The role of micro-RNA -221 and -222 in the pathophysiology of viral myocarditis

Introduction and background
Viral myocarditis is a major health problem: up to 40% of sudden death in young people is due to myocarditis – adverse inflammation – in the heart (1). A lot of viruses can cause myocarditis, but especially rhinoviruses and enteroviruses are responsible pathogens (2). 
Cardiac inflammation plays a central role in myocarditis and heart failure, mediating cardiac hypertrophy, myocyte death and fibrosis. Identification of targets that explain why one person does get adverse cardiac inflammation, whereas many others are spared, is crucial to better understand this disease and to develop new therapeutic tools (3).
The discovery of micro-RNAs (miRs), a new class of small non-coding RNAs, is among the major scientific breakthroughs in recent years. They are crucial players in the regulation of gene expression by binding to messenger RNAs (mRNAs) and inhibiting their translation into protein (4).
 Global goals of this project
MiRs have been implicated in the pathophysiology of cancer, infectious disorders, and hypertensive and ischemic heart disease (5). However, their involvement in cardiac inflammation during viral myocarditis is largely unknown. The overall objective of this project is to unravel the precise biological roleof specific inflammation-mediated miR-221 and -222 in cardiac injury and dysfunction following viral infection, and in cardiac hypertrophy and fibrosis development. Whereas recent studies (6) mainly focused on the role of cardiomyocyte- and fibroblast-derived miRs in hypertrophy, fibrosis and arrhythmias, this project will unravel the biological role – and determine their upstream regulation – of inflammation-mediated miR-221 and -222 in cardiac inflammatory disease. 
 
Preliminary data
In a mouse model of human Coxsackievirus B3 (CVB3) induced myocarditis, the comparison of miR profiles at baseline using a miR microarray resulted in the identification and further validation via quantitativertPCR of miR-221 and -222 as inflammation-related miRs, beside miR-146,-155 and -223 (7). We validated miRNA-221 and -222 as relevant targets mediating cardiac inflammation in viral myocarditis. However, their pathophysiologic role in cardiac inflammation is largely unknown.
 
Recently, we obtained clear evidence that the absence of miR-155 (part of another project) in mice resulted in a higher mortality, with increased cardiac inflammation and injury in CVB3-myocarditis. Whereas lack of miR-155 in mice did not affect cardiac development or function at baseline, it resulted in increased cardiac inflammation and mortality compared to wild type littermates in CVB3-myocarditis (8). Fifty two percent (12/23) of miR-155 knockout mice died within 8 days after CVB3-infection compared to 6 percent of wild types (1/15), with increased cardiacinflammation and injury but a similar CVB3-load  in miR-155 knockout mice. 
 
Hypothesis and specific aims of this project
Both miR-221 and -222 are implicated in cancer formation and T-cell function, and have been found to modulate myogenesis (9). They are both up regulated by ERK1/2, a cell signaling pathway which is crucial in cardiac hypertrophy and fibrosis. Further encouraged by the preliminary data described above, and well-knowing that raised levels of the inflammation-related miR-221 and -222 are present in both viral myocarditis and hypertensive HD, we postulate that:
       MiR-221 and -222 are key regulators of cardiac inflammation in viral myocarditis.  Therefore we will unravel the precise biological function of miR-221 and -222 in knockout animals and by using antagomirs. We will also determine their mRNA-targets and pathways, and study their upstream regulation, explaining their possible implication in viral myocarditis.
       MiR-221 and -222 may be diagnostic tools and prognostic biomarkers for the severity of heart failure in viral heart disease. We aim to translate our findings to human viral myocarditis by studying miR levels in blood samples and inflammatory cardiac tissue biopsies. We will study whether miR-221 or -222 expression is raised in the blood of patients with myocarditis compared to controls, and whether quantification of miR-221 or -222 signifies a prognostic value in HF patients.
Methodology
1a. What is the biological role of miR-221 and -222 in heart failure? Do they protect against exaggerated inflammation and tissue damage?
The severity of cardiac inflammation, fibrosis, tissue damage and dysfunction will be measured in miR-221 or miR-222 gene-inactivated mice (currently available) and compared to wild type mice. We will also use the commercially available antagomir-approach for these miRs, allowing an efficient and time-dependent way of miR-silencing (10). To obtain miR-221 and -222 overexpression or knockdown (with miR-spounges), an adeno-associated virus (AAV-mediated) method will be used (11). The following experimental conditions will be evaluated: (a) angiotensin-II induced hypertension, 7 and 28 days; (b) human Coxsackievirus B3-induced myocarditis, 7 and 35 days; (c) myocardial infarction by persistent left coronary ligation, 7 and 14 days.
Heart function will be quantified with ultrasound- and electrocardiography, and by invasive intracardial pressure measurements. After obduction, hearts will be prepared for histological and molecular investigations. Haematoxylin-eosin staining, Siriusred (collagen) staining, immunohistochemistry of CD45-leucocytes, CD3-,CD4-, and CD8-lymfocytes, CD68-macrophages, CD31-capillars, and α-smooth muscle cell-actin coronary arteries. Expression of miR-221 and -222 in the heart will be evaluated with rtPCR and in situ hybridization.
 
