Assessment of the Cardiac Noncoding Transcriptome by Single-Cell RNA Sequencing Identifies FIXER, a Conserved Profibrogenic Long Noncoding RNA.

BACKGROUND: Cardiac fibroblasts have crucial roles in the heart. In particular, fibroblasts differentiate into myofibroblasts in the damaged myocardium, contributing to scar formation and interstitial fibrosis. Fibrosis is associated with heart dysfunction and failure. Myofibroblasts therefore represent attractive therapeutic targets. However, the lack of myofibroblast-specific markers has precluded the development of targeted therapies. In this context, most of the noncoding genome is transcribed into long noncoding RNAs (lncRNAs). A number of lncRNAs have pivotal functions in the cardiovascular system. lncRNAs are globally more cell-specific than protein-coding genes, supporting their importance as key determinants of cell identity. METHODS: In this study, we evaluated the value of the lncRNA transcriptome in very deep single-cell RNA sequencing. We profiled the lncRNA transcriptome in cardiac nonmyocyte cells after infarction and probed heterogeneity in the fibroblast and myofibroblast populations. In addition, we searched for subpopulation-specific markers that can constitute novel targets in therapy for heart disease. RESULTS: We demonstrated that cardiac cell identity can be defined by the sole expression of lncRNAs in single-cell experiments. In this analysis, we identified lncRNAs enriched in relevant myofibroblast subpopulations. Selecting 1 candidate we named FIXER (fibrogenic LOX-locus enhancer RNA), we showed that its silencing limits fibrosis and improves heart function after infarction. Mechanitically, FIXER interacts with CBX4, an E3 SUMO protein ligase and transcription factor, guiding CBX4 to the promoter of the transcription factor RUNX1 to control its expression and, consequently, the expression of a fibrogenic gene program.. FIXER is conserved in humans, supporting its translational value. CONCLUSIONS: Our results demonstrated that lncRNA expression is sufficient to identify the various cell types composing the mammalian heart. Focusing on cardiac fibroblasts and their derivatives, we identified lncRNAs uniquely expressed in myofibroblasts. In particular, the lncRNA FIXER represents a novel therapeutic target for cardiac fibrosis.

Transcriptomic Analysis of Extracellular Vesicles in the Search for Novel Plasma and Thrombus Biomarkers of Ischemic Stroke Etiologies.

Accurate etiologic diagnosis provides an appropriate secondary prevention and better prognosis in ischemic stroke (IS) patients; still, 45% of IS are cryptogenic, urging us to enhance diagnostic precision. We have studied the transcriptomic content of plasma extracellular vesicles (EVs) (n = 21) to identify potential biomarkers of IS etiologies. The proteins encoded by the selected genes were measured in the sera of IS patients (n = 114) and in hypertensive patients with (n = 78) and without atrial fibrillation (AF) (n = 20). IGFBP-2, the most promising candidate, was studied using immunohistochemistry in the IS thrombi (n = 23) and atrium of AF patients (n = 13). In vitro, the IGFBP-2 blockade was analyzed using thromboelastometry and endothelial cell cultures. We identified 745 differentially expressed genes among EVs of cardioembolic, atherothrombotic, and ESUS groups. From these, IGFBP-2 (cutoff > 247.6 ng/mL) emerged as a potential circulating biomarker of embolic IS [OR = 8.70 (1.84-41.13) p = 0.003], which was increased in patients with AF vs. controls (p < 0.001) and was augmented in cardioembolic vs. atherothrombotic thrombi (p < 0.01). Ex vivo, the blockage of IGFBP-2 reduced clot firmness (p < 0.01) and lysis time (p < 0.001) and in vitro, diminished endothelial permeability (p < 0.05) and transmigration (p = 0.06). IGFBP-2 could be a biomarker of embolic IS and a new therapeutic target involved in clot formation and endothelial dysfunction.

Heart failure in chronic kidney disease: the emerging role of myocardial fibrosis.

