University of Groningen
Circulating microRNAs in heart failure
Vegter, Eline Lizet
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Vegter, E. L. (2017). Circulating microRNAs in heart failure. Rijksuniversiteit Groningen.
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Circulating microRNAs in heart failure
Circulating microRNAs in heart failure ISBN: 978-94-9268-392-2
© Copyright 2017 - E.L. Vegter
All rights are reserved. No part of this publication may be reproduced, stored in a re-trieval system, or transmitted in any form or by any means, without the written permis-sion of the author.
The financial support of the Graduate School of Medical Sciences and the University of Groningen for the publication of this thesis is gratefully acknowledged.
Cover design: Sara ten Broeke en Rik Vegter
Circulating microRNAs
in heart failure
Proefschrift
ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen
op gezag van de
rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op woensdag 18 oktober 2017 om 14.30 uur
door
Eline Lizet Vegter
geboren op 27 december 1988 te Venlo
Promotor
Prof. dr. A.A. Voors
Copromotor
Dr. P. van der Meer
Beoordelingscommissie
Prof. dr. J. van der Velden Prof. dr. L.J. de Windt Prof. dr. M.P. van den Berg
Financial support by the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged. The research described in this thesis was supported by a grant from the Dutch Heart Foundation (CVON 2011-11).
Paranimfen
Dr. J.C. Tanis Drs. L.V. de Vries
TABLE of CoNTENTs
Chapter 1 Introduction and aims 9
Chapter 2 MicroRNAs in heart failure: from biomarker to target for therapy Eur J Heart Fail. 2016 May;18(5):457-68
23
Chapter 3 Signature of circulating microRNAs in patients with acute heart failure
Eur J Heart Fail. 2016 Apr;18(4):414-23
53
Chapter 4 Use of biomarkers to establish potential role and function of circulating microRNAs in acute heart failure
Int J Cardiol. 2016 Dec 1;224:231-239
77
Chapter 5 Associations between volume status and circulating microRNAs in acute heart failure
Eur J Heart Fail. 2017 Aug;19(8):1077-1078.
115
Chapter 6 Low circulating microRNA levels in heart failure patients are associated with atherosclerotic disease and cardiovascular-related rehospitalizations
Clin Res Cardiol. 2017 Aug;106(8):598-609.
123
Chapter 7 Rodent heart failure models do not reflect the human circulating microRNA signature in heart failure
Plos One. 2017 May 5;12(5):e0177242
153
Chapter 8 General discussion and future perspectives 185
Appendices Nederlandse samenvatting 209
Dankwoord 221
About the author 227
Chapter 1
Introduction and aims
Introduction
11
1
HEART fAiLuREHeart failure is a major public health concern and affects 1-2% of the adult population.1
The European Society of Cardiology defined heart failure as a clinical syndrome charac-terized by typical symptoms (such as dyspnea and fatigue) and signs (pulmonary rales, ankle oedema, elevated jugular venous pressure), accompanied by structural and/or functional cardiac abnormality, resulting in a reduced cardiac output and/or elevated intracardiac pressures at rest or during stress. The aetiology of heart failure is diverse, including cardiac abnormalities caused by for example valve defects, congenital heart disease and myocarditis, as well as arrhythmias and systemic diseases such as hyper-tension, diabetes and anaemia. Although it is likely that multiple factors contribute to the development of heart failure, ischaemic heart disease is known as the most com-mon cause of heart failure in the Western world.2 Ischaemic heart disease results from a
reduced blood supply from the coronary arteries to the myocardium, most often caused by atherosclerosis. This in turn leads to a mismatch between oxygen demand and sup-ply, which eventually can lead to ventricular remodeling and impaired cardiac function. Acute heart failure is characterized by the acute onset of severe signs and symptoms of heart failure requiring immediate treatment, while physicians generally speak of chronic heart failure when patients are stable and treated for previously diagnosed heart failure. Heart failure can be further categorized in 3 main groups based on left ven-tricular ejection fraction (LVEF); heart failure with a reduced LVEF (<40%), also known as HFrEF, heart failure with a mid-range LVEF of 40-49% (HFmrEF) and heart failure with a preserved LVEF of ≥50% (HFpEF).1 These 3 groups represent patients with different
underlying characteristics and do not respond in a similar way to therapy.3
In the last decades several advancements in heart failure treatment have been made, such as the development of neurohormonal antagonists (beta-blockers, ACE inhibi-tors, angiotensin II receptor blockers and mineralocorticoid receptor antagonists), and more recently the new drug sacubitril/valsartan, an angiotensin receptor-neprilysin inhibitor.1,4 Besides medication, device therapy including the implantable
cardioverter-defibrillator (ICD) and cardiac resynchronization therapy (CRT) has shown to improve survival in selected patient groups.1 Despite these much-needed improvements, the
prognosis of heart failure is poor. Five-year mortality in heart failure ranges between 41-60%,2 while more recent data on 1-year mortality report 7-17%.5 When a patient is
hospitalized with acute heart failure, the 5-year mortality rate increases to more than 75%.6
As the prognosis of heart failure patients clearly remains unsatisfactory, new treatment strategies are needed. In search for new drug targets, it is important to understand the underlying pathophysiological mechanisms in heart failure. Several processes have
12 Chapter 1
been discovered with crucial involvement in the development and progression of the disease, for example the neurohormonal adaptation system; the keystone of the presently available heart failure pharmacotherapy. These drugs are mainly focused on targeting cell-surface or intracellular mineralocorticoid receptors, eventually attenuat-ing cardiac remodelattenuat-ing and cardiac dysfunction.7,8 Other approaches focusing on
intra-cellular molecular signalling pathways may lead to additional benefit ideally resulting in reduced mortality and rehospitalisation rates. Currently there is a rapidly growing interest in the genetic and epigenetic background of heart failure patients, providing detailed information on the cellular level. Gene therapy can interfere with these mo-lecular processes by modulating the production and/or activity of important regulatory proteins in heart failure. Pharmacogenomics may therefore be a promising new area of interest and may eventually emerge as new treatment strategy in heart failure.
GENE ExPREssioN ANd EPiGENETiCs
A large part of the human body consists of proteins and they play a central role in the function of important (patho)physiological processes. Proteins are molecules made of amino acids and the information to produce these proteins lies within portions of the DNA (deoxyribonucleic acid), called genes. DNA consists of two strands which are both formed of nucleotides. These nucleotides contain the nucleobase adenine (A), thymine (T), cytosine (C) or guanine (G), deoxyribose (a sugar) and a phosphate group, and form sequences holding the genes containing the biologically important information for the production of proteins.9 The nucleobases of the 2 strands join together according to
base complementary (A with T and C with G). DNA gets transcribed into RNA (ribonucleic acid) and the mature transcript (messenger RNA, or mRNA) leaves the nucleus. This mRNA can then be translated into amino acids to form proteins.10 This process from
gene to gene product (most often proteins) is called gene expression. Gene expression of specific genes can by quantified by measuring the mRNA transcripts in a particular cell type or tissue and can give an impression of the regulation of important (patho) physiological processes.
