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lncExACT1 and DCHS2 Regulate Physiological and Pathological Cardiac Growth

  • Massachusetts General Hospital and Harvard Medical School
  • EdiGene Biotechnology USA Inc.

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Background: The heart grows in response to pathological and physiological stimuli. The former often precedes cardiomyocyte loss and heart failure; the latter paradoxically protects the heart and enhances cardiomyogenesis. The mechanisms underlying these differences remain incompletely understood. While long noncoding RNAs (lncRNAs) are important in cardiac development and disease, less is known about their roles in physiological hypertrophy or cardiomyogenesis. Methods: RNA sequencing was applied to hearts from mice after eight weeks voluntary exercise-induced physiological hypertrophy and cardiomyogenesis or transverse aortic constriction (TAC) for two or eight weeks to induce pathological hypertrophy or heart failure. The top lncRNA candidate was overexpressed in hearts with adeno-associated virus (AAV) vectors and inhibited with antisense locked nucleic acid (LNA)-GapmeRs to examine its function. Downstream effectors were identified through promoter analyses and binding assays. The functional roles of a novel downstream effector, dachsous cadherin-related 2 (DCHS2), were examined through transgenic overexpression in zebrafish and cardiac-specific deletion in Cas9-knockin mice. Results: We identified exercise-regulated cardiac lncRNAs, termed lncExACTs. lncExACT1 was evolutionarily conserved and decreased in exercised hearts but increased in human and experimental heart failure. Cardiac lncExACT1 overexpression caused pathological hypertrophy and heart failure, while lncExACT1 inhibition induced physiological hypertrophy and cardiomyogenesis, protecting against cardiac fibrosis and dysfunction. lncExACT1 functioned by regulating microRNA-222, calcineurin signaling, and Hippo/Yap1 signaling through DCHS2. Cardiomyocyte DCHS2 overexpression in zebrafish induced pathological hypertrophy and impaired cardiac regeneration, promoting scarring after injury. In contrast, murine DCHS2 deletion induced physiological hypertrophy and promoted cardiomyogenesis. Conclusions: These studies identify lncExACT1-DCHS2 as a novel pathway regulating cardiac hypertrophy and cardiomyogenesis. lncExACT1-DCHS2 acts as a master switch toggling the heart between physiological and pathological growth to determine functional outcomes, providing a potentially tractable therapeutic target for harnessing the beneficial effects of exercise.
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1218 April 19, 2022 Circulation. 2022;145:1218–1233. DOI: 10.1161/CIRCULATIONAHA.121.056850
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Correspondence to: Anthony Rosenzweig, MD, Corrigan Minehan Heart Center, Division of Cardiology, Massachusetts General Hospital, GRB810, 55 Fruit Street,
Boston, MA 02114. Email
Supplemental Material is available at
For Sources of Funding and Disclosures, see page 1232.
© 2022 American Heart Association, Inc.
lncExACT1 and DCHS2 Regulate Physiological
and Pathological Cardiac Growth
Haobo Li , PhD; Lena E. Trager, BA; Xiaojun Liu, PhD; Margaret H. Hastings, PhD; Chunyang Xiao, PhD;
Justin Guerra , BS; Samantha To, BS; Guoping Li, PhD; Ashish Yeri, PhD; Rodosthenis Rodosthenous, ScD;
Michael G. Silverman , MD; Saumya Das , MD, PhD; Amrut V. Ambardekar , MD; Michael R. Bristow , MD, PhD;
Juan Manuel González-Rosa, PhD; Anthony Rosenzweig , MD
BACKGROUND: The heart grows in response to pathological and physiological stimuli. The former often precedes cardiomyocyte
loss and heart failure; the latter paradoxically protects the heart and enhances cardiomyogenesis. The mechanisms underlying
these differences remain incompletely understood. Although long noncoding RNAs (lncRNAs) are important in cardiac
development and disease, less is known about their roles in physiological hypertrophy or cardiomyogenesis.
METHODS: RNA sequencing was applied to hearts from mice after 8 weeks of voluntary exercise-induced physiological
hypertrophy and cardiomyogenesis or transverse aortic constriction for 2 or 8 weeks to induce pathological hypertrophy or
heart failure. The top lncRNA candidate was overexpressed in hearts with adeno-associated virus vectors and inhibited with
antisense locked nucleic acid–GapmeRs to examine its function. Downstream effectors were identified through promoter
analyses and binding assays. The functional roles of a novel downstream effector, dachsous cadherin-related 2 (DCHS2),
were examined through transgenic overexpression in zebrafish and cardiac-specific deletion in Cas9-knockin mice.
RESULTS: We identified exercise-regulated cardiac lncRNAs, called lncExACTs. lncExACT1 was evolutionarily conserved and
decreased in exercised hearts but increased in human and experimental heart failure. Cardiac lncExACT1 overexpression
caused pathological hypertrophy and heart failure; lncExACT1 inhibition induced physiological hypertrophy and
cardiomyogenesis, protecting against cardiac fibrosis and dysfunction. lncExACT1 functioned by regulating microRNA-222,
calcineurin signaling, and Hippo/Yap1 signaling through DCHS2. Cardiomyocyte DCHS2 overexpression in zebrafish
induced pathological hypertrophy and impaired cardiac regeneration, promoting scarring after injury. In contrast, murine
DCHS2 deletion induced physiological hypertrophy and promoted cardiomyogenesis.
CONCLUSIONS: These studies identify lncExACT1-DCHS2 as a novel pathway regulating cardiac hypertrophy and cardiomyogenesis.
lncExACT1-DCHS2 acts as a master switch toggling the heart between physiological and pathological growth to determine
functional outcomes, providing a potentially tractable therapeutic target for harnessing the beneficial effects of exercise.
Key Words: exercise heart failure Hippo signaling pathway hypertrophy RNA, long noncoding Yap1 protein, human
Editorial, see p 1234
Heart failure (HF) is a growing cause of morbidity
and mortality.1 Prognosis remains poor for many
patients with HF despite the best available treat-
ments.1 Physical activity is associated with a lower risk of
HF, 2 although exercise training itself can induce cardiac
hypertrophy. In animal models, exercise training paradoxi-
cally reduces the hypertrophy, dysfunction, and fibrosis
seen in response to pathological stimuli.3 Such observa-
tions suggest that although pathological hypertrophy and
physiological hypertrophy appear similar, they may reflect
fundamentally different underlying mechanisms that exist
in dynamic tension. This model is supported by their distinct
gene expression profiles, which diverge early and include
sentinel differences in expression of markers such as ANP
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
(atrial natriuretic peptide), BNP (brain natriuretic pep-
tide), PGC1α (peroxisome proliferator–activated receptor
gamma coactivator 1-α), C/EBPβ, and myosin heavy chain
isoforms, often used to distinguish these states.4,5
Others have suggested that the difference between
physiological and pathological hypertrophy is simply one of
degree,6 recognizing that exercise is intermittent whereas
disease states are generally constant. Some work has
also suggested that the transition from early, compensated
hypertrophy to HF hinges on other factors such as failure of
angiogenesis needed to support growth of the myocardium.7
Of course, these possibilities are not mutually exclusive.
Another central distinction is that pathological hyper-
trophy is associated with death of cardiomyocytes,8
which are not replaced because of the limited regen-
erative capacity of the adult heart.9 In contrast, exercise
protects against cardiomyocyte death and induces car-
diomyogenesis in the adult mammalian heart.10 Identify-
ing pathways capable of recapitulating these benefits of
exercise with a feasible path to translation would have
important clinical implications.
Using multi-isotope imaging mass spectrometry, we
demonstrated that 8 weeks of voluntary wheel run-
ning increased cardiomyogenesis in adult mice almost
5-fold.10 microRNA (miR)-222, an exercise-induced
microRNA that mitigates adverse remodeling after isch-
emic injury,11 was necessary for exercise-induced cardio-
myogenesis10 and cardiac growth.11 However, miR-222
overexpression was not sufficient to induce cardiomyo-
genesis or physiological hypertrophy in vivo,11 suggesting
the importance of other pathways.
Here we compared cardiac long noncoding RNAs
(lncRNAs) altered in physiological hypertrophy with
pathological hypertrophy or HF. We identified a novel
set of cardiac lncRNAs, called lncExACTs (long non-
coding exercise-associated cardiac transcripts). The
only lncExACTs significantly changed in both the physi-
ological and pathological models changed in opposite
directions. One of these, lncExACT1, was uniquely
downregulated by exercise and upregulated in patho-
logical hypertrophy and HF. Overexpression of lncEx-
ACT1 was sufficient to induce pathological hypertrophy
and HF, whereas its inhibition recapitulated multiple
exercise phenotypes, including physiological hypertro-
phy, increased markers of cardiomyogenesis, and pro-
tection against HF. Mechanistic studies revealed that
lncExACT1 acts by binding miR-222 and modulating
Hippo/Yap signaling through transcriptional regulation
of its genomic neighbor, dachsous cadherin-related 2
(DCHS2). Inhibition of lncExACT1 in vivo by systemic
delivery of antisense oligonucleotides effectively medi-
ated these benefits, underscoring the potential transla-
tional implications of these observations.
The data, methods, and study materials used to conduct the
research will be available from the corresponding author on
reasonable request.
Animals studies were conducted in accordance with the
National Institutes of Health’s Guide for the Care and Use of
Laboratory Animals and approved by the Massachusetts General
Hospital Institutional Animal Care and Use Committee. Human
studies were approved by the Partners or Colorado Multicenter
Clinical Perspective
What Is New?
Long noncoding exercise-associated cardiac tran-
script (lncExACT1) is a conserved long noncod-
ing RNA that decreases in exercised hearts but
increases in failing hearts from animals and people.
• lncExACT1 overexpression induces pathological
hypertrophy and cardiac dysfunction, whereas its
inhibition induces physiological growth and pro-
tects against dysfunction and fibrosis.
What Are the Clinical Implications?
lncExACT1 inhibition reduces cardiomyocyte loss
and scar formation while improving cardiac function
after pathological stress, including ischemic injury
and pressure-overload.
Interventions such as modified nucleic acid anti-
sense drugs, which have been approved by the US
Food and Drug Administration for other indications,
can effectively inhibit cardiac lncExACT1 to miti-
gate cardiac dysfunction and heart failure in animal
models and warrant further investigation as a thera-
peutic strategy.
