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 Epigenetics in
Cardiovascular Disease
 Translational Epigenetics Series
Trygve Tollefsbol - Series Editor
Transgenerational Epigenetics
Edited by Trygve O. Tollefsbol, 2014
Personalized Epigenetics
Edited by Trygve O. Tollefsbol, 2015
Epigenetic Technological Applications
Edited by Y. George Zheng, 2015
Epigenetic Cancer Therapy
Edited by Steven G. Gray, 2015
DNA Methylation and Complex Human Disease
By Michel Neidhart, 2015
Epigenomics in Health and Disease
Edited by Mario F. Fraga and Agustin F. F Fernández, 2015
Epigenetic Gene Expression and Regulation
Edited by Suming Huang, Michael Litt and C. Ann
Blakey, 2015
Epigenetic Biomarkers and Diagnostics
Edited by Jose Luis Garcı́a-Gim
enez, 2015
Drug Discovery in Cancer Epigenetics
Edited by Gerda Egger and Paola Barbara
Arimondo, 2015
Medical Epigenetics
Edited by Trygve O. Tollefsbol, 2016
Chromatin Signaling and Diseases
Edited by Olivier Binda and Martin Fernandez-Zapico, 2016
Genome Stability
Edited by Igor Kovalchuk and Olga Kovalchuk, 2016
Chromatin Regulation and Dynamics
Edited by Anita G€
ond€or, 2016
Neuropsychiatric Disorders and Epigenetics
Edited by Dag H. Yasui, Jacob Peedicayil and Dennis R.
Grayson, 2016
Polycomb Group Proteins
Edited by Vincenzo Pirrotta, 2016
Epigenetics and Systems Biology
Edited by Leonie Ringrose, 2017
Cancer and Noncoding RNAs
Edited by Jayprokas Chakrabarti and Sanga Mitra, 2017
Nuclear Architecture and Dynamics
Edited by Christophe Lavelle and Jean-Marc Victor, 2017
Epigenetic Mechanisms in Cancer
Edited by Sabita Saldanha, 2017
Epigenetics of Aging and Longevity
Edited by Alexey Moskalev and Alexander
M. Vaiserman, 2017
The Epigenetics of Autoimmunity
Edited by Rongxin Zhang, 2018
Epigenetics in Human Disease, Second Edition
Edited by Trygve O. Tollefsbol, 2018
Epigenetics of Chronic Pain
Edited by Guang Bai and Ke Ren, 2018
Epigenetics of Cancer Prevention
Edited by Anupam Bishayee and Deepak Bhatia, 2018
Computational Epigenetics and Diseases
Edited by Loo Keat Wei, 2019
Pharmacoepigenetics
Edited by Ramón Cacabelos, 2019
Epigenetics and Regeneration
Edited by Daniela Palacios, 2019
Chromatin Signaling and Neurological Disorders
Edited by Olivier Binda, 2019
Transgenerational Epigenetics, Second Edition
Edited by Trygve Tollefsbol, 2019
Nutritional Epigenomics
Edited by Bradley Ferguson, 2019
Prognostic Epigenetics
Edited by Shilpy Sharma, 2019
Epigenetics of the Immune System
Edited by Dieter Kabelitz, 2020
Stem Cell Epigenetics
Edited by Eran Meshorer and Giuseppe Testa, 2020
Epigenetics Methods
Edited by Trygve Tollefsbol, 2020
Histone Modifications in Therapy
Edited by Pedro Castelo-Branco and Carmen Jeronimo, 2020
Environmental Epigenetics in Toxicology and Public Health
Edited by Rebecca Fry, 2020
Developmental Human Behavioral Epigenetics
Edited by Livio Provenzi and Rosario Montirosso, 2020
 Translational Epigenetics
Epigenetics in
Cardiovascular Disease
Volume 24

Series Editor
Trygve Tollefsbol
Comprehensive Cancer Center, Comprehensive Center for Healthy Aging,
University of Alabama at Birmingham, Birmingham, AL, United States

Edited by
Yvan Devaux
Head, Cardiovascular Research Unit, Luxembourg Institute of Health, Strassen, Luxembourg

Emma Louise Robinson

School of Medicine, Division of Cardiology, University of Colorado Denver,
Anschutz Medical Campus, Aurora, CO, United States
CARIM School for Cardiovascular Diseases, Faculty of Health, Medicine and Life Sciences,
Maastricht University, Maastricht, The Netherlands
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Contributors
Bence Ágg
Cardiometabolic and MTA-SE System Pharmacology Research Group, Department of
Pharmacology and Pharmacotherapy, Semmelweis University, Budapest; Pharmahungary Group,
Szeged; Heart and Vascular Center, Semmelweis University, Budapest, Hungary
Parisa Aghagolzadeh
Experimental Cardiology Unit, Division of Cardiology, Department of Cardiovascular Medicine,
University of Lausanne Medical School, Lausanne, Switzerland
Chukwuemeka George Anene-Nzelu
Genome Institute of Singapore; Cardiovascular Disease Translational Research Programme,
National University Health System, National University of Singapore Yong Loo Lin School of
Medicine, Singapore, Singapore
Johannes Backs
Institute of Experimental Cardiology, University of Heidelberg; DZHK (German Centre for
Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany
Ferran Barb

e
Translational Research in Respiratory Medicine, University Hospital Arnau de Vilanova and Santa
Maria, IRB Lleida, Lleida; CIBER of Respiratory Diseases (CIBERES), Institute of Health Carlos III,
Madrid, Spain
Fay Betsou
IBBL (Integrated Biobank of Luxembourg), Dudelange, Luxembourg
Stephanie Bezzina Wettinger
Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta, Msida,
MSD2080, Malta
Andrei Codreanu
Hospital Center of Luxembourg, Strassen, Luxembourg
Yvan Devaux
Cardiovascular Research Unit, Luxembourg Institute of Health, Luxembourg, Luxembourg
Christoph Dieterich
German Center for Cardiovascular Research (DZHK)—Partner site Heidelberg/Mannheim;
Section of Bioinformatics and Systems Cardiology, Klaus Tschira Institute for Integrative
Computational Cardiology and Department of Internal Medicine III, University Hospital Heidelberg,
Heidelberg, Germany
Javier Durán
Institute of Experimental Cardiology, University of Heidelberg; DZHK (German Centre for
Cardiovascular Research), Partner Site Heidelberg/Mannheim, Heidelberg, Germany
Rosienne Farrugia
Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta, Msida,
MSD2080, Malta

xvii
xviii Contributors

Kyriacos Felekkis
Department of Life and Health Sciences, School of Sciences and Engineering, University of Nicosia,
Nicosia, Cyprus
P

eter Ferdinandy
Cardiometabolic and MTA-SE System Pharmacology Research Group, Department of
Pharmacology and Pharmacotherapy, Semmelweis University, Budapest; Pharmahungary Group,
Szeged, Hungary
Roger S-Y Foo
Genome Institute of Singapore; Cardiovascular Disease Translational Research Programme,
National University Health System, National University of Singapore Yong Loo Lin School of
Medicine, Singapore, Singapore
Eleftheria Galatou
Department of Life and Health Sciences, School of Sciences and Engineering, University of Nicosia,
Nicosia, Cyprus
David de Gonzalo-Calvo
Translational Research in Respiratory Medicine, University Hospital Arnau de Vilanova and Santa
Maria, IRB Lleida, Lleida; CIBER of Respiratory Diseases (CIBERES), Institute of Health Carlos III,
Madrid, Spain
Simona Greco
Molecular Cardiology Laboratory, IRCCS Policlinico San Donato, Milan, Italy
Johannes Grillari
TAmiRNA GmbH; Austrian Cluster for Tissue Regeneration, Medical University of Vienna; Ludwig
Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria
Hakan Gunes
Faculty of Medicine, Department of Cardiology, Kahramanmaras Sutcu Imam University,
Kahramanmaras, Turkey
Matthias Hackl
TAmiRNA GmbH; Austrian Cluster for Tissue Regeneration, Medical University of Vienna, Vienna,
Austria
Nazha Hamdani
Department of Molecular and Experimental Cardiology; Department of Cardiology, St. Josef-
Hospital and Bergmannsheil; Department of Clinical Pharmacology; Institute of Physiology, Ruhr
University Bochum, Bochum, Germany
Lutz Hein
Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine; BIOSS
Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany
Carlos Hermenegildo
Department of Physiology, Faculty of Medicine and Dentistry, University of Valencia, and INCLIVA
Biomedical Research Institute, Valencia, Spain
Eduardo Iglesias-Guti
errez
Department of Functional Biology, Physiology, University of Oviedo; Health Research Institute of
the Principality of Asturias (ISPA), Oviedo, Spain
 Contributors xix

Benedetta Izzi
Department of Epidemiology and Prevention, IRCCS NEUROMED, Pozzilli, IS, Italy
Kornelia Jaquet
Department of Molecular and Experimental Cardiology; Department of Cardiology, St. Josef-
Hospital and Bergmannsheil; Department of Clinical Pharmacology, Ruhr University Bochum,
Bochum, Germany
Amela Jusic
Department of Biology, Faculty of Natural Sciences and Mathematics, University of Tuzla, Tuzla,
Bosnia and Herzegovina; Cardiovascular Research Unit, Luxembourg Institute of Health,
Luxembourg, Luxembourg
Kanita Karad-uzovi

c-Hadžiabdi

c
Department of Engineering, International University of Sarajevo, Sarajevo, Bosnia and Herzegovina
Gabriela M. Kuster
Department of Biomedicine, University Hospital Basel and University of Basel; Department of
Cardiology, University Hospital Basel, Basel, Switzerland
Alisia Madè
Molecular Cardiology Laboratory, IRCCS Policlinico San Donato, Milan, Italy
Federica De Majo
Department of Molecular Genetics, Faculty of Science and Engineering; CARIM School for
Cardiovascular Diseases, Faculty of Health, Medicine and Life Sciences; Maastricht University,
Maastricht, The Netherlands
Fabio Martelli
Molecular Cardiology Laboratory, IRCCS Policlinico San Donato, Milan, Italy
Andreas M€ugge
Department of Molecular and Experimental Cardiology; Department of Cardiology, St. Josef-
Hospital and Bergmannsheil, Ruhr University Bochum, Bochum, Germany
Vivien Ngo
Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University
of Freiburg, Freiburg, Germany
Susana Novella
Department of Physiology, Faculty of Medicine and Dentistry, University of Valencia, and INCLIVA
Biomedical Research Institute, Valencia, Spain
Ana Bel

en Paes
Department of Physiology, Faculty of Medicine and Dentistry, University of Valencia, and INCLIVA
Biomedical Research Institute, Valencia, Spain
Christos Papaneophytou
Department of Life and Health Sciences, School of Sciences and Engineering, University of Nicosia,
Nicosia, Cyprus
Thierry Pedrazzini
Experimental Cardiology Unit, Division of Cardiology, Department of Cardiovascular Medicine,
University of Lausanne Medical School, Lausanne, Switzerland
xx Contributors

Antje Peters
€nster, Mu
Department of Genetic Epidemiology, Institute of Human Genetics, University of Mu €nster,
Germany
Lucı́a Pinilla
Translational Research in Respiratory Medicine, University Hospital Arnau de Vilanova and Santa
Maria, IRB Lleida, Lleida; CIBER of Respiratory Diseases (CIBERES), Institute of Health Carlos III,
Madrid, Spain
Emma Louise Robinson
School of Medicine, Division of Cardiology, University of Colorado Denver, Anschutz Medical
Campus, Aurora, CO, United States
Elisabeth Semmelrock
TAmiRNA GmbH, Vienna, Austria
Justus Stenzig
Department of Experimental Pharmacology and Toxicology, University Medical Centre Hamburg-
Eppendorf, Hamburg, Germany
Maarten Vanhaverbeke
Cardiovascular Medicine, University Hospitals Leuven, Leuven, Belgium
Mirko V€
olkers
German Center for Cardiovascular Research (DZHK)—Partner site Heidelberg/Mannheim;
Department of Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany
Leon J. De Windt
Department of Molecular Genetics, Faculty of Science and Engineering; CARIM School for
Cardiovascular Diseases, Faculty of Health, Medicine and Life Sciences; Maastricht University,
Maastricht, The Netherlands
Johannes Winkler
Department of Cardiology, Medical University of Vienna, Vienna, Austria
Angela Xuereb Anastasi
Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta, Msida,
MSD2080, Malta
Mehmet Birhan Yilmaz
Faculty of Medicine, Department of Cardiology, Dokuz Eylul University, Izmir, Turkey
Preface

Yvan Devauxa and Emma Louise Robinsonb,c

Cardiovascular Research Unit, Luxembourg Institute of Health, Strassen, Luxembourga School of Medicine, Division of
Cardiology, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, United Statesb CARIM School for
Cardiovascular Diseases, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht,
The Netherlandsc

On behalf of the EU-CardioRNA COST Action CA17129.

The burden of cardiovascular disease

Cardiovascular disease (CVD) remains a major cause of disability and death worldwide. According to
the 2019 statistics of the World Health Organization (WHO), 17.9 million people die each year from
CVD, which represents almost a third of all deaths globally.1 Despite significant improvements in
health care, CVD continues to represent a major socioeconomic burden. Risk factors for CVD are either
modifiable, such as tobacco use, physical inactivity, excessive food intake, or acquired. Genetic pre-
dispositions to CVD have been extensively investigated, yet most large association studies remain at
the DNA (genomic) level, searching for associations between nucleotide polymorphisms and the risk of
developing a CVD or having a poor clinical outcome after an acute cardiovascular event (e.g., wors-
ening of an acute condition, developing comorbidities affecting other organs, or death). More recently,
a better knowledge of the mechanisms regulating gene and protein expression allowed a diversification
of these association studies, now focusing also on modifications occurring at the DNA, chromatin, and
RNA levels. Those modifications not altering nucleic acid sequences have been grouped under the term
“epigenetics,” derived from the Greek “epi” meaning “over, around, on top.” It is now accepted that
epigenetic modifications play a major role in regulating cardiovascular homeostasis, CVD
development, and progression.

Central dogma of molecular biology

Cellular and tissue structure, morphology, biochemistry, and function are determined by the profile of
proteins and RNA molecules present. The original central dogma of molecular biology—as defined by
Sir Francis Crick in 1957—states that DNA is copied into RNA in a process called transcription, with
RNA then being exported from the nucleus for protein synthesis at the ribosomes, in a process called
translation (Fig. 1). However, we now know that some RNA molecules are functional as RNA mol-
ecules in their own right and are not used to encode proteins, accounting for at least 80% of the tran-
scribed genome.2 This family of RNAs is known as noncoding RNAs (ncRNAs). The expression of
protein-coding RNAs (messenger RNAs) and ncRNAs is determined by the rate of transcription.
The transcription is under the control of epigenetic mechanisms. Epigenetic mechanisms involve

xxi
xxii Preface

FIG. 1
An illustration of the central dogma of molecular biology, the flow of biochemical information from DNA to RNA
and protein.
Adapted from Genome Research Limited.
 Preface xxiii

chemical modification either to the DNA or RNA bases themselves or to the proteins (histones) that
package DNA into chromatin.
Epigenetic mechanisms include:

– DNA cytosine methylation and its derivatives,

– Covalent modifications on amino acids of histones,
– Noncoding RNAs that mediate chromatin structure and regulate other epigenetic modifiers,
– Higher-order chromatin structure and scaffolding, and
– Covalent modifications on RNA nucleotides (known specifically as the epitranscriptome).

These epigenetic modifications act in concert with each other to determine the protein and RNA pro-
files at any given point in time.3

Epigenetic mechanisms
Epigenetic mechanisms regulate differentiation, development, homeostasis, aging, and disease. Epige-
netic marks are laid down in differentiation and development to program the transcriptome from plur-
ipotency to determine cell fate and define the differentiated cell state. Both in a replication-dependent
as well as replication-independent manner, the epigenetic landscape is not static (unlike the genome)
but is dynamic throughout replicative and chronological aging. Importantly, the epigenome is respon-
sive to environmental cues such as redox and metabolic or neurohumoral signaling. Wholesale remo-
deling of DNA methylation, histone modification, noncoding RNA, and RNA modification profiles has
been described in the heart and vasculature in CVD.4–7
Epigenetic marks (DNA, RNA, and chromatin modifications) are mediated by epigenetic modi-
fiers. Epigenetic modifiers can be further described according to their function—epigenetic writers,
erasers, and readers. These are also dynamically regulated in disease processes. The addition or re-
moval of epigenetic marks, accompanied by binding of epigenetic readers and ncRNAs, in turn reg-
ulates gene expression. It does so by altering the chromatin structure at a particular location in the
genome; affecting the accessibility of enhancers, promoters, and gene bodies to transcription factors
and RNA polymerases; as well as creating platforms for long-distance regulatory genomic interactions.
In the case of the epitranscriptome, RNA stability, localization, and function can be modulated.
This book addresses the current knowledge and understanding of how epigenetic modifications and
modifiers regulate gene expression and different biological processes in the initiation and progression
of CVD in the heart and vasculature. It also evaluates the use of epigenetic marks in diagnostic and
epigenetic modifiers emerging as therapeutic targets.

Epigenetic mechanisms as biomarkers and treatment targets in CVD

Biomarkers are molecules that can be easily and quantitatively measured in biological samples and
give information on the presence (diagnostic biomarkers) of or evolution (prognostic biomarkers)
of diseases. Traditionally, most biomarkers have been searched for in the bloodstream and belong
xxiv Preface

to the family of proteins. As an example, troponins—contractile proteins contained in cardiomyocytes,

the cells responsible for heart contractility—are used as diagnostic biomarkers of acute myocardial
infarction, since they are released in the blood upon the damage of the cardiac tissue following rupture
of blood supply due to obstruction of a coronary artery (also known as heart attack or myocardial in-
farction). Diversification of biomarker studies led to the discovery that not only proteins (or peptides)
can be used as biomarkers, but also changes affecting epigenetic mechanisms that can be quantified in
biological fluids or tissue biopsies. Noncoding RNAs for instance, many of which can be found in the
bloodstream, are emerging as potential novel biomarkers of CVD. The most widely investigated have
been short 21–25 nucleotide ncRNAs, called microRNAs (miRNAs), and their potential to help in per-
sonalizing health care has been proposed.8 Other types of epigenetic mechanisms will also be addressed
in this book, for both their biomarker value and their therapeutic potential. Indeed, since biomarkers
reflect disease progression, they may also have therapeutic potential, as recently discussed.9
The use of epigenetic mechanisms to design novel drugs to treat patients, almost as their use as
biomarkers, is only in its infancy, and further work and studies are needed to fully address their po-
tential to be used in precision medicine. Hopes and limitations of the use of epigenetic mechanisms
to help in implementing personalized medicine in the cardiovascular field will be discussed in
this book.

The EU-CardioRNA COST Action: networking to advance science

Networking and sharing of complementary expertise is essential in biomedical research. Only syner-
gistic multicenter investigations have the potential to decipher the mechanisms of complex and mul-
tifactorial diseases such as CVD. In this context, researchers from different horizons teamed up in a
networking initiative funded by COST. COST (European Cooperation in Science and Technology;
https://www.cost.eu) is a H2020-funded organization for research and innovation networks. COST
networking tools—known as Actions—are research initiatives across Europe and beyond. They help
researchers to grow their ideas in any science and technology field, such as biomedical research. The
EU-CardioRNA COST Action CA17129 (https://cardiorna.eu) started in October 2018 for a duration
of four years. The main goal of this Action is to catalyze the research on the role of transcriptomics in
CVD through networking activities and collaborative exchange of expertise. The Action aims at a bet-
ter knowledge of how epigenetics and more precisely regulatory RNA molecules affect CVD devel-
opment.10 Ultimately, this gain of knowledge is expected to allow the development of novel
biomarkers and drugs to improve health care of CVD patients. The Action organizes yearly meetings
to discuss the most recent scientific signs of progress in the field. Through the funding of Short-Term
Scientific Missions (STSMs) between partner laboratories, the Action aims to favor staff mobility,
technology transfer, and the setup of synergistic multicenter research projects. The Action provides
Inclusiveness Target Country (ITC) grants to help young investigators from less research-intensive
countries to attend scientific conferences and discuss their data with their peers. Dissemination activ-
ities as well as scientific communication outreach activities are key components of the Action. An over-
all presentation of EU-CardioRNA’s Action goals and organization has been published.11 This book on
Epigenetics in Cardiovascular Disease is a concrete example of such dissemination activities, which
has been possible by a joint effort of more than 25 EU-CardioRNA partners from 15 countries.
 Preface xxv

Presentation of the book and chapters

EU-CardioRNA COST Action members have designed this book tackling the emerging and complex
topic of the role of epigenetics in CVD. An introductory section will set the stage, presenting the latest
data available on the burden of CVD, a state-of-the-art overview of the different epigenetic mecha-
nisms, as well as an in-depth view of the intracellular molecular pathways regulating epigenetics. A
main section has been then assembled to gather the current knowledge of the role of epigenetic mech-
anisms in CVD. The focus will be on DNA methylation, histone modifications, post-transcriptional
RNA modifications, regulatory RNAs, splicing regulation, cardiac transcriptomic remodeling, sex dif-
ferences, and the role of epigenetics in cardiac development, and induced pluripotent stem cells. The
next section will address the potential of epigenetic mechanisms to be used as CVD biomarkers, from
the value of peripheral DNA and RNA to be translated to clinical practice, to the promise of long
ncRNAs and circular RNAs as heart failure biomarkers. Effect of physical exercise on epigenetic reg-
ulation as well as the added value of artificial intelligence in clinical decision making will be presented
and discussed. A section on therapeutic potential will present the novel strategies for modulating epi-
genetic mechanisms in CVD, as well as an overview of emerging epigenetic cardiovascular drugs. A
section will present methodological issues in RNA-sequencing and single-cell RNA-seq techniques,
will provide recommendations for best experimental practices in RNA research and biomarker devel-
opment, and will discuss the potential of bioinformatics analysis for target identification. We will fi-
nally provide our vision of the potential of epigenetic mechanisms to help in the management of CVD
patients, and we will propose directions for future work.
The epigenetics field in cardiovascular research is only in its infancy, and we expect that it will lead
to the discovery of novel drugs and biomarkers for patient’s benefit.

Acknowledgments
This article is based upon the work from EU-CardioRNA COST Action CA17129 (www.cardiorna.eu)
supported by COST (European Cooperation in Science and Technology). ELR is supported by a Dutch
Heart Foundation (Hartstichting) CVON EARLY-HFPEF-2015 consortium grant (Dutch Heart Foun-
dation) and a CVON RECONNECT Young Talent Programme award. YD is funded by the National
Research Fund (grants # C14/BM/8225223 and C17/BM/11613033), the Ministry of Higher Education
and Research, and the Fondation Coeur—Daniel Wagner of Luxembourg.

References
1. http://origin.who.int/cardiovascular_diseases/en/.
2. Azodi M, et al. The missing “lnc” between genetics and cardiac disease. Noncoding RNA. 2020;6(1).
3. Unfinished symphony j Nature [Internet]. [cited 2020 March 2019]. Available from: https://www-nature-com.
kuleuven.ezproxy.kuleuven.be/articles/441143a.
4. Berulava T, et al. Changes in m6A RNA methylation contribute to heart failure progression by modulating
translation. Eur J Heart Fail. 2020;22(1):54–66.
xxvi Preface

5. Gilsbach R, et al. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and dis-
ease. Nat Commun. 2014;5:5288.
6. Hermans-Beijnsberger S, van Bilsen M, Schroen B. Long non-coding RNAs in the failing heart and vascu-
lature. Noncoding RNA Res. 2018;3(3):118–130.
7. Kaneda R, et al. Genome-wide histone methylation profile for heart failure. Genes Cells. 2009;14(1):69–77.
8. Goretti E, Wagner DR, Devaux Y. miRNAs as biomarkers of myocardial infarction: a step forward towards
personalized medicine? Trends Mol Med. 2014;20(12):716–725.
9. Huang C-K, Kafert-Kasting S, Thum T. Preclinical and clinical development of noncoding RNA therapeutics
for cardiovascular disease. Circul Res. 2020;126(5):663–678.
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CA17129. Noncoding RNA. 2019;5.
 CHAPTER

The ever-growing burden of

cardiovascular disease
1
Mehmet Birhan Yilmaza and Hakan Gunesb
Faculty of Medicine, Department of Cardiology, Dokuz Eylul University, Izmir, Turkeya Faculty of Medicine, Department
of Cardiology, Kahramanmaras Sutcu Imam University, Kahramanmaras, Turkeyb

1.1 Introduction
Cardiovascular diseases, which comprise ischemic cardiac disease, stroke, heart failure, peripheral
artery disease, and other vascular diseases, are a leading cause of mortality and morbidity in the world
and contribute significantly to impaired quality of life. In 2017, CVD resulted in 17.8 million death and
35.6 million-year disability worldwide. Awareness of prevalence and incidence of CVD and risk
factors can provide us with the opportunity for prevention and management of the disease and can lead
to improved survival.1

1.2 Financial load of cardiovascular disease

Cardiovascular disease (CVD), as a multifaceted disease with multiple stages, is the major contributor
of early death all over the world, as about one-third of all deaths are attributable to CVD, and this mea-
sure is foreseen to rise further in this decade.2 There are at least three significant domains of CVD:
epidemiological burden, which considers frequency in overall population and productive ages; eco-
nomic burden, which constitutes direct and indirect costs; and disability burden, which accounts for
several consequences of these diseases. Cardiovascular health services constitute a significant part
of the overall health budget of member countries of the European Society of Cardiology (ESC).
The overall burden of health expenditures related to CVD can be brought under two subheadings of
fiscal and economic burden. ESC countries diverge significantly when it comes to CVD-related fiscal
burden since health systems, structural organizations, and per capita income differ between these coun-
tries significantly. According to 2016 reports, health expenditure per person in Kirghizstan was
reported to be 240 US Dollars, whereas in Switzerland, this measure reached 7900 US Dollars.2
According to the ESC Atlas report, in 2016, the ratio of current health expenditure to gross domestic
product (GDP) ranged from 3.5% to 12.2% in the ESC countries with Germany, France, and Switzer-
land leading the list and with Romania, Turkey, Egypt, and Kazakhstan at the bottom of the list by
reserving <5% of GDP on health care.2
Simply by considering economic strength and local purchasing power, health expenditures of
high-income countries quadruple that of low-income countries. The fiscal burden of CVD, evaluated
Epigenetics in Cardiovascular Disease. https://doi.org/10.1016/B978-0-12-822258-4.00008-0
Copyright # 2021 Elsevier Inc. All rights reserved.
3
4 Chapter 1 The ever-growing burden of cardiovascular disease

by the data derived from Eurostat and the Organization for Economic Cooperation and Development
(OECD), was shown to yield more than 10% of total health expenditures in 2016.2 Of note, majority
was shown to be related to hospitalizations and pharmacological therapy. In Health Expenditures by
Diseases and Conditions (HEDIC) Project, which includes several European countries in 2013, CVD
was shown to cover the highest ratio of all health expenditures, yielding almost 16% of all expen-
ditures by verifying OECD measures. The ratio of health expenditures reserved for CVD was notified
to be an important part of total expenditures although there is a decreasing trend in recent years.
When it comes to the economic burden of CVD, it was calculated to be 169 billion Euros accounting
for 62% of all health expenditures.2 In 2015, this was calculated to rise to 210 billion Euros, and of
this cost, 53% went to health costs, 26% went to productivity losses, and 21% went to off-the-record
care.2 Among healthcare costs, in-hospital care of CVD patients accounts for 51% and the medica-
tion costs account for 21%. On the contrary, the total cost of ischemic heart disease was calculated to
be approximately 59 billion Euros and this was followed up by cerebrovascular disease-related costs
of 45 billion Euros.2 In the ENH study, per capita cost for CVD was considered thoroughly and it was
shown that it was 48,000 Euro in Bulgaria and 365,000 Euro in Finland.2 In the light of these findings,
it could be stated that CVD accounts for a significant portion of healthcare expenditures of ESC
countries with regard to financial situation and economic power. Cardiovascular disease accounts
for a significant part of healthcare expenditures in the USA, quite similar to that in ESC countries.
In 2015, 102.7 million people (41.5% of the whole population) were noted to suffer from CVD and
this number is expected to rise to 131.2 million people by the year 2035. With half of its population
suffering from CVD, the USA was calculated to spend 318 billion US Dollars in 2015, and this is
expected to rise to 749 billion US Dollars by the year 2035. Cardiovascular disease has important
consequences all over the world considering individuals, healthcare systems, and population level.
World Heart Federation estimated that the 963 billion US Dollars of the total global financial burden
of CVD to rise 1044 billion US Dollars by 2030.2 Hence, it is an important subsistence as it gives
shape to epidemiological and economic future.

1.3 Risk factors and health(y) behaviors

In the Framingham Heart Study, significant predisposing factors are identified for the development
of CVD.3 These risk factors are validated in many other studies and, hence, generate frequently used
predictive models. In the INTERHEART study, it was shown that there were risk factors (Fig. 1.1)
and lifestyle habits that lead to acute myocardial infarction and elimination or control of these
risk factors was shown to yield a significant decrease in cardiovascular events.4 World Health
Organization targeted to decrease the risk factors, such as hypertension, dyslipidemia, obesity,
smoking, alcohol, diet, and sedentary lifestyle till 2025.2 Controlling risk factors and lifestyle is
of great importance with regard to the prevention of CVD. When the prevalence of these risk factors
and life habits is thoroughly taken into consideration, it is not a big surprise to see morbidity and
mortality of CVD remains on the top list in the following decades of the ESC countries. Risk factors
and lifestyle habits for the development of CVD are reviewed individually in the following.
 1.3 Risk factors and health(y) behaviors 5

FIG. 1.1
Population attributable risk for CVD.

1.3.1 Risk factors

1.3.1.1 Hypertension
Hypertension is defined as office systolic blood pressure of more than and equal to 140 mmHg or di-
astolic blood pressure of more than and equal to 90 mmHg on two occasions or taking antihypertensive
medication. Hypertension is one of the most frequent chronic medical conditions that increase the risk
of impairment in the function of heart, brain, kidney, eye, and other organs. The World Health Orga-
nization states that 1 in 4 men and 1 in 5 women in the world have hypertension that contributes to
premature mortality worldwide.
It is well established that there is a continuous and linear relationship between increasing blood
pressure levels and myocardial infarction and stroke.5 International surveys designated that between
1990 and 2015, the prevalence of hypertension was shown to increase significantly and contributed
not only to mortality but also to morbidity.6 In the INTERHEART study, it was shown that 22% of
all acute myocardial infarction cases are related to hypertension and it was also noted that the presence
of hypertension increased the risk of myocardial infarction at least twofold.7 The prevalence of hyper-
tension differs according to gender and income levels of countries. In 2015, median age-standardized
(MAS) hypertension prevalence all over ESC countries was calculated to be 24.8%.2 Of note, the high-
est prevalence was reported in Croatia and the lowest was reported in the UK (32.4% versus 15.2%). It
is also important to note that the prevalence of hypertension in women is lower than in men in ESC
countries (22.3% versus 27%). Income-based comparison of ESC countries also yielded interesting
results such that the prevalence of hypertension was higher in low-to-middle-income countries com-
pared to high-income countries (23.8% versus 15.7%).2 This finding was primarily driven by the fact
6 Chapter 1 The ever-growing burden of cardiovascular disease

that the prevalence of hypertension decreased from 1980 to 2015 in all ESC countries (35.3% versus
24.2%) although it was much more striking in high-income countries.2 As an important contributor of
CVD-related morbidity and mortality, hypertension has not only been better recognized and managed
in high-income countries, but also achieved considerable progress in low-to-middle-income countries.7
In light of these findings, WHO announced its wish to decrease the prevalence of hypertension by 25%
by the year 2025.2 Management of hypertension has a critical role in the primary and secondary
prevention of CVD. This is particularly valid for patients with diabetes mellitus, renal failure, and
vascular disease by achieving strict control of blood pressure. Of note, this achievement can yield a
significant decrease in the prevalence of CVD.

1.3.1.2 Cholesterol
Cholesterol, particularly LDL cholesterol, is a significant predictor of CVD, and increased serum con-
centration of LDL cholesterol is linearly related to increased risk for CVD.8 Among people without a
history of CVD, i.e., in primary prevention, statin-related 1 mmol/L decrease in LDL cholesterol can
decrease CVD risk by 15%.9 Just like hypertension, the prevalence of high cholesterol (i.e., hypercho-
lesterolemia) also differs by gender or income levels of countries. High total cholesterol is reported in
more than 50% of high-income European countries, whereas it is less than 30% in Asian or African
countries. In 2008, MAS hypercholesterolemia prevalence was 15.6% in women versus 14.3% in
men in the ESC countries. The prevalence of hypercholesterolemia in women from high-income coun-
tries raised to 18.8% (IQR 16.5%–20.8%), compared to 11.8% (IQR 9.6%–13%) prevalence of women
from low-income countries. Besides, the prevalence was 18.1% among men from high-income coun-
tries, and among men from low-income countries, hypercholesterolemia prevalence was just 8.7%.2 Of
note, similar decreasing trends in the prevalence of hypercholesterolemia was observed in both high-
income and low-income countries. Effective management of hyperlipidemia, not surprisingly, can
decrease not only the prevalence but also the incidence of CVD.

1.3.1.3 Diabetes mellitus

Diabetes mellitus is defined as fasting plasma glucose more than and equal to 126 mg/dL or casual
plasma glucose more than and equal to 200 mg/dL together with diabetes symptoms or HbA1c level
of more than and equal to 6.5%.10 There are sixty million patients with diabetes mellitus (DM) in
Europe and 422 million patients with DM in the world, with the majority of type 2 DM according
to WHO.11 In the last 30 years, irrespective of gender- and income-based distribution, the prevalence
of DM has been significantly increasing all over the world. This increase is linked to an increased prev-
alence of obesity and overweight people in association with high caloric nutrition and physical inac-
tivity leading to a sedentary lifestyle. The median prevalence of DM in 2017 is 6.8% across ESC
member countries, with the lowest frequency in Estonia, Ireland, and Lithuania and the highest
frequencies in Serbia, Albania, North Macedonia, Montenegro, Bosnia and Herzegovina, Turkey, Leb-
anon, Egypt, and Libya.2 Mortality risk of patients with DM is traditionally known to be twice as much
as those without DM although disparity has been reported to be less apparent recently.12 More than half
of these deaths belong to cardiovascular system diseases, particularly ischemic cardiac disease and
cerebrovascular diseases. Hence, despite the decreasing prevalence of hypertension and hypercholes-
terolemia, an increase in DM prevalence seems to compensate for any benefit and even further increase
in CVD and associated outcomes.
 1.3 Risk factors and health(y) behaviors 7

1.3.1.4 Obesity
Obesity, in the general population, is calculated as the body mass index (BMI), in which an individual’s
weight in kilograms is divided by the square of height in meters. Obesity is present when body mass
index is more than 30 kg/m2, reported by WHO along with increasing BMI categories increasing the
severity of obesity. Of note, a person with a BMI more than and equal to 25 kg/m2 is considered over-
weight. The prevalence of obesity is rising not only in developed but also in developing countries.
Liberalism in global trading, rapid urbanization, sedentary lifestyle changes, and increased consump-
tion of animal-derived oils and sugar has brought about obesity as the epidemic of the new century.13 In
the world, the number of obese people exceeded the number of lean people. In ESC countries, every one
in five individuals is obese. In the ESC countries, MAS BMI is reported to be 26.6 kg/m2 (IQR
26.1–27.1) with similar numbers in both genders. Global BMI Mortality Collaboration reported in
2016 that every 5 kg/m2 increase in BMI among people with BMI
25.0 kg/m2 increased the risk of
death by 1.39-folds.14 Although the prevalence of obesity in high-income countries is higher than that
in low-income countries, both groups yielded a significant increase in the prevalence of obesity. In
1980, obesity prevalence was 9.6% (IQR 8.2%–11.9%), and this number in 2016 has raised to
22.6% (IQR 20.9%–25.8%).2 An increase in the prevalence of obesity along with DM significantly
contributes to an uncontrollable increase in CVD.

1.3.2 Health behaviors

1.3.2.1 Smoking
Smoking or tobacco use has been defined as the single most preventable health risk by every authority
in the world. Smoking has been associated with many types of cancer and CVD and remains as the
major indicator of mortality, particularly premature mortality in the European countries.15 Every year,
about 6 million people succumbed to death secondary to smoking-related diseases. Smoking
prevalence in 2014 was reported to be 21% in men and women with age more than 15 years in the
ESC countries.2 The prevalence in the ESC countries ranges from 11.9% in Sweden to 36.1% in Lith-
uania. Smoking has been more prevalent in men than in women. The prevalence of male smokers is
higher in low-income countries compared to high-income countries, whereas the prevalence of female
smokers is higher in the high-income countries compared to low-income countries. In the ESC
countries, smoking prevalence decreased from 28% to 21% by about 25%.2

1.3.2.2 Alcohol
Alcohol consumption is defined as the per capita consumption of pure alcohol in liters per year and is a
European Core Health Indicator and target of WHO.16 Excessive alcohol consumption is responsible
for one in every ten deaths among adults and, hence, is an important reason of premature death in the
USA. In the EU countries, excessive alcohol consumption is also an important contributor to premature
death after smoking and alcohol-abuse-related hypertension is responsible for 60% of all alcohol-
related deaths. In 2016, 66.6% of adults in the ESC countries reported to consume alcohol in the last
year with a median alcohol consumption of 10.2 L/capita/year.2 Of note, significant differences exist in
different countries with the highest consumption in Portugal, Belgium, Bulgaria, France, Germany,
Latvia, Republic of Moldova, Romania, and Slovenia, and the lowest consumption in Algeria,
Azerbaijan, Egypt, Israel, Lebanon, Libya, Morocco, Syria, Tunisia, and Turkey. Of note, there is
no level at which chronic alcohol consumption can be beneficial, according to GBD 2016 Alcohol
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that five thousand, belongs to me by rights. Oh, never mind, I’m
going along. Do you hear?” he shouted, his fists raised, “I’m going
along, I tell you. There ain’t a man of you big enough to stop me.
Let’s see you try and stop me going. Let’s see you once, any two of
you.” He filled the barroom with his clamor.
“Lord love you, come along, then,” said the sheriff.
The posse rode out of Keeler that same night. The keeper of the
general merchandise store, from whom Marcus had borrowed a
second pony, had informed them that Cribbens and his partner,
whose description tallied exactly with that given in the notice of
reward, had outfitted at his place with a view to prospecting in the
Panamint hills. The posse trailed them at once to their first camp at
the head of the valley. It was an easy matter. It was only necessary
to inquire of the cowboys and range-riders of the valley if they had
seen and noted the passage of two men, one of whom carried a bird-
cage.
Beyond this first camp the trail was lost, and a week was wasted in
a bootless search around the mine at Gold Gulch, whither it seemed
probable the partners had gone. Then a traveling peddler, who
included Gold Gulch in his route, brought in the news of a wonderful
strike of gold-bearing quartz some ten miles to the south on the
western slope of the range. Two men from Keeler had made a strike,
the peddler had said, and added the curious detail that one of the
men had a canary bird in a cage with him.
The posse made Cribbens’ camp three days after the
unaccountable disappearance of his partner. Their man was gone,
but the narrow hoof-prints of a mule, mixed with those of huge hob-
nailed boots, could be plainly followed in the sand. Here they picked
up the trail and held to it steadily till the point was reached where,
instead of tending southward, it swerved abruptly to the east. The
men could hardly believe their eyes.
“It ain’t reason,” exclaimed the sheriff. “What in thunder is he up
to? This beats me! Cutting out into Death Valley at this time of year!”
“He’s heading for Gold Mountain over in the Armagosa, sure.”
 The men decided that this conjecture was true. It was the only
inhabited locality in that direction. A discussion began as to the
further movements of the posse.
“I don’t figure on going into that alkali sink with no eight men and
horses,” declared the sheriff. “One man can’t carry enough water to
take him and his mount across, let alone eight. No, sir. Four couldn’t
do it. No, three couldn’t. We’ve got to make a circuit round the valley
and come up on the other side and head him off at Gold Mountain.
That’s what we got to do, and ride like blazes to do it, too.”
But Marcus protested with all the strength of his lungs against
abandoning the trail now that they had found it. He argued that they
were now but a day and a half behind their man. There was no
possibility of their missing the trail—as distinct in the white alkali as
in snow. They could make a dash into the valley, secure their man,
and return long before their water failed them. He, for one, would not
give up the pursuit, now that they were so close. In the haste of the
departure from Keeler the sheriff had neglected to swear him in. He
was under no orders. He would do as he pleased.
“Go on, then, you darn fool,” answered the sheriff. “We’ll cut on
round the valley, for all that. It’s a gamble he’ll be at Gold Mountain
before you’re half-way across. But if you catch him, here”—he
tossed Marcus a pair of handcuffs—“put ’em on him and bring him
back to Keeler.”
Two days after he had left the posse, and when he was already far
out in the desert, Marcus’s horse gave out. In the fury of his
impatience he had spurred mercilessly forward on the trail, and on
the morning of the third day found that his horse was unable to
move. The joints of his legs seemed locked rigidly. He would go his
own length, stumbling and interfering, then collapse helplessly upon
the ground with a pitiful groan. He was used up.
Marcus believed himself to be close upon McTeague now. The
ashes at his last camp had still been smoldering. Marcus took what
supplies of food and water he could carry, and hurried on. But
McTeague was farther ahead than he had guessed, and by evening
of his third day upon the desert Marcus, raging with thirst, had drunk
his last mouthful of water and had flung away the empty canteen.
“If he ain’t got water with um,” he said to himself, as he pushed on,
“if he ain’t got water with um, I’ll be in a bad way. I will, for a fact.”

At Marcus’s shout McTeague looked up and around him. For the

instant he saw no one. The white glare of alkali was still unbroken.
Then his swiftly rolling eyes lighted upon a head and shoulder that
protruded above the low crest of the break directly in front of him. A
man was there, lying at full length upon the ground, covering him
with a revolver. For a few seconds McTeague looked at the man
stupidly, bewildered, confused, as yet without definite thought. Then
he noticed that the man was singularly like Marcus Schouler. It was
Marcus Schouler. How in the world did Marcus Schouler happen to
be in that desert? What did he mean by pointing a pistol at him that
way? He’d best look out or the pistol would go off. Then his thoughts
readjusted themselves with a swiftness born of a vivid sense of
danger. Here was the enemy at last, the tracker he had felt upon his
footsteps. Now at length he had “come on” and shown himself, after
all those days of skulking. McTeague was glad of it. He’d show him
now. They two would have it out right then and there. His rifle! He
had thrown it away long since. He was helpless. Marcus had ordered
him to put up his hands. If he did not, Marcus would kill him. He had
the drop on him. McTeague stared, scowling fiercely at the leveled
pistol. He did not move.
“Hands up!” shouted Marcus a second time. “I’ll give you three to
do it in. One, two—” Instinctively McTeague put his hands above his
head.
Marcus rose and came towards him over the break.
“Keep ’em up,” he cried. “If you move ’em once I’ll kill you, sure.”
He came up to McTeague and searched him, going through his
pockets; but McTeague had no revolver; not even a hunting knife.
“What did you do with that money, with that five thousand dollars?”
 “It’s on the mule,” answered McTeague, sullenly.
Marcus grunted, and cast a glance at the mule, who was standing
some distance away, snorting nervously, and from time to time
flattening his long ears.
“Is that it there on the horn of the saddle, there in that canvas
sack?” Marcus demanded.
“Yes, that’s it.”
A gleam of satisfaction came into Marcus’s eyes, and under his
breath he muttered: “Got it at last.”
He was singularly puzzled to know what next to do. He had got
McTeague. There he stood at length, with his big hands over his
head, scowling at him sullenly. Marcus had caught his enemy, had
run down the man for whom every officer in the state had been
looking. What should he do with him now? He couldn’t keep him
standing there forever with his hands over his head.
“Got any water?” he demanded.
“There’s a canteen of water on the mule.”
Marcus moved toward the mule and made as if to reach the bridle-
rein. The mule squealed, threw up his head, and galloped to a little
distance, rolling his eyes and flattening his ears.
Marcus swore wrathfully.
“He acted that way once before,” explained McTeague, his hands
still in the air. “He ate some loco-weed back in the hills before I
started.”
For a moment Marcus hesitated. While he was catching the mule
McTeague might get away. But where to, in heaven’s name? A rat
could not hide on the surface of that glistening alkali, and besides, all
McTeague’s store of provisions and his priceless supply of water
were on the mule. Marcus ran after the mule, revolver in hand,
shouting and cursing. But the mule would not be caught. He acted as
if possessed, squealing, lashing out, and galloping in wide circles,
his head high in the air.
 “Come on,” shouted Marcus, furious, turning back to McTeague.
“Come on, help me catch him. We got to catch him. All the water we
got is on the saddle.”
McTeague came up.
“He’s eatun some loco-weed,” he repeated. “He went kinda crazy
before.”
“If he should take it into his head to bolt and keep on running—”
Marcus did not finish. A sudden great fear seemed to widen
around and inclose the two men. Once their water gone, the end
would not be long.
“We can catch him all right,” said the dentist. “I caught him once
before.”
“Oh, I guess we can catch him,” answered Marcus, reassuringly.
Already the sense of enmity between the two had weakened in the
face of a common peril. Marcus let down the hammer of his revolver
and slid it back into the holster.
The mule was trotting on ahead, snorting and throwing up great
clouds of alkali dust. At every step the canvas sack jingled, and
McTeague’s bird-cage, still wrapped in the flour bags, bumped
against the saddle-pads. By and by the mule stopped, blowing out
his nostrils excitedly.
“He’s clean crazy,” fumed Marcus, panting and swearing.
“We ought to come up on him quiet,” observed McTeague.
“I’ll try and sneak up,” said Marcus; “two of us would scare him
again. You stay here.”
Marcus went forward a step at a time. He was almost within arm’s
length of the bridle when the mule shied from him abruptly and
galloped away.
Marcus danced with rage, shaking his fists, and swearing horribly.
Some hundred yards away the mule paused and began blowing and
snuffing in the alkali as though in search of food. Then, for no
reason, he shied again, and started off on a jog trot toward the east.
“We’ve got to follow him,” exclaimed Marcus, as McTeague came
up. “There’s no water within seventy miles of here.”
Then began an interminable pursuit. Mile after mile, under the
terrible heat of the desert sun, the two men followed the mule,
racked with a thirst that grew fiercer every hour. A dozen times they
could almost touch the canteen of water, and as often the distraught
animal shied away and fled before them. At length Marcus cried:
“It’s no use, we can’t catch him, and we’re killing ourselves with
thirst. We got to take our chances.” He drew his revolver from its
holster, cocked it, and crept forward.
“Steady now,” said McTeague; “it won’t do to shoot through the
canteen.”
Within twenty yards Marcus paused, made a rest of his left
forearm and fired.
“You got him,” cried McTeague. “No, he’s up again. Shoot him
again. He’s going to bolt.”
Marcus ran on, firing as he went. The mule, one foreleg trailing,
scrambled along, squealing and snorting. Marcus fired his last shot.
The mule pitched forward upon his head, then, rolling sideways, fell
upon the canteen, bursting it open and spilling its entire contents into
the sand.
Marcus and McTeague ran up, and Marcus snatched the battered
canteen from under the reeking, bloody hide. There was no water
left. Marcus flung the canteen from him and stood up, facing
McTeague. There was a pause.
“We’re dead men,” said Marcus.
McTeague looked from him out over the desert. Chaotic desolation
stretched from them on either hand, flaming and glaring with the
afternoon heat. There was the brazen sky and the leagues upon
leagues of alkali, leper white. There was nothing more. They were in
the heart of Death Valley.
 “Not a drop of water,” muttered McTeague; “not a drop of water.”
“We can drink the mule’s blood,” said Marcus. “It’s been done
before. But—but—” he looked down at the quivering, gory body “—
but I ain’t thirsty enough for that yet.”
“Where’s the nearest water?”
“Well, it’s about a hundred miles or more back of us in the
Panamint hills,” returned Marcus, doggedly. “We’d be crazy long
before we reached it. I tell you we’re done for. We ain’t ever going to
get outa here.”
“Done for?” murmured the other, looking about stupidly. “Done for,
that’s the word. Done for? Yes, I guess we’re done for.”
“What are we going to do now?” exclaimed Marcus, sharply, after
a while.
“Well, let’s be moving along—somewhere.”
“Where, I’d like to know? What’s the good of moving on?”
“Wat’s the good of stopping here?”
There was a silence.
“Lord, it’s hot,” said the dentist, finally, wiping his forehead with the
back of his hand. Marcus ground his teeth.
“Done for,” he muttered; “done for.”
“I never was so thirsty,” continued McTeague. “I’m that dry I can
hear my tongue rubbing against the roof of my mouth.”
“Well, we can’t stop here,” said Marcus, finally; “we got to go
somewhere. We’ll try and get back, but it ain’t no manner of use.
Anything we want to take along with us from the mule? We can—”
Suddenly he paused. In an instant the eyes of the two doomed
men had met as the same thought simultaneously rose in their
minds. The canvas sack with its five thousand dollars was still tied to
the horn of the saddle.
Marcus had emptied his revolver at the mule, and though he still
wore his cartridge belt, he was for the moment as unarmed as
McTeague.
“I guess,” began McTeague, coming forward a step, “I guess even
if we are done for, I’ll take—some of my truck along.”
“Hold on,” exclaimed Marcus, with rising aggressiveness. “Let’s
talk about that. I ain’t so sure about who that—who that money
belongs to.”
“Well, I am, you see,” growled the dentist.
The old enmity between the two men, their ancient hate, was
flaming up again.
“Don’t try an’ load that gun either,” cried McTeague, fixing Marcus
with his little eyes.
“Then don’t lay your finger on that sack,” shouted the other.
“You’re my prisoner, do you understand? You’ll do as I say.” Marcus
had drawn the handcuffs from his pocket, and stood ready with his
revolver held as a club. “You soldiered me out of that money once,
and played me for a sucker, an’ it’s my turn now. Don’t you lay your
finger on that sack.”
Marcus barred McTeague’s way, white with passion. McTeague did
not answer. His eyes drew to two fine, twinkling points, and his
enormous hands knotted themselves into fists, hard as wooden
mallets. He moved a step nearer to Marcus, then another.
Suddenly the men grappled, and in another instant were rolling
and struggling upon the hot, white ground. McTeague thrust Marcus
backward until he tripped and fell over the body of the dead mule.
The little bird-cage broke from the saddle with the violence of their
fall, and rolled out upon the ground, the flour-bags slipping from it.
McTeague tore the revolver from Marcus’s grip and struck out with it
blindly. Clouds of alkali dust, fine and pungent, enveloped the two
fighting men, all but strangling them.
McTeague did not know how he killed his enemy, but all at once
Marcus grew still beneath his blows. Then there was a sudden last
return of energy. McTeague’s right wrist was caught, something
clicked upon it, then the struggling body fell limp and motionless with
a long breath.
As McTeague rose to his feet, he felt a pull at his right wrist;
something held it fast. Looking down, he saw that Marcus in that last
struggle had found strength to handcuff their right wrists together.
Marcus was dead now; McTeague was locked to the body. All about
him, vast, interminable, stretched the measureless leagues of Death
Valley.
McTeague remained stupidly looking around him, now at the
distant horizon, now at the ground, now at the half-dead canary
chittering feebly in its little gilt prison.—Arranged from “McTeague.”
Copyright by Doubleday, Page & Co., New York, and used by kind
permission.

