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Am J Physiol Heart Circ Physiol 310: H269 –H278, 2016.
First published November 25, 2015; doi:10.1152/ajpheart.00717.2014.
LRRC10 is required to maintain cardiac function in response to pressure
overload
Matthew J. Brody,1,2 Li Feng,3 Adrian C. Grimes,3 Timothy A. Hacker,3 Timothy M. Olson,4
Timothy J. Kamp,1,3 X Ravi C. Balijepalli,3 and Youngsook Lee1,2
1
Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin, Madison,
Wisconsin; 2Molecular and Environmental Toxicology Center, School of Medicine and Public Health, University of
Wisconsin, Madison, Wisconsin; 3Department of Medicine, School of Medicine and Public Health, University of Wisconsin,
Madison, Wisconsin; and 4Cardiovascular Genetics Research Laboratory and Division of Pediatric Cardiology, Department
of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, Minnesota
Submitted 7 October 2014; accepted in final form 18 November 2015
leucine-rich repeat containing 10; pressure overload; cardiomyopathy;
eccentric hypertrophy
NEW & NOTEWORTHY
Absence of leucine-rich repeat containing protein (LRRC)10
sensitizes mice to severe cardiac dysfunction and decompensation after pressure overload without altering myocardial
fibrosis or cardiomyocyte death, suggesting a unique requirement for LRRC10 in maintaining cardiac functional
performance. These results suggest important roles for
LRRC10 under conditions of pressure overload, such as
hypertension or aortic stenosis.
Address for reprint requests and other correspondence: Y. Lee, Dept. of Cell
and Regenerative Biology, Univ. of Wisconsin, 1111 Highland Ave., 4431
Wisconsin Institutes for Medical Research, Madison, WI 53706 (e-mail:
[email protected]).
http://www.ajpheart.org
CARDIOMYOCYTE HYPERTROPHY in response to extracellular stimuli, such as neurohumoral or growth factor stimulation of
membrane-bound receptors (13, 23, 44), or in response to
mechanical stretch or strain that is sensed by mechanosensory
machinery embedded in the Z-disc cytoskeleton, and sarcolemma (25, 38, 49). At the whole organ level, the heart
undergoes hypertrophy as an adaptive mechanism to maintain
cardiac output and reduce ventricular wall stress (20, 23, 38).
While initially beneficial, prolonged cardiac hypertrophy becomes maladaptive and progresses to cardiac dilation, decompensation, heart failure, and/or sudden death (20, 23, 38).
Cardiac hypertrophy can be induced by a number of cardiovascular diseases, such as myocardial infarction or chronic
hypertension (23, 37). Indeed, high blood pressure is a common precursor to heart disease, particularly in the United
States, where chronic hypertension is widespread (18, 21, 42).
Despite increased awareness and treatment of hypertension, recent
epidemiological studies have suggested that its prevalence continues to rise, affecting ⬃55– 65 million adults in the United
States alone (18, 21). Therefore, understanding the factors that are
required to maintain cardiac function in the setting of pressure
overload is critical for developing efficacious therapeutic strategies to treat heart disease.
Cardiac remodeling can be characterized as concentric or
eccentric. Concentric hypertrophy occurs in pressure overload,
such as from chronic hypertension or aortic valve stenosis, or
in inherited hypertrophic cardiomyopathy induced by mutations in genes encoding sarcomeric proteins (22, 37, 38, 46,
49). Concentric hypertrophy results from the addition of sarcomeres in parallel within cardiomyocytes, resulting in greater
myocyte cell surface area, disproportionate growth in cardiomyocyte width relative to length, and thickening of the ventricular wall (26, 52). In contrast, eccentric cardiac hypertrophy
is caused by serial addition of sarcomeres, resulting in a
disproportionate increase in the length relative to the width of
cardiomyocytes that causes ventricular dilation (26, 52). Eccentric cardiac hypertrophy occurs in conditions of volume
overload, such as valvular regurgitation or after myocardial
infarction, and in inherited cardiomyopathies associated with a
wide range of genes, including many cytoskeletal and Z-disc
genes (8, 11, 19, 26, 29, 37, 50, 56). Therefore, the heart
hypertrophies in response to a variety of pathological stimuli;
however, the nature and resulting geometry of cardiac remodeling are stimulus dependent.
Leucine-rich repeat containing protein (LRRC)10 is a cardiac-specific member of the LRRC superfamily, which have in
0363-6135/16 Copyright © 2016 the American Physiological Society
H269
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Brody MJ, Feng L, Grimes AC, Hacker TA, Olson TM, Kamp
TJ, Balijepalli RC, Lee Y. LRRC10 is required to maintain cardiac
function in response to pressure overload. Am J Physiol Heart Circ
Physiol 310: H269 –H278, 2016. First published November 25, 2015;
doi:10.1152/ajpheart.00717.2014.—We previously reported that the
cardiomyocyte-specific leucine-rich repeat containing protein
(LRRC)10 has critical functions in the mammalian heart. In the
present study, we tested the role of LRRC10 in the response of the
heart to biomechanical stress by performing transverse aortic constriction on Lrrc10-null (Lrrc10⫺/⫺) mice. Mild pressure overload induced severe cardiac dysfunction and ventricular dilation in
Lrrc10⫺/⫺ mice compared with control mice. In addition to dilation
and cardiomyopathy, Lrrc10⫺/⫺ mice showed a pronounced increase
in heart weight with pressure overload stimulation and a more dramatic loss of cardiac ventricular performance, collectively suggesting
that the absence of LRRC10 renders the heart more disease prone with
greater hypertrophy and structural remodeling, although rates of
cardiac fibrosis and myocyte dropout were not different from control
mice. Lrrc10⫺/⫺ cardiomyocytes also exhibited reduced contractility
in response to ␤-adrenergic stimulation, consistent with loss of cardiac
ventricular performance after pressure overload. We have previously
shown that LRRC10 interacts with actin in the heart. Here, we show
that His150 of LRRC10 was required for an interaction with actin, and
this interaction was reduced after pressure overload, suggesting an
integral role for LRRC10 in the response of the heart to mechanical
stress. Importantly, these experiments demonstrated that LRRC10 is
required to maintain cardiac performance in response to pressure
overload and suggest that dysregulated expression or mutation of
LRRC10 may greatly sensitize human patients to more severe cardiac
disease in conditions such as chronic hypertension or aortic stenosis.
