ArticlePDF Available

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


Abstract and Figures

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.
This content is subject to copyright. Terms and conditions apply.
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 signicant increases in the lateral wall compared with sedentary mice. Concordantly
in this same region, the hearts local microstructural tissue helicity is also selectively
increased in exercised mice. Quantication of CITED4 expression and microstructural tissue
helicity reveals a signicant 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 hearts microstructural helicity revealing how a
key molecular regulator of cardiac remodeling manifests into downstream local tissue-level
changes. 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:;
COMMUNICATIONS BIOLOGY | (2022) 5:656 | | /commsbio 1
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Exercise has expansive benets for the adult heart by pro-
tecting it from cardiovascular disease1. These benecial
effects arise from changes in metabolism, skeletal muscles,
peripheral vessels, and the heart itself2. Specically in the heart,
exercise induces cardiac structural remodeling in the form of
hypertrophy25, which occurs with an increase in the length and
width of cardiomyocytes2,3. Aside from producing benecial
myocardial hypertrophy, some transcription factors and cell
proliferation markers have also been implicated in producing
cardioprotective effects2,4,6. Specically, 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
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 conrmed 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 failure1114.
This indicates that the therapeutic abilities of exercise are not
specic 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 hearts 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 hearts microstructural helicity.
Recent studies have shown that the hearts microstructural
helicity can be non-invasively quantied by diffusion tensor
magnetic resonance imaging (DT-MRI) in mice1618 and
patients1923. 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 brosis21,2629 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-
nicant clinical and scientic 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 dene 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 efciently interrogate the whole
heart while also spatially co-registering quantications of
CITED4 expression with myocardial microstructural tissue heli-
city. Furthermore, we used cardiomyocyte-specic CITED4
knock out mice (C4KO) to demonstrate the crucial role CITED4
plays in necessitating these microstructural changes following
Exercise from wheel running. Free wheel-running was used to
exercise wild-type mice (n=7), C4KO mice (n=7) and /mice
(n=6) to induce cardiac remodeling while sedentary groups of
wild-type mice (n=7), C4KO mice (n=13) and /mice
(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 [277.0450.8] km. The average distance run
per day by the end of the 8 weeks of the exercise was 6.028
[4.3977.336] km/day (Fig. 2a, b). Over the eight-week period, the
sedentary group had a signicantly 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 signicant
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 hearts 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 signicantly
(p=0.0024) increased (19.9%) in the exercise cohort compared
with the sedentary cohort (Fig. 4d). Regional cardiac tissue heli-
city analyses revealed signicant (p=0.048) increases (17.1%)
within the septal region (AHA segment 2 and 3) and even greater
signicant (p=0.0007) increases (21.5%) in the lateral wall (AHA
segments 5 and 6) when comparing exercise with sedentary
cohorts (Fig. 4ad). 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 uorescent light
microscopy and the tissue level CITED4 expression normalized to
DAPI nuclei counts was quantied (Fig. 3bf). 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
2COMMUNICATIONS BIOLOGY | (2022) 5:656 | | /commsbio
Content courtesy of Springer Nature, terms of use apply. Rights reserved
(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 insignicant (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. 1ac, 2).
Signicant increases in the CITED4/DAPI ratio were present
within varying AHA sections within the three transmural layers
(Supplemental Figs. 1ad, 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-specic dele-
tion of CITED4 (C4KO). Cardiac tissue helicity was signicantly
reduced in both C4KO sedentary and exercise groups compared
to the /sedentary group (31.7%, p=0.0024) and /exercise
group (40.3%, p< 0.0001, Fig. 6a, b). In the /group, exercise
produced a trending increase in cardiac tissue helicity similar to
Fig. 2 Wheel running promotes signicant 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 groups 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-specic CITED4 knock out (C4KO) exercise (n=7), C4KO sedentary (n=13), /exercise (n=6) and /sedentary (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 uorescent light microscope.
COMMUNICATIONS BIOLOGY | (2022) 5:656 | | /commsbio 3
Content courtesy of Springer Nature, terms of use apply. Rights reserved
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 /groups were expressing CITED4 as
expected. The cardiomyocyte-specic knock out of CITED4
produced a negligible expression of CITED4 (Fig. 6c, d). Mean-
while, the expression of CITED4 in the /was comparable to
cohort 1 (Fig. 6c, d).