Gene expression can be regulated and this may have important consequences for pro-tein synthesis and subsequently the development of diseases. These gene expression modifying mechanisms are all collectively known as epigenetics. The Oxford dictionary defines epigenetics as “the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself”, or in other words; a change in phenotype without a change in genotype. At the moment, several different gene expression modifying mechanisms have been described including DNA methyla-tion, chromatin remodeling, histone modifications and non-coding RNA mechanisms.11
Introduction
13
1
Non-coding RNAs are transcribed from DNA, but are not translated into proteins. This is the large majority of all transcripts, as only 2% is translated into proteins. In the early days, these non-coding transcripts were regarded to as non-functional, however it is now clear that non-coding RNAs have several functions in regulatory mechanisms of gene expression and function. Non-coding RNAs can be divided into short non-coding RNAs (< 30 nucleotides) and long non-coding RNAs (>200 nucleotides).
MiCRoRNAs
The microRNAs (miRNAs) belong to the short non-coding RNAs and were first described in the roundworm Caenorhabditis elegans by Lee et al.12 and Wightman et al.,13 in 1993.
They showed that the gene lin-4 was not coding for proteins but instead produced a short RNA of 22 nucleotides long, which was later found to be capable of binding to lin-14 and thereby reducing the production of the LIN-14 protein. In 2001, several more miRNAs were discovered in C. elegans and could also be identified in other invertebrates, vertebrates and humans.14-16 These milestone papers paved the way for several years of
continuously increasing miRNA research (Figure 1). So far, 2588 mature miRNAs have been discovered in humans (www.mirbase.org).
In the nucleus, primary miRNAs (pri-miRNAs) are transcribed from DNA. These long stemloop structures with single stranded RNA extensions on both ends are then cleaved by processing proteins DGR8 and Drosha, resulting in precursors hairpin miRNAs (pre-miRNAs). The pre-miRNAs leave the nucleus and are further processed by Dicer in the cytoplasm of the cell. This results in the loss of the hairpin structure and a mature miRNA is formed. These mature miRNAs can then be cleaved by helicase, creating 2 separate
0 2000 4000 6000 8000 10000 12000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 Cou nts Year
figure 1. Number of PubMed publications containing the search term “microRNA” from the year 2001 to 2016
14 Chapter 1
miRNA strands. The active miRNA strand binds to an Ago protein which can be loaded into a RNA-induced silencing complex (RISC). This complex is capable of binding to the complementary 3’-untranslated region of the mRNA strand, and with perfect comple-mentary the mRNA transcript will degrade. With incomplete binding the mRNA will not degrade but repressed translationally.17 Figure 1 in Chapter 2 graphically presents the
miRNA biogenesis and function. Thus, by targeting the complementary mRNA, miRNAs are able to regulate gene expression at the post-transcriptional level.
The current understanding is that miRNAs function intracellularly, and hence in tissue. Dysregulation of miRNAs have been linked to specific diseases and underly-ing pathways. Of all mature miRNAs, a small minority was found to be organ specific, such as miR-122 (which is only expressed in the liver) or miR-9 (expression limited to the brain).18 MiRNAs with high specificity for the heart are miR-208, miR-133, miR-1 and
miR-499, which are all associated with cardiac development and disease.19 For example,
deletion of the mature miR-1-2 sequence in mouse embryonic stem cells resulted in ventricular septum abnormalities, rapid heart dilatation, rhythm disturbances and ventricular dysfunction.20 MiR-208a was shown to be required for normal cardiac
con-duction and contraction, while upregulated levels were found to be related to cardiac hypertrophy, fibrosis and a disturbed electrical conduction in mice.21 As such, methods
were developed to alter miRNA expression in order to inhibit or stimulate their gene expression-modifying function. Several pre-clinical studies were published to date and have shown promising results in the cardiovascular field, although no clinical trials have entered the arena yet.22
Circulating microRNAs
Although miRNAs have a gene silencing function within cells, in 2008, researchers dis-covered extracellular miRNAs in blood23 and later also in other body fluids such as urine,
saliva and cerebrospinal fluid.24 In this thesis, we specifically focus on miRNAs in blood
plasma. These miRNAs are protected from early degradation by RNases in the circulation since they are either bound to proteins or encapsulated by exosomes, apoptotic bodies or other microparticles. Even extreme conditions such as boiling and long-term storage do not affect circulating miRNA levels.25 As it appeared that miRNAs can be transported
through the circulation,26 the hypothesis that miRNAs may exert paracrine functions led
to a plethora of studies investigating this possibility. Indeed, several studies showed that miRNAs are able to change gene expression and thereby protein synthesis in tar-get cells in controlled, experimental settings.27,28 Besides their function as cell-to-cell
communicators, the stability and relative straightforward measurability prompted the investigation of circulating miRNAs as biomarkers of disease, also in heart failure.
Biomarkers can reflect biological processes in health and disease, and are therefore helpful tools in the management of disease. In heart failure, the biomarkers N-terminal
Introduction
15
1
pro b-type natriuretic peptide (NT-proBNP) and b-type natriuretic peptide (BNP) can help establishing the diagnosis of heart failure and are currently the gold standard.1
Additionally, several other, novel biomarkers such as soluble ST2, growth differentiation factor 15 (GDF-15) and galectin-3 have been described to have a role in heart failure and mainly reported as prognosticators of survival.29 However, biomarkers may also be
used to evaluate therapy and can provide insight into underlying disease mechanisms. Circulating miRNAs could add value to the already available heart failure biomarkers and have been detected in blood and plasma of patients with cardiovascular disease.
Distinct circulating miRNA patterns were reported for patients with myocardial infarc-tion in comparison to control subjects, and also in cardiac arrhythmias and hyperten-sion.30 In heart failure, most studies focused on differential miRNA expression in chronic
heart failure patients compared to healthy subjects,31-33 while only few research groups
directed their attention to acute heart failure. In these relatively small studies, circu-lating miRNAs capable of differentiating patients presenting at the emergency room with dyspnea owing to heart failure and breathlessness caused by other causes were described.34,35 Some groups tried to use circulating miRNAs to distinguish HFpEF from
HFrEF, and identified specific differential miRNA patterns, although very few overlap was found between these studies.35-37 Because of the lack of robust circulating miRNA
studies in acute heart failure, we focused on the investigation of a circulating miRNA profile in acute heart failure patients. In addition, the association between circulating miRNAs and outcome in acute heart failure has not been described before.
Although the proper identification of the most differentially expressed circulating miRNAs in heart failure is the first step, understanding the findings is a crucial next stage in the study of circulating miRNAs. These miRNAs may represent several clinical factors and disease processes in heart failure, however, information on this topic is limited. Increasing our knowledge concerning the potential meaning of these miRNAs in the circulation of heart failure patients is therefore one of the primary aims of this thesis.
AiMs ANd ouTLiNE of THE THEsis
The main aims of this thesis are as follows:
1) Discover differentially expressed circulating miRNAs in acute heart failure patients 2) Provide additional information regarding clinical factors and disease processes
these circulating miRNAs may reflect
16 Chapter 1
In Chapter 2 we discuss the current knowledge of miRNAs in heart failure, specifically
focusing on circulating miRNAs and their release and uptake from the circulation, their role in paracrine cell signalling and biomarker potential. Further, we describe miRNAs with important functions in cardiac hypertrophy and fibrosis and discuss the possibili-ties of inhibiting or overexpressing these miRNAs in the context of future drug targets.
