Exercise-induced CITED4 expression is necessary for regional remodeling of cardiac microstructural tissue helicity

Abstract

Both exercise-induced molecular mechanisms and physiological cardiac remodeling have been previously studied on a whole heart level. However, the regional microstructural tissue effects of these molecular mechanisms in the heart have yet to be spatially linked and further elucidated. We show in exercised mice that the expression of CITED4, a transcriptional co-regulator necessary for cardioprotection, is regionally heterogenous in the heart with preferential significant increases in the lateral wall compared with sedentary mice. Concordantly in this same region, the heart’s local microstructural tissue helicity is also selectively increased in exercised mice. Quantification of CITED4 expression and microstructural tissue helicity reveals a significant correlation across both sedentary and exercise mouse cohorts. Furthermore, genetic deletion of CITED4 in the heart prohibits regional exercise-induced microstructural helicity remodeling. Taken together, CITED4 expression is necessary for exercise-induced regional remodeling of the heart’s microstructural helicity revealing how a key molecular regulator of cardiac remodeling manifests into downstream local tissue-level changes. Expression of transcription factor CITED4 is necessary for exercise-induced regional remodeling of the heart’s microstructural helicity, revealing how a key molecular regulator of cardiac remodeling mediates local tissue-level changes.

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ARTICLE
Exercise-induced CITED4 expression is necessary
for regional remodeling of cardiac microstructural
tissue helicity
Robert A. Eder 1, Maaike van den Boomen 1,2,3,4, Salva R. Yurista 1,4, Yaiel G. Rodriguez-Aviles 1,5,
Mohammad Rashedul Islam1,4, Yin-Ching Iris Chen2,4, Lena Trager1, Jaume Coll-Font1,2,4, Leo Cheng2,4,
Haobo Li 1,4, Anthony Rosenzweig 1,4,6, Christiane D. Wrann 1,4,7 ✉& Christopher T. Nguyen 1,2,4,8,9 ✉
Both exercise-induced molecular mechanisms and physiological cardiac remodeling have
been previously studied on a whole heart level. However, the regional microstructural tissue
effects of these molecular mechanisms in the heart have yet to be spatially linked and further
elucidated. We show in exercised mice that the expression of CITED4, a transcriptional co-
regulator necessary for cardioprotection, is regionally heterogenous in the heart with pre-
ferential significant increases in the lateral wall compared with sedentary mice. Concordantly
in this same region, the heart’s local microstructural tissue helicity is also selectively
increased in exercised mice. Quantification of CITED4 expression and microstructural tissue
helicity reveals a significant correlation across both sedentary and exercise mouse cohorts.
Furthermore, genetic deletion of CITED4 in the heart prohibits regional exercise-induced
microstructural helicity remodeling. Taken together, CITED4 expression is necessary for
exercise-induced regional remodeling of the heart’s microstructural helicity revealing how a
key molecular regulator of cardiac remodeling manifests into downstream local tissue-level
changes.
https://doi.org/10.1038/s42003-022-03635-y OPEN
1Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA 02129, USA. 2Martinos Center for Biomedical Imaging, Massachusetts
General Hospital, Charlestown, MA 02129, USA. 3Department of Radiology, University Medical Center Groningen, University of Groningen, Hanzeplein 1,
9713 GZ Groningen, The Netherlands. 4Harvard Medical School, Boston, MA 02129, USA. 5Ponce Health Sciences University, School of Medicine, Ponce, PR
00716, USA. 6Massachusetts General Hospital, Cardiology Division and Corrigan Minehan Heart Center, Boston, MA 02114, USA. 7McCance Center for
Brain Health, Massachusetts General Hospital, Boston, MA 02114, USA. 8Division of Health Sciences and Technology, Harvard-Massachusetts Institute of
Technology, Cambridge, MA 02139, USA. 9Cardiovascular Innovation Research Center, Heart, Vascular, and Thoracic Institute, Cleveland Clinic, Cleveland,
Ohio 44195, USA. ✉email: cwrann@mgh.harvard.edu;nguyenc6@ccf.org
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Exercise has expansive benefits for the adult heart by pro-
tecting it from cardiovascular disease1. These beneficial
effects arise from changes in metabolism, skeletal muscles,
peripheral vessels, and the heart itself2. Specifically in the heart,
exercise induces cardiac structural remodeling in the form of
hypertrophy2–5, which occurs with an increase in the length and
width of cardiomyocytes2,3. Aside from producing beneficial
myocardial hypertrophy, some transcription factors and cell
proliferation markers have also been implicated in producing
cardioprotective effects2,4,6. Specifically, CBP/p300-interacting
transactivators with E [glutamic acid]/D [aspartic acid]-rich-
carboxyl terminal domain 4 (CITED4) is known to play a key role
in the exercised-induced cell hypertrophy pathways7,8. The
upregulated CITED4 is involved in physiological heart growth as
a downstream effector of transcriptional pathways resulting in
cell growth and proliferation2,9. Triggered by exercise, although
not exclusive to exercise7, CITED4 has shown to globally increase
in the cardiac tissue as measured by quantitative polymerase
chain reaction (qPCR)2,7. Importantly, studies using conditional
deletion of CITED4 in cardiomyocytes have demonstrated that it
is necessary for adaptive cardiac remodeling in response to
exercise7.
