Cardiac rupture is a fatal complication of acute myocardial infarction lacking treatment. Here, acute myocardial infarction resulted in rupture in wild-type mice and in mice lacking tissue-type plasminogen activator, urokinase receptor, matrix metalloproteinase stromelysin-1 or metalloelastase. Instead, deficiency of urokinase-type plasminogen activator (u-PA-/-) completely protected against rupture, whereas lack of gelatinase-B partially protected against rupture. However, u-PA-/- mice showed impaired scar formation and infarct revascularization, even after treatment with vascular endothelial growth factor, and died of cardiac failure due to depressed contractility, arrhythmias and ischemia. Temporary administration of PA inhibitor-1 or the matrix metalloproteinase-inhibitor TIMP-1 completely protected wild-type mice against rupture but did not abort infarct healing, thus constituting a new approach to prevent cardiac rupture after acute myocardial infarction.

Abstract:In the past few years, the crucial role of different micro-RNAs (miRNAs) in the cardiovascular system has been widely recognized. Recently, it was discovered that extracellular miRNAs circulate in the bloodstream and that such circulating miRNAs are remarkably stable. This has raised the possibility that miRNAs may be probed in the circulation and can serve as novel diagnostic markers. Although the precise cellular release mechanisms of miRNAs remain largely unknown, the first studies revealed that these circulating miRNAs may be delivered to recipient cells, where they can regulate translation of target genes. In this review, we will discuss the nature of the stability of miRNAs that circulate in the bloodstream and discuss the available evidence regarding the possible function of these circulating miRNAs in distant cell-to-cell communication. Furthermore, we summarize and discuss the usefulness of circulating miRNAs as biomarkers for a wide range of cardiovascular diseases such as myocardial infarction, heart failure, atherosclerosis, hypertension, and type 2 diabetes mellitus. One of the major challenges in cardiovascular research is the identification of reliable biomarkers that can be measured routinely in easily accessible samples, such as plasma. Because of their stability in the circulation, miRNAs are currently being explored for their potential as biomarkers for cardiovascular disease. Thus far, distinctive patterns of circulating miRNAs have been found for myocardial infarction, 11 heart failure (HF), 12 atherosclerotic disease, 13 type 2 diabetes mellitus (DM), 14 and hypertension. 15 In the present review, we will discuss the available evidence of the cellular release mechanisms and the nature of the stability of miRNAs in the bloodstream (eg, microparticles, RNA-binding proteins, and HDL). Next, we will discuss the available evidence for a possible function of miRNAs in cell-to-cell communication. Finally, we will review the current knowledge about circulating miRNAs as putative biomarkers in cardiovascular disease. Cellular Release and Stability of Extracellular miRNAsAs early as 1972, it was reported that intact extracellular RNA could be detected in plasma despite the presence of ribonucleases that were expected to destroy any freely circulating RNA. 16 It has therefore been suggested that this extracellular RNA, including miRNAs, is somehow shielded to prevent its degradation. As discussed in the sections below, evidence is now accumulating that miRNAs are protected against degradation by being packaged in lipid vesicles or by being associated with protein or lipoprotein complexes (Figure). Plasma miRNAs in MicroparticlesEl-Hefnawy et al 17 were among the first to show that plasma RNA is protected from degradation by its inclusion in protein or lipid vesicles. Depending on their size and mode of release from cells, these particles are known as exosomes, microvesicles (MVs), or apoptotic bodies. 18 Exosomes are small vesicles (50 -100 nm) that originate from the endosome and are release...

Rationale: Aberrant expression profiles of circulating microRNAs (miRNAs) have been described in various diseases and provide high sensitivity and specificity. We explored circulating miRNAs as potential biomarkers in patients with heart failure (HF). Objective: The goal of this study was to determine whether miRNAs allow to distinguish clinical HF not only from healthy controls but also from non-HF forms of dyspnea. Methods and Results: A miRNA array was performed on plasma of 12 healthy controls and 12 HF patients. From this array, we selected 16 miRNAs for a second clinical study in 39 healthy controls and in 50 cases with reports of dyspnea, of whom 30 were diagnosed with HF and 20 were diagnosed with dyspnea attributable to non-HF-related causes. This revealed that miR423-5p was specifically enriched in blood of HF cases and receiver-operator-characteristics (ROC) curve analysis showed miR423-5p to be a diagnostic predictor of HF, with an area under the curve of 0.91 (P<0.001). Five other miRNAs were elevated in HF cases but also slightly increased in non-HF dyspnea cases. Conclusion: We identify 6 miRNAs that are elevated in patients with HF, among which miR423-5p is most strongly related to the clinical diagnosis of HF. Recent studies have unveiled powerful and unexpected roles for microRNAs (miRNAs) in cardiovascular diseases, including HF. There are estimated to be more than 1000 different miRNAs, many of which are expressed in a tissue and cell-specific manner. 3 It was discovered only recently that miRNAs are also abundantly present in blood, where they can be detected in plasma, platelets, and erythrocytes, as well as in nucleated blood cells. 4 Aberrant expression profiles of miRNAs have been identified in blood of subjects with sickle cell anemia, prostate cancer, lung cancer and myocardial injury. 4 -6 This led us to hypothesize that miRNA profiling can also be used for diagnostic approaches in HF.Here we explored whether circulating miRNAs can be used as biomarkers in patients with HF. We first performed miRNA arrays on RNA isolated from plasma and selected 16 miRNAs expressed differentially in HF patients. Next, we evaluated these miRNAs in a second group of patients, consisting of 50 cases with reports of dyspnea, of whom 30 were diagnosed with HF (HF cases) and 20 were diagnosed to have dyspnea attributable to non-HF causes (non-HF cases). One circulating miRNA in particular, miR423-5p, was able to distinguish HF cases from non-HF cases.In conclusion, we demonstrate a number of miRNAs as putative biomarkers for HF, in particular miR423-5p. MethodsHuman plasma samples were obtained with informed consent under a general waiver by the Academic Medical Center institutional review board for the proper secondary use of human material. For the dyspnea registry, plasma samples were obtained as part of a multicenter effort involving 3 centers in The Netherlands. Experiments described were performed on samples obtained at the Academic Medical Center. For a detailed description of the dyspnea registry,...