Nonstandard Abbreviations and Acronyms
AAV adeno-associated virus
ANP atrial natriuretic peptide
BNP brain natriuretic peptide
DCHS2 dachsous cadherin-related 2
EF ejection fraction
GFP green fluorescent protein
gRNA guide RNA
HF heart failure
LNA locked nucleic acid
lncExACT long noncoding exercise-associated
cardiac transcript
miR microRNA
ORF open reading frame
p-Yap1 S127-phosphorylated Yap1
PCG1α peroxisome proliferator–activated recep-
tor gamma coactivator 1-α
sORF small open reading frame
TAC transverse aortic constriction
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
Institutional Review Boards. A detailed description of the meth-
ods and supporting data are available in the Data Supplement.
Statistical Analysis
Data are presented as mean±SEM unless otherwise indi-
cated and were analyzed with GraphPad Prism 8 (GraphPad
Software). We used an unpaired 2-tailed Student t test or, for
assessment of multiple groups, a 1-way ANOVA with Tukey
post hoc test as indicated. In Figure 1E, we used a pair-
wise Wilcoxon rank- sum test with Bonferroni correction. In
Figure 4K, we used a repeated-measures ANOVA. Values of
P<0.05 were considered significant.
Identification and Characterization of Exercise-
Associated Cardiac lncRNAs
RNA sequencing was performed on hearts from seden-
tary mice and mice subjected to voluntary running for 8
weeks, which induces physiological cardiac growth and
cardiomyogenesis.10 For comparison, RNA sequencing
was also performed on hearts from mice subjected to
transverse aortic constriction (TAC) for 2 or 8 weeks to in-
duce pathological cardiac hypertrophy or HF, respective-
ly. Principal component analysis plots using all lncRNAs
from the treatment groups (normalized to corresponding
controls) showed that lncRNAs clearly distinguish these
states (Figure S1A). Twenty-five lncRNAs were differen-
tially expressed (11 downregulated and 14 upregulated)
in hearts from exercised compared with sedentary mice
(Figure 1A). We named these lncExACTs. Of these, 6 also
changed in the pathological models, in each case in a di-
rection opposite to that seen with exercise (Figure 1B
and 1C). One of these could not be reliably detected
by quantitative reverse transcription–polymerase chain
reaction, but the other 5 were validated by quantitative
reverse transcription–polymerase chain reaction in inde-
pendent cohorts (Figure 1C). Among them, lncExACT1
Figure 1. Identification of exercise-associated cardiac lncRNAs (lncExACTs).
A, Volcano plot showing lncRNA RNA sequencing results in hearts from exercised compared with sedentary mice; n=4 mice per group. B, Venn
diagram of differentially regulated cardiac lncRNAs in exercised (Run) mice and animals with transverse aortic constriction (TAC)–induced left
ventricular hypertrophy (TAC-LVH) or heart failure (TAC-HF); n=4 mice/group. C, Quantitative reverse transcription–polymerase chain reaction
(QRT-PCR) measurement of cardiac long noncoding exercise-associated cardiac transcript (lncExACT) 1 through lncExACT5 expression
in control (Ctrl), exercised, TAC-LVH, or TAC-HF mice. *P<0.05 vs Ctrl by 1-way ANOVA with post hoc Tukey; n=4 to 5 mice per group. D,
lncExACT1 expression measured by QRT-PCR is increased in hearts from patients with heart failure (HF) compared with control subjects; n=12
per group. P<0.001 by Student t test. E, Plasma lncExACT1 determined by droplet digital polymerase chain reaction is increased in patients
with HF with reduced ejection fraction (HFrEF; n=18) or HF with preserved ejection fraction (HFpEF; n=16) compared with patients with
supraventricular tachycardiac (SVT) without HF (n=8). Data shown as mean±SEM. P=0.006 and P=0.032 by pairwise Wilcoxon rank-sum test
with Bonferroni correction.
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
was chosen for detailed study as the only lncExACT al-
tered in both the physiological and pathological models
that was downregulated in exercised hearts, suggesting
that antisense inhibition could have therapeutic benefits.
The full-length lncExACT1 sequence (ENS-
MUSG00000074517) was confirmed by rapid ampli-
fication of cDNA ends. lncExACT1 is highly conserved
across mammalian species. The mouse ortholog of
lncExACT1 exhibits 67% nucleotide identity with its
human counterpart (NONHSAG111911; Figure S1B).
Of note, DCHS2 is the closest protein-coding gene in
both species (overlapping in mice and within 136 bp in
humans). The Coding Potential Assessment Tool failed to
identify any major open reading frames (ORF) with trans-
lational potential longer than 100 amino acids; however,
2 small ORFs (sORFs; sORF1 is 306 bp and sORF2 is
525bp) were identified in lncExACT1. To test whether
these sORFs have peptide-coding potential, full-length
lncExACT1 or the sORFs were cloned upstream of the
FLAG coding sequence in p3xFLAG-CMV and trans-
fected into HEK293T cells. No FLAG-tagged peptides
were detected (Figure S1C), suggesting that these
sORFs are not translated at a significant level. More-
over, constructs were generated in which a mutant green
fluorescent protein (GFP) ORF (the start codon ATG-
GTT is mutated to ATTGTT) was fused to the 3 end of
the sORFs and the lncExACT1 sequence upstream of
each sORF. Substantial expression of GFP fusion pro-
tein was observed in GFP wild-type but not in mutant
GFP or mutant GFP fused with sORF1 or sORF2. These
results further argue against significant translation (Fig-
ure S1D), although very low translation levels cannot be
completely excluded.
lncExACT1 is expressed by many tissues (Figure
S1E), including the heart, in which expression is com-
parable in cardiomyocytes (35.59±3.60 copies per cell)
and noncardiomyocytes (28.23±4.60 copies per cell;
Figure S1F). Although lncExACT1 is present in both car-
diomyocyte nuclei and cytoplasm, it is 2.5 times more
abundant in the nucleus (Figure S1G). To see whether
lncExACT1 expression is affected by relevant stimuli,
primary cardiomyocytes were exposed to insulin-like
growth factor-1, which induces physiological cardiac
growth through phosphoinositide 3-kinase12 and Akt.13,14
Insulin-like growth factor-1 reduced lncExACT1 with a
gene expression pattern consistent with physiological
hypertrophy (Figure S1H). Phosphoinositide 3-kinase
inhibition increased lncExACT1 and reversed the physi-
ological expression pattern seen with insulin-like growth
factor-1 treatment alone (Figure S1H), suggesting that
insulin-like growth factor-1 inhibits lncExACT1 through
phosphoinositide 3-kinase activation. In contrast, treat-
ment with phenylephrine, which induces pathological
hypertrophy through mitogen-activated protein kinases,15
increased lncExACT1 expression with a gene expression
pattern consistent with pathological hypertrophy. These
changes and lncExACT1 induction were attenuated by
c-Jun N-terminal kinase inhibition (Figure S1I and S1J),
whereas mitogen-activated protein kinase and p38 inhi-
bition did not block lncExACT1 induction.
In hearts explanted from patients with nonischemic
cardiomyopathy and reduced systolic function (ejection
fraction [EF], 22.3±9.0%; n=12), lncExACT1 expres-
sion was increased 1.8-fold compared with nonfailing
(unused donor) hearts (EF, 67.7±7.2%; n=12; P<10−11)
from otherwise similar subjects (50% female and mean
age of 57 years for both; P>0.7) (Figure 1D; P<0.001).
In the entire population, cardiac lncExACT1 levels were
weakly correlated with left ventricular EF, but this asso-
ciation was driven by the lower levels in controls. In HF,
lncExACT1 expression varied for unclear reasons and
did not correlate with pre-explantation left ventricu-
lar EF (Figure S1K and S1L). In separate cohorts, we
examined circulating lncExACT1 levels by droplet digital
polymerase chain reaction in plasma from patients with
HF with reduced EF (n=16; EF, 22.3±8.92%; P<10−14)
and HF with preserved EF (n=18; EF, 64.7±7.76%)
compared with plasma from patients without HF and
structurally normal hearts who presented with supraven-
tricular tachycardia (n=8; EF, 60.8±2.3%). Circulating
lncExACT1 increased 2.9-fold in plasma from patients
with HF with reduced EF (P=0.032 versus supraven-
tricular tachycardia) and 3.4-fold in patients with HF with
preserved EF (P=0.006 versus supraventricular tachy-
cardia; Figure 1E). Together, these data underscore the
potential clinical relevance of lncExACT1.
lncExACT1 Overexpression Induces
Pathological Cardiac Hypertrophy
In primary cardiomyocytes, lentiviral expression of lncEx-
ACT1 increased cardiomyocyte size and induced a gene
expression pattern characteristic of pathological hyper-
trophy, including increased ANP, BNP, β/α-myosin heavy
chain ratio, and C/EBPβ and decreased PGC1α (Figure
S2A–S2C). Overexpression of lncExACT1 did not alter
cardiomyocyte proliferation but enhanced proliferation
of noncardiomyocytes, made up predominantly of fibro-
blasts (Figure S2D). In primary cardiomyocytes, lentiviral
expression of lncExACT1 decreased DSCR1, an endog-
enous inhibitor of calcineurin, a well-established driver
of pathological hypertrophy,16 while increasing protein
expression of CnA (the catalytic subunit of calcineurin)
and nuclear NFATc3 (downstream effector of calcineurin;
Figure S2E). Inhibition of calcineurin with FK506 par-
tially attenuated lncExACT1-induced pathological gene
expression (Figure S2F). These results suggest that
lncExACT1 may induce pathological hypertrophy at least
in part through activation of calcineurin signaling.
We next injected a cardiotropic adeno-associated
virus (AAV9; 2×1012 genome copies/mouse) encod-
ing lncExACT1 driven by a cardiac-specific promoter
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
through the tail veins of wild-type mice. AAV9-lncEx-
ACT1 increased cardiac lncExACT1 gene expression
7-fold at 16 weeks compared with control vector-
injected mice (Figure 2A), which is somewhat but
not dramatically greater than the 2-5-fold increase
seen in human and experimental HF. AAV9-lncEx-
ACT1 increased expression both in the cytoplasm
and in the nucleus (Figure S2G), where lncExACT1
is more abundant (Figure S2H). lncExACT1 expres-
sion was sufficient to increase heart weight relative to
tibial length and relative wall thickness (Figure 2B and
2C). Fractional shortening was reduced (Figure 2D),
consistent with impaired systolic function, and lung
weight relative to tibial length was modestly increased
(Figure 2E), consistent with HF. Of note, chamber size
was reduced rather than increased as commonly seen
in TAC-HF (Figure 2F and Table S1). These changes
were associated with a pathological gene expres-
sion pattern (Figure 2G). lncExACT1 overexpression
increased cardiomyocyte size but did not affect mark-
ers of cardiomyocyte proliferation (Figure 2H and 2I
and Figure S2I and S2J).