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
conrmed 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 quantication 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 magnied 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. bdHelicity differences within
the septum, lateral wall and averaged across all AHA sections. eMagnied representative images of DAPI and CITED4 within the septal and lateral wall
regions within exercised and sedentary mice. fhCITED4 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 groups distribution of values.
4COMMUNICATIONS BIOLOGY | (2022) 5:656 | | /commsbio
Content courtesy of Springer Nature, terms of use apply. Rights reserved
provide further insight into how molecular expression can
manifest into gross anatomical and physiological remodeling. To
the best our knowledge, this is the rst 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 ber
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 signicant changes to the helicity of the myo-
cardial bers. 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 signicantly correlated with CITED4/DAPI expression. acSimple 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%
condence interval.
Fig. 6 Cardiomyocyte-specic knock out of CITED4 impacts regional tissue helicity changes. a Helix angle changes differ signicantly between groups.
bAn analysis of helicity between all groups of cohort 2. cImages of DAPI and CITED4 staining of representative mice from cohort 2. dQuantication 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 groups
distribution of values.
COMMUNICATIONS BIOLOGY | (2022) 5:656 | | /commsbio 5
Content courtesy of Springer Nature, terms of use apply. Rights reserved
changes to helicity shown in this study further demonstrate the
structural cardiac remodeling benets 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-invasively1923,
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. Specically, increases in
CITED4 within the lateral wall correlate with cardiac remodeling
revealed by DT-MRI-based microstructural changes. Although,
qPCR already showed signicant 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 modications with the local transcriptional
changes. The implications of this CITED4 local expression may
serve to guide more targeted delivery of CITED47or other
modiers to cardiac tissue, such as Irisin33,34.
While CITED4 is known to play a role in tissue proliferation
and hypertrophy2,79, 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/EBPβs 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 conrm this hypothesis2,7.
Furthermore, this CITED4 pathway provides a potential link to
the microstructural changes revealed through DT-MRI. The
specic changes in helicity present between the exercise and
sedentary groups might be explained, in part, by these exercised-
induced molecular changes.
The specic 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 specically upregulates CITED4, while
downregulating C/EBPβto produce benecial cardiomyocyte
growth and tissue remodeling from micro to macro and from
structural to functional2,8,35. In this study, DT-MRI analyses
conrm that the trends in increased CITED4 expression occur in
conjunction with the widespread structural changes marked by
increased helicity. Our results specically 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 specic lateral wall increases in tissue volume found with DT-
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-specic
deletion of CITED4, there were signicant 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 signicant structural remodeling
of the cardiac tissue in both the wild-type mice of cohort 1 and
the /mice 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 signicant
reduction in helicity in both the C4KO sedentary and exercise
groups compared to the /sedentary. Presumably, helicity
should have been returned to the same level as the /sedentary
but was detrimentally reduced when cardiomyocyte-specic
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 signicant 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 rst cohort of animals. Further,
the knockdown of CITED4 in cohort 2 was specic only to the
cardiomyocytes. However, this should not have impacted the
results as CITED4 is expressed at very low levels in broblasts
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 conrmed the hypothesis that exercise leads
to left ventricular microstructural changes in the hearts 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 hearts microstructure. These ndings 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 inuence cardiac remodeling in humans.
Animal studies. All animal procedures were approved by the Institutional Animal
Care and Use Committee (IACUC) of our institute. Mice were maintained in a
specic 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 rst cohort, A
total of fourteen seven-week-old male wild-type C57BL/6J mice (Jackson
6COMMUNICATIONS BIOLOGY | (2022) 5:656 | | /commsbio
Content courtesy of Springer Nature, terms of use apply. Rights reserved
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 oxed mice (/) and hemizygous αMHC-Cre mice as described
previously7. Only male CITED4 KO mice and its control littermates (/mice)
were used and randomized into four groups: CITED4 KO sedentary (n=13),
CITED4 KO exercise (n=7), CITED4 /sedentary (n=7), and CITED4 /
exercise (n=6). The mice from these groups were 812 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 isourane
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 peruoropolyether (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 myober 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 modied weighted
least squares t. Mean LV helicity was calculated for each short-axis slice by
automatically segmenting the LV into ve 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 ash 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 Scientic, USA). RNAscope®
in situ hybridization (Advanced Cell Diagnostics INC., U.S.) was performed on 1
mid-ventricular slice per heart following the manufacturers instructions. In short,
samples were pretreated by heating the slides at 60 oC for 30 min and post xed 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
uorescent 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
quantied 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 signicant. 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 gures are provided in Supplementary Data 1.