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ABSENCE OF LRRC10 EXACERBATES HYPERTENSIVE CARDIOMYOPATHY
EXPERIMENTAL PROCEDURES
Animals. Lrrc10⫺/⫺ mice have been previously described (8, 39).
Experiments used Lrrc10⫺/⫺ mice and littermate or age-matched WT
control mice maintained on the same C57BL/6 genetic background (8,
39). Mice were euthanized by cervical dislocation to harvest tissue for
biochemical and histological analyses. All procedures were performed
in accordance with the National Institutes of Health (NIH) Guide for
the Care and Use of Laboratory Animals and University of Wisconsin
Research Animal Resource Center policies and approved by the
Institutional Animal Care and Use Committee of the University of
Wisconsin (protocol no. M01461).
TAC and echocardiography. TAC was performed to induce pressure overload hypertrophy as previously described (6, 10). Briefly,
3-mo-old mice were anesthetized by face mask administration of 3%
isofluorane and then intubated and placed on a ventilator (Harvard
Apparatus) with supplemental O2 and 1.5% isoflurane using a tidal
volume of 0.2 ml and a respiratory rate of 110 breaths/min. The chest
cavity was entered in the second intercostal space at the upper sternal
border through a small incision, and aortic constriction was performed
by tying a 7– 0 nylon suture ligature against a 27-gauge needle. The
needle was then removed, and the pneumothorax was evacuated and
extubated. Subcutaneous buprenorphine (0.8 mg/kg) was administered for pain relief, and mice were allowed to recover in a heated
chamber with 100% O2. Pressure gradients across the constriction
were estimated from Doppler echocardiography using the modified
Bernoulli equation. Animals were euthanized, and tissues harvested
for analysis after 28 days of TAC.
Transthoracic echocardiography was performed on mice under 1%
isofluorane gas anesthesia using a Visual Sonics 770 ultrasonograph
with a 30-MHz transducer (RMV 707B, Visual Sonics, Toronto, ON,
Canada) as previously (8, 22). Two-dimensional guided M-mode
images of the left ventricle (LV) were acquired at the tips of the
papillary muscles. Doppler experiments at the level of the aortic
constriction were also acquired. The LV mass-to-body weight ratio,
LV internal diameter (LVID) at diastole, thickness of the posterior
walls at diastole, and isovolumetric relaxation time were recorded.
Parameters were measured over at least three consecutive cycles (8,
10, 58).
Histology and immuohistochemistry. Hematoxylin and eosin staining was done as previously described (8). Masson’s trichrome (Sigma)
and TUNEL (Millipore) staining of cardiac sections prepared from
paraffin-embedded hearts were performed to detect collagen deposition and apoptosis, respectively, according to the manufacturer’s
instructions. Cardiomyocyte cross-sectional area was measured in
wheat germ agglutinin (conjugated to Oregon green 488, 10 ␮g/ml,
Invitrogen)-stained cardiac sections costained with phalloidin (conjugated to Alexa fluor 546, 165 nM, Invitrogen). Cross-sectional area
was evaluated in at least 300 cardiomyocytes/animal from identical
areas of the LV (8). Fibrosis was quantified as previously described
(12) using images taken from the LV free wall of trichrome-stained
cardiac sections, and percent fibrosis was averaged from seven ⫻20
images per heart. Imaging of heart sections was done on a Zeiss
Axiovert 200 microscope with Zeiss AxioCam. Morphometric quantitation was performed with NIH ImageJ software.
For cryosectioning, hearts isolated 4 wk after TAC or sham
surgeries were transferred into 30% sucrose in PBS for 1 h and then
placed in a 1:1 mixture of 30% sucrose in PBS and OCT compound
for an additional hour. Hearts were then embedded in 100% OCT
compound and frozen on dry ice before being sectioned at 10 ␮m on
a Leica CM3050S cryostat. For immunohistochemistry, sections were
fixed in acetone for 15 min at ⫺20°C and blocked in PBS with 0.1%
Tween 20 (PBST), 5% normal goat serum, and 2 mg/ml BSA for 1 h.
Sections were incubated with LRRC10 (NBP1-81704, Novus Biologicals) and ␣-sarcomeric actin (A-2172, Sigma), ␣-actinin (A-7811,
Sigma), or ryanodine receptor (MA3-925, Thermo Pierce) primary
antibodies at a 1:200 dilution for 1 h followed by the appropriate
secondary Alexa fluor antibodies (Molecular Probes) in PBST for 90
min. Slides were mounted using Prolong Gold Antifade Reagent with
4’,6-diamidino-2-phenylindole (Life Technologies). Images were acquired and processed using a fluorescent microscope (ECLIPSE 90i,
Nikon), NIS-Elements software suite (Nikon), and Photoshop in
Adobe Creative Suite.
Generation of LRRC10 mutants and glutathione-S-transferase pulldown assays. Repeat Conservation Mapping (24) was used to identify
the residues in LRRC10 that are likely to be important for proteinprotein interactions. A model of the predicted structure of mouse
LRRC10 was created with 3D Jigsaw (4) and visualized with First
Glance in Jmol to map the location of putative functionally important
amino acids within LRRC10. To produce the glutathione-S-transferase (GST)-LRRC10 fusion protein, mouse Lrrc10 was amplified by
PCR and inserted in frame into the pGEX-2T vector containing
NH2-terminal GST (GE Healthcare) (8). Mutant GST-LRRC10 fusion
proteins were generated using the QuikChange II XL Site-Directed
Mutagenesis Kit (Agilent Technologies) to mutate the WT GSTLRRC10 sequence. All new constructs were subjected to diagnostic
digestion and confirmed by DNA sequencing. The oligonucleotides
used to generate point mutations in LRRC10 were as follows:
LRRC10 Y104A forward 5=-GAAACAGCTCTGCATCCTCGC-
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common LRR motifs that function as protein interaction domains (31, 33). LRRC10 has no other known functional domains other than its LRRs (28), suggesting that it mediates its
biological functions by interacting with other proteins. Lrrc10
is expressed exclusively in cardiomyocytes and is dramatically
upregulated after birth, with robust expression maintained in
the adult heart (7, 28), implying important roles in the postnatal
heart. LRRC10 interacts with actin and ␣-actinin in the heart
(8) and localizes at the dyad region where the Z-disc comes
into close juxtaposition to the T-tubule and sarcoplasmic reticulum (8, 28). This places LRRC10 at a mechanosensitive
signaling hub in cardiomyocytes (19).