The association of transcriptional coactivators with sequence-specific DNA-binding proteins provides versatility and specificity to gene regulation and expands the regulatory potential of individual cis-regulatory DNA sequences. Members of the myocardin family of coactivators activate genes involved in cell proliferation, migration, and myogenesis by associating with serum response factor (SRF). The partnership of myocardin family members and SRF also controls genes encoding components of the actin cytoskeleton and confers responsiveness to extracellular growth signals and intracellular changes in the cytoskeleton, thereby creating a transcriptional-cytoskeletal regulatory circuit. These functions are reflected in defects in smooth muscle differentiation and function in mice with mutations in myocardin family members. This article reviews the functions and mechanisms of action of the myocardin family of coactivators and the physiological significance of transcriptional coactivation in the context of signal-dependent and cell-type-specific gene regulation.The instructions for gene expression are "hard wired" into DNA sequence in the form of cis-regulatory elements that bind sequence-specific transcription factors and confer specialized patterns of gene expression (Howard and Davidson 2004). However, the binding of transcription factors to their cognate sites is often insufficient to account for the patterns of expression of their target genes. Indeed, it is not uncommon for a transcription factor to control genes with distinct or even mutually exclusive expression patterns. Many transcription factors are also relatively weak transcriptional activators and function by recruiting coactivators (or corepressors) that do not bind DNA directly but regulate transcription in a DNA sequence-specific manner by associating with DNA-bound factors (Spiegelman and Heinrich 2004). Thus, the association of DNA-binding proteins with accessory factors provides specificity and versatility to gene regulation and expands the regulatory potential of individual cis-regulatory elements.

Abstract-Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease processes, including tumor growth and metastasis, arthritis, and periodontal disease. It is now becoming increasingly clear that extracellular matrix degradation by MMPs is also involved in the pathogenesis of cardiovascular disease, including atherosclerosis, restenosis, dilated cardiomyopathy, and myocardial infarction. Administration of synthetic MMP inhibitors in experimental animal models of these cardiovascular diseases significantly inhibits the progression of, respectively, atherosclerotic lesion formation, neointima formation, left ventricular remodeling, pump dysfunction, and infarct healing. This review focuses on the role of MMPs in cardiovascular disease, in particular myocardial infarction and the subsequent progression to heart failure. MMPs, which are present in the myocardium and capable of degrading all the matrix components of the heart, are the driving force behind myocardial matrix remodeling. The recent finding that acute pharmacological inhibition of MMPs or deficiency in MMP-9 attenuates left ventricular dilatation in the infarcted mouse heart led to the proposal that MMP inhibitors could be used as a potential therapy for patients at risk for the development of heart failure after myocardial infarction. Although these promising results encourage the design of clinical trials with MMP inhibitors, there are still several unresolved issues. This review describes the biology of MMPs and discusses new insights into the role of MMPs in several cardiovascular diseases. Attention will be paid to the central role of the plasminogen system as an important activator of MMPs in the remodeling process after myocardial infarction. Finally, we speculate on the use of MMP inhibitors as potential therapy for heart failure. Key Words: myocardial infarction Ⅲ therapy Ⅲ matrix metalloproteinase inhibition M yocardial infarction (MI) leads to complex architectural alterations involving both the infarcted and noninfarcted myocardium. Dilatation of the left ventricle and infarct thinning, also called infarct expansion, are the most prominent structural changes in the infarct region. 1 Patients exhibiting extensive infarct expansion after MI are more likely to experience complications, such as the development of congestive heart failure, aneurysm formation, and myocardial rupture. 2 The extent of ventricular dilatation after MI is related to several factors such as the magnitude of the initial Original