The other data that support the ndings of this study are available from the
corresponding author, upon reasonable request.
Received: 14 January 2022; Accepted: 23 June 2022;
1. Vujic, A. et al. Exercise induces new cardiomyocyte generation in the adult
mammalian heart. Nat. Commun. 9,19 (2018).
2. Bezzerides, V. J. et al. CITED4 induces physiologic hypertrophy and promotes
functional recovery after ischemic injury. JCI Insight 1,114 (2016).
3. Bernardo, B. C., Weeks, K. L., Pretorius, L. & McMullen, J. R. Molecular
distinction between physiological and pathological cardiac hypertrophy:
Experimental ndings and therapeutic strategies. Pharmacol. Ther. 128,
191227 (2010).
4. Liu, X. et al. MiR-222 is necessary for exercise-induced cardiac growth and
protects against pathological cardiac remodeling. Cell Metab. 21, 584595
5. DeMaria, A. N., Neumann, A., Lee, G., Fowler, W. & Mason, D. T. Alterations
in ventricular mass and performance induced by exercise training in man
evaluated by echocardiography. Circulation 57, 237244 (1978).
6. Akazawa, H. & Komuro, I. Roles of cardiac transcription factors in cardiac
hypertrophy. Circ. Res. 92, 10791088 (2003).
7. Lerchenmüller, C. et al. CITED4 protects against adverse remodeling in
response to physiological and pathological stress. Circ. Res. 631646. https:// (2020).
8. Boström, P. et al. C/EBPβcontrols exercise-induced cardiac growth and
protects against pathological cardiac remodeling. Cell 143, 10721083
9. Bragança, J. et al. Human CREB-binding protein/p300-interacting
transactivator with ED-rich tail (CITED) 4, a new member of the CITED
family, functions as a co-activator for transcription factor AP-2. J. Biol. Chem.
277, 85598565 (2002).
10. Ryall, K. A., Bezzerides, V. J., Rosenzweig, A. & Saucerman, J. J. Phenotypic
screen quantifying differential regulation of cardiac myocyte hypertrophy
identies CITED4 regulation of myocyte elongation. J. Mol. Cell. Cardiol. 72,
7484 (2014).
11. HART, G. Cardiac hypertrophy and failure. Cardiovasc. Res. 25, 176176
12. Frey, N. & Olson, E. N. Cardiac hypertrophy: the good, the bad, and the ugly.
Annu. Rev. Physiol. 65,4579 (2003).
13. Tham, Y. K., Bernardo, B. C., Ooi, J. Y. Y., Weeks, K. L. & McMullen, J. R.
Pathophysiology of cardiac hypertrophy and heart failure: signaling pathways
and novel therapeutic targets. Arch. Toxicol. 89, 14011438 (2015).
14. Berenji, K., Drazner, M. H., Rothermel, B. A. & Hill, J. A. Does load-induced
ventricular hypertrophy progress to systolic heart failure? Am. J. Physiol. -
Hear. Circ. Physiol. 289,816 (2005).
15. Streeter, D. D., Spotnitz, H. M., Patel, D. P., Ross, J. & Sonnenblick, E. H. Fiber
orientation in the canine left ventricle during diastole and systole. Circ. Res.
24, 339347 (1969).
16. Lee, S. E. et al. Three-dimensional cardiomyocytes structure revealed by
diffusion tensor imaging and its validation using a tissue-clearing technique.
Sci. Rep. 8,111 (2018).
17. Angeli, S., Befera, N., Peyrat, J., Calabrese, E. & Johnson, G. A. A high-resolution
cardiovascular magnetic resonance diffusion tensor map from ex-vivo C57BL / 6
murine hearts. 114. (2014)
18. Healy, L. J., Jiang, Y. & Hsu, E. W. Quantitative comparison of myocardial
ber structure between mice, rabbit, and sheep using diffusion tensor
cardiovascular magnetic resonance. J. Cardiovasc. Magn. Reson.13,74
COMMUNICATIONS BIOLOGY | (2022) 5:656 | | /commsbio 7
Content courtesy of Springer Nature, terms of use apply. Rights reserved
19. Nielles-Vallespin, S. et al. Assessment of myocardial microstructural dynamics
by in vivo diffusion tensor cardiac magnetic resonance. J. Am. Coll. Cardiol. (2017).