Heart failure (HF) is one of the main causes of morbidity and mortality in patients with chronic kidney disease (CKD). Decreased glomerular filtration rate is associated with diffuse deposition of fibrotic tissue in the myocardial interstitium [i.e. myocardial interstitial fibrosis (MIF)] and loss of cardiac function. MIF results from cardiac fibroblast-mediated alterations in the turnover of fibrillary collagen that lead to the excessive synthesis and deposition of collagen fibres. The accumulation of stiff fibrotic tissue alters the mechanical properties of the myocardium, thus contributing to the development of HF. Accumulating evidence suggests that several mechanisms are operative along the different stages of CKD that may converge to alter fibroblasts and collagen turnover in the heart. Therefore, focusing on MIF might enable the identification of fibrosis-related biomarkers and targets that could potentially lead to a new strategy for the prevention and treatment of HF in patients with CKD. This article summarizes current knowledge on the mechanisms and detrimental consequences of MIF in CKD and discusses the validity and usefulness of available biomarkers to recognize the clinical-pathological variability of MIF and track its clinical evolution in CKD patients. Finally, the currently available and potential future therapeutic strategies aimed at personalizing prevention and reversal of MIF in CKD patients, especially those with HF, will be also discussed.

Lipocalin-2 and Calprotectin Potential Prognosis Biomarkers in Peripheral Arterial Disease.

OBJECTIVE: Peripheral arterial disease (PAD) is the most prevalent cardiovascular (CV) condition globally. Despite the high CV risk of PAD patients, no reliable predictors of adverse clinical evolution are yet available. In this regard, previous transcriptomic analyses revealed increased expression of calprotectin (S100A8/A9) and lipocalin-2 (LCN2) in circulating extracellular vesicles (EVs) of patients with PAD. The aim of this study was to determine the prognostic value of LCN2 and calprotectin for CV risk assessment in PAD. METHODS: LCN2 and the S100A9 subunit of calprotectin were examined in human femoral plaques by immunohistochemistry and qPCR. LCN2 and calprotectin were determined by ELISA in PAD (CHN cohort, n = 331, Fontaine II-IV, serum), and PAD diagnosed by population based screening (VIVA trial, n = 413, the majority Fontaine 0-I, plasma). Patients were followed up for a mean of four years, recording the primary outcomes; CV death or amputation in the CHN cohort and CV death or major lower limb events (MALE) in the VIVA population. Secondary outcomes were all cause death or amputation, and all cause death or MALE, respectively. RESULTS: LCN2 and S100A9 were detected in human plaques in regions rich in inflammatory cells. LCN2 and calprotectin levels were 70% and 64% lower in plasma than in serum. In the CHN cohort, high serum levels of LCN2 and calprotectin increased the risk of primary and secondary outcomes 5.6 fold (p < .001) and 1.8 fold (p = .034), respectively, after covariable adjustment. Similarly, elevated plasma levels of LCN2 and calprotectin increased by three fold the risk of primary and secondary outcomes (p < .001) in the VIVA cohort. Moreover, addition of the combined variable to basal models, considering clinically relevant risk factors, improved reclassification for the primary outcome in both cohorts (p ≤ .024). CONCLUSION: Combined assessment of the inflammatory biomarkers LCN2 and calprotectin might be useful for risk stratification in advanced and early PAD.

The effect of spironolactone on cardiovascular function and markers of fibrosis in people at increased risk of developing heart failure: the heart 'OMics' in AGEing (HOMAGE) randomized clinical trial.