Lrrc10-null (Lrrc10⫺/⫺) mice exhibit perinatal defects in
cardiac function and moderate dilated cardiomyopathy in
adulthood (8). We have also recently reported that the cardiomyocyte-specific expression of Lrrc10 is regulated by
Nkx2.5, GATA4, and serum response factor (7). Importantly,
heterozygous missense mutations in LRRC10 have been recently identified in human dilated cardiomyopathy patients
(43). This strongly supports our working model that LRRC10 is
a dilated cardiomyopathy candidate gene in humans. However,
the roles of LRRC10 in the context of pathological stimulation,
such as pressure overload, remain unknown.
In the present study, we performed transverse aortic constriction (TAC) on Lrrc10⫺/⫺ mice to test the hypothesis that
LRRC10 is important for the response of the heart to mechanical stress. Lrrc10⫺/⫺ mice exhibit severely deteriorated cardiac function after 4 week of mild pressure overload stimulation, whereas cardiac function in wild-type (WT) mice is
preserved. Interestingly, Lrrc10⫺/⫺ hearts undergo a normal
concentric growth response and show similar increases in
apoptosis or fibrosis in response to TAC compared with control
hearts. These data indicate that LRRC10 is uniquely required
to maintain cardiac contractile performance but is not necessary for concentric cardiac remodeling in the setting of pressure overload. Additionally, we demonstrate that His150 of
LRRC10 mediates its interaction with actin in the heart and
that the interaction of LRRC10 with actin is reduced after
pressure overload, suggesting a potential mechanosensing
function for LRRC10.
ABSENCE OF LRRC10 EXACERBATES HYPERTENSIVE CARDIOMYOPATHY
RESULTS
Lrrc10⫺/⫺ mice exhibit increased cardiac growth in response to pressure overload. To test the role of LRRC10 in the
response of the heart to biomechanical stress, we performed
TAC on mice to induce pressure overload hypertrophy. The
cardiac structure and function of Lrrc10⫺/⫺ mice were evaluated after 28 days of pressure overload stimulation compared
with WT control mice. Lrrc10⫺/⫺ mice underwent greater
cardiac growth and ventricular dilation than WT mice after
TAC as observed by histology (Fig. 1A). The heart weight-tobody weight ratios indicated a modest increase in cardiac
growth in Lrrc10⫺/⫺ mice at baseline (Fig. 1B), as previously
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CCTGG-3=; LRRC10 W127A forward 5=-GAACCTACGGACCCTGGCGCTCGAAT-3=; and LRRC10 H150A forward 5=-TCTCCTTAAAACCCTGGCTGCCGGTTC-3=.
GST, GST-LRRC10, or mutant GST-LRRC10 proteins were expressed in Esherichia coli BL21 (DE3, Merck) and purified with
glutathione agarose (Sigma) as previously described (8, 41). Pulldown
assays were performed with purified rabbit skeletal muscle ␣-actin
(AKL95, Cytoskeleton) exactly as previously described (8). For
pulldown of heart extracts, adult WT mouse hearts were homogenized
in extraction buffer [50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1%
Triton X-100, 0.4% sodium deoxycholate, and protease inhibitors],
and lysates were diluted in lysate incubation buffer [20 mM Tris·HCl
(pH 8.0), 100 mM NaCl, 1 mM EDTA, and 1% Triton X-100] and
incubated with GST, GST-LRRC10, or GST-LRRC10H150A for 2 h.
Proteins bound to beads were then washed in lysate incubation buffer,
resolved by SDS-PAGE, and immunoblotted.
Quantitative real-time PCR, Western blot analysis, and
immunoprecipitation. Quantitative real-time PCR was performed using FastStart SYBR Green Master (Roche) on a Bio-Rad iCycler as
previously described (8). Data were generated using the standard
curve method and normalized to 18S rRNA expression levels. Primers
were evaluated by melt curve analysis to ensure specific amplification
of a single, desired amplicon. Primer sequences for 18S, atrial natriurtic factor (ANF; Nppa), and ␤-myosin heavy chain (␤-MHC; Myh7)
have been previously published (8).
Immunoblot analysis was performed using standard methods with
primary antibodies for LRRC10 (28) and ␣-sarcomeric actin (Sigma)
as previously described (8, 9). For coimmunoprecipitation, cardiac
lysates were made in extraction buffer as described for GST pulldown
assays from mice 4 wk after sham or TAC surgery and diluted in
lysate incubation buffer containing rabbit IgG (Santa Cruz Biotechnology) or anti-LRRC10 (1) coupled to protein G Dynabeads (Invitrogen) and incubated overnight at 4°C. Protein G beads were then
washed three times with lysate incubation buffer, and immunoprecipitated proteins were eluted, resolved by SDS-PAGE, and immunoblotted as described above (9).
Myocyte fractional shortening. Myocyte contractility was measured simultaneously in freshly isolated ventricular myocytes from
WT and Lrrc10⫺/⫺ mice. Isolated cardiomyocytes were incubated
with Tyrode solution (142 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8
mM CaCl2, 5 mM glucose, 5 mM HEPES, and 0.33 mM Na2HPO4,
pH 7.4 with NaOH) in a temperature-controlled chamber at 37°C.