Thus, lncExACT1 expression is sufficient to induce
cardiac hypertrophy most consistent with pathological
hypertrophy given the changes in gene expression and
the development of cardiac dysfunction and HF. More-
over, there was no evidence of increased cardiomyo-
genesis, a hallmark of exercise-induced physiological
lncExACT1 Inhibition Induces Physiological
Cardiac Hypertrophy
Because lncExACT1 is reduced in the exercised heart,
we next examined whether knockdown of lncExACT1
could mimic exercise-induced physiological cardiac
growth. In primary cardiomyocytes, transfection with 2
locked nucleic acid (LNA) antisense oligonucleotides
(GapmeR No. 1 and 2) specific to different regions of
lncExACT1 each led to a 50% reduction in lncExACT1
transcript levels (Figure S3A). Paradoxically, lncExACT1
knockdown increased cardiomyocyte size, as did over-
expression (Figure S3B). However, in contrast with
overexpression, lncExACT1 knockdown induced a gene
expression pattern consistent with physiological hyper-
trophy (Figure S3C). Inhibition of lncExACT1 increased
the number of cardiomyocytes (Figure S3B) and in-
creased markers of proliferation in cardiomyocytes while
suppressing these markers in noncardiomyocytes, pre-
dominantly fibroblasts (Figure S3B, S3D, and S3E).
To examine the effects of lncExACT1 inhibition in vivo,
GapmeR No. 1 was injected via the tail vein into mice.
Two weeks of lncExACT1 GapmeR treatment reduced
cardiac lncExACT1 expression by 50% (Figure 3A),
Figure 2. lncExACT1 overexpression induces pathological hypertrophy.
A, Quantitative reverse transcription–polymerase chain reaction (QRT-PCR) measurement of long noncoding exercise-associated cardiac
transcript 1 (lncExACT1) in mouse hearts 16 weeks after injection with adeno-associated virus (AAV)–lncExACT1 (lncExACT1) or AAV–
green fluorescent protein (Con). B, Heart weight (HW) relative to tibial length (TL). C, Relative wall thickness (RWT). D, Fractional shortening
(FS). E, Lung weight (LW) relative to tibial length (TL). F, Left ventricular end-diastolic internal dimension (LVIDd). G, QRT-PCR measurement
of hypertrophy markers in the heart. H, Quantification of cardiomyocyte area from wheat germ agglutinin (WGA)–stained heart sections.
I, Quantification of EdU and pericentriolar material-1 (PCM1) double-positive cardiomyocytes (CMs) in heart sections. Data shown as
mean±SEM.*P<0.05, **P<0.01 by Student t test.
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
comparable to the 70% reduction seen in exercised
hearts (Figure 1C). lncExACT1 GapmeR reduced lncEx-
ACT1 expression in both cardiomyocytes and fibroblasts,
with higher efficiency in cardiomyocytes (Figure S3F).
Inhibition of lncExACT1 increased heart weight relative
to tibial length by 23%, increased relative wall thick-
ness by 24% (Figure 3B and 3C), and improved cardiac
function (Figure 3D and Table S2) without affecting lung
weight relative to tibial length or chamber dimensions
(Figure 3E and 3F), similar to the changes seen in exer-
cised mice (data not shown). Inhibition of lncExACT1
induced a gene expression pattern most consistent
with physiological hypertrophy (Figure 3G). Inhibition of
lncExACT1 increased cardiomyocyte size (Figure 3H)
comparably to lncExACT1 overexpression (Figure 2H).
However, lncExACT1 inhibition also increased markers
of cardiomyocyte proliferation including increased EdU,
Ki67, and phosphorylated histone H3 in cells also posi-
tive for pericentriolar material-1 (a marker of cardiomyo-
cyte nuclei17; Figure 3I–3K). Taken together, these data
demonstrate that lncExACT1 inhibition is sufficient to
induce physiological cardiac hypertrophy and markers of
Figure 3. lncExACT1 inhibition induces physiological hypertrophy.
A, Quantitative reverse transcription–polymerase chain reaction (QRT-PCR) measurement of long noncoding exercise-associated cardiac
transcript 1 (lncExACT1) in hearts from mice injected with LNA-GapmeR-Control (Con) or LNA-GapmeR-lncExACT1 (Gap) for 2 weeks. B,
Heart weight (HW) relative to tibial length (TL). C, Relative wall thickness (RWT). D, Fractional shortening (FS). E, Lung weight (LW) relative to
tibial length (TL). F, Left ventricular end-diastolic internal dimension (LVIDd). G, QRT-PCR measurement of hypertrophy markers in the heart. H,
Quantification of cardiomyocyte area from wheat germ agglutinin (WGA)–stained heart sections. I, Quantification of EdU, pericentriolar material-1
(PCM1) double-positive cardiomyocytes (CMs) in stained heart sections. J, Quantification of Ki67 and PCM1 double-positive cardiomyocytes
in stained heart sections. K, Quantification of phosphorylated histone H3 (pHH3) and PCM1 double-positive cardiomyocytes in stained heart
sections. Data shown as mean±SEM. *P<0.05, **P<0.01 by Student t test.
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
Figure 4. lncExACT1 inhibition protects against pathological hypertrophy, cardiac dysfunction, and fibrosis.
A, Quantitative reverse transcription–polymerase chain reaction (QRT-PCR) measurement of long noncoding exercise-associated cardiac
transcript 1 (lncExACT1) expression in hearts from mice subjected to sham operation (Sham), transverse aortic constriction with LNA-GapmeR-
Control injection (TAC), or TAC with LNA-GapmeR-lncExACT1 (TAC+Gap). B, Heart weight (HW) relative to tibial length (TL). C, Fractional
shortening (FS). D, Left ventricular end-diastolic internal dimension (LVIDd). E, Relative wall thickness (RWT). F, QRT-PCR measurement
of hypertrophy markers in the heart. G, Quantification of fibrotic area from Masson trichrome–stained heart sections. H, Quantification of
cardiomyocyte area from heart sections with wheat germ agglutinin (WGA) staining. I, Quantification of Ki67 (Continued )
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
cardiomyogenesis, similar to those induced by 8 weeks
of exercise.
lncExACT1 Inhibition Protects the Heart Against
Adverse Remodeling
Given that exercise protects the heart against pathologi-
cal stress,18,19 we asked whether lncExACT1 inhibition
protects against pressure-overload (TAC). Treatment with
lncExACT1-specific GapmeR for 6 weeks completely
blocked the TAC-induced increase in cardiac lncEx-
ACT1 (Figure 4A). lncExACT1 inhibition also mitigated
the TAC-induced increase in heart weight (Figure 4B)
and improved cardiac function while reducing chamber
dilatation and wall thinning (Figure 4C–4E and Table
S3). lncExACT1 inhibition partially reversed pathological
gene expression (Figure 4F) while reducing cardiac fi-
brosis 2-fold and blocking the increase in cardiomyocyte
size seen after TAC (Figure 4G and 4H). Thus, although
lncExACT1 inhibition induces physiological hypertrophy
at baseline, it actually reduces pathological hypertrophy,
suggesting a dynamic tension between physiological and
pathological hypertrophy that is mediated by lncExACT1.
Ki67 and phosphorylated histone H3, markers of prolif-
eration, were modestly increased in cardiomyocytes after
TAC, consistent with prior reports,20,21 and were further
increased by lncExACT1 inhibition (Figure 4I).
We next examined whether lncExACT1 inhibition miti-
gated adverse remodeling after ischemic injury. In mice
subjected to myocardial ischemia/reperfusion injury,
injection of lncExACT1-specific GapmeR for 7 weeks
starting after reperfusion reduced the ischemia/reper-
fusion injury–induced increase in cardiac lncExACT1
compared with control GapmeR-injected mice (Fig-
ure 4J and 4K). lncExACT1 inhibition increased markers
of cardiomyocyte proliferation, EdU incorporation, and
phosphorylated histone H3 staining in the infarct border
zone (Figure 4L) and reduced cardiomyocyte apoptosis
(Figure 4N). lncExACT1 inhibition did not affect the initial
injury, as reflected in the reduced fractional shortening
seen at 24 hours (Figure 4K). However, the benefits of
lncExACT1 inhibition became apparent after 4 weeks
(Figure 4K), suggesting that improved initial cardiomyo-
cyte survival may not be the dominant factor. Together,
these results indicate that inhibition of lncExACT1 is suf-
ficient to protect the heart against pathological hypertro-
phy, cardiac dysfunction, and adverse remodeling, likely
through a combination of enhancing cardiomyocyte pro-
liferation and survival culminating in dramatically reduced
cardiac fibrosis.
lncExACT1 Binds MiR-222
lncRNAs can act as competitive endogenous RNAs
binding miRNAs, typically in the cytoplasm,22 to inhibit
their actions. Bioinformatic analyses with ENCORI iden-
tified 5 miRNAs as potentially binding lncExACT1 (Fig-
ure S4A). We examined lncExACT1 binding in primary
cardiomyocytes by quantitative reverse transcription–
polymerase chain reaction after pulldown of biotinylated
probes specific for the sense or antisense strands of
lncExACT1. Only miR-876-5p and miR-222-3p demon-
strated detectable binding to lncExACT1. miR-876-5p,
but not miR-222-3p, also bound lncExACT1 antisense
although at a lower level (Figure S4B). Sequences cor-
responding to the predicted wild-type or mutated miRNA
binding sites of lncExACT1 were inserted 3 of a lucif-
erase reporter and cotransfected into cardiomyocytes
with the related miRNA, miR-876-5p or miR-222-3p
(Figure S4C). The miR-222-3p mimic reduced luciferase
activity of the construct with wild-type but not mutated
putative miR-222 binding sites. In contrast, transfection
with miR-876-5p did not affect luciferase activity with ei-
ther the wild-type or mutated binding site (Figure S4D).
miR-222 inhibition reversed some of the physiological
gene expression pattern otherwise seen with lncEx-
ACT1 knockdown and blocked the increase in cell size
seen with lncExACT1 knockdown (Figure S4E and S4F)
without affecting lncExACT1 knockdown–induced car-
diomyocyte proliferation (Figure S4G). Taken together,
these data suggest that miR-222, which we previously
demonstrated is necessary for exercise-induced cardiac
growth,11 uniquely among the bioinformatic candidates,
binds lncExACT1 and contributes to the physiological
cardiomyocyte hypertrophy but not the proliferation seen
with lncExACT1 inhibition. From these observations and
our prior work demonstrating that even massive miR-222
overexpression is not sufficient to induce hypertrophy or
cardiomyogenesis in the adult heart, we sought to identi-
fy other effectors contributing the phenotypes observed,
particularly the changes in cardiomyocyte proliferation.
lncExACT1 Regulates DCHS2 and Yap1
lncRNAs often regulate expression of nearby genes,23
so we examined DCHS2, the protein-coding gene clos-
Figure 4 Continued. and phosphorylated histone H3 (pHH3) and pericentriolar material-1 (PCM1) double-positive cardiomyocytes in
heart sections. J, QRT-PCR measurement of lncExACT1 expression in hearts from mice subjected to sham operation (Sham), myocardial
ischemia/reperfusion (IR) with LNA-GapmeR-control injection, or IR with LNA-GapmeR-lncExACT1 (IR+Gap). K, Fractional shortening (FS). L,
Quantification of EdU and pHH3 and PCM1 double-positive cardiomyocytes (CMs) at the infarct border zone in heart sections. M, Quantification
of fibrotic area in Masson trichrome–stained heart sections. N, Quantification of terminal deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL) and cardiac troponin I (cTnI) double-positive cardiomyocytes in heart sections. Data shown as mean±SEM. IRI indicates ischemia/
reperfusion injury. *P<0.05. **P<0.01. In I, *P<0.05 vs Sham, #P<0.05 vs TAC by 1-way ANOVA with post hoc Tukey. In K, *P<0.05 vs Sham,
#P<0.05 vs IR+Gap by repeated-measures ANOVA.