AIMS: To investigate the effects of spironolactone on fibrosis and cardiac function in people at increased risk of developing heart failure. METHODS AND RESULTS: Randomized, open-label, blinded-endpoint trial comparing spironolactone (50 mg/day) or control for up to 9 months in people with, or at high risk of, coronary disease and raised plasma B-type natriuretic peptides. The primary endpoint was the interaction between baseline serum galectin-3 and changes in serum procollagen type-III N-terminal pro-peptide (PIIINP) in participants assigned to spironolactone or control. Procollagen type-I C-terminal pro-peptide (PICP) and collagen type-1 C-terminal telopeptide (CITP), reflecting synthesis and degradation of type-I collagen, were also measured. In 527 participants (median age 73 years, 26% women), changes in PIIINP were similar for spironolactone and control [mean difference (mdiff): -0.15; 95% confidence interval (CI) -0.44 to 0.15 µg/L; P = 0.32] but those receiving spironolactone had greater reductions in PICP (mdiff: -8.1; 95% CI -11.9 to -4.3 µg/L; P < 0.0001) and PICP/CITP ratio (mdiff: -2.9; 95% CI -4.3 to -1.5; <0.0001). No interactions with serum galectin were observed. Systolic blood pressure (mdiff: -10; 95% CI -13 to -7 mmHg; P < 0.0001), left atrial volume (mdiff: -1; 95% CI -2 to 0 mL/m2; P = 0.010), and NT-proBNP (mdiff: -57; 95% CI -81 to -33 ng/L; P < 0.0001) were reduced in those assigned spironolactone. CONCLUSIONS: Galectin-3 did not identify greater reductions in serum concentrations of collagen biomarkers in response to spironolactone. However, spironolactone may influence type-I collagen metabolism. Whether spironolactone can delay or prevent progression to symptomatic heart failure should be investigated.

Natural Compound Library Screening Identifies New Molecules for the Treatment of Cardiac Fibrosis and Diastolic Dysfunction.

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.

Deficiency of Procollagen C-Proteinase Enhancer 1 in Mice has No Major Impact on Cardiac Collagen and Function Under Basal Conditions.

ABSTRACT: Maturation of fibrillar collagen is known to play a crucial role in the pathophysiology of myocardial fibrosis. Procollagen C-proteinase enhancer 1 (PCPE1) has a key role in procollagen maturation and collagen fibril formation. The phenotype of both male and female PCPE1 knock-out mice was investigated under basal conditions to explore the potential of PCPE1 as a therapeutic target in heart failure. Global constitutive PCPE1-/- mice were generated. Serum procollagen I C-terminal propeptide, organ histology, and cutaneous wound healing were assessed in both wild type (WT) and PCPE1-/- mice. In addition, the cardiac expression of genes involved in collagen metabolism was investigated and the total and insoluble cardiac collagen contents determined. Cardiac function was evaluated by echocardiography. No differences in survival, clinical chemistry, or organ histology were observed in PCPE1-/- mice compared with WT. Serum procollagen I C-terminal propeptide was lower in PCPE1-/- mice. Cardiac mRNA expression of Bmp1, Col1a1, Col3a1, and Loxl2 was similar, whereas Tgfb and Loxl1 mRNA levels were decreased in PCPE1-/- mice compared with sex-matched WT. No modification of total or insoluble cardiac collagen content was observed between the 2 strains. Ejection fraction was slightly decreased in PCPE1-/- male mice, but not in females. Finally, wound healing was not altered in PCPE1-/- mice. PCPE1 deficiency does not trigger any major liabilities and does not affect cardiac collagen content nor its function under basal conditions. Further studies are required to evaluate its role under stressed conditions and determine its suitability as a therapeutic target for heart failure.

Omics phenotyping in heart failure: the next frontier.

This state-of-the-art review aims to provide an up-to-date look at breakthrough omic technologies that are helping to unravel heart failure (HF) disease mechanisms and heterogeneity. Genomics, transcriptomics, proteomics, and metabolomics in HF are reviewed in depth. In addition, there is a thorough, expert discussion regarding the value of omics in identifying novel disease pathways, advancing understanding of disease mechanisms, differentiating HF phenotypes, yielding biomarkers for diagnosis or prognosis, or identifying new therapeutic targets in HF. The combination of multiple omics technologies may create a more comprehensive picture of the factors and physiology involved in HF than achieved by either one alone and provides a rich resource for predictive phenotype modelling. However, the successful translation of omics tools as solutions to clinical HF requires that the observations are robust and reproducible and can be validated across multiple independent populations to ensure confidence in clinical decision-making.