This differs from the pathological remodeling in heart failure
where the length of cardiomyocytes increases disproportionately
to width10. Structural remodeling is a complex yet dynamic
process triggered by changes in the cardiac workload which cause
either physiological or pathological hypertrophic growth resulting
in a spatially altered architecture of the myocardium1. While
previous studies have confirmed that exercise-induced cardio-
myogenesis can naturally occur within mature mammalian hearts
leading ultimately to improved cardiac function1, maladaptive
growth has been linked to an increased risk of heart failure11–14.
This indicates that the therapeutic abilities of exercise are not
specific to cell growth alone but may result in potential down-
stream changes to tertiary tissue structures that form the
microstructural helicity of the heart. The heart’s microstructural
helicity has been predominantly characterized tediously through
complex histological sectioning15 revealing cardiomyocytes are
helically oriented smoothly transitioning from left-handedness to
right-handedness throughout the transmural tissue layers of the
myocardium. Owing to the challenges to performing such com-
plex histology, there has yet to be studies that elucidate the
potential impact of exercise-induced molecular mechanisms on
the heart’s microstructural helicity.
Recent studies have shown that the heart’s microstructural
helicity can be non-invasively quantified by diffusion tensor
magnetic resonance imaging (DT-MRI) in mice16–18 and
patients19–23. Detecting the spatial distributions of the diffusion
of water molecules enables DT-MRI to map the underlying
orientation of cardiomyocytes in each imaging voxel24,25. Fur-
thermore, DT-MRI can uncover these microstructural changes to
aid in the detection of myocardial fibrosis21,26–29 and has also
been used to assess myocardial regeneration and microstructural
changes by cell-based therapies30,31. In addition, previous studies
have shown strong utility of DT-MRI in detecting cardiac
structural changes associated with hypertrophic cardiomyopathy
(HCM) and ventricular arrythmias22 demonstrating the sig-
nificant clinical and scientific potential of this tool. DT-MRI is
well poised to characterize the microstructural impact of exercise
and can map how exercise-induced hypertrophy manifests at the
microstructural level.
Spatially linking the molecular mechanism of CITED4 to
microstructural helical remodeling would require meticulous
sectioning of the heart to perform thousands of qPCR experi-
ments. However recently, new approaches to RNA in situ
hybridization (RNA-FISH) have enabled the quantitative analysis
of the spatial distribution of gene expression32. RNA-FISH could
be used to map the spatial distribution of CITED4 as a physio-
logical proliferation marker to further elucidate its role in
microstructural tissue alterations in exercise-induced cardiac
remodeling. Furthermore, counterstaining for nuclei with DAPI
would allow quantitative measurements of CITED4 expression
for a given region of interest.
In this study, we aim to define the spatial interactions between
the exercise-induced molecular mechanism of CITED4 with
tissue-level microstructural helical remodeling in exercised and
sedentary mice. By imploring two novel imaging technologies,
RNA-FISH and DT-MRI, we can efficiently interrogate the whole
heart while also spatially co-registering quantifications of
CITED4 expression with myocardial microstructural tissue heli-
city. Furthermore, we used cardiomyocyte-specific CITED4
knock out mice (C4KO) to demonstrate the crucial role CITED4
plays in necessitating these microstructural changes following
exercise.
