Paracrine signalling by cardiac calcitonin controls atrial fibrogenesis and arrhythmia.

Atrial fibrillation, the most common cardiac arrhythmia, is an important contributor to mortality and morbidity, and particularly to the risk of stroke in humans1. Atrial-tissue fibrosis is a central pathophysiological feature of atrial fibrillation that also hampers its treatment; the underlying molecular mechanisms are poorly understood and warrant investigation given the inadequacy of present therapies2. Here we show that calcitonin, a hormone product of the thyroid gland involved in bone metabolism3, is also produced by atrial cardiomyocytes in substantial quantities and acts as a paracrine signal that affects neighbouring collagen-producing fibroblasts to control their proliferation and secretion of extracellular matrix proteins. Global disruption of calcitonin receptor signalling in mice causes atrial fibrosis and increases susceptibility to atrial fibrillation. In mice in which liver kinase B1 is knocked down specifically in the atria, atrial-specific knockdown of calcitonin promotes atrial fibrosis and increases and prolongs spontaneous episodes of atrial fibrillation, whereas atrial-specific overexpression of calcitonin prevents both atrial fibrosis and fibrillation. Human patients with persistent atrial fibrillation show sixfold lower levels of myocardial calcitonin compared to control individuals with normal heart rhythm, with loss of calcitonin receptors in the fibroblast membrane. Although transcriptome analysis of human atrial fibroblasts reveals little change after exposure to calcitonin, proteomic analysis shows extensive alterations in extracellular matrix proteins and pathways related to fibrogenesis, infection and immune responses, and transcriptional regulation. Strategies to restore disrupted myocardial calcitonin signalling thus may offer therapeutic avenues for patients with atrial fibrillation.

Proteomic Atlas of Atherosclerosis: The Contribution of Proteoglycans to Sex Differences, Plaque Phenotypes, and Outcomes.

BACKGROUND: Using proteomics, we aimed to reveal molecular types of human atherosclerotic lesions and study their associations with histology, imaging, and cardiovascular outcomes. METHODS: Two hundred nineteen carotid endarterectomy samples were procured from 120 patients. A sequential protein extraction protocol was employed in conjunction with multiplexed, discovery proteomics. To focus on extracellular proteins, parallel reaction monitoring was employed for targeted proteomics. Proteomic signatures were integrated with bulk, single-cell, and spatial RNA-sequencing data, and validated in 200 patients from the Athero-Express Biobank study. RESULTS: This extensive proteomics analysis identified plaque inflammation and calcification signatures, which were inversely correlated and validated using targeted proteomics. The inflammation signature was characterized by the presence of neutrophil-derived proteins, such as S100A8/9 (calprotectin) and myeloperoxidase, whereas the calcification signature included fetuin-A, osteopontin, and gamma-carboxylated proteins. The proteomics data also revealed sex differences in atherosclerosis, with large-aggregating proteoglycans versican and aggrecan being more abundant in females and exhibiting an inverse correlation with estradiol levels. The integration of RNA-sequencing data attributed the inflammation signature predominantly to neutrophils and macrophages, and the calcification and sex signatures to smooth muscle cells, except for certain plasma proteins that were not expressed but retained in plaques, such as fetuin-A. Dimensionality reduction and machine learning techniques were applied to identify 4 distinct plaque phenotypes based on proteomics data. A protein signature of 4 key proteins (calponin, protein C, serpin H1, and versican) predicted future cardiovascular mortality with an area under the curve of 75% and 67.5% in the discovery and validation cohort, respectively, surpassing the prognostic performance of imaging and histology. CONCLUSIONS: Plaque proteomics redefined clinically relevant patient groups with distinct outcomes, identifying subgroups of male and female patients with elevated risk of future cardiovascular events.

Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling.

BACKGROUND: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. METHODS: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. RESULTS: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin ß1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of ß-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that ß1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with ß-blockers had a distinct cardiac ECM profile. CONCLUSIONS: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that ß-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

Cathepsin A contributes to left ventricular remodeling by degrading extracellular superoxide dismutase in mice.

