Melissa Swinnen

MicroRNA‐18 and microRNA‐19 regulate CTGF and TSP‐1 expression in age‐related heart failure

To understand the process of cardiac aging, it is of crucial importance to gain insight into the age‐related changes in gene expression in the senescent failing heart. Age‐related cardiac remodeling is known to be accompanied by changes in extracellular matrix (ECM) gene and protein levels. Small noncoding microRNAs regulate gene expression in cardiac development and disease and have been implicated in the aging process and in the regulation of ECM proteins. However, their role in age‐related cardiac remodeling and heart failure is unknown. In this study, we investigated the aging‐associated microRNA cluster 17–92, which targets the ECM proteins connective tissue growth factor (CTGF) and thrombospondin‐1 (TSP‐1). We employed aged mice with a failure‐resistant (C57Bl6) and failure‐prone (C57Bl6 × 129Sv) genetic background and extrapolated our findings to human age‐associated heart failure. In aging‐associated heart failure, we linked an aging‐induced increase in the ECM proteins CTGF and TSP‐1 to a decreased expression of their targeting microRNAs 18a, 19a, and 19b, all members of the miR‐17–92 cluster. Failure‐resistant mice showed an opposite expression pattern for both the ECM proteins and the microRNAs. We showed that these expression changes are specific for cardiomyocytes and are absent in cardiac fibroblasts. In cardiomyocytes, modulation of miR‐18/19 changes the levels of ECM proteins CTGF and TSP‐1 and collagens type 1 and 3. Together, our data support a role for cardiomyocyte‐derived miR‐18/19 during cardiac aging, in the fine‐tuning of cardiac ECM protein levels. During aging, decreased miR‐18/19 and increased CTGF and TSP‐1 levels identify the failure‐prone heart.

Absence of SPARC results in increased cardiac rupture and dysfunction after acute myocardial infarction

The matricellular protein SPARC (secreted protein, acidic and rich in cysteine, also known as osteonectin) mediates cell–matrix interactions during wound healing and regulates the production and/or assembly of the extracellular matrix (ECM). This study investigated whether SPARC functions in infarct healing and ECM maturation after myocardial infarction (MI). In comparison with wild-type (WT) mice, animals with a targeted inactivation of SPARC exhibited a fourfold increase in mortality that resulted from an increased incidence of cardiac rupture and failure after MI. SPARC-null infarcts had a disorganized granulation tissue and immature collagenous ECM. In contrast, adenoviral overexpression of SPARC in WT mice improved the collagen maturation and prevented cardiac dilatation and dysfunction after MI. In cardiac fibroblasts in vitro, reduction of SPARC by short hairpin RNA attenuated transforming growth factor β (TGF)–mediated increase of Smad2 phosphorylation, whereas addition of recombinant SPARC increased Smad2 phosphorylation concordant with increased Smad2 phosphorylation in SPARC-treated mice. Importantly, infusion of TGF-β rescued cardiac rupture in SPARC-null mice but did not significantly alter infarct healing in WT mice. These findings indicate that local production of SPARC is essential for maintenance of the integrity of cardiac ECM after MI. The protective effects of SPARC emphasize the potential therapeutic applications of this protein to prevent cardiac dilatation and dysfunction after MI.

Inhibition of Urokinase-Type Plasminogen Activator or Matrix Metalloproteinases Prevents Cardiac Injury and Dysfunction During Viral Myocarditis

Background— Acute viral myocarditis is an important cause of cardiac failure in young adults for which there is no effective treatment apart from general heart failure therapy. The present study tested the hypothesis that increased expression of the proteinases urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) is implicated in cardiac inflammation, injury, and subsequent failure during Coxsackievirus-B3 (CVB3)–induced myocarditis. Methods and Results— First, we showed increased expression and activity of uPA and MMP-9 in wild-type mice at 7 days of CVB3-induced myocarditis. Targeted deletion of uPA, which resulted in reduced MMP activity and cytokine expression or inhibition of MMPs by adenoviral gene overexpression of tissue inhibitor of metalloproteinases-1, decreased cardiac inflammation and reduced myocardial necrosis at 7 days and decreased cardiac fibrosis at 35 days after CVB3 infection. Importantly, loss of uPA or MMP activity prevented CVB3-induced cardiac dilatation and dysfunction, as determined by serial echocardiography. Conclusions— Loss of uPA or MMP activity reduces the cardiac inflammatory response after CVB3 infection, thereby protecting against cardiac injury, dilatation, and failure during CVB3-induced myocarditis.