Although some studies identified a pattern of differentially expressed circulating miRNAs in heart failure, most studies were conducted in a small number of chronic heart failure patients and few validated their findings in independent patient cohorts. Therefore, we aimed in Chapter 3 to identify a miRNA signature in several different
heart failure cohorts representing different stages of acute and chronic heart failure. While the identification of these differentially expressed miRNAs in plasma is the first step in the study of heart failure-related circulating miRNAs, not much is known regarding their origin, role and function in the circulation of heart failure patients. In the next chapters, we try to provide more background on our previous findings described in Chapter 3, in which we show that the most differentially expressed circulating miRNAs in heart failure are downregulated. We explore several different hypotheses in Chapter 4, 5 and 6, as presented in Figure 2. In Chapter 4 we investigate the previously identified
miRNAs in relation to other, well-known biomarkers. We also sought to find associations between these circulating miRNAs, their potential targets and relevant cardiac-related pathways. In Chapter 5 we focus on another import aspect of acute heart failure, namely
dilution and fluid overload. We explored whether volume status was associated to cir-culating miRNA levels by relating changes in haemoconcentration to changes in miRNA levels in patients with acute heart failure. We also investigated the hypothesis that these heart failure-related miRNAs may be associated to vascular processes. In general, the current concept is that miRNAs in the circulation mainly derive from blood cells. Further,
Downregulated circulating miRNAs in heart failure Volume status Cardiac related
pathways dysfunction Vascular
Introduction
17
1
in heart failure it has been shown that the most abundantly expressed miRNAs in the circulation of do not originate from the heart, but from blood and endothelial cells. Therefore, we aimed in Chapter 6 to measure the heart failure-related set of miRNAs
in another cohort of heart failure patients and investigated the potential relation with vascular dysfunction as reflected in heart failure patients with atherosclerotic disease. Further, we assessed the associations between circulating miRNA levels and biomarkers reflecting atherosclerosis-related processes including angiogenesis, inflammation and endothelial dysfunction.
In order to gain insight into the potential function of these circulating miRNAs, proper animal models suitable for further mechanistic follow-up studies are needed. However, very few circulating miRNAs identified in human heart failure have been measured in animal models of heart failure. In Chapter 7, we report about 3 mechanistically
differ-ent roddiffer-ent heart failure models in which we investigate our set of previously described heart failure-related circulating miRNAs.
Finally, in Chapter 8 we discuss the main findings and conclusions and place our
re-sults in perspective. Moreover, we describe the future directions and new developments in non-coding RNA research.
18 Chapter 1 REfERENCEs
1. Ponikowski P, Voors AA, Anker SD, Bueno H, Cleland JG, Coats AJ, Falk V, Gonzalez-Juanatey JR, Harjola VP, Jankowska EA, Jessup M, Linde C, Nihoyannopoulos P, Parissis JT, Pieske B, Riley JP, Rosano GM, Ruilope LM, Ruschitzka F, Rutten FH, van der Meer P, Authors/Task Force Members. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC)Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur Heart J 2016; 37: 2129-2200.
2. Mosterd A, Hoes AW. Clinical epidemiology of heart failure. Heart 2007; 93: 1137-1146.
3. Paulus WJ, van Ballegoij JJ. Treatment of heart failure with normal ejection fraction: an inconve-nient truth! J Am Coll Cardiol 2010; 55: 526-537.
4. McMurray JJ, Packer M, Desai AS, Gong J, Lefkowitz MP, Rizkala AR, Rouleau JL, Shi VC, Solomon SD, Swedberg K, Zile MR, PARADIGM-HF Investigators and Committees. Angiotensin-neprilysin inhibition versus enalapril in heart failure. N Engl J Med 2014; 371: 993-1004.
5. Maggioni AP, Dahlstrom U, Filippatos G, Chioncel O, Crespo Leiro M, Drozdz J, Fruhwald F, Gulles-tad L, Logeart D, Fabbri G, Urso R, Metra M, Parissis J, Persson H, Ponikowski P, Rauchhaus M, Voors AA, Nielsen OW, Zannad F, Tavazzi L, Heart Failure Association of the European Society of Cardiology (HFA). EURObservational Research Programme: regional differences and 1-year follow-up results of the Heart Failure Pilot Survey (ESC-HF Pilot). Eur J Heart Fail 2013; 15: 808-817. 6. Goldberg RJ, Ciampa J, Lessard D, Meyer TE, Spencer FA. Long-term survival after heart failure: a
contemporary population-based perspective. Arch Intern Med 2007; 167: 490-496.
7. Iwaz JA, Lee E, Aramin H, Romero D, Iqbal N, Kawahara M, Khusro F, Knight B, Patel MV, Sharma S, Maisel AS. New Targets in the Drug Treatment of Heart Failure. Drugs 2016; 76: 187-201.
8. Kaye DM, Krum H. Drug discovery for heart failure: a new era or the end of the pipeline? Nat Rev
Drug Discov 2007; 6: 127-139.
9. Watson JD, Crick FH. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. J.D. Watson and F.H.C. Crick. Published in Nature, number 4356 April 25, 1953. Nature 1974; 248: 765.
10. Alberts B, Bray D, Hopkin K. From DNA to protein: how cells read the genome. Essential Cell
Biol-ogy. 3rd ed. New York (NY): Garland Science; 2009. p. 231-260.
11. Gibney ER, Nolan CM. Epigenetics and gene expression. Heredity (Edinb) 2010; 105: 4-13.
12. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75: 843-854.
13. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993; 75: 855-862.
14. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. Identification of novel genes coding for small expressed RNAs. Science 2001; 294: 853-858.
15. Lau NC, Lim LP, Weinstein EG, Bartel DP. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 2001; 294: 858-862.
16. Lee RC, Ambros V. An extensive class of small RNAs in Caenorhabditis elegans. Science 2001; 294: 862-864.
17. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 2014; 15: 509-524. 18. Ludwig N, Leidinger P, Becker K, Backes C, Fehlmann T, Pallasch C, Rheinheimer S, Meder B,
Stahler C, Meese E, Keller A. Distribution of miRNA expression across human tissues. Nucleic Acids
Introduction
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19. Chistiakov DA, Orekhov AN, Bobryshev YV. Cardiac-specific miRNA in cardiogenesis, heartfunc-tion, and cardiac pathology (with focus on myocardial infarction). J Mol Cell Cardiol 2016; 94: 107-121.
20. Zhao Y, Ransom JF, Li A, Vedantham V, von Drehle M, Muth AN, Tsuchihashi T, McManus MT, Schwartz RJ, Srivastava D. Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2. Cell 2007; 129: 303-317.
21. Callis TE, Pandya K, Seok HY, Tang RH, Tatsuguchi M, Huang ZP, Chen JF, Deng Z, Gunn B, Shu-mate J, Willis MS, Selzman CH, Wang DZ. MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice. J Clin Invest 2009; 119: 2772-2786.
22. Duygu B, de Windt LJ, da Costa Martins PA. Targeting microRNAs in heart failure. Trends
Cardio-vasc Med 2016; 26: 99-110.
23. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Note-boom J, O’Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentle-man R, Vessella RL, Nelson PS, Martin DB, Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 2008; 105: 10513-10518.
24. Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, Galas DJ, Wang K. The microRNA spectrum in 12 body fluids. Clin Chem 2010; 56: 1733-1741.
25. Chen X, Ba Y, Ma L, Cai X, Yin Y, Wang K, Guo J, Zhang Y, Chen J, Guo X, Li Q, Li X, Wang W, Zhang Y, Wang J, Jiang X, Xiang Y, Xu C, Zheng P, Zhang J, Li R, Zhang H, Shang X, Gong T, Ning G, Wang J, Zen K, Zhang J, Zhang CY. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 2008; 18: 997-1006.
26. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007; 9: 654-659.