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
est to lncExACT1 in both mice and humans. Paralleling
lncExACT1, cardiac DCHS2 expression increased in pa-
tients and mice with HF but decreased in exercise (Fig-
ure 5A). Moreover, cardiac DCHS2 decreased in mice af-
ter lncExACT1 knockdown with GapmeR and increased
with AAV-lncExACT1 expression (Figure 5A). Similar
effects were seen in primary cardiomyocytes, whereas
DCHS2 expression or knockdown did not affect lncEx-
ACT1 expression (Figure S5A and S5B). These data
suggest that DCHS2 expression may be regulated by
lncExACT1. To determine whether lncExACT1 directly
regulates DCHS2 transcription, we performed analy-
ses of the DCHS2 promoter. Sequences correspond-
ing to 500, 1000, or 1500 bp upstream of the DCHS2
transcriptional start site were inserted into a luciferase
reporter and transfected into primary cardiomyocytes
in combination with either lncExACT1 overexpression
or inhibition. lncExACT1 expression increased whereas
lncExACT1 knockdown decreased the activity of pro-
moter constructs that included 1000 or 1500 bp 5 of
DCHS2 but not the construct that included only 500 bp
(Figure 5B). Consistent with this, chromatin oligo-affin-
ity precipitation with oligonucleotides corresponding to
lncExACT1 sense sequence compared with oligonucle-
otides corresponding to lncExACT1 antisense sequence
(as negative controls) pulled down DCHS2 promoter
fragments detected by quantitative reverse transcrip-
tion–polymerase chain reaction corresponding to 500 to
1000 bp and 1000 to 1500 bp but not 0 to 500 bp up-
stream of DCHS2 (Figure 5C). These data suggest that
lncExACT1 binds sequences between 500 and 1500
bp upstream of DCHS2 and positively regulates DCHS2
transcription. Because this occurs with exogenously ex-
pressed lncExACT1, it can occur in trans.
In primary cardiomyocytes, siRNA knockdown of
DCHS2 (Figure S5C) induced an increase in cardio-
myocyte proliferation as reflected by EdU incorpora-
tion (Figure 5D). DCHS2 knockdown also increased
cardiomyocyte size with a physiological gene expres-
sion pattern (Figure 5E and Figure S5D). In contrast,
although overexpression of DCHS2 with a CRISPR/
dCas9 activation system also increased cardiomyocyte
size, this was associated with a pathological hypertro-
phy gene expression pattern and reduced markers of
cardiomyocyte proliferation (Figure S5E–S5H). Thus,
similar to lncExACT1, DCHS2 inhibition induces physi-
ological cardiomyocyte hypertrophy and proliferation,
whereas its overexpression induces pathological car-
diomyocyte hypertrophy.
We then examined the interacting effects of DCHS2
and lncExACT1 in cardiomyocytes. DCHS2 knockdown
prevented the pathological gene expression pattern oth-
erwise seen with lncExACT1 overexpression (Figure 5F),
and DCHS2 expression blocked the physiological gene
expression pattern seen with lncExACT1 knockdown
(Figure S5I). Moreover, DCHS2 knockdown increased
proliferation markers in cardiomyocytes overexpressing
lncExACT1, whereas DCHS2 overexpression prevented
the increase in cardiomyocyte proliferation induced
by lncExACT1 inhibition (Figure 5G and Figure S5J).
Furthermore, either knockdown or overexpression of
DCHS2 prevented the increase in cardiomyocyte size
seen with lncExACT1 overexpression (Figure 5H) and
knockdown (Figure S5K), respectively. Together these
data suggest that DCHS2 is necessary and sufficient for
the effects of lncExACT on cardiomyocyte growth and
proliferation in vitro.
Although it has no known role in the heart, in other
systems, DCHS2 modulates Hippo/Yap1 signaling,24
a highly conserved regulator of size and proliferation
in many organs, including the heart.25 Hippo activa-
tion ultimately induces phosphorylation and cytoplas-
mic sequestration of Yap1, which reduces proliferation,
whereas its inhibition culminates in dephosphorylation
and nuclear localization of Yap1, driving cell cycle pro-
gression.26 Total nuclear Yap1 was increased whereas
S127-phosphorylated cytoplasmic Yap1 (p-Yap1) was
reduced in exercised hearts. The reverse was seen in
failing hearts after TAC27,28 (Figure S6A). lncExACT1
inhibition in vivo also increased total nuclear Yap1 and
reduced cytoplasmic p-Yap1. Conversely lncExACT1
overexpression increased cytoplasmic p-Yap1 and
decreased total nuclear Yap1 (Figure S6B). Similarly,
either lncExACT1 or DCHS2 overexpression increased
cytoplasmic p-Yap1 and reduced total nuclear Yap1 in
primary cardiomyocytes (Figure S6C). In contrast, lncEx-
ACT1 or DCHS2 inhibition in primary cardiomyocytes
reduced cytoplasmic p-Yap1 and increased nuclear total
Yap1 (Figure S6D). These data suggest that physiologi-
cal hypertrophy enhances but pathological hypertrophy
inhibits Yap1 transcriptional activity, likely through lncEx-
ACT1-DCHS2 signaling. Consistent with this model,
knockdown of either lncExACT1 or DCHS2 in cardio-
myocytes increased expression of multiple downstream
targets of Yap1,26,29 whereas lncExACT1 or DCHS2
expression had the opposite effect (Figure S6E and
S6F). Taken together, these data implicate lncExACT1
and DCHS2 as novel regulators of cardiac Yap1, which
likely contributes to exercise-induced heart growth and
DCHS2 Overexpression Induces Pathological
Cardiac Hypertrophy and Impairs Cardiac
Regeneration in Zebrafish
To examine the role of DCHS2 in vivo, we turned to
the zebrafish model, known for its remarkable ability
to regenerate the heart after cardiac injury through
cardiomyocyte proliferation.30 We generated ze-
brafish constitutively expressing human DCHS2 in
cardiomyocytes (Figure 6A). Compared with controls,
cardiac overexpression of DCHS2 increased cardio-
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
myocyte size and expression of ANP and BNP, consis-
tent with pathological cardiac hypertrophy (Figure 6B
and 6C). Seven days after cardiac cryoinjury, many
proliferating cardiomyocytes (nxk2.5 and PCNA posi-
tive) were evident in the injured area and border zone
in wild-type animals. In contrast, significantly fewer
were seen in DCHS2-expressing animals (Figure 6D).
We then asked whether the DCHS2-induced defect in
cardiomyocyte proliferation would translate into long-
term regeneration defects. At 90 days after cardiac
cryoinjury, all hearts (12 of 12) from wild-type animals
had repaired the myocardial wall and showed only
minimal residual fibrosis. In contrast, animals overex-
pressing DCHS2 developed substantially more fibro-
sis (Figure 6E). These results indicate that DCHS2
impairs cardiac regeneration and promotes scarring
after injury in zebrafish. Cardiac DCHS2 overexpres-
sion also reduced total nuclear Yap1 and increased cy-
toplasmic p-Yap1 (Figure 6F and 6G). Together, these
results indicate that cardiac overexpression of DCHS2
in zebrafish promotes pathological cardiac hypertro-
phy in unperturbed hearts and impairs cardiomyocyte
proliferation and regeneration, increasing fibrosis after
cryoinjury. As in the mammalian studies, it seems likely
that these effects are mediated through suppression
of nuclear Yap1.
DCHS2 Knockdown Promotes Physiological
Cardiac Hypertrophy
To examine the effects of DCHS2 inhibition in vivo, we
returned to the adult murine model, which has limited
cardiomyocyte proliferation at baseline. We injected
Cas9 knock-in mice31,32 with AAV9 vectors encoding
either control guide RNAs (gRNAs), gRNAs targeting
DCHS2 exon 2 (gRNA1), or exon 3 (gRNA2), along
with a cardiac-specific troponin promoter-driven Cre
expression cassette (Figure 7A). Eight weeks after
AAV injection, cardiac DCHS2 was reduced by 50%
in mice with gRNA1 or gRNA2 (Figure 7B), which is
comparable to the reduction seen in exercised hearts
(Figure 5F). Reduced cardiac DCHS2 led to increased
heart weight relative to tibial length (Figure 7C) and
increased cardiomyocyte size (Figure 7D) without af-
fecting lung weight relative to tibial length (Figure 7E).
Reduced cardiac DCHS2 also improved cardiac func-
tion and increased relative wall thickness (Figure 7F and
7G) without a significant change in chamber dimension
(Figure 7H). Reduced DCHS2 induced a gene expres-
sion pattern suggestive of physiological hypertrophy
(Figure 7I). These changes were qualitatively similar to
those seen with comparable lncExACT1 knockdown
but smaller quantitatively. Notably, DCHS2 knockdown
Figure 5. lncExACT1 works through DCHS2.
