Sweet,yet underappreciated: Proteoglycans and extracellular matrix remodeling in heart disease

Extracellular matrix remodeling is extensive in several heart diseases and hampers cardiac filling, often leading to heart failure. Proteoglycans have over the last two decades emerged as molecules with important roles in matrix remodeling and fibrosis in the heart. Here we discuss and review current literature on proteoglycans that have been studied in cardiac remodeling. The small leucine rich proteoglycans (SLRPs) are located within the extracellular matrix and are organizers of the matrix structure. Membrane-bound proteoglycans, such as syndecans and glypicans, act as receptors and direct cardiac fibroblast signaling. Recent studies indicate that proteoglycans are promising as diagnostic biomarkers for cardiac fibrosis, and that they may provide new therapeutic strategies for cardiac disease.     

Cross your heart? Collagen cross-links in cardiac health and disease

Extracellular matrix (ECM) remodeling occurs in response to various cardiac insults including infarction, pressure overload and dilated myopathies. Each type of remodeling necessitates distinct types of ECM turnover and deposition yet an increase in myocardial fibrillar collagen content is appreciated as a contributing feature to cardiac dysfunction in each of these pathologies. In addition, aging, is also associated with increases in cardiac collagen content. The importance of characterizing differences in ECM composition and processes used by cardiac fibroblasts in the assembly of fibrotic collagen accumulation is critical for the design of strategies to reduce and ultimately regress cardiac fibrosis. Collagen cross-linking is one factor that influences collagen deposition and insolubility with direct implications for tissue properties such as stiffness. In this review, three different types of collagen cross-links shown to be important in cardiac fibrosis will be discussed; those catalyzed by lysyl oxidases, those catalyzed by transglutaminases, and those that result from non-enzymatic modification by the addition of advanced glycation end products. Insight into cellular mechanisms that govern collagen cross-linking in the myocardium will provide novel pathways for exploring new treatments to treat diseases associated with cardiac fibrosis.     

Understanding cardiac extracellular matrix remodeling to develop biomarkers of myocardial infarction outcomes

Cardiovascular Disease (CVD) is the most common cause of death in industrialized countries, and myocardial infarction (MI) is a major CVD with significant morbidity and mortality. Following MI, the left ventricle (LV) undergoes a wound healing response to ischemia that results in extracellular matrix (ECM) scar formation to replace necrotic myocytes. While ECM accumulation following MI is termed cardiac fibrosis, this is a generic term that does not differentiate between ECM accumulation that occurs in the infarct region to form a scar that is structurally necessary to preserve left ventricle (LV) wall integrity and ECM accumulation that increases LV wall stiffness to exacerbate dilation and stimulate the progression to heart failure. This review focuses on post-MI LV ECM remodeling, targeting the discussion on ECM biomarkers that could be useful for predicting MI outcomes.     

A novel therapeutic strategy for alleviating atrial remodeling by targeting exosomal miRNAs in atrial fibrillation

Atrial fibrillation (AF) is one of the most frequent cardiac arrhythmias, and atrial remodeling is related to the progression of AF. Although several therapeutic approaches have been presented in recent years, the continuously increasing mortality rate suggests that more advanced strategies for treatment are urgently needed. Exosomes regulate pathological processes through intercellular communication mediated by microribonucleic acid (miRNA) in various cardiovascular diseases (CVDs). Exosomal miRNAs associated with signaling pathways have added more complexity to an already complex direct cell-to-cell interaction. Exosome delivery of miRNAs is involved in cardiac regeneration and cardiac protection. Recent studies have found that exosomes play a critical role in the diagnosis and treatment of cardiac fibrosis. By improving exosome stability and modifying surface epitopes, specific pharmaceutical agents can be supplied to improve tropism and targeting to cells and tissues in vivo. Exosomes harboring miRNAs may have clinical utility in cell-free therapeutic approaches and may serve as prognostic and diagnostic biomarkers for AF. Currently, limitations challenge pharmaceutic design, therapeutic utility and in vivo targeted delivery to patients. The aim of this article is to review the developmental features of AF associated with exosomal miRNAs and relate them to underlying mechanisms.     