27. Pegtel DM, Cosmopoulos K, Thorley-Lawson DA, van Eijndhoven MA, Hopmans ES, Lindenberg JL, de Gruijl TD, Wurdinger T, Middeldorp JM. Functional delivery of viral miRNAs via exosomes. Proc
Natl Acad Sci U S A 2010; 107: 6328-6333.
28. Bang C, Batkai S, Dangwal S, Gupta SK, Foinquinos A, Holzmann A, Just A, Remke J, Zimmer K, Zeug A, Ponimaskin E, Schmiedl A, Yin X, Mayr M, Halder R, Fischer A, Engelhardt S, Wei Y, Schober A, Fiedler J, Thum T. Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy. J Clin Invest 2014; 124: 2136-2146.
29. Schmitter D, Cotter G, Voors AA. Clinical use of novel biomarkers in heart failure: towards person-alized medicine. Heart Fail Rev 2014; 19: 369-381.
30. Romaine SP, Tomaszewski M, Condorelli G, Samani NJ. MicroRNAs in cardiovascular disease: an introduction for clinicians. Heart 2015; 101: 921-928.
31. Vogel B, Keller A, Frese KS, Leidinger P, Sedaghat-Hamedani F, Kayvanpour E, Kloos W, Backe C, Thanaraj A, Brefort T, Beier M, Hardt S, Meese E, Katus HA, Meder B. Multivariate miRNA signa-tures as biomarkers for non-ischaemic systolic heart failure. Eur Heart J 2013; 34: 2812-2822. 32. Voellenkle C, van Rooij J, Cappuzzello C, Greco S, Arcelli D, Di Vito L, Melillo G, Rigolini R, Costa E,
Crea F, Capogrossi MC, Napolitano M, Martelli F. MicroRNA signatures in peripheral blood mono-nuclear cells of chronic heart failure patients. Physiol Genomics 2010; 42: 420-426.
33. Corsten MF, Dennert R, Jochems S, Kuznetsova T, Devaux Y, Hofstra L, Wagner DR, Staessen JA, Heymans S, Schroen B. Circulating MicroRNA-208b and MicroRNA-499 reflect myocardial damage in cardiovascular disease. Circ Cardiovasc Genet 2010; 3: 499-506.
34. Tijsen AJ, Creemers EE, Moerland PD, de Windt LJ, van der Wal AC, Kok WE, Pinto YM. MiR423-5p as a circulating biomarker for heart failure. Circ Res 2010; 106: 1035-1039.
20 Chapter 1
35. Ellis KL, Cameron VA, Troughton RW, Frampton CM, Ellmers LJ, Richards AM. Circulating microR-NAs as candidate markers to distinguish heart failure in breathless patients. Eur J Heart Fail 2013; 15: 1138-1147.
36. Wong LL, Armugam A, Sepramaniam S, Karolina DS, Lim KY, Lim JY, Chong JP, Ng JY, Chen YT, Chan MM, Chen Z, Yeo PS, Ng TP, Ling LH, Sim D, Leong KT, Ong HY, Jaufeerally F, Wong R, Chai P, Low AF, Lam CS, Jeyaseelan K, Richards AM. Circulating microRNAs in heart failure with reduced and preserved left ventricular ejection fraction. Eur J Heart Fail 2015; 17: 393-404.
37. Watson CJ, Gupta SK, O’Connell E, Thum S, Glezeva N, Fendrich J, Gallagher J, Ledwidge M, Grote-Levi L, McDonald K, Thum T. MicroRNA signatures differentiate preserved from reduced ejection fraction heart failure. Eur J Heart Fail 2015; 17: 405-415.
Chapter 2
MicroRNAs in heart failure: from
biomarker to target for therapy
Eline L. Vegter Peter van der Meer Leon J. de Windt Yigal M. Pinto Adriaan A. Voors
24 Chapter 2 ABsTRACT
MicroRNAs (miRNAs) are increasingly recognized to play important roles in cardio-vascular diseases, including heart failure. These small, non-coding RNAs have been identified in tissue and are involved in several pathophysiological processes related to heart failure, such as cardiac fibrosis and hypertrophy. As a result, miRNAs have be-come interesting novel drug targets, leading to the development of miRNA mimics and antimirs. MicroRNAs are also detected in the circulation, and are proposed as potential diagnostic and prognostic biomarkers in heart failure. However, their role and function in the circulation remains to be resolved. Here, we review the potential roles of miRNAs as circulating biomarkers and as targets for therapy.
MicroRNAs in heart failure
25
2
iNTRoduCTioNMicroRNAs (miRNAs) have been studied intensively since their discovery more than two decades ago, which led to a drastic change in our understanding of regulatory epigenetic processes. MicroRNAs (~22 nucleotides in length) are involved in several cell processes by repressing messenger RNA (mRNA) translation mainly via binding at the complementary 3’-untranslated region, thus modulating gene expression at the post-transcriptional level. In cardiac development, miRNAs are needed for the forma-tion of normal, funcforma-tional heart tissue and a variety of miRNAs have been discovered as important regulators of several phases in cardiac development.1-3
The importance of miRNAs was originally demonstrated in embryonic development by modifications in Dicer, an enzyme involved in miRNA processing. Dicer1 gene target-ing in mice resulted in early embryonic death.4 Furthermore, cardiac-specific deletion
of Dicer shortly after embryonic heart formation resulted in heart failure and eventually death.1 Deletion of Dicer in the postnatal myocardium induced cardiac remodeling,
increased atrial size and resulted in early lethality.2 These results led to an increasing
number of studies identifying specific miRNAs associated with several phases of cardiac development, including miR-1 and miR-133, which will be discussed in this review in more detail. Interestingly, the miRNAs associated with the pathophysiology of heart fail-ure were found to be closely similar to the miRNAs involved in the fetal gene program.5
The activation of the fetal gene program results in adaptive processes in the heart, which eventually lead to heart failure.
Functional miRNA studies reported that a variety of miRNAs play a role in pathogenic mechanisms leading to heart failure, such as remodeling, hypertrophy, apoptosis and hypoxia.6,7 Furthermore, in response to the progression of heart failure, miRNAs were
shown to behave in a dynamic and stage-specific way. For example, decreased levels of miRNAs (including miR-1 and miR-133a) were found in transgenic hypertrophic cardiomyopathy (HCM) mice before development of the disease, while an increasing number of miRNAs exhibited a dysregulated and mainly upregulated pattern in the later stages towards end-stage heart failure.8 Others demonstrated that the aetiology of
heart failure (ischaemic, aortic stenosis or idiopathic cardiomyopathy) was associated with differential expressed miRNA patterns.9 Thus, compelling evidence suggests that
miRNAs play an active role in the onset and progression of heart failure.
CiRCuLATiNG MiCRoRNAs
Based on current knowledge, the effects of miRNAs on repressing mRNA translation take place inside cells. However, in 2008, miRNAs were discovered outside cells and
26 Chapter 2
in circulating blood.10-12 These extracellular circulating miRNAs are remarkably stable,
even under several extreme conditions such as repeated freeze-thaw cycles, boiling and long-term storage.10-12 This is because of the protective effect of their carriers against
RNases in the circulation, including protein-complexes, exosomes, apoptotic bodies and other microvesicles. These findings led to an increasing number of studies investigating the potential of miRNAs as circulating biomarkers for disease, including heart failure. In the following section, the potential roles of miRNAs as biomarkers in the diagnosis, prognosis and treatment of heart failure will be discussed.