In the heart, the serine carboxypeptidase cathepsin A (CatA) is distributed between lysosomes and the extracellular matrix (ECM). CatA-mediated degradation of extracellular peptides may contribute to ECM remodeling and left ventricular (LV) dysfunction. Here, we aimed to evaluate the effects of CatA overexpression on LV remodeling. A proteomic analysis of the secretome of adult mouse cardiac fibroblasts upon digestion by CatA identified the extracellular antioxidant enzyme superoxide dismutase (EC-SOD) as a novel substrate of CatA, which decreased EC-SOD abundance 5-fold. In vitro, both cardiomyocytes and cardiac fibroblasts expressed and secreted CatA protein, and only cardiac fibroblasts expressed and secreted EC-SOD protein. Cardiomyocyte-specific CatA overexpression and increased CatA activity in the LV of transgenic mice (CatA-TG) reduced EC-SOD protein levels by 43%. Loss of EC-SOD-mediated antioxidative activity resulted in significant accumulation of superoxide radicals (WT, 4.54 µmol/mg tissue/min; CatA-TG, 8.62 µmol/mg tissue/min), increased inflammation, myocyte hypertrophy (WT, 19.8 µm; CatA-TG, 21.9 µm), cellular apoptosis, and elevated mRNA expression of hypertrophy-related and profibrotic marker genes, without affecting intracellular detoxifying proteins. In CatA-TG mice, LV interstitial fibrosis formation was enhanced by 19%, and the type I/type III collagen ratio was shifted toward higher abundance of collagen I fibers. Cardiac remodeling in CatA-TG was accompanied by an increased LV weight/body weight ratio and LV end diastolic volume (WT, 50.8 µl; CatA-TG, 61.9 µl). In conclusion, CatA-mediated EC-SOD reduction in the heart contributes to increased oxidative stress, myocyte hypertrophy, ECM remodeling, and inflammation, implicating CatA as a potential therapeutic target to prevent ventricular remodeling.

Comparative Analysis of Circulating Noncoding RNAs Versus Protein Biomarkers in the Detection of Myocardial Injury.

RATIONALE: Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), circular RNAs (circRNAs), and long noncoding RNAs (lncRNAs), are proposed novel biomarkers of myocardial injury. Their release kinetics have not been explored without confounding by heparin nor has their relationship to myocardial protein biomarkers. OBJECTIVE: To compare ncRNA types in heparinase-treated samples with established and emerging protein biomarkers for myocardial injury. METHODS AND RESULTS: Screening of 158 circRNAs and 21 lncRNAs in human cardiac tissue identified 12 circRNAs and 11 lncRNAs as potential biomarkers with cardiac origin. Eleven miRNAs were included. At low spike-in concentrations of myocardial tissue, significantly higher regression coefficients were observed across ncRNA types compared with cardiac troponins and cMyBP-C (cardiac myosin-binding protein C). Heparinase treatment of serial plasma and serum samples of patients undergoing transcoronary ablation of septal hypertrophy removed spurious correlations between miRNAs in non-heparinase-treated samples. After transcoronary ablation of septal hypertrophy, muscle-enriched miRNAs (miR-1 and miR-133a) showed a steeper and earlier increase than cardiac-enriched miRNAs (miR-499 and miR-208b). Putative cardiac lncRNAs, including LIPCAR (long intergenic noncoding RNA predicting cardiac remodeling and survival), did not rise, refuting a predominant cardiac origin. Cardiac circRNAs remained largely undetectable. In a validation cohort of acute myocardial infarction, receiver operating characteristic curve analysis revealed noninferiority of cardiac-enriched miRNAs, but miRNAs failed to identify cases presenting with low troponin values. cMyBP-C was validated as a biomarker with highly sensitive properties, and the combination of muscle-enriched miRNAs with high-sensitive cardiac troponin T and cMyBP-C returned the highest area under the curve values. CONCLUSIONS: In a comparative assessment of ncRNAs and protein biomarkers for myocardial injury, cMyBP-C showed properties as the most sensitive cardiac biomarker while miRNAs emerged as promising candidates to integrate ncRNAs with protein biomarkers. Sensitivity of current miRNA detection is inferior to cardiac proteins but a multibiomarker combination of muscle-enriched miRNAs with cMyBP-C and cardiac troponins could open a new path of integrating complementary characteristics of different biomarker types.

