Download Low intrinsic exercise capacity in rats predisposes to - AJP

Am J Physiol Heart Circ Physiol 304: H729–H739, 2013.
First published December 21, 2012; doi:10.1152/ajpheart.00638.2012.
Low intrinsic exercise capacity in rats predisposes to age-dependent cardiac
remodeling independent of macrovascular function
Rebecca H. Ritchie,1* Chen Huei Leo,2 Chengxue Qin,1 Erin J. Stephenson,2 Marissa A. Bowden,1
Keith D. Buxton,1 Sarah J. Lessard,2 Donato A. Rivas,2 Lauren G. Koch,3 Steven L. Britton,3
John A. Hawley,2 and Owen L. Woodman2*
1
Baker IDI Heart and Diabetes Institute, Melbourne, Australia; 2The School of Medical Sciences, Royal Melbourne Institute
of Technology University, Bundoora, Australia; and 3Department of Anesthesiology, University of Michigan, Ann Arbor, Michigan
Submitted 27 August 2012; accepted in final form 17 December 2012
cardiomyocyte hypertrophy; cardiac fibrosis; insulin resistance;
EDHF; low-capacity runner; metabolic syndrome; resistance arteries
THE METABOLIC SYNDROME is a polygenic disorder that includes
obesity, insulin resistance, type 2 diabetes, dyslipidemia, hypertension, and impaired glycemic control. The prevalence of
this disorder is dramatically increasing and is strongly linked to
cardiovascular diseases (23). The presence of cardiovascular
risk factors that constitute the metabolic syndrome is correlated
with impairments in aerobic capacity and vascular endothelial
function, as well as increased heart failure risk, all of which are
*R. H. Ritchie and O. L. Woodman are co-senior authors on this work.
Address for reprint requests and other correspondence: J. A. Hawley,
Exercise Metabolism Research Group, The School of Medical Sciences, RMIT
Univ., Bundoora, VIC 3083, Australia (e-mail: [email protected]).
http://www.ajpheart.org
strong independent predictors of mortality (13, 30, 37, 48).
Furthermore, the myocardial and vascular abnormalities associated with the metabolic syndrome in general, and the associated impairments in insulin signaling, likely include alterations in myocardial structure (through cardiomyocyte hypertrophy, cardiac fibrosis, and/or impairments in myocardial
perfusion). These cardiac changes, in combination with induction of systemic endothelial dysfunction (potentially elevating
afterload), may well be contributing factors to later development of cardiac dysfunction and failure (48). How these
cardiovascular abnormalities progress in the context of a reduction in habitual physical activity, however, remain unresolved.
In 2001, Koch and Britton (20) developed a novel rodent model
based on differences in intrinsic exercise capacity. Rats were
selectively bred to yield divergent phenotypes with either low
(LCR) or high (HCR) intrinsic running capacity. When compared
with HCR, LCR rats have reduced whole body insulin sensitivity,
glucose tolerance, and hyperlipidemia, indicative of poor metabolic health (26, 43, 46, 49). Furthermore, LCR rats exhibit
modest but significantly higher systolic blood pressure (SBP)
compared with HCR, particularly in males (6, 49). While this may
be a result of concomitant impairments in endothelial nitric oxide
(NO)-dependent relaxation (49), endothelial function has yet to be
studied in the LCR microvasculature. Skeletal muscle impairments are also evident in LCR with regard to insulin sensitivity,
responsiveness to ␤-adrenoceptor activation, and levels of key
proteins for mitochondrial function (3, 20, 26). Modest but significant impairments in myocardial systolic and diastolic function
are observed in cardiomyocytes isolated from LCR compared
with HCR rats (49). Presumably, these differences are attributable
to abnormalities in myocardial mitochondrial protein content (3),
similar to those previously seen in skeletal muscle in LCR rats, in
addition to a relative myocardial downregulated expression of
genes controlling lipid versus glucose as an energy substrate (4).
Taken collectively, it is evident that there is a close association between impaired muscle metabolic function and low
intrinsic exercise capacity and that this relationship increases
cardiovascular risk. In the context of the LCR phenotype, it
remains to be resolved whether the myocardial and/or skeletal
muscle metabolic defects precede the impairment in exercise
capacity or vice versa. Whether these differences in both the
heart and vasculature between the LCR and HCR phenotypes
are evident at the onset of early adulthood or further develop
with age and the extent to which they differ from the nonselected founder rats from which the two selected lines were
derived have not been fully elucidated. In the present study, we
thus tested the hypotheses that the cardiovascular phenotype of
0363-6135/13 Copyright © 2013 the American Physiological Society
H729
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Ritchie RH, Leo CH, Qin C, Stephenson EJ, Bowden MA, Buxton
KD, Lessard SJ, Rivas DA, Koch LG, Britton SL, Hawley JA,
Woodman OL. Low intrinsic exercise capacity in rats predisposes to
age-dependent cardiac remodeling independent of macrovascular function. Am J Physiol Heart Circ Physiol 304: H729 –H739, 2013. First
published December 21, 2012; doi:10.1152/ajpheart.00638.2012.—Rats
selectively bred for low (LCR) or high (HCR) intrinsic running
capacity simultaneously present with contrasting risk factors for
cardiovascular and metabolic disease. However, the impact of these
phenotypes on left ventricular (LV) morphology and microvascular
function, and their progression with aging, remains unresolved. We
tested the hypothesis that the LCR phenotype induces progressive
age-dependent LV remodeling and impairments in microvascular
function, glucose utilization, and ␤-adrenergic responsiveness, compared with HCR. Hearts and vessels isolated from female LCR (n ⫽
22) or HCR (n ⫽ 26) were studied at 12 and 35 wk. Nonselected
N:NIH founder rats (11 wk) were also investigated (n ⫽ 12). LCR had
impaired glucose tolerance and elevated plasma insulin (but not
glucose) and body-mass at 12 wk compared with HCR, with early LV
remodeling. By 35 wk, LV prohypertrophic and glucose transporter
GLUT4 gene expression were up- and downregulated, respectively.