Mechanical stress regulates the mechanotransduction and metabolism of cardiac fibroblasts in fibrotic cardiac diseases

Fibrotic cardiac diseases are characterized by myocardial fibrosis that results in maladaptive cardiac remodeling. Cardiac fibroblasts (CFs) are the main cell type responsible for fibrosis. In response to stress or injury, intrinsic CFs develop into myofibroblasts and produce excess extracellular matrix (ECM) proteins. Myofibroblasts are mechanosensitive cells that can detect changes in tissue stiffness and respond accordingly. Previous studies have revealed that some mechanical stimuli control fibroblast behaviors, including ECM formation, cell migration, and other phenotypic traits. Further, metabolic alteration is reported to regulate fibrotic signaling cascades, such as the transforming growth factor-β pathway and ECM deposition. However, the relationship between metabolic changes and mechanical stress during fibroblast-to-myofibroblast transition remains unclear. This review aims to elaborate on the crosstalk between mechanical stress and metabolic changes during the pathological transition of cardiac fibroblasts.     

Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-β1 (TGF-β), decorin, and collagen in cardiomyopathy and heart failure. J. Cell. Biochem. 68:403–410, 1998. © 1998 Wiley-Liss, Inc.     

Eicosapentaenoic acid prevents atrial fibrillation associated with heart failure in a rabbit model

Atrial fibrillation (AF) is associated with morbidity and mortality of heart failure. Eicosapentaenoic acid (EPA), which is contained in fish oil, was shown to reduce the risk of cardiovascular diseases. We investigated the effects of EPA on AF associated with heart failure in a rabbit model. Rabbits were subjected to ventricular tachypacing (VTP) for 4 wk with or without EPA treatment. Continuous VTP induced heart failure status in these rabbits. The duration of AF (DAF) induced by burst pacing was analyzed by electrophysiological studies. VTP resulted in increased DAF following burst pacing. EPA treatment attenuated increased DAF. Atrial fibrosis increased in response to VTP, accompanied by extracellular signal-regulated kinase (ERK) phosphorylation and transforming growth factor-β1 (TGF-β1) expression in the atrium. Treatment with EPA attenuated atrial fibrosis, ERK phosphorylation, and TGF-β1 expression in response to VTP. EPA treatment increased adiponectin as an anti-inflammatory adipokine and decreased tumor necrosis factor-α as a proinflammatory adipokine in the atrium and epicardial adipose tissues. EPA attenuated VTP-induced AF promotion and atrial remodeling, which was accompanied by modulating the profiles of adipokine production from epicardial adipose tissue. EPA may be useful for prevention and treatment of AF associated with heart failure.     

Urinary bladder matrix promotes site appropriate tissue formation following right ventricle outflow tract repair

The current prevalence and severity of heart defects requiring functional replacement of cardiac tissue pose a serious clinical challenge. Biologic scaffolds are an attractive tissue engineering approach to cardiac repair because they avoid sensitization associated with homograft materials and theoretically possess the potential for growth in similar patterns as surrounding native tissue. Both urinary bladder matrix (UBM) and cardiac ECM (C-ECM) have been previously investigated as scaffolds for cardiac repair with modest success, but have not been compared directly. In other tissue locations, bone marrow derived cells have been shown to play a role in the remodeling process, but this has not been investigated for UBM in the cardiac location, and has never been studied for C-ECM. The objectives of the present study were to compare the effectiveness of an organ-specific C-ECM patch with a commonly used ECM scaffold for myocardial tissue repair of the right ventricle outflow tract (RVOT), and to examine the role of bone marrow derived cells in the remodeling response. A chimeric rat model in which all bone marrow cells express green fluorescent protein (GFP) was generated and used to show the ability of ECM scaffolds derived from the heart and bladder to support cardiac function and cellular growth in the RVOT. The results from this study suggest that urinary bladder matrix may provide a more appropriate substrate for myocardial repair than cardiac derived matrices, as shown by differences in the remodeling responses following implantation, as well as the presence of site appropriate cells and the formation of immature, myocardial tissue.     