Glycoproteomic Analysis of the Aortic Extracellular Matrix in Marfan Patients.

OBJECTIVE: Marfan syndrome (MFS) is caused by mutations in FBN1 (fibrillin-1), an extracellular matrix (ECM) component, which is modified post-translationally by glycosylation. This study aimed to characterize the glycoproteome of the aortic ECM from patients with MFS and relate it to aortopathy. Approach and Results: ECM extracts of aneurysmal ascending aortic tissue from patients with and without MFS were enriched for glycopeptides. Direct N-glycopeptide analysis by mass spectrometry identified 141 glycoforms from 47 glycosites within 35 glycoproteins in the human aortic ECM. Notably, MFAP4 (microfibril-associated glycoprotein 4) showed increased and more diverse N-glycosylation in patients with MFS compared with control patients. MFAP4 mRNA levels were markedly higher in MFS aortic tissue. MFAP4 protein levels were also increased at the predilection (convexity) site for ascending aorta aneurysm in bicuspid aortic valve patients, preceding aortic dilatation. In human aortic smooth muscle cells, MFAP4 mRNA expression was induced by TGF (transforming growth factor)-ß1 whereas siRNA knockdown of MFAP4 decreased FBN1 but increased elastin expression. These ECM changes were accompanied by differential gene expression and protein abundance of proteases from ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family and their proteoglycan substrates, respectively. Finally, high plasma MFAP4 concentrations in patients with MFS were associated with a lower thoracic descending aorta distensibility and greater incidence of type B aortic dissection during 68 months follow-up. CONCLUSIONS: Our glycoproteomics analysis revealed that MFAP4 glycosylation is enhanced, as well as its expression during the advanced, aneurysmal stages of MFS compared with control aneurysms from patients without MFS.

Extracellular Matrix Proteomics Reveals Interplay of Aggrecan and Aggrecanases in Vascular Remodeling of Stented Coronary Arteries.

BACKGROUND: Extracellular matrix (ECM) remodeling contributes to in-stent restenosis and thrombosis. Despite its important clinical implications, little is known about ECM changes post-stent implantation. METHODS: Bare-metal and drug-eluting stents were implanted in pig coronary arteries with an overstretch under optical coherence tomography guidance. Stented segments were harvested 1, 3, 7, 14, and 28 days post-stenting for proteomics analysis of the media and neointima. RESULTS: A total of 151 ECM and ECM-associated proteins were identified by mass spectrometry. After stent implantation, proteins involved in regulating calcification were upregulated in the neointima of drug-eluting stents. The earliest changes in the media were proteins involved in inflammation and thrombosis, followed by changes in regulatory ECM proteins. By day 28, basement membrane proteins were reduced in drug-eluting stents in comparison with bare-metal stents. In contrast, the large aggregating proteoglycan aggrecan was increased. Aggrecanases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family contribute to the catabolism of vascular proteoglycans. An increase in ADAMTS-specific aggrecan fragments was accompanied by a notable shift from ADAMTS1 and ADAMTS5 to ADAMTS4 gene expression after stent implantation. Immunostaining in human stented coronary arteries confirmed the presence of aggrecan and aggrecan fragments, in particular, at the contacts of the stent struts with the artery. Further investigation of aggrecan presence in the human vasculature revealed that aggrecan and aggrecan cleavage were more abundant in human arteries than in human veins. In addition, aggrecan synthesis was induced on grafting a vein into the arterial circulation, suggesting an important role for aggrecan in vascular plasticity. Finally, lack of ADAMTS-5 activity in mice resulted in an accumulation of aggrecan and a dilation of the thoracic aorta, confirming that aggrecanase activity regulates aggrecan abundance in the arterial wall and contributes to vascular remodeling. CONCLUSIONS: Significant differences were identified by proteomics in the ECM of coronary arteries after bare-metal and drug-eluting stent implantation, most notably an upregulation of aggrecan, a major ECM component of cartilaginous tissues that confers resistance to compression. The accumulation of aggrecan coincided with a shift in ADAMTS gene expression. This study provides the first evidence implicating aggrecan and aggrecanases in the vascular injury response after stenting.

Role of ADAMTS-5 in Aortic Dilatation and Extracellular Matrix Remodeling.

