Cardiomyocyte contractile dysfunction in the APPswe/PS1dE9 mouse model of Alzheimer's disease.

Abstract

Ample clinical and experimental evidence indicated that patients with Alzheimer's disease display a high incidence of cardiovascular events. This study was designed to examine myocardial histology, cardiomyocyte shortening, intracellular Ca(2+) homeostasis and regulatory proteins, electrocardiogram, adrenergic response, endoplasmic reticulum (ER) stress and protein carbonyl formation in C57 wild-type (WT) mice and an APPswe/PS1dE9 transgenic (APP/PS1) model for Alzheimer's disease.
Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR), maximal velocity of shortening and relengthening (+/-dL/dt), intracellular Ca(2+) transient rise and decay.
Little histological changes were observed in APP/PS1 myocardium. Cardiomyocytes from APP/PS1 but not APP or PS1 single mutation mice exhibited depressed PS, reduced+/-dL/dt, normal TPS and TR compared with WT mice(.) Rise in intracellular Ca(2+) was lower accompanied by unchanged resting/peak intracellular Ca(2+) levels and intracellular Ca(2+) decay in APP/PS1 mice. Cardiomyocytes from APP/PS1 mice exhibited a steeper decline in PS at high frequencies. The responsiveness to adrenergic agonists was dampened although beta(1)-adrenergic receptor expression was unchanged in APP/PS1 hearts. Expression of the Ca(2+) regulatory protein phospholamban and protein carbonyl formation were downregulated and elevated, respectively, associated with unchanged SERCA2a, Na(+)-Ca(2+) exchanger and ER stress markers in APP/PS1 hearts. Our further study revealed that antioxidant N-acetylcysteine attenuated the contractile dysfunction in APP/PS1 mice.
Our results depicted overt cardiomyocyte mechanical dysfunction in the APP/PS1 Alzheimer's disease model, possibly due to oxidative stress.