Increased expression of syndecan-1 protects against cardiac dilatation and dysfunction after myocardial infarction

Background— The cell-associated proteoglycan syndecan-1 (Synd1) closely regulates inflammation and cell-matrix interactions during wound healing and tumorigenesis. The present study investigated whether Synd1 may also regulate cardiac inflammation, matrix remodeling, and function after myocardial infarction (MI). Methods and Results— First, we showed increased protein and mRNA expression of Synd1 from 24 hours on, reaching its maximum at 7 days after MI and declining thereafter. Targeted deletion of Synd1 resulted in increased inflammation and accelerated, yet functionally adverse, infarct healing after MI. In concordance, adenoviral gene expression of Synd1 protected against exaggerated inflammation after MI, mainly by reducing transendothelial adhesion and migration of leukocytes, as shown in vitro. Increased inflammation in the absence of Synd1 resulted in increased monocyte chemoattractant protein-1 expression, increased activity of matrix metalloproteinase-2 and -9, and decreased activity of tissue transglutaminase, associated with increased collagen fragmentation and disorganization. Exaggerated inflammation and adverse matrix remodeling in the absence of Synd1 increased cardiac dilatation and impaired systolic function, whereas gene overexpression of Synd1 reduced inflammation and protected against cardiac dilatation and failure. Conclusions— Increased expression of Synd1 in the infarct protects against exaggerated inflammation and adverse infarct healing, thereby reducing cardiac dilatation and dysfunction after MI in mice.

Absence of Thrombospondin-2 Causes Age-Related Dilated Cardiomyopathy

Background— The progressive shift from a young to an aged heart is characterized by alterations in the cardiac matrix. The present study investigated whether the matricellular protein thrombospondin-2 (TSP-2) may affect cardiac dimensions and function with physiological aging of the heart. Methods and Results— TSP-2 knockout (KO) and wild-type mice were followed up to an age of 60 weeks. Survival rate, cardiac function, and morphology did not differ at a young age in TSP-2 KO compared with wild-type mice. However, >55% of the TSP-2 KO mice died between 24 and 60 weeks of age, whereas <10% of the wild-type mice died. In the absence of TSP-2, older mice displayed a severe dilated cardiomyopathy with impaired systolic function, increased cardiac dilatation, and fibrosis. Ultrastructural analysis revealed progressive myocyte stress and death, accompanied by an inflammatory response and replacement fibrosis, in aging TSP-2 KO animals, whereas capillary or coronary morphology or density was not affected. Importantly, adeno-associated virus-9 gene–mediated transfer of TSP-2 in 7-week-old TSP-2 KO mice normalized their survival and prevented dilated cardiomyopathy. In TSP-2 KO animals, age-related cardiomyopathy was accompanied by increased matrix metalloproteinase-2 and decreased tissue transglutaminase-2 activity, together with impaired collagen cross-linking. At the cardiomyocyte level, TSP-2 deficiency in vivo and its knockdown in vitro decreased the activation of the Akt survival pathway in cardiomyocytes. Conclusion— TSP-2 expression in the heart protects against age-dependent dilated cardiomyopathy.