Abstract-The myocardium of the failing heart undergoes a number of structural alterations, most notably hypertrophy of cardiac myocytes and an increase in extracellular matrix proteins, often seen as primary fibrosis. Connective tissue growth factor (CTGF) is a key molecule in the process of fibrosis and therefore seems an attractive therapeutic target. Regulation of CTGF expression at the promoter level has been studied extensively, but it is unknown how CTGF transcripts are regulated at the posttranscriptional level. Here we provide several lines of evidence to show that CTGF is importantly regulated by 2 major cardiac microRNAs (miRNAs), miR-133 and miR-30. First, the expression of both miRNAs was inversely related to the amount of CTGF in 2 rodent models of heart disease and in human pathological left ventricular hypertrophy. Second, in cultured cardiomyocytes and fibroblasts, knockdown of these miRNAs increased CTGF levels. Third, overexpression of miR-133 or miR-30c decreased CTGF levels, which was accompanied by decreased production of collagens. Fourth, we show that CTGF is a direct target of these miRNAs, because they directly interact with the 3Ј untranslated region of CTGF. Taken together, our results indicate that miR-133 and miR-30 importantly limit the production of CTGF. We also provide evidence that the decrease of these 2 miRNAs in pathological left ventricular hypertrophy allows CTGF levels to increase, which contributes to collagen synthesis. In conclusion, our results show that both miR-133 and miR-30 directly downregulate CTGF, a key profibrotic protein, and thereby establish an important role for these miRNAs in the control of structural changes in the extracellular matrix of the myocardium.

When considering the pathological steps in the progression from cardiac overload towards the full clinical syndrome of heart failure, it is becoming increasingly clear that the extracellular matrix (ECM) is an important determinant in this process. Chronic pressure overload induces a number of structural alterations, not only hypertrophy of cardiomyocytes but also an increase in ECM proteins in the interstitium and perivascular regions of the myocardium. When this culminates in excessive fibrosis, myocardial compliance decreases and electrical conduction is affected. Altogether, fibrosis is associated with an increased risk of ventricular dysfunction and arrhythmias. Consequently, anti-fibrotic strategies are increasingly recognized as a promising approach in the prevention and treatment of heart failure. Thus, dissecting the molecular mechanisms underlying the development of cardiac fibrosis is of great scientific and therapeutic interest. In this review, we provide an overview of the available evidence supporting the general idea that fibrosis plays a causal role in deteriorating cardiac function. Next, we will delineate the signalling pathways importantly governed by transforming growth factor β (TGFβ) in the control of cardiac fibrosis. Finally, we will discuss the recent discovery that miRNAs importantly regulate cardiac fibrosis.

M utations in the RNA-binding motif protein 20 (RBM20) have been shown to cause a clinically aggressive form of dilated cardiomyopathy (DCM).1 A next-generation sequencing study in a large cohort of idiopathic DCM patients revealed that RBM20 belongs to the most frequently affected genes in DCM.2 RBM20 is essential for proper splicing of a large number of genes, and loss of RBM20 induces splicing defects in, for example, titin.3 These splicing defects in titin are thought to be an important reason why these mutations in RBM20 cause DCM. 3,4 However, the pathophysiological role of RBM20 mutations may not be limited to abnormal splicing. Given its essential role in splicing, we hypothesized that RBM20 may also regulate other splicing-dependent processes, like the formation of circular RNAs (circRNAs). If so, this would be of importance because it adds a novel potential disease mechanism. Editorial, see p 966 In This Issue, see p 965Despite their discovery over 20 years ago, circRNAs have only recently been recognized as a novel class of noncoding RNA molecules. Because of their unusual properties, they were presumed to be by-products of aberrant RNA splicing.5 Decades later, next-generation sequencing has revealed that thousands of endogenous circRNAs are expressed in mammals, including the cardiovascular system, 6 and that some of these circRNAs are even more abundant than their linear counterparts. Rationale: RNA-binding motif protein 20 (RBM20) is essential for normal splicing of many cardiac genes, and loss of RBM20 causes dilated cardiomyopathy. Given its role in splicing, we hypothesized an important role for RBM20 in forming circular RNAs (circRNAs), a novel class of noncoding RNA molecules.Objective: To establish the role of RBM20 in the formation of circRNAs in the heart. Methods and Results: Here, we performed circRNA profiling on ribosomal depleted RNA from human hearts and identified the expression of thousands of circRNAs, with some of them regulated in disease. Interestingly, we identified 80 circRNAs to be expressed from the titin gene, a gene that is known to undergo highly complex alternative splicing. We show that some of these circRNAs are dynamically regulated in dilated cardiomyopathy but not in hypertrophic cardiomyopathy. We generated RBM20-null mice and show that they completely lack these titin circRNAs. In addition, in a cardiac sample from an RBM20 mutation carrier, titin circRNA production was severely altered. Interestingly, the loss of RBM20 caused only a specific subset of titin circRNAs to be lost. These circRNAs originated from the RBM20-regulated I-band region of the titin transcript. Conclusions:We show that RBM20 is crucial for the formation of a subset of circRNAs that originate from the I-band of the titin gene. We propose that RBM20, by excluding specific exons from the pre-mRNA, provides the substrate to form this class of RBM20-dependent circRNAs. Khan et al circRNAs in the Human Heart 997CircRNAs are produced by the canonical spliceosome machinery, by back-splicing of e...