Results
Exercise from wheel running. Free wheel-running was used to
exercise wild-type mice (n=7), C4KO mice (n=7) and fl/flmice
(n=6) to induce cardiac remodeling while sedentary groups of
wild-type mice (n=7), C4KO mice (n=13) and fl/flmice
(n=7) were not provided access to running wheels (Fig. 1). The
average cumulative total kilometers ran by the exercise group up
to 8 weeks was 371.9 [277.0–450.8] km. The average distance run
per day by the end of the 8 weeks of the exercise was 6.028
[4.397–7.336] km/day (Fig. 2a, b). Over the eight-week period, the
sedentary group had a significantly greater increase in body
weight in comparison to the exercise group (p=0.017) (Fig. 2c).
Analyses of the wall thickness of the left ventricle and left ven-
tricular (LV) mass in the exercise group demonstrated significant
increases (47.7%, p=0.0002 and 9.5%, p=0.004 respectively)
recapitulating previously described exercise-induced cardiac
remodeling (Fig. 2d, e).
Exercise induces microstructural remodeling of regional car-
diac tissue helicity. After 8 weeks, the sedentary and exercise
mouse hearts were perfused and extracted for non-invasive
ex vivo DT-MRI to reveal the heart’s tissue microstructure. Fiber
orientation was mapped for each voxel, and regional cardiac
tissue helicity was calculated across the transmural wall for each
American Heart Association (AHA) segment as the gradient of
the helix angle15 normalized to the percent heart wall depth from
the endocardium (innermost layer) to the epicardium (outermost
layer) (Fig. 3a). Total cardiac tissue helicity was significantly
(p=0.0024) increased (19.9%) in the exercise cohort compared
with the sedentary cohort (Fig. 4d). Regional cardiac tissue heli-
city analyses revealed significant (p=0.048) increases (17.1%)
within the septal region (AHA segment 2 and 3) and even greater
significant (p=0.0007) increases (21.5%) in the lateral wall (AHA
segments 5 and 6) when comparing exercise with sedentary
cohorts (Fig. 4a–d). This underlying remodeling of cardiac tissue
helicity in the exercise cohort was predominantly driven by the
increase in the helix angle of the endocardial layer (Fig. 4a).
Cardiac regional heterogeneity in exercise-induced modulation
of CITED4 expression. Following DT-MRI, the intact hearts were
sectioned and then stained during RNA-FISH for CITED4 and
DAPI. These sections were imaged under fluorescent light
microscopy and the tissue level CITED4 expression normalized to
DAPI nuclei counts was quantified (Fig. 3b–f). The exercise group
revealed a trend towards an increased (59%) total CITED4/DAPI
ratio (Fig. 4h, p=0.051). A regional analysis revealed an increased
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(71.7%) CITED4/DAPI ratio in the lateral wall (including AHA
segments 5 and 6) between the exercise and sedentary groups
(Fig. 4e, g, p=0.048). However, while there was also an increase
in the CITED4/DAPI ratio (32.9%) of the septal region (including
AHA segments 2 and 3), the difference between the exercise and
sedentary group was insignificant (Fig. 4e, f, p=0.22). Further
spatial comparisons of the CITED4/DAPI ratio within the epi-,
mid- and endocardium of each AHA section revealed further
heterogeneity of CITED4 expression (Supplemental Figs. 1a–c, 2).
Significant increases in the CITED4/DAPI ratio were present
within varying AHA sections within the three transmural layers
(Supplemental Figs. 1a–d, 2).
Exercise-induced modulation of CITED4 expression is spatially
linked to microstructural remodeling of cardiac tissue helicity.
To investigate the spatial relationship between CITED4 expres-
sion and microstructural remodeling of cardiac tissue helicity,
interclass correlations were performed on septal/lateral wall
regional segments and total tissue level. Moderate correlations
were found within the septal and lateral regions between
CITED4/DAPI ratio and cardiac tissue helicity (R2=0.78,
p< 0.0001 and R2=0.50, p< 0.0001 respectively) (Fig. 5). Sub-
stantial correlations were found between the total CITED4/DAPI
ratio and total cardiac tissue helicity (R2=0.87, p< 0.0001).