1b. What are the mRNA-targets of miR-221 and -222 in inflammatory cells, fibroblasts and cardiomyocytes? To address which pathways and targets are really functional, and may explain the phenotype observed in in vivo studies, we will combine in vivo and in vitro approaches of pathway analysis and specific target confirmation. We will combine transcriptome, miRNAome and proteome, in order to get gene network analyses and informatics-driven reconstruction of complex interactions. We will perform promoter analysis using Genomatix software (mRNAs and miRs), and we will link mRNA expression to microRNA expression profiles. We intend to investigate whether ERK-1 and/or -2 is a target pathway for miR-221 and -222 in cardiomyocytes versus fibroblasts. We will also investigate and confirm presumed targets by means of TargetScan and MiRanda. Predicted targets for miR-221 and -222 in myocarditis and hypertensive HD could be elements of the cytoskeleton {myosin, actin and sarcoglycan (hypertrophy)}, matrix metalloproteinase-3 (MMP-3)which activates MMP-9 (inflammation and dilatation), smad-2 and -3, involved in transforming growth factor β (fibrosis). These targets will be validated in vitro in miR-221 and -222 (a) overexpressing(by means of lentiviral overexpression), (b) knockdown (using antagomirs), and (c) control cell populations (i.e. monocytes, fibroblasts and cardiomyocytes). Finally, luciferase reporter experiments, wherein 3’-UTR of target-mRNA is ligated to luciferase reporters, will give us theability to confirm these experiments. All necessary logistic and experimental infrastructure is available in the lab.
 
2.  Are miR-221 and -222 useful diagnostic tools and biomarkers for inflammation and for heart failure?
Different groups reported that miRNAs are circulating freely in human plasma and serum with marked biostability. Serum profiling performed in routine blood tests opens a window of opportunity for miR biomarker determinations, as RNA detection methods have much higher sensitivity and specificity than currently used protein detection assays (12).
We will quantify miR expression in blood and cardiac muscle samples of patients with different kinds of heart failure, compared to a control population. In biopsies, we routinely isolate mRNA (for determination of RNA viruses such as CVB3) in such a way that miRs can also be measured. We will use biopsies of patients with (a) acute ischemia; (b) hypertension; (c) viral myocarditis (in cooperation with prof. dr. A. Frustaci, Rome and prof. dr. H. Schultheiss, Berlin); and (d) control hearts. Already more than 250 blood and cardiac muscle biopsies have been sampled, obtained from patients of the clinicalheart failure unit (UZ Leuven) with ischemic (n=65), hypertensive (n=50) or viral heart disease (n=150).
 
Expected results
We expect that our study of miR-221 and -222 in cardiac pathology will lead to a better understanding of their role in inflammatory cardiomyopathies and of viral myocarditis in particular, to a better physiopathological view of hypertrophy and fibrosis in heart failure, to a non-invasive biomarker of heart failure and of viral myocarditis, and eventually toa potential therapeutic target in these settings (13).
 
Embedding of the project
Research will be performed in contribution withprof. dr. Peter Carmeliet of the Vesalius Research Center, Flemish Institute for Biotechnology, KULeuven. Bench-to-bedside transformation will be achieved via cooperation with the clinical department of Cardiology UZ Leuven, under guidance of prof. dr. Frans Van de Werf. International collaboration with CARIM (Cardiovascular Research Institute Maastricht) is guaranteed thanks to prof. dr. Stephane Heymans (Maastricht and Leuven), who will help to coordinate this research project.
 
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