OBJECTIVE: Thoracic aortic aneurysm (TAA), a degenerative disease of the aortic wall, is accompanied by changes in the structure and composition of the aortic ECM (extracellular matrix). The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases has recently been implicated in TAA formation. This study aimed to investigate the contribution of ADAMTS-5 to TAA development. APPROACH AND RESULTS: A model of aortic dilatation by AngII (angiotensin II) infusion was adopted in mice lacking the catalytic domain of ADAMTS-5 (Adamts5Δcat). Adamts5Δcat mice showed an attenuated rise in blood pressure while displaying increased dilatation of the ascending aorta (AsAo). Interestingly, a proteomic comparison of the aortic ECM from AngII-treated wild-type and Adamts5Δcat mice revealed versican as the most upregulated ECM protein in Adamts5Δcat mice. This was accompanied by a marked reduction of ADAMTS-specific versican cleavage products (versikine) and a decrease of LRP1 (low-density lipoprotein-related protein 1). Silencing LRP1 expression in human aortic smooth muscle cells reduced the expression of ADAMTS5, attenuated the generation of versikine, but increased soluble ADAMTS-1. A similar increase in ADAMTS-1 was observed in aortas of AngII-treated Adamts5Δcat mice but was not sufficient to maintain versican processing and prevent aortic dilatation. CONCLUSIONS: Our results support the emerging role of ADAMTS proteases in TAA. ADAMTS-5 rather than ADAMTS-1 is the key protease for versican regulation in murine aortas. Further studies are needed to define the ECM substrates of the different ADAMTS proteases and their contribution to TAA formation.

Analysis of Mitochondrial Proteins in the Surviving Myocardium after Ischemia Identifies Mitochondrial Pyruvate Carrier Expression as Possible Mediator of Tissue Viability.

The endogenous mechanisms contributing to tissue survival following myocardial infarction are not fully understood. We investigated the alterations in the mitochondrial proteome after ischemia-reperfusion (I/R) and its possible implications on cell survival. Mitochondrial proteomic analysis of cardiac tissue from an in vivo porcine I/R model found that surviving tissue in the peri-infarct border zone showed increased expression of several proteins. Notably, these included subunits of the mitochondrial pyruvate carrier (MPC), namely MPC1 and MPC2. Western blot, immunohistochemistry, and mRNA analysis corroborated the elevated expression of MPC in the surviving tissue. Furthermore, MPC1 and MPC2 protein levels were found to be markedly elevated in the myocardium of ischemic cardiomyopathy patients. These findings led to the hypothesis that increased MPC expression is cardioprotective due to enhancement of mitochondrial pyruvate uptake in the energy-starved heart following I/R. To test this, isolated mouse hearts perfused with a modified Krebs buffer (containing glucose, pyruvate, and octanoate as metabolic substrates) were subjected to I/R with or without the MPC transport inhibitor UK5099. UK5099 increased myocardial infarction and attenuated post-ischemic recovery of left ventricular end-diastolic pressure. However, aerobically perfused control hearts that were exposed to UK5099 did not modulate contractile function, although pyruvate uptake was blocked as evidenced by increased cytosolic lactate and pyruvate levels. Our findings indicate that increased expression of MPC leads to enhanced uptake and utilization of pyruvate during I/R. We propose this as a putative endogenous mechanism that promotes myocardial survival to limit infarct size.

Oxidant-induced Interprotein Disulfide Formation in Cardiac Protein DJ-1 Occurs via an Interaction with Peroxiredoxin 2.