Myocardial fibrosis in asymptomatic and symptomatic chronic severe primary mitral regurgitation and relationship to tissue characterisation and left ventricular function on cardiovascular magnetic resonance.

BACKGROUND: Myocardial fibrosis occurs in end-stage heart failure secondary to mitral regurgitation (MR), but it is not known whether this is present before onset of symptoms or myocardial dysfunction. This study aimed to characterise myocardial fibrosis in chronic severe primary MR on histology, compare this to tissue characterisation on cardiovascular magnetic resonance (CMR) imaging, and investigate associations with symptoms, left ventricular (LV) function, and exercise capacity. METHODS: Patients with class I or IIa indications for surgery underwent CMR and cardiopulmonary exercise testing. LV biopsies were taken at surgery and the extent of fibrosis was quantified on histology using collagen volume fraction (CVFmean) compared to autopsy controls without cardiac pathology. RESULTS: 120 consecutive patients (64 ± 13 years; 71% male) were recruited; 105 patients underwent MV repair while 15 chose conservative management. LV biopsies were obtained in 86 patients (234 biopsy samples in total). MR patients had more fibrosis compared to 8 autopsy controls (median: 14.6% [interquartile range 7.4-20.3] vs. 3.3% [2.6-6.1], P < 0.001); this difference persisted in the asymptomatic patients (CVFmean 13.6% [6.3-18.8], P < 0.001), but severity of fibrosis was not significantly higher in NYHA II-III symptomatic MR (CVFmean 15.7% [9.9-23.1] (P = 0.083). Fibrosis was patchy across biopsy sites (intraclass correlation 0.23, 95% CI 0.08-0.39, P = 0.001). No significant relationships were identified between CVFmean and CMR tissue characterisation [native T1, extracellular volume (ECV) or late gadolinium enhancement] or measures of LV function [LV ejection fraction (LVEF), global longitudinal strain (GLS)]. Although the range of ECV was small (27.3 ± 3.2%), ECV correlated with multiple measures of LV function (LVEF: Rho = - 0.22, P = 0.029, GLS: Rho = 0.29, P = 0.003), as well as NTproBNP (Rho = 0.54, P < 0.001) and exercise capacity (%PredVO2max: R = - 0.22, P = 0.030). CONCLUSIONS: Patients with chronic primary MR have increased fibrosis before the onset of symptoms. Due to the patchy nature of fibrosis, CMR derived ECV may be a better marker of global myocardial status. Clinical trial registration Mitral FINDER study; Clinical Trials NCT02355418, Registered 4 February 2015, https://clinicaltrials.gov/ct2/show/NCT02355418.

Association of left atrium voltage amplitude and distribution with the risk of atrial fibrillation recurrence and evolution after pulmonary vein isolation: An ultrahigh-density mapping study.

INTRODUCTION: Ultrahigh-density-voltage mapping (uHDV M) is a new tool that can add new insights into the pathophysiology of atrial fibrillation (AF). The aim of this study was to evaluate the performance of uHDV M in predicting postablation AF recurrence (AFR). METHODS AND RESULTS: We included 98 consecutive patients undergoing pulmonary vein isolation for AF (40.8% persistent) using an uHDV M system and followed for 1 year. The left atrium (LA) mean voltage (Vm ) and the Vslope (slope of the voltage histogram calculated by linear interpolation, with the relative frequency on the vertical axis and the bipolar potential on the horizontal axis) were calculated from 12 567 ± 5486 points per map. Patients with AFR (N = 29) had lower Vm and higher Vslope as compared with patients without AFR (N = 69). Receiver operating characteristic curves identified Vm as the strongest predictor of AFR, with a higher incidence of AFR in patients with Vm 0.758 mV (57.6%) or lower than patients with Vm higher than 0.758 mV (15.4%; P < .0001). Among patients with Vm  higher than 0.758 mV, patients with Vslope 0.637 or higher exhibited higher (P = .043) AFR incidence (31.3%) than patients with Vslope lower than 0.637 (10.2%). This classification showed incremental predictive value over relevant covariables. Vm values were lower and Vslope values were higher in patients that progressed from paroxysmal to persistent AF. Patients with Vslope 0.637 or higher had a 14.2% incidence of postablation atypical atrial flutter, whereas patients with Vslope lower than 0.637 did not present this outcome. CONCLUSIONS: The risk of AFR, atrial flutter, and progression from paroxysmal to persistent AF can be detected by quantitative analysis of LA uHDV M identifying diverse patterns of atrial substrate alterations.