Meox2/Tcf15 Heterodimers Program the Heart Capillary Endothelium for Cardiac Fatty Acid Uptake

Background— Microvascular endothelium in different organs is specialized to fulfill the particular needs of parenchymal cells. However, specific information about heart capillary endothelial cells (ECs) is lacking. Methods and Results— Using microarray profiling on freshly isolated ECs from heart, brain, and liver, we revealed a genetic signature for microvascular heart ECs and identified Meox2/Tcf15 heterodimers as novel transcriptional determinants. This signature was largely shared with skeletal muscle and adipose tissue endothelium and was enriched in genes encoding fatty acid (FA) transport–related proteins. Using gain- and loss-of-function approaches, we showed that Meox2/Tcf15 mediate FA uptake in heart ECs, in part, by driving endothelial CD36 and lipoprotein lipase expression and facilitate FA transport across heart ECs. Combined Meox2 and Tcf15 haplodeficiency impaired FA uptake in heart ECs and reduced FA transfer to cardiomyocytes. In the long term, this combined haplodeficiency resulted in impaired cardiac contractility. Conclusions— Our findings highlight a regulatory role for ECs in FA transfer to the heart parenchyma and unveil 2 of its intrinsic regulators. Our insights could be used to develop new strategies based on endothelial Meox2/Tcf15 targeting to modulate FA transfer to the heart and remedy cardiac dysfunction resulting from altered energy substrate usage.

Syndecan-1 Amplifies Angiotensin II–Induced Cardiac Fibrosis

Syndecan-1 (Synd1) is a transmembrane heparan sulfate proteoglycan that functions as a coreceptor for various growth factors and modulates signal transduction. The present study investigated whether Synd1, by affecting growth factor signaling, may play a role in hypertension-induced cardiac fibrosis and dysfunction. Expression of Synd1 was increased significantly in mouse hearts with angiotensin II–induced hypertension, which was spatially related to cardiac fibrosis. Angiotensin II significantly impaired fractional shortening and induced cardiac fibrosis in wild-type mice, whereas these effects were blunted in Synd1-null mice. Angiotensin II significantly increased cardiac expression of connective tissue growth factor and collagen type I and III in wild-type mice, which was blunted in Synd1-null mice. These findings were confirmed in vitro, where angiotensin II induced the expression of both connective tissue growth factor and collagen I in fibroblasts. The absence of Synd1 in either Synd1-null fibroblasts, after knockdown of Synd1 by short hairpin RNA, or after inhibition of heparan sulfates by protamine attenuated this increase, which was associated with reduced phosphorylation of Smad2. In conclusion, loss of Synd1 reduces cardiac fibrosis and dysfunction during angiotensin II–induced hypertension.

Osteoglycin Prevents Cardiac Dilatation and Dysfunction After Myocardial Infarction Through Infarct Collagen Strengthening

Rationale: To maintain cardiac mechanical and structural integrity after an ischemic insult, profound alterations occur within the extracellular matrix. Osteoglycin is a small leucine-rich proteoglycan previously described as a marker of cardiac hypertrophy. Objective: To establish whether osteoglycin may play a role in cardiac integrity and function after myocardial infarction (MI). Methods and Results: Osteoglycin expression is associated with collagen deposition and scar formation in mouse and human MI. Absence of osteoglycin in mice resulted in significantly increased rupture-related mortality with tissue disruption, intramyocardial bleeding, and increased cardiac dysfunction, despite equal infarct sizes. Surviving osteoglycin null mice had greater infarct expansion in comparison with wild-type mice because of impaired collagen fibrillogenesis and maturation in the infarcts as revealed by electron microscopy and collagen polarization. Absence of osteoglycin did not affect cardiomyocyte hypertrophy in the remodeling remote myocardium. In cultured fibroblasts, osteoglycin knockdown or supplementation did not alter transforming growth factor-&bgr; signaling. Adenoviral overexpression of osteoglycin in wild-type mice significantly improved collagen quality, thereby blunting cardiac dilatation and dysfunction after MI. In osteoglycin null mice, adenoviral overexpression of osteoglycin was unable to prevent rupture-related mortality because of insufficiently restoring osteoglycin protein levels in the heart. Finally, circulating osteoglycin levels in patients with heart failure were significantly increased in the patients with a previous history of MI compared with those with nonischemic heart failure and correlated with survival, left ventricular volumes, and other markers of fibrosis. Conclusions: Increased osteoglycin expression in the infarct scar promotes proper collagen maturation and protects against cardiac disruption and adverse remodeling after MI. In human heart failure, osteoglycin is a promising biomarker for ischemic heart failure.