The role and responses of the dimeric DJ-1 protein to cardiac oxidative stress is incompletely understood. H2O2 induces a 50-kDa DJ-1 interprotein homodimer disulfide, known to form between Cys-53 on each subunit. A trimeric 75-kDa DJ-1 complex that mass spectrometry shows contained 2-Cys peroxiredoxin also formed and precedes the appearance of the disulfide dimer. These observations may represent peroxiredoxin sensing and transducing the oxidant signal to DJ-1. The dimeric disulfide DJ-1 complex was stabilized by auranofin, suggesting that thioredoxin recycles it in cells. Higher concentrations of H2O2 concomitantly induce DJ-1 Cys-106 hyperoxidation (sulfination or sulfonation) in myocytes, perfused heart, or HEK cells. An oxidation-resistant C53A DJ-1 shows potentiated H2O2-induced Cys-106 hyperoxidation. DJ-1 also forms multiple disulfides with unknown target proteins during H2O2 treatment, the formation of which is also potentiated in cells expressing the C53A mutant. This suggests that the intersubunit disulfide induces a conformational change that limits Cys-106 forming heterodisulfide protein complexes or from hyperoxidizing. High concentrations of H2O2 also induce cell death, with DJ-1 Cys-106 sulfonation appearing causal in these events, as expressionof C53A DJ-1 enhanced both Cys-106 sulfonation and cell death. Nonetheless, expression of the DJ-1 C106A mutant, which fully prevents hyperoxidation, also showed exacerbated cell death responses to H2O2 A rational explanation for these findings is that DJ-1 Cys-106 forms disulfides with target proteins to limit oxidant-induced cell death. However, when Cys-106 is hyperoxidized, formation of these potentially protective heterodimeric disulfide complexes is limited, and so cell death is exacerbated.

Glycoproteomics Reveals Decorin Peptides With Anti-Myostatin Activity in Human Atrial Fibrillation.

BACKGROUND: Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). METHODS: Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. RESULTS: ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. CONCLUSIONS: This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.

Role of miR-195 in aortic aneurysmal disease.

RATIONALE: Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 and miR-15 families have been implicated in regulating the vascular extracellular matrix. OBJECTIVE: Our aim was to assess the effect of the miR-15 family on aortic aneurysm development. METHODS AND RESULTS: Among the miR-15 family members, miR-195 was differentially expressed in aortas of apolipoprotein E-deficient mice on angiotensin II infusion. Proteomics analysis of the secretome of murine aortic smooth muscle cells, after miR-195 manipulation, revealed that miR-195 targets a cadre of extracellular matrix proteins, including collagens, proteoglycans, elastin, and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect, particularly in regulating collagens. Systemic and local administration of cholesterol-conjugated antagomiRs revealed better inhibition of miR-195 compared with miR-29b in the uninjured aorta. However, in apolipoprotein E-deficient mice receiving angiotensin II, silencing of miR-29b, but not miR-195, led to an attenuation of aortic dilation. Higher aortic elastin expression was accompanied by an increase of matrix metalloproteinases 2 and 9 in mice treated with antagomiR-195. In human plasma, an inverse correlation of miR-195 was observed with the presence of abdominal aortic aneurysms and aortic diameter. CONCLUSIONS: We provide the first evidence that miR-195 may contribute to the pathogenesis of aortic aneurysmal disease. Although inhibition of miR-29b proved more effective in preventing aneurysm formation in a preclinical model, miR-195 represents a potent regulator of the aortic extracellular matrix. Notably, plasma levels of miR-195 were reduced in patients with abdominal aortic aneurysms suggesting that microRNAs might serve as a noninvasive biomarker of abdominal aortic aneurysms.

Extracellular matrix secretion by cardiac fibroblasts: role of microRNA-29b and microRNA-30c.

RATIONALE: MicroRNAs (miRNAs), in particular miR-29b and miR-30c, have been implicated as important regulators of cardiac fibrosis. OBJECTIVE: To perform a proteomics comparison of miRNA effects on extracellular matrix secretion by cardiac fibroblasts. METHODS AND RESULTS: Mouse cardiac fibroblasts were transfected with pre-/anti-miR of miR-29b and miR-30c, and their conditioned medium was analyzed by mass spectrometry. miR-29b targeted a cadre of proteins involved in fibrosis, including multiple collagens, matrix metalloproteinases, and leukemia inhibitory factor, insulin-like growth factor 1, and pentraxin 3, 3 predicted targets of miR-29b. miR-29b also attenuated the cardiac fibroblast response to transforming growth factor-ß. In contrast, miR-30c had little effect on extracellular matrix production but opposite effects regarding leukemia inhibitory factor and insulin-like growth factor 1. Both miRNAs indirectly affected cardiac myocytes. On transfection with pre-miR-29b, the conditioned medium of cardiac fibroblasts lost its ability to support adhesion of rat ventricular myocytes and led to a significant reduction of cardiac myocyte proteins (α-actinin, cardiac myosin-binding protein C, and cardiac troponin I). Similarly, cardiomyocytes derived from mouse embryonic stem cells atrophied under pre-miR-29 conditioned medium, whereas pre-miR-30c conditioned medium had a prohypertrophic effect. Levels of miR-29a, miR-29c, and miR-30c, but not miR-29b, were significantly reduced in a mouse model of pathological but not physiological hypertrophy. Treatment with antagomiRs to miR-29b induced excess fibrosis after aortic constriction without overt deterioration in cardiac function. CONCLUSIONS: Our proteomic analysis revealed novel molecular targets of miRNAs that are linked to a fibrogenic cardiac phenotype. Such comprehensive screening methods are essential to define the concerted actions of miRNAs in cardiovascular disease.