Immunomodulation by adoptive regulatory T-cell transfer improves Coxsackievirus B3-induced myocarditis.

Regulatory T (Treg) cells offer new therapeutic options for controlling undesired systemic and local immune responses. The aim of the current study was to determine the impact of therapeutic Treg administration on systemic and cardiac inflammation and remodeling in coxsackievirus B3 (CVB3) -induced myocarditis. Therefore, syngeneic Treg cells were applied intravenously in CVB3-infected mice 3 d after infection. Compared with CVB3 + PBS mice, CVB3 + Treg mice exhibited lower left ventricular (LV) chemokine expression, accompanied by reduced cardiac presence of proinflammatory Ly6ChighCCR2highCx3Cr1low monocytes and higher retention of proinflammatory Ly6CmidCCR2highCx3Cr1low monocytes in the spleen. In addition, splenic myelopoiesis was reduced in CVB3 + Treg compared with CVB3 + PBS mice. Coculture of Treg cells with splenocytes isolated from mice 3 d post-CVB3 infection further demonstrated the ability of Treg cells to modulate monocyte differentiation in favor of the anti-inflammatory Ly6ClowCCR2lowCx3Cr1high subset. Treg-mediated immunomodulation was paralleled by lower collagen 1 protein expression and decreased levels of soluble and insoluble collagen in LV of CVB3 + Treg compared with CVB3 + PBS mice. In agreement with these findings, LV systolic and diastolic function was improved in CVB3 + Treg mice compared with CVB3 + PBS mice. In summary, adoptive Treg transfer in the inflammatory phase of viral-induced myocarditis protects the heart against inflammatory damage and fibrosis via modulation of monocyte subsets.-Pappritz, K., Savvatis, K., Miteva, K., Kerim, B., Dong, F., Fechner, H., Müller, I., Brandt, C., Lopez, B., González, A., Ravassa, S., Klingel, K., Diez, J., Reinke, P., Volk, H.-D., Van Linthout, S., Tschöpe, C. Immunomodulation by adoptive regulatory T-cell transfer improves Coxsackievirus B3-induced myocarditis.

Reappraising myocardial fibrosis in severe aortic stenosis: an invasive and non-invasive study in 133 patients.

Aims: To investigate myocardial fibrosis (MF) in a large series of severe aortic stenosis (AS) patients using invasive biopsy and non-invasive imaging. Methods and results: One hundred thirty-three patients with severe, symptomatic AS accepted for surgical aortic valve replacement underwent cardiovascular magnetic resonance (CMR) with late gadolinium enhancement (LGE) and extracellular volume fraction (ECV) quantification. Intra-operative left ventricular (LV) biopsies were performed by needle or scalpel, yielding tissue with (n = 53) and without endocardium (n = 80), and compared with 10 controls. Myocardial fibrosis occurred in three patterns: (i) thickened endocardium with a fibrotic layer; (ii) microscopic scars, with a subendomyocardial predominance; and (iii) diffuse interstitial fibrosis. Collagen volume fraction (CVF) was elevated (P < 0.001) compared with controls, and higher (P < 0.001) in endocardium-containing samples with a decreasing CVF gradient from the subendocardium (P = 0.001). Late gadolinium enhancement correlated with CVF (P < 0.001) but not ECV. Both LGE and ECV correlated independently (P < 0.001) with N-terminal pro-brain natriuretic peptide and high-sensitivity-troponin T. High ECV was also associated with worse LV remodelling, left ventricular ejection fraction and functional capacity. Combining high ECV and LGE better identified patients with more adverse LV remodelling, blood biomarkers and histological parameters, and worse functional capacity than each parameter alone. Conclusion: Myocardial fibrosis in severe AS is complex, but three main patterns exist: endocardial fibrosis, microscars (mainly in the subendomyocardium), and diffuse interstitial fibrosis. Neither histological CVF nor the CMR parameters ECV and LGE capture fibrosis in its totality. A combined, multi-parametric approach with ECV and LGE allows best stratification of AS patients according to the response of the myocardial collagen matrix.