Exercised C4KO mice exhibit blunted microstructural remo-
deling of cardiac tissue helicity. To understand whether CITED4
was necessary for microstructural remodeling, we performed DT-
MRI and RNA-FISH on mice with cardiomyocyte-specific dele-
tion of CITED4 (C4KO). Cardiac tissue helicity was significantly
reduced in both C4KO sedentary and exercise groups compared
to the fl/flsedentary group (−31.7%, p=0.0024) and fl/flexercise
group (−40.3%, p< 0.0001, Fig. 6a, b). In the fl/flgroup, exercise
produced a trending increase in cardiac tissue helicity similar to
Fig. 2 Wheel running promotes significant changes in body weight, heart mass, and thickness. a The average distance cohort 1 ran per day in kilometers
(km). bThe average total km cohort 2 ran through 8-weeks. cThe body weight changes between the sedentary and exercise groups of cohort 1. dThe
difference in wall thickness between the sedentary and exercise groups of cohort 1. eThe difference in left ventricular (LV) mass between the exercise and
sedentary groups. Unpaired two-tailed t-test *P< 0.05, **P< 0.01, ***P< 0.001. Data are presented min to max. Within each box, horizontal black and red
lines denote median values; boxes extend from the 25th to the 75th percentile of each group’s distribution of values.
Fig. 1 Characterization of the experimental set-up. Cohort 1 consisted of wild-type sedentary (n=7) and wild-type exercise (n=7) mice. Cohort 2
included cardiomyocyte-specific CITED4 knock out (C4KO) exercise (n=7), C4KO sedentary (n=13), fl/flexercise (n=6) and fl/flsedentary (n=7)
mice. These groups were housed for 8 weeks, and the exercise group had free access to a running wheel. The hearts were perfused and excised after the
8-week holding period. The hearts were imaged using 14 T magnetic resonance imaging (MRI) followed by RNA-FISH of a whole axial slice of the left
ventricle. These slides were imaged and analyzed with a fluorescent light microscope.
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the wild types in previous experiments (25.1%, p=0.06). We
measured the levels of CITED4 expression in these groups to
ensure that the C4KO and fl/flgroups were expressing CITED4 as
expected. The cardiomyocyte-specific knock out of CITED4
produced a negligible expression of CITED4 (Fig. 6c, d). Mean-
while, the expression of CITED4 in the fl/flwas comparable to
cohort 1 (Fig. 6c, d).
Discussion
In this study, the spatial distribution and upregulation of the
physiological growth marker CITED4 were determined in exer-
cised cardiac mouse tissue using RNA-FISH and compared with
DT-MRI-based microstructural tissue biomarkers. The trend
towards a total increase in the CITED4/DAPI ratio in the exer-
cised heart tissue was in line with previous reports using
qPCR2,7,8. However, RNA-FISH also allowed for a more targeted
spatial analysis which revealed increased CITED4/DAPI ratios
primarily within the lateral wall. Furthermore, the ex vivo ana-
tomical cardiac MRI of both the sedentary and exercised hearts
confirmed the increase in left ventricular mass by exercise1,2and
the myocardial microstructural characterization revealed the
microstructural alterations characterized by helicity within the
left ventricle of the exercised mice. The direct spatial correlation
of these microstructural tissues and molecular changes could
Fig. 3 Helix angle analysis and RNA-FISH imaging and quantification of exercise and sedentary mouse hearts. a Helix angles change from the
endocardium to epicardium between sedentary and exercised mice. bA visual representation of the location of the six AHA sections and a zoomed in
images of the complete cross-section across each transmural layer. cComplete image of sectioned mouse tissue with AHA sections overlayed.
dCITED4 signal isolated within mid-myocardium. eDAPI signal isolated and magnified in the mid-myocardium. fCITED4 and DAPI signal imaged from
mid-myocardium overlayed.
Fig. 4 Wheel running in mice promotes physiological remodeling in the heart in coordination with spatial upregulations of CITED4. a Representative
DT-MRI images of helicity changes from the endocardium to epicardium between sedentary and exercised mice in cohort 1. b–dHelicity differences within
the septum, lateral wall and averaged across all AHA sections. eMagnified representative images of DAPI and CITED4 within the septal and lateral wall
regions within exercised and sedentary mice. f–hCITED4 signal in sedentary and exercised mice within the septum, lateral wall and averaged across all
AHA sections. Unpaired two-tailed t-test. *P< 0.05, **P< 0.01, ***P< 0.001. Data are presented min to max. Within each box, horizontal black and red
lines denote median values; boxes extend from the 25th to the 75th percentile of each group’s distribution of values.