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Cardiomyocyte Contractile Dysfunction in the APPswe/
PS1dE9 Mouse Model of Alzheimer’s Disease
Subat Turdi, Rui Guo, Anna F. Huff, Eliza M. Wolf, Bruce Culver, Jun Ren*
Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming College of Health Sciences, Laramie, Wyoming,
United States of America
Abstract
Objectives: Ample clinical and experimental evidence indicated that patients with Alzheimer’s disease display a high
incidence of cardiovascular events. This study was designed to examine myocardial histology, cardiomyocyte shortening,
intracellular Ca2+homeostasis and regulatory proteins, electrocardiogram, adrenergic response, endoplasmic reticulum (ER)
stress and protein carbonyl formation in C57 wild-type (WT) mice and an APPswe/PS1dE9 transgenic (APP/PS1) model for
Alzheimer’s disease.
Methods: Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-
relengthening (TR), maximal velocity of shortening and relengthening (6dL/dt), intracellular Ca2+transient rise and decay.
Results: Little histological changes were observed in APP/PS1 myocardium. Cardiomyocytes from APP/PS1 but not APP or
PS1 single mutation mice exhibited depressed PS, reduced6dL/dt, normal TPS and TR compared with WT mice.Rise in
intracellular Ca2+was lower accompanied by unchanged resting/peak intracellular Ca2+levels and intracellular Ca2+decay in
APP/PS1 mice. Cardiomyocytes from APP/PS1 mice exhibited a steeper decline in PS at high frequencies. The responsiveness
to adrenergic agonists was dampened although b1-adrenergic receptor expression was unchanged in APP/PS1 hearts.
Expression of the Ca2+regulatory protein phospholamban and protein carbonyl formation were downregulated and
elevated, respectively, associated with unchanged SERCA2a, Na+-Ca2+exchanger and ER stress markers in APP/PS1 hearts.
Our further study revealed that antioxidant N-acetylcysteine attenuated the contractile dysfunction in APP/PS1 mice.
Conclusions: Our results depicted overt cardiomyocyte mechanical dysfunction in the APP/PS1 Alzheimer’s disease model,
possibly due to oxidative stress.
Citation: Turdi S, Guo R, Huff AF, Wolf EM, Culver B, et al. (2009) Cardiomyocyte Contractile Dysfunction in the APPswe/PS1dE9 Mouse Model of Alzheimer’s
Disease. PLoS ONE 4(6): e6033. doi:10.1371/journal.pone.0006033
Editor: Leszek Wojnowski, University Mainz, Germany
Received February 12, 2009; Accepted May 24, 2009; Published June 24, 2009
Copyright: ? 2009 Turdi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by the University of Wyoming Northern Rockies Regional INBRE (5P20RR016474). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: jren@uwyo.edu
Introduction
Alzheimer’s disease (AD) is a devastating neurodegenerative
disease leading to memory loss and cognitive impairment.
Accumulation of amyloid b-peptide (Ab) and neurofibrillary
tangles are considered hallmarks for neuropathological changes
in AD. At this time, neither the precise pathogenesis nor radical
cure is available for AD. Although majority of AD cases are
idiopathic, an autosomal dominant disorder triggered by mutation
in the b-amyloid precursor protein (APP), presenilin 1 (PS1) or
presenilin 2 (PS2) has been identified to be responsible for familial
AD (FAD) [1]. Interestingly, AD is associated with a high
prevalence of heart dysfunction in the elderly . Recent data has
indicated a close tie between heart dysfunction and AD. A number
of studies have revealed that the onset and progression of AD are
closely associated with hypertension, atherosclerosis, diabetes
mellitus, hypoxia, myocardial infarction and the presence of
ApoE4 allele [2–10]. However, limited information is available
with regards to the cardiovascular function in AD. Given that the
AD genes PS1 and PS2 are also expressed in the heart [11], it may
be speculated that mutations in these genes in familial AD affect
cardiac function. Presenilins have been suggested to play a key role
in the regulation of cardiovascular function. PS1-deficient mice
exhibit ventricular septal defect and double outlet right ventricle in
the mutant hearts [12]. PS2 also play an important role in cardiac
excitation-contraction coupling by interacting with ryanodine
receptor type 2 (RyR2) [13]. PS1 and PS2 mutations are
associated with a higher risk of dilated cardiomyopathy and heart
failure in AD patients [14].
The recent development of transgenic mouse models of AD has
provided an important avenue for AD research. Double transgenic
mice overexpressing FAD-linked amyloid precursor protein with
Swedish mutation and PS-1 with deletion of exon 9 (APPswe/
PS1dE9) demonstrated early Ab deposition as early as 4–6 months
of age that correspond to a form of early onset AD [15]. At 9
months of age, APPswe/PS1dE9 transgenic mice display high
levels of amyloid b-peptide and numerous amyloid b-peptide
deposits [16]. Furthermore, the APPswe/PS1dE9 mice exhibit a
consistent b-amyloid accumulation early on and age-associated
cognitive decline, consolidating the model for AD [17]. Although
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many studies have used this transgenic line to investigate aspects of
neurodegenerative lesions in AD, no information is available with
regards to the cardiac function of this mouse model of AD.
Therefore, we took advantage of this mouse model of AD to
examine cardiomyocyte mechanical function and underlying
mechanisms. Since intracellular Ca2+homeostasis and endoplas-
mic reticulum (ER) stress are known to be closely associated with
cardiomyocyte contractile dysfunction [18,19], key intracellular
Ca2+regulatory proteins such as sarco(endo)plasmic reticulum
Ca2+-ATPase (SERCA), phospholamban (PLB) and Na+-Ca2+
exchanger (NCX) as well as crucial protein markers of ER stress
including inositol requiring enzyme-1 (IRE-1), calregulin and Bip
(GRP78) were also monitored in myocardium of APP/PS1
transgenic and wild-type C57BL/6 mice.
Methods
Experimental animals
All animal procedures were conducted in accordance with
humane animal care standards outlined in the NIH Guide for the
Care and Use of Experimental and were approved the University of
Wyoming AnimalCare and UseCommittee.Inbrief,10-month-old
male C57BL/6 (WT), APPswe/PS1dE9 (APP/PS1) transgenic
mice and mice with transgenic expression of only APPswe (APP) or
PS1dE9 (PS1) (Jackson Laboratory, Bar Harbor, Maine, USA) were
maintained on a 12:12-h light-dark cycle, with free access to food
and water. Production and characterization of the APP/PS1 mouse
line were described in detail previously [15]. In brief, APPswe/