Thrombospondin-2 prevents cardiac injury and dysfunction in viral myocarditis through the activation of regulatory T-cells

AIMS Thrombospondin-2 (TSP-2) modulates matrix integrity and myocyte survival in the hypertensive or ageing heart. Whether TSP-2 may affect cardiac inflammation and injury, in particular during acute viral myocarditis, is completely unknown. METHODS AND RESULTS Therefore, mortality, cardiac inflammation, and function were assessed in TSP-2-null (KO) and wild-type (WT) mice in human Coxsackie virus B3 (CVB3)-induced myocarditis. TSP-2 KO had an increased mortality when compared with WT mice during viral myocarditis. The absence of TSP-2 resulted in increased cardiac inflammation and injury at 14 days, which resulted in depressed systolic function [fractional shortening (FS); 34 ± 2.6 in WT vs. 24 ± 1.8 in KO mice, P< 0.05] and increased cardiac dilatation (end-diastolic dimensions, mm; 3.7 ± 0.09 in WT vs. 4.8 ± 0.06 in KO mice, P< 0.05) 35 days post-infection. Lack of TSP-2 resulted in a decreased activation of the anti-inflammatory T-regulatory cells, as indicated by a lower number of CD25-positive T-cells, and significantly decreased gene expression of regulatory T-cell markers, Foxp3 and CTLA-4. Finally, overexpression of TSP-2 in WT hearts using cardiotropic vectors derived from adeno-associated virus serotype 9 (AAV9) inhibited cardiac inflammation and injury at 14 days and improved cardiac function at 35 days post-CVB3 infection when compared with control AAV9. CONCLUSION TSP-2 has a protective role against cardiac inflammation, injury, and dysfunction in acute viral myocarditis.

Absence of thrombospondin-2 increases cardiomyocyte damage and matrix disruption in doxorubicin-induced cardiomyopathy

Clinical use of the antineoplastic agent doxorubicin (DOX) is limited by its cardiomyocyte toxicity. Attempts to decrease cardiomyocyte injury showed promising results in vitro, but failed to reduce the adverse effects of DOX in vivo, suggesting that other mechanisms contribute to its cardiotoxicity as well. Evidence that DOX also induces cardiac injury by compromising extracellular matrix integrity is lacking. The matricellular protein thrombospondin-2 (TSP-2) is known for its matrix-preserving function, and for modulating cellular function. Here, we investigated whether TSP-2 modulates the process of doxorubicin-induced cardiomyopathy (DOX-CMP). TSP-2-knockout (TSP-2-KO) and wild-type (WT) mice were treated with DOX (2 mg/kg/week) for 12 weeks to induce DOX-CMP. Mortality was significantly increased in TSP-2-KO compared to WT mice. Surviving DOX-treated TSP-2-KO mice had depressed cardiac function compared to WT animals, accompanied by increased cardiomyocyte apoptosis and matrix damage. Enhanced myocyte damage in the absence of TSP-2 was associated with impaired activation of the Akt signaling pathway in TSP-2-KO compared to WT. The absence of TSP-2, in vivo and in vitro, reduced Akt activation both under non-treated conditions and after DOX. Importantly, inhibition of Akt phosphorylation in cardiomyocytes significantly reduced TSP-2 expression, unveiling a unique feedback loop between Akt and TSP-2. Finally, enhanced matrix disruption in DOX-treated TSP-2-KO hearts went along with increased matrix metalloproteinase-2 levels. Taken together, this study is the first to provide evidence for the implication of the matrix element TSP-2 in protecting against DOX-induced cardiac injury and dysfunction.