Yigal M. Pinto

miR-133 and miR-30 Regulate Connective Tissue Growth Factor. Implications for a Role of MicroRNAs in Myocardial Matrix Remodeling

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 3′ 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.

Circulating MicroRNAs Novel Biomarkers and Extracellular Communicators in Cardiovascular Disease

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.

Current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the European Society of Cardiology Working Group on Myocardial and Pericardial Diseases

In this position statement of the ESC Working Group on Myocardial and Pericardial Diseases an expert consensus group reviews the current knowledge on clinical presentation, diagnosis and treatment of myocarditis, and proposes new diagnostic criteria for clinically suspected myocarditis and its distinct biopsy-proven pathogenetic forms. The aims are to bridge the gap between clinical and tissue-based diagnosis, to improve management and provide a common reference point for future registries and multicentre randomised controlled trials of aetiology-driven treatment in inflammatory heart muscle disease.

Galectin-3 Marks Activated Macrophages in Failure-Prone Hypertrophied Hearts and Contributes to Cardiac Dysfunction

Background—Inflammatory mechanisms have been proposed to be important in heart failure (HF), and cytokines have been implicated to add to the progression of HF. However, it is unclear whether such mechanisms are already activated when hypertrophied hearts still appear well-compensated and whether such early mechanisms contribute to the development of HF. Methods and Results—In a comprehensive microarray study, galectin-3 emerged as the most robustly overexpressed gene in failing versus functionally compensated hearts from homozygous transgenic TGRmRen2-27 (Ren-2) rats. Myocardial biopsies obtained at an early stage of hypertrophy before apparent HF showed that expression of galectin-3 was increased specifically in the rats that later rapidly developed HF. Galectin-3 colocalized with activated myocardial macrophages. We found galectin-3–binding sites in rat cardiac fibroblasts and the extracellular matrix. Recombinant galectin-3 induced cardiac fibroblast proliferation, collagen production, and cyclin D1 expression. A 4-week continuous infusion of low-dose galectin-3 into the pericardial sac of healthy Sprague-Dawley rats led to left ventricular dysfunction, with a 3-fold differential increase of collagen I over collagen III. Myocardial galectin-3 expression was increased in aortic stenosis patients with depressed ejection fraction. Conclusions—This study shows that an early increase in galectin-3 expression identifies failure-prone hypertrophied hearts. Galectin-3, a macrophage-derived mediator, induces cardiac fibroblast proliferation, collagen deposition, and ventricular dysfunction. This implies that HF therapy aimed at inflammatory responses may need to be targeted at the early stages of HF and probably needs to antagonize multiple inflammatory mediators, including galectin-3.

MiR423-5p As a Circulating Biomarker for Heart Failure

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. These 6 circulating miRNAs provide attractive candidates as putative biomarkers for HF.

Molecular mechanisms that control interstitial fibrosis in the pressure-overloaded heart

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.

Conditional Dicer Gene Deletion in the Postnatal Myocardium Provokes Spontaneous Cardiac Remodeling

Background— Dicer, an RNAse III endonuclease critical for processing of pre-microRNAs (miRNAs) into mature 22-nucleotide miRNAs, has proven a useful target to dissect the significance of miRNAs biogenesis in mammalian biology. Methods and Results— To circumvent the embryonic lethality associated with germline null mutations for Dicer, we triggered conditional Dicer loss through the use of a tamoxifen-inducible Cre recombinase in the postnatal murine myocardium. Targeted Dicer deletion in 3-week-old mice provoked premature death within 1 week accompanied by mild ventricular remodeling and dramatic atrial enlargement. In the adult myocardium, loss of Dicer induced rapid and dramatic biventricular enlargement, accompanied by myocyte hypertrophy, myofiber disarray, ventricular fibrosis, and strong induction of fetal gene transcripts. Comparative miRNA profiling revealed a set of miRNAs that imply causality between miRNA depletion and spontaneous cardiac remodeling. Conclusions— Overall, these results indicate that modifications in miRNA biogenesis affect both juvenile and adult myocardial morphology and function.