Proteomics analysis of cardiac extracellular matrix remodeling in a porcine model of ischemia/reperfusion injury.

BACKGROUND: After myocardial ischemia, extracellular matrix (ECM) deposition occurs at the site of the focal injury and at the border region. METHODS AND RESULTS: We have applied a novel proteomic method for the analysis of ECM in cardiovascular tissues to a porcine model of ischemia/reperfusion injury. ECM proteins were sequentially extracted and identified by liquid chromatography tandem mass spectrometry. For the first time, ECM proteins such as cartilage intermediate layer protein 1, matrilin-4, extracellular adipocyte enhancer binding protein 1, collagen α-1(XIV), and several members of the small leucine-rich proteoglycan family, including asporin and prolargin, were shown to contribute to cardiac remodeling. A comparison in 2 distinct cardiac regions (the focal injury in the left ventricle and the border region close to the occluded coronary artery) revealed a discordant regulation of protein and mRNA levels; although gene expression for selected ECM proteins was similar in both regions, the corresponding protein levels were much higher in the focal lesion. Further analysis based on >100 ECM proteins delineated a signature of early- and late-stage cardiac remodeling with transforming growth factor-ß1 signaling at the center of the interaction network. Finally, novel cardiac ECM proteins identified by proteomics were validated in human left ventricular tissue acquired from ischemic cardiomyopathy patients at cardiac transplantation. CONCLUSION: Our findings reveal a biosignature of early- and late-stage ECM remodeling after myocardial ischemia/reperfusion injury, which may have clinical utility as a prognostic marker and modifiable target for drug discovery.

Inhibition of miR-199a-3p in a murine hypertrophic cardiomyopathy (HCM) model attenuates fibrotic remodeling.

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 heart failure and sudden death. The mechanisms through which diverse HCM-causing mutations cause cardiac dysfunction remain mostly unknown and their identification may reveal new therapeutic avenues. MicroRNAs (miRNAs) 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 murine primary cardiac fibroblasts and analyzed the conditioned media by proteomics, we found that several extracellular matrix (ECM) proteins (e.g., TSP2, FBLN3, COL11A1, LYOX) were differentially secreted (data are available via ProteomeXchange with identifier PXD042904). We confirmed our proteomics findings by qPCR analysis of selected mRNAs and demonstrated that miR-199a-3p mimic expression in cardiac fibroblasts drives upregulation of ECM gene expression, 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 fibroblasts. Conclusions: Altogether, our results suggest that miR-199a-3p may contribute to cardiac fibrosis in HCM through its actions in cardiac fibroblasts. 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.

Reduced secretion of neuronal growth regulator 1 contributes to impaired adipose-neuronal crosstalk in obesity.

While the endocrine function of white adipose tissue has been extensively explored, comparatively little is known about the secretory activity of less-investigated fat depots. Here, we use proteomics to compare the secretory profiles of male murine perivascular depots with those of canonical white and brown fat. Perivascular secretomes show enrichment for neuronal cell-adhesion molecules, reflecting a higher content of intra-parenchymal sympathetic projections compared to other adipose depots. The sympathetic innervation is reduced in the perivascular fat of obese (ob/ob) male mice, as well as in the epicardial fat of patients with obesity. Degeneration of sympathetic neurites is observed in presence of conditioned media of fat explants from ob/ob mice, that show reduced secretion of neuronal growth regulator 1. Supplementation of neuronal growth regulator 1 reverses this neurodegenerative effect, unveiling a neurotrophic role for this protein previously identified as a locus associated with human obesity. As sympathetic stimulation triggers energy-consuming processes in adipose tissue, an impaired adipose-neuronal crosstalk is likely to contribute to the disrupted metabolic homeostasis characterising obesity.