Myocyte length was measured using a video-based microscope camera system (IonOptix, Milton, MA). After cells had been allowed to
settle down for 5 min, stable rod-shaped cardiomyocytes with clear
striations were field stimulated for 5 min at 0.5 Hz using 2-ms pulses
at twice of the threshold voltages applied to two remote platinum
electrodes placed in the perfusing solution by myopacer (IonOptix).
Only myocytes with a resting sarcomere length of ⬎1.7 ␮m that
followed the field stimulation protocol 1:1 were used. Once the steady
stimulation had been established, myocytes were stimulated at frequencies of 0.5, 1, and 2 Hz separately for at least 10 continuous
contractions at each stimulation frequency. Isoproterenol (Iso; 10 nM)
was then applied through the perfusion solution, and recording was
repeated under a similar pacing stimulation. Myocyte length was
calculated by performing fast Fourier transforms of the images, and
the resulting plot of the calculated myocyte length was recorded. The
indexes used to describe myocyte contractility were fractional shortening (FS), time to peak (in ms), and time to 50% relaxation. All
indexes were analyzed offline using IonWizard (IonOptix) and were
obtained by averaging 10 steady-state transients for each myocyte.
Statistical analyses. All results are expressed as means ⫾ SE
except for those in Fig. 7, where results are expressed as means ⫾ SD.
All statistical analysis was performed by a Student’s t-test unless
otherwise stated. P values of ⬍0.05 were considered statistically
significant.
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Fig. 1. Response of leucine-rich repeat containing (LRRC)10-deficient
(Lrrc10⫺/⫺) hearts to pressure overload stimulation. Control [wild type (WT)]
and Lrrc10⫺/⫺ mice at 3 mo of age were subjected to 28 days of transverse
aortic constriction (TAC) or sham surgery to test the response to biomechanical stress. A: hematoxylin and eosin (H&E) staining and whole images of WT
and Lrrc10⫺/⫺ hearts in response to TAC. Scale bars ⫽ 1 mm. B: wet heart
weight-to-body weight ratios (HW/BW) in sham- and TAC-operated WT and
Lrrc10⫺/⫺ mice (n ⫽ 8 –12). C: left ventricular (LV) pressure gradient
demonstrating a similar pressure overload stimulus in WT and Lrrc10⫺/⫺
mice. D and E: expression of the hypertrophy marker genes atrial natriuretic
factor (ANF; Nppa; D) and ␤-myosin heavy chain (␤-MHC; Myh7; B) as
evaluated by quantitative PCR (n ⫽ 5–9). Results are normalized to 18S rRNA
expression. *P ⬍ 0.05; **P ⬍ 0.01.
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ABSENCE OF LRRC10 EXACERBATES HYPERTENSIVE CARDIOMYOPATHY
significantly reduced in WT mice in response to TAC (Fig. 2,
C and D).
Comparison of echocardiographic functional parameters before and after 28 days of TAC revealed that Lrrc10⫺/⫺ hearts
underwent much greater reductions in cardiac functional performance due to pressure overload (Fig. 2, E and F). Isovolumetric relaxation time was significantly increased in Lrrc10⫺/⫺
hearts after TAC (18.90 ⫾ 1.01 ms in Lrrc10⫺/⫺ hearts
compared with 16.69 ⫾ 0.72 ms in WT hearts, P ⬍ 0.05),
indicating reduced diastolic function in Lrrc10⫺/⫺ hearts and
suggesting poor cardiac performance and functional decompensation after pressure overload. Lrrc10⫺/⫺ mice had significantly elevated heart rates after TAC (Table 1), resulting in a
maintenance of cardiac output (data not shown) despite a
marked reduction in cardiac function (Fig. 2, C–F). Representative M-mode images showed modest dilated cardiomyopathy
in sham-operated Lrrc10⫺/⫺ mice versus WT mice but greatly
exacerbated cardiomyopathy and LV dilation in Lrrc10⫺/⫺
mice after TAC (Fig. 2G). In contrast, cardiac structure and
function were largely preserved in WT mice after TAC (Fig.
2G and Table 1).
Closer examination of hematoxylin and eosin-stained cardiac sections revealed that Lrrc10⫺/⫺ hearts exhibited overtly
normal cardiac morphology in response to pressure overload
without histological abnormalities such as myocyte disarray or
alterations in myocyte size between WT and Lrrc10⫺/⫺ hearts
(Fig. 3A, top). No differences in fibrosis were detected between
WT and Lrrc10⫺/⫺ hearts after TAC by Masson’s trichrome
staining (Fig. 3A, bottom, and B). Measurements of myocyte
cross-sectional area from wheat germ agglutinin-stained cardiac sections demonstrated similar increases in WT and
Lrrc10⫺/⫺ mice in response to TAC (Fig. 3, C and D). This
suggests that LRRC10 is dispensable for appropriate concentric LV hypertrophy in response to pressure overload. To
further evaluate the nature of cardiac remodeling after pressure
Fig. 2. Lrrc10⫺/⫺ mice have greatly exacerbated cardiomyopathy in response to pressure overload stimulation. A–F: cardiac function in control (WT) and
Lrrc10⫺/⫺ mice subjected to TAC was evaluated by
echocardiography. A–D: measurements of LV inner
diameters at systole (LVIDs; A) and diastole (LVIDd;
B) as well as the percent fractional shortening (FS; C)
and percent ejection fraction (EF; D) 28 days after TAC
or sham surgery. E and F: percent change in FS (E) and
percent change in EF (F) after 28 days of TAC compared with preoperative values. n ⫽ 7–11. G: representative M-mode images. *P ⬍ 0.05; **P ⬍ 0.01; ***P ⬍
0.001.
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reported (8). However, in response to TAC, Lrrc10⫺/⫺ mice
underwent substantially more cardiac enlargement than WT
mice (54% increase in the heart weight-to-body weight ratio
compared with 34% in WT mice; Fig. 1B). Control and
Lrrc10⫺/⫺ mice experienced an equivalent, relatively mild (see
DISCUSSION) pressure overload stimulus from the TAC procedure (Fig. 1C), indicating that differences in cardiac growth or
function after TAC are due to the absence of LRRC10.