20. Das, A. et al. Insight INto Myocardial Microstructure of Athletes and
Hypertrophic Cardiomyopathy Patients Using Diffusion Tensor Imaging. J.
Magn. Reson. Imaging 110. (2020).
21. Von Deuster, C. et al. Studying dynamic myober aggregate reorientation in
dilated cardiomyopathy using in vivo magnetic resonance diffusion tensor
imaging. Circ. Cardiovasc. Imaging
116.005018 (2016).
22. Ariga, R. et al. Identication of myocardial disarray in patients with
hypertrophic cardiomyopathy and ventricular arrhythmias. J. Am. Coll.
Cardiol. 73, 24932502 (2019).
23. Mekkaoui, C. et al. Diffusion tractography of the entire left ventricle by using
free-breathing accelerated simultaneous multisection imaging. Radiology 282,
850856 (2017).
24. Pierpaoli, C. & Basser, P. J. Toward a quantitative assessment of diffusion
anisotropy. Magn. Reson. Med. 36, 893906 (1996).
25. Mekkaoui, C., Reese, T. G., Jackowski, M. P., Bhat, H. & Sosnovik, D. E.
Diffusion MRI in the heart. NMR Biomed.30, e3426 (2017).
26. Nguyen, C. et al. In vivo contrast free chronic myocardial infarction
characterization using diffusion-weighted cardiovascular magnetic resonance.
J. Cardiovasc. Magn. Reson. 16,110 (2014).
27. Nguyen, C. et al. Contrast-free detection of myocardial brosis in
hypertrophic cardiomyopathy patients with diffusion-weighted cardiovascular
magnetic resonance. J. Cardiovasc. Magn. Reson.17, 107 (2015).
28. Pop, M. et al. Quantication of brosis in infarcted swine hearts by ex vivo
late gadolinium-enhancement and diffusion-weighted MRI methods. Phys.
Med. Biol. 58, 50095028 (2013).
29. Ferreira, P. F. et al. In vivo cardiovascular magnetic resonance diffusion tensor
imaging shows evidence of abnormal myocardial laminar orientations and
mobility in hypertrophic cardiomyopathy. J. Cardiovasc. Magn. Reson. 16,87
30. Nguyen, C. T., Dawkins, J., Bi, X., Marbán, E. & Li, D. Diffusion tensor cardiac
magnetic resonance reveals exosomes from cardiosphere-derived cells
preserve myocardial ber architecture after myocardial infarction. JACC Basic
Transl. Sci. 3,97109 (2018).
31. Sosnovik, D. E. et al. Microstructural impact of ischemia and bone marrow-
derived cell therapy revealed with diffusion tensor magnetic resonance
imaging tractography of the heart in vivo. Circulation 129, 17311741 (2014).
32. Wang, F. et al. RNAscope: a novel in situ RNA analysis platform for formalin-
xed, parafn-embedded tissues. J. Mol. Diagnostics 14,2229 (2012).
33. Wang, Z. et al. Irisin protects heart against ischemia-reperfusion injury
through a SOD2-dependent mitochondria mechanism HHS public access. J.
Cardiovasc. Pharm. 72, 259269 (2018).
34. Liao, Q. et al. Irisin exerts a therapeutic effect against myocardial infarction via
promoting angiogenesis. Acta Pharmacol. Sin. 40, 13141321 (2019).
35. Arany, Z. et al. Transverse aortic constriction leads to accelerated heart failure
in mice lacking PPAR-γcoactivator 1α.Proc. Natl Acad. Sci. USA 103,
1008610091 (2006).
36. Nguyen, C. T. et al. Freebreathing diffusion tensor MRI of the whole left
ventricle using secondorder motion compensation and multitasking
respiratory motion correction. Magn. Reson. Med.
mrm.28611 (2020).
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 nal manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material
available at
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.
Reprints and permission information is available at
Publishers note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional afliations.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The images or other third party
material in this article are included in the articles Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not included in the
articles Creative Commons license and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license, visit
© The Author(s) 2022, corrected publication 2022
8COMMUNICATIONS BIOLOGY | (2022) 5:656 | | /commsbio
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
Description of the cardiac myofiber architecture in pathological or even physiological conditions is essential for image-based modeling in electrophysiology or mechanical studies. While diffusion tensor imaging (DTI) is one of the best modalities to capture myofiber orientation of large mammalian hearts, validations of putative myofiber’s main orientation from DTI in whole hearts of large mammals is limited. First we design an experimental protocol for sheep (N = 1) and human (N = 1) whole hearts that combine a standardized sample preparation with high-resolution diffusion MRI at 600 µm3 using low angular resolution (6 directions) followed by a tissue air-drying approach coupled with X-ray imaging at 42 µm3. Secondly, we propose a standardized post-processing pipeline for symmetric multimodal mapping allowing the comparison of myofiber orientation computed from DTI and structure tensor imaging (STI), respectively. We then identified region-of-interest (ROI) exhibiting small or sharp spatial variations in myofiber orientation and compared the putative myofiber orientation for both methods. In conclusion, we show a good correspondence of structural features between the two imaging modalities and identify new unexpected and complex cardiomyocytes organization such as oscillating patterns or clear separation of opposing fiber-bundles.KeywordsCardiac myofiber architectureDiffusion MRIMicroCTLarge mammalian whole heartsRegistration
Full-text available
Background: Hypertrophic cardiomyopathy (HCM) remains the commonest cause of sudden cardiac death among young athletes. Differentiating between physiologically adaptive left ventricular (LV) hypertrophy observed in athletes' hearts and pathological HCM remains challenging. By quantifying the diffusion of water molecules, diffusion tensor imaging (DTI) MRI allows voxelwise characterization of myocardial microstructure. Purpose: To explore microstructural differences between healthy volunteers, athletes, and HCM patients using DTI. Study type: Prospective cohort. Population: Twenty healthy volunteers, 20 athletes, and 20 HCM patients. Field strength/sequence: 3T/DTI spin echo. Assessment: In-house MatLab software was used to derive mean diffusivity (MD) and fractional anisotropy (FA) as markers of amplitude and anisotropy of the diffusion of water molecules, and secondary eigenvector angles (E2A)-reflecting the orientations of laminar sheetlets. Statistical tests: Independent samples t-tests were used to detect statistical significance between any two cohorts. Analysis of variance was utilized for detecting the statistical difference between the three cohorts. Statistical tests were two-tailed. A result was considered statistically significant at P ≤ 0.05. Results: DTI markers were significantly different between HCM, athletes, and volunteers. HCM patients had significantly higher global MD and E2A, and significantly lower FA than athletes and volunteers. (MDHCM = 1.52 ± 0.06 × 10-3 mm2 /s, MDAthletes = 1.49 ± 0.03 × 10-3 mm2 /s, MDvolunteers = 1.47 ± 0.02 × 10-3 mm2 /s, P < 0.05; E2AHCM = 58.8 ± 4°, E2Aathletes = 47 ± 5°, E2Avolunteers = 38.5 ± 7°, P < 0.05; FAHCM = 0.30 ± 0.02, FAAthletes = 0.35 ± 0.02, FAvolunteers = 0.36 ± 0.03, P < 0.05). HCM patients had significantly higher E2A in their thickest segments compared to the remote (E2Athickest = 66.8 ± 7, E2Aremote = 51.2 ± 9, P < 0.05). Data conclusion: DTI depicts an increase in amplitude and isotropy of diffusion in the myocardium of HCM compared to athletes and volunteers as reflected by increased MD and decreased FA values. While significantly higher E2A values in HCM and athletes reflect steeper configurations of the myocardial sheetlets than in volunteers, HCM patients demonstrated an eccentric rise in E2A in their thickest segments, while athletes demonstrated a concentric rise. Further studies are required to determine the diagnostic capabilities of DTI. Evidence level: 1 TECHNICAL EFFICACY STAGE: 2.