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provide further insight into how molecular expression can
manifest into gross anatomical and physiological remodeling. To
the best our knowledge, this is the first study that investigates the
interplay between regional gene expression and microstructural
tissue remodeling yielding insight on how tissue level remodeling
may manifest from molecular mechanisms.
DT-MRI is a powerful tool to determine small scale fiber
orientation changes associated with the disease pathology30,31.In
this study, DT-MRI of the exercised mice revealed similar
microstructural changes to previous results in myocardial
infarction mouse models treated with cell therapy injections31,
which included significant changes to the helicity of the myo-
cardial fibers. However, in this study, these changes to micro-
structural helicity are tied directly to exercise-induced structural
remodeling, which is shown to result in improved outcomes
following cardiac injury and disease30. These exercise-induced
Fig. 5 Regional helicity changes are significantly correlated with CITED4/DAPI expression. a–cSimple linear regressions of CITED4/DAPI signal and
helicity within the septum, lateral wall and averaged across all AHA sections. Black dots represent sedentary animals and red dots represent exercised
animals. The blue line represents the total simple linear regression of both groups. *P< 0.05, **P< 0.01, ***P< 0.001. Data presented with mean and 95%
confidence interval.
Fig. 6 Cardiomyocyte-specific knock out of CITED4 impacts regional tissue helicity changes. a Helix angle changes differ significantly between groups.
bAn analysis of helicity between all groups of cohort 2. cImages of DAPI and CITED4 staining of representative mice from cohort 2. dQuantification of
CITED4/DAPI using RNA-FISH with cohort 2. Two-way ANOVA used for multivariant analyses *P< 0.05, **P< 0.01, ***P< 0.001. Data are presented min
to max. Within each box, horizontal black and red lines denote median values; boxes extend from the 25th to the 75th percentile of each group’s
distribution of values.
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changes to helicity shown in this study further demonstrate the
structural cardiac remodeling benefits of exercise, and in con-
junction with past work30, help to demonstrate the structural
changes produced by exercise that may be cardioprotective. Given
DT-MRI can be performed in vivo and non-invasively19–23,
potentially these CITED4-induced myocardial microstructural
changes could be explored to see if they are cardioprotective in a
rescue preclinical study.
Conventional qPCR has routinely been used as the standard
procedure for determining total CITED4 expression1,2,8. How-
ever, in this study, we were able to demonstrate the difference in
the spatial distribution of CITED4 expression across the six dif-
ferent AHA regions of the heart and within the three different
transmural layers using RNA-FISH. Specifically, increases in
CITED4 within the lateral wall correlate with cardiac remodeling
revealed by DT-MRI-based microstructural changes. Although,
qPCR already showed significant increases in CITED4 expression
within exercised cardiac tissue2,7, the use of RNA-FISH-based
spatial information in this study helped to directly link the
microstructural tissue modifications with the local transcriptional
changes. The implications of this CITED4 local expression may
serve to guide more targeted delivery of CITED47or other
modifiers to cardiac tissue, such as Irisin33,34.
While CITED4 is known to play a role in tissue proliferation
and hypertrophy2,7–9, it is inversely regulated by the transcription
factor C/EBPβ2and manipulation of CITED4 levels has been
shown to impact cardiomyocyte proliferation8. Following exer-
cise, a reduction in C/EBPβleads to an increase in cell growth and
division2and at the same time C/EBPβmay also be involved in
the induction of the PGC1αpathway, which prevents cardiac
dysfunction following hypertrophy2,8. The inhibition of CITED4
by C/EBPβled to the hypothesis that C/EBPβ’s impact on
reducing physiological growth may be directly linked to the
inhibition of CITED48. Data presented within this study, as well
as recent studies, seem to further confirm this hypothesis2,7.
Furthermore, this CITED4 pathway provides a potential link to
the microstructural changes revealed through DT-MRI. The
specific changes in helicity present between the exercise and
sedentary groups might be explained, in part, by these exercised-
induced molecular changes.
