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-β1 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.

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 (Adamts5 ^ΔCat ). 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 Adamts5 ^ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin β1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5 ^ΔCat 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 β-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that β1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with β-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 β-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.

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.

Since mitochondria are the principal source of reactive oxygen species (ROS), these organelles may play an important role in ischemic cardiomyopathy (IC) development. The mitochondrial genome may influence this disease. The aim of the present study was to test the relationship between IC development and the impact of single nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) defining the mitochondrial haplogroups in a population study. Ten major European haplogroups were identified by using the single base extension technique and by polymerase chain reaction-restriction fragment length polymorphism. Frequencies and Odds Ratios for the association between IC patients (n = 358) and healthy controls (n = 423) were calculated. No convincing associations between classical risk factors for ischemic cardiomyopathy development and haplogroups were found. However, compared to healthy controls, the prevalence of haplogroup H was significantly higher in IC patients (40.0% vs 50.0%, p-value = 0.039) while the frequency of haplogroup J was significantly lower (11.1% vs 5.6%, p-value = 0.048). The analysis of the SNPs characterizing the European mtDNA haplogroups showed that the m.7028C allele (40.0% vs 50.0%, p-value = 0.005) and m.14766C allele (43.0% vs 54.2%, p-value = 0.002) were overrepresented in IC patients, meanwhile the m.10398G allele (19.8% vs 13.1%, p-value = 0.015) and m.4216C allele (22.2% vs 16.5%, p-value = 0.044) were found as protective factors against IC. Our results showed that the haplogroups H and J were found as a risk and protective factors for ischemic cardiomyopathy development, respectively.

Idiopathic dilated cardiomyopathy (IDC) is a structural heart disease with strong genetic background. The different single nucleotide polymorphisms (SNPs) that constitute mitochondrial haplogroups could play an important role in IDC progression. The aim of this study was to test frequencies of mitochondrial haplogroups in healthy controls (n=422) and IDC patients (n=304) of a Caucasian Spanish population. To achieve this, ten major European haplogroups were identified. Frequencies and Odds Ratios for the association between IDC and haplogroups were calculated in both groups. We found that compared to healthy controls, the prevalence of haplogroup H was significantly higher in IDC patients (40.0% vs 50.7%, p-value=0.040).