Full-text available
Background: Myocardial disarray is a likely focus for fatal arrhythmia in hypertrophic cardiomyopathy (HCM). This microstructural abnormality can be inferred by mapping the preferential diffusion of water along cardiac muscle fibres using diffusion tensor cardiac magnetic resonance (DT-CMR) imaging. Fractional anisotropy (FA) quantifies directionality of diffusion in three dimensions. We hypothesised that FA would be reduced in HCM due to disarray and fibrosis which may represent the anatomical substrate for ventricular arrhythmia. Objectives: This study sought to assess FA as a non-invasive in vivo biomarker of HCM myoarchitecture and its association with ventricular arrhythmia. Methods: 50 HCM patients (47 ± 15y, 77% male) and 30 healthy controls (46 ± 16y, 70% male) underwent DT-CMR in diastole, cine, late gadolinium enhancement (LGE) and extracellular volume (ECV) imaging at 3T. Results: Diastolic FA was reduced in HCM compared to controls (0.49 ± 0.05 v 0.52 ± 0.03, p=0.0005). Controls had a mid-wall ring of high FA. In HCM, this ring was disrupted by reduced FA, consistent with published histology demonstrating that disarray and fibrosis invade circumferentially-aligned mid-wall myocytes. LGE and ECV were significant predictors of FA, in line with fibrosis contributing to low FA. Yet FA adjusted for LGE and ECV remained reduced in HCM (p=0.028). FA in the hypertrophied segment was reduced in HCM patients with ventricular arrhythmia compared to patients without (n=15; 0.41 ± 0.03 v 0.46 ± 0.06, p=0.007). A decrease in FA of 0.05 increased odds of ventricular arrhythmia by 2.5 (95% CI 1.2 to 5.3; p=0.015) in HCM and remained significant even after correcting for LGE, ECV and wall thickness (p=0.036). Conclusion: DT-CMR assessment of left ventricular myoarchitecture matched patterns reported previously on histology. Low diastolic FA in HCM was associated with ventricular arrhythmia and is likely to represent disarray after accounting for fibrosis. We propose that diastolic FA could be the first in vivo marker of disarray in HCM and a potential independent risk factor.
Full-text available
We characterized the microstructural response of the myocardium to cardiovascular disease using diffusion tensor imaging (DTI) and performed histological validation by intact, un-sectioned, three-dimensional (3D) histology using a tissue-clearing technique. The approach was validated in normal (n = 7) and ischemic (n = 8) heart failure model mice. Whole heart fiber tracking using DTI in fixed ex-vivo mouse hearts was performed, and the hearts were processed with the tissue-clearing technique. Cardiomyocytes orientation was quantified on both DTI and 3D histology. Helix angle (HA) and global HA transmurality (HAT) were calculated, and the DTI findings were confirmed with 3D histology. Global HAT was significantly reduced in the ischemic group (DTI: 0.79 ± 0.13°/% transmural depth [TD] and 3D histology: 0.84 ± 0.26°/%TD) compared with controls (DTI: 1.31 ± 0.20°/%TD and 3D histology: 1.36 ± 0.27°/%TD, all p < 0.001). On direct comparison of DTI with 3D histology for the quantitative assessment of cardiomyocytes orientation, significant correlations were observed in both per-sample (R2 = 0.803) and per-segment analyses (R2 = 0.872). We demonstrated the capability and accuracy of DTI for mapping cardiomyocytes orientation by comparison with the intact 3D histology acquired by tissue-clearing technique. DTI is a promising tool for the noninvasive characterization of cardiomyocytes architecture.
Full-text available
Loss of cardiomyocytes is a major cause of heart failure, and while the adult heart has a limited capacity for cardiomyogenesis, little is known about what regulates this ability or whether it can be effectively harnessed. Here we show that 8 weeks of running exercise increase birth of new cardiomyocytes in adult mice (~4.6-fold). New cardiomyocytes are identified based on incorporation of 15N-thymidine by multi-isotope imaging mass spectrometry (MIMS) and on being mononucleate/diploid. Furthermore, we demonstrate that exercise after myocardial infarction induces a robust cardiomyogenic response in an extended border zone of the infarcted area. Inhibition of miR-222, a microRNA increased by exercise in both animal models and humans, completely blocks the cardiomyogenic exercise response. These findings demonstrate that cardiomyogenesis can be activated by exercise in the normal and injured adult mouse heart and suggest that stimulation of endogenous cardiomyocyte generation could contribute to the benefits of exercise.