No differences in LV ␤-adrenoceptor expression or cAMP content
between phenotypes were observed. Macrovascular endothelial function was predominantly nitric oxide (NO)-mediated in both phenotypes and remained intact in LCR for both age-groups. In contrast,
mesenteric arteries microvascular endothelial function, which was
impaired in LCR rats regardless of age. At 35 wk, endothelial-derived
hyperpolarizing factor-mediated relaxation was impaired whereas the
NO contribution to relaxation is intact. Furthermore, there was reduced ␤2-adrenoceptor responsiveness in both aorta and mesenteric
LCR arteries. In conclusion, diminished intrinsic exercise capacity
impairs systemic glucose tolerance and is accompanied by progressive
development of LV remodeling. Impaired microvascular perfusion is
a likely contributing factor to the cardiac phenotype.
H730
LOW RUNNING CAPACITY AND PROGRESSIVE CARDIAC REMODELING
the young adult LCR rat is 1) impaired relative to both control
N:NIH and HCR animals and 2) worsens with aging. Regional
differences in vascular reactivity and the relative contribution
of endothelial-derived NO versus endothelial-derived hyperpolarizing factor (EDHF) to vasorelaxation were also investigated.
METHODS
Table 1. Systemic characteristics of HCR, control founder, and LCR rats at 12 and 35 wk of age
12-wk-Old Rats
n
Age, wk
Body weight, g
Heart weight, mg
Heart weight:body weight, mg/g
Fasting blood glucose, mM
Fasting plasma insulin, ng/ml
35-wk-Old Rats
HCR
Founders
LCR
HCR
LCR
12
12 ⫾ 0.1
168 ⫾ 4
621 ⫾ 16
3.71 ⫾ 0.07
6.48 ⫾ 0.16
0.39 ⫾ 0.01§
12
11 ⫾ 0.1
199 ⫾ 4***,†
720 ⫾ 57
3.60 ⫾ 0.25
6.34 ⫾ 0.12
0.41§§
12
12 ⫾ 0.1
214 ⫾ 4***
717 ⫾ 18
3.34 ⫾ 0.05
6.54 ⫾ 0.14
0.62 ⫾ 0.07§*
14
34.5 ⫾ 0.5
237 ⫾ 6
760 ⫾ 22
3.21 ⫾ 0.06
3.87 ⫾ 0.22
ND
10
34.5 ⫾ 1.0
268 ⫾ 5#
821 ⫾ 19
3.06 ⫾ 0.06
4.87 ⫾ 0.18##
ND
Values are means ⫾ SE. HCR, high capacity runner; LCR, low capacity runner; *P ⬍ 0.05 and ***P ⬍ 0.001 vs. HCR; †P ⬍ 0.05 vs. LCR in 12-wk-old
rats (one-way ANOVA; Dunnett’s multiple comparison) and #P ⬍ 0.05 and ##P ⬍ 0.005 vs. HCR in 35-wk-old rats (unpaired t-test). ND, not determined. §Data
for n ⫽ 7/group only. §§Samples from all rats combined to yield sufficient serum for analysis.
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Animal model. LCR and HCR rats were bred from genetically
heterogeneous N:NIH stock rats by artificial selection as previously
reported (20). The present study used female rats from generations 16,
20, and 22, as the body weight differences between phenotypes are
less marked in females than in males at the same age (16), and hence
body weight is less likely to represent a confounding influence to our
observations. Rats were phenotyped for intrinsic running capacity at
11 wk of age (26, 43, 49) and were studied in two age groups, young
(⬃12 wk) and mature adulthood (⬃35 wk). Conscious fasting glucose
tolerance tests were determined (26, 43, 49) before euthanasia. All
animal experimentation procedures were carried out with the approval
of animal ethics committees from Royal Melbourne Institute of
Technology University and the University of Michigan. The investigation conformed to the Guidelines for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH
Publication No. 85-23, Revised 1996). All reagents were purchased
from Sigma-Aldrich (St. Louis, MO) and dissolved in distilled water
except where indicated.
Tissue collection and histology. Following a 5-h fast, animals were
weighed, anesthetized with pentobarbital sodium (60 mg/kg), and
killed by exsanguination. Blood samples (⬃60 ␮l, via tail cut) were
collected for determination of glucose (One Touch glucometer,
Roche, Sydney, NSW, Australia) and plasma insulin (Rat insulin
ELISA, ALPCO Immunoassays, No. 80-INSRT-E01). Hearts were
removed and rinsed in ice-cold Krebs buffer, consisting of (in mM)
118 NaCl, 4.7 KCl, 1.18 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 11.1
D-glucose, and 1.6 CaCl2, and lightly blotted and wet weight determined. Hearts were cut at the horizontal short-axis plane, and the
middle portion of the left ventricle was fixed in 10% neutral buffered
formalin (Australian Biostain, Melbourne, Australia) and paraffinembedded for histological analysis of 4-␮m cross sections via hematoxylin-eosin and Sirius red staining (17, 18, 41). Aorta and mesenteric arteries were also promptly removed on euthanasia and immediately placed in ice-cold Krebs containing indomethacin (10 ␮M,
dissolved in 0.1 M sodium carbonate), a nonselective cyclooxygenase
(COX) inhibitor, to limit prostanoid synthesis. Superoxide generation
was quantified using lucigenin (5 ␮M)-enhanced chemiluminescence
in fresh left ventricular (LV) and aortic tissues (17, 35, 41, 42).
Results were normalized to tissue weight and expressed as counts per
milligram of tissue. Remaining basal and apical portions of the
ventricle were snap frozen in liquid nitrogen and stored at ⫺80°C for
biochemical analysis.
Assessment of cardiac remodeling. Cardiomyocyte hypertrophy
was determined using cell width from hematoxylin-eosin sections and
on ␤-myosin heavy chain expression; cardiac fibrosis was assessed on
collagen deposition in Sirius red-stained sections (17, 18, 41). Cardiac
gene expression of glucose transporters (GLUT1, GLUT4) and ␤1adrenoceptors (via real-time PCR), as well as cardiac content of the
␤-adrenoceptor second messenger cAMP [via enzyme immunoassay,
Cayman Chemical, Ann Arbor, MI (28)], were also determined in
addition to markers of cardiac remodeling. RNA was extracted from
frozen ventricle as previously described (42). DNase-treated RNA
was reverse transcribed (Taqman Reverse Transcription reagent, Applied Biosystems, Mulgrave, VIC, Australia) using the Gene-Amp
PCR system 9700 (Applied Biosystems) to produce cDNA template at
20 ng/␮l (42, 44). Gene expression was quantitated using SYBR
Green chemistry with the FAST 7500 v. 2.0.5 sequence detection
system with ribosomal 18s as the endogenous control (Applied Biosystems). All primers were generated from rat-specific sequences
published on GenBank at previously determined optimal concentrations (24, 44). The comparative ⌬-⌬ cycle threshold method was used
to analyze changes in gene expression as a relative fold change to
HCR rats (42).