Increased Cardiac Expression of Tissue Inhibitor of Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-2 Is Related to Cardiac Fibrosis and Dysfunction in the Chronic Pressure-Overloaded Human Heart

Background—Alterations in the balance of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) are involved in left ventricular (LV) remodeling. Whether their expression is related to interstitial fibrosis or LV dysfunction in patients with chronic pressure overload–induced LV hypertrophy, however, is unknown. Methods and Results—Therefore, cardiac biopsies were taken in 36 patients with isolated aortic stenosis (AS) and in 29 control patients without LV hypertrophy. Microarray analysis revealed significantly increased mRNA expression of collagen types I, III, and IV and transcripts involved in collagen synthesis, including procollagen endopeptidase and lysine and proline hydroxylases, in AS compared with control patients. Collagen deposition was greater in AS than in control patients and was most pronounced in AS patients with severe diastolic dysfunction. Cardiac mRNA expression of TIMP-1 and TIMP-2 was significantly increased in AS compared with control patients (mRNA transcript levels normalized to GAPDH: TIMP-1, 0.67±0.1 in AS versus 0.37±0.08 in control patients; TIMP-2, 9.5±2.6 in AS versus 1.6±0.4 in control patients; P<0.05 for both) but did not differ significantly for MMP-1, -2, or -9. Cardiac TIMP-1 and -2 transcripts were significantly related to the degree of interstitial fibrosis and proportional to diastolic dysfunction in AS patients. Conclusions—Cardiac expression of TIMP-1 and TIMP-2 is significantly increased in chronic pressure-overloaded human hearts compared with controls and is related to the degree of interstitial fibrosis.

Impaired Renal Clearance Explains Elevated Troponin T Fragments in Hemodialysis Patients

Background—Patients with severe renal dysfunction often have unexplained elevated serum concentrations of cardiac troponin T (cTnT). We investigated whether in vivo fragmentation of cTnT could explain these increases. Methods and Results—cTnT, creatine kinase isoenzyme MB, and myoglobin serum concentrations were measured in all 63 dialysis patients of our in-hospital dialysis department. A highly sensitive immunoprecipitation assay, followed by electrophoresis and Western blotting, was used to extract and concentrate cTnT and its possible fragments from serum of these 63 hemodialysis patients. Although creatine kinase isoenzyme MB values excluded recent ischemic myocardial events in 55 of the 63 cases, cTnT fragments ranging in size from 8 to 25 kDa were present in the serum samples of all dialysis patients. Conclusions—cTnT is fragmented into molecules small enough to be cleared by the kidneys of healthy subjects. Impaired renal function causes accumulation of these cTnT fragments and is very likely the cause of the unexplained elevations of serum cTnT found in patients with severe renal failure.

Risk Factors for Malignant Ventricular Arrhythmias in Lamin A/C Mutation Carriers A European Cohort Study

OBJECTIVES The purpose of this study was to determine risk factors that predict malignant ventricular arrhythmias (MVA) in Lamin A/C (LMNA) mutation carriers. BACKGROUND LMNA mutations cause a variety of clinical phenotypes, including dilated cardiomyopathy and conduction disease. Many LMNA mutation carriers have a poor prognosis, because of a high frequency of MVA and progression to end-stage heart failure. However, it is unclear how to identify mutation carriers that are at risk for MVA. METHODS In this multicenter cohort of 269 LMNA mutation carriers, we evaluated risk factors for MVA, defined as sudden cardiac death, resuscitation, and appropriate implantable cardioverter-defibrillator (ICD) treatment. RESULTS In a median follow-up period of 43 months (interquartile range: 17 to 101 months), 48 (18%) persons experienced a first episode of MVA: 11 persons received successful cardiopulmonary resuscitation, 25 received appropriate ICD treatment, and 12 persons died suddenly. Independent risk factors for MVA were nonsustained ventricular tachycardia, left ventricular ejection fraction <45% at the first clinical contact, male sex, and non-missense mutations (ins-del/truncating or mutations affecting splicing). MVA occurred only in persons with at least 2 of these risk factors. There was a cumulative risk for MVA per additional risk factor. CONCLUSIONS Carriers of LMNA mutations with a high risk of MVA can be identified using these risk factors. This facilitates selection of LMNA mutation carriers who are most likely to benefit from an ICD.