PoTENTiAL CLiNiCAL APPLiCATioN foR MiCRoRNAs As BioMARkERs iN HEART fAiLuRE
Biomarkers are used in heart failure for several purposes. They play an important role in the diagnosis of heart failure and are used to determine the cause of heart failure. Fur-ther, many biomarkers can be used as prognostic markers and in some circumstances guide the choice, intensity, and the response to therapy. Finally, biomarkers may pro-vide additional insight about specific pathophysiological mechanisms in heart failure.13
As there is strong evidence that miRNAs play a role in the onset and progression of heart failure, and because of their stability in plasma, miRNAs are interesting potential novel biomarkers in heart failure.
MicroRNAs as diagnostic biomarkers
Although B-type natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) are currently used as the gold standard in ruling out and confirming the diagnosis of heart failure,14 circulating miRNAs have been increasingly studied as
poten-tial diagnostic biomarkers. However, in order to be used as biomarkers for the diagnosis of heart failure, they should either outperform natriuretic peptides, or have an additive value. For the diagnosis of heart failure, the sensitivity of natriuretic peptides is high, but their specificity to detect heart failure leaves room for improvement. Several stud-ies investigated the potential of circulating miRNAs for the diagnosis of heart failure (Table 1). In chronic heart failure, multiple miRNAs have been described as candidates for future diagnostic biomarkers in heart failure.15-18 A few studies have reported on
cir-culating miRNAs that were able to distinguish patients with breathlessness due to heart failure and other causes of dyspnea. Tijsen et al. found miR-423-5p to be differential expressed between heart failure patients and healthy controls, and patients with other causes of dyspnea.19 Other studies also described differentially expressed circulating
miRNAs in acute heart failure, including low levels of miR-103, miR-142-3p, miR-30b and miR-342-3p,20 and high levels of miR-499.21 A recent study by our group identified a panel
MicroRNAs in heart failure
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Table 1. Circulating microRNAs (miRNAs) associated with the diagnosis of heart failure
miRNA Regulation in heart failure References
diagnosis
Acute heart failure Compared with controls
miR-18a-5p Decreased 22 miR-26b-5p Decreased 22 miR-27a-3p Decreased 22 miR-30b Decreased 20 miR-30e-5p Decreased 22 miR-103 Decreased 20 miR-106a-5p Decreased 22 miR-142-3p Decreased 20 miR-199a-3p Decreased 22 miR-342-3p Decreased 20 miR-423-5p Increased 19 miR-499 Increased 21 miR-652-3p Decreased 22
Chronic heart failure
miR-22 Increased 16 miR-30c Decreased 25 miR-92b Increased 16 miR-107 Decreased 17 miR-122* Increased 18 miR-139 Decreased 17 miR-142-5p Decreased 17 miR-146a Decreased 25 miR-183-3p Decreased 26 miR-190a Decreased 26 miR-193b-3p Decreased 26 miR-193b-5p Decreased 26 miR-203 Decreased 15 miR-210 Increased 15 miR-211-5p Decreased 26 miR-221 Decreased 25 miR-320a Increased 16 miR-328 Decreased 25 miR-375 Decreased 25 Increased 15 miR-423-5p Increased 16
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of acute heart failure-specific miRNAs, in which decreased miRNA levels were observed in acute heart failure patients compared with healthy controls and patients with an acute exacerbation of chronic obstructive pulmonary disease.22
In plasma of patients diagnosed with HCM without heart failure symptoms, miR-29a, among others, was found to be significantly upregulated and the only miRNA to corre-late with both LV hypertrophy and fibrosis.23 These results suggest that this miRNA may
function as a biomarker for remodeling processes in HCM. The specificity of miR-29a for HCM was confirmed by Derda et al, demonstrating that miR-29a was able to differ-entiate between hypertrophic obstructive cardiomyopathy (HOCM), hypertrophic non-obstructive cardiomyopathy (HNCM), senile amyloidosis and aortic stenosis.24 MiR-29a
was positively correlated with the interventricular septum size, which is a parameter for remodeling processes including hypertrophy and fibrosis.
Recent evidence suggests that miRNAs might discriminate between heart failure with a reduced ejection fraction (HFrEF) and heart failure with a preserved ejection fraction (HFpEF). To date, 3 studies reported differential levels of several circulating miRNAs in HFrEF and HFpEF, with only a few similarities.20,25,26 Differentially expressed miRNAs
be-Table 1. Circulating microRNAs (miRNAs) associated with the diagnosis of heart failure (continued)
miRNA Regulation in heart failure References
miR-494 Decreased 26 miR-520d-5p Increased 18 miR-558 Decreased 18 miR-671-5p Increased 26 miR-1180 Increased 15 miR-1233 Increased 26 miR-1908 Increased 15
HFpEF Compared with controls Compared with HFrEF
miR-30c Decreased Decreased 25
miR-125a-5p Increased Increased 26
miR-146a Decreased Decreased 25
miR-190a Decreased Decreased 26
miR-221 Decreased Increased 25
miR-328 Decreased Increased 25
miR-375 Decreased Increased 25
miR-550a-5p Increased Decreased 26
miR-638 Decreased Increased 26
HFpEF indicates heart failure with preserved ejection fraction, HFrEF; heart failure with reduced ejection fraction.
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tween HFpEF and HFrEF might not only be relevant for diagnostic purposes, but might provide a better insight into their differential pathophysiology as well.
Prognostic value of microRNAs
In heart failure, large numbers of biomarkers are predictors of outcome. However, a limited number of studies focused on the prognostic value of circulating miRNAs in pa-tients with acute and chronic heart failure; these are summarized in Table 2. Circulating miRNAs have been studied more extensively in relation to the prognosis after myocar-dial infarction. Several circulating miRNAs or a combination of miRNAs in patients with acute myocardial infarction were associated with impaired LV contractility,27
remodel-ing28,29 and risk of (cardiovascular) death or heart failure.28,30-32
Qiang et al. measured miRNAs in endothelial progenitor cells (derived from mono-nuclear cells from the circulation) in 106 heart failure patients and found that low levels of miR-126 were associated with cardiovascular death in ischaemic heart failure Table 2. Circulating microRNAs (miRNAs) associated with the prognosis and response to therapy in heart failure patients
miRNA Regulation in heart failure Endpoint References
Prognosis Compared with
controls
miR-18a-5p Decreased 180 day all-cause mortality 22
miR-126 Increased Cardiovascular death after 2 years 33
miR-508-5p Increased Cardiovascular death after 2 years 33
miR-652-3p Decreased 180-day all-cause mortality 22
Response to therapy Compared with
controls
After successful therapy
miR-1 Increased Decreased 3 months after LVAD 15
miR-26b-5p Decreased Increased 12 months after CRT 35
miR-29a-3p Decreased Increased
miR-30d Increased Decreased 6 months after CRT 36
miR-30e-5p Decreased Increased 12 months after CRT 35
miR-92a-3p Decreased Increased
miR-145-5p Decreased Increased
miR-208a/208b Increased Decreased 3 months after LVAD 15
miR-483-3p Increased Decreased 3, 6, 9 and 12 months after LVAD,
change in NT-proBNP
34
miR-499 Increased Decreased 3 months after LVAD 15
miR-1202 Increased Decreased 3 months after LVAD, change in
NT-proBNP
34
CRT; cardiac resynchronization therapy, LVAD, left ventricular assist device; NT-proBNP, N-terminal pro-brain natriuretic peptide.