Vascular function experiments. Aortae and third-order branches of
rat mesenteric artery (internal diameter, ⬃200 –300 ␮m) were cleared
of fat and connective tissue and cut into ⬃2 mm-long ring segments.
Aortic rings were mounted in standard organ baths and allowed to
equilibrate for 60 min at a resting tension of 1 g. Mesenteric artery
rings were mounted in a Mulvany-style small vessel myograph (Danish Myo Technology, Aarhus, Denmark) and allowed to stabilize at
zero tension for 15 min. After stabilization, the mesenteric arteries
were normalized to 90 –100 mmHg and all vascular reactivity experiments were performed at 37°C and bubbled with carbogen (95% O2
and 5% CO2) as previously described (25, 31, 36). Briefly, after
maximum contraction and assessment of endothelial integrity were
established, arteries were precontracted to ⬃50% of maximum with
phenylephrine (0.1–3 ␮M) and cumulative concentration-response
curves to acetylcholine (ACh, 0.1 nM–10 ␮M, BDH Chemicals,
Poole, Dorset, UK) and sodium nitroprusside (SNP, 0.01 nM–10 ␮M)
obtained in vessels from both age groups were studied.
The contribution of endothelial-derived NO to ACh responses was
examined after 20 min incubation with NG-nitro-L-arginine [L-NNA,
100 ␮M, nonselective NO synthase (NOS) inhibitor] and 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 ␮M, soluble guanylate
cyclase inhibitor, Cayman Chemical). The role of EDHF in ACh
responses was examined using the combination of 1-[(2-chlorophenyl)(diphenyl)methyl]-1H-pyrazole (TRAM-34, 1 ␮M, selective
blocker of intermediate-conductance Ca2⫹-activated K⫹ channels)
and apamin (1 ␮M, inhibitor of small-conductance Ca2⫹-activated
K⫹ channels). Stock solutions of both ODQ and TRAM-34 were
prepared in dimethyl sulfoxide before dilution in Krebs buffer. Cumulative concentration-response curves to the selective ␤2-adrenocep-
H731
Blood [glucose], mM
20
***
HCR
LCR
15
**
10
5
0
0
30
90
60
AUC for blood [glucose], AU
LOW RUNNING CAPACITY AND PROGRESSIVE CARDIAC REMODELING
1500
**
1000
500
0
HCR
120
LCR
Time (mins) post glucose i.p.
RESULTS
Systemic characteristics. The systemic characteristics of the
three cohorts under investigation are shown in Table 1. At ⬃12
wk of age, LCR rats are similar in body mass and heart mass to
founder animals. HCR rats were significantly smaller than either
LCR or founders, and this difference was maintained at 35 wk of
age. No significant differences in heart mass or heart mass index
were observed. Elevated fasting plasma insulin (but not blood
glucose) and impaired glucose tolerance were evident in the
cohort of young LCR compared with HCR rats (Fig. 1), indicative
of some degree of systemic insulin resistance. By 35 wk of age,
blood glucose levels in LCR rats were significantly elevated
compared with their HCR counterparts.
LCR is associated with progressive cardiac structural
remodeling. At ⬃12 wk of age, LCR rats exhibit cardiomyocyte
hypertrophy in the intact heart relative to age-matched HCR
counterparts (Fig. 2). Cardiomyocyte size increased with age to
a similar extent in both LCR and HCR rats, such that the
relative increase in cardiomyocyte width for the LCR rat was
similar at 12 and 35 wk (Fig. 2B, right). Furthermore, on
two-way ANOVA, both age and phenotype were associated
with significant differences in cardiomyocyte size (both, P ⬍
0.0001), although the interaction was not significant. Hyper-
A
12 wks old
Founders
B
LCR
20.0
**
17.5
**
15.0
12.5
10.0
HCR
Founders
LCR
35 wks old
cardiomyocyte width ( µ m)
cardiomyocyte width ( µ m)
12 wks old
HCR
LCR
20.0
LCR
**
17.5
15.0
12.5
10.0
HCR
LCR
∆ cardiomyocyte width ( µ m)
HCR
35 wks old
2.5
2.0
1.5
1.0
0.5
0.0
12 wks
35 wks
Fig. 2. LCR exhibit cardiomyocyte hypertrophy. Increased cardiomyocyte width is evident on histological analysis of left ventricular (LV) cross sections stained
with hematoxylin-eosin in the intact LCR heart relative to HCR rats at both 12 and 35 wk of age. A: representative sections (hematoxylin-eosin stain;
magnification, ⫻400). Scale bars show 20 ␮m. B: cardiomyocyte width pooled data. The LCR-induced increase in cardiomyocyte size (⌬cardiomyocyte size
relative to age-matched HCR) is comparable at both ages (n ⫽ 9 –14 per group). **P ⬍ 0.01 vs. age-matched HCR rats (on one-way ANOVA at 12 wk and
unpaired t-test at 35 wk of age).
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tor agonist fenoterol (1 nM–100 ␮M) were constructed in precontracted mesenteric artery and aorta as described above.
Data and statistical analyses. All results are expressed as means ⫾
SE, where n represents the number of animals per group. The area
under the curve for glucose tolerance was calculated using Prism
version 5.0 (GraphPad Software, San Diego, CA). Concentrationresponse curves from rat isolated arteries were computer fitted to a
sigmoidal curve using nonlinear regression (Prism version 5.0) to
calculate the sensitivity of each agonist (EC50). Maximum relaxation
(Rmax) to ACh or SNP was measured as a percentage of precontraction to phenylephrine. Group data for pEC50, Rmax, glucose tolerance
area under the curve, cardiac histology, gene expression, and cAMP
were compared via one-way ANOVA (with post hoc analysis using
Dunnett’s test) or Student’s unpaired t-test, as appropriate. Two-way
ANOVA was also used to compare differences in cardiomyocyte size,
cardiac collagen deposition, and microvascular sensitivity to ACh
across phenotype and age. P ⬍ 0.05 was considered statistically
significant.