Necroptosis is required for atrial fibrillation and involved in aerobic exercise-conferred cardioprotection

Necroptosis, a novel programmed cell death, plays a critical role in the development of fibrosis, yet its role in atrial fibrillation (AF) remains elusive. Mounting evidence demonstrates that aerobic exercise improves AF-related symptoms and quality of life. Therefore, we explored the role of necroptosis in AF pathogenesis and exercise-conferred cardioprotection. A mouse AF model was established either by calcium chloride and acetylcholine (CaCl₂-Ach) administration for 3 weeks or high-fat diet (HFD) feeding for 12 weeks, whereas swim training was conducted 60 min/day, for 3-week duration. AF susceptibility, heart morphology and function and atrial fibrosis were assessed by electrophysiological examinations, echocardiography and Masson's trichrome staining, respectively. Both CaCl₂-Ach administration and HFD feeding significantly enhanced AF susceptibility (including frequency and duration of episodes), left atrial enlargement and fibrosis. Moreover, protein levels of necroptotic signaling (receptor-interacting protein kinase 1, receptor-interacting protein kinase 3, mixed lineage kinase domain-like protein and calcium/calmodulin-dependent protein kinase II or their phosphorylated forms) were markedly elevated in the atria of AF mice. However, inhibiting necroptosis with necrostatin-1 partly attenuated CaCl₂-Ach (or HFD)-induced fibrosis and AF susceptibility, implicating necroptosis as contributing to AF pathogenesis. Finally, we found 3-week swim training inhibited necroptotic signaling, consequently decreasing CaCl₂-Ach-induced AF susceptibility and atrial structural remodeling. Our findings identify necroptosis as a novel mechanism in AF pathogenesis and highlight that aerobic exercise may confer benefits on AF via inhibiting cardiac necroptosis.     

Interaction between cell and extracellular matrix in heart disease: multiple roles of tenascin-C in tissue remodeling

The heart remodels myocardial tissue in physiological and pathological response. The cell-extracellular matrix (ECM) interaction provides not only structural and mechanical support but also important biological signaling during tissue remodeling. Among various ECM molecules, tenascin-C (TNC) is well known as a regulator of multiple cellular functions during embryogenesis, wound healing or cancer progression. In the heart, TNC appears in several important steps of embryonic development such as the initial differentiation of cardiomyocytes or coronary vasculo/angiogenesis, but it is not detected in a normal adult myocardium. However, TNC is found to re-express after myocardial injury and may regulate cellular behavior during tissue remodeling by modulating the attachment of cardiomyocytes to connective tissue, by enhancing migration and differentiation of myofibroblasts, and by inducing matrix metallo-proteinases. TNC also interacts with other ECM molecules and may modulate progression of fibrosis. Furthermore, transient and site specific expression of TNC closely associated with myocardial injury and inflammation suggests not only its key roles during tissue remodeling but also that TNC can be a marker for myocardial disease activity.     

Inhibition of nuclear factor κB regresses cardiac hypertrophy by modulating the expression of extracellular matrix and adhesion molecules

Myocardial remodeling denotes a chronic pathological condition of dysfunctional myocardium that occurs in cardiac hypertrophy (CH) and heart failure (HF). Reactive oxygen species (ROS) are major initiators of excessive collagen and fibronectin deposition in cardiac fibrosis. Increased production of ROS and nuclear factor κB (NF-κB) activation provide a strong link between oxidative stress and extracellular matrix (ECM) remodeling in cardiac hypertrophy. The protective inhibitory actions of pyrrolidine dithiocarbamate (PDTC), a pharmacological inhibitor of NF-κB and a potent antioxidant, make this a good agent to evaluate the role of inhibition of NF-κB and prevention of excessive ECM deposition in maladaptive cardiac remodeling during HF. In this report, we used a transgenic mouse model (Myo-Tg) that has cardiac-specific overexpression of myotrophin. This overexpression of myotrophin in the Myo-Tg model directs ECM deposition and increased NF-κB activity, which result in CH and ultimately HF. Using the Myo-Tg model, our data showed upregulation of profibrotic genes (including collagen types I and III, connective tissue growth factor, and fibronectin) in Myo-Tg mice, compared to wild-type mice, during the progression of CH. Pharmacological inhibition of NF-κB by PDTC in the Myo-Tg mice resulted in a significant reduction in cardiac mass, NF-κB activity, and profibrotic gene expression and improved cardiac function. To the best of our knowledge, this is the first report of ECM regulation by inhibition of NF-κB activation by PDTC. The study highlights the importance of the NF-κB signaling pathway and therapeutic benefits of PDTC treatment in cardiac remodeling.     

