Restoration of Cardiomyogenesis in Aged Mouse Hearts by Voluntary Exercise

Abstract

Background:
The human heart has limited capacity to generate new cardiomyocytes and this capacity declines with age. Because loss of cardiomyocytes may contribute to heart failure, it is crucial to explore stimuli of endogenous cardiac regeneration to favorably shift the balance between loss of cardiomyocytes and the birth of new cardiomyocytes in the aged heart. We have previously shown that cardiomyogenesis can be activated by exercise in the young adult mouse heart. Whether exercise also induces cardiomyogenesis in aged hearts, however, is still unknown. Here, we aim to investigate the effect of exercise on the generation of new cardiomyocytes in the aged heart.

Methods:
Aged (20-month-old) mice were subjected to an 8-week voluntary running protocol, and age-matched sedentary animals served as controls. Cardiomyogenesis in aged hearts was assessed on the basis of 15N-thymidine incorporation and multi-isotope imaging mass spectrometry. We analyzed 1793 cardiomyocytes from 5 aged sedentary mice and compared these with 2002 cardiomyocytes from 5 aged exercised mice, followed by advanced histology and imaging to account for ploidy and nucleation status of the cell. RNA sequencing and subsequent bioinformatic analyses were performed to investigate transcriptional changes induced by exercise specifically in aged hearts in comparison with young hearts.

Results:
Cardiomyogenesis was observed at a significantly higher frequency in exercised compared with sedentary aged hearts on the basis of the detection of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes. No mononucleated/diploid 15N-thymidine-labeled cardiomyocyte was detected in sedentary aged mice. The annual rate of mononucleated/diploid 15N-thymidine-labeled cardiomyocytes in aged exercised mice was 2.3% per year. This compares with our previously reported annual rate of 7.5% in young exercised mice and 1.63% in young sedentary mice. Transcriptional profiling of young and aged exercised murine hearts and their sedentary controls revealed that exercise induces pathways related to circadian rhythm, irrespective of age. One known oscillating transcript, however, that was exclusively upregulated in aged exercised hearts, was isoform 1.4 of regulator of calcineurin, whose regulation and functional role were explored further.

Conclusions:
Our data demonstrate that voluntary running in part restores cardiomyogenesis in aged mice and suggest that pathways associated with circadian rhythm may play a role in physiologically stimulated cardiomyogenesis.

Full text

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Circulation
Circulation. 2022;146:00–00. DOI: 10.1161/CIRCULATIONAHA.121.057276 August 2, 2022
Circulation is available at www.ahajournals.org/journal/circ
Correspondence to: Anthony Rosenzweig, MD, Massachusetts General Hospital, Cardiology Division, 55 Fruit Street, Boston, MA 02114, Email arosenzweig@partners.
org or Richard T. Lee, MD, Harvard University, Sherman Fairchild Building, Rm 159, 7 Divinity Avenue, Cambridge, MA 02138. Email richard_lee@harvard.edu.
*C. Lerchenmüller and A. Vujic contributed equally.
†A. Rosenzweig and R.T. Lee supervised equally.
Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/CIRCULATIONAHA.121.057276
For Sources of Funding and Disclosures, see page xxx.
© 2022 American Heart Association, Inc.
ORIGINAL RESEARCH ARTICLE
Restoration of Cardiomyogenesis in Aged Mouse
Hearts by Voluntary Exercise
Carolin Lerchenmüller , MD*; Ana Vujic, PhD*; Sonja Mittag , MD; Annie Wang, BS; Charles P. Rabolli , BS;
Chiara Heß ; Fynn Betge ; Ashraf Y. Rangrez, PhD; Malay Chaklader, PhD; Christelle Guillermier, PhD;
Frank Gyngard, PhD; Jason D. Roh , MD; Haobo Li , PhD; Matthew L. Steinhauser , MD; Norbert Frey, MD;
Beverly Rothermel , PhD; Christoph Dieterich, PhD; Anthony Rosenzweig , MD†; Richard T. Lee , MD†
BACKGROUND: The human heart has limited capacity to generate new cardiomyocytes and this capacity declines with age.
Because loss of cardiomyocytes may contribute to heart failure, it is crucial to explore stimuli of endogenous cardiac
regeneration to favorably shift the balance between loss of cardiomyocytes and the birth of new cardiomyocytes in the
aged heart. We have previously shown that cardiomyogenesis can be activated by exercise in the young adult mouse heart.
Whether exercise also induces cardiomyogenesis in aged hearts, however, is still unknown. Here, we aim to investigate the
effect of exercise on the generation of new cardiomyocytes in the aged heart.
METHODS: Aged (20-month-old) mice were subjected to an 8-week voluntary running protocol, and age-matched sedentary
animals served as controls. Cardiomyogenesis in aged hearts was assessed on the basis of 15N-thymidine incorporation and
multi-isotope imaging mass spectrometry. We analyzed 1793 cardiomyocytes from 5 aged sedentary mice and compared
these with 2002 cardiomyocytes from 5 aged exercised mice, followed by advanced histology and imaging to account
for ploidy and nucleation status of the cell. RNA sequencing and subsequent bioinformatic analyses were performed to
investigate transcriptional changes induced by exercise specifically in aged hearts in comparison with young hearts.
RESULTS: Cardiomyogenesis was observed at a significantly higher frequency in exercised compared with sedentary aged
hearts on the basis of the detection of mononucleated/diploid 15N-thymidine–labeled cardiomyocytes. No mononucleated/
diploid 15N-thymidine–labeled cardiomyocyte was detected in sedentary aged mice. The annual rate of mononucleated/
diploid 15N-thymidine–labeled cardiomyocytes in aged exercised mice was 2.3% per year. This compares with our previously
reported annual rate of 7.5% in young exercised mice and 1.63% in young sedentary mice. Transcriptional profiling of young
and aged exercised murine hearts and their sedentary controls revealed that exercise induces pathways related to circadian
rhythm, irrespective of age. One known oscillating transcript, however, that was exclusively upregulated in aged exercised
hearts, was isoform 1.4 of regulator of calcineurin, whose regulation and functional role were explored further.
CONCLUSIONS: Our data demonstrate that voluntary running in part restores cardiomyogenesis in aged mice and suggest that
pathways associated with circadian rhythm may play a role in physiologically stimulated cardiomyogenesis.
Key Words: age factors ◼ circadian rhythm ◼ exercise ◼ heart failure ◼ muscle development
Average life expectancy increased in the past cen-
tury throughout the world,1 and it is expected that
≈25% of the European and Northern American
population and ≈15% of the worldwide population will
be >65 years by the year 2050.2 Age is an indepen-
dent risk factor for the development of cardiovascular
diseases, and heart failure represents a leading cause
of hospitalization and death in older adults.3 Aging leads
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to structural and functional changes in the heart that are
similar to those seen in the failing heart, suggesting that
processes intrinsic to cardiac aging may contribute to
the pathophysiology of heart failure.4 Multiple processes,
including increased interstitial fibrosis, decreased mito-
chondrial function, chronic inflammation, and maladap-
tive cardiomyocyte hypertrophy, are thought to contribute
to the development of heart failure in older adults.4 The
loss of cardiomyocytes through increased cardiomyocyte
apoptosis and decreased generation of new cardiomyo-
cytes poses a risk for the development of cardiovascular
diseases, in particular, heart failure.5–9
In a previous study, we showed that exercise stimu-
lates the endogenous generation of new cardiomyocytes
in adult mouse hearts. We applied multi-isotope imag-
ing mass spectrometry (MIMS) to left ventricular heart
sections combined with cardiomyocyte nuclei tracing
and ploidy assessment after continuous application of
15N-thymidine during an 8-week voluntary wheel running
protocol in adult mice.10 We found that exercise increased
cardiomyogenesis by ≈4.6-fold in young mice compared
with sedentary controls. However, whether exercise can
also induce the formation of new cardiomyocytes in the
aged heart remains unclear.
Here, we demonstrate that endogenous cardiomyo-
genesis can be stimulated by exercise in aged mice. We
could not detect baseline cardiomyogenesis in our cohort
of aged sedentary animals, in contrast with previous mea-
surements of stable baseline cardiomyogenesis in young
sedentary hearts.8,10 Exercise was unable to completely
restore the rate of cardiomyogenesis lost with age on the
basis of our past studies.8,10 However, given the overall
reduced number of cardiomyocytes in aged hearts, the
relative change could potentially have a profound effect on
cardiac function.4,7 Our finding suggested that understand-
ing the pathways involved might allow insights into stimu-
lation of cardiomyogenesis for aged hearts. To this end,
through global exercise-induced transcriptional programs
in young and aged mouse hearts, we found that exercise
induces gene expression patterns related to circadian
rhythm. Because aging is characterized by decreased
central clock function, which, in turn, contributes to car-
diovascular disease in older adults, we were intrigued to
find an oscillating gene, RCAN1.4 (isoform 1.4 of regula-
tor of calcineurin), to be induced with exercise exclusively
in aged hearts. RCAN1 is known to influence intracellular
signaling primarily by regulating calcineurin activity and
was reported to play a role not only for cardiac remodeling
and angiogenesis, but also cardiomyogenesis.11–13
METHODS
All supporting data are available within the article. Detailed
methods are described in the Supplemental Material.
Animal Studies
For this study, 20-month-old aged C57BL/6J male mice were
used for the primary investigations. For additional experiments
and comparison with young mice, 10- to 12-week-old male mice
were also investigated. All mice were studied and cared for in
accordance with protocols approved by the Institutional Animal
Care and Use Committee of Faculty of Arts and Science, Harvard
University (protocol number 16-05-273), Massachusetts
General Hospital (protocol number 2015N000029), and
regional authorities (Regierungspräsidium) Karlsruhe, Germany.
Data and Code Availability
Datasets are deposited and publicly available at the following
link: https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA811435.
Statistical Analyses
Statistical testing was performed using Prism 8.4.2. (Graphpad)
if not indicated otherwise.
Clinical Perspective
What Is New?
• Endogenous cardiomyogenesis can be stimulated
by exercise in aged hearts.
• Comparative global transcriptional analyses
reveal exercise- and age-specific changes in gene
programs.
• The regulator of calcineurin RCAN1.4 (isoform
1.4 of regulator of calcineurin) was found specifi-
cally induced with exercise in aged hearts and was
accompanied by reduced calcineurin activity.
What Are the Clinical Implications?
• Exercise-induced cardiomyogenesis may counter
increased cardiomyocyte loss and reduced cardio-
myogenic capacity, enabling the development of
cardiovascular diseases in elderly patients.
• New insights into exercise-induced transcriptional
programs of young and aged hearts build a founda-
tion to better understand the beneficial effects of
exercise for the heart.
Nonstandard Abbreviations and Acronyms
CCND1 Cyclin D1
CEBP CCAAT/enhancer binding protein
CITED Cbp/p300-interacting transactivator 4
HIPK homeodomain-interacting protein kinase
MIMS multi-isotope imaging mass
spectrometry
NFAT nuclear factor of activated T cells
NRVM neonatal rat ventricular myocytes
PGC 1α-peroxisome proliferator-activated
receptor gamma coactivator 1-alpha
RCAN regulator of calcineurin
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Unpaired, 2-tailed Student t tests were used comparing 2
groups at 1 time point. When assessing multiple groups, 1-way
ANOVA was used with the Sidak or Tukey posttest for mul-
tiple comparisons; multiplicity-adjusted P values are reported
for each comparison. Time course analysis was assessed using
repeated-measures 2-way ANOVA with Sidak posttest for mul-
tiple comparisons. Data comparing event rates were tested with
a Fisher exact test. Results are presented as mean±SEM; sig-
nificance was assigned for P<0.05. For all data, n equals the
number of biological replicates of animals used per experiment.
The number of animals used for each group was determined on
the basis of total empirical data and anticipated completeness
of datasets and was sufficient to detect differences in experi-
mental outcomes, if present.
RESULTS
Voluntary Running Induces Physiological
Cardiac Hypertrophy in Aged Mice
Cardiac systolic function (ie, fractional shortening) was
not changed after 8 weeks of voluntary wheel running
when assessed by echocardiography in aged exercised
animals compared with sedentary controls (Figure 1A),
suggesting the absence of maladaptive cardiac remodel-
ing. However, in previous studies investigating the exer-
cise response of young animals, a mild exercise-induced
increase in fractional shortening was observed.10,14 Echo-
cardiographic imaging also revealed an increase in left
ventricular (LV) posterior wall thickness and septal wall
thickness (Figure 1B) in mice subjected to voluntary run-
ning indicative of exercise-induced hypertrophic LV re-
modeling. Cardiac weight normalized to tibial length or
body weight after 8 weeks of voluntary wheel running
supported the echocardiographic data, showing in-
creased heart weights (Figure 1C) and cardiomyocyte hy-
pertrophy (cross-sectional area; Figure 1D) in exercised
mice compared with sedentary mice. In addition, gene
expression analysis showed typical changes indicating
physiological cardiac remodeling, for example, decreased
β-myosin heavy chain expression accompanied with a
≈2-fold increase in α-myosin heavy chain to β-myosin
heavy chain ratio without differences in atrial natriuretic
peptide/brain natriuretic peptide expression (Figure 1E).
Unlike in young mice, in which exercise induces differ-
ential expression of CEBP/β (CCAAT/enhancer binding
protein), CITED4 (Cbp/p300-interacting transactivator
4), PGC1α (1α-peroxisome proliferator-activated re-
ceptor gamma coactivator 1-alpha), and microRNA 222
targets HIPK1 and HIPK2 (homeodomain-interacting
protein kinase), among others, we did not observe these
changes in aged exercised mice, suggesting that ex-
ercise may induce physiological hypertrophic changes
through different mechanisms in the aged heart (Fig-
ure 1E). Aged mice provided with a running wheel for
8 weeks ran 2.4 km/24 h on average (Figure 1F). We
previously reported that young mice from the same strain
ran 5.57 km/24 hours on average when subjected to the
voluntary wheel running protocol,10 demonstrating a de-
cline in voluntary running with age. Taken together, these
data show that voluntary wheel running overall results in
typical exercise-induced hypertrophy in aged mice, de-
spite a reduced daily exercise load and different physi-
ological gene expression alterations.
Voluntary Running Increases the Number of
New Cardiomyocytes in the Aged Heart
The goal of this study was to investigate whether vol-
untary wheel running increases the generation of new
cardiomyocytes in aged mice, in comparison with our
previously reported values in young mice, using identi-
cal techniques with mice from the same genetic back-
ground.10 We used MIMS analysis of tissue sections to
track and quantify the incorporation of 15N-thymidine
in cardiomyocyte nuclei in aged mouse hearts af-
ter 8 weeks of voluntary running or sedentary activ-
ity (Figure 2A).8,10 Simultaneous NanoSIMS imaging
of 12C14N,31P, and 32S ions at suborganelle resolution
allowed the identification of cardiomyocytes by char-
acteristic ultrastructures such as sarcomeres, 1 or 2
prominent nucleoli, and the granular nuclear chromatin
pattern identified by high phosphorus content.10 With
particular relevance to the labeling of replicating ge-
nomic DNA, the high 31P content of chromatin is colo-
calized with the quantitative 15N-thymidine isotopic ratio
(Figure 2B). Furthermore, all counts were subsequently
validated by advanced histological imaging analysis.10
We analyzed 1793 cardiomyocytes from 5 aged sed-
entary mice and compared these with 2002 cardiomy-
ocytes from 5 aged exercised mice. We found 1.20%
(0.0214% per day) 15N-thymidine–positive cardiomyo-
cyte nuclei in exercised animals compared with 0.06%
(0.0011% per day) 15N-thymidine–positive cardiomyo-
cyte nuclei in sedentary animals, indicating an exercise-
induced increase in DNA synthesis during the labeling
period in aged mice (Figure 2C). Although MIMS pro-
vides high-fidelity quantification of cell cycle activity,
in isolation it cannot distinguish between polyploidiza-
tion or multinucleation. Therefore, we performed multi-
modal interrogation of adjacent sections for each of the
15N-thymidine–positive cardiomyocyte nucleus, to char-
acterize ploidy status and nucleation. We considered
only mononucleated, diploid, and 15N-thymidine–pos-
itive cardiomyocytes as potential new cardiomyocytes.
To determine nucleation, we performed cardiomyocyte
tracing on serial Periodic acid Schiff–stained sections
in both directions of each thymidine-labeled cardiomyo-
cyte and determined the number of nuclei contained in
each positive cell. We found 67% of thymidine-positive
cardiomyocytes to be mononucleated (0.80% thymi-
dine-positive mononucleated cardiomyocytes among
all counted cardiomyocytes; Figure 2D). To account
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Figure 1. Voluntary wheel running induces physiological cardiac adaptation in aged mice.
Echocardiographic analysis showed preserved fractional shortening (FS) (A), with increased anterior and posterior wall thickness (B) in systole
and diastole (LVAWs/d, LVPWs/d) in exercised aged animals compared with aged sedentary controls (n=7–11 mice/group, ****P<0.0001,
***P=0.0001, **P=0.0081, Student t-test). C, Both heart weight to tibia length (HW/TL) and heart weight to body weight (HW/BW) ratios were
increased in exercised aged mice (n=11–15 mice/group, HW/TL ****P<0.0001, HW/BW ***P=0.0007 Student t-test). D, Likewise, cardiomyocyte
cross-sectional area analyzed from wheat germ-agglutinin–stained transverse left ventricular sections showed an increase in cardiomyocyte size
with exercise (>100 cardiomyocytes were counted per group, n=4 mice/group, *P<0.05, Student t-test). E, Anticipated switch of α/β-myosin
heavy chain gene (MHC) expression after 8 weeks of voluntary wheel running was also detected. PGC1-α was downregulated after 8 weeks
(n=5 mice/group, *P<0.05, Student t-test). F, Running activity (running distance per 24 hours) was measured for each mouse for 8 weeks. Aged
mice ran on average 2.4 km per 24 hours (n=4 mice/group). All data are presented as mean±SEM. ANP indicates atrial natriuretic peptide;
BNP, brain natriuretic peptide; CEBP, CCAAT/enhancer binding protein; CITED, Cbp/p300-interacting transactivator 4; HIPK, homeodomain-
interacting protein kinase; PGC, 1α-peroxisome proliferator-activated receptor gamma coactivator 1-alpha; and SED, sedentary.
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Figure 2. Exercise stimulates an increase in mononuclear diploid15N-thymidine–labeled cardiomyocytes in the aged mouse heart.
15N-Thymidine was administered continuously for 8 weeks to aged mice (20 months old at the beginning of the experiment) undergoing
voluntary wheel running or sedentary activity. A, Representative images of the myocardium in sedentary aged mouse hearts, labeled with15N-
thymidine. Mass 14N image (Left) shows histological details such as cell architecture, whereas the hue-saturation intensity-image (mosaics,
Right) demonstrates nuclear 15N-thymidine labeling. 15N-Thymidine labeling was detected predominantly in noncardiomyocyte cells in aged
sedentary hearts. Scale bar, 10 μm. B, Representative image of 15N-thymidine–labeled cardiomyocyte (yellow asterisks), together with 1
nonlabeled cardiomyocyte (white asterisk) and 1 labeled noncardiomyocyte (yellow arrow) in the myocardium of an aged runner. The scale
ranges from blue, where the ratio is equivalent to natural ratio (0.37%, expressed as 0% above natural ratio [enrichment over natural ratio]), to
red, where the ratio is 150% above natural ratio. 15N-Thymidine has labeled the nucleus, whereas the cytoplasm is at the natural abundance
level. Scale bar, 10 μm. C, Data presented as comparison of the percentages (%) of 15N-labeled cardiomyocyte nuclei in exercised to sedentary
aged hearts. Exercise increases cardiomyocyte cell cycle activity (sedentary:exercise=0.06:1.20%; >1700 cardiomyocytes from 4 mice/group
were counted, ****P<0.0001, Fisher exact test). Of 1.20% 15N-labeled cardiomyocyte nuclei, 0.35% were mononuclear and diploid. D, Periodic
acid Schiff staining and fluorescent in situ hybridization (Y-chromosome) were performed on serial adjacent sections in both directions from the
15N-thymidine–labeled cardiomyocyte to define the number of nuclei and ploidy status in each 15N-thymidine–labeled cardiomyocyte. The table
shows absolute numbers and percentages (%) of 15N-thymidine–labeled cardiomyocytes, and the absolute numbers and percentages (%) of
mononucleate/diploid cells among all 15N-thymidine–labeled cardiomyocytes from each group, as well (sedentary:exercised=0.00:0.35%, n=4
mice per group, *P<0.05, Fisher exact test). CM indicates cardiomyocyte; and SED, sedentary.
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for ploidy status, we then performed in situ hybridiza-
tion targeting the Y-chromosome in sections adjacent
to mononucleated thymidine-positive cardiomyocytes
and found that 44% of those were diploid, consistent
with the ploidy state of a new cardiomyocyte. Taken
together, we observed a significantly higher frequency
of mononucleated/diploid 15N-thymidine–labeled car-
diomyocytes (0.35% of all counted cardiomyocytes in
aged exercised hearts, whereas no mononucleated/
diploid 15N-thymidine–positive cardiomyocytes were
detected in sedentary controls (Figure 2D) after the
8-week labeling period. The calculated annual rate of
mononucleated/diploid 15N-thymidine–labeled cardio-
myocytes in aged exercised mice was 2.3% per year.
This compares with our previously reported annual rate
of 7.5% in young exercised mice and 1.63% in seden-
tary mice. Thus, exercised aged mice reached a level of
cardiomyocyte generation that was numerically higher
than young sedentary mice, but exercise in aged mice
did not restore cardiomyocyte generation to the level
previously reported in young exercised mice.10
In conjunction with the increase in cardiomyo-
cyte DNA synthesis, we also detected an increase in
15N-thymidine–labeled noncardiomyocytes in aged
exercised hearts compared with sedentary controls
(Figure 3A and 3B). We have previously shown that a
fraction of 15N-thymidine–labeled noncardiomyocytes
was attributable to an exercise-induced neo-angio-
genic response in young animals.10 Therefore, we next
analyzed capillary density by counting the number of
CD31-positive endothelial cells (capillaries) per cardio-
myocyte in LV cross sections in an independent cohort
of aged exercised mice (Figure 3C and 3D). Consistent
with previous data, we found an increase in the number
of capillaries per cardiomyocyte after exercise training
in aged mice (Figure 3C).15
We further sought to investigate fibroblasts in the LV
myocardium in exercised versus sedentary aged animals.
We found a trend toward an increase in vimentin-positive
fibroblasts in aged exercised hearts. Therefore, it is likely
that also fibroblast proliferation accounts for a trend of
increased 15N-thymidine–labeled noncardiomyocytes in
exercised aged hearts (Figure 3E and 3F). To exclude
excess extracellular matrix deposition, we analyzed fibro-
sis by Masson trichrome staining. As depicted in Fig-
ure 3H, gross histological imaging showed interstitial and
perivascular fibrosis in aged hearts from both sedentary
and exercised mice, but the degree of fibrosis was not
significantly different in exercised hearts (Figure 3G).
Exercise-Induced Myocardial Remodeling Is
Associated With the Induction of Circadian
Rhythm Pathways Independent of Age
We next sought to identify pathways that potentially
drive cardiomyogenesis in response to exercise in
aged and young hearts. Thus, we performed RNA se-
quencing analysis in heart samples from both young
and aged exercised mice, and their age-matched
sedentary controls, as well. We assessed differential
gene expression with a 2-factor model including an
interaction term to identify differentially expressed
genes (y ~ age+exercise+interaction [age:exercise],
false discovery rate<0.05) as shown in Figure 4A. We
identified 42 genes of interest in our model, which
are called significant in 1 exercise-related model term
(false discovery rate<0.05; Figure 4A). We first inves-
tigated genes significantly regulated by exercise irre-
spective of age (30 genes, green circle, Figure 4A)
for enriched biological pathways using g:Profiler.16 We
found that a common exercise-induced term in both
aged and young animals was associated with circa-
dian rhythm (KEGG). Among genes enriched in this
pathway were core clock components Arntl (BMAL1),
Npas2, and Per3, for example, also shown hierarchi-
cally clustered in a heatmap (Figure 4B).
Transcriptome Analysis Reveals Increased
RCAN1 Expression in Aged Exercised Mice
Given the difference in exercise-induced cardiomyogen-
esis between aged and young animals, we next sought
to investigate genes that differed when comparing aged
and young hearts with exercise. Therefore, we systemati-
cally investigated genes significantly regulated in our in-
teraction term analysis (21 genes, red circle, Figure 4A).
One gene, Rcan1, that was exclusively regulated
in aged exercised hearts compared with young exer-
cised hearts (no overlap red circle, Figure 4A), was
previously found to oscillate largely over the course
of the day in a circadian manner in healthy hearts.17
RCAN1 was also recently shown to be involved in the
regulation of new cardiomyocyte generation,12 prompt-
ing further interrogation of RCAN1 in aged exercised
hearts. We first validated its differential regulation by
quantitative polymerase chain reaction in an indepen-
dent cohort in addition to the hearts used for RNA
sequencing analysis (Figure 5A). Transcript variant
1.4 of Rcan1 (Rcan1.4) was consistently and signifi-
cantly upregulated by exercise ≈4-fold in aged cohorts,
whereas transcript variant 1.1 was unchanged. Cardiac
Rcan1.1 and Rcan1.4 were not significantly altered by
exercise in young hearts in either cohort (Figure 5A).
We next isolated cardiomyocytes and noncardiomyo-
cytes from young and aged hearts to determine the
primary expression pattern of Rcan1 in the heart.
Pure isolation was confirmed by mRNA expression
of Troponin T (Tnnt, mostly expressed in cardiomyo-
cytes, Figure S1A) and expression of Collagen 1a
(Col1α, mostly expressed in fibroblasts, Figure S1B).
We could confirm that both Rcan1.1 (Figure 5B) and
Rcan1.4 (Figure 5C) were predominantly expressed
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Figure 3. Angiogenic and fibrotic response in the aged exercised heart.
A, Exercise increases the 15N-thymidine–labeled noncardiomyocyte fraction (aged sedentary:exercised=23.3:34.5%) (>10 000
noncardiomyocytes from 4 mice/group were counted, ****P<0.0001, Fisher exact test). B, Representative images of 15N-thymdine–labeled
noncardiomyocyte nuclei in aged mouse hearts (mosaic, Right, white arrow). Mass 14N images (Left) show histological details such as localization
and structure. Scale bar, 10 μm. C, Number of CD31 (PECAM1 [platelet and endothelial cell adhesion molecule 1]) positive capillaries per
cardiomyocyte after voluntary wheel running (>100 cardiomyocytes counted, n=4 mice/group, *P<0.05, Student t-test; Left). D, Representative
images of endothelial CD31 (PECAM1) staining (red), in combination with wheat germ agglutinin (WGA, green) to determine the capillary density
(capillaries/cardiomyocyte). Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm (Right). E, Expression of vimentin in aged exercised
and sedentary mouse hearts, shown as % Vimentin-positive area/total area (n=4 mice/group, P=0.07, Student t-test; Left). F, Representative
images of vimentin staining (red), in combination with WGA (green). Nuclei were counterstained with DAPI (blue; Right). G, Cardiac fibrosis was
not significantly affected by exercise in aged mice, n=4 mice/group, Student t-test). H, Representative Masson trichrome–stained images from
aged sedentary and exercised mice. Scale bar, 500 μm. Collagen-rich areas stained blue (black arrows). Data presented as mean±SEM. CM
indicates cardiomyocyte; DAPI, 4′,6-diamidino-2-phenylindole; and SED, sedentary.
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in cardiomyocytes in young and aged hearts, whereas
expression in noncardiomyocytes was relatively low or
undetectable in the case of Rcan1.4, in comparison.
Rcan1.1 and 1.4 expression were not statistically sig-
nificantly different between young and aged hearts at
baseline (Figure 5A–5C). Increased exercise induced
RCAN1.4, but not RCAN1.1, protein expression was
confirmed in hearts from aged exercised hearts com-
pared with hearts from aged sedentary mice (Figure 5D
and 5E). These results indicated that exercise induces
distinct gene expression changes in aged compared
with young exercised hearts.
Exercise Reduces Calcineurin Activity in Aged
Hearts
RCAN1 is a known regulator of calcineurin activity in car-
diomyocytes. Increased RCAN1 expression with reduced
calcineurin activity has previously been reported to sup-
port cardiomyogenesis in neonatal and adult hearts.12
Therefore, we sought to investigate calcineurin activity
in aged exercised hearts compared with aged sedentary
hearts. Activated calcineurin dephosphorylates NFAT
(nuclear factor of activated T cells) transcription fac-
tors, thereby inducing their translocation to the nucleus.
Figure 4. Differentially regulated
genes in exercised hearts.
Global gene expression in the heart was
determined by using RNA sequencing
in young (8 weeks) and aged mice
(20 months) euthanized 8 weeks after
voluntary wheel running or sedentary
activity (n=4 per group). A, Venn diagram
comparing functionally enriched biological
genes in young and aged mice exposed
to either exercise or sedentary activity.
Differentially expressed genes unique
to age (blue), exercise (green), or the
interaction thereof (exercise:age; red) are
shown. B, Heat map depicts hierarchically
clustered differentially regulated genes
in the myocardium of young and aged
exercised mice, compared with their age-
matched sedentary controls by either age,
exercise, or the interaction thereof (n=8
mice per treatment, 4 young and 4 aged).
Sed indicates sedentary.
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Nuclear localization of NFAT can therefore be used as an
indirect measure of calcineurin activity. We used immu-
nofluorescence staining to determine endogenous nu-
clear localization of NFATc1 in cardiomyocytes from aged
exercised and sedentary hearts. We found significantly
reduced NFATc1 nuclear staining in aged exercised
hearts compared with aged sedentary hearts, indicating
reduced calcineurin activity with exercise (Figure 6A and
6B). These findings confirm a role for the calcineurin-
NFAT signaling axis for myocardial adaptation to exer-
cise in aged hearts.
RCAN1.4 Expression Stimulates the Induction
of Cell Cycle Markers in Aged Cardiomyocytes
As previously reported, exercise reverses the reduction
of cell cycle or cell division pathways in the aged hearts.15
In our RNA sequencing dataset, we also found the term
Cell Cycle significantly regulated for the interaction
age:exercise (gene set enrichment analysis; Figure S2A).
Among the differentially expressed genes in aged exer-
cised hearts (Figure 4B), we found Cell Division Cycle
Associated 2 gene (Cdc2a), which is known to promote
Figure 5. Exercise induces expression of RCAN1.4 in aged hearts.
A, Validation of increased Rcan1.1 and Rcan1.4 gene expression by quantitative polymerase chain reaction analysis in exercised aged hearts in
both the hearts that were analyzed by RNA sequencing and an independent exercised cohort (n=7–9 mice/group, *P<0.05, Student t-test). B
and C, Rcan1.1 and Rcan1.4 gene expression was quantified from isolated young (2 months old) and aged (20 months old) cardiomyocytes and
noncardiomyocytes (n=8 mice/group, *P<0.05, **P<0.01, ****P<0.0001, 1-way ANOVA). D and E, RCAN1.1 (≈37 kDa) and RCAN1.4 (≈25 kDa)
protein levels were determined in aged sedentary and exercised mouse hearts by immunoblotting. RCAN1.4 protein levels were increased in aged
exercised hearts (n=3 mice/group, *P<0.05, Student t-test). Vinculin (≈100 kDa) was used as a protein loading control. All data are presented as
mean±SEM. CM indicates cardiomyocyte; RCAN, regulator of calcineurin; and SED, sedentary.
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Lerchenmüller et al Restoration of Cardiomyogenesis in Aged Mouse Hearts by Voluntary Exercise
Figure 6. Exercise reduces calcineurin activity in aged hearts, and RCAN1.4 overexpression in aged cardiomyocytes increases
cardiomyocyte cell cycle markers.
A, The fraction of NFATc1 positive cardiomyocyte nuclei per section area (%) was quantified in aged mouse hearts. Exercise treatment
reduced NFATc1 expression in cardiomyocyte nuclei, compared with sedentary control hearts (n=4 mice/group, **P<0.001, Student t-test). B,
Representative images from aged sedentary and exercised mouse hearts show NFATc1 (red) stained nuclei (DAPI, blue), in combination with
wheat germ agglutinin (WGA, green). Scale bar, 100 μm. C, Validation of cell cycle regulators Cdca2 and Ccnd1 mRNA expression in aged
sedentary and exercised mouse hearts (n=8 mice/group, *P<0.05, **P<0.01, Student t-test). D, Isolated murine aged cardiomyocytes (20 months)
were infected with RCAN1.1 (AdV-RCAN1.1) and RCAN1.4 (AdV-RCAN1.4) and LacZ control (AdV-LacZ) adenoviruses, respectively, and Cdc2a
and Ccnd1 mRNA expression was analyzed (n=3–6 replicates per treatment from a total of 6 hearts, *P<0.05, 1-way ANOVA). E, NRVM were
infected with RCAN1.1 (AdV-RCAN1.1) and RCAN1.4 (AdV-RCAN1.4) and LacZ control (AdV-LacZ) adenoviruses, respectively, and Ccnd1
mRNA expression was analyzed after stimulation with indicated serum concentrations (n=4, *P<0.05, **P<0.01, ***P<0.001, 2-way ANOVA). F,
NRVM were infected with RCAN1.1 (AdV-RCAN1.1) and RCAN1.4 (AdV-RCAN1.4) and LacZ control (AdV-LacZ) adenoviruses, (Continued )
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proliferation in other tissues, for example, in the liver or
malignant diseases such as colorectal cancer, by activat-
ing cyclin D1 (Ccnd1).18 We confirmed upregulated Cd-
c2a in aged exercised hearts, and also found increased
Ccnd1 expression in these tissues compared with aged
sedentary hearts, suggesting a role for cell cycle regula-
tors in our model as well (Figure 6C). We next sought to
test whether adenoviral overexpression of either Rcan1.1
or Rcan1.4 stimulates cardiomyocyte cell cycle activity
in isolated murine aged cardiomyocytes. We found that
both Cdc2a and Ccnd1 expression were increased spe-
cifically by adenoviral Rcan1.4 expression (Figure 6D).
We confirmed sufficient and specific adenoviral (multi-
plicity of infection 50) overexpression of RCAN1.1 and
RCAN1.4 protein in cultured neonatal rat ventricular
myocytes (NRVM; Figure S3A).
NRVM treatment with RCAN1.4 adenovirus did not
lead to increased Ccnd1 expression compared with
LacZ-treated NRVM with serum stimulation. However,
RCAN1.1 expression showed significantly decreased
Ccnd1 expression compared with both LacZ control and
RCAN1.4-treated cells, suggesting (as in isolated aged
cardiomyocytes) isotype-specific regulation of cell cycle
in NRVM (Figure 6E). As depicted in Figure S3A, a simi-
lar pattern was detected when analyzing protein expres-
sion of the mitosis marker pHH3 (phospho-histone-H3)
in RCAN1.1/1.4 NRVM after serum stimulation (Figure
S3A). A similar increase in Ki67 staining with serum
stimulation in RCAN1.1/1.4 NRVM was detected when
compared with control LacZ NRVM, in the absence of
increased cell counts, further suggesting that RCAN1
overexpression in NRVM did not lead to increased car-
diomyocyte cytokinesis during the observed time (Fig-
ure 6G and 6H). In summary, our data show that RCAN1.4
is sufficient to stimulate cardiomyocyte cell cycle activity
in aged cardiomyocytes in vitro, suggesting a particular
susceptibility to RCAN1-calcineurin signaling.
Confirming the complex role of RCAN1 in cardiomyo-
cyte function, we found that, paradoxically, small interfer-
ing RNA–mediated Rcan1 gene silencing in NRVM led
to significantly increased expression of the proliferation
marker Ki67 (Figure S3B and S3C), and an increase in
the cell number (Figure S3D) in low serum (2% fetal
bovine serum) conditions when compared with negative
control (scramble; scr) small interfering RNA–treated
cells, as well. We found that the chosen small interfer-
ing RNA was more effective in depleting Rcan1.4 mRNA
and protein, although both isoforms were reduced
(Figure S4A–S4E). When NRVMs were stimulated with
insulin-like growth factor 1, an agonist for physiological
cellular remodeling, Rcan1.4 (but not Rcan1.1) mRNA
expression was significantly reduced in NRVM (Figure
S5A and S5B). Stimulation of pathological remodeling
with phenylephrine, on the other hand, did not change
Rcan1 expression compared with vehicle-treated control
cells (Figure S5A and S5B). These results were con-
firmed for RCAN1.1 and RCAN1.4 protein as well (Fig-
ure S5C and S5D), suggesting a stimulus-dependent
isoform preference in the heart. In summary, our results
underline a complex isoform-specific, potentially age-,
stimulation-, and condition-dependent RCAN1 signaling
in cardiomyocytes that influences cardiomyocyte prolif-
eration and cardiomyogenesis.
DISCUSSION
In this study, we used continuous long-term 15N-thy-
midine labeling and NanoSIMS (MIMS) analysis in
combination with advanced histological analyses to
demonstrate that 8 weeks of voluntary running not only
results in physiological exercise adaptations in the aged
heart, but also in part restores cardiomyogenic activity.
This is of particular interest because the loss of car-
diomyocytes increases with age,5 whereas the already
limited extent of mammalian cardiomyogenesis further
declines with age.6–9 The imbalance between cardio-
myocyte loss and generation may contribute to heart
failure.4,5 More than 80% of patients with heart failure
are individuals older than the age of 65 years,1,4 which
is why it is of particular interest to find a way to stimu-
late endogenous cardiomyocyte generation to favorably
shift the balance between the loss of cardiomyocytes
and the birth of new cardiomyocytes in the aged heart.
Various exogenous stimulation strategies have been
used to stimulate cardiomyocyte cell cycle reentry,19–21
but limitations remain particularly with respect to cell
specificity. Increased cell cycle activity in many other
cells poses a risk for neoplasm formation. Exercise,
however, is not only beneficial for cardiovascular out-
comes,22,23 but also is well studied as a preventive and
therapeutic measure in cancer, for example.24
Determining the rate of cardiomyocyte renewal in
adult hearts is technically challenging. The technique
applied in the present study is reliably and unambigu-
ously able to determine the rate of cardiomyogenesis
per time period by combining quantitative isotope-based
Figure 6 Continued. respectively, and pHH3 (phospho-Histone-H3) protein expression was analyzed after stimulation with indicated serum
concentrations (n=4, *P<0.05, **P<0.01, ***P<0.001****P<0.0001, 2-way ANOVA). G, NRVM were infected with RCAN1.1 (AdV-RCAN1.1) and
RCAN1.4 (AdV-RCAN1.4) and LacZ control (AdV-LacZ) adenoviruses, respectively, and Ki67-positive cardiomyocytes (immunofluorescence
staining) were counted and normalized to total cardiomyocyte cell count per slide after treatment with indicated serum concentrations (n=4,
***P<0.001, 2-way ANOVA). H, Cell count of NRVM after infection with RCAN1.1 (AdV-RCAN1.1) and RCAN1.4 (AdV-RCAN1.4) and LacZ
control (AdV-LacZ) adenoviruses followed by indicated serum stimulation (n=4, *P<0.05, ***P<0.001, 2-way ANOVA). All data are presented as
mean±SEM. CCND1 indicates cyclin D1; CDCA2, cell division cycle associated 2; DAPI, 4′,6-diamidino-2-phenylindole; NFAT, nuclear factor of
activated T cells; NRVM, neonatal rat ventricular myocytes; and SED, sedentary.
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DNA synthesis (15N-thymidine incorporation) together
with whole cell nucleation and ploidy analysis. MIMS
allows us to quantitatively distinguish DNA synthesis
(15N-thymidine levels >100% above natural ratio) from
DNA repair (15N-thymidine levels <1.5% above natural
ratio).8 By using this technique, we note that we rarely see
2 15N-labeled cardiomyocytes adjacent to one another.10
This is possible because of the 2-dimensional thin sec-
tions used in MIMS and missing out-of-plane cells, but it
is also possible that daughter cells migrate differently or
that 1 daughter cell has not survived. With our present
methods, we cannot distinguish between these possibili-
ties. We have previously shown, applying the same tech-
niques as in the present study, that exercise increases
cardiomyogenesis in healthy young adult mice at a pro-
jected annual rate of 7.5% versus 1.63% in sedentary
conditions (≈4.6-fold increase).10 Cardiomyogenesis in
aged hearts, in general, is thought to be much lower than
in young hearts,6,7 which is consistent with the data pre-
sented here for sedentary animals. The relative increase
in exercise-induced cardiomyogenesis in aged animals is
at least as high as in young animals. However, the abso-
lute number of new cardiomyocytes in exercised aged
hearts is less than in exercised young hearts (0.35% in
exercised aged mice compared with 1.15% in exercised
young mice).10 The reduced running activity of aged
animals may contribute to the lower absolute number
of new cardiomyocytes in exercised aged hearts. How-
ever, post hoc analysis of the correlation of 15N-positive
cardiomyocytes to the distance run in young exercised
cohorts we previously reported10 did not reveal a linear
relationship, suggesting that the critical difference may
not be attributable to less distance run (Figure S6). It is
important to mention, however, that our previous project
was not designed to answer that question specifically,
the sample size is small, and a threshold effect cannot be
fully excluded, which is why this research question will be
given specific attention in future projects. Also, given the
very low baseline cardiomyocyte 15N-thymidine incorpo-
ration in aged mice, one could speculate that aged hearts
might be capable of an even greater relative increase
in cardiomyogenesis if they were to exercise more and
started at an earlier age. A study performed in humans
showed that exercise exerted the most benefits to
reverse the effects of cardiac aging and prevent cardio-
vascular disease when started by late middle age (before
65), when the heart retains some plasticity.25 Accordingly,
effects on exercise-induced cardiomyogenesis could be
similarly larger, when started before 18 months of age
(<65 in human years).26 However, we have previously
shown that regular exercise improved exercise capacity,
diastolic function, and contractile reserves, while reduc-
ing pulmonary congestion in a mouse model of heart
failure with preserved ejection fraction even in very old
mice. These changes were accompanied by reversal
of the age-dependent reduction in transcriptome cell
cycle or cell division pathways, which were shown to be
a hallmark of aging.15 Whether the exercise-dependent
increase in cardiomyogenesis would be sufficient to
counteract aging-related loss of cardiomyocytes remains
to be elucidated. Taken together, our findings prompted
us to investigate pathways involved that might enhance
cardiomyogenesis in aged hearts.
Both in the whole transcriptome, and in quantita-
tive polymerase chain reaction analyses of well-known
exercise-regulated genes, as well, we found similarities
in exercise-induced genes, but also unique age-depen-
dent differences. It is likely that pathways activated or
inhibited because of aging may affect expression of
genes in response to exercise (eg, different baseline),
some of which could be important for cardiomyogenesis.
Comparing global exercise-induced transcriptional pro-
grams in young and aged hearts revealed that exercise
induced expression of genes related to circadian rhythm,
irrespective of age. Aging is characterized by decreased
function of the central clock that can contribute to age-
related pathologies, including cardiovascular disease.27,28
Running wheel exercise has previously been shown to
reset some age-dependent declines in clock activity
through regulation of peripheral clock protein expression
that governs physiological circadian rhythms.29,30 A direct
link between cell cycle activity and circadian entrainment
has previously been reported. For example, it has been
shown that the circadian clockwork can control cell-
division cycles in the liver directly,31 and in the heart, it
has been shown that cardiac neurons use clock genes
to control myocyte proliferation.32 One limitation of our
study is that the bulk RNA sequencing used allows lim-
ited insight into the transcriptome of single cells, even
less into differentially expressed genes in rare cells.
However, our study suggests that changes to the cardiac
environment, for example, improved circadian entrain-
ment attributable to exercise or exercise-induced cardiac
cell cycle activity, might help to facilitate cytokinesis in a
few cardiomyocytes.
One gene that was previously found not only oscil-
lating over the course of the day in a circadian manner
in healthy hearts,17 but was also recently shown to be
involved in the regulation of new cardiomyocyte genera-
tion, and therefore caught our attention, is RCAN1.4.12
RCAN1.4 was exclusively induced with exercise in aged
hearts. We hypothesized that the modulation of RCAN1
might influence the cardiomyogenic response to exer-
cise in aged hearts. RCAN1 is primarily known as a
regulator of calcineurin-NFAT signaling.33,34 It has been
demonstrated that increased calcineurin/NFAT signal-
ing reactivates the fetal gene program and, in particular,
increased levels of NFAT are detected in failing and aged
hearts.35,36 There are 2 major RCAN1 transcript variants:
RCAN1.1 and RCAN1.4. We show that both isoforms
are abundantly expressed in cardiomyocytes compared
with other cardiac cell types. However, RCAN1.4 was
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Lerchenmüller et al Restoration of Cardiomyogenesis in Aged Mouse Hearts by Voluntary Exercise
the dominant transcript variant found in aged exercised
hearts. We found significantly reduced NFAT nuclear
staining in aged exercised hearts, indicating reduced cal-
cineurin activity. It has previously been reported that the
heart is protected from ischemia-reperfusion injury during
the daytime when RCAN1.4 levels peak, and that RCAN1
protects from ischemia-reperfusion injury through calci-
neurin inhibition.17 These findings and our data are in line
with Nguyen et al12 who reported that transgenic over-
expression of calcineurin led to reduced regenerative
capacity in a mouse model of neonatal apical resection,
whereas concomitant RCAN1 overexpression reversed
that phenotype. Transgenic overexpression of RCAN1
alone inhibited calcineurin activity and enhanced cardio-
myocyte proliferation also in adult hearts after myocardial
infarction. Cell cycle was also among the differentially
expressed pathways in aged exercised hearts, as previ-
ously reported in aged hearts in another exercise model.15
We found that Cdc2a is an interesting gene to validate in
our aged exercised cohort because Cdc2a (Cell Division
Cycle Associated 2) was previously reported as an essen-
tial regulator of cell proliferation by activating Ccnd1 in
other tissues.31 We found that both Cdc2a and Ccnd1 are
increased in hearts from aged exercised mice. We then
confirmed that Rcan1.4 expression in isolated aged car-
diomyocytes led to increased Cdc2a and Ccnd1 expres-
sion as well, whereas Rcan1.1 did not result in the same
degree of Ccnd1 expression.
NRVM treatment with Rcan1.4 adenovirus did not lead
to increased Ccnd1 expression with serum stimulation,
whereas Rcan1.1 expression even showed significantly
decreased Ccnd1 expression compared with both con-
trol and Rcan1.4-treated cells, suggesting (as in isolated
aged cardiomyocytes) isotype-specific regulation of cell
cycle in NRVM. Similarly, Rcan1.1/1.4 overexpression
did not lead to increased mitosis marker pHH3 and pro-
liferation marker Ki67 expression in NRVM after serum
stimulation compared with control LacZ. The absence of
increased cell counts further suggested that Rcan1 over-
expression in NRVM did not cause elevated cardiomyo-
cyte cytokinesis during the observed time. Paradoxically,
when treating NRVMs with insulin-like growth factor 1 as
a physiological stimulus of cardiomyocyte hypertrophy/
proliferation, we found that decreased Rcan1.4 expres-
sion in vitro and depletion of RCAN1 in NRVMs resulted
in increased cardiomyocyte proliferation. Although the
role of RCAN1 in cardiomyogenesis might not seem
straightforward, it is important to note that opposing
effects regarding the role of RCAN1 have been reported
before, for example, on cardiac hypertrophy13,37 and
angiogenesis.11,38,39 RCAN1 function might depend on its
expression level,40 phosphorylation status,40,41 isoform,39
and cellular context.42 Overall, our data indicate that
RCAN1.4 expression in aged exercised hearts is accom-
panied by reduced calcineurin activity and that RCAN1.4.
is sufficient to stimulate cardiomyocyte cell cycle activity
in aged cardiomyocytes in vitro, which is in line with previ-
ous studies.12 The paradoxical result that RCAN1 deple-
tion could also lead to increased proliferation underlines
the complex, multifaceted RCAN1 signaling in cardio-
myocytes. Future studies with manipulation of RCAN1 in
aged mice will be interesting and required to decipher its
role in the context of the aged heart.
Our results collectively demonstrate that voluntary
running restores cardiomyogenic capacity in aged hearts.
Aged and young exercised hearts share common molec-
ular pathways related to cell cycle progression. However,
differentially regulated pathways, like those involving the
regulator of calcineurin, RCAN1.4, might be able to fur-
ther modulate the low absolute number of new cardio-
myocytes in aged hearts. Our study adds more insight
into the stimulation of endogenous cardiomyogenesis
through exercise and provides the foundation for explor-
ing new potentially targetable pathways.
ARTICLE INFORMATION
Received August 30, 2021; accepted May 24, 2022.
Affiliations
Department of Cardiology, University Hospital Heidelberg, Germany (C.L., S.M.,
C.P.R., C.H., F.B., A.Y.R., N.F., C.D.). German Center for Cardiovascular Research
(DZHK), Partner Site Heidelberg/Mannheim, Germany (C.L., S.M., F.B., N.F., C.D.).
Cardiology Division and Corrigan Minehan Heart Center, Massachusetts General
Hospital, Boston (C.L., S.M., C.P.R., J.D.R., H.L., A.R.). Department of Stem Cell and
Regenerative Biology and the Harvard Stem Cell Institute, Harvard University,
Cambridge, MA (A.V., A.W., R.T.L.). Department of Internal Medicine, Division of
Cardiology (M.C., B.R.), Department of Molecular Biology (B.R.), The University
of Texas Southwestern Medical Center, TX. Harvard Medical School, Boston, MA
(C.G., F.G., J.D.R., H.L., M.L.S., A.R.). Center for NanoImaging and Division of Ge-
netics, Brigham and Women’s Hospital, Cambridge, MA (C.G., F.G., M.L.S.). Aging
Institute, University of Pittsburgh School of Medicine, PA (C.G., F.G., M.L.S.). Divi-
sion of Cardiovascular Medicine, Department of Medicine, Brigham and Women’s
Hospital, Boston, MA (R.T.L.).
Acknowledgments
The authors thank Y. Iwamoto and D. Capen of the Center for Systems Biol-
ogy at Massachusetts General Hospital for pathology and histology services.
They also thank K. Mühlburger at University Hospital Heidelberg for technical
assistance and J. Shelton and Dr Nguyen at UT Southwestern Medical Center
for technical input.
Sources of Funding
Dr Lerchenmüller was funded by the German Center for Cardiovascular Research
(DZHK), the Medical Faculty of Heidelberg University Medical School (Germany),
and the Else-Kröner-Fresenius Stiftung. Dr Mittag was funded by the German
Center for Cardiovascular Research (DZHK). Dr Gyngard was funded by the Ger-
man Cardiac Society (DGK). Dr Li was supported by the American Heart Associa-
tion (AHA; 20CDA35310184). Dr Roh was supported by the National Institutes
of Health (NIH; K76AG064328). Dr Lee was funded by the NIH (HL117986,
HL119230, HL137710, and HL122987) and the Leducq Foundation. Dr Rosen-
zweig was funded by the NIH (R35HL15531 and R01AG061034) and the AHA
(16SFRN31720000).
Disclosures
Dr Lee is a co-founder, scientific advisory board member, and private equity hold-
er of Elevian, Inc. He is also a member of the scientific advisory board of Revidia
Therapeutics, Inc., and a consultant to BlueRock Therapeutics.
Supplemental Material
Supplemental Methods
Figures S1–S6
References 43–50
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