Full-text available
Highlights • CDCEXO are RNA-laden nanoparticles that reduce scarring, halt adverse remodeling, and preserve cardiac function in rodents and pigs after MI. • The therapeutic effects of CDCEXO on myocardial fiber architecture and how it relates to preserved cardiac function and reduced scarring remain unclear. • After intramyocardial CDCEXO treatment in MI pigs, DT-CMR elucidated myocardial fiber architecture was preserved indicated by the unchanged helix angle transmurality. • Scar size measured by conventional CMR combined with helix angle transmurality measured by DT-CMR demonstrated significant improvement in the prediction of cardiac function. • DT-CMR is a powerful technology for myocardial regenerative therapy evaluation revealing unique insight into the myocardium’s microstructure.
Full-text available
Background: Cardiomyocytes are organized in microstructures termed sheetlets that reorientate during left ventricular thickening. Diffusion tensor cardiac magnetic resonance (DT-CMR) may enable noninvasive interrogation of in vivo cardiac microstructural dynamics. Dilated cardiomyopathy (DCM) is a condition of abnormal myocardium with unknown sheetlet function. Objectives: This study sought to validate in vivo DT-CMR measures of cardiac microstructure against histology, characterize microstructural dynamics during left ventricular wall thickening, and apply the technique in hypertrophic cardiomyopathy (HCM) and DCM. Methods: In vivo DT-CMR was acquired throughout the cardiac cycle in healthy swine, followed by in situ and ex vivo DT-CMR, then validated against histology. In vivo DT-CMR was performed in 19 control subjects, 19 DCM, and 13 HCM patients. Results: In swine, a DT-CMR index of sheetlet reorientation (E2A) changed substantially (E2A mobility ∼46°). E2A changes correlated with wall thickness changes (in vivo r(2) = 0.75; in situ r(2) = 0.89), were consistently observed under all experimental conditions, and accorded closely with histological analyses in both relaxed and contracted states. The potential contribution of cyclical strain effects to in vivo E2A was ∼17%. In healthy human control subjects, E2A increased from diastole (18°) to systole (65°; p < 0.001; E2A mobility = 45°). HCM patients showed significantly greater E2A in diastole than control subjects did (48°; p < 0.001) with impaired E2A mobility (23°; p < 0.001). In DCM, E2A was similar to control subjects in diastole, but systolic values were markedly lower (40°; p < 0.001) with impaired E2A mobility (20°; p < 0.001). Conclusions: Myocardial microstructure dynamics can be characterized by in vivo DT-CMR. Sheetlet function was abnormal in DCM with altered systolic conformation and reduced mobility, contrasting with HCM, which showed reduced mobility with altered diastolic conformation. These novel insights significantly improve understanding of contractile dysfunction at a level of noninvasive interrogation not previously available in humans.
Purpose We aimed to develop a novel free‐breathing cardiac diffusion tensor MRI (DT‐MRI) approach, M2‐MT‐MOCO, capable of whole left ventricular coverage that leverages second‐order motion compensation (M2) diffusion encoding and multitasking (MT) framework to efficiently correct for respiratory motion (MOCO). Methods Imaging was performed in 16 healthy volunteers and 3 heart failure patients with symptomatic dyspnea. The healthy volunteers were scanned to compare the accuracy of interleaved multislice coverage of the entire left ventricle with a single‐slice acquisition and the accuracy of the free‐breathing conventional MOCO and MT‐MOCO approaches with reference breath‐hold DT‐MRI. Mean diffusivity (MD), fractional anisotropy (FA), helix angle transmurality (HAT), and intrascan repeatability were quantified and compared. Results In all subjects, free‐breathing M2‐MT‐MOCO DT‐MRI yielded DWI of the entire left ventricle without bulk motion‐induced signal loss. No significant differences were seen in the global values of MD, FA, and HAT in the multislice and single‐slice acquisitions. Furthermore, global quantification of MD, FA, and HAT were also not significantly different between the MT‐MOCO and breath‐hold, whereas conventional MOCO yielded significant differences in MD, FA, and HAT with MT‐MOCO and FA with breath‐hold. In heart failure patients, M2‐MT‐MOCO DT‐MRI was feasible yielding higher MD, lower FA, and lower HAT compared with healthy volunteers. Substantial agreement was found between repeated scans across all subjects for MT‐MOCO. Conclusion M2‐MT‐MOCO enables free‐breathing DT‐MRI of the entire left ventricle in 10 min, while preserving quantification of myocardial microstructure compared to breath‐held and single‐slice acquisitions and is feasible in heart failure patients.