Matrix metalloproteinases and their function in myocardium

A significant number of myocardial diseases are accompanied by increased synthesis and degradation of the extracellular matrix (ECM) as well as by changed maturation and incorporation of ECM components. Important groups of enzymes responsible for both normal and pathological processes in ECM remodeling are matrix metaloproteinases (MMPs). These enzymes share a relatively conserved structure with a number of identifiable modules linked to their specific functions. The most important function of MMPs is the ability to cleave various ECM components; including such rigid molecules as fibrillar collagen molecules. The amount and activity of MMPs in cardiac tissue are regulated by a range of activating and inhibiting processes. Although MMPs play multifarious roles in many myocardial diseases, here we have focused on their function in ischemic cardiac tissue, dilated cardiomyopathy and hypertrophied cardiac tissue. The inhibition of MMPs by means of synthetic inhibitors seems to be a promising strategy in cardiac disease treatment. Their effects on diseased cardiac tissue have been successfully tested in several experimental studies.     

Changes in extra cellular matrix remodelling and re-expression of fibronectin and tenascin-C splicing variants in human myocardial tissue of the right atrial auricle: implications for a targeted therapy of cardiovascular diseases using human SIP format antibodies

Cardiovascular diseases are accompanied by changes in the extracellular matrix (ECM) including the re-expression of fibronectin and tenascin-C splicing variants. Using human recombinant small immunoprotein (SIP) format antibodies, a molecular targeting of these proteins is of therapeutic interest. Tissue samples of the right atrial auricle from patients with coronary artery disease and valvular heart disease were analysed by PCR based ECM gene expression profiling. Moreover, the re-expression of fibronectin and tenascin-C splicing variants was investigated by immunofluoerescence labelling. We demonstrated changes in ECM gene expression depending on histological damage or underlying cardiac disease. An increased expression of fibronectin and tenascin-C mRNA in association to histological damage and in valvular heart disease compared to coronary artery disease could be shown. There was a distinct re-expression of ED-A containing fibronectin and A1 domain containing tenascin-C detectable with human recombinant SIP format antibodies in diseased myocardium. ED-A containing fibronectin showed a clear vessel positivity. For A1 domain containing tenascin-C, there was a particular positivity in areas of interstitial and perivascular fibrosis. Right atrial myocardial tissue is a valuable model to investigate cardiac ECM remodelling. Human recombinant SIP format antibodies usable for an antibody-mediated targeted delivery of drugs might offer completely new therapeutic options in cardiac diseases.     

Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer

Dynamic remodeling of the extracellular matrix (ECM) is essential for development, wound healing and normal organ homeostasis. Life-threatening pathological conditions arise when ECM remodeling becomes excessive or uncontrolled. In this Perspective, we focus on how ECM remodeling contributes to fibrotic diseases and cancer, which both present challenging obstacles with respect to clinical treatment, to illustrate the importance and complexity of cell-ECM interactions in the pathogenesis of these conditions. Fibrotic diseases, which include pulmonary fibrosis, systemic sclerosis, liver cirrhosis and cardiovascular disease, account for over 45% of deaths in the developed world. ECM remodeling is also crucial for tumor malignancy and metastatic progression, which ultimately cause over 90% of deaths from cancer. Here, we discuss current methodologies and models for understanding and quantifying the impact of environmental cues provided by the ECM on disease progression, and how improving our understanding of ECM remodeling in these pathological conditions is crucial for uncovering novel therapeutic targets and treatment strategies. This can only be achieved through the use of appropriate in vitro and in vivo models to mimic disease, and with technologies that enable accurate monitoring, imaging and quantification of the ECM.     