The specific role of CITED4 in regulating gene transcription,
appears to impact exercise-induced cardiac growth, as it has been
found to be a physiological marker for cardiac growth which is
also seen in the increase in cardiac wall thickness and LV mass in
this study2,8. Although more studies must be performed to fur-
ther elucidate this pathway, it appears C/EBPβand CITED4 are
intrinsically tied to the production of exercise-induced cardiac
growth2,8,35. Exercise specifically upregulates CITED4, while
downregulating C/EBPβto produce beneficial cardiomyocyte
growth and tissue remodeling from micro to macro and from
structural to functional2,8,35. In this study, DT-MRI analyses
confirm that the trends in increased CITED4 expression occur in
conjunction with the widespread structural changes marked by
increased helicity. Our results specifically indicate an increased
amount of CITED4 expression within the lateral wall of the left
ventricle. This upregulation of CITED4 within the lateral wall of
the left ventricle could also help to elucidate the mechanism for
the specific lateral wall increases in tissue volume found with DT-
MRI.
Utilizing the CITED4 KO mice, we demonstrated the necessary
role that CITED4 plays in cardiac microstructural changes the
following exercise thereby addressing a key gap in knowledge
between the molecular mechanism of exercise and how it man-
ifests into tissue level changes. In mice with cardiac-specific
deletion of CITED4, there were significant differences in the DT-
MRI biomarker of helicity, indicating that CITED4 is required for
cardiac structural changes to take place. In addition, without
active expression of CITED4, exercise resulted in no differences
within these DT-MRI markers, further proving that CITED4
expression is necessary for exercise-induced structural changes.
Meanwhile, we demonstrated that active expression of CITED4
coupled with exercise, results in significant structural remodeling
of the cardiac tissue in both the wild-type mice of cohort 1 and
the fl/flmice of cohort 2. These results highlight the necessary
and required role CITED4 plays in cardiac remodeling following
exercise. Our results indicate that CITED4 also plays an essential
role in the baseline helicity formation as shown by the significant
reduction in helicity in both the C4KO sedentary and exercise
groups compared to the fl/flsedentary. Presumably, helicity
should have been returned to the same level as the fl/flsedentary
but was detrimentally reduced when cardiomyocyte-specific
CITED4 was knocked out.
Finally, we want to emphasize that the novel imaging tech-
nologies used in the study are generalizable and can be used to
spatially link other molecular mechanisms to microstructural
tissue remodeling. The novel imaging technologies could also be
used to investigate other cardiovascular diseases as well as the
impact of targeted molecular therapies such as mRNA. We
believe our study marks a potential new platform to examine how
molecular mechanisms manifest into downstream tertiary tissue
structures, which may play a critical role in how to scale up the
impact of molecular therapeutics.
However, this study has several potential limitations. The small
sample size of the exercise and sedentary mouse groups is a
possible limitation. Despite this limitation, a significant correla-
tion was found between the upregulation CITED4 and changes in
myocardial microstructure. Comparing cohort 1 and 2 had lim-
itations, as in cohort 2 we only had access to mid-ventricular
slices of the cardiac tissue. This prevented us from doing some of
the same analyses we did for the first cohort of animals. Further,
the knockdown of CITED4 in cohort 2 was specific only to the
cardiomyocytes. However, this should not have impacted the
results as CITED4 is expressed at very low levels in fibroblasts
and other noncardiomyocytes7,8. Finally, the co-registration of
CITED4 expression analyzed through RNAscope and helicity
measured with DT-MRI could be a possible limitation. Future
studies should pursue a more advanced method of co-registration
between mRNA expression and DT-MRI markers.
Overall, this study confirmed the hypothesis that exercise leads
to left ventricular microstructural changes in the heart’s helicity
and that CITED4 plays a necessary role in this process. We
demonstrated that the expression of CITED4 following exercise
differs by cardiac regions, which can explain the adaptive patterns
of cardiac remodeling determined by cardiac DT-MRI. Further-
more, we showed deletion of the CITED4 expression in trans-
genic mice leads to complete prohibition of the exercise-induced
remodeling of the heart’s microstructure. These findings serve as
a fundamental basis for understanding exercise-induced hyper-
trophic changes at a molecular and microstructural level and
motivate further evaluation of exercise and cardiovascular health.
Future studies should explore the impacts of exercise on CITED4
using large animal models to gain a better understanding of how
exercise may influence cardiac remodeling in humans.