Extracellular Matrix Profiling and Disease Modelling in Engineered Vascular Smooth Muscle Cell Tissues.

Aortic smooth muscle cells (SMCs) have an intrinsic role in regulating vessel homeostasis and pathological remodelling. In two-dimensional (2D) cell culture formats, however, SMCs are not embedded in their physiological extracellular matrix (ECM) environment. To overcome the limitations of conventional 2D SMC cultures, we established a 3D in vitro model of engineered vascular smooth muscle cell tissues (EVTs). EVTs were casted from primary murine aortic SMCs by suspending a SMC-fibrin master mix between two flexible silicon-posts at day 0 before prolonged culture up to 14 days. Immunohistochemical analysis of EVT longitudinal sections demonstrated that SMCs were aligned, viable and secretory. Mass spectrometry-based proteomics analysis of murine EVT lysates was performed and identified 135 matrisome proteins. Proteoglycans, including the large aggregating proteoglycan versican, accumulated within EVTs by day 7 of culture. This was followed by the deposition of collagens, elastin-binding proteins and matrix regulators up to day 14 of culture. In contrast to 2D SMC controls, accumulation of versican occurred in parallel to an increase in versikine, a cleavage product mediated by proteases of the A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family. Next, we tested the response of EVTs to stimulation with transforming growth factor beta-1 (TGFß-1). EVTs contracted in response to TGFß-1 stimulation with altered ECM composition. In contrast, treatment with the pharmacological activin-like kinase inhibitor (ALKi) SB 431542 suppressed ECM secretion. As a disease stimulus, we performed calcification assays. The ECM acts as a nidus for calcium phosphate deposition in the arterial wall. We compared the onset and extent of calcification in EVTs and 2D SMCs cultured under high calcium and phosphate conditions for 7 days. Calcified EVTs displayed increased tissue stiffness by up to 30 % compared to non-calcified controls. Unlike the rapid calcification of SMCs in 2D cultures, EVTs sustained expression of the calcification inhibitor matrix Gla protein and allowed for better discrimination of the calcification propensity between independent biological replicates. In summary, EVTs are an intuitive and versatile model to investigate ECM synthesis and turnover by SMCs in a 3D environment. Unlike conventional 2D cultures, EVTs provide a more relevant pathophysiological model for retention of the nascent ECM produced by SMCs.

Gene expression profiles following intracoronary injection of mesenchymal stromal cells using a porcine model of chronic myocardial infarction.

BACKGROUND AIMS: We evaluated the therapeutic potential of injection of in vitro differentiated bone marrow mesenchymal stromal cells (MSC) using a swine model. METHODS AND RESULTS: Myocardial infarction was induced by coronary occlusion. Three groups (n = 5 each) were analyzed: one group received an injection of 17.8 ± 9.3 × 10(6) 5-azacytidine-treated allogeneic MSC 1 month after infarction; a placebo group received an injection of medium; and controls were kept untreated. After 4 weeks, heart samples were taken from three infarcted areas, interventricular septa, ventricles and atria. Gene expression profiles of genes related to contractility (Serca2a), fibrosis (Col1a1), cardiomyogenesis (Mef2c, Gata4 and Nkx2.5) and mobilization of stem cells (Sdf1, Cxcr4 and c-kit) were compared by quantitative real-time PCR (qRT-PCR). Gene expression profiles varied in different heart areas. Thus Serca2a expression was reduced in infarcted groups in all heart regions except for the left ventricles, where Col1a1 was overexpressed. The expression of genes related to cardiomyogenesis decreased in the infarcted zones and left atria compared with healthy hearts. Interestingly, increased expression of Cxcr4 was detected in infarcted regions of MSC-treated pigs compared with the placebo group. CONCLUSIONS: Infarction induced changes in expression of genes involved in various biologic processes. Genes involved in cardiomyogenesis were downregulated in the left atrium. The intracoronary injection of MSC resulted in localized changes in the expression of Cxcr4.