p-SMAD2/3 and DICER promote pre-miR-21 processing during pressure overload-associated myocardial remodeling.

Transforming growth factor-ß (TGF-ß) induces miR-21 expression which contributes to fibrotic events in the left ventricle (LV) under pressure overload. SMAD effectors of TGF-ß signaling interact with DROSHA to promote primary miR-21 processing into precursor miR-21 (pre-miR-21). We hypothesize that p-SMAD-2 and -3 also interact with DICER1 to regulate the processing of pre-miR-21 to mature miR-21 in cardiac fibroblasts under experimental and clinical pressure overload. The subjects of the study were mice undergoing transverse aortic constriction (TAC) and patients with aortic stenosis (AS). In vitro, NIH-3T3 fibroblasts transfected with pre-miR-21 responded to TGF-ß1 stimulation by overexpressing miR-21. Overexpression and silencing of SMAD2/3 resulted in higher and lower production of mature miR-21, respectively. DICER1 co-precipitated along with SMAD2/3 and both proteins were up-regulated in the LV from TAC-mice. Pre-miR-21 was isolated bound to the DICER1 maturation complex. Immunofluorescence analysis revealed co-localization of p-SMAD2/3 and DICER1 in NIH-3T3 and mouse cardiac fibroblasts. DICER1-p-SMAD2/3 protein-protein interaction was confirmed by in situ proximity ligation assay. Myocardial up-regulation of DICER1 constituted a response to pressure overload in TAC-mice. DICER mRNA levels correlated directly with those of TGF-ß1, SMAD2 and SMAD3. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with TGF-ß1, SMAD2, and SMAD3. Our results support that p-SMAD2/3 interacts with DICER1 to promote pre-miR-21 processing to mature miR-21. This new TGFß-dependent regulatory mechanism is involved in miR-21 overexpression in cultured fibroblasts, and in the pressure overloaded LV of mice and human patients.

Potential role of microRNA-10b down-regulation in cardiomyocyte apoptosis in aortic stenosis patients.

MicroRNAs have been associated with cardiomyocyte apoptosis, a process involved in myocardial remodelling in aortic valve (Av) stenosis (AS). Our aim was to analyse whether the dysregulation of myocardial microRNAs was related to cardiomyocyte apoptosis in AS patients. Endomyocardial biopsies were obtained from 28 patients with severe AS (based on pressure gradients and Av area) referred for Av replacement and from necropsies of 10 cardiovascular disease-free control subjects. AS patients showed an increased (P<0.001) cardiomyocyte apoptotic index (CMAI) compared with controls. Two clusters of patients were identified according to the CMAI: group 1 (CMAI ≤ 0.08%; n=16) and group 2 (CMAI > 0.08%; n=12). Group 2 patients presented lower cardiomyocyte density (P<0.001) and ejection fraction (P<0.05), and higher troponin T levels (P<0.05), prevalence of heart failure (HF; P<0.05) and NT-proBNP levels (P<0.05) than those from group 1. miRNA expression profile analysed in 5 patients randomly selected from each group showed 64 microRNAs down-regulated and 6 up-regulated (P<0.05) in group 2 compared with group 1. Those microRNAs with the highest fold-change were validated in the full two groups corroborating that miR-10b, miR-125b-2* and miR-338-3p were down-regulated (P<0.05) in group 2 compared with group 1 and control subjects. These three microRNAs were inversely correlated (P<0.05) with the CMAI. Inhibition of miR-10b induced an increase (P<0.05) of apoptosis and increased expression (P<0.05) of apoptosis protease-activating factor-1 (Apaf-1) in HL-1 cardiomyocytes. In conclusion, myocardial down-regulation of miR-10b may be involved in increased cardiomyocyte apoptosis in AS patients, probably through Apaf-1 up-regulation, contributing to cardiomyocyte damage and to the development of HF.