A, Quantitative reverse transcription–polymerase chain reaction (QRT-PCR) measurement of dachsous cadherin-related 2 (DCHS2) in hearts
from patients with heart failure (HF) and controls (n=12 per group) and in mice with HF or long noncoding exercise-associated cardiac transcript
1 (lncExACT1) overexpression, or exercise (Run) or lncExACT1 inhibition (Gap). B, Luciferase activity driven by DCHS2 promotor fragments in
neonatal rat ventricular myocytes (NRVMs) with lncExACT1 overexpression (OE) or knockdown (KD). C, QRT-PCR measurement with primers
targeting different regions of DCHS2 promoter in complex from pulldown with probes targeting sense and antisense sequence of lncExACT1. D,
Quantification of EdU and troponin double-positive cardiomyocytes (CMs) by flow cytometry. E, Quantification of cardiomyocyte size in NRVMs
treated with scrambled control or DCHS2 siRNA. F, QRT-PCR measurement of hypertrophy gene markers; n=3 per group. G, Quantification of
EdU-positive cardiomyocytes by flow cytometry. H, Quantification of cardiomyocyte size after lncExACT1 OE or DCHS2 KD in NRVMs. *P<0.05.
**P<0.01. In C and D, *P<0.05 vs Control, #P<0.05 vs lncExACT1 KD; in A and C through E, by Student t test; and in B and F through H, by
1-way ANOVA with post hoc Tukey. Data shown as mean±SEM.
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
increased cardiomyocyte proliferation, as indicated by
Ki67 and phosphorylated histone H3 in pericentriolar
material-1–positive cells (Figure 7J and JK), to a simi-
lar degree as seen with lncExACT1 knockdown (Fig-
ure 3). Consistent with the in vitro and zebrafish studies,
knockdown of DCHS2 increased nuclear total Yap1 but
reduced cytoplasmic p-Yap1 expression in the heart
(Figure 7L). Knockdown of DCHS2 reduced cytoplas-
mic protein expression of p-MST1/2 (Thr183/Thr180)
core regulatory proteins that work upstream of Yap1
in the Hippo pathway (Figure 7M).33 Taken together,
these data demonstrate that knockdown of DCHS2 in
the adult mammalian heart is sufficient to induce physi-
ological cardiac hypertrophy and evidence of cardiomyo-
cyte proliferation. Although the cardiomyocyte growth is
modest, the increase in markers of proliferation is quan-
titatively similar to and thus likely sufficient to account
for that seen with lncExACT1 knockdown. The corre-
sponding changes in nuclear Yap1 observed are likely
Figure 6. DCHS2 overexpression induces pathological cardiac hypertrophy and reduces regenerative capacity in zebrafish.
A, Representative image from apex region of an adult transgenic zebrafish heart immunostained for tropomyosin (red), green fluorescent
protein (GFP; green), and nuclei (DPAI, blue). B, Representative image and quantification of cardiomyocytes size isolated from wild-type control
(CTRL; white arrow) or dachsous cadherin-related 2 (DCHS2) overexpression (hGFP-DCHS2; yellow arrow) zebrafish. C, Quantitative reverse
transcription–polymerase chain reaction measurement of ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide) in hearts from
CTRL and hGFP-DCHS2 zebrafish. D, Representative images and quantification of nkx2.5 and PCNA double-positive cardiomyocytes in hearts
at 7 days post injury (dpi) from CTRL and hGFP-DCHS2 zebrafish. E, Representative images and quantification of fibrosis in hearts at 60 dpi
from CTRL and hGFP-DCHS2 zebrafish. F and G, Representative images and quantification of nuclear (Nu) total (t-) Yap1 and cytoplasmic
(Cyto) S127-phosphorylated Yap1 (p-Yap1) protein expression in hearts from CTRL and hGFP-DCHS2 zebrafish. Data shown as mean±SEM.
*P<0.05, **P<0.01 by Student t test.
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
Figure 7. DCHS2 knockdown promotes physiological cardiac hypertrophy.
A, Schematic of experimental strategy used to knockdown dachsous cadherin-related 2 (DCHS2) in the hearts in vivo. B, Quantitative
reverse transcription–polymerase chain reaction (QRT-PCR) measurement of cardiac DCHS2 in Cas9-knockin mice with injection
of adeno-associated virus (AAV9) carrying gRNA1 or gRNA2. C, Heart weight (HW) relative to tibial length (TL). D, Quantification of
cardiomyocyte area from wheat germ agglutinin (WGA)–stained heart sections. E, Lung weight (LW) relative to tibial length (TL). F,
Fractional shortening (FS). G, Relative wall thickness (RWT). H, Left ventricular end-diastolic internal dimension (LVIDd). I, QRT-PCR
measurement of hypertrophy gene markers. J, Quantification of Ki67. K, Quantification of phosphorylated histone H3 (pHH3), pericentriolar
material-1 (PCM1) double-positive cardiomyocytes in stained heart sections. L and M, Representative images and quantification of nuclear
(Nu) total (t-) Yap1, cytoplasmic (Cyto) S127-phosphorylated Yap1 (p-Yap1), phosphorylated (p-) MST1/2, and total (t-) MST1 (MST1)
protein expressions in hearts from mice injected with AAV9 carrying control (Con), gRNA1, or gRNA2. Data shown as mean±SEM. ANP
indicates atrial natriuretic peptide; BNP, brain natriuretic peptide; and PGC1α, peroxisome proliferator–activated receptor gamma coactivator
1-α. *P<0.05 by 1-way ANOVA with post hoc Tukey.
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
to explain the proliferation changes seen with DCHS2
manipulation in both zebrafish and mammalian models.
Understanding how exercise promotes cardiac health
and whether the responsible mechanisms can be feasibly
targeted has important fundamental and clinical implica-
tions. The data presented here identify a set of cardiac
lncRNAs (lncExACTs) that are dynamically regulated by
exercise and play an important role in determining the
outcome of cardiac growth. A small number of the lncEx-
ACTs identified were also altered in pathological hyper-
trophy or HF. In every case, the change in exercise was
opposite of that observed in disease models, underscor-
ing the distinct nature of these responses despite super-
ficial similarities. Among these, lncExACT1 was uniquely
downregulated in exercise and highly conserved across
species. lncExACT1 expression was sufficient to induce
pathological hypertrophy and HF. Inhibition of lncEx-
ACT1 was sufficient to induce multiple exercise-related
cardiac phenotypes, including physiological hypertrophy,
improved cardiac function, protection against HF and fi-
brosis, and signs of cardiomyocyte proliferation.
A surprising finding was that both lncExACT1 over-
expression and inhibition induced cardiac hypertrophy.
However, several lines of evidence suggest that lncEx-
ACT1 overexpression produced pathological growth, in
contrast to the physiological growth induced by inhibi-
tion. First, lncExACT1 gain and loss of function induced
distinct patterns in expression of the sentinel markers
of pathological versus physiological growth.4,5 Second,
lncExACT1 inhibition increased markers of cardio-
myocyte proliferation, a hallmark of exercise-induced
but not pathological growth, both at baseline and in
disease models. lncExACT1 overexpression did not
affect markers of cardiomyocyte proliferation in vivo.
Last, and perhaps most important, lncExACT1 over-
expression culminated in cardiac dysfunction and HF,
whereas inhibition actually improved cardiac function
modestly at baseline and more significantly after patho-
logical stress. Taken together, these data strongly sup-
port the model that the cardiomyocyte growth induced
by lncExACT1 overexpression and the growth induced
by inhibition are fundamentally different, representing
pathological and physiological growth, respectively. Of
note, the expression level of lncRNAs is low compared
with protein coding genes. Prior work suggests that for
lncRNA cis effects, an abundance of 1 to 10 molecules
per cell can be sufficient,34 whereas for trans action,
an abundance of 10 to 1000 has been reported.35 We
found there are 35.59±3.60 copies of lncExACT1 per
cardiomyocyte at baseline, which increases by 4- to
8-fold in HF and decreases 2- to 3-fold in exercise.
These changes could plausibly account for its regula-
tory effects on DCHS2 and miR-222. To the best of our
knowledge, this is a unique example of a molecule with
an expression that toggles the heart between patho-
logical and physiological growth with very different
functional outcomes and suggests that targeting lncEx-
ACT1 may hold therapeutic promise.
The adult mammalian heart has a very limited capac-
ity for cardiomyogenesis, estimated at 1% per year in
both humans36 and mice.37 This limitation has important
clinical consequences because heart disease is often
associated with cardiomyocyte loss.38 To date, exer-
cise is the only physiological stimulus demonstrated
to increase cardiomyogenesis in the adult mammalian
heart.10 Although prior work demonstrated that miR-
222 is necessary for this effect, miR-222 alone was not
sufficient to recapitulate cardiac exercise phenotypes,
including cardiomyogenesis.10,11 Collectively, the studies
presented here implicate a novel pathway including the
lncRNA, lncExACT1, and its effector, DCHS2, as candi-
date regulators of exercise-induced cardiomyogenesis.
The zebrafish studies demonstrate a role for DCHS2 in
regulating cardiomyocyte proliferation and regeneration
with important consequences for healing and scar for-
mation. These findings resonate with the murine data
in which lncExACT1 inhibition increased markers of
cardiomyocyte proliferation after TAC and in the border
zone after ischemia/reperfusion injury, with a corre-
sponding reduction in late scar formation and improved
cardiac function. However, the murine studies presented
rely on surrogate markers of DNA synthesis, which can-
not conclusively establish cardiomyogenesis or its role in
the functional benefits seen.
The observed effects on fibrosis are consistent with
prior work demonstrating that exercise itself reduces
cardiac fibrosis after aortic constriction or ischemic
injury.39 The 2-fold reduction in fibrosis seen with lncEx-
ACT1 knockdown after TAC or ischemia/reperfusion
injury could be secondary to effects in cardiomyocytes,
including increased cardiomyocyte survival and prolifera-
tion leading to less replacement fibrosis. This model is
supported by the zebrafish studies in which cardiomyo-
cyte-specific expression of DCHS2 reduced cardiomyo-
cyte proliferation, impairing cardiac regeneration and
culminating in greater fibrosis (Figure 6). This pathway
also may have direct effects on fibroblast proliferation
opposite of its effects in cardiomyocytes, as suggested
in our in vitro studies (Figure S3). Although lncExACT1
knockdown was moderately less efficient in fibroblasts
compared with cardiomyocytes, this seems unlikely to
account for the qualitatively opposite effect on prolifera-
tion. It seems likely that a combination of all these effects
contributes to the reduced fibrosis and cardiac benefits
of lncExACT1 or DCHS2 inhibition.