Methods
Animal studies. All animal procedures were approved by the Institutional Animal
Care and Use Committee (IACUC) of our institute. Mice were maintained in a
specific pathogen-free environment at an in-house animal facility. The mice were
kept under 12-h light/12-h dark cycles at a constant room temperature (22 °C).
Mice had access to a standard diet of food and water ad libitum.
Study design. Two cohorts of mice were used in this study. In the first cohort, A
total of fourteen seven-week-old male wild-type C57BL/6J mice (Jackson
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Laboratory, USA) were housed in the same facility. The mice were housed indi-
vidually in cages with (exercise, n=7) or without (sedentary, n=7) free access to
running wheel (Starr Life Sciences, USA) in the same facility (Fig. 1). The running
activity of these seven mice was tracked using a revolution counter that tracked
running activity data every hour. The mice were weighed again after four and
eight weeks.
In the second cohort, CITED4 knockout (KO) mice were generated by breeding
CITED4 floxed mice (fl/fl) and hemizygous αMHC-Cre mice as described
previously7. Only male CITED4 KO mice and its control littermates (fl/flmice)
were used and randomized into four groups: CITED4 KO sedentary (n=13),
CITED4 KO exercise (n=7), CITED4 fl/flsedentary (n=7), and CITED4 fl/fl
exercise (n=6). The mice from these groups were 8–12 weeks old. All mice were
held in the same room in the animal facility. The running group (n=13) was
housed in cages with stainless steel running wheels (Starr Life Sciences, USA)
(Fig. 1). The remaining mice (n=20) belonged to the sedentary group and were
housed identically, except without access to a running wheel.
After eight weeks of running, the mice were euthanized using isoflurane
anesthesia followed by cardiac puncture and perfused with 4% paraformaldehyde
(PFA, Electron Microscopy Sciences, USA). Hearts were extracted and stored in 4%
PFA. For cohort 1, MRI scans were taken of the whole heart. For cohort 2, a mid-
ventricular slice of 1 mm width was taken for imaging and sectioning.
MRI acquisition. After initial perfusion, the hearts and sections were placed in a 15-
ml Falcontube and submerged in perfluoropolyether (Solvay Specialty Polymers, Italy)
for 1 month +/−3 weeks. Diffusion tensor magnetic resonance imaging (DT-MRI) of
the mouse hearts was performed on a 14T MRI scanner (Bruker Avance III HD, PV
6.0.1) with a 20 mm birdcage coil. Twelve diffusion-weighted (b=500 s/mm2)and
four non-diffusion-weighted (b=0s/mm
2) single spin-echo MRI images were
acquired with the following imaging parameters: repetition time =1500 ms, echo
time =12.37 ms, number of averages=3, acquisition matrix =128 × 128 × 80, spatial
resolution =140 μm×140μm, slice thickness =140 μm, receiver bandwidth =7100
Hz, diffusion duration =7 ms, total scan time 6 h 24 m 0 s. DT-MRI was acquired in a
stack of short-axis reaching from the base to the apex and the 4- and 2-chamber
long axis.
MRI image analysis. The DT-MRI-derived cardiac helicity serves as the readout
parameters of myofiber organization and architecture investigated at the three
transmural zones (epicardium, mid-myocardium and endocardium). Pixel-wise
values of helix angle were calculated using custom software in Matlab (Mathworks,
Natick, MA)36. Tensor reconstruction was calculated using a modified weighted
least squares fit. Mean LV helicity was calculated for each short-axis slice by
automatically segmenting the LV into five transmural concentric rings, following
which the slope was extracted from the linear regression of the mean helix angle for
each ring against the transmural depth from the endocardium to the epicardium.
RNA in situ hybridization. Following DT-MRI, the hearts were cleaned with saline
and dehydrated within 10, 20, and 30% sucrose solutions. The hearts were then
embedded in Optimal Cutting Temperature (OCT) gel (Sakura Finetek INC., USA)
and flash frozen before being stored at −80 °C for 24 h. They have subsequently
sectioned in 10 μm short-axis slices at the midventricular level on a Leica CM3050
S (Leica Biosystems, Germany) research cryostat and placed on Superfrost
microscope slides (Fisherbrand®, (ThermoFisher Scientific, USA). RNAscope®
in situ hybridization (Advanced Cell Diagnostics INC., U.S.) was performed on 1
mid-ventricular slice per heart following the manufacturer’s instructions. In short,
samples were pretreated by heating the slides at 60 oC for 30 min and post fixed in
4% PFA at 4 oC for 15 min. This was followed by dehydration in 50, 70, and 100%
ethyl alcohol (EtOH) for 5 min each and repeated once more with 100% EtOH.