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patients, while high levels of miR-508a-5p were associated with cardiovascular death in non-ischaemic heart failure.33 We recently found that decreases in 18a-5p and
miR-652-3p during a hospitalization for heart failure was predictive for 180-day mortality.22
MicroRNAs as biomarkers for response to therapy
Patients with severe end-stage heart failure who receive a left ventricular assist device (LVAD), undergo a sudden unloading of the heart. Morley-Smith et al. showed that circulating and myocardial miR-483-3p was increased in patients after LVAD support. Interestingly, levels of circulating miR-1202 before LVAD implantation were able to distinguish responders from non-responders.34 Another study also showed a change in
several circulating miRNA levels after LVAD implantation, with a decreased levels of the myomirs miR-208a/208b, miR-499 and miR-1 after 3 months.15
In response to cardiac resynchronization therapy (CRT), Marfella et al. found five miRNAs (miR-26b-5p, miR-145-5p, miR-92a-3p, miR-30e-5p and miR-29a-3p) in higher levels in the circulation of responding patients, compared with non-responders.35 In a
recent study comprising 61 patients receiving CRT, higher levels of miR-30d were found in responders to CRT therapy (LVEF increase of >10%) after 6 months.36
An animal study identified increased levels of miR-16, miR-20b, miR-93, miR-106b, miR-223 and miR-423-5p in plasma from rats with hypertension-induced heart failure compared with controls. After treatment with an antimir for miR-208a and/or an angiotensin-converting enzyme (ACE) inhibitor, these miRNAs (except for miR-19b) normalized partly or completely after 8 weeks, suggesting that circulating miRNAs can react in a dynamic way in response to therapy, potentially indicating efficacy of treat-ment.37
Taken together, several studies have shown the prognostic capacities of circulating miRNAs in heart failure (Table 2) and indicated a potential role for predicting response to LVAD, CRT and pharmacological therapy. Although these studies need to be validated in larger independent cohorts, this potential role of circulating miRNAs is of interest and might lead to a more individualized approach to treating patients with heart failure.
Limitations of circulating microRNA studies and future considerations
However, the present studies do not yet provide sufficient evidence for a clinical use of miRNAs as biomarkers in heart failure. First, not all studies started with a large miRNA panel screen in order to select the most differentially expressed miRNAs. An unbiased approach is needed to identify the miRNAs of interest for further testing in extended cohorts. Second, it is important to compare the predictive diagnostic and prognostic value of the miRNAs to established heart failure biomarkers such as NT-proBNP and/ or BNP to assess the individual predictive value and determine the additional value of the miRNA on top of natriuretic peptides. Some studies indeed report on miRNAs which
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(in combination with natriuretic peptides) outperform NT-proBNP or BNP alone in discriminating heart failure patients from non-heart failure patients,18,20 but the lack of
validation of their diagnostic predictive value in other, independent cohorts make these results difficult to interpret. Third, most studies have relatively small patient numbers, which decreases statistical power, therefore larger studies should be conducted to verify the diagnostic and prognostic potential of miRNAs.
The lack of consistency in the available studies regarding the most differential circu-lating miRNAs in heart failure is intriguing. Several factors may be contributing to these differences. Anticoagulants in blood collection tubes can be of influence, since heparin has been shown to cause difficulties in polymerase chain reaction (PCR) amplification, in contrast to ethylenediaminetetraacetic acid (EDTA) or citrate collection tubes.38,39
Moreover, the material analysed (whole blood, serum or plasma) may contain differ-ent miRNA levels, as was demonstrated by several groups.10,40,41 Further, the presence
of blood cells in plasma and serum may contribute to higher levels of certain miRNAs, therefore appropriate plasma handling (e.g. centrifuging steps) is crucial. As there is currently no gold standard for measuring circulating miRNAs, variability due to the isolation protocol is therefore a plausible explanation. Among the available techniques to measure miRNAs in plasma such as microarrays, RNA sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR), qRT-PCR is most commonly used. Several companies have developed qRT-PCR kits to measure miRNAs in blood, plasma and other body fluids, in which differences in the miRNA extraction techniques may lead to different findings.42 Furthermore, different methods of internal
normaliza-tion with housekeeping or reference genes were reported, although to date no global and standardized method has been proposed. Exogenous, synthetic miRNAs are com-monly used as stable reference miRNAs to normalize the PCR data obtained from the miRNAs of interest. For example, synthetic Caenorhabditis elegans-derived miRNAs can be spiked-in before miRNA extraction to control for sample quality and extraction efficiency. Endogenous miRNAs and other small non-coding RNAs as data normalizers require stable expression levels under various conditions in the cohorts investigated. Owing to variability in clinical characteristics and comorbidities, well-performing en-dogenous references in certain patient populations might not be suitable for miRNA profiling studies in other patient cohorts. Therefore, general accepted standardized protocols, techniques and a well-performing normalization method are needed before clinical use of miRNAs as biomarkers can be considered.
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CiRCuLATiNG MiCRoRNAs ANd THEiR PoTENTiAL RoLE ANd fuNCTioN
Although there is increasing interest in circulating miRNAs in heart failure, there are still major uncertainties about their origin and function in the circulation. Some speculate that in heart failure, cells die and release miRNAs into the circulation, which could lead to higher levels of circulating miRNAs compared with controls. However, lower miRNA levels are frequently found in the circulation of patients with heart failure, arguing against this speculation. This might be explained by the possibility that cells take up miRNAs from the circulation to restore deleterious intracellular mechanisms related to the progression of heart failure. On the other hand, it is also likely that several deregu-lated circulating miRNAs in heart failure might not even come directly from the heart. Therefore, before using miRNAs as circulating biomarkers, it is highly desirable to un-derstand 1) how these miRNAs are released into the circulation, 2) if they play an active role in the circulation and 3) whether these circulating miRNAs reflect tissue levels.
How are microRNAs released into the circulation?
Primary miRNAs are transcribed in the nucleus after which several processing steps fol-low (Figure 1). The mature miRNA is formed after processing by Dicer in the cytoplasm of the cell. In the circulation, miRNAs were discovered in various ways; in conjunction with exosomes and other microvesicles,43 apoptotic bodies,44 HDL particles45 and other
RNA-binding proteins.46 The exact mechanisms underpinning release of miRNAs into the
extracellular space are not known, although some mechanisms involving extracellular vesicles have been described. Precursor and mature miRNAs packaged into microvesicles can leave the cells by blebbing of the plasma membrane. Exosomes containing miRNAs can be formed in the cell after which they are transported to the cell membrane and released into the extracellular compartments and circulation. The ceramide-dependent pathway, which is under the influence of neutral sphingomyelinase 2 (nSMase2), has been described to be involved in the release of miRNAs via exomes outside the cell.47
The precise sorting mechanisms that determine when and how miRNAs are selected to leave the cell have not yet been established. However, the membrane-associated RNA-induced silencing complex (RISC) was found to regulate miRNA loading into exo-somes.48 Further, the heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) has
been reported to recognize miRNAs by sequence motifs which then control the loading of miRNAs into exosomes.49 RNA-binding proteins such as Argonaut 2 (Ago2),
nucleo-phosmin 1 (NPM1) and high-density lipoprotein (HDL) are also capable of transporting miRNAs outside the cell, although the exact mechanisms are still unclear.45,46
MicroRNAs in heart failure 33
2
Ago Cytoplasm Cytoplasm Extracellular space Nucleus pre-miRNA mature miRNA micro-vesicles apoptotic bodies miRNA-protein complexes helicase mRNA HDL particles Ago RISC exosomes pri-miRNA DNA Drosha Dicer DGR8 Ago2 Ago2 Ago2figure 1. Mechanisms of microRNA (miRNA) processing and transportation. Primary miRNAs (pri-miRNAs) are transcribed from DNA, after which Drosha and DiGeorge syndrome chromosomal region 8 (DGR8) cleave the pri-miRNAs into precursor hairpin miRNAs (pre-miRNAs). Outside the nucleus, the pre-miRNAs are fur-ther processed by Dicer, which results in the loss of the hairpin structure and the formation of the mature miRNA. MiRNAs can act directly on mRNA targets in the cytoplasm or are released into the extracellular space and circulation by the shedding of exosomes and other microvesicles. Recipient cells can engulf the exosomes and the microvesicles can fuse with the recipient cell membrane to establish intracellular com-munication. Other carriers of miRNAs in the circulation are high-density lipoprotein (HDL) particles, apop-totic bodies and miRNA-protein complexes, mostly with Argonaute2 (Ago2). In the recipient cell, miRNAs can be cleaved by helicase after which an Ago protein binds to the active miRNA strand. The miRNA-Ago complex will be loaded into a RNA-induced silencing complex (RISC) which can bind to the target mRNA. With perfect complementary sequences, the mRNA transcript will degrade, while with incomplete binding the mRNA will be repressed translationally.