These studies also implicate DCHS2, not previously
known to have a role in the heart, as an important modu-
lator of cardiac growth and cardiomyocyte proliferation,
as well as a downstream effector of lncExACT1. lncEx-
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
ACT1 binds the promoter of DCHS2, positively regulating
its transcription. DCHS2 has been demonstrated in other
systems to work with FAT as a ligand-receptor system
in regulating cell proliferation through Hippo/Yap sig-
naling.24,40 Consistent with this, we found that exercise,
as well as inhibition of cardiac lncExACT1 or DCHS2,
increased nuclear Yap1 and expression of its downstream
targets. Our results suggest that DCHS2 may regulate
Yap1, at least in part, through inhibiting MST1/2 phos-
phorylation, although the precise mechanism remains to
be elucidated. To the best of our knowledge, these are
also the first studies to implicate Hippo/Yap1 signaling,
a recognized regulator of cardiomyogenesis in the adult
heart,25 in exercise-induced cardiomyogenesis.10
The role of Hippo/Yap1 signaling in the cardiac hyper-
trophy observed is less clear. Prior studies demonstrated
that postnatal activation of cardiac Yap1 did not increase
cardiac or cardiomyocyte size,41,42 suggesting that Yap1
activation is not sufficient to induce physiological cardiac
growth. In contrast, cardiac deletion of Yap1 reduced
hypertrophy in response to pressure overload.43 Taken
together, the previous work and our data suggest that the
effects observed on cardiac hypertrophy are likely medi-
ated by Yap1-independent pathways. In vitro studies sug-
gest that lncExACT1 binding of miR-222, which we had
previously shown is necessary for cardiac growth11 and
exercise-induced cardiomyogenesis10 in vivo, contributes
to the effects of lncExACT1 in physiological hypertrophy
(Figure S4). In vitro studies also suggest that calcineurin
signaling may contribute to the pathological hypertrophy
and HF seen with lncExACT1 overexpression. We think
it is also likely that other downstream contributors remain
to be uncovered and will be a focal point for future work.
lncRNAs have well-documented roles in cardiac devel-
opment,44,45 pathological hypertrophy,46,47 and HF.48,49 How-
ever, less is known about their roles in exercise. A recent
report by Gao and colleagues50 identified an lncRNA,
CPhar, induced by exercise in murine hearts. CPhar was
not one of the lncRNAs detected in our screen, perhaps
reflecting differences in the time points (3 weeks versus 8
weeks) and exercise models (swim versus voluntary run-
ning). It is interesting to note that the prior study dem-
onstrated that CPhar was necessary for exercise-induced
hypertrophy and, when expressed before ischemic injury,
significantly mitigated cardiac dysfunction.50
We believe the present work adds to this literature in
important conceptual and practical ways. First, by com-
prehensively comparing cardiac lncRNAs dynamically
regulated in both exercise and pathological states, we
were able to identify a subset of lncRNAs altered in both
(but in opposing ways), which speaks to the fundamen-
tal differences between these states. This lncRNA set is
likely enriched for functional candidates contributing to
both states and worthy of evaluation as therapeutic tar-
gets. Second, mimicking the change in lncExACT1 seen
in exercise is sufficient to recapitulate many exercise-
induced cardiac phenotypes in vivo, including evidence
of cardiomyogenesis, whereas it appears that CPhar
expression (similar to miR-222) was not sufficient to
reproduce these general phenotypes or cardiomyo-
genesis in vivo.50 Pathways sufficient to reproduce the
benefits of exercise, including cardiomyogenesis, are
particularly unusual and valuable.
lncExACT1 has other strategic advantages as a ther-
apeutic target. It is highly (70%) conserved between
mice and humans and is altered similarly in human
and experimental HF, whereas most lncRNAs are not
well conserved. It is important to note that benefits are
observed with lncExACT1 inhibition rather than overex-
pression. Multiple modified nucleic acid antisense drugs
have been approved by the US Food and Drug Admin-
istration, including some using the LNA chemistry used
here,51 and both large animal and early clinical studies
support the feasibility of using LNA-antisense delivery
to inhibit noncoding cardiac RNAs.52,53 These practical
advantages enabled us to demonstrate a substantive
reduction in cardiac scar and improvement in function
even when inhibitor was delivered after reperfusion, a
clinically relevant time frame. In contrast, targets requiring
anticipatory overexpression can be challenging to trans-
late clinically given the generally unanticipated nature of
ischemic injury. Nevertheless, both studies reinforce the
message that much can be learned from lncRNAs medi-
ating the benefits of exercise.
These studies identify the lncExACT1-DCHS2 pathway
as a crucial and previously unrecognized regulator of both
physiological and pathological cardiomyocyte growth, in-
tegrating the dynamic tension between these states and
influencing clinically relevant outcomes in each. Target-
ing this pathway therapeutically warrants further investi-
gation in a range of cardiac diseases.
Received August 2, 2021; accepted January 5, 2022.
Corrigan-Minehan Heart Center and Cardiology Division, Massachusetts General
Hospital, Harvard Medical School, Boston (H.L., L.E.T., X.L., M.H.H., C.X., J.G., S.T.,
G.L., A.Y., R.R., M.G.S., S.D., J.M.G.-R., A.R.). Division of Cardiology, University of
Colorado Anschutz Medical Campus, Aurora (A.V.A., M.R.B.).
The authors thank Drs Ling Li (Cardiovascular Research Center, Massachusetts
General Hospital) for technical support, James Rhee (Department of Anesthesiol-
ogy, Massachusetts General Hospital) for sharing RILPL1-Flag tag plasmid, and
Yoshiko Iwamoto (Center for Systems Biology, Massachusetts General Hospital)
for helping with sample staining. H.L. and A.R. conceptualized and designed the
study. H.L., M.H.H., and A.R. wrote the article. H.L. and X.L. performed animal ex-
periments. H.L., G.L., and L.E.T. performed cell culture experiments. S.T and J.M.G.
performed zebrafish experiments. A.Y. and J.G. analyzed the RNA sequencing
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Li et al LncExACT1 and DCHS2 in Cardiac Hypertrophy
data. R.R., M.G.S., SD, A.V.A., and M.B. provided clinical samples. C.X. performed
animal surgeries. A.R. supervised the study.
Sources of Funding
This research is supported by the National Institutes of Health (R01AG061034,
R35HL155318 to Dr Rosenzweig), American Heart Association
(20CDA35310184 to Dr H. Li, 19CDA34660207 to Dr Gonzalez-Rosa,
16SFRN31720000 to Dr Rosenzweig, 19POST34381027 to Dr G. Li), National
Institutes of Health/National Center for Advancing Translational Science Colo-
rado CTSA (UL1 TR002535 to Drs Ambardekar and Bristow), Alan Hassenfeld
Research Scholar Award to Dr Gonzalez-Rosa, and Sarnoff Cardiovascular Re-
search Foundation Fellowship to L.E. Trager, and reflects a collaboration across
American Heart Association Strategically Focused Research Networks Heart
Failure Centers.
Drs H. Li and Rosenzweig are inventors on a pending patent (63/058,268: Inhibi-
tion of lncExACT1 to Treat Heart Disease) submitted by Massachusetts General
Hospital. The other authors report no conflicts.
Supplemental Material
Supplemental Materials and Methods
Tables S1–S5
Figures S1–S6
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... It mediates various physiological and pathological processes by recruiting transcription factors that act as microRNA sponges, regulate the translation of various mRNAs, silence transcription, and reprogram epigenetic processes [8,9]. Recent studies have reported that LncRNAs play important roles in cardiovascular disease and are reliable diagnostic indicators as well as therapeutic targets for various cardiovascular diseases, including cardiac hypertrophy [10][11][12]. Accumulating evidence suggests that LncRNAs are dysregulated in cardiac hypertrophy and contribute to sarcomere construction, calcium channeling, and mitochondrial homeostasis in hypertrophic cardiomyopathy [1,13,14]. However, the role of LncRNAs in the treatment and diagnosis of cardiac hypertrophy remains unclear and requires further investigation. ...
... While it was decreased in exercised hearts, its expression was increased in exercised hearts and cardiac hypertrophy increased in human and experimental heart failure. LncExACT1, interacted with dachsous cadherin related 2 (DCHS2), and this interaction plays an important role in the transformation between physiological and pathological growth to determine the functional outcomes [12]. Thus, our findings showed that LncKCND1 plays a crucial role in cardiac hypertrophy concurs with previous reports of pathological cardiac hypertrophy. ...
Full-text available
Cardiac hypertrophy is a common structural remodeling in many cardiovascular diseases. Recently, long non-coding RNAs (LncRNAs) were found to be involved in the physiological and pathological processes of cardiac hypertrophy. In this study, we found that LncRNA KCND1 (LncKCND1) was downregulated in both transverse aortic constriction (TAC)-induced hypertrophic mouse hearts and Angiotensin II (Ang II)-induced neonatal mouse cardiomyocytes. Further analyses showed that the knockdown of LncKCND1 impaired cardiac mitochondrial function and led to hypertrophic changes in cardiomyocytes. In contrast, overexpression of LncKCND1 inhibited Ang II-induced cardiomyocyte hypertrophic changes. Importantly, enhanced expression of LncKCND1 protected the heart from TAC-induced pathological cardiac hypertrophy and improved heart function in TAC mice. Subsequent analyses involving mass spectrometry and RNA immunoprecipitation assays showed that LncKCND1 directly binds to YBX1. Furthermore, overexpression of LncKCND1 upregulated the expression level of YBX1, while silencing LncKCND1 had the opposite effect. Furthermore, YBX1 was downregulated during cardiac hypertrophy, whereas overexpression of YBX1 inhibited Ang II-induced cardiomyocyte hypertrophy. Moreover, silencing YBX1 reversed the effect of LncKCND1 on cardiomyocyte mitochondrial function and its protective role in cardiac hypertrophy, suggesting that YBX1 is a downstream target of LncKCND1 in regulating cardiac hypertrophy. In conclusion, our study provides mechanistic insights into the functioning of LncKCND1 and supports LncKCND1 as a potential therapeutic target for pathological cardiac hypertrophy.
... They identified potential lncRNAs that were differentially expressed and significantly associated with each phenotype. Their results revealed an exercise-regulated cardiac lncRNA that was named lncExACTs [47]. This lncRNA was evolutionarily conserved and was reduced in exercised hearts despite augmented expression in human and experimental models of heart failure. ...
... In contrast, DCHS2 inhibition in a mouse model led to controlled hypertrophy. This study, therefore, established the novel lncExACT1/DCHS2 pathway as an important regulator of cardiac development [47]. ...