microRNA-122 down-regulation may play a role in severe myocardial fibrosis in human aortic stenosis through TGF-ß1 up-regulation.

miRNAs (microRNAs) have been shown to play a role in myocardial fibrosis. The present study was designed to analyse whether alterations in miRNA expression contribute to the progression of myocardial fibrosis in AS (aortic valve stenosis) patients through up-regulation of the pro-fibrotic factor TGF-ß1 (transforming growth factor-ß type 1). Endomyocardial biopsies were obtained from 28 patients with severe AS, and from the necropsies of 10 control subjects. AS patients presented increased myocardial CVF (collagen volume fraction) and TGF-ß1 compared with the controls, these parameters being correlated in all patients. Patients were divided into two groups by cluster analysis according to their CVF: SF (severe fibrosis; CVF >15%; n=15) and non-SF (CVF ≤15%; n=13). TGF-ß1 was increased in patients with SF compared with those with non-SF. To analyse the involvement of miRNAs in SF, the miRNA expression profile of 10 patients (four with non-SF and six with SF) was analysed showing that 99 miRNAs were down-regulated and 19 up-regulated in the SF patients compared with the non-SF patients. Those miRNAs potentially targeting TGF-ß1 were validated by real-time RT (reverse transcription)-PCR in the whole test population, corroborating that miR-122 and miR-18b were down-regulated in patients with SF compared with those with non-SF and the control subjects. Additionally, miR-122 was inversely correlated with the CVF, TGF-ß1 and the TGF-ß1-regulated PCPE-1 (procollagen C-terminal proteinase enhancer-1) in all patients. Experiments in human fibroblasts demonstrated that miR-122 targets and inhibits TGF-ß1. In conclusion, for the first time we show that myocardial down-regulation of miR-122 might be involved in myocardial fibrosis in AS patients, probably through TGF-ß1 up-regulation.

Atrial fibrillation and biomarkers of myocardial fibrosis in heart failure.

OBJECTIVES: Alterations of collagen metabolism present in heart failure promote the fibrotic substrate for the development of atrial fibrillation (AF). Myocardial collagen I synthesis and degradation can be assessed indirectly by circulating biomarkers such as the carboxy terminal propeptide (PICP) and carboxy-terminal telopeptide (CITP), respectively. DESIGN: We examined myocardial collagen type-I metabolism in 143 patients with systolic heart failure (New York Heart Association Class 2-4) in relation to coexisting AF. RESULTS: Mean age was 75 years, blood pressure 134/80 mm Hg, ejection fraction 34%, serum PICP 81 µg/L and CITP 8.3 µg/L, and median plasma brain natriuretic peptide 215 pg/L; 77 were in AF. PICP and CITP were related to left atrial diameter (r = 0.22, P = 0.013, and r = 0.26, P = 0.003) and CITP to pulmonary capillary wedge pressure and C-reactive protein (r = 0.19, P = 0.044, and r = 0.29, P = 0.003). A logistic regression suggested that PICP (odds ratio per 1 µg/L change 1.01, P = 0.012) and left ventricular end-diastolic volume (odds ratio per 1 mL change 0.98, P < 0.001) were independently associated with coexisting AF. CONCLUSION: Collagen type-I metabolism is associated to left atrial size. Heart failure patients with coexisting AF exhibit more altered collagen type-I metabolism than patients in sinus rhythm. This might represent more severe atrial and ventricular fibrosis.

High variability in assays of blood markers of collagen turnover in cardiovascular disease: Implications for research and clinical practice.