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do microRNAs play an active role in the circulation?
An increasing number of studies have shown that miRNAs can be secreted into the circulation by one cell and taken up by another cell, suggesting a role in cell-to-cell communication. Exosomes, microvesicles and other extracellular vesicles can transfer miRNAs to recipient cells by fusion with the recipient cell membrane (Figure 1). In some cases, the recipient cells contain higher levels of specific miRNAs than the cells from which the exosomes originated, suggesting a process that actively stimulates specific miRNA transfer via exosomes. Interestingly, protein synthesis in recipient cells was found to change, suggesting a regulating role of miRNAs in these cells.50-52 Heart
failure-related research on miRNA communication through exosomes was conducted by Bang et al.53 They found that cardiac fibroblasts excreted exosomes containing several
miRNA passenger strands, of which miR-21-3p was able to induce hypertrophy in recipi-ent cardiomyocytes.
Although increasing evidence has emerged regarding the mechanistic processes of circulating miRNAs in extracellular vesicles, several critical notes can be placed. It is proposed that in order to exert an effect in a cell, a substantial amount of miRNA should be transferred, which means numerous exosomes should fuse with the recipient cell in order to obtain miRNA levels that can regulate protein synthesis.54,55 As exosomes
also carry other particles within their membranes, the change in gene expression of the target cell might not exclusively be related to miRNA function.56-58 Furthermore, because
these studies are conducted in a controlled, experimental setting, this might not ac-curately reflect the (patho)physiology of miRNA transport in the human body.59
Other extracellular vesicles reported as transporting miRNAs are apoptotic bodies; vesicles derived from apoptotic cells. Zernecke et al. investigated miRNAs in apoptotic bodies derived from endothelial cells in atherosclerosis and found a set of miRNAs in apoptotic bodies similar to those found in their cells of origin, suggesting a paracrine function.44 MiR-126 was enriched in these apoptotic bodies and was found to function
as regulator of vascular endothelial growth factor (VEGF).
High-density lipoproteins are also able to transport miRNAs between cells. Vickers et al. found miRNAs bound to HDL particles which are not found within exosomes, suggest-ing that the mode of export of miRNAs might be specific to cell types.45 Furthermore,
HDL particles from patients with hypercholesterolemia contained increased levels of miR-105, compared with healthy subjects. Adding these miR-105 enriched particles to cultured hepatocytes led to a change in gene expression of multiple genes, mostly puta-tive targets of miR-105.
Although the transport of miRNAs within extracellular vesicles and HDL is being increasingly studied, it is believed that the majority of the miRNAs in the circulation (95-99%) are transported as miRNA-protein complexes, mostly bound to Argonaute (Ago) proteins, protecting them from degradation by RNases in the circulation.60-62 It is
hy-MicroRNAs in heart failure
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pothesized that these miRNA-Ago complexes are byproducts of cell death, which makes potential paracrine miRNA signaling less convincing.60,61 However, the exact transport
of miRNA-protein complexes is largely unknown and it is unclear whether cells are able to engulf these miRNAs from the circulation in order to function as post-transcriptional regulators in recipient cells. Thus, more extensive research especially involving these miRNA-protein complexes would be valuable.
Are circulating microRNA levels reflecting tissue levels?
In heart failure, similar responses in miRNA levels in the circulation and in myocardial tissue have been observed. For example, Tijsen et al. found higher levels of miR-423-5p in both the circulation of heart failure patients and in post-mortem cardiac tissue of patients with dilated cardiomyopathy.19 To determine whether differentially expressed
circulating miRNAs in heart failure might be derived from the heart, several groups in-vestigated the transcoronary gradients of miRNA levels. The transcoronary gradient of miR-423-5p was found to be higher in heart failure patients than in controls, suggesting that this miRNA may be predominantly derived from the heart.63 In contrast,
miR-423-5p levels did not differ in the femoral arteries, veins and coronary sinus between the two groups, which may be due to the influence of pharmacotherapy on miRNA levels or the small population size. In patients with an acute coronary syndrome, De Rosa et al. showed increased transcoronary concentration gradients of miR-133a and miR-499, which suggests that these miRNAs were likely released by the heart.64 Further, these
miRNA levels correlated with myocardial injury biomarker high-sensitivity troponin T. A recent study in patients receiving CRT reported significantly increased miR-30d levels in coronary sinus blood compared to peripheral blood, suggesting a cardiac origin of this miRNA.36
However, the current concept is that blood cells are the major contributors to the circulating miRNA pool, which was demonstrated by Pritchard et al.40 They showed that
different types of blood cells are the main origin of previously described circulating miR-NAs in cancer. Akat et al. showed that the differences in miRNA expression in myocardial tissue in severe heart failure patients compared with healthy controls are, in general, not reflected in circulating miRNA levels, suggesting that the heart may be an unlikely origin of the most abundant circulating miRNAs.15 Moreover, most circulating miRNAs in
this study were highly abundant in haemopoietic cells and endothelial cells; only 0.1% consisted of muscle and cardiac-specific miRNAs. However, these circulating myomirs changed comparably with the miRNA expression in myocardial tissue, which confirms that the origin of these cardiac-specific miRNAs probably lies in the heart. Higher levels of these circulating myomirs were found in severe heart failure patients compared with controls, suggesting these miRNAs could still be potential biomarker candidates in heart failure, despite their low abundance in the circulating miRNA pool. These results
36 Chapter 2
indicate that although certain circulating miRNAs such as the myomirs can be tissue specific, the majority of the abundant circulating miRNAs originate from blood or endo-thelial cells. Consequently, the search for cardiac-derived circulating miRNAs as novel biomarkers in heart failure might be challenging because of their low concentrations in the circulation. However, we hypothesize that the most differentially expressed circu-lating miRNAs in heart failure may be a consequence of a systemic (vascular) response to an overloaded heart along with an impaired perfusion of multiple organs, which may lead to a differential miRNA response. These miRNAs may therefore serve as potential biomarkers in heart failure.