Full-text available
Noncoding RNAs (ncRNAs) play fundamental roles in cardiac development and cardiovascular diseases (CVDs), which are a major cause of morbidity and mortality. With advances in RNA sequencing technology, the focus of recent research has transitioned from studies of specific candidates to whole transcriptome analyses. Thanks to these types of studies, new ncRNAs have been identified for their implication in cardiac development and CVDs. In this review, we briefly describe the classification of ncRNAs into microRNAs, long ncRNAs, and circular RNAs. We then discuss their critical roles in cardiac development and CVDs by citing the most up-to-date research articles. More specifically, we summarize the roles of ncRNAs in the formation of the heart tube and cardiac morphogenesis, cardiac mesoderm specification, and embryonic cardiomyocytes and cardiac progenitor cells. We also highlight ncRNAs that have recently emerged as key regulators in CVDs by focusing on six of them. We believe that this review concisely addresses perhaps not all but certainly the major aspects of current progress in ncRNA research in cardiac development and CVDs. Thus, this review would be beneficial for readers to obtain a recent picture of key ncRNAs and their mechanisms of action in cardiac development and CVDs.
... Another study showed that lncExACT exhibited decreased expression in hearts of mice undergoing exercise. Moreover, lncExACT1 inhibition could induce physiological hypertrophy mimicking exercise-enhanced cardio-myogenesis [71]. Taken together, non-coding RNAs such as miRNAs and lncRNAs play an important role in exercise-induced cardiomyocyte proliferation. ...
Full-text available
Exercise has well-recognized beneficial effects on the whole body. Previous studies suggest that exercise could promote tissue regeneration and repair in various organs. In this review, we have summarized the major effects of exercise on tissue regeneration primarily mediated by stem cells and progenitor cells in skeletal muscle, nervous system, and vascular system. The protective function of exercise-induced stem cell activation under pathological conditions and aging in different organs have also been discussed in detail. Moreover, we have described the primary molecular mechanisms involved in exercise-induced tissue regeneration, including the roles of growth factors, signaling pathways, oxidative stress, metabolic factors, and non-coding RNAs. We have also summarized therapeutic approaches that target crucial signaling pathways and molecules responsible for exercise-induced tissue regeneration, such as IGF1, PI3K, and microRNAs. Collectively, the comprehensive understanding of exercise-induced tissue regeneration will facilitate the discovery of novel drug targets and therapeutic strategies.
... The immune responserelated genes included ESR1 [48,49], ITLN1 [50,51], AHNAK2 [52,53], FFAR2 [54][55][56], IL20RA [57,58], and VEGFC [59,60]. The myocardial growth and development-related genes included ESR1 [61,62], ITLN1 [63,64], EYA2 [65,66], VEGFC [67], and DCHS2 [68]. The myocardial conduction and electrophysiology-related genes included ESR1 [69], KCNIP1 [70,71], KCNA5 [72,73], and AHNAK2 [74]. ...
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Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease. However, a detailed DNA methylation (DNAme) landscape has not yet been elucidated. Our study combined DNAme and transcriptome profiles for HCM myocardium and identify aberrant DNAme associated with altered myocardial function in HCM. The transcription of methylation-related genes did not significantly differ between HCM and normal myocardium. Nevertheless, the former had an altered DNAme profile compared with the latter. The hypermethylated and hypomethylated sites in HCM tissues had chromosomal distributions and functional enrichment of correlated genes differing from those of their normal tissue counterparts. The GO analysis of network underlying the genes correlated with DNAme alteration and differentially expressed genes (DEGs) shows functional clusters centred on immune cell function and muscle system processes. In KEGG analysis, only the calcium signalling pathway was enriched either by the genes correlated with changes in DNAme or DEGs. The protein-protein interactions (PPI) underlying the genes altered at both the DNAme and transcriptional highlighted two important functional clusters. One of these was related to the immune response and had the estrogen receptor-encoding ESR1 gene as its node. The other cluster comprised cardiac electrophysiology-related genes. Intelliectin-1 (ITLN1), a component of the innate immune system, was transcriptionally downregulated in HCM and had a hypermethylated site within 1500 bp upstream of the ITLN1 transcription start site. Estimates of immune infiltration demonstrated a relative decline in immune cell population diversity in HCM. A combination of DNAme and transcriptome profiles may help identify and develop new therapeutic targets for HCM. ARTICLE HISTORY
Cardiac fibrosis is a common pathological feature of cardiac remodelling process with disordered expression of multiple genes and eventually lead to heart failure. Emerging evidence suggests that long noncoding RNAs (lncRNAs) have emerged as critical regulators of various biological processes. However, the exact mechanisms of lncRNAs as mediators in cardiac fibrosis have not been fully elucidated. This study aimed to profile the lncRNA expression pattern in human cardiac fibroblasts (HCFs) with cardiac fibrosis. We treated HCFs with transforming growth factor-β (TGF-β) to induce their activation. Then, strand-specific RNA-seq was performed to profile and classify lncRNAs; and perform functional analysis in HCFs. We study the transformation of HCFs with molecular and cell biology methods. Among all identified lncRNA candidates, 176 and 526 lncRNAs were upregulated and downregulated respectively in TGF-β-stimulated HCFs compared with controls. Functional analyses revealed that the target genes of differentially expressed lncRNAs were mainly related to focal adhesion, metabolic pathways, Hippo signaling pathway, PI3K-Akt signaling pathway, regulation of actin cytoskeleton, and hypertrophic cardiomyopathy. As a representative, novel lncRNAs NONHSAG005537 and NONHSAG017620 inhibited the proliferation, migration, invasion, and transformation of HCFs induced by TGF-β. Collectively, our study established the expression signature of lncRNAs in cardiac fibrosis and demonstrated the cardioprotective role of NONHSAG005537 and NONHSAG017620 in cardiac fibrosis, providing a promising target for anti-fibrotic therapy.
Purpose of review: Here, we review recent findings on the role of long noncoding RNAs (lncRNAs) in cardiovascular disease (CVD). In addition, we highlight some of the latest findings in lncRNA biology, providing an outlook for future avenues of lncRNA research in CVD. Recent findings: Recent publications provide translational evidence from patient studies and animal models for the role of specific lncRNAs in CVD. The molecular effector mechanisms of these lncRNAs are diverse. Overall, cell-type selective modulation of gene expression is the largest common denominator. New methods, such as single-cell profiling and CRISPR/Cas9-screening, reveal additional novel mechanistic principles: For example, many lncRNAs establish RNA-based spatial compartments that concentrate effector proteins. Also, RNA modifications and splicing features can be determinants of lncRNA function. Summary: lncRNA research is passing the stage of enumerating lncRNAs or recording simplified on-off expression switches. Mechanistic analyses are starting to reveal overarching principles of how lncRNAs can function. Exploring these principles with decisive genetic testing in vivo remains the ultimate test to discern how lncRNA loci, by RNA motifs or DNA elements, affect CVD pathophysiology.
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As a prevalent epigenetic modification, the role of m6A has been increasingly highlighted in the alteration of numerous RNAs implicated with multiple biological processes, such as formation, export, translation, and degradation. With further the understanding of m6A, accumulating evidence shows that m6A modification similarly affects metabolic process of non-coding genes. But the specifical interplay of m6A and ncRNAs (non-coding RNAs) in gastrointestinal cancers still lacks complete discussion. Thus, we analyzed and summarized how ncRNAs affect the regulators of m6A and by what means the expression of ncRNAs is altered via m6A in gastrointestinal cancers. We focused on the effect of the interaction of m6A and ncRNAs on the molecular mechanisms of malignant behavior in gastrointestinal cancers, revealing more possibilities of ncRNAs for diagnosis and treatment in term of epigenetic modification.
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Copy number variations (CNVs) have long been recognized as pathogenic factors for congenital heart disease (CHD). Few CHD associated CNVs could be interpreted as dosage effect due to disruption of coding sequences. Emerging evidences have highlighted the regulatory roles of long noncoding RNAs (lncRNAs) in cardiac development. Whereas it remains unexplored whether lncRNAs within CNVs (CNV-lncRNAs) could contribute to the etiology of CHD associated CNVs. Here we constructed coexpression networks involving CNV-lncRNAs within CHD associated CNVs and protein coding genes using the human organ developmental transcriptomic data, and showed that CNV-lncRNAs within 10 of the non-syndromic CHD associated CNVs clustered in the most significant heart correlated module, and had highly correlated coexpression with multiple key CHD genes. HSALNG0104472 within 15q11.2 region was identified as a hub CNV-lncRNA with heart-biased expression and validated experimentally. Our results indicated that HSALNG0104472 should be a main effector responsible for cardiac defects of 15q11.2 deletion through regulating cardiomyocytes differentiation. Our findings suggested that CNV-lncRNAs could potentially contribute to the pathologies of a maximum proportion of 68.4% (13/19) of non-syndromic CHD associated CNVs. These results indicated that explaining the pathogenesis of CHD associated CNVs should take account of the noncoding regions.
Changes in human genome-wide long noncoding RNAs (lncRNAs) associated with air pollution are unknown. This study aimed to investigate the effect of air pollution on human exosomal lncRNAs. A randomized, crossover trial was conducted among 35 healthy adults. Participants were allocated to 4 h exposure in road (high air pollution) and park (low air pollution) sessions in random order with a 2 week washout period. RNA sequencing was performed to measure lncRNAs. Differential lncRNAs were identified using a linear mixed-effect model. Mean concentrations of air pollutants such as ultrafine particles (UFP), black carbon (BC), carbon monoxide (CO), and nitrogen dioxide (NO2) were 2-3 times higher in the road than those in the park. Fifty-five lncRNAs [false discovery rate (FDR) < 0.05] including lncRNA NORAD, MALAT1, and H19 were changed in response to air pollution exposure. We found that 54 lncRNAs were associated with CO, 49 lncRNAs with UFP, 49 lncRNAs with BC, 48 lncRNAs with NO2, and 4 lncRNAs with PM2.5 (FDR < 0.05). These differential lncRNAs participated in dozens of pathways including cardiovascular signaling, epithelial cell proliferation, inflammation, and transforming growth factor. This trial for the first time profiled changes of human exosomal lncRNAs following air pollution. Our findings revealed multiple biological processes moderated by lncRNAs and provided epigenetic insights into cardiovascular effects of air pollution.
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Subcellular localization of RNAs has gained attention in recent years as a prevalent phenomenon that influences numerous cellular processes. This is also evident for the large and relatively novel class of long noncoding RNAs (lncRNAs). Because lncRNAs are defined as RNA transcripts >200 nucleotides that do not encode protein, they are themselves the functional units, making their subcellular localization critical to their function. The discovery of tens of thousands of lncRNAs and the cumulative evidence involving them in almost every cellular activity render assessment of their subcellular localization essential to fully understanding their biology. In this review, we summarize current knowledge of lncRNA subcellular localization, factors controlling their localization, emerging themes, including the role of lncRNA isoforms and the involvement of lncRNAs in phase separation bodies, and the implications of lncRNA localization on their function and on cellular behavior. We also discuss gaps in the current knowledge as well as opportunities that these provide for novel avenues of investigation.