Rationale: Cardiac CITED4 is induced by exercise and is sufficient to cause physiological hypertrophy and mitigate adverse ventricular remodeling after ischemic injury. However, the role of endogenous CITED4 in response to physiological or pathological stress is unknown. Objective: To investigate the role of CITED4 in murine models of exercise and pressure overload. Methods and Results: We generated cardiomyocyte-specific CITED4 knockout mice (C4KO) and subjected them to an intensive swim exercise protocol as well as transverse aortic constriction (TAC). Echocardiography, western blotting, qPCR, immunohistochemistry, immunofluorescence, and transcriptional profiling for mRNA and miRNA expression were performed. Cellular crosstalk was investigated in vitro. CITED4 deletion in cardiomyocytes did not affect baseline cardiac size or function in young adult mice. C4KO mice developed modest cardiac dysfunction and dilation in response to exercise. After TAC, C4KOs developed severe heart failure with left ventricular dilation, impaired cardiomyocyte growth accompanied by reduced mammalian target of rapamycin (mTOR) activity and maladaptive cardiac remodeling with increased apoptosis, autophagy, and impaired mitochondrial signaling. Interstitial fibrosis was markedly increased in C4KO hearts after TAC. RNAseq revealed induction of a pro-fibrotic miRNA network. miR30d was decreased in C4KO hearts after TAC and mediated crosstalk between cardiomyocytes and fibroblasts to modulate fibrosis. miR30d inhibition was sufficient to increase cardiac dysfunction and fibrosis after TAC. Conclusions: CITED4 protects against pathological cardiac remodeling by regulating mTOR activity and a network of miRNAs mediating cardiomyocyte to fibroblast crosstalk. Our findings highlight the importance of CITED4 in response to both physiological and pathological stimuli.
Irisin, a myokine, is cleaved from the extracellular portion of fibronectin domain-containing 5 protein in skeletal muscle and myocardium and secreted into circulation as a hormone during exercise. Irisin has been found to exert protective effects against lung and heart injuries. However, whether irisin influences myocardial infarction (MI) remains unclear. In this study we investigated the therapeutic effects of irisin in an acute MI model and its underlying mechanisms. Adult C57BL/6 mice were subjected to ligation of the left anterior descending coronary artery and treated with irisin for 2 weeks after MI. Cardiac function was assessed using echocardiography. We found that irisin administration significantly alleviated MI-induced cardiac dysfunction and ventricular dilation at 4 weeks post-MI. Irisin significantly reduced infarct size and fibrosis in post-MI hearts. Irisin administration significantly increased angiogenesis in the infarct border zone and decreased cardiomyocyte apoptosis, but did not influence cardiomyocyte proliferation. In human umbilical vein endothelial cells (HUVEC), irisin significantly increased the phosphorylation of ERK, and promoted the migration of HUVEC detected in wound-healing and transwell chamber migration assay. The effects of irisin were blocked by the ERK inhibitor U0126. In conclusion, irisin improves cardiac function and reduces infarct size in post-MI mouse heart. The therapeutic effect is associated with its pro-angiogenic function through activating ERK signaling pathway.
Irisin, a muscle-origin protein derived from the extracellular domain of the fibronectin domain-containing 5 protein (FNDC5), has been shown to modulate mitochondria welfare through paracrine action. Here we test the hypothesis that irisin contributes to cardioprotection after myocardial infarction by preserving mitochondrial function in cardiomyocytes. Animal model studies show that intravenous administration of exogenous irisin produces dose-dependent protection against ischemia/reperfusion (I/R)-induced injury to the heart as reflected by the improvement of left-ventricular ejection fraction and the reduction in serum level of cTnI (n=15, P<0.05). I/R-induced apoptosis of cardiomyocytes is reduced after irisin treatment. The irisin-mediated protection has, at least in part, an effect on mitochondrial function because administration of irisin increases irisin staining in the mitochondria of the infarct area. Irisin also reduces I/R-induced oxidative stress as determined by mitochondrial membrane potential evaluation and superoxide FLASH event recording (n=4, P<0.05). The interaction between irisin and SOD2 plays a key role in the protective process because irisin treatment increases SOD activity (n=10, P<0.05) and restores the mitochondria-localization of SOD2 in cardiomyocytes (n=5, P<0.05). These results demonstrate that irisin plays a protective role against I/R injury to the heart. Targeting the action of irisin in mitochondria presents a novel therapeutic intervention for myocardial infarction.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.