Early induction of matrix metalloproteinase-9 transduces signaling in human heart end stage failure

Extracellular matrix (ECM) turnover is regulated by matrix metalloproteinases (MMPs) and plays an important role in cardiac remodeling. Previous studies from our lab demonstrated an increase in gelatinolytic-MMP-2 and -9 activities in endocardial tissue from ischemic cardiomyopathic (ICM) and idiopathic dilated cardiomyopathic (DCM) hearts. The signaling mechanism responsible for the left ventricular (LV) remodeling, however, is unclear. Administration of cardiac specific inhibitor of metalloproteinase (CIMP) prevented the activation of MMP-2 and -9 in ailing to failing myocardium. Activation of MMP-2 and -9 leads to induction of proteinase activated receptor-1 (PAR-1). We hypothesize that the early induction of MMP-9 is a key regulator for modulating intracellular signaling through activation of PAR and various downstream events which are implicated in development of cardiac fibrosis in an extracellular receptor mediated kinase-1 (ERK-1) and focal adhesion kinase (FAK) dependent manner. To test this hypothesis, explanted human heart tissues from ICM and DCM patients were obtained at the time of orthotopic cardiac transplants. Quantitative analysis of MMP-2 and -9 gelatinolytic activities was made by real-time quantitative zymography. Gel phosphorylation staining for PAR-1 showed a significant increase in ICM hearts. Western blot and RT-PCR analysis and in-situ labeling, showed significant increased expression of PAR-1, ERK-1and FAK in ICM and DCM. These observations suggest that the enhanced expression and potentially increased activity of LV myocardial MMP-9 triggers the signal cascade instigating cardiac remodeling. This early mechanism for the initiation of LV remodeling appears to have a role in end-stage human heart failure.     

Chronic administration of hexarelin attenuates cardiac fibrosis in the spontaneously hypertensive rat

Cardiac fibrosis is a hallmark of heart disease and plays a vital role in cardiac remodeling during heart diseases, including hypertensive heart disease. Hexarelin is one of a series of synthetic growth hormone secretagogues (GHSs) possessing a variety of cardiovascular effects via action on GHS receptors (GHS-Rs). However, the role of hexarelin in cardiac fibrosis in vivo has not yet been investigated. In the present study, spontaneously hypertensive rats (SHRs) were treated with hexarelin alone or in combination with a GHS-R antagonist for 5 wk from an age of 16 wk. Hexarelin treatment significantly reduced cardiac fibrosis in SHRs by decreasing interstitial and perivascular myocardial collagen deposition and myocardial hydroxyproline content and reducing mRNA and protein expression of collagen I and III in SHR hearts. Hexarelin treatment also increased matrix metalloproteinase (MMP)-2 and MMP-9 activities and decreased myocardial mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 in SHRs. In addition, hexarelin treatment significantly attenuated left ventricular (LV) hypertrophy, LV diastolic dysfunction, and high blood pressure in SHRs. The effect of hexarelin on cardiac fibrosis, blood pressure, and cardiac function was mediated by its receptor, GHS-R, since a selective GHS-R antagonist abolished these effects and expression of GHS-Rs was upregulated by hexarelin treatment. In summary, our data demonstrate that hexarelin reduces cardiac fibrosis in SHRs, perhaps by decreasing collagen synthesis and accelerating collagen degradation via regulation of MMPs/TIMP. Hexarelin-reduced systolic blood pressure may also contribute to this reduced cardiac fibrosis in SHRs. The present findings provided novel insights and underscore the therapeutic potential of hexarelin as an antifibrotic agent for the treatment of cardiac fibrosis.     

MiR-34a/miR-93 target c-Ski to modulate the proliferaton of rat cardiac fibroblasts and extracellular matrix deposition in vivo and in vitro

Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-β signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-β signaling, has been reported to suppress TGF-β1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-β1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-β1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-β1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-β1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis.