MicroRNAs As Important Regulators of Exercise Adaptation

MicroRNAs as Important Regulators of Exercise Adaptation
Gustavo J.J. Silva, Anja Bye, Hamid el Azzouzi, Ulrik Wisløff
PII: S0033-0620(17)30090-7
DOI: doi: 10.1016/j.pcad.2017.06.003
Reference: YPCAD 821
To appear in: Progress in Cardiovascular Diseases
Received date: 25 June 2017

Accepted date: 25 June 2017
Please cite this article as: Silva Gustavo J.J., Bye Anja, el Azzouzi Hamid, Wisløff Ulrik,
MicroRNAs as Important Regulators of Exercise Adaptation, Progress in Cardiovascular
Diseases (2017), doi: 10.1016/j.pcad.2017.06.003
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MicroRNAs as Important Regulators of Exercise Adaptation
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Gustavo J. J. Silvaa, Anja Byea, Hamid el Azzouzib, Ulrik Wisløffa,c*
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aK.G. Jebsen Center of Exercise in Medicine at the Department of Circulation and
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Medical Imaging, Norwegian University of Science and Technology (NTNU),
Trondheim, Norway.
bDepartment of Cardiology, University Medical Center Utrecht-UMCU, Utrecht,
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The Netherlands.
cSchool of Human Movement & Nutrition Sciences, University of Queensland,
Australia.
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Short Title: MicroRNA and Exercise

Disclosures/COI: None
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* Address reprint requests and correspondence to: Ulrik Wisløff, K. G. Jebsen

Center of Exercise in Medicine, Norwegian University of Science and Technology
(NTNU). Prinsesse Kristinas gt. 3, 3rd floor, 7006 Trondheim, Norway.
E-mail address: ulrik.wisloff@ntnu.no (U. Wisløff)
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Abstract
A significant body of evidence supports the protective role of exercise training
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(ET) in cardiovascular diseases, skeletal muscle dystrophies, several types of
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cancer, Alzheimer disease or even in the recovery of spinal cord injury. In spite
of this, the molecular mechanisms underlying the beneficial effects of exercise
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training are not well understood and remain elusive. Several mechanisms have
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been proposed in the past, but more recently microRNAs (miRNAs), small non-
coding RNA molecules involved in a variety of basic biological processes that

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negatively modulate gene expression, recognized as important regulatory
molecules. In this review, we highlight recent advances on the miRNA

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involvement in the benefits of ET. Here, we assess the role of microRNAs
expressed in the heart, in the skeletal muscle, detected in the circulation (serum
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and plasma), and in other conditions (e.g., spinal cord injury). Additionally, the
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long-term effects of diverse ET modalities (e.g., running, cycling, resistance
training) in the cardiac miRNA profile are properly addressed.

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Keywords: microRNA, exercise training, heart, skeletal muscle, spinal cord

injury, brain
Abbreviations and Acronyms
AUC = Area under the curve
CAD = Coronary artery disease
CRF = Cardiorespiratory fitness

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CV = Cardiovascular
CVD = Cardiovascular disease
ET = Exercise training
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HF = Heart failure
hsa = Homo sapiens (human)
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HUNT = The Nord-Trøndelag Health Study
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HF = Heart failure
KEGG = Kyoto Encyclopedia of Genes and Genomes
dKO = Double knockout

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LNA = Locked nucleic acid
LV = Left ventricular
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LVH = Left ventricular hypertrophy
mmu = Mus musculus (mouse)

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miRNA = MicroRNA
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myomiRNA = Skeletal muscle microRNA
ncRNA = noncoding RNA

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NK = Natural killer cells
pre-miRNA = Precursor-miRNA
pri-miRNA = Primary microRNA transcripts
PA = Physical activity
Pmax = maximum skeletal muscle force
RAAS = Renin-angiotensin-aldosterone system
RIPC = Remote ischemic preconditioning
rno = Rattus norvegicus (rat)
ROC = Receiver Operating Characteristic

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RT = Resistance training
SHR = Spontaneously hypertensive rats
SNP = Single nucleotide polymorphism
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UTR = 3' untranslated region
VO2max = Maximal oxygen uptake
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Exercise Training
The lack of physical activity (PA) contributes to the worldwide rise in life-style
related diseases, such as cardiovascular (CV) disease (CVD) and metabolic
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diseases, and diabetes1. In line with this observation, large-scale epidemiological
studies performed in healthy subjects and in patients with CVD demonstrate that
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low aerobic exercise capacity is a stronger predictor of mortality than other
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established risk factors2-4. Conversely, high PA levels and cardiorespiratory
fitness (CRF) are associated with low risk of CVD and mortality3, 5, 6. It has also
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been demonstrated that CRF, measured as maximal oxygen uptake (VO2max), is a
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good indicator of CV health, and a strong predictor of CVD mortality in healthy
individuals and in patients with CVD3, 7-11. Levels of PA that increases CRF may
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therefore be one of the most important modifiable life-style interventions
indicated for preventing or controlling CV, metabolic or skeletal muscle diseases,

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and has been strongly recommended for both healthy individuals and for
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patients with CVD to improve CV health and reduce risk of premature
mortality12-14.
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Exercise training (ET) results in a large number of effects that benefit the subject
in both health and disease. Adaptations to endurance training include increased
VO2max, improved lipid profile, improved endothelial function, and increased
abundance of capillaries, mitochondria and metabolic enzymes in different
organs and systems15-18. However, a great majority of the studies in the exercise
field have focused on CV and skeletal muscle adaptations8, 11, 19-32. In the heart, a
central feature of regular ET is improved systolic and diastolic function and
larger cardiac output33-36. The long-term effects of ET include cardiomyocyte

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shortening and rates of contraction-relaxation improvements, providing a
cellular basis for a better organ function37. In contrast, CVD in general induce
cardiac dysfunction with reduced ejection fraction and lower cardiac output,
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which is also associated with impaired cardiomyocyte contractile function38-41.
Experimental studies of cellular effects of regular ET appear therefore to
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translate to humans, also when exercise training is applied therapeutically in
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CVD. Such studies suggest restored contractility and attenuated pathological
growth as important cellular mechanisms for the beneficial effects of PA in heart
failure (HF)37, 42-46.

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However, it has been shown that aerobic ET not only improves cardiac function
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in patients with CVD, but also promotes a broad range of benefits in the skeletal
muscle. In fact, skeletal muscle abnormities induced by CVD has been extensively
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described, including rarefaction, type I to type II fibers switch, impaired

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metabolism, and atrophy47-51. Mainly, ET can prevent skeletal muscle atrophy,
increase in type I fiber distribution, and improve metabolic status47, 48, 51-55. At
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the molecular level, over the past few years, we have come to better understand
the intracellular signaling pathways mediating physiological adaptations induced
by both acute and chronic ET, and its benefits against pathological conditions.
Accordingly, recent efforts have identified intracellular mechanisms underlying
both cardiac and skeletal muscle abnormalities in CVD, and promising targets
such as metabolic enzymes and calcium handling-related proteins37, 42, 43, 52-54, 56-
61.
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MicroRNAs
In the early 1990s, a novel class of genes, known as microRNAs (miRNAs), have
emerged as powerful agents that control the expression of a vast gene pool in a
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sequence–specific manner and lead to post-transcriptional regulation62-65. The
laboratories of Dr. Victor Ambros and Dr. Gary Ruvkun discovered
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simultaneously the first miRNA, lin-4, in 1993 while studying post-embryonic
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developmental events in C. elegans66, 67. Actually, the lin-4 gene itself was initially
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identified in 1981 in a Cell paper by Marty Chalfie, Bob Horvitz, and John
Sulston68. At that time, these authors described the detailed cell lineage analysis,
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but not the molecular identity, of two genes that affect C. elegans developmental
timing, lin-4 and unc-86. Later, what both Ambros’s and Ruvkun’s groups have
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done was describe that lin-4 transcripts (of approximately 22 and 61 nt)
containing sequences complementary to a repeated sequence element in the 3'

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untranslated region (UTR) of lin-14 mRNA, suggesting that lin-4 regulates lin-14
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translation via an antisense RNA-RNA interaction. Since the first description of
their solid regulatory functions, miRNAs have been detected from plants to
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humans, and have revealed a formidable mediator of a broad range of cellular
adaptive processes such as differentiation, proliferation, apoptosis and
metabolism62, 65, 69.
MicroRNA Biogenesis
MiRNA biogenesis is a very complex process and involves multiple steps as
miRNAs can be transcribed as from non-coding regions (intergenic) or within the
protein coding genes (intragenic)62. Initiated by the RNA polymerase II62, their
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transcription results in the generation of primary transcripts (pri-miRNA), which
can encode individual miRNAs (monocistronic) or multiple miRNAs
(polycistronic)70. These pri-miRNAs are then processed into 60~70 nucleotides
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precursor-miRNAs (pre-miRNAs) by the RNase III endonuclease Drosha and its
cofactor Pasha62. As Pasha recognizes RNA stem loop structures and the
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neighboring single stranded sequences, this results in recruitment and cleavage
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by Drosha62. The pre-miRNAs are then exported to the cytosol by the Exportin-5
(XPO5) and processed into a ~22 nucleotide complex by DICER, whereby one of
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the duplex strands is degraded and the other becomes the mature miRNA. This
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mature miRNA is incorporated into the RNA-induced silencing complex (RISC)
allowing this complex to identify and bind to the 3’ or 5’UTR of the target mRNAs,
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resulting in its degradation or repression of protein translation62, 65.

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In contrast to miRNAs, little is known about ET-induced modulation on miRNA

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biogenesis. Recently, a study of an acute bout of endurance exercise in untrained
men showed increased gene expression of components of the miRNA biogenesis

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machinery. Acute endurance exercise increased mRNA of members of the
endonuclease III enzymes Drosha and DICER as well as the miRNA export
protein, Exportin 571.
Circulating MicroRNAs as New Biomarkers for Healthy and Disease
Conditions
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In 2008, independent groups72-74 demonstrated at the same time that miRNAs
could be detected in the circulation and are remarkably stable in biofluids,
followed by demonstrations in other body fluids like urine, cerebrospinal fluid,
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and saliva75. Several studies have since then shown that miRNAs can be secreted
into the bloodstream by different organs like the heart, vascular endothelial cells,
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skeletal muscle, liver and the brain74, 76-90. Although not yet fully understood, the
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mechanisms by which the miRNAs are detectable in biofluids are commencing to
be deciphered. Evidence has suggested that miRNAs are packaged in
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microparticles (exosomes, microvesicles, and apoptotic bodies)91-93 or associated
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with RNA-binding proteins (e.g., Argonaute 2)94 or lipoprotein complexes (high-
density lipoprotein)95. Some circulating miRNAs might just be waist products of

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ongoing physiological or pathological processes in tissue. However there is
evidence indicating that certain circulating miRNAs may have important

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functions as intercellular communicators77, 95-97. For instance, miRNA

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communication has been demonstrated between endothelial cells and
endothelial apoptotic bodies, smooth muscle cells and cardiomyocytes93, 98, 99.
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Interestingly, several studies have reported the association of changes in
circulating miRNAs levels with altered physiological states such as with aging, PA,
and pregnancy77, 78, 87, 89, 100-103. Due to their stability in blood, miRNAs have
emerged as promising clinical biomarkers of disease73, 74, 80, 82, 83, 89, 104-108. Our
group performed a prospective nested case-control study, with 10-year
observation period, and identified several circulating miRNAs (let-7d-5p, let-7g-
5p, miR-26a-5p, miR-29c-3p, miR-103a-3p, miR-106a-5p, miR-148b-3p, miR-
151a-5p, miR-424-5p, miR-660-5p) that predict future fatal acute myocardial
infarction in healthy participants from the Nord-Trøndelag Health (HUNT)

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Study109. Additionally, a miRNA model added to the Framingham Risk Score for
global CVD risk prediction revealed that 5 (let-7g-5p, miR-106a-5p, miR-144-3p,
miR-424-5p and miR-660-5p) out of the 10 miRNAs listed above significantly
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increased the area under the curve (AUC) obtained from the Receiver Operating
Characteristic (ROC)-curves analysis, from 0.72 (95% CI: [0.61, 0.82]) to 0.91
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(95% CI: [0.85, 0.96])109. Therefore, assessment of CVD risk supported by novel
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circulating biomarkers, such as miRNAs, is important to stratifying the
individuals at high-risk, to optimizing treatment strategies, and to enhancing our
understanding of the underlying biology.

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Role of MicroRNAs in Exercise
MiRNAs have been also found essential as mediators of processes associated

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with ET adaptation including cardiac110-113 and skeletal muscle114, 115
hypertrophies, angiogenesis116, 117, atherosclerosis118, neuron regeneration119,

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metabolism/CRF85, 120, 121. Additionally, miRNAs seem to play important roles in

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the acute71, 77, 88, 122 and chronic76, 86, 123 endurance exercise, resistance (RT)
exercise90, 124, in athletes (i.e., versus marathon runners78, 85), performed on a
treadmill125 or using a swimming110, 112, 113, 116, 117 protocol; in animal models (i.e.,
mouse, rats, etc)112, 117, 120, in individuals/patient cohorts76, 78, 86, 123, as well as, in
the general population121, 126. Here, we review emerging features of miRNA-
target interactions involved in the benefits of ET in the cardiac and skeletal
muscle tissues, as well as, in the spinal cord injury and in the immune system
(Fig 1). Furthermore, we will discuss the changes in the circulating microRNA
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levels induced by exercise-trained, and genetic variants influencing mechanisms
of post-transcriptional regulation by miRNAs.
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The current review efforts are therefore aimed at elucidating the role of non-
coding RNAs, such as miRNAs, with respect to beneficial effects of ET (acute and
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chronic) on different systems (i.e., heart, skeletal muscle, etc.), and in
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physiological and pathological (i.e., hypertension, HF, spinal cord injury, etc.)
conditions.
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The Influence of Exercise on Cardiac MicroRNAs
In the last years, the use of different large-scale screening approaches provided
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the tools to uncover the entire cardiac transcriptome landscape, including
mRNAs, miRNAs, long noncoding RNAs and other RNA molecules, suggesting
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that changes in the transcriptome homeostasis are critically associated with

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different processes in the heart127. With regard to the role of miRNAs in the
embryonic heart development, several candidates have been implicated in the

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past years128-132. The role of miRNAs in myocardial development was first
assessed in the fruit fly D. melanogaster. It was demonstrated that the
overexpression of the orthologue of miR-1 in D. melanogaster (dmiR-1) in cardiac
mesoderm results in fewer cardiac cells. Conversely, loss-of-function approach
for miR-1 revealed to be uniformly lethal, showing that dmiR-1 is involved in
maintaining muscle gene expression and determination of specific cardiac cell
types from pluripotent progenitors130, 131. Additionally, the targeted deletion of
the Dicer gene, which is critical for processing of pre-miRNAs into their mature
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form in the heart, provokes spontaneous cardiac remodeling, dilated
cardiomyopathy and HF, reflecting the apparent role of miRNAs to modulate
multiple targets and interfere in the embryonic heart development128, 129. In the
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adult myocardium, several studies have demonstrated the involvement of
miRNAs in a number of physiological110-113, 116, 117 and pathological125, 127, 133-140
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conditions. Changes in cardiac miRNA expression levels have been associated
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with cardiac stress and development of cardiac hypertrophy due to pressure
overload133-135, 141, myocardial infarction139, and also, in humans, end-stage HF127,
142, 143.
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Aerobic ET leads to a physiological, nonpathological left ventricular (LV)

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hypertrophy (LVH). However, in order to study the beneficial effects of ET, a
number of experimental strategies have been established, such as treadmill37, 44,

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61, 144, 145 and voluntary wheel146, 147 running, swimming117, 148, 149 and RT150-152.
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These approaches have been applied to numerous animal models with various
backgrounds. However, important differences exist between these experimental

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approaches, which may affect the interpretation of the results.
Most of the studies performed in rats and mice have applied continuous
treadmill running, characterized by fixed or progressively increasing speed,
inclination, and duration during the session (Table 1 and 2). Different treadmill
running protocols have been developed, lasting from weeks to months, with
individual running session durations ranging from minutes to hours and running
speeds ranging 10-97 m/min, and with the treadmill inclinations ranging 0-25°
(0-47 %)45, 46, 153, 154. Care and colleagues125 assessed the role of cardiac miRNAs
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in the three murine models of cardiac hypertrophy, i.e. transverse aortic arch–
constricted mice, transgenic mice with selective cardiac overexpression of a
constitutively active mutant of the Akt kinase, and ET performed in rats.
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Interestingly, cardiac hypertrophy in all three models (pathological or
physiological cardiac hypertrophy) resulted in reduced expression of both hsa-
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miR-133 and hsa-miR-1.
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Conversely, swimming exercise protocols induce different responses in terms of
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acute and chronic hemodynamic changes compared to running protocols149.
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Additionally, swimming is recognized for its efficiency in inducing myocardial
hypertrophy and a significant increase in LV end-diastolic volume in rats148.

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Therefore, Ma and colleagues111 demonstrated that as swimming exercise-
induced physiological cardiac hypertrophy is correlated with the modulation of

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few miRNAs (rno-miR-21, rno-miR-124, rno-miR-144, and rno-miR-145) that

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has components of the PI3K/AKT/mTOR signaling pathway (Pik3a, Pten, and
Tsc2) as validated targets. Fernandes and colleagues155 studied, in females

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Wistar rats, the effects of two different swimming ET regiments, moderate (60
min/day, 5x/wks for 10 wks with 5% body overload) and high (60 min/day,
5x/wks for 10 wks with 5% body overload, 2-3 times a day) volumes, on
microRNA profile and their targets. Both swimming ET regiments altered the
expression of cardiac miRNAs targeting renin-angiotensin aldosterone system
(RAAS) components and were associated with the development of LVH.
Swimming exercise increased rno-miR-27a and rno-miR-27b, targeting
angiotensin converting enzyme (Ace) gene, and decreased rno-miR-143,
targeting Ace type 2 (Ace2), in the heart. da Silva Jr and colleagues116 showed
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that swimming ET also induced an increase in rno-miR-126 which target Spred-1
(associated with angiogenesis), which decreased after 10 weeks of ET. Although
Pi3kr2 is also a well-validated target for rno-miR-126, swimming exercised rats
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did not change its expression.
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The involvement of different members of the miR-29 family in the cardiac
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hypertrophy induced by swimming exercise training has been also assessed.
Rno-miR-29c expression increased in trained rats and was inversely correlated
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with ColIaI and ColIIIaI expression and OH-proline concentration, which is
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relevant to the improved left ventricle compliance observed113. Members of the
miR-29 family (miR-29a, miR-29b-1, miR-29b-2, miR-29c) seem to orchestrate a

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complex network of signaling pathways regulating deposition of extracellular
matrix proteins in different tissues and pathological conditions156. In fact, miR-

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29 family has been implicated in development of cardiac139, liver157, renal158 and

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pulmonary159 fibrosis. Moreover, it has been described that the cardiac
remodeling associated with hypertrophic cardiomyopathy in patients

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determines a release of hsa-miR-29a into the bloodstream to be significantly
associated with both hypertrophy and fibrosis. This data suggest that this miRNA
family has the potential to be used as a circulating biomarker for myocardial
remodeling assessment in patients with hypertrophic cardiomyopathy160.
Interestingly, ET seems to modify the expression levels of the different members
of the miR-29 family under pathological conditions. Swimming ET restores
cardiac rno-miR-29ac levels and prevents collagen deposition in the border zone,
as well as, in the remote myocardium, of the infarcted hearts, which may
contribute to the improvement in ventricular function induced by swimming

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ET112. The studies described above collectively suggest that swimming ET in
experimental animal models seems to change the cardiac levels of several
miRNAs (miR-27a, -27b, -143, -29ac, and -126) influencing a broad spectrum of
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targets, from components of the RAAS to the Raf-1/Erk1/2 or collagen pathways;
and, associated with different phenotypes, tissue remodeling, angiogenesis, etc.
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Nevertheless, the long-term effects of ET on cardiac miRNA expression are
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particularly dependent on the modalities, e.g., motorized treadmills, voluntary
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running wheel, and swimming or RT. Figure 2A compiled all the results
presented in the current review (Table 1 and 2) and shows a lack of overlap
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regarding cardiac miRNAs profile in different animal models (mice and rats).
This suggest that the molecular and cellular adaptations in the heart, at least at
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the miRNA level, are distinct according to the type of ET. Importantly, the
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current review sheds some lights in the especificities of exercise training
modalities and miRNA expression, and hopes to help scientists to choose the
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most appropriate ET modality and/or animal model for their investigation.

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Nevertheless, any inference regarding the influence of ET on cardiac miRNAs is
limited to data obtained from preclinical studies (mostly in small rodents). No
clinical study has been published so far, uncovering the effects of ET on the
cardiac miRNA profile. Changes in cardiac miRNA expression levels have been,
however, described in CVD patients. A few years ago, a randomized clinical trial
study was conducted by our group and demonstrated that remote ischemic
preconditioning (RIPC) preserves mitochondrial function and influences miRNA
expression in atrial myocardium during coronary bypass surgery161. MiR-133a

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and miR-133b were found upregulated after aortic cross-clamping in both RIPC
and control groups, whereas miR-1 was upregulated in control only, and miR-
338-3p expression level was higher in RIPC versus control after aortic cross-
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clamping.
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In line with these ideas, several biotech and pharmaceutical companies have
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invested in clinical trials on miRNA-based therapy in management of cancer,
metabolic diseases, chronic infections and mycosis (for reference cite162). In one
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of the first reports, miR‑122‑targeted locked nucleic acids (LNAs) inhibitor
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(Mirvirasen, Santaris Pharma A/S and Hoffmann‑La Roche) was used to treat
Hepatitis C-infected patients, in a multicentre phase IIa clinical trial, and

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demonstrated a significant reduction in viral titres163. More recently, a
multicentre phase I clinical trial (ongoing) has been conducted to treat

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Cutaneous T cell lymphoma and mycosis fungoides patients by using

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antimiR‑155 LNA-modified antisense inhibitor (MRG-106, miRagen
Therapeutics). In addition, antimiR‑103/-107 (RG-125/ AZD4076, Regulus

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Therapeutics) is in use to treat patients with type 2 diabetes and non-alcoholic
fatty liver diseases, in a single-centre phase I/IIa clinical trial (ongoing).
Skeletal Muscle MicroRNAs
Although miRNAs have been implicated in skeletal muscle development,
regeneration, and function, our understanding of the molecular mechanisms

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underlying the regulation of skeletal muscle mass by miRNAs is still limited.
Exercise, nutrition, and disease affect skeletal muscle mass postnatally, and they
were also recently shown to alter skeletal muscle miRNA expression164, 165.
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Several of miRNAs have been implicated in these processes (Tables 1, 2 and 3).
For instance, miR-206166, and miR-1115 and miR-133115 are grouped as
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“myomiRNAs” or myocytes miRNAs, as they are specific to or enrich the skeletal
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muscle tissue and play important roles in the regulation of muscle development
and differentiation167.
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Either loss- and gain-of-function experiments have shown that miR-208b and
miR-499 play important and redundant roles in the specification of muscle fiber
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identity by activating slow and repressing fast myofiber gene programs168. The
mmu-miR-208b-/-; mmu-miR-499-/- double knockout (dKO) mouse presented a

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marked loss of slow myofibers in the soleus, and also reduced expression of slow
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-MHC at the protein and mRNA levels. Conversely, muscle-specific
overexpression of mmu-miR-499 was sufficient to induce a complete conversion

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of all fast myofibers in soleus to a slow, type I phenotype168.
Similarly to what has been described in the heart, miRNAs can mediate the
exercise training-induced changes in the skeletal muscle phenotype. In fact,
exercise can rapidly and transiently regulate several miRNAs in the skeletal
muscle. Russell and colleagues71 have performed measurements in muscle
biopsies from healthy untrained males at rest, after a single bout of moderate-
intensity endurance cycling and after chronic endurance cycling training. In the
3h period following the acute exercise bout hsa-miR-1, hsa-miR-133a, hsa-miR-

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133-b and hsa-miR-181a were all increased. Hsa-miR-9, hsa-miR-23a, hsa-miR-
23b and hsa-miR-31 were decreased. In contrast, short-term ET regime
increased hsa-miR-1 and hsa-miR-29b, while decreasing hsa-miR-31 in the
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skeletal muscle biopsies. Additionally, luciferase reporter activity assay validated
both HDAC4 and NRF1 as direct targets for hsa-miR-31. In another study, Nielsen
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and colleagues86 demonstrated that hsa-miR-1 and hsa-miR-133a expression
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levels in the vastus lateralis of healthy individuals were significantly upregulated
in response to a single bout of cycle ergometer exercise at 65% of Pmax86. On the
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other hand, 12 weeks of endurance ET on a cycle ergometer resulted in a
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decrease in the hsa-miR-1, hsa-miR-133a, hsa-miR-133b, and hsa-miR-206.
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Of interest, miR-1 and miR-133, clustered on the same chromosomal loci, are
transcribed together in a tissue-specific manner during development and have

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distinct roles in the modulating skeletal muscle proliferation and

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differentiation169. Therefore, miR-1 and miR-133 are both involved in cardiac-
related125, 140, 161 and skeletal-related169-171 human diseases.

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Moreover, mmu-miR-494 levels significantly decrease after endurance ET in
C57BL/6J mice, accompanied by an increase in expression of mtTfa and Foxj3
proteins. These results suggest that miR-494 regulates mitochondrial biogenesis
by downregulating mtTfa and Foxj3 during myocyte differentiation and skeletal
muscle adaptation to ET172.
The benefits of swimming ET in the skeletal muscle vasculature have also been
studied in an experimental model of arterial hypertension (i.e., spontaneously

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hypertensive rats, SHR). Fernandes and colleagues117 investigated the miRNAs
profile after ET, and the changes in the vascularization in the skeletal muscle. It
was found that ET reduced rno-miR-16 and rno-miR-21 expression and elevated
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vascular endothelial growth factor (Vegf) and Bcl-2 levels, key proteins in
angiogenesis and apoptosis, respectively. Moreover, rno-miR-126 level, which
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was reduced in the hypertensive sedentary rats, was normalized by ET in the
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hypertensive rats accompanied by changes in the phosphoinositol-3 kinase
regulatory subunit 2 (Pi3kr2) levels117.
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Nevertheless, human subjects show considerable variation in the extent of ET
adaptation. Several factors, e.g.. age, sex, race, and initial fitness level, are known
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to play a role. In addition, genetics may certainly explain a large variability in the
ET response12, 173, 174. In order to verify the role of miRNAs in the ET

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responsiveness Davidsen and colleagues114 collected biopsies from the vastus

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lateralis muscle of healthy men, stratified according to their capacity to gain
muscle mass with RT (i.e., low and high responders), and showed a differential
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expression profile in hsa-miR-378, hsa-miR-29a, hsa-miR-26a, and hsa-miR-
451114. Interestingly, miR-133a/b and miR-1 expression levels, also known as
myomiRNAs, were found unaffected after a 12-wk RT program. The authors also
examined the changes in the mRNA expression levels of three genes (IGF-I, VEGF-
A, eIF4E2) that were predicted as targets of the miRNAs found in the study.
Taken together, the data suggested that miRNAs play an important role in
regulating the translation of key pathways responsible for skeletal muscle
growth in response to RT in high and low responder individuals.

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Taken together, the studies reported in the present review suggest that
myomiRNAs play an important role in the skeletal muscle adaptations to ET in
both human health and disease. Precisely, ET can modify the levels of several
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miRNAs, e.g.. miR-1, miR-16, miR-21, miR-26a, miR-29a, miR-126, miR-133a,
miR-133b, and miR-206, miR-378, miR-451, and miR-494. Coordinated changes
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in the expression levels of these myomiRNA contribute to skeletal muscle
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adaptations to acute and chronic ET.
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Circulating MicroRNAs
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Recently, miRNAs have arisen as promising circulating biomarkers of disease, as
large amounts of stable miRNAs can enter the circulation106. Increased

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circulating levels of hsa-miR-1 have been associated with myocardial

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infarction175, while increased circulating levels of hsa-miR-423 have been
associated with HF176. Furthermore, increased circulating levels of the cardiac

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muscle-enriched hsa-miR-208 have been reported in patients with stable
coronary artery disease (CAD) and with myocardial damage caused by CVD80-82,
107. Elevation in another myocardial-enriched miRNA, hsa-miR-133a, has been
reported in the plasma of patients with stable CAD81. Additionally, increased
serum levels of hsa-miR-210 have been reported in patients with atherosclerosis
and kidney disease84, 177. On the other hand, several miRNAs have also been
reported to be downregulated in patients with stable CAD81, like the endothelial-

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associated hsa-miR-126, hsa-miR-17, and hsa-miR-92a; the inflammation-
associated hsa-miR-155; and the smooth muscle-enriched hsa-miR-145.
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With regard to physiological conditions, during the last few years there has been
a rise in the number of studies on circulating miRNAs in the bloodstream
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induced by both acute and chronic exercise76-79, 85, 90, 121, 123, 178. Release of
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miRNAs into the circulation after acute exhaustive exercise is likely to be a result
of exercise-induced tissue stress and/or damage. For instance, Baggish and
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colleagues77 reported that vascular endothelial cells are capable of releasing high
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levels of both hsa-miR-21 and hsa-miR-222 into the circulation immediately
after acute exhaustive cycling exercise.

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The same authors77 highlighted the rapid mobilization of circulating miRNAs in

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response to a cardiopulmonary exercise test, as they reported a rapid

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upregulation of miR-146a, miR-222, miR-21, and miR-221 in the setting of acute
exercise (cardiopulmonary exercise test), followed by a restoration of the plasma

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levels in some of the circulating miRNAs (miR-146a, miR-222 and miR-21) after
1 h of rest following acute exercise. Moreover, Nielsen and colleagues123
performed a screening for miRNAs in the circulation from young healthy men
before and after an acute endurance exercise bout (60 min cycle ergometer
exercise bout at 65% of Pmax) and following endurance ET (60-120 min/section
on cycle ergometer, 5x/wk, 12 wks, 55-91% of Pmax). These authors
demonstrated that ET orchestrates a dynamic regulation of miRNAs in the
circulation. For instance, immediately after the acute exercise bout, hsa-miR-
106a, hsa-miR-221, hsa-miR-30b, hsa-miR-151-5p, hsa-let-7i, hsa-miR-146, hsa-

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miR-652 and hsa-miR-151-3p were significantly downregulated in the
circulation, suggesting a bloodstream clearance as a result of uptake from
surrounding tissues. One hour after the acute bout, 5 miRNAs (miR-338-3p, miR-
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330-3p, miR-223, miR-139-5p, miR-143) were found upregulated and after three
hours only hsa-miR-1 was found increased.
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A few studies have also reported changes in circulating miRNAs after long-term
exercise training protocols (Figs 1 and 2; Table 3). Baggish and colleagues77
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studied subjects that performed moderate-intensity endurance ET (rowing) for 3
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months and reported increased basal levels of hsa-miR-21, hsa-miR-146a, hsa-
miR-221 and hsa-miR-222. Another study by Taurino and colleagues178 reported

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that long-term endurance ET (10 weeks) caused an increase in both hsa-miR-92a
and hsa-miR-92b circulating levels in patients with CAD.

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Uhlemann and colleagues103 showed that the mode of ET is important for the
miRNA-signature in the bloodstream. Whereas different protocols of endurance

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ET led to an acute increase in the endothelial-specific hsa-miR-126, RT caused an
increase in the skeletal muscle-specific hsa-miR-133. In another study, miRNAs
levels were measured in serum samples from twelve healthy males performing a
single resistance exercise session (bench press and leg press), consisting of five
sets of 10 repetitions at 70% of maximum strength, with a 1 min rest between
each set. Three days after the resistance exercise session the authors found
increased levels of hsa-miR-149 and decreased levels of hsa-miR-146a and hsa-
miR-22190. Comparing the acute against the chronic effects of exercise on the
miRNAs levels in the circulation, different results have been reported (Fig 2B).
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Nielsen and colleagues123 showed that chronic ET induced changes in a
completely different set of miRNAs in the circulation compared to an acute bout.
Differently, Aoi and colleagues76 found that hsa-miR-486 levels were
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significantly decreased following both acute and chronic exercise.
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Circulating miRNAs have also been the subject of a study in athletes78, 85.
Fourteen male marathon runners were investigated for myomiRNAs and
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inflammation related miRNAs in the plasma before, directly after, and 24h after a
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marathon run85. It was found that hsa-miR-1, hsa-miR-133a, and hsa-miR-206
correlated to aerobic performance parameters, such as VO2max and running

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speed at individual anaerobic lactate threshold. Interestingly, hsa-miR-1 showed
a moderate negative correlation with fractional shortening, whereas hsa-miR-

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133a was positively correlated with the thickness of intraventricular septum.

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None of the miRNAs correlated with cardiac injury markers, such as troponin T,
troponin I, and pro-brain natriuretic peptide85. Baggish and colleagues78

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evaluated the levels of muscle-enriched miRNAs (hsa-miR-1, hsa-miR-133a, hsa-
miR-499–5p), cardiac tissue (hsa-miR-208a), and the vascular endothelium (hsa-

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miR-126), as well as those important in inflammation (hsa-miR-146a) in male
marathon runners at rest, immediately after a marathon (42-km foot race), and
24 h after the race. All the measured miRNAs were found elevated after the
marathon, but hsa-miR-1, hsa-miR-133a, hsa-miR-208a and hsa-miR-146a were
the most robustly upregulated. More recently, it has been reported in data from
the Munich Marathon Study showing that both elite and non-elite runners
exhibited increased plasma levels of miR-30a mmediately after a marathon.

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Interestingly, miR-1 and miR-133a plasma levels also increased but showed
significant changes only in elite runners179.
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Changes in the miRNAs levels in the circulation not only reflect the effects of
both acute and chronic exercise, but also associate with inherited CRF. A recent
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study by Bye and colleagues121 reported that circulating levels of hsa-miR-210
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and hsa-miR-222 were significantly higher in individuals with low VO2max
compared to individuals with high VO2max. Furthermore, hsa-miR-21 was
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elevated in male participants with low VO2max. However, in this particular study
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no correlations were found between hsa-miR-210, hsa-miR-21 and hsa-miR-222
and frequency, intensity, and duration of self-reported regular ET. Another study
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conducted by Baggish and colleagues77 reported a positive correlation between
the circulating levels of hsa-miR-146a and VO2max in 10 male athletic subjects.

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These findings strengthen the hypothesis that CRF biomarkers also have a
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potential as circulating markers of future CV health independent of traditional
biomarkers.
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Taken together the studies reported that even brief bouts of exercise
substantially perturb the cellular homeostasis and dramatically change the
circulating miRNA signature. These changes are dynamic over short periods of
time in acute exercise, as well as over longer periods of time of endurance ET.
Furthermore, detection of miRNAs in the bloodstream after acute and chronic
exercise seems to be due to selective secretion rather than generalized passive
release due to muscle damage, for example. Therefore, detection of miRNAs in
the bloodstream may become a reliable marker for CRF or a diagnosis and
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prognosis marker for chronic diseases (i.e. CVD). In fact, the diagnostic and
prognostic value of circulating miRNAs is currently exploited by several groups
around the world74, 80-82, 89, 104, 160, 177. However, the mechanisms underlying the
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exercise-induced miRNA secretion in the bloodstream remain unclear. The vast
majority of the circulating miRNA data available in the literature is restricted to
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associations uncovered by observational studies, limiting any mechanistic
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inference. The synergy of mechanistic (preclinical) and multicentric, prospective
large-scale studies is therefore required to determine the potential use of
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circulating miRNAs as biomarkers for the development of complex traits and
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diseases.
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MicroRNAs and Single Nucleotide Polymorphisms (SNPs)

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More recently, a myriad of studies180, 181 have been focused on identification and
description of single nucleotide polymorphisms (SNPs) located in non-coding

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regions and their role in the gene regulation in association with different
pathological conditions. Particularly, genetic variants influencing miRNA post-
transcriptional regulation have been investigated182-184. Single nucleotide
polymorphisms can influence miRNAs function in several ways, depending on
where positioned: if located in the pre-miRNA (outside the seed sequence); or, in
a 3’UTR of a target gene; or, in miRNAs promoter region; or, if a mutation
generates an alternative polyA site; or, even, if a gene responsible for the miRNA
biogenesis is mutated (i.e., Drosha, Dicer, and Argonaute).

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Interestingly, miRNA sequence variation can broadly affect miRNA function, even
when the nucleotide mismatch is located far away from the critical 5’ seed
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sequence generally considered to drive mRNA target recognition and
suppression. In an elegant study, Dorn and colleagues184 showed that a mutation
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outside the seed sequence of hsa-miR-499 alters cardiac mRNA targeting and
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end-organ function. Son and colleagues181 reported that a promoter
polymorphism (rs1042522) of pri-miR-34b/c is associated with an increase in
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the risk of hepatocellular carcinoma in the Korean population. In addition,
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genetic variations in the miRNA-processing machinery component (DROSHA)
can affect miRNA expression levels and lung cancer-specific survival185.

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Furthermore, AGO1 rs636832 A>G might also determine the susceptibility to
lung cancer, suggesting that the AGO1 and/or miRNAs might be involved in the
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development of lung cancer186.

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SNPs can interfere directly in the post-transcriptional regulation of miRNAs

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when is positioned in the mRNA target sites. Amin and colleagues182
demonstrated that rs2519184, rs8234, and rs10798 variants in the 3’UTR of the
KCNQ1 (Kv7.1 potassium channel) modify disease severity in patients with long
QT Syndrome type 1 in an allele-specific manner. Therefore, these authors
suggest that assessing these 3’UTR SNPs can predict disease severity of a given
KCNQ1 mutation in one family. Moreover, it has been shown that a functional
variant (rs2507800) in the 3’UTR of angiopoietin-1 gene might reduce risk of
stroke by interfering with the binding efficiency of hsa-miR-211180. Similarly, a
rs3735590 variant of PON1 3’UTR interferes with hsa-miR-616 binding to

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increase the risk of ischemic stroke and carotid atherosclerosis187. Skeletal
muscle phenotypes seem to be equally influenced by genetic variants in miRNA
target site. Clop and colleagues183 verified that a mutation in the myostatin
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(GDF8) 3’UTR creates a target site for hsa-miR-1 and hsa-miR-206, affecting
muscularity in sheep.
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Polyadenylation signals can, for example, be created or disrupted by SNPs
altering gene regulation. Thomas and colleagues188, matching RNA-seq data to
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miRNA expression profile, showed an association between alternative polyA site
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strength (signal and GU-content) and loss of miRNA target sites, allelic imbalance,
and transcript expression. Accordingly, alternative polyA motifs created by SNPs

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can affect miRNA-based gene regulation and thereby are potential disease-
causative variants.
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More recently, we were able to describe a new genetic variant (rs540) within a
miRNA target site in the TMEM8A (transmembrane protein 8A) 3’UTR associated
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with VO2max in adult healthy males126. Interestingly, a point mutation in the
TMEM8A 3’UTR creates a new target site for hsa-let-7a-3p and affects TMEM8A
gene expression through miRNA regulation (Table 3).
Other Conditions
MiRNAs changes induced by exercise training have been investigated in a few
other conditions or pathological diseases, e.g. spinal cord injury, senescence and
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the immune system. Here we detailed the exercise-induced miRNAs regulation in
those particular conditions (Fig 1; Tables 1 and 2).
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Spinal cord injury changes in the skeletal muscles are characterized by decrease
in gross muscle size, myofiber atrophy, and altered expression of myosin heavy
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chain proteins, and lead to loss of strength and endurance of the muscle189, 190.
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With regard to therapeutic treatment of spinal cord injury, it has generally been
accepted that exercise is an effective therapy that maintains muscle mass191,
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stabilizes rhythmic firing patterns of lumbar motoneurons192, 193 and improves
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functional motor and sensory recovery194, 195 after spinal cord injury. Over the
past years, we have witnessed some profiling attempts to uncover the miRNAs
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involved in this process. Liu and colleagues196 screened for miRNAs associated
with the damage to the spinal cord and treatment using exercise in an
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experimental model. Briefly, the spinal cord injury was experimentally induced
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in Sprague-Dawley rats by exposing the dorsal surface of spinal cord at the level
of the tenth thoracic vertebra and aspiration of the spinal cord through a
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micropipette in order to create a 2 mm long complete transection lesion; ET
consisted of a passive form of hind limb cycling exercise (45 rpm, 30 min, 2x per
day, starting 5 days after the spinal cord transection, 5 days per week, for 20
days). Spinal cord injury induced a significant decrease in the rno-miR-15b level.
Five days of exercise (passive form of hind limb exercise) after the injury
resulted in a significant increase in the expression of rno-miR-21 and an even
larger reduction of rno-miR-15b. In addition, changes in the microRNA levels
were accompanied by changes in the expression of target genes of these miRNAs
and a significant increase in the expression of mRNA for the anti-apoptotic

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marker Bcl-2196. In a subsequent article, the same group showed that the activity-
dependent plasticity in the injured spinal cord is modulated, at least in part,
through miRNAs (rno-miR-21 and rno-miR-199a) in spinal cord tissue that
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regulates Pten and mTor signaling and may indicate that exercise (cycle
ergometer, 5 days/week for 31 days) leads to an increase in the regenerative
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potential of neurons affected by a spinal cord injury119. Therefore, these studies
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provide evidences that cycling ET is a potent modulator of miRNAs expression
that affect both growth-associated Pten/mTor and apoptosis-associated
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pathways after spinal cord injury in rats. These pathways are a critical regulator
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of protein synthesis, axon regeneration and plasticity in the damaged central
nervous system.
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Exercise is beneficial for healthy aging and can improve cognitive function in
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older people, as well as people with existing neurological diseases, such as

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multiple sclerosis, Parkinson's and Alzheimer's disease197-199. However, the
cellular and molecular mechanisms underlying the beneficial effects of ET in

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people with neurological conditions are poorly understood. Recently, Cosín-
Tomás and colleagues200 studied the miRNAs expression profile in SAMP8 mice
and the modulation by exercise in the central nervous system, more precisely in
the hippocampus. The senescence-accelerated mouse prone (SAMP8) is an
animal model of age-related cognitive decline. The learning and memory
disturbances develop spontaneously and are due to overproduction of amyloid
precursor protein in the central nervous system, a similar mechanism
responsible for the pathophysiology of the Alzheimer’s disease201. The authors
found 84 differentially expressed miRNAs in the hippocampus that are suitable

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as senescence markers in this neurological condition. Furthermore, 8 weeks of
voluntary running wheel potentiated (mmu-miR-28a, mmu-miR-98, mmu-miR-
148b, mmu-miR-7a and mmu-miR-15b) the expression levels of few miRNAs, as
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well as restored (mmu-miR-105 and mmu-miR-133b) the transcription of other
miRNAs. Additionally, exercise training led to an increase in several neurogenic
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factors (i.e., Igf1, Bdnf, etc.) in the circulation and in the hippocampus in the
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SAMP8 strain. Data suggests that miRNAs are involved in the downregulation of
the target genes that control accelerated senescence. Therefore, miRNAs are
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increasingly recognized to play valuable roles in different pathological diseases
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and the development of new therapeutic strategies for senescence and
neurodegeneration.
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Exercise seems to also boost the immune system. In fact, it has been well
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demonstrated that ET modulates several parameters of both natural and

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acquired immunity202, 203, reducing the number of infections, inhibiting
tumourigenesis, and enhancing resistance to tumor implantation56, 202-205.

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Exercise seems to affect the immune system according to type, duration and
intensity of exercise, training or nutritional status, and environmental conditions
(temperature or altitude). In terms of mechanisms, a single brief bout of exercise
causes substantial changes in the expression of gene pathways in different
immune cells and inflammatory mediators. Recent publications have highlighted
the involvement of miRNAs in regulating the immune response induced by
exercise87, 88, 102, 118. Radom-Aizik and colleagues88 investigated the effects of an
acute bout of exercise (ten 2-min bouts of cycle ergometer exercise, at 76% of
VO2max) in the mRNA/miRNA transcriptional levels in circulating neutrophils.

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Neutrophils are the most abundant type of white blood cells in mammals,
comprising about 40-75% of all white blood cells, and form an essential part of
the innate immune system. Exercise seems to lead not just to increased numbers
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of neutrophils in the circulation, but also to substantial changes in expression
gene206. Pathway analysis revealed an enrichment of the gene associated with
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important inflammatory mechanisms (Ubiquitin-mediated proteolysis pathway,
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the JAK/STAT signaling pathway and the Hedgehog signaling pathway)88.
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In a subsequent study, Radom-Aizik and colleagues87 evaluated the effects of
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acute exercise (brief bouts of heavy exercise) on miRNAs expression in
circulating peripheral blood mononuclear cells, leukocytes, natural killer (NK)

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cells, and monocytes. The authors reported altered expression level of 34
miRNAs, mostly related to inflammatory processes. Among others, they reported

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a downregulation of hsa-miR-125b, hsa-let-7e and upregulation of hsa-miR-132.

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Furthermore, another paper by Radom-Aizik and colleagues102 studied the
effects of acute exercise on miRNA expression in natural killers cells. Briefly, NK

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cells are unique innate immune cells that increase up to fivefold in the circulating
blood with brief exercise and are known to play a key role in first-response
defense against pathogens and in cancer immunosurveillance. In total, 13
individuals have been submitted to an acute bout of exercise (cycle ergometer,
10x 2-min bouts, 1-min rest interval between each bout, 77% of VO2peak) and 23
differently expressed miRNAs and 986 genes altered by exercise have been
identified (Table 3)102. The authors identified 7 KEGG pathways (p53 signaling
pathway, melanoma, glioma, pathways in cancer, adherens junction, and focal
adhesion) found enriched with genes.

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More recently, the same group evaluated the transcriptomic
(mRNAs/microRNAs) profile in circulating monocytes immediately after a single
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bout of exercise118. In total, 20 healthy men were submitted to an acute exercise
session (ten 2-min bouts of cycle ergometer exercise at 82% of VO2max
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interspersed with 1-min rest) and 894 genes and 19 miRNAs were found
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differently expressed. The authors believe that the changes found in the
mRNAs/miRNAs are likely to direct monocytes in an anti-inflammatory, anti-
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atherogenic pathway. These data support the hypothesis that exercise affects the
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gene and miRNA expression pattern in different population of circulating cell
lines (monocytes, NK, neutrophils, etc.) and suggest mechanisms through which
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both acute and chronic exercise training could alter the immune system.
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Concluding Remarks and Future Perspectives
Altogether, we have underlined the role of miRNAs as essential mediators of

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processes associated with exercise adaptation including cardiac and skeletal
muscle hypertrophies, angiogenesis, atherosclerosis, neuron regeneration,
metabolism, and CRF207, 208. Although a considerable number of preclinical
investigations on miRNA therapeutics have been conducted over the past years,
only a small number has so far moved into clinical application. Therefore,
specifically within the Exercise Physiology field, we can list few important points
that have to be properly addressed: First, a better understanding of miRNA
dynamic changes over shorts periods of time in acute exercise, as well as, over
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longer periods of time of endurance ET is not fully known; Secondly, we here
stressed how critical is the selection of ET protocols/modalities, as well as, a
choice of an animal model, regarding miRNA adjustments; Thus, a more detailed
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description of miRNA-mediated gene regulation induced by ET, especially into
preclinical and large clinical setups, is indispensable. Additionally, it is equally
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essential to address other more fundamental questions regarding miRNAs
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function, e.g. miRNA biogenesis (for referece see12) and the role of genetics (for
reference see179). Equally important, it is highly desirable to elucidate the use of
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miRNAs as circulating biomarkers, such as, miRNA release mechanisms (e.g.,
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extracellular vesicles, exosomes, and ceramide-dependent pathway)(for
reference see207, 208) and miRNA active role (e.g., miRNA communication through
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exosomes). Finally, in the near future it will be important to expand the miRNA
field of investigation towards other regulatory noncoding RNA (ncRNA)

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molecules (for reference see209). A better undertanding of these mechanisms can

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guide us to prevent and treat CVD, by uncovering new potencial therapeutic
targets. For instance, an in vivo therapeutic approach using a combination of key

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miRNAs, named “miR combo”210-212, has shown promising results.
Acknowledgments
We gratefully acknowledge Nina Zisko for critically proof-reading of the
manuscript. This research was supported by grants from the K.G. Jebsen
Foundation, The Research Council of Norway, and The Norwegian Council of
Cardiovascular Disease.
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Figure Legends
Fig 1 - miRNAs up and downregulated by exercise training in different tissues
and in the circulation. * other conditions included spinal cord lesion and brain
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phenotypes.
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Fig 2 - Effects of acute and chronic exercise training on miRNAs. (A) Exercise
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training modality (resistance training vs. aerobic exercise training on motorized
treadmill vs. voluntary running wheel vs. swimming exercise training)
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influencing miRNAs expression in the heart; (B) Acute and chronic exercise
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inducing changes in circulating miRNAs.
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miR-1 miR-126
miR-17 miR-133b
miR-19b miR-144
miR-21 miR-145
miR-27a miR-150
miR-1 miR-181
miR-27b miR-208a
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miR-29a miR-216a miR-23a miR-200c miR-20a miR-146a
miR-29c miR-222 miR-27a miR-376a miR-21 miR-221 miR-7a miR-98
miR-30b miR-484 miR-107 miR-377 miR-103 miR-222 miR-15b miR-105
miR-30e miR-488 miR-126 miR-499b miR-107 miR-376a miR-21 miR-133b
miR-96 miR-136 miR-558 miR-126 miR-28a miR-148b
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Heart Skeletal Muscle Circulation Other Conditions*
(serum/plasma)
miR-1 miR-181a miR-1 miR-486 let-7d miR-185 miR-15b miR-199a
miR-22 miR-191a miR-16 miR-494 miR-16 miR-192
miR-99b miR-208a miR-21 miR-509 miR-21 miR-193b
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miR-100 miR-208b miR-23 miR-520a miR-25 miR-342
miR-124 miR-214 miR-28 miR-548an miR-27a miR-486
miR-143 miR-425 miR-30d miR-628 miR-28 miR-766
miR-133a miR-653 miR-148a
miR-133b miR-670
miR-204 miR-696
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miR-206 miR-761
miR-330 miR-889
miR-345 miR-1245a
miR-375 miR-1270
miR-378 miR-1280
miR-449c miR-1322
miR-483 miR-3180
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Fig 1
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A Cardiac miRs Treadmill

Resistance
Swimming
↓mir-425 ↑ miR-96
↓ miR-214
↑ miR-216a
↓ miR-1 ↑ miR-1 ↑ miR-484
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↓ miR-22 ↑ miR-17 ↑ miR-488
↓ miR-99b ↑ miR-19b
↓ miR-100 ↑ miR-21
↓ miR-124 ↑ miR-27a
↓ miR-143 ↑ miR-27b
↓ miR-181a ↑ miR-29a
Voluntary wheel
↓ miR-191a ↑ miR-29c
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↓ miR-208a ↑ miR-30b
↓ miR-208b ↑ miR-30e
↓ miR-214 ↑ miR-126
↑ miR-150
↑ miR-133b
↑ miR-222
↑ miR-144
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↑ miR-145
↑ miR-208a
B c-miR (plasma)
Acute
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↑ miR-1 Chronic
↑ miR-133a
↑ miR-133b
↑ miR-139
↑ miR-143
↑ miR-181b ↓ let-7d
↑ miR-206 ↓ miR-16
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↓ let-7i
↑ miR-208b ↓ miR-21
↓ miR-30b ↑ miR-20a
↑ miR-214 ↑ miR-21 ↓ miR-25
↓ miR-106a ↑ miR-103
↑ miR-223 ↑ miR-146a ↓ miR-27a
↓ miR-146a ↑ miR-107
↑ miR-330 ↑ miR-221 ↓ miR-28
↓ miR-151 ↑ miR-126
↑ miR-338 ↑ miR-222 ↓ miR-148a
↓ miR-221 ↑ miR-376a
↑ miR-485 ↓ miR-185
↓ miR-652
↑ miR-509 ↓ miR-342
↑ miR-517a ↓ miR-766
↑ miR-518f
↑ miR-520f
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↑ miR-522
↑ miR-553
↑ miR-888
Fig 2
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Table 1. Reported changes in miRNA expression in mouse models.
miRNA
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Phenotype
Target Organ/System Type of Exercise* Reference
Up Down Influenced
mmu-miR-150 ___ ___ Heart Cardiac hypertrophy Voluntary wheel (35 days) 213
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Hipk1 Cardiomyocyte growth 214
mmu-miR-222 ___ Heart Voluntary wheel (3 wks)
Hmbox1 and proliferation
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Bcl-2
mmu-miR-21 p53 Swimming (2 times/day, 5 215
mmu-miR-1 Heart Cardiac apoptosis
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mmu-miR-30b Pdcd4 days/week, 8 weeks)
Drp-1
Swimming (90 min, 2
Cardiac growth
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Timp-3 times/day, 21 days) or 216
mmu- miR-17 ___ Heart and myocyte
Pten Voluntary wheel exercise (21
proliferation
days)
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mmu-miR-1 Skeletal muscle Acute exercise bout
mmu-miR-107 mmu-miR-23 Pgc-1α (quadriceps ___ (motorized treadmill, 15 122
mmu-miR-181 femoris) m/min for 90 min)
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Chronic training (motorized
___ mmu-miR-696 Pgc-1α CE
Skeletal muscle
(gastrocnemius)
Metabolism
treadmill, 5 times/wk, 18-32
m/min, 20-60 min per
120
session, 4 wk)
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Pgc-1α Skeletal muscle treadmill, 5 days/wk, 28 217
___ mmu-miR-761 Metabolism
p-Mk2 (gastrocnemius) m/min, 45 min per session, 4
wk)
Nrf1
Mitochondrial
mmu-miR-494 Bim Skeletal muscle 218
___ biogenesis and Voluntary wheel (8 wks)
mmu-miR-696 Bcl-xl (gastrocnemius)
apoptosis
Pgc-1α
Acute (motorized treadmill,
Skeletal muscle
Igf-1 Mitochondrial 14 m/min, 10% grade, 60 219
mmu-miR-133a ___ (extensor
Akt biogenesis min) or Chronic (motorized
digitorum longus)
treadmill, 14 m/min, 10%
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grade, 60 min, 5 d/wk, 6 wks)

exercises
Pten
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mmu-miR-23a Skeletal muscle Resistance exercise (muscle 220
___ Casp7 Muscle atrophy
mmu-miR-27a (tibialis anterior) overload)
FoxO1
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mmu-miR-192 Circulation 221
___ ___ ___ treadmill, 1 h/d, 5 d/wk,
mmu-miR-193b (serum)
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22m/min, 5 wks)
mmu-miR-7a
mmu-miR-15b
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mmu-miR-28a
Hdac3 Brain Voluntary wheel running
mmu-miR-98 ___ Senescence 200
Hdac5 (hippocampus) (8 weeks)
mmu-miR-105
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mmu-miR-133b
mmu-miR-148b
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Table 2. Reported changes in miRNA expression in rat models.
miRNA
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Phenotype
Up Down Influenced
Swimming (60 min, 5
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rno-miR-27a Ace
rno-miR-143 Heart Cardiac hypertrophy days/week, 5% caudal body 110
rno-miR-27b Ace2
weight workload, 10 wks)
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Swimming (60 min, 5 d/wk,
ColIaI
rno-miR-29c ___ Heart Ventricular compliance 5% caudal body weight 113
ColIIIaI
workload, 10 wks)
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Swimming (60 min, 5 d/wk,
Spred-1
rno-miR-126 ___ Heart Angiogenesis 5% caudal body weight 116
Pi3kr2
workload, 10 wks)
MA
rno-miR-21 Pten Swimming (60 min, 5 d/wk,
rno-miR-144 rno-miR-124 Pik3a Heart Cardiac hypertrophy 5% caudal body weight 111
rno-miR-145 Tsc2 workload, 8 wks)
ED
Cardiac fibrosis Swimming (60 min, 5 d/wk,
rno-miR-29a ColIaI
___ Heart induced by myocardial 5% caudal body weight 112
rno-miR-29c ColIIIaI
infarction workload, 10 wks)
PT
Swimming (60 min, 5
Ncx 222
rno-miR-1 rno-miR-214 Heart Ca+2 handling days/wk, 3% caudal body
Serca2a
miR-22
CE weight workload, 10 wks)
rno-miR-19b Igf1
miR-99b Swimming (90 min, 2 x/day,
rno-miR-30e PI3/Akt/mTor
AC
miR-100 Heart Apoptosis 5 d/week, 5% caudal body 223

rno-miR-208a Mapk
miR-181a weight workload, 8 wks)
rno-miR-133b p53
miR-191a
Med13
Pur Swimming (60 min, 5
rno-miR-208a Cardiac hypertrophy 224
___ Sox6 Heart days/week, 5% caudal body
rno-miR-208b and metabolism
Hp1 weight workload, 10 wks)
Sp3
rno-miR-96
rno-miR-216a Treadmill (1 h/d, 5 d/wk, 15 225
rno-mir-425 ___ Heart Cardiad protection
rno-miR-484 m/min, 10 wks)
rno-miR-488
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40
Resistance exercise (4 × 12
___ rno-miR-214 Serca2a Heart Ca+2 handling bouts, 5 x/wk, 8 wks, 80% 226
1-repetition maximum)
PT
Swimming (60 min, 5
rno-miR-16 Bcl-2 Skeletal Muscle
rno-miR-126 Vascularization days/week, 4% caudal body 117
rno-miR-21 Pi3kr2 (soleus)
RI
weight workload, 10 weeks)
Pten
SC
Pdcd4 Cycling (5 days/week, 45
rno-miR-21 rno-miR-15b Ras Spinal cord Apoptosis rpm, 2x 30 min sessions, 10- 196
Casp7 31 days)
NU
Casp9
Cycling (5 days/week, 45
Pten
rno-miR-21 rno-miR-199a Spinal cord Neuron regeneration rpm, 2x 30 min sessions, 10- 119
MA
Mtor
31 days)
ED
PT
CE
AC
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41
Table 3. Reported changes in miRNA expression in human.
miRNA
PT
Phenotype
Up Down Influenced
hsa-miR-1 hsa-miR-9
RI
Acute exercise bout (cycle
hsa-miR-133a hsa-miR-23a HDAC4 Skeletal Muscle
___ ergometer, 60 min, 70% 71
hsa-miR-133b hsa-miR-23b NRF1 (vastus lateralis)
SC
VO2peak)
hsa-miR-181a hsa-miR-31
hsa-miR-1 Acute resistance exercise
Skeletal Muscle
___ hsa-miR-23a ___ ___ (one-legged knee extension, 227
NU
(vastus lateralis)
hsa-miR-133a 45 min, 60% of wattmax)
Acute resistance exercise
hsa-miR-1
Skeletal Muscle (two-legged knee extension, 8
MA
___ hsa-miR-133a ___ ___ 228
(vastus lateralis) sets of 10 repetitions, 70% of
hsa-miR-206
their 1 RM)
hsa-miR-1 Chronic training (cycle
ED
hsa-miR-133a Skeletal Muscle ergometer, 5x/wk, 12 wks,
___ ___ ___ 86
hsa-miR-133b (vastus lateralis) 60-120min/section, 55-91%
hsa-miR-206 of Pmax)
PT
Chronic resistance exercise
Skeletal Muscle (leg press, knee extension, leg
___ hsa-miR-1 IGF-1 Hypertrophy 124
CE
(vastus lateralis) curl, hip extension, 3x/8–10
repetitions, 12 wks)
Muscle regeneration,
AC
hsa-miR-1 HDAC4 Skeletal Muscle gene transcription and 71

___ Endurance (cyclying, 10 days)
hsa-miR-29b NRF1 (vastus lateralis) mitochondrial
biogenesis
MCT (cycling, 30 min, 80%
lactate threshold), HIIT
hsa-miR-133a (cycling, 10 times/2 min,
Skeletal Muscle 229
___ hsa-miR-378 mTORC1 ___ 120% lactate threshold) or
(vastus lateralis)
hsa-miR-486 Resistance exercise (8 x 5 leg
press repetitions, 80% of 1-
repetition maximum)
hsa-miR-136 hsa-miR-28 Skeletal Muscle Hypertrophy Chronic resistance exercise (8 230
___
hsa-miR-200c hsa-miR-30d (vastus lateralis) (high or low x 5 leg press repetitions, 80%
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42
hsa-miR-376a hsa-miR-204 responders) of 1-repetition maximum, 12

hsa-miR-377 hsa-miR-330 wks)
hsa-miR-499b hsa-miR-345
PT
hsa-miR-558 hsa-miR-375
hsa-miR-449c
RI
hsa-miR-483
hsa-miR-509
SC
hsa-miR-520a
hsa-miR-548an
hsa-miR-628
NU
hsa-miR-653
hsa-miR-670
hsa-miR-889
MA
hsa-miR-1245a
hsa-miR-1270
hsa-miR-1280
hsa-miR-1322
ED
hsa-miR-3180
hsa-miR-26a
Skeletal Muscle
PT
hsa-miR-451 hsa-miR-29a ___ Skeletal mass gain High vs. low responders 114
(vastus lateralis)
hsa-miR-378
hsa-miR-125a hsa-let-7i
CE
hsa-miR-181b hsa-miR-17
hsa-miR-193a hsa-miR-18a
AC
hsa-miR-197 hsa-miR-18b
hsa-miR-212 hsa-miR-20a
hsa-miR-223 hsa-miR-20b Acute exercise bout (cycle
Circulating
hsa-miR-340 hsa-miR-22 ergometer, 10x 2-min bouts,
___ (neutrophils Immune system 88
hsa-miR-365 hsa-miR-93 1-min rest interval between
cells)
hsa-miR-485 hsa-miR-96 each bout, 76% VO2peak)
hsa-miR-520d hsa-miR-107
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43
hsa-miR-363
PT
hsa-miR-660
hsa-let-7e
RI
hsa-miR-23b
SC
hsa-miR-15a hsa-miR-99a
hsa-miR-26b hsa-miR-125b
NU
Circulating
hsa-miR-140 hsa-miR-130a Acute exercise bout (cycle
(peripheral
hsa-miR-181a hsa-miR-145 ergometer, 10x 2-min bouts,
MA
___ blood Immune system 87
hsa-miR-181b hsa-miR-151 1-min rest interval between
mononuclear
hsa-miR-181c hsa-miR-199a each bout, 76% VO2peak)
cells)
ED
PT
hsa-miR-584
hsa-miR-652
hsa-miR-let-7f-1 hsa-miR-let-7f-1
CE
Circulating
(peripheral Chronic exercise training
hsa-miR-21 hsa-miR-21 VO2peak response to 231
___ blood (running, 3 x/wk, 60mi, 18
AC
hsa-miR-29c hsa-miR-29c exercise training

mononuclear wks)
cells
hsa-miR-7 hsa-let-7e
hsa-miR-29a hsa-miR-126
hsa-miR-29b hsa-miR-130a
hsa-miR-29c hsa-miR-151 Acute exercise bout (cycle
Circulating
hsa-miR-30e hsa-miR-199a ergometer, 10x 2-min bouts,
___ (natural killers Immune system 102
hsa-miR-142 hsa-miR-221 1-min rest interval between
cells)
hsa-miR-192 hsa-miR-223 each bout, 77% VO2peak)
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44
hsa-miR-15a
hsa-miR-29b
hsa-miR-29c
PT
hsa-miR-30e
hsa-miR-23b
hsa-miR-140
hsa-miR-130a Acute exercise bout (10x 2
RI
hsa-miR-324 Circulating
hsa-miR-151 ___ Immune system min bouts of cycle ergometer 118
hsa-miR-338 (Monocytes)
hsa-miR-199a exercise, 82% VO2max)
SC
hsa-miR-362
hsa-miR-221
hsa-miR-532
hsa-miR-660
NU
hsa-miR-1202
hsa-miR-1305
Circulation
MA
(skeletal muscle Acute exercise bout
hsa-miR-133b 232
___ ___ releases CRF (treadmill, 40 min, 1% grade,
hsa-miR-181a
extracellular 80% VO2max)
vesicles)
ED
Cardiac rehabilitation
hsa-miR-92a Circulating
___ ___ ___ programme (60 min, 2/wk, 178
hsa-miR-92b (whole blood)
PT
10 wks)
hsa-miR-1
Circulating Endurance athletes (runners,
hsa-miR-486 ___ ___ VO2max 233
hsa-miR-494
CE
(whole blood) cyclists, and triathletes)
hsa-miR-21
Acute exhaustive
hsa-miR-146a
AC
cardiopulmonary exercise
hsa-miR-221
test
hsa-miR-222
Circulating
___ ___ ___ 77
hsa-miR-20a (plasma)
Chronic exercise training
hsa-miR-21
(rowing training, 5 km, 1-3 h
hsa-miR-146a
per session, 20-24
hsa-miR-221
strokes/min, 90 days)
hsa-miR-222
hsa-miR-1
Acute exercise bout (30-min
___ ___ ___ downhill and uphill walking 79
hsa-miR-133b (plasma)
exercises)
hsa-miR-181b
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45
hsa-miR-208b
hsa-miR-214
hsa-let-7i Acute exercise bout (60min
PT
hsa-miR-139 hsa-miR-30b cycle ergometer exercise bout
hsa-miR-143 hsa-miR-106a at 65% of Pmax)
RI
SC
hsa-miR-652
Circulating
___ ___ Chronic training (cycle 123
(plasma)
NU
hsa-miR-103 hsa-let-7d ergometer, 5x/wk, 12 wks,
hsa-miR-107 hsa-miR-21 60-120min/section, 55-91%
hsa-miR-25 of Pmax)
MA
hsa-miR-148a
hsa-miR-185
hsa-miR-342
hsa-miR-766
ED
hsa-miR-1
hsa-miR-133a
PT
hsa-miR-133b
hsa-miR-206 Acute HIIE (motorized
hsa-miR-485 treadmill, 7 x 4 min, ∼85–
hsa-miR-509
___ ___
CE
Circulating
___
95% of HRmax) vs. VICE 234
hsa-miR-517a (plasma) (motorized treadmill,
hsa-miR-518f matched distance covered
AC
hsa-miR-520f during the HIIE trial)

hsa-miR-522
hsa-miR-553
hsa-miR-888
High-Volume Training (130
min, 55% peak power output)
hsa-miR-21 Circulating vs. High-Intensity Training
___ ___ ___ 235
hsa-miR-126 (plasma) (4×4 min, 95% peak power
output) vs. Sprint-Interval
Training (4×30s all-out).
hsa-miR-16 Circulating Aerobic exercise training
hsa-miR-376a ___ ___ 236
hsa-miR-27a (plasma) (motorized treadmill, 4
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46
hsa-miR-28 days/wk, 5 months)
hsa-miR-140
PT
Circulating High vs. low responders
hsa-miR-221 ___ ___ Body weight 237
(plasma) (losses in body weight)
hsa-miR-223
RI
hsa-miR-1
hsa-miR-133a
Circulating
SC
hsa-miR-206 ___ ___ VO2max Marathon run 85
(plasma)
hsa-miR-208b
hsa-miR-499
NU
hsa-miR-1
hsa-miR-126
MA
___ ___ ___ Marathon run 78
hsa-miR-134 (plasma)
hsa-miR-146a
hsa-miR-499
Acute exercise bout (single
ED
bout of steady-state cycling
Circulating exercise, 70% VO2max, 60min)
___ hsa-miR-486 ___ ___ 76
PT
(serum)
Chronic exercise (cycling
training 3 d/wk, 4 wks)
CE Acute resistance exercise
(bench press and leg press, 5
hsa-miR-149 ___ ___ sets of 10 repetitions, 70% of
AC
90
hsa-miR-221 (serum)
maximum strength, 1 min rest
between sets)
hsa-miR-19a Acute resistance exercise
hsa-miR-19b bout (3 times, bilateral knee
hsa-miR-20a p-AKTSer473 Circulating extension exercise + bilateral
___ ___ 238
hsa-miR-26b p-S6K1Thr389 (serum) leg press exercise, 10
hsa-miR-143 repetitions, 80% of 1-
hsa-miR-195 repetition maximum.)
Acute exhaustive exercise
hsa-miR-21
Circulating (cycle ergometer, starting
hsa-miR-378 ___ ___ ___ 239
(serum) from 20 J/s for 2 min and
hsa-miR-940
increased by 5J/s every 30s,
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47
60 rpm)
Chronic training (cycle
hsa-miR-192 Circulation 221
___ ___ ___ ergometer, 60-85% VO2max, 2
PT
hsa-miR-193b (serum)
times/wk, 16 wks)
hsa-miR-210 Circulating
hsa-miR-21 ___ VO2max High vs. low VO2max 121
RI
hsa-miR-222 (serum)
SC
Genetic
Markers
hsa-let-7a TMEM8A SNP (rs540) VO2max ___ 126
NU
MCT, moderate-intensity continuous training; HIIT, high-intensity interval training.
HIIE, high-intensity interval exercise; VICE, Vigorous-intensity continuous exercise.
MA
CRF, Cardiorespiratory fitness.
ED
PT
CE
AC
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MicroRNAs As Important Regulators of Exercise Adaptation

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MicroRNAs As Important Regulators of Exercise Adaptation

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Gustavo J.J. Silva, Anja Bye, Hamid el Azzouzi, Ulrik Wisløff

To appear in: Progress in Cardiovascular Diseases

Received date: 25 June 2017

MicroRNAs as Important Regulators of Exercise Adaptation

Short Title: MicroRNA and Exercise

* Address reprint requests and correspondence to: Ulrik Wisløff, K. G. Jebsen

A significant body of evidence supports the protective role of exercise training

of this, the molecular mechanisms underlying the beneficial effects of exercise

coding RNA molecules involved in a variety of basic biological processes that

molecules. In this review, we highlight recent advances on the miRNA

involvement in the benefits of ET. Here, we assess the role of microRNAs

long-term effects of diverse ET modalities (e.g., running, cycling, resistance

training) in the cardiac miRNA profile are properly addressed.

Keywords: microRNA, exercise training, heart, skeletal muscle, spinal cord

Abbreviations and Acronyms

AUC = Area under the curve

CAD = Coronary artery disease

CRF = Cardiorespiratory fitness

CVD = Cardiovascular disease

hsa = Homo sapiens (human)

KEGG = Kyoto Encyclopedia of Genes and Genomes

dKO = Double knockout

LVH = Left ventricular hypertrophy

mmu = Mus musculus (mouse)

myomiRNA = Skeletal muscle microRNA

ncRNA = noncoding RNA

NK = Natural killer cells

pri-miRNA = Primary microRNA transcripts

Pmax = maximum skeletal muscle force

RAAS = Renin-angiotensin-aldosterone system

RIPC = Remote ischemic preconditioning

rno = Rattus norvegicus (rat)

ROC = Receiver Operating Characteristic

SHR = Spontaneously hypertensive rats

SNP = Single nucleotide polymorphism

VO2max = Maximal oxygen uptake

related diseases, such as cardiovascular (CV) disease (CVD) and metabolic

therefore be one of the most important modifiable life-style interventions

indicated for preventing or controlling CV, metabolic or skeletal muscle diseases,

patients with CVD to improve CV health and reduce risk of premature

in both health and disease. Adaptations to endurance training include increased

VO2max, improved lipid profile, improved endothelial function, and increased

abundance of capillaries, mitochondria and metabolic enzymes in different

central feature of regular ET is improved systolic and diastolic function and

larger cardiac output33-36. The long-term effects of ET include cardiomyocyte

shortening and rates of contraction-relaxation improvements, providing a

Experimental studies of cellular effects of regular ET appear therefore to

growth as important cellular mechanisms for the beneficial effects of PA in heart

failure (HF)37, 42-46.

described, including rarefaction, type I to type II fibers switch, impaired

metabolism, and atrophy47-51. Mainly, ET can prevent skeletal muscle atrophy,

the intracellular signaling pathways mediating physiological adaptations induced

Accordingly, recent efforts have identified intracellular mechanisms underlying

laboratories of Dr. Victor Ambros and Dr. Gary Ruvkun discovered

containing sequences complementary to a repeated sequence element in the 3'

translation via an antisense RNA-RNA interaction. Since the first description of

humans, and have revealed a formidable mediator of a broad range of cellular

adaptive processes such as differentiation, proliferation, apoptosis and

metabolism62, 65, 69.

MiRNA biogenesis is a very complex process and involves multiple steps as

miRNAs can be transcribed as from non-coding regions (intergenic) or within the

transcription results in the generation of primary transcripts (pri-miRNA), which