After this sample preparation, they were processed with the RNAscope®multiplex
fluorescent reagent kit V2 (Advanced Cell Diagnostics INC., U.S.) using the Mm-
CITED4 probe (Advanced Cell Diagnostics INC., U.S.). All slides were DAPI
(Advanced Cell Diagnostic INC., U.S.) stained prior to cover slipping. Samples
were imaged on a Zeiss Axio Imager.A2 microscope at 20× (Carl Zeiss AG, Ger-
many). For cohort 1, images were taken in six separate segments following the
American Heart Association (AHA) heart segmentation of a mid-ventricular slice.
These six-segment images were obtained by stitching 20× cross-sectional images
together using Zen 2.3 Pro software (Carl Zeiss AG, Germany). In cohort 2, single
images at 20× were taken from the mid-ventricular slice.
RNA-FISH image analysis. Images of the RNA-FISH assays were processed and
quantified using an in-house thresholding and counting software written in
MATLAB (MATLAB 2019a, MathWorks, USA). In cohort 1, three ROIs were
created within each of the six segments of each heart that represent the epi-, myo-,
and endocardium. For cohort 2, whole images were analyzed. The images were
processed to a threshold that accurately isolated and counted CITED4 and DAPI
signals and manually adjusted to insure the inclusion of all. This resulted in the
number of CITED4 and DAPI signals per AHA segment per epi-, myo- and
endocardial transmural layer. The CITED4 expression was normalized by the
DAPI count to determine the CITED4 expression per cell nuclei.
Statistics and reproducibility. Unpaired t-tests were used for single group
comparisons of normally distributed data. Two-way ANOVA followed by Tukey
post hoc was used for multivariant comparisons of normally distributed data.
Simple linear regressions were used to assess the correlation of DT-MRI-based
microstructural biomarkers with CITED4/DAPI expression ratio. pvalues < 0.05
were considered statistically significant. Statistical analysis was performed using
GraphPad Prism8 software (San Diego, CA).
Reporting summary. Further information on research design is available in the Nature
Research Reporting Summary linked to this article.
Data availability
The source data for the graphs in the main figures are provided in Supplementary Data 1.
The other data that support the findings of this study are available from the
corresponding author, upon reasonable request.
Received: 14 January 2022; Accepted: 23 June 2022;
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Acknowledgements
We would like to thank the MGH Research Institute Division of Clinical Research
Service for providing statistical consultation. We would also like to thank BioHues
Digital (Toronto, Ontario) for creating original illustration of the study methods in Figs.
1 and 3. This work was supported by grants from the National Institutes of Health
(R01HL151704, R01HL159010, R01HL135242 to C.N., and R35HL155318 to A.R.), the
American Heart Association (20CDA35310184 to H.L.), the Sarnoff Cardiovascular
Research Foundation (Fellowship to L.T.), an MGH Corrigan Minehan Spark Award (to
C.N.), and the Hassenfeld Foundation (to C.N.). C.D.W. was supported by the NIH
(NS087096, NS117694) and the Hassenfeld Clinical Scholar Award.
Author contributions
R.E., Y.R., C.N., C.W., and A.R. designed the study; R.E., M.v.d.B., S.Y., A.R., C.W., and
C.N. interpreted the results; C.N., L.C., and Y.C. performed the CMR and analyzed the
data; MI., L.T., and H.L. provided experimental and analytical support; R.E., M.v.d.B.,
S.Y., J.C., and C.N. conducted functional analysis; R.E., S.Y., and Y.R. performed bio-
chemical experiments and analyzed the data; R.E., M.v.d.B., S.Y., and C.N. wrote the
manuscript with input from all authors; all authors have contributed to the editing and
review of the final manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s42003-022-03635-y.
Correspondence and requests for materials should be addressed to Christiane D. Wrann
or Christopher T. Nguyen.
Peer review information Communications Biology thanks Michael Dodd and the other,
anonymous, reviewer(s) for their contribution to the peer review of this work. Primary
Handling Editors: Tami Martino and Christina Karlsson Rosenthal.
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