MICHAEL STROGOFF, COURIER OF THE CZAR

By Jules Verne
The door of the imperial cabinet was opened and General Kissoff
was announced.
“The courier?” inquired the Czar eagerly.
“He is here, sire,” replied General Kissoff.
“Let him come in,” said the Czar.
In a few moments Michael Strogoff, the courier, entered. The Czar
fixed a penetrating look upon him without uttering a word. Then in an
abrupt tone—
“Thy name?”
“Michael Strogoff, sire.”
“Thy rank?”
“Captain in the corps of Couriers to the Czar.”
“Thou dost know Siberia?”
“I am a Siberian.”
 “A native of—?”
“Omsk, sire.”
“Hast thou relations there?”
“Yes, sire, my aged mother.”
The Czar suspended his questions for a moment; then pointed to
a letter which he held in his hand:
“Here is a letter which I charge thee, Michael Strogoff, to deliver
into the hands of the Grand Duke, and to no one but him.”
“I will deliver it, sire.”
“The Grand Duke is at Irkutsk. Thou wilt have to traverse a
rebellious country, invaded by Tartars, whose interest it will be to
intercept this letter.”
“I will traverse it.”
“Above all, beware of the traitor, Ivan Ogareff, who will perhaps
meet thee on the way.”
“I will beware of him.”
“Wilt thou pass through Omsk?”
“Sire, that is my route.”
“If thou dost see thy mother, there will be the risk of being
recognized. Thou must not see her!”
Michael Strogoff hesitated a moment, and then said:
“I will not see her.”
“Swear to me that nothing will make thee acknowledge who thou
art, nor whither thou art going.”
“I swear it.”
“Michael Strogoff, take this letter. On it depends the safety of all
Siberia, and perhaps the life of my brother, the Grand Duke.”
“This letter shall be delivered to His Highness, the Grand Duke.”
 “Go, thou, for God, for the Czar, and for your native land.”
The courier saluted his sovereign and that very night set out to
fulfill his perilous mission. All went well until he reached Omsk.
Compelled to stop here for food and a change of horses, he was
about to leave the posting house to continue his journey when
suddenly a cry made him tremble—a cry which penetrated to the
depths of his soul—and these two words rushed into his ear: “My
son!”
His mother, the old woman, Marfa, was before him! Trembling, she
smiled upon him and stretched forth her arms to him. Michael
Strogoff stepped forward; he was about to throw himself—when the
thought of duty, the serious danger to himself, and his mother, in this
unfortunate meeting, stopped him, and so great was his self-
command that not a muscle of his face moved. There were twenty
people in the public room, and among them perhaps spies, and was
it not known that the son of Marfa Strogoff belonged to the corps of
Couriers to the Czar? Michael Strogoff did not move.
“Michael!” cried his mother.
“Who are you, my good woman?”
“Who am I? Dost thou no longer know thy mother?”
“You are mistaken; a resemblance deceives you.”
Marfa went up to him, and looking straight into his eyes, said: “Art
thou not the son of Peter and Marfa Strogoff?”
Michael would have given his life to have locked his mother in his
arms. But if he yielded now it was all over with him, with her, with his
mission, with his oath. Completely master of himself, he closed his
eyes that he might not see the inexpressible anguish of his mother.
“I do not know, in truth, what it is you say, my good woman.”
“Michael!”
“My name is not Michael. I never was your son! I am Nicholas
Kopanoff, a merchant of Irkutsk.”
 And suddenly he left the room, while for the last time the words
echoed in his ears,—
“My son! My son!”
Michael Strogoff by a desperate effort had gone. He did not heed
his old mother, who had fallen back almost inanimate on a bench.
But when the postmaster hastened to assist her, the aged woman
raised herself. Suddenly the thought occurred to her: She denied by
her own son. It was impossible! As for being deceived, it was equally
impossible. It was certainly her son whom she had just seen; and if
he had not recognized her it was because he had some strong
reason for acting thus. And then, her mother-feelings arising within
her, she had only one thought: Can I unwittingly have ruined him?
“I am mad,” she said to her interrogators. “This young man was
not my son; he had not his voice. Let us think no more of it. If we do,
I shall end in finding him everywhere.”
This scene, however, was immediately reported to Ivan Ogareff,
who was stationed in the town. He at once arrested Michael Strogoff,
and then had Marfa brought before him. Marfa, standing before Ivan
Ogareff, drew herself up, crossed her arms on her breast, and
waited. “You are Marfa Strogoff?” asked Ogareff.
“Yes.”
“Do you retract what you said a few hours ago?”
“No.”
“Then you do not know that your son, Michael Strogoff, Courier to
the Czar, has passed through Omsk?”
“I do not know it.”
“And the man whom you thought you recognized as your son was
not your son?”
“He was not my son.”
“And since then, you have seen him among the prisoners?”
“No.”
 “If he were pointed out to you, would you recognize him?”
“No.”
“Listen! Your son is here, and you shall immediately point him out
to me.”
“No.”
“All these men will file before you, and if you do not show me
Michael Strogoff, you shall receive as many blows from the knout as
men shall have passed before you.”
On an order from Ogareff, the prisoners filed one by one past
Marfa, who was immovable as a statue, and whose face expressed
only perfect indifference. Michael was to all appearances unmoved,
but the palms of his hands bled under the nails which were pressed
into the flesh.
Marfa, seized by two soldiers, was forced on her knees on the
ground. Her dress torn off left her back bare. A saber was placed
before her breast at a few inches’ distance. If she bent beneath her
sufferings, her breast would be pierced by the sharp steel. The Tartar
drew himself up and waited.
“Begin,” said Ogareff.
The whip whistled through the air, but, before it fell, a powerful
hand stopped the Tartar’s arm. Ivan Ogareff had succeeded.
“Michael Strogoff!” cried he.
“Himself!” said Michael, and raising the knout, he struck Ogareff a
blow across the face.
“Blow for blow!” said he.
Twenty soldiers threw themselves on Michael and in another
instant he would have been slain, but Ogareff stopped them.
“This man is reserved for the Emir’s judgment. Search him.”
The letter bearing the imperial arms was found in Michael’s
bosom; he had not time to destroy it. It was handed to Ogareff.
Michael was then led before the Emir.
 “Your forehead to the ground!” exclaimed Ogareff.
“No!”
Two soldiers tried to make him bend, but were themselves laid on
the ground by a blow from Michael’s fist.
“Who is this prisoner?” asked the Emir.
“A Russian spy,” answered Ogareff.
In asserting that Michael was a spy, he knew that the sentence
would be terrible. The Emir made a sign, at which all bowed low their
heads. Then he pointed to the Koran, which was brought to him. He
opened the sacred book, and placing his finger on one of its pages,
read in a loud voice a verse ending in these words: “And he shall no
more see the things of this earth.”
“Russian spy, you have come to see what is going on in the Tartar
camp; then look while you may! You have seen for the last time. In
an instant your eyes will be for ever shut to the light of day.”
Michael’s fate was to be not death, but blindness. He was going to
be blinded in the Tartar fashion, with a hot saber-blade passed
before his eyes.
The Emir’s orders executed, Ivan Ogareff approached Michael,
drew from his pocket the Imperial letter, opened it and held it up
before the face of the Czar’s courier, saying with supreme irony:
“Read, now, Michael Strogoff, read, and go and repeat at Irkutsk
what you have read. The true Courier of the Czar is henceforth Ivan
Ogareff.”
The Emir retired with his train. Ivan followed after, and sightless
Michael was left alone to his fate. One thought possessed him. He
must somehow arrive at Irkutsk before the traitor and warn the
Grand Duke of the intended deception.
Some months later Michael Strogoff had reached his journey’s
end! He was in Irkutsk. Hastening to the governor’s palace to see
the Grand Duke, he meets in a waiting-room Ivan Ogareff, the traitor.
The latter must act quickly. Ogareff arose, and thinking he had an
immeasureable advantage over the blind man threw himself upon
him. But with one hand Michael grasps the arm of his enemy and
hurls him to the ground. Ogareff gathers himself together like a tiger
about to spring, and utters not a word. The noise of his footsteps, his
very breathing, he tries to conceal from the blind man. At last, with a
spring, he drives his sword full blast at Michael’s breast. An
imperceptible movement of the blind man’s knife turns aside the
blow. Michael is not touched, and coolly waits a second attack. Cold
drops stand on Ogareff’s brow; he draws back a step and again
leaps forward. But like the first, this attempt fails. Michael’s knife has
parried the blow from the traitor’s useless sword. Mad with rage and
terror, he gazes into the wide-open eyes of the blind man. Those
eyes which seem to pierce to the bottom of his soul, and which do
not, cannot, see, exercise a sort of dreadful fascination over him.
Suddenly Ogareff utters a cry: “He sees! He sees!”
“Yes, I see. Thinking of my mother, the tears which sprang to my
eyes saved my sight. I see the mark of the knout which I gave you,
traitor and coward! I see the place where I am about to strike you!
Defend your life! It is a duel I offer you! My knife against your sword!”
Ogareff now feels that he is lost, but, mustering up all his courage,
he springs forward. The two blades cross, but at a touch from
Michael’s knife the sword flies in splinters, and the wretch, stabbed
to the heart, falls lifeless to the ground.
At the same moment the door is thrown open, and the Grand
Duke, accompanied by some of his officers, enters. The Grand Duke
advances. In the body lying on the ground he recognizes the man
whom he believes to be the Czar’s Courier. Then in threatening
voice:
“Who killed this man?”
“I,” answered Michael.
“Thy name? Who dares kill the servant of my brother, the Czar’s
Courier?”
“That man, your highness, is not a Courier of the Czar! He is Ivan
Ogareff!”
 “Ivan Ogareff!”
“Yes, Ivan the traitor.”
“But who are you, then?”
“Michael Strogoff.”