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Aims Cardiac microRNA-132-3p (miR-132) levels are increased in patients with heart failure (HF) and mechanistically drive cardiac remodelling processes. CDR132L, a specific antisense oligonucleotide, is a first-in-class miR-132 inhibitor that attenuates and even reverses HF in preclinical models. The aim of the current clinical Phase 1b study was to assess safety, pharmacokinetics, target engagement, and exploratory pharmacodynamic effects of CDR132L in patients on standard-of-care therapy for chronic ischaemic HF in a randomized, placebo-controlled, double-blind, dose-escalation study (NCT04045405). Methods and results Patients had left ventricular ejection fraction between ≥30% and <50% or amino terminal fragment of pro-brain natriuretic peptide (NT-proBNP) >125 ng/L at screening. Twenty-eight patients were randomized to receive CDR132L (0.32, 1, 3, and 10 mg/kg body weight) or placebo (0.9% saline) in two intravenous infusions, 4 weeks apart in four cohorts of seven (five verum and two placebo) patients each. CDR132L was safe and well tolerated, without apparent dose-limiting toxicity. A pharmacokinetic/pharmacodynamic dose modelling approach suggested an effective dose level at ≥1 mg/kg CDR132L. CDR132L treatment resulted in a dose-dependent, sustained miR-132 reduction in plasma. Patients given CDR132L ≥1 mg/kg displayed a median 23.3% NT-proBNP reduction, vs. a 0.9% median increase in the control group. CDR132L treatment induced significant QRS narrowing and encouraging positive trends for relevant cardiac fibrosis biomarkers. Conclusion This study is the first clinical trial of an antisense drug in HF patients. CDR132L was safe and well tolerated, confirmed linear plasma pharmacokinetics with no signs of accumulation, and suggests cardiac functional improvements. Although this study is limited by the small patient numbers, the indicative efficacy of this drug is very encouraging justifying additional clinical studies to confirm the beneficial CDR132L pharmacodynamic effects for the treatment of HF.
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Intercalated discs (ICD), specific cell-to-cell contacts that connect adjacent cardiomyocytes, ensure mechanical and electrochemical coupling during contraction of the heart. Mutations in genes encoding ICD components are linked to cardiovascular diseases. Here, we show that loss of Xinβ, a newly-identified component of ICDs, results in cardiomyocyte proliferation defects and cardiomyopathy. We uncovered a role for Xinβ in signaling via the Hippo-YAP pathway by recruiting NF2 to the ICD to modulate cardiac function. In Xinβ mutant hearts levels of phosphorylated NF2 are substantially reduced, suggesting an impairment of Hippo-YAP signaling. Cardiac-specific overexpression of YAP rescues cardiac defects in Xinβ knockout mice-indicating a functional and genetic interaction between Xinβ and YAP. Our study reveals a molecular mechanism by which cardiac-expressed intercalated disc protein Xinβ modulates Hippo-YAP signaling to control heart development and cardiac function in a tissue specific manner. Consequently, this pathway may represent a therapeutic target for the treatment of cardiovascular diseases.
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Antisense oligonucleotides (ASOs) bind sequence specifically to the target RNA and modulate protein expression through several different mechanisms. The ASO field is an emerging area of drug development that targets the disease source at the RNA level and offers a promising alternative to therapies targeting downstream processes. To translate ASO-based therapies into a clinical success, it is crucial to overcome the challenges associated with off-target side effects and insufficient biological activity. In this regard, several chemical modifications and diverse delivery strategies have been explored. In this review, we systematically discuss the chemical modifications, mechanism of action, and optimized delivery strategies of several different classes of ASOs. Further, we highlight the recent advances made in development of ASO-based drugs with a focus on drugs that are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical applications. We also discuss various promising ASO-based drug candidates in the clinical trials, and the outstanding opportunity of emerging microRNA as a viable therapeutic target for future ASO-based therapies.
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Despite proven efficacy of pharmacotherapies targeting primarily global neurohormonal dysregulation, heart failure (HF) is a growing pandemic with increasing burden. Treatments mechanistically focusing at the cardiomyocyte level are lacking. MicroRNAs (miRNA) are transcriptional regulators and essential drivers of disease progression. We previously demonstrated that miR-132 is both necessary and sufficient to drive the pathological cardiomyocytes growth, a hallmark of adverse cardiac remodelling. Therefore, miR-132 may serve as a target for HF therapy. Here we report further mechanistic insight of the mode of action and translational evidence for an optimized, synthetic locked nucleic acid antisense oligonucleotide inhibitor (antimiR-132). We reveal the compound’s therapeutic efficacy in various models, including a clinically highly relevant pig model of HF. We demonstrate favourable pharmacokinetics, safety, tolerability, dose-dependent PK/PD relationships and high clinical potential for the antimiR-132 treatment scheme.
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BACKGROUND The American Heart Association, in conjunction with the National Institutes of Health, annually reports on the most up-to-date statistics related to heart disease, stroke, and cardiovascular risk factors, including core health behaviors (smoking, physical activity, diet, and weight) and health factors (cholesterol, blood pressure, and glucose control) that contribute to cardiovascular health. The Statistical Update presents the latest data on a range of major clinical heart and circulatory disease conditions (including stroke, congenital heart disease, rhythm disorders, subclinical atherosclerosis, coronary heart disease, heart failure, valvular disease, venous disease, and peripheral artery disease) and the associated outcomes (including quality of care, procedures, and economic costs). METHODS The American Heart Association, through its Statistics Committee, continuously monitors and evaluates sources of data on heart disease and stroke in the United States to provide the most current information available in the annual Statistical Update. The 2020 Statistical Update is the product of a full year’s worth of effort by dedicated volunteer clinicians and scientists, committed government professionals, and American Heart Association staff members. This year’s edition includes data on the monitoring and benefits of cardiovascular health in the population, metrics to assess and monitor healthy diets, an enhanced focus on social determinants of health, a focus on the global burden of cardiovascular disease, and further evidence-based approaches to changing behaviors, implementation strategies, and implications of the American Heart Association’s 2020 Impact Goals. RESULTS Each of the 26 chapters in the Statistical Update focuses on a different topic related to heart disease and stroke statistics. CONCLUSIONS The Statistical Update represents a critical resource for the lay public, policy makers, media professionals, clinicians, healthcare administrators, researchers, health advocates, and others seeking the best available data on these factors and conditions.
Background: Exercise training's benefits in cardiovascular system have been well accepted, however, the underlying mechanism remains to be explored. Here, we report the initial functional characterization of an exercise-induced cardiac physiological hypertrophy associated novel lncRNA. Methods: Using lncRNA microarray profiling, we identified lncRNAs in contributing the modulation of exercise-induced cardiac growth that we termed Cardiac Physiological hypertrophy associated regulator (CPhar). Mice with Adeno-associated virus serotype 9 (AAV9) driving CPhar overexpression and knockdown were used in in-vivo experiments. Swim training was used to induce physiological cardiac hypertrophy in mice and ischemia reperfusion injury (IR/I) surgery was conducted to investigate the protective effects of CPhar in mice. To investigate the mechanisms of CPhar's function, we performed various analysis including RTqPCR, western blot, histology, cardiac function (by echocardiography), functional rescue experiments, mass spectrometry, in vitro RNA transcription, RNA pull down, RNA immunoprecipitation, chromatin immunoprecipitation assay, luciferase reporter assay, and coimmunoprecipitation assays. Results: We screened the lncRNAs in contributing the modulation of exercise-induced cardiac growth via lncRNA microarray profiling and found that CPhar was increased with exercise and was necessary for exercise-induced physiological cardiac growth. Gain- and loss- of function of CPhar regulated the expression of proliferation markers, hypertrophy, and apoptosis in cultured neonatal mouse cardiomyocytes (NMCMs). Overexpression of CPhar prevented myocardial ischemia reperfusion injury and cardiac dysfunction in vivo . We identified DDX17 as a binding partner of CPhar in regulating CPhar downstream factor ATF7 by sequestering C/EBPβ. Conclusions: Our study of this lncRNA CPhar provides new insights into the regulation of exercise-induced cardiac physiological growth, demonstrating the cardioprotective role of CPhar in the heart, as well as expanding our mechanistic understanding of lncRNA function.
Revealing the interactions of long noncoding RNAs (LncRNAs) with specific genomic regions is of basic importance to explore the mechanisms by which they regulate gene expression. Chromatin oligo-affinity precipitation (ChOP) technique was the first method developed to analyze the association of LncRNAs with genomic regions in the chromatin context. The first step of the procedure is cell cross-linking, aimed at stabilizing the RNA–protein–DNA complexes. Next, after chromatin fragmentation, the RNA complexes are pulled down through hybridization with antisense oligonucleotides tagged with biotin and purification with anti-biotin antibody. After extensive wash, the RNA-interacting chromatin is eluted by RNase treatment. Subsequent protein elimination and DNA purification allow to retrieve DNA fragments for following analyses such as qPCR or sequencing.
Activin type II receptor (ActRII) ligands have been implicated in muscle wasting in aging and disease. However, the role of these ligands and ActRII signaling in the heart remains unclear. Here, we investigated this catabolic pathway in human aging and heart failure (HF) using circulating follistatin-like 3 (FSTL3) as a potential indicator of systemic ActRII activity. FSTL3 is a downstream regulator of ActRII signaling, whose expression is up-regulated by the major ActRII ligands, activin A, circulating growth differentiation factor-8 (GDF8), and GDF11. In humans, we found that circulating FSTL3 increased with aging, frailty, and HF severity, correlating with an increase in circulating activins. In mice, increasing circulating activin A increased cardiac ActRII signaling and FSTL3 expression, as well as impaired cardiac function. Conversely, ActRII blockade with either clinical-stage inhibitors or genetic ablation reduced cardiac ActRII signaling while restoring or preserving cardiac function in multiple models of HF induced by aging, sarcomere mutation, or pressure overload. Using unbiased RNA sequencing, we show that activin A, GDF8, and GDF11 all induce a similar pathologic profile associated with up-regulation of the proteasome pathway in mammalian cardiomyocytes. The E3 ubiquitin ligase, Smurf1, was identified as a key downstream effector of activin-mediated ActRII signaling, which increased proteasome-dependent degradation of sarcoplasmic reticulum Ca ²⁺ ATPase (SERCA2a), a critical determinant of cardiomyocyte function. Together, our findings suggest that increased activin/ActRII signaling links aging and HF pathobiology and that targeted inhibition of this catabolic pathway holds promise as a therapeutic strategy for multiple forms of HF.