AIMS: Fibrosis is a common feature of many chronic diseases, including heart failure, which can have deleterious effects on cardiac structure and function that are associated with adverse outcomes. By-products of collagen synthesis and degradation, such as carboxy- and amino-terminal pro- or telo-peptides of collagen type I and III (PICP, PINP, PIIINP, and CITP) have been extensively investigated as markers of fibrosis. Although the majority of studies report on the reproducibility of their assay results, there is no a comparison of biomarker assays across studies. Therefore, we conducted a systematic review adhering to PRISMA guidelines. METHODS AND RESULTS: The search terms employed in Medline were: 'collagen AND cardiac' or 'collagen AND heart'. This query yielded a total of 1049 articles. Thereafter, specific search criteria were applied: (i) original English-language papers; (ii) human studies; (iii) in-vivo investigations; and (iv) blood/serum/plasma samples. Overall, 89 studies were identified (42 on PIIINP, 32 on PICP, 29 on CITP, and 17 on PINP). The range of reported values for PIIINP was between 0.06 to 11 800 µg/l; for PICP 0.006 to 1265 µg/l; for CITP 0.3 to 5450 µg/l; for PINP 0.15 to 80 µg/l. Extreme variations in values for fibrosis biomarkers were observed across studies, especially when different assays were used, but also with the same assays. CONCLUSIONS: Our findings show that it is challenging to ascertain normal ranges or compare studies for the measurement of fibrosis biomarkers. Given the potential implications for clinical practice and current lack of awareness of these issues, this subject warrants comprehensive acknowledgement and understanding.

Myocardial Interstitial Fibrosis in Hypertensive Heart Disease: From Mechanisms to Clinical Management.

Hypertensive heart disease (HHD) can no longer be considered as the beneficial adaptive result of the hypertrophy of cardiomyocytes in response to pressure overload leading to the development of left ventricular hypertrophy. The current evidence indicates that in patients with HHD, pathological lesions in the myocardium lead to maladaptive structural remodeling and subsequent alterations in cardiac function, electrical activity, and perfusion, all contributing to poor outcomes. Diffuse myocardial interstitial fibrosis is probably the most critically involved lesion in these disorders. Therefore, in this review, we will focus on the histological characteristics, the mechanisms, and the clinical consequences of myocardial interstitial fibrosis in patients with HHD. In addition, we will consider the most useful tools for the noninvasive diagnosis of myocardial interstitial fibrosis in patients with HHD, as well as the most effective available therapeutic strategies to prevent its development or facilitate its regression in this patient population. Finally, we will issue a call to action for the need for more fundamental and clinical research on myocardial interstitial fibrosis in HHD.

Generation of Self-Induced Myocardial Ischemia in Large-Sized Cardiac Spheroids without Alteration of Environmental Conditions Recreates Fibrotic Remodeling and Tissue Stiffening Revealed by Constriction Assays.

A combination of human-induced pluripotent stem cells (hiPSCs) and 3D microtissue culture techniques allows the generation of models that recapitulate the cardiac microenvironment for preclinical research of new treatments. In particular, spheroids represent the simplest approach to culture cells in 3D and generate gradients of cellular access to the media, mimicking the effects of an ischemic event. However, previous models required incubation under low oxygen conditions or deprived nutrient media to recreate ischemia. Here, we describe the generation of large spheroids (i.e., larger than 500 µm diameter) that self-induce an ischemic core. Spheroids were generated by coculture of cardiomyocytes derived from hiPSCs (hiPSC-CMs) and primary human cardiac fibroblast (hCF). In the proper medium, cells formed aggregates that generated an ischemic core 2 days after seeding. Spheroids also showed spontaneous cellular reorganization after 10 days, with hiPSC-CMs located at the center and surrounded by hCFs. This led to an increase in microtissue stiffness, characterized by the implementation of a constriction assay. All in all, these phenomena are hints of the fibrotic tissue remodeling secondary to a cardiac ischemic event, thus demonstrating the suitability of these spheroids for the modeling of human cardiac ischemia and its potential application for new treatments and drug research.