THE TIGER’S CAVE

AN ADVENTURE AMONG THE MOUNTAINS OF QUITO
On leaving the Indian village, we continued to wind round
Chimborazo’s wide base; but its snow-crowned head no longer
shone above us in clear brilliancy, for a dense fog was gathering
gradually around it. Our guides looked anxiously towards it, and
announced their apprehensions of a violent storm. We soon found
that their fears were well founded. The fog rapidly covered and
obscured the whole of the mountain; the atmosphere was
suffocating, and yet so humid that the steelwork of our watches was
covered with rust, and the watches stopped. The river beside which
we were traveling rushed down with still greater impetuosity; and
from the clefts of the rocks which lay on the left of our path were
suddenly precipitated small rivulets that bore the roots of trees and
innumerable serpents along with them. These rivulets often came
down so suddenly and violently that we had great difficulty in
preserving our footing. The thunder at length began to roll, and
resounded through the mountainous passes with the most terrific
grandeur. Then came the vivid lightning, flash following flash—
above, around, beneath—everywhere a sea of fire. We sought a
momentary shelter in a cleft of the rocks, while one of our guides
hastened forward to seek a more secure asylum. In a short time he
returned, and informed us that he had discovered a spacious cavern,
which would afford us sufficient protection from the elements. We
proceeded thither immediately, and with great difficulty, and not a
little danger, at last got into it.
The noise and raging of the storm continued with so much
violence that we could not hear the sound of our voices. I had placed
myself near the entrance of the cave, and could observe, through the
opening, which was straight and narrow, the singular scene without.
The highest cedar-trees were struck down, or bent like reeds;
monkeys and parrots lay strewed upon the ground, killed by the
falling branches; the water had collected in the path we had just
passed, and hurried along it like a mountain stream. From everything
I saw I thought it extremely probable that we should be obliged to
pass some days in this cavern. When the storm, however, had
somewhat abated, our guides ventured out in order to ascertain if it
were possible to continue our journey. The cave in which we had
taken refuge was extremely dark, so that if we moved a few paces
from the entrance we could see no more than an inch before us; and
we were debating as to the propriety of leaving it, even before the
Indians came back, when we suddenly heard a singular rumbling or
growling at the farther end of the cavern, which instantly fixed all our
attention. Wharton and myself listened anxiously, but our daring and
inconsiderate young friend Lincoln, together with my huntsman, crept
about upon their hands and knees, and endeavored to discover, by
groping, from whence the sound proceeded. They had not advanced
far into the cavern before we heard them utter an exclamation of
surprise; and they returned to us, each carrying in his arms an
animal singularly marked, and about the size of a cat, seemingly of
great strength and power, and furnished with immense fangs. The
eyes were of a green color; strong claws were upon their feet; and a
blood-red tongue hung out of their mouths. Wharton had scarcely
glanced at them, when he exclaimed, in consternation, “Good God!
we have come into the den of a—.” He was interrupted by a fearful
cry of dismay from our guides, who came rushing precipitately
towards us, calling out, “A tiger! a tiger!” and at the same time, with
extraordinary rapidity, they climbed up a cedar-tree, which stood at
the entrance of the cave, and hid themselves among the branches.
After the first sensation of horror and surprise, which rendered me
motionless for a moment, had subsided, I grasped my firearms.
Wharton had already regained his composure and self-possession;
and he called to us to assist him instantly in blocking up the mouth of
the cave with an immense stone, which fortunately lay near it. The
sense of approaching danger augmented our strength, for we now
distinctly heard the growl of the ferocious animal, and we were lost
beyond redemption if it reached the entrance before we could get it
closed. Ere this was done, we could distinctly see the tiger bounding
towards the spot and stooping in order to creep into his den by the
narrow opening. At this fearful moment our exertions were
successful, and the great stone kept the wild beast at bay. There
was a small open space, however, left between the top of the
entrance and the stone, through which we could see the head of the
animal, illuminated by its glowing eyes, which rolled, glaring with
fury, upon us. Its frightful roaring, too, penetrated to the depths of the
cavern, and was answered by the hoarse growling of the cubs, which
Lincoln and Frank had now tossed from them. Our ferocious enemy
attempted first to remove the stone with his powerful claws, and then
to push it with his head from its place; and these efforts, proving
abortive, served only to increase his wrath. He uttered a
tremendous, heart-piercing howl, and his flaming eyes darted light
into the darkness of our retreat.
“Now is the time to fire at him,” said Wharton, with his usual
calmness; “aim at his eyes; the ball will go through his brain, and we
shall then have a chance to get rid of him.”
Frank seized his double-barreled gun, and Lincoln his pistols; the
former placed the muzzle within a few inches of the tiger, and Lincoln
did the same. At Wharton’s command, they both drew the triggers at
the same moment, but no shot followed. The tiger, who seemed
aware that the flash indicated an attack upon him, sprang growling
from the entrance, but, feeling himself unhurt, immediately turned
back again, and stationed himself in his former place. The powder in
both pieces was wet. Frank and Lincoln, therefore, proceeded to
draw the useless charges while Wharton and myself hastened to
seek our powder-flask. It was so extremely dark that we were
obliged to grope about the cave; and, at last, coming in contact with
the cubs, we heard a rustling noise, as if they were playing with
some metal substance, which we soon discovered was the cannister
we were looking for. Most unfortunately, however, the animals had
pushed off the lid with their claws, and the powder had been strewed
over the damp earth and rendered entirely useless. This fearful
discovery excited the greatest consternation.
 “All is now over,” said Wharton. “We have only now to choose
whether we shall die of hunger, together with these animals who are
shut up along with us, or open the entrance to the blood-thirsty
monster without, and so make a quicker end of the matter.”
So saying, he placed himself close beside the stone, which, for the
moment, defended us, and looked undauntedly upon the lightning
eyes of the tiger.
Lincoln raved and swore; and Frank took a piece of strong cord
from his pocket and hastened to the farther end of the cave—I knew
not with what design. We soon, however, heard a low, stifled
groaning; and the tiger, who had heard it also, became more restless
and disturbed than ever. He went backwards and forwards before
the entrance of the cave, in the most wild, impetuous manner, then
stood still, and stretching out his neck in the direction of the forest,
broke forth into a deafening howl. Our two Indian guides took
advantage of this opportunity to discharge several arrows from the
tree. The animal was struck more than once, but the light weapons
bounded back harmless from his thick skin. At length, however, one
of them struck him near the eye, and the arrow remained sticking in
the wound. He now broke anew into the wildest fury, and sprang at
the tree, and tore it with his claws, as if he would have dragged it to
the ground. But, having at length succeeded in getting rid of the
arrow, became more calm, and laid himself down as before in front
of the cave.
Frank now returned from the lower end of the den, and a glance
showed us what he had been doing. In each hand, and dangling
from the end of a string, were the two cubs. He had strangled them,
and before we were aware what he intended, he threw them through
the opening to the tiger. No sooner did the animal perceive them,
than he gazed earnestly upon them, and began to examine them
closely, turning them cautiously from side to side. As soon as he
became aware that they were dead, he uttered so piercing a howl of
sorrow that we were obliged to put our hands to our ears. When I
upbraided my huntsman for the cruel action he had so rashly
committed, I perceived by his blunt and abrupt answers that he also
had lost all hope of rescue from our impending fate, and, that, under
these circumstances, the ties between master and servant were
dissolved. For my own part, without knowing why, I could not help
believing that some unexpected assistance would yet rescue us from
so horrible a fate. Alas! I little anticipated the sacrifice that my rescue
was to cost.
The thunder had now ceased, and the storm had sunk to a gentle
gale; the songs of the birds were again heard in the neighboring
forest, and the sunbeams sparkled in the drops that hung from the
leaves. We saw through the aperture that all nature was reviving
after the wild war of elements which had so recently taken place; but
the contrast only made our situation the more terrible. We were in a
grave from which there was no deliverance; and a monster, worse
than the fabled Cerberus, kept watch over us. The tiger had laid
himself down beside his whelps. He was a beautiful animal, of great
size and strength, and his limbs being stretched out at their full
length, displayed his immense power of muscle. A double row of
great teeth stood far enough apart to show his large red tongue, from
which the white foam fell in large drops. All at once, another roar was
heard at a distance, and the tiger immediately rose and answered it
with a mournful howl. At the same instant, our Indians uttered a
shriek, which announced that some new danger threatened us. A
few moments confirmed our worst fears, for another tiger, not quite
so large as the former, came rapidly towards the spot where we
were.
“This enemy will prove more cruel than the other,” said Wharton;
“for this is the female, and she knows no pity for those who deprive
her of her young.”
The howls which the tigress gave when she had examined the
bodies of her cubs, surpassed everything of the horrible that we had
yet heard; and the tiger mingled his mournful cries with hers.
Suddenly her roaring was lowered to a hoarse growling, and we saw
her anxiously stretch out her head, extend her wide and smoking
nostrils, and look as if she were determined to discover immediately
the murderers of her young. Her eyes quickly fell upon us, and she
made a spring forward with the intention of penetrating to our place
of refuge. Perhaps she might have been enabled by her immense