly with a decrease in longitudinal (r = 0.47, p < 0.001) and radial (r = - 0.28, p < 0.05) strain. In athletes, attenuation of strain values is associated with cardiac hypertrophy and differ between soccer players and triathletes. Further studies are needed to investigate whether it is an adaptive or maladaptive change of the heart induced by intense athletic training.

S100A4 as a Target of the E3-Ligase Asb2ß and Its Effect on Engineered Heart Tissue.

Background: S100A4 has recently emerged as an important player in cardiac disease, affecting phenotype development in animal models of myocardial infarction and pathological cardiac hypertrophy, albeit it is unclear whether S100A4 exerts a detrimental or beneficial function. The goal of the current study was to analyze S100A4 expression in models of cardiac pathology, investigate its degradation by the ubiquitin-proteasome system (UPS), and furthermore examine the functional effects of S100A4 levels in a 3D model of engineered heart tissue (EHT). Methods and Results: S100A4 mRNA and protein levels were analyzed in different models of cardiac pathology via quantitative RT-PCR and Western blot, showing a higher S100A4 steady-state protein concentration in hearts of Mybpc3-knock-in (KI) hypertrophic cardiomyopathy (HCM) mice. COS-7 cells co-transfected with plasmids encoding mutant (MUT) Asb2ß lacking the E3 ligase activity in combination with V5-tagged S100A4 plasmid presented higher S100A4-V5 protein steady-state concentrations than cells co-transfected with the Asb2ß wild type (WT) plasmid. This effect was blunted by treatment with the specific proteasome inhibitor epoxomicin. Adeno-associated virus serotype 6 (AAV6)-mediated S100A4 overexpression in a 3D model of EHT did not affect contractile parameters. Immunofluorescence analysis showed a cytosolic and partly nuclear expression pattern of S100A4. Gene expression analysis in EHTs overexpressing S100A4-V5 showed markedly lower steady-state concentrations of genes involved in cardiac fibrosis and pathological cardiac hypertrophy. Conclusion: We showed that S100A4 protein level is higher in cardiac tissue of Mybpc3-KI HCM mice probably as a result of a lower degradation by the E3 ligase Asb2ß. While an overexpression of S100A4 did not alter contractile parameters in EHTs, downstream gene expression analysis points toward modulation of signaling cascades involved in fibrosis and hypertrophy.