Fig. 1. Low intrinsic running capacity (LCR) exhibit impaired systemic glucose tolerance. Both the
time course of glucose utilization and the area
under the curve (AUC) for this time course were
significantly impaired in female LCR rats relative
to aged-matched high intrinsic running capacity
(HCR) rats at 12 wk of age, following a glucose
load (2 g/kg ip) in fasted animals (n ⫽ 7 per group).
AU, arbitrary units. ***P ⬍ 0.001 and **P ⬍ 0.01,
LCR vs. HCR (at the same time point on two-way
ANOVA or on AUC on unpaired t-test).
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LOW RUNNING CAPACITY AND PROGRESSIVE CARDIAC REMODELING
LVβ–myosin heavy chain
35 wks old
2
1
0
HCR
Founders
LCR
LCR
**
3
2
1
0
HCR
LCR
∆LVβ-myosin heavy chain:18S
expression (AU)
3
LVβ-myosin heavy chain:18S
expression (fold HCR)
LVβ-myosin heavy chain:18S
expression (fold HCR)
12 wks old
**
2.5
2.0
1.5
1.0
0.5
0.0
12 wks
35 wks
trophic gene expression progressively increased with age in
LCR rats relative to their HCR counterparts. At 12 wk, ␤-myosin heavy chain expression in LCR rats was not significantly
different to HCR or founder control rats (P ⫽ 0.1, Fig. 3, left).
By 35 wk, ␤-myosin heavy chain expression in LCR rats was
markedly elevated compared with age-matched HCR rats (P ⬍
0.005, Fig. 3). Cardiac collagen deposition was elevated in
LCR compared with HCR rats, at both ages studied. At 12 wk
of age, cardiac collagen deposition was 2.7 ⫾ 0.2-fold in LCR
relative to age-matched HCR rats, with some suggestion of
age-dependent progression of cardiac fibrosis (Fig. 4). On
two-way ANOVA, however, only phenotype (and not age)
tended to remain associated with differences in cardiac collagen deposition (P ⫽ 0.07). Despite this clear cardiac remodeling evident in LCR rats, no phenotype differences in cardiac
generation of superoxide were seen (Table 2).
Cardiac GLUT4 is downregulated with inherent LCR. LCR
rats exhibit a degree of systemic insulin resistance (Table 1 and
Fig. 1). There was no impact of the LCR phenotype on GLUT4
expression at 12 wk (Fig. 5A, left), but GLUT4 was significantly downregulated in the LCR myocardium by 35 wk (Fig.
5A, middle and right). In contrast, the LCR phenotype does not
impact on cardiac GLUT1 expression at either 12 or 35 wk
(Fig. 5B). GLUT1 and GLUT4 expression in founders mimicked that seen in the HCR phenotype.
LCR selectively impairs endothelial function in resistance
vessels. For both selected lines and age-groups studied, ACh
elicited concentration-dependent vasorelaxation in isolated
aorta, a large conduit vessel, and in mesenteric artery, a smaller
resistance vessel (Fig. 6). Vascular sensitivity to ACh was
significantly impaired in LCR mesenteric arteries, at 12 and 35
wk (Fig. 6A), compared with HCR arteries. Furthermore, on
A
12 wks old
HCR
35 wks old
Founders
15
***
***
10
5
0
HCR
Founders
LCR
35 wks old
LCR
Cardiac collagen content (AU)
Cardiac collagen content (AU)
12 wks old
HCR
LCR
**
15
10
5
0
HCR
LCR
Cardiac collagen content (AU)
B
LCR
*
15
10
5
0
12 wks
35 wks
Fig. 4. LCR exhibit cardiac fibrosis. Increased cardiac collagen deposition is evident on histological analysis of ventricular cross sections stained with Sirius red
in the intact LCR heart relative to HCR rats at both 12 and 35 wk of age. A: representative sections (magnification, ⫻200). Scale bars show 40 ␮m. B: quantitation
of collagen deposition pooled data. The LCR-induced increase in cardiac collagen deposition is evident at both ages but was further increased in LCR at 35 wk
of age (n ⫽ 8 –14 per group). ***P ⬍ 0.001, **P ⬍ 0.01, and *P ⬍ 0.05 (on one-way ANOVA at 12 wk and unpaired t-test at 35 wk of age).
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Fig. 3. Hypertrophic gene expression in LCR rats progressively increases with age. LV ␤-myosin heavy chain expression relative to the housekeeping gene 18S
in either LCR or control founder rats relative to age-matched HCR rats was not significantly different at 12 wk of age (left), but LCR upregulated ␤-myosin heavy
chain expression at 35 wk of age (middle). The relative increase in LCR above age-matched HCRs is shown (right) (n ⫽ 5–12 per group). **P ⬍ 0.005 vs
age-matched HCR rats (unpaired t-test).
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LOW RUNNING CAPACITY AND PROGRESSIVE CARDIAC REMODELING
Table 2. Impact of low vs. high inherent running capacity
on cardiac ␤-adrenergic signaling and superoxide
generation in 35-wk-old rats
LV ␤1-adrenoceptor expression, AU
LV cAMP content, pmol/ml
LV superoxide, counts/mg tissue
Aortic superoxide, counts/mg tissue
HCR (n)
LCR (n)
0.86 ⫾ 0.1 (7)
42.6 ⫾ 3.1 (8)
530 ⫾ 70 (15)
1,360 ⫾ 220 (17)
1.23 ⫾ 0.3 (6)
36.4 ⫾ 1.6 (9)
540 ⫾ 80 (11)
1,380 ⫾ 150 (14)
Values are means ⫾ SE. LV, left ventricular; AU, arbitrary units.
DISCUSSION
Much of the recent evidence suggesting that low intrinsic
aerobic capacity is associated with increased risk factors for
cardiovascular disease has emerged from a novel rodent model
of inherited LCR and impaired metabolic health. Using this
model, we now provide new information regarding the impact
A: LV GLUT4
35 wks old
1.0
0.5
0.0
HCR
Founders
1.5
LCR
0.00
*
∆LV GLUT4:18S
expression (AU)
LV GLUT4:18S
expression (fold HCR)
LV GLUT4:18S
expression (fold HCR)
12 wks old
1.5
1.0
0.5
0.0
LCR
-0.25
-0.50
-0.75
HCR
LCR
12 wks
**
35 wks
B: LV GLUT1
35 wks old
1.0
0.5
0.0
HCR
Founders
LCR
LCR
1.5
0.50
∆LV GLUT1:18S
expression (AU)
1.5
LV GLUT1:18S
expression (fold HCR)
LV GLUT1:18S
expression (fold HCR)
12 wks old
1.0
0.5
0.0
0.25
0.00
-0.25
-0.50
HCR
LCR
12 wks
35 wks
Fig. 5. Cardiac glucose transporter GLUT4 gene expression in LCR rats progressively increases with age. A: LV GLUT4 expression relative to the housekeeping
gene 18S in LCR relative to age-matched HCR or control founder rats was not significantly different at 12 wk of age (left), but LCR upregulated GLUT4
expression at 35 wk of age (middle). The relative increase in LCR above age-matched HCRs is shown (right). B: LV GLUT1 expression relative to the
housekeeping gene 18S in LCR relative to age-matched HCR or control founder rats was not significantly different at either 12 wk (left) or 35 wk of age (middle).
The relative increase in LCR above age-matched HCRs is shown (right) (n ⫽ 8 –12 per group). *P ⬍0.05 and **P ⬍0.005 vs. age-matched HCR rats (unpaired
t-test).
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two-way ANOVA, phenotype remained associated with significant differences in sensitivity to ACh (P ⬍ 0.0005); a similar
but nonsignificant trend for age to influence sensitivity to ACh
was also seen (P ⫽ 0.09). In contrast, endothelial function was
intact in LCR aorta (Fig. 6B), and superoxide generation was
not different (Table 2). No differences in maximal relaxation
elicited by ACh in either vessel were observed. No phenotype
differences were evident on responses to the endotheliumindependent vasorelaxant SNP in either vessel regardless of
age (n ⫽ 3, P ⫽ 0.087, Fig. 7).
EDHF is impaired in LCR resistance vessels. The relative roles
of endothelium-derived NO and EDHF to endothelium-dependent
vasorelaxation were determined in rat isolated aorta and mesenteric artery at 35 wk (Fig. 8). In both HCR (Fig. 8A) and LCR
(Fig. 8B) mesenteric arteries, the presence of L-NNA ⫹ ODQ did
not significantly affect either the sensitivity (pEC50, Fig. 8C) or
Rmax (Fig. 8D) to ACh. Furthermore, the impairment in microvascular endothelial function evident in LCR rats persisted in the
presence of L-NNA ⫹ ODQ, indicating that EDHF-type relax-
ation is impaired in LCR mesenteric arteries. In contrast, blockade
of Ca2⫹-activated K⫹ channels with TRAM-34 ⫹ apamin markedly shifted the concentration-response curve to ACh rightward in
both HCR (Fig. 8A) and LCR (Fig. 8B) mesenteric arteries,
revealing a significant contribution of EDHF in resistance vessels
of both phenotypes. As also shown in Fig. 8C, mesenteric sensitivity to ACh in the presence of TRAM-34 ⫹ apamin is similar in
HCR and LCR, indicating that NO makes a similar contribution to
mesenteric relaxation in the two phenotypes. As shown in Fig. 8,
E and F, in direct contrast to results obtained in the microvasculature, concentration-response curves to ACh in aortic rings from
both HCR and LCR rats at 35 wk of age are abolished by L-NNA,
implicating a predominant role for NO in macrovascular function
regardless of phenotype.
␤-Adrenergic signaling is impaired in LCR vasculature. The
selective ␤2-adrenoceptor agonist fenoterol elicited concentration-dependent vasorelaxation in both rat isolated mesenteric
artery (Fig. 9A) and aorta (Fig. 9B) at 12 wk. The sensitivity
(pEC50) to fenoterol, however, was significantly impaired in
both micro- and macrovasculature in LCR animals. In contrast,
no differences in either expression of the predominant cardiac
subtype ␤1-adrenoceptors or content of its second messenger
cAMP were observed (Table 2).
H734
LOW RUNNING CAPACITY AND PROGRESSIVE CARDIAC REMODELING
A: Mesenteric artery
12 w ks old
12 w ks old
9
*
50
75
7
HCR
LCR
Founders
-10
-9
*
8
0
Relaxation (%)
pEC50
25
100
-7
-6
HCR
-5
50
Founders LCR
8
7
75
HCR
LCR
100
6
-8
*
25
-10
-9
Log [ACh] M
6
-8
-7
-6
HCR
-5
LCR
Log [ACh] M
35 wks old
12 w ks old
35 wks old
12 w ks old
9
9
0
50
8
7
75
100
-9
-8
-7
-6
-5
50
HCR
Founders LCR
Log [ACh] M
8
7
75
LCR
HCR
100
6
-10
25
pEC50
pEC50
25
Relaxation (%)
0
-10
-9
6
-8
-7
-6
-5
HCR
LCR
Log [ACh] M
Fig. 6. Endothelial function is impaired in LCR rat mesenteric arteries. Concentration-response curves and sensitivity (pEC50) to the endothelium-dependent
dilator ACh in both mesenteric (A) and aortic (B) rings from HCR, LCR, and control founder rats at either at 12 wk of age (left) or 35 wk of age (right).
Endothelial function is selectively impaired in mesenteric arteries (but not aorta) in LCR relative to HCR, regardless of age (n ⫽ 4 –12 per group). *P ⬍ 0.05.
of these phenotypes on LV morphology and microvascular
function and how this is affected with age in young adult
females. Our key findings provide evidence that impaired
intrinsic exercise capacity is accompanied by early LV remodeling (evident at 12 wk of age, Figs. 2 and 4) with concomitant
early microvascular endothelial dysfunction (Figs. 6A and 8).
This cardiovascular phenotype was associated with impaired
glucose tolerance and elevated plasma insulin and body weight
but precedes elevation in blood glucose (Table 1 and Fig. 1).
Subsequent progression to upregulated LV hypertrophic gene
expression and downregulated myocardial GLUT4 expression
was evident with age (Figs. 3 and 5). Macrovascular function,
however, remained intact (Fig. 6B). Although microvascular
␤2-adrenoceptor responsiveness was reduced in LCR rats (Fig.
9), no phenotypic differences in LV ␤-adrenoceptor expression
or activity (as estimated on LV cAMP content) were apparent
(Table 2). These findings raise the possibility that impaired
microvascular perfusion is a contributing factor to the cardiac
phenotype associated with an inherited low capacity for exercise.
Previous proteomic and array studies (3, 4) suggested that
LCR may exhibit a predisposition for LV remodeling, but
those studies focused on older animals (30 –50 wk of age).
Confirmation of morphological changes specifically on LV
histology, protein content, or gene expression using real-time
PCR or other semiquantitative approaches was not available.
Recent work has now suggested that more mature LCR rats
(⬃30 wk of age) exhibit upregulated cardiac collagen deposition (6). Following enzymatic isolation, cardiomyocytes from
older LCR also tend to be shorter and wider than those from
their HCR counterparts (4, 19). Here we demonstrate that the
onset of cardiac fibrosis in the LCR phenotype occurs considerably earlier and is evident already by 12 wk. In addition, we
demonstrate that concomitant cardiomyocyte hypertrophy is
not an artefact of enzymatic isolation but is manifest in the
heart in situ (Fig. 2). Interestingly, increased LV cardiomyocyte width is also manifest at the same early time point as
collagen deposition (Fig. 4). Furthermore, the temporal upregulation of both hypertrophic gene expression (Fig. 3) and
LV collagen content (Fig. 4) suggests that cardiac remodeling
in the LCR continues to progress with age. The LV remodeling
evident in even relatively young LCR rats (at 12 wk of age) is
associated with significant cardiac fibrosis (Figs. 2 and 4), with
subsequent alterations at the level of hypertrophic and GLUT4
gene expression evident by 35 wk of age (Fig. 3). Thus the
cardiomyocyte hypertrophy observed in this phenotype more
closely resembles a pathological (as opposed to a physiological) cardiac growth response (31), even at 12 wk of age. This
is also in agreement with the loss of myocardial transverse T
tubules (19, 21) and blunted cardiomyocyte contractile function (15) evident in older LCR rats, as well as the cardiac
phenotype of rodent models of insulin resistance/type 2 diabetes (15, 39, 42).
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B: Aorta
Relaxation (%)
35 wks old
pEC50
0
Relaxation (%)
35 wks old
9
H735
LOW RUNNING CAPACITY AND PROGRESSIVE CARDIAC REMODELING
A: Mesenteric artery
12 w ks old
10
0
9.0
50
75
8
Founders
HCR
LCR
100
-11
-10
-9
9
7
-8
-7
-6
50
8.0
75
7.5
100
7.0
-11
HCR Founders LCR
-5
8.5
25
pEC50
25
Relaxation (%)
0
pEC50
Relaxation (%)
35 wks old
35 wks old
12 w ks old
-10
-9
-8
-7
-6
HCR
-5
LCR
Log [SNP] M
Log [SNP] M
12 w ks old
35 wks old
9.0
0
9.0
8.5
25
50
75
8.0
7.5
100
7.0
-11
-10
-9
-8
-7
-6
-5
8.5
25
pEC 50
Relaxation (%)
0
pEC50
Relaxation (%)
35 wks old
12 w ks old
50
75
7.5
100
7.0
125
HCR
Founders LCR
8.0
-11
-10
-9
-8
-7
-6
-5
HCR
LCR
Log [SNP] M
Log [SNP] M
Fig. 7. Vascular nitric oxide (NO) responsiveness is intact in LCR rat arteries. Concentration-response curves and sensitivity (pEC50) to the endotheliumindependent dilator sodium nitroprusside (SNP) in both mesenteric (A) and aortic (B) rings from HCR, LCR, and control founder rats at either at 12 wk of age
(left) or 35 wk of age (right) (n ⫽ 3– 6 per group).
There is strong evidence that maintaining an increased level
of physical activity is a key intervention favoring health benefits and longevity in patients with insulin resistance and
diabetes (5, 14). The current data suggest that a low capacity
for aerobic exercise can be inherited (20), and that this inherited trait per se represents a setting of metabolic syndrome
(26). Insulin resistance is an antecedent of type 2 diabetes, and
both scenarios are associated with cardiac upregulation of
reactive oxygen species (ROS) generation and systemic oxidative stress (17, 33, 35, 39, 42). Given that superoxide is a key
trigger of both cardiomyocyte hypertrophy (24, 40, 41) and
impaired vasoreactivity (9), its upregulation is widely regarded
as a contributing factor to cardiovascular abnormalities in
insulin resistance (9, 12, 22, 34, 39). In the present study,
impaired systemic glucose tolerance and hyperinsulinemia
were already evident at 12 wk of age in LCR rats, relative to
their HCR counterparts (Table 1 and Fig. 1), but sensitivity to
insulin was not specifically investigated. Insulin resistance is
clearly evident in LCR rats by 30 wk of age (26). Surprisingly,
LV and vascular superoxide generation were not different
between LCR and HCR rats at 35 wk of age (Table 2),
suggesting that the cardiovascular phenotype evident in mature
LCR may not be a downstream consequence of ROS upregulation. We are unable to preclude a transient increase in LV and
vascular superoxide generation at an earlier age as a potential
trigger of early remodeling, as this was not determined at 12
wk of age in the present study. At the level of endogenous
cardiovascular antioxidant enzyme activity, the counterbalance
to ROS generation, is similarly lacking from current knowledge, although trends for reduced antioxidant enzyme activity
have been reported in LCR skeletal muscle (47).
In the present study, macrovascular function (i.e., in large
conduit vessels) was determined ex vivo in rat isolated aortae.
Contrary to a previous report from carotid arteries, a moderatesized conduit vessel (49), we did not observe any differences in
endothelium-dependent (i.e., in response to ACh, Fig. 6B) or
-independent vasorelaxation (i.e., in response to SNP, Fig. 7B)
in aortae isolated from LCR rats relative to HCR in either age
group. We were thus unable to attribute the changes in cardiac
remodeling to macrovascular abnormalities. Given that these
studies were performed in the presence of the COX inhibitor
indomethacin, the abolition of ACh vasodilatation responses
by the nonselective inhibition of NOS indicates that NO is the
sole endothelium-derived macrovascular vasorelaxant mediator in both phenotypes (29).
This study provides the first insights into endothelial function in smaller LCR arteries. In direct contrast to the macrovasculature, microvascular endothelial function (determined ex
vivo in third-order mesenteric arteries) was significantly impaired in LCR rats at both ages studied (Fig. 6A) without
impact on endothelium-independent vasorelaxation (Fig. 7A).
NO and EDHF both contribute to vascular tone in vivo (1, 7,
25, 29, 50); our observations that the contribution of EDHF
was greater in the rat microvasculature than the macrovasculature ex vivo (Fig. 8) are consistent with the mouse mesenteric
microvasculature in vivo (50). Our studies revealed a signifi-
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B: Aorta
LOW RUNNING CAPACITY AND PROGRESSIVE CARDIAC REMODELING
B:
HCR Mesenteric ar ter y
0
50
Control
75
TRAM-34+apamin
100
-9
-8
Control
L-NAME+ODQ
75
TRAM-34+apamin
100
L-NAME+ODQ+
TRAM-34+apamin
-10
50
-7
-6
L-NAME+ODQ+
TRAM-34+apamin
-5
-10
-9
Log [ACh] M
*
*
*
Rm ax to ACh vasorelaxation
6
*
E:
D:
Mesenter ic sensitivity
HCR
LCR
*
pEC50 to ACh vasorelaxation
C:
7
8
9
Control
L-NNA
+ODQ
TRAM -34 L-NNA+ODQ+
+apamin TRAM -34+apamin
-7
-6
-5
Mesenter ic maximal relaxation
*
100
*
80
60
40
20
0
Control
F:
HCR aor ta
L-NNA
+ODQ
TRAM -34 L-NNA+ODQ+
+apamin TRAM -34+apamin
LCR aorta
0
*
25
50
75
Control
L-NNA
100
-10
-9
Relaxation (%)
0
Relaxation (%)
-8
Log [ACh] M
25
50
75
Control
L-NNA
100
-8
-7
-6
-5
-10
Log [ACh] M
cant contribution of EDHF to endothelium-dependent vasorelaxation in these smaller vessels (considerably greater than that
attributable to NO), as indicated by the significantly greater
sensitivity to inhibition of Ca2⫹-activated K⫹ channels [with
TRAM-34 ⫹ apamin (29)] than to inhibition of NO/cGMP
signaling (with L-NNA ⫹ ODQ, Fig. 8). This greater reliance
on EDHF over NO was observed in both HCR and LCR
mesenteric arteries. Indeed, the role of endothelial-derived NO
in smaller resistance vessels is revealed once the EDHF component of ACh vasodilatation is removed (by the combination
of TRAM-34 ⫹ apamin)—the residual vasodilatation response
is attributed to endothelial-derived NO, as we can confirm by
the combined addition of L-NNA ⫹ ODQ and TRAM-34 ⫹
apamin (Fig. 8C), confirming that endothelial-derived NO and
EDHF account for all of the microvascular sensitivity to ACh
in both phenotypes studied. Furthermore, the sensitivity to
ACh in mesenteric arteries in the face of combined NOS, COX,
and cGMP inhibition was significantly greater in microvessels
-9
-8
-7
-6
-5
Log [ACh] M
from HCR to that seen from LCR, indicative of reduced EDHF
responses in LCR microvasculature. NO, particularly that derived from the endothelium, is a negative regulator of cardiomyocyte hypertrophy [for review, see Ritchie et al. (40)], in
addition to its well-known role as a critical regulator of
vascular tone (10). Although differences in NOS expression
were not determined in the present study, our findings indicate
that there were no phenotype differences in vascular NOS
activity. Rather, these data suggest that impairment at the level
of EDHF-type relaxation mediates microvascular dysfunction
in LCR rats, which is in accordance with settings of type 2
diabetes, where EDHF-dependent responses are similarly impaired in resistance arteries (2, 51).
An increase in afterload as a result of hypertension is an
important driver of LV hypertrophy (40). Based on previous
evidence of only a modest level of hypertension in female LCR
rats, SBP was not measured in the present study. Differences in
SBP female LCR relative to HCR rats range from negligible
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Fig. 8. Endothelium-derived hyperpolarizing
factor (EDHF) is impaired in LCR resistance
vessels. Concentration-response curves to the
endothelium-dependent vasodilator ACh in
mesenteric rings from both HCR (A) and LCR
(B) rats at 35 wk of age are unaffected by
NG-nitro-L-arginine (L-NNA) ⫹ 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; inhibitors of NO synthase and soluble guanylyl
cyclase) but are significantly impaired by 1-[(2chlorophenyl)(diphenyl)methyl]-1H-pyrazole
(TRAM-34) ⫹ apamin (Ca2⫹-activated K⫹
channel inhibitors), implicating a role for EDHF
in microvascular function (n ⫽ 4 per group).
C: mesenteric sensitivity to ACh (pEC50). D: maximal relaxation to ACh (Rmax). Concentrationresponse curves to ACh in aortic rings from both
HCR (E) and LCR (F) rats at 35 wk of age are
abolished by L-NNA, implicating a predominant role for NO in macrovascular function,
regardless of phenotype (n ⫽ 3–11 per group).
*P ⬍ 0.05.
*
*
L-NAME+ODQ
25
*
25
Relaxation (%)
Relaxation (%)
0
LCR Mesenteric arter y
*
A:
*
H736
LOW RUNNING CAPACITY AND PROGRESSIVE CARDIAC REMODELING
A: Mesenteric artery
25
pEC50
Relaxation (%)
*
6
0
50
75
-9
-8
**
7
8
HCR
LCR
Founders
100
9
-7
-6
-5
HCR
-4
Founders
LCR
Log [Fenoterol] M
B: Aorta
NS
pEC50
25
50
**
Fig. 9. ␤-Adrenergic signaling is impaired in
LCR vasculature. Concentration-response
curves to the ␤2-adrenoceptor agonist fenoterol
in mesenteric (A) and aortic (B) rings from HCR,
LCR, and control founder rats at 12 wk of age
(n ⫽ 7–11 per group). *P ⬍ 0.05 and **P ⬍
0.01 (one-way ANOVA). NS, not significant.
7
8
75
100
9
-9
-8
-7
-6
-5
-4
HCR
Founders
LCR
Log [Fenoterol] M
(6) to about 10 mmHg (49) at 24 –30 wk of age, considerably
older that the young adult females studied here, unlikely to be
a sufficiently large enough stimulus to drive cardiac remodeling. For comparison, male spontaneously hypertensive rats
exhibit an ⬃40 mmHg greater SBP than their normotensive
counterparts even at 12 wk of age (8); such a substantial
pressure gradient in contrast is clearly sufficient to drive a
cardiac hypertrophic response (45). As both phenotypes age,
this difference becomes more marked but still does not exceed
⬃20 mmHg, as shown in female LCR relative to female HCR
rats at both 2 yr of age (21). Together, these findings suggest
that only modest effects on SBP are evident in LCR and that
these emerge later in life and may be more pronounced in
males than females.
Although macrovascular endothelial function was intact in
LCR rats (Fig. 6), we demonstrate that ␤-adrenergic responsiveness is impaired in LCR aorta and mesenteric artery at 12
wk of age (Fig. 9). This finding is consistent with previous
observations in skeletal muscle (26, 27). In contrast, no phenotypic differences in mesenteric responses to isoproterenol
were observed (results not shown). Despite comparable net
microvascular ␤-adrenergic responsiveness in LCR and HCR
rats, differences were observed on the relative contribution of
different ␤-adrenoceptor subtypes to vasorelaxation. No corresponding phenotypic differences in basal myocardial ␤-adrenoceptor actions were apparent (Table 2), although LV responses to ␤-adrenoceptor stimulation was beyond the scope of
the current study.
LV systolic and diastolic function has previously been reported to be impaired in sedentary LCR rats compared with
their HCR counterparts, both in vivo and at the level of the
isolated cardiomyocyte (4, 6, 15, 49). This evidence, like that
for cardiac remodeling, has largely been obtained in older
animals (20 –30 wk of age) and whether LV dysfunction is
impaired in younger animals remains to be determined. Often,
changes at the level of LV diastolic function are more marked
than systolic parameters (6, 15), suggesting that LCR, like
other models of metabolic syndrome and type 2 diabetes, may
predispose to earlier onset of abnormalities in myocardial
relaxation rather than contractile dysfunction (39). If permitted
to proceed to senescence (ⱖ2 yr of age), marked dysfunction in
both systolic and diastolic function are evident (21). Interestingly, the NOS1 isoform of NOS (as distinct from the NOS3
isoform likely mediating macrovascular function) has been
implicated in both the systolic and diastolic abnormalities in
LCR cardiomyocyte mechanical function (15), using the selective NOS1 inhibitor N5-(1-imino-3-butenyl)-L-ornitine. Given
the extent of cardiac remodeling already evident at 12 wk of
age in LCR rats, we speculate that the age of onset of cardiac
dysfunction may occur earlier than first thought, particularly if
the extent of microvascular dysfunction evident in the coronary
vascular bed at this age mirrors that in the mesenteric circulation. The reduced cardiac index and coronary flow rates reported in LCR even in the absence of a significant LV systolic
fraction (6, 16) indicate that interrogation of myocardial perfusion abnormalities particularly at the level of the microvasculature (analogous to our work here in mesenteric arteries) is
warranted. While our findings of impaired mesenteric microvascular function cannot necessarily be extrapolated to changes
at the level of coronary microvascular function, such abnormalities could account for the impairments in myocardial
perfusion often evident in metabolic syndrome.
The prevalence of the metabolic syndrome is dramatically
increasing and is strongly linked to cardiovascular diseases
(23). This link persists in females, where it represents a
powerful risk factors for cardiovascular disease, even before
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6
0
Relaxation (%)
H737
H738
LOW RUNNING CAPACITY AND PROGRESSIVE CARDIAC REMODELING
5.
6.
7.
8.
9.
10.
11.
ACKNOWLEDGMENTS
We thank Ritu Bala and Amy Alexander for technical assistance. The LCR
and HCR model is maintained at the University of Michigan and can be made
available for collaborative study (contact: [email protected] or lgkoch
@umich.edu).
Present addresses: S. J. Lessard, Joslin Diabetes Center, 1 Joslin Pl.,
Boston, MA 02215; D. A. Rivas, John Mayer USDA HNRCA at Tufts
University, 711 Washington St., Boston, MA 02111-1524.
GRANTS
12.
13.
14.
15.
This work was supported by National Health and Medical Research Council
(NHMRC) of Australia Project Grant ID526638 (to R. H. Ritchie) and
National Heart Foundation Grant-in-Aid G-09M-4348 (to J. A. Hawley and
O. L. Woodman) and supported in part by the Victorian Government’s
Operational Infrastructure Support Program. R. H. Ritchie is an NHMRC
Senior Research Fellow (ID472673). The LCR-HCR rat model system was
funded by the National Center for Research Resources Grant R24 RR017718
and is currently supported by the Office of Research Infrastructure Programs/OD Grant ROD012098A from the National Institutes of Health.
16.
17.
18.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
R.H.R., J.A.H., and O.L.W. conception and design of research; R.H.R.,
C.H.L., C.Q., M.A.B., K.D.B., and O.L.W. analyzed data; R.H.R., C.H.L.,
J.A.H., and O.L.W. interpreted results of experiments; R.H.R. prepared figures; R.H.R. and O.L.W. drafted manuscript; R.H.R., C.H.L., C.Q., E.J.S.,
L.G.K., S.L.B., J.A.H., and O.L.W. edited and revised manuscript; R.H.R.,
C.H.L., C.Q., E.J.S., M.A.B., K.D.B., S.J.L., D.A.R., L.G.K., S.L.B., J.A.H.,
and O.L.W. approved final version of manuscript; C.H.L., C.Q., E.J.S.,
M.A.B., K.D.B., S.J.L., and D.A.R. performed experiments.
19.
20.
21.
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