Expression of the hypertrophy marker genes Nppa (ANF)
and Myh7 (␤-MHC) were analyzed by quantitative PCR. As
previously reported (8), adult Lrrc10⫺/⫺ hearts exhibited a
mild induction of ANF and robust increase in ␤-MHC expression without hypertrophic stimulation (Fig. 1, D and E). In
response to pressure overload, Lrrc10⫺/⫺ mice induced ANF
expression to a similar extent as WT mice (Fig. 1D). ␤-MHC
expression was elevated in Lrrc10⫺/⫺ hearts compared with
WT hearts at baseline and after TAC (Fig. 1E).
Deletion of Lrrc10 greatly exacerbates cardiac dysfunction
in response to pressure overload. Next, we tested the hypothesis that LRRC10 is important for cardiac function in response
to pressure overload by performing echocardiography on control and Lrrc10⫺/⫺ mice after TAC or sham surgery.
Lrrc10⫺/⫺ mice underwent significant increases in LVID during systole (Fig. 2A) and diastole (Fig. 2B) in response to TAC,
whereas LV dimensions were not altered in WT mice (Fig. 2,
A and B). These data indicate that pressure overload greatly
exacerbates ventricular dilation in Lrrc10⫺/⫺ mice compared
with WT mice. At baseline, Lrrc10⫺/⫺ mice had moderate
dilated cardiomyopathy with increased LVID at diastole and
systole and decreased FS and ejection fraction compared with
WT mice, as we have previously reported (8). However, after
TAC, cardiac contractility was severely compromised in
Lrrc10⫺/⫺ mice, with drastically reduced FS (Fig. 2C and
Table 1) and ejection fraction (Fig. 2D and Table 1) compared
with sham-operated mice, whereas these parameters were not
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ABSENCE OF LRRC10 EXACERBATES HYPERTENSIVE CARDIOMYOPATHY
Table 1. Echocardiographic assessment of cardiac structure and function in Lrrc10⫺/⫺ mice after pressure overload
Sham Operation
Transaortic Constriction
WT
Number of mice/group
LVIDd, mm
LVPWd, mm
LVIDs, mm
LVPWs, mm
LVAWd, mm
LVAWs, mm
LV volume at diastole, ␮l
LV volume at systole, ␮l
Ejection fraction, %
Fractional shortening, %
LV mass, mg
LV mass/body weight, mg/g
Heart rate, beats/min
7
4.32 ⫾ 0.15
0.72 ⫾ 0.03
3.03 ⫾ 0.17
1.06 ⫾ 0.04
0.71 ⫾ 0.02
1.05 ⫾ 0.04
85.09 ⫾ 6.55
37.22 ⫾ 4.79
57.12 ⫾ 2.95
30.01 ⫾ 1.93
114.55 ⫾ 6.18
3.81 ⫾ 0.21
496 ⫾ 28
8
4.65 ⫾ 0.07*
0.59 ⫾ 0.03**
3.75 ⫾ 0.11**
0.81 ⫾ 0.05***
0.59 ⫾ 0.03**
0.81 ⫾ 0.05**
99.96 ⫾ 3.31*
60.76 ⫾ 4.14**
39.36 ⫾ 3.16***
19.26 ⫾ 1.82***
103.07 ⫾ 5.64
3.70 ⫾ 0.19
499 ⫾ 23
WT
Lrrc10⫺/⫺
11
4.34 ⫾ 0.11
0.77 ⫾ .04
3.20 ⫾ 0.14
1.10 ⫾ 0.03
0.77 ⫾ 0.05
1.16 ⫾ 0.04#
85.76 ⫾ 5.54
42.39 ⫾ 4.51
51.34 ⫾ 2.83
26.38 ⫾ 1.69
127.96 ⫾ 8.47
4.38 ⫾ 0.38
493 ⫾ 19
11
5.09 ⫾ 0.16***#
0.72 ⫾ .03##
4.46 ⫾ 0.25***#
0.91 ⫾ 0.04***
0.72 ⫾ .02##
0.92 ⫾ 0.04***
125.28 ⫾ 9.21***#
94.91 ⫾ 12.45***#
27.14 ⫾ 4.68***#
13.09 ⫾ 2.35***#
156.17 ⫾ 12.70*##
5.48 ⫾ 0.46*##
542 ⫾ 16*
Values are means ⫾ SE. Shown is the evaluation of cardiac structural and functional parameters by echocardiography in leucine-rich repeat containing
10-deficient (Lrrc10⫺/⫺) and control wild-type (WT) mice at various ages. LVIDd and LVIDs, left ventricular (LV) inner diameter at diastole and systole,
respectively; LVPWd and LVPWs, LV posterior wall at diastole and systole, respectively; LVAWd and LVAWs, LV anterior wall at diastole and systole,
respectively; EF, ejection fraction; FS, fractional shortening. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 compared with WT mice subjected to the same treatment;
#P ⬍ 0.05 and ##P ⬍ 0.01 compared with sham-operated mice of the same genotype.
overload, the relative wall thickness or ratio of LV wall and
septal thickness to LV chamber radius (H/R ratio) was calculated from echocardiography data. The reduced H/R ratios in
Lrrc10⫺/⫺ versus WT mice after sham surgery indicated eccentric cardiac growth and dilation at baseline (Fig. 3E), as
previously reported (8). Lrrc10⫺/⫺ mice exhibited a robust
concentric hypertrophy response to pressure overload stimulation, as evidenced by the significant increase in the H/R ratio
after TAC (Fig. 3E), consistent with myofiber area measurements (Fig. 3D).
Despite severely impaired cardiac function (Fig. 2),
Lrrc10⫺/⫺ hearts underwent a similar degree of cardiac fibrosis
after pressure overload compared with WT hearts, as detected
by Masson’s trichrome staining (Fig. 3A, bottom). Moreover,
no difference in the induction of the fibrotic marker gene
connective tissue growth factor (Ctgf) was detected between
WT and Lrrc10⫺/⫺ hearts with or without TAC surgery by
quantitative PCR (data not shown), consistent with histological
analyses (Fig. 3A). There was no difference in cardiomyocyte
apoptosis in Lrrc10⫺/⫺ hearts compared with WT control
hearts 28 days after TAC or sham surgery (Fig. 4, A and B),
indicating that apoptosis does not exacerbate pressure overload-induced cardiomyopathy in Lrrc10⫺/⫺ mice. Therefore,
although cardiac functional performance is drastically reduced
in Lrrc10⫺/⫺ mice after pressure overload, the extent of cardiac fibrosis and apoptosis are unchanged compared with WT
mice.
His150 of LRRC10 mediates binding to actin. Some actinbinding proteins are critical for the response of the heart to
biomechanical stress (5, 17, 34, 55), suggesting that localization to the Z-disc or actin cytoskeleton within the cardiomyocyte provides an optimal environment for sensing or transduc-
Fig. 3. Analysis of cardiac growth characteristics and
fibrosis in Lrrc10⫺/⫺ mice in response to pressure
overload. A: histological examination of WT and
Lrrc10⫺/⫺ heart sections by H&E and trichrome staining. Scale bars ⫽ 50 ␮m. B: quantification of fibrosis
from trichrome staining in A. Values are averages of
n ⫽ 3– 4 animals. C and D: myocyte cell surface
cross-sectional area (CSA) evaluated in wheat germ
agglutinin (WGA)-stained cardiac sections revealed
similar increases in WT and Lrrc10⫺/⫺ hearts in response to TAC, indicating that Lrrc10⫺/⫺ mice undergo
normal concentric hypertrophy in response to pressure
overload. Values are averages of n ⫽ 3– 4 animals. At
least 300 myocytes were evaluated per heart. Scale bars ⫽
10 ␮m. E: ratios of LV wall and septal thickness to LV
chamber radius (H/R ratios) or relative wall thicknesses
were calculated from echocardiography data. The decreased H/R ratios in sham-operated Lrrc10⫺/⫺ hearts
indicate dilation or eccentric cardiac growth, and the increased H/R ratios after TAC indicate a normal concentric
growth in response to TAC. n ⫽ 7–11. *P ⬍ 0.05; **P ⬍
0.01.
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Measurement
Lrrc10⫺/⫺
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ABSENCE OF LRRC10 EXACERBATES HYPERTENSIVE CARDIOMYOPATHY
ing signals in response to mechanical stretch or strain. We have
shown that LRRC10 interacts with ␣-actin in the heart and all
actin isoforms in vitro (8), suggesting that LRRC10 serves as
a pan-actin-binding protein. LRRCs physically interact with
other proteins via hydrophobic residues on the interior concave
face of the solenoid-shaped structure imparted by the LRR
domains (31–33). To probe further into mechanisms mediating
interactions of LRRC10 with actin, we used Repeat Conservation Mapping, a computational method to predict functional
sites in LRR domains (24), to predict protein-protein interaction sites in LRRC10. Residues Tyr104, Trp127, and His150 were
identified as putative functional sites in LRRC10, which
mapped to the interior concave face of the predicted protein
Fig. 5. His150 of LRRC10 is critical for its interaction with actin. A: model of the predicted structure of mouse LRRC10 created in 3D Jigsaw and visualized
with First Glance in Jmol. Depicted in yellow are the residues Tyr104 (Y104), Trp127 (W127), and His150 (H150), which were predicted to be functional sites
with Repeat Conservation Mapping. B, top: glutathione-S-transferase (GST) pulldown assays indicated that residue His150 of LRRC10 is critical for its physical
interaction with actin. Purified ␣-actin was incubated with GST or GST fusion proteins containing LRRC10 or a mutant LRRC10 containing Tyr104, Trp127, or
His150 mutated to alanine followed by immunoblot analysis for ␣-actin. Bottom, Coomassie-stained gels demonstrating equal loading conditions. C: His150 of
LRRC10 is required for the interaction with actin in the heart. Mouse heart extract was incubated with GST, GST-LRRC10, or GST-LRRC10H150A and then
immunoblotted for ␣-actin, indicating that GST-LRRC10 but not GST or the GST-LRRC10H150A mutant interacts with actin in cardiac lysates. D: models of
the predicted structures of WT LRRC10 and mouse LRRC10H150A.
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Fig. 4. Apoptosis is unaltered in Lrrc10⫺/⫺ hearts in response to pressure
overload. A: TUNEL staining of WT and Lrrc10⫺/⫺ heart sections after TAC
(TUNEL, green; phalloidin, red; and nuclei, blue). Scale bars ⫽ 50 ␮m. B:
quantification of TUNEL-positive cardiomyocytes in sham- and TAC-operated
WT and Lrrc10⫺/⫺ hearts.
structure of LRRC10 (Fig. 5A). To determine if these amino
acids are critical for the interaction of LRRC10 with actin,
Tyr104, Trp127, or His150 were mutated to alanine, and in vitro
pulldown assays were performed with GST or a GST-LRRC10
fusion protein and purified ␣-actin (Fig. 5B). As expected, GST
fusion protein containing WT LRRC10 interacted with actin,
whereas GST alone did not. Mutation of Tyr104 or Trp127 did
not affect the interaction of LRRC10 with actin. In contrast,
mutation of His150 of LRRC10 abrogated its interaction with
actin (Fig. 5B), indicating that His150 of LRRC10 is essential
for actin binding. To test if His150 is important for the interaction of LRRC10 with actin in the heart, mouse heart extracts
were incubated and pulled down with GST, GST-LRRC10, or
GST-LRRC10H150A and then immunoblotted for ␣-actin (Fig.
5C). WT LRRC10 interacted with ␣-actin in the heart, as
previously reported (8), but mutation of His150 abolished the
interaction of LRRC10 with actin (Fig. 5C), indicating that
His150 of LRRC10 is a functional actin-binding site. Predictive
modeling of the structures of WT LRRC and mutant
LRRC10H150A illustrated a similar protein conformation (Fig.
5D), suggesting mutation of this histidine to alanine did not
change the overall conformation of LRRC10 but specifically
altered the actin-binding motif within LRRC10.
We next determined if the interaction of LRRC10 with actin
in the heart is altered in response to biomechanical stress. We
performed immunoprecipitation with an LRRC10 antibody on
cardiac lysates from sham or TAC-operated mice and immunoblotted for actin. The results demonstrated a reduced interaction of LRRC10 with actin in TAC hearts (Fig. 6A), indicating that binding of LRRC10 to actin is reduced during pressure
overload and that the association of LRRC10 with actin thin
filaments is dynamically regulated in response to mechanical stress. To determine if LRRC10 undergoes a substantial
change in subcellular localization after pressure overload,
immunohistochemistry was performed on cardiac sections
from sham or TAC mice. Intracellular localization of
LRRC10 appeared normal in TAC hearts compared with
ABSENCE OF LRRC10 EXACERBATES HYPERTENSIVE CARDIOMYOPATHY
H275
DISCUSSION
sham-operated hearts (Fig. 6B). Despite the reduced interaction of LRRC10 and ␣-actin, LRRC10 properly colocalized in a striated pattern with ␣-actin (Fig. 6B) as well as the
Z-disc protein ␣-actinin and the ryanodine receptor, a sarcoplasmic reticulum protein, in both control and pressureoverloaded hearts (data not shown). These data indicate that
LRRC10 remains at or near the dyad region under pressure
overload, maintaining its close proximity to the Z-disc,
sarcoplasmic reticulum, and T-tubule.
Myocyte contractility. To determine if the absence of
LRRC10 alters cardiomyocyte contractile function, single cell
contractility was evaluated in myocytes isolated from WT and
Lrrc10⫺/⫺ hearts. The impact of loss of LRRC10 on myocyte
contractile function was determined by measuring myocyte
shortening (FS) under field stimulation. Baseline FS was not
significantly different between myocytes from WT and
Lrrc10⫺/⫺ mice at all stimulation frequencies (0.5, 1, and 2 Hz;
Fig. 7, A and B). However, after perfusion with 10 nM Iso, FS
was significantly increased at all stimulation frequencies in WT
myocytes, whereas FS in ventricular myocytes from Lrrc10⫺/⫺
hearts did not increase in response to Iso (Fig. 7, A and B),
demonstrating that Lrrc10⫺/⫺ myocytes exhibit a defective
contractile response to ␤-adrenergic stimulation. Furthermore,
we analyzed the ratio of FS after and before Iso treatment and
found that this ratio was significantly reduced in Lrrc10 ⫺/⫺
myocytes compared with WT myocytes at 1 and 2 Hz (Fig.
7C), further highlighting the impaired contractile response of
Lrrc10⫺/⫺ myocytes in response to Iso. The time to peak
contraction and time of 50% relaxation were not altered in
Lrrc10⫺/⫺ and WT myocytes under basal conditions and with
Iso perfusion (Fig. 7, D and E).
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Fig. 6. Reduced interaction of LRRC10 with actin after pressure overload. A:
heart extracts from TAC- or sham-operated mice were immunoprecipitated
with an LRRC10 antibody and immunoblotted for ␣-actin, revealing a reduced
association of LRRC10 and ␣-actin after pressure overload. IP, immunoprecipitation. *IgG light chain. B: immunohistochemistry for LRRC10 (green)
and ␣-actin (red) was performed on cardiac sections of mice subjected to sham
or TAC surgery, indicating normal colocalization of LRRC10 with ␣-actin
near the Z-disc after pressure overload. 4=,6-Diamidino-2-phenylindole was
used as a nuclear stain in the overlays (blue). Scale bar ⫽ 10 ␮m.
Pressure overload occurs in response to cardiovascular diseases, including hypertension and LV outflow tract obstruction,
and results in concentric cardiac hypertrophy that, when prolonged, becomes maladaptive and can progress to congestive
heart failure (14, 27, 36, 37, 42). Pressure overload activates
mechanosensitive signals that increase myocardial mass via the
addition of sarcomeres in parallel, resulting in radial growth of
cardiomyocytes and increased LV wall thickness (26, 27, 37,
49). Here, we tested the hypothesis that LRRC10 is important
for the response of the heart to mechanical stress and pressure
overload hypertrophy.
LRRC10 is a cardiomyocyte-specific factor required for
proper cardiac function (1, 7, 8, 28), and mutations in the
LRRC10 gene have been recently linked to human dilated
cardiomyopathy (43). We demonstrated that Lrrc10⫺/⫺ mice
exhibit severe cardiac dysfunction in response to TAC and that
Lrrc10⫺/⫺ myocytes have reduced single cell contractility in
response to adrenergic stimulation, indicating that LRRC10 is
indispensable for the response of the mammalian heart to
biomechanical stress. We have previously shown that LRRC10
interacts with actin and ␣-actinin (8), positioning it in an ideal
location to sense or transduce signals in response to biomechanical stress. Embryonic hearts of Lrrc10⫺/⫺ mice upregulate a number of cytoskeletal genes critical for the response of
the heart to mechanical stress (3, 8, 35, 47), suggesting that the
absence of LRRC10 may render the heart sensitive to stretch or
strain. Here, we identified His150 of LRRC10 as necessary for
its interaction with actin and showed that this interaction is
reduced after pressure overload. Mechanistically, how the
interaction of LRRC10 with actin is reduced after TAC is
unclear. However, the maintenance of a similar immunolocalization pattern that colocalizes with ␣-actin, ␣-actinin, and the
sarcoplasmic reticulum indicates that LRRC10 remains within
the dyad region in close proximity to actin. It is possible that
posttranslational modifications or biomechanical signals in
response to pressure overload result in the displacement of
LRRC10 from actin thin filaments and/or association with
other interacting factors or protein complexes at the Z-disc,
costamere, sarcoplasmic reticulum, or T-tubule. Indeed, the
close proximity of structures within the dyad region (45) makes
it so that localization of LRRC10 at the Z-disc, T-tubule, or
dyadic cleft is indistinguishable by immunohistochemistry.
His150 of LRRC10 is located on its interior concave face (Fig.
5A). LRRCs commonly bind other interacting factors on this
interior face of the ␣/␤-horseshoe fold imparted by LRRs (31,
32), so posttranslational modifications and/or binding of other
factors to LRRC10 near His150 may be critical for its regulation. Future studies will investigate molecular defects in the
LRRC10 His150Ala mutant as well as LRRC10 mutations
recently identified in human dilated cardiomyopathy (43).
Nonetheless, LRRC10 may serve as a cardiomyocyte-specific scaffolding protein to tether important structural or signaling molecules to mediate appropriate responses to mechanical stress. Z-disc proteins such as muscle LIM protein (2, 29),
Cypher (56, 57), and telethonin (29, 30) are thought to compose a mechanosensory signalosome that allows the cardiomyocyte to internally sense and respond to mechanical stretch or
strain. Moreover, LRRC39, a striated muscle-specific LRRC,
H276
ABSENCE OF LRRC10 EXACERBATES HYPERTENSIVE CARDIOMYOPATHY
acts as a mechanosensor at the cardiomyocyte M-line by
regulating serum response factor-dependent transcription (51).
Importantly, Lrrc10⫺/⫺ mice develop severe dilated cardiomyopathy in response to 4 wk of mild pressure overload
stimulation. In our hands, performance of TAC to surgically
induce pressure overload resulted in a pressure gradient across
the aortic constriction of ⬍50 mmHg, which caused concentric
hypertrophy with preserved cardiac function in control mice
that did not progress to decompensation or present clinical
signs of heart failure. This is a relatively mild stimulus compared with other TAC models that achieve a greater aortic
pressure gradient and induce a robust hypertrophy response
and decompensation of ventricular performance in control
mice within 4 wk of pressure overload stimulation (15, 40, 48).
Therefore, our model of mild pressure overload serves as a
good pathophysiological model of chronic hypertension, a
condition that occurs in nearly one-third of Americans (18, 21).
Lrrc10⫺/⫺ mice exhibit severe cardiomyopathy and decompensated ventricular performance in response to mild pressure
overload stimulation, yet are still capable of undergoing appropriate concentric cardiac hypertrophy. This suggests that
LRRC10 is uniquely required to maintain functional performance but is not necessary for concentric cardiomyocyte
growth in response to pressure overload. Lrrc10⫺/⫺ hearts
become very dilated after TAC, indicative of eccentric growth,
but also undergo pressure overload-induced concentric LV
remodeling, with increased myocyte cross-sectional area and
increased H/R ratios. Therefore, Lrrc10⫺/⫺ hearts likely undergo a combination of eccentric and concentric remodeling in
response to TAC. We have previously reported that unstimulated adult Lrrc10⫺/⫺ cardiomyocytes are longer than WT
cardiomyocytes (8), suggesting eccentric growth by the addition of sarcomeres in series, consistent with ventricular dilation. Thus, pressure overload worsens cardiac function in the
absence of LRRC10, which may further increase neuroendocrine drive, providing a positive feedback loop that further
enhances cardiac growth and exacerbates cardiac dysfunction.
The fact that Lrrc10⫺/⫺ mice exhibit greatly diminished
cardiac function but a normal concentric hypertrophy response
to TAC suggests that cardiac contractility is specifically compromised in the absence of LRRC10. Here, we demonstrated
that Lrrc10⫺/⫺ mice display reduced cardiac function after
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Fig. 7. Reduced contractility in Lrrc10⫺/⫺ myocytes after
␤-adrenergic stimulation. A: representative myocyte shortening traces of WT and Lrrc10⫺/⫺ myocytes paced at
frequencies of 0.5, 1, and 2 Hz. B: quantification of myocyte
FS in WT and Lrrc10⫺/⫺ myocytes at baseline and in
response to 10 nM isoproterenol (Iso) treatment. C: ratio of
FS after Iso treatment relative to before Iso treatment.
Iso-to-baseline ratios were calculated from FS values measured from individual cardiomyocytes after and before Iso
perfusion, respectively. D and E: time to peak contraction
(D) and time of 50% relaxation (E) in Lrrc10⫺/⫺ and WT
myocytes under basal conditions and with Iso perfusion.
Data are means ⫾ SD; n ⫽ 18 myocytes from 5 WT mice
and 15 myocytes from 4 Lrrc10⫺/⫺ mice. *P ⬍ 0.05.
ABSENCE OF LRRC10 EXACERBATES HYPERTENSIVE CARDIOMYOPATHY
ACKNOWLEDGMENTS
Present address of M. J. Brody: Cincinnati Children’s Hospital, 240 Albert
Sabin Way, MLC7020, Cincinnati, OH, 45229.
Present address of L. Feng: Department of Cardiology, Beijing Anzhen
Hospital, Capital University of Medical Sciences, Beijing, China.
GRANTS
This work was supported by National Institutes of Health (NIH) Grant
HL-067050 and American Heart Association (AHA) Grant 12GRNT12070021
(to Y. Lee), AHA Predoctoral Fellowship 11PRE5580012 (to M. J. Brody),
and NIH Molecular and Environmental Toxicology Training Grant
T32ES007015 and Grant HL-105713 (to R. C. Balijepalli).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: M.J.B., A.C.G., and Y.L. conception and design of
research; M.J.B., L.F., A.C.G., and T.A.H. performed experiments; M.J.B. and
A.C.G. analyzed data; M.J.B., T.A.H., T.J.K., R.C.B., and Y.L. interpreted
results of experiments; M.J.B. prepared figures; M.J.B. and Y.L. drafted
manuscript; M.J.B., T.A.H., T.M.O., T.J.K., R.C.B., and Y.L. edited and
revised manuscript; M.J.B., L.F., T.A.H., T.M.O., T.J.K., R.C.B., and Y.L.
approved final version of manuscript.
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ABSENCE OF LRRC10 EXACERBATES HYPERTENSIVE CARDIOMYOPATHY