THERAPEuTiC MiCRoRNA-BAsEd sTRATEGiEs iN HEART fAiLuRE
The discovery of circulating miRNAs has opened windows for novel drug development by administration of extracellular miRNAs. Several studies have shed light on the role of miRNAs in the maladaptive processes involved in heart failure, such as hypertrophy and fibrosis. Here, we discuss the most intensely studied miRNAs in cardiac hypertro-phy and fibrosis and the potential of miRNA-based therapies to inhibit or reverse these processes. Further, we elaborate on the difficulties in moving forward towards clinical application of miRNA mimics and antimirs in heart failure patients.
MicroRNA mimics and antimirs
In general, low expression levels of miRNAs can be restored with miRNA mimics, which are synthetic double-stranded oligonucleotides resembling precursor miRNAs. In target cells they are cleaved into functional single-stranded miRNAs where they are able to bind to the 3’ untranslated region of the mRNA, leading to unsuccessful translation into proteins. MiRNA mimics can be administered exogenously using adeno-associated viruses (AAVs), subcutaneously and directly into the circulation. MiRNA mimics have to undergo the same processes as double-stranded pre-miRNA, therefore chemical modi-fications to optimize their specific delivery, uptake in the recipient cell and regulating function are more challenging for miRNA mimics than for antimirs.65
Antimirs are single-stranded antisense oligonucleotide molecules with varying chemical modifications to resist ectonucleases and endonucleases. They directly block the miRNA function by binding to single-stranded mature miRNAs thus preventing their binding to target mRNAs. Antimirs can be administered intravenously, subcutaneously and in the intraperitoneal space. In the circulation, antimirs remain stable owing to chemical modification processes.66,67 Enhanced uptake by target cells has been achieved
by adding cholesterol particles to the antimirs.67,68 Other modification processes to
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(LNA) modifications, non-nucleotide ZEN (N,N-diethyl-4(4-nitronaphthalen-1-ylazo)-phenylamine) modifications, conjugation to N-acetylgalactosamine sugars and the use of miRNA sponges have been recently reviewed by Philippen et al.69 The majority
of antimirs can be taken up into tissue within hours and animal models have shown that antimirs remain stable in the intracellular space for months.70 However, functional
effects of the antimirs may be expected only after several days or weeks.71
Although, to date, no clinical trials with antimirs or miRNA mimics in the cardiovas-cular field are ongoing, pre-clinical trials have been conducted using miRNA-based therapies in animal heart failure models.
Hypertrophy
A variety of miRNAs have been associated with cardiac hypertrophy, of which miR-1 has been described as one of the key regulating miRNAs in this process (Figure 2). MiR-1 is highly abundant in the heart and deletion of this miRNA causes serious cardiac de-fects.1,72,73 Sayed et al. demonstrated that in hearts of mice undergoing transverse aortic
constriction (TAC), miR-1 is downregulated even before development of hypertrophy.74
Elia et al. identified insulin-like growth factor-1 (IGF-1) and IGF-1 receptor as targets of miR-1, controlling cell growth and differentiation.75 Further, twinfilin-1, a cytoskeleton
regulatory protein, was also identified as miR-1 target.76 The heart-type fatty
acid-binding protein-3 (FABP3) was also found to be targeted by miR-1, and plasma FABP3 levels functioned as an indirect biomarker of miR-1 expression.77
The use of an antimir for miR-1 induced cardiac hypertrophy in neonatal rat ventricu-lar cardiomyocytes, while overexpression of miR-1 was capable of inhibiting cardiac hypertrophy by regulating calmodulin-encoding genes and genes implicated in calcium handling mechanisms.78 Interestingly, intravenous administration of a miRNA mimic
using an AAV expressing miR-1 in rats with left ventricular hypertrophy induced by pres-sure overload resulted in regression of cardiac hypertrophy, reduction of fibrosis and apoptosis, and improved calcium signaling.79 These results suggest that miR-1 might
play a role in the development of hypertrophy and that this process may be reversed with a miR-1 mimic.
The myomir miR-133 exists within the same transcriptional unit as miR-1 and is also highly abundant in the myocardium. In both animal and human models, miR-133 was identified as regulator of cardiac hypertrophy, with lower levels in heart failure and car-diac hypertrophy compared with controls (Figure 2).73,76,80-82 The prohypertrophic
phos-phatase calcineurin was found to regulate this miRNA, resulting in a parallel increase of calcineurin and decrease of miR-133 in both in vivo and in vitro cardiac hypertrophy.82
This miRNA also plays an important role in β-adrenergic receptor signaling with its ability to target multiple effectors of this pathway.83 In vitro and in vivo overexpression
38 Chapter 2 Figure-2 460pts X 219pts 3rd Proof December 21, 2015 IGF -1 + IGF r ↑ ↓ incr
eased cell siz
e Akt ↑ Spr y1 ↓ → Er k/MAP kinase activit y ↑ Fibr osis Hyper tr oph y PTEN ↓ Smad7 ↓ TGF -β III ↓ ↓ TGF -β1 ↑ TGF -β1 ↑ TGF -β /Smad signaling ↑ TGF -β /Smad 3 signaling ↑ LIF ↑ IGF -1 ↑ PTX-3 ↑ NF AT ↑ ← calcineur in ↑ RhoA ↑ Cdc42 ↑ NelfA /WHSC2 ↑ calmodulin ↑ Gat a4 ↑ Mef2a ↑ cyt osk elet on regulat or y pr ot ein twinfi lin-1 ↑ β1 AR signal tr ansduction ↑ ↓ apopt osis COL1A1 ↑ COL1A2 ↑ COL3A1 ↑ FBN1 ↑ ELN ↑ miR-29 ↓ Akt ↑ MMP -2 ↑ miR-1 33 ↓ miR-1 ↓ miR-21 ↑ figur e 2. Micr oRNAs with ke y roles in car diac fibr osis and hypertr ophy in response to car diac injur y or overlo ad. MiR -1, miR -21, miR -29 and miR -133 ar e pr esent ed with their known tar ge ts. The expr ession of miR -1, miR -29 and miR -133 is downr egulat ed in car diac tissue in response to car diac injur y or overlo ad, le ading to a decr eased ne gative re gulation of their mRNA tar ge ts. MiR -21 is upr egulat ed in response to car diac injur y or overlo ad, resulting in an incr eased ne gative re gulation of the corr e-sponding tar ge ts. This in turn activ at es pathw ays contributing to car diac fibr osis and hypertr ophy . β 1AR, be ta-1 adr ener gic rec ept or; Cdc42, cell division contr ol pr o-tein 42 homolog; COL1A1, collag en type I, alpha 1; COL1A2, collag en type I, alpha 2; COL3A1, collag en type III, alpha 1; ELN, elastin; ERK, extr ac ellular signal-r egulat ed kinase; FBN1, fibrillin 1; Gat a4, GA TA binding pr ot ein 4; IGF-1; insulin-lik e gr owth fact or -1; IGF r, insulin-lik e gr owth fact or rec ept or; LIF , leuk emia inhibit or y fact or; MAP , mit og en-activ at ed pr ot ein; Me f2a, myoc yt e enhanc er fact or 2a; MMP -2, matrix me tallopr ot einase -2; NelfA/WHSC2, ne gative elong ation fact or complex member A/Wolf -Hir schhorn syndr ome candidat e 2; NF AT , nucle ar fact or of activ at ed T-c ells; PTEN, phosphat ase and tensin homolog; PT X-3, pentr axin-3; RhoA, ras homolog gene family
member A; Smad 3, Smad f
amily member 3; Smad7, Smad f
amily member 7; Spr y1, spr outy homolog 1; T GF-β, tr ansf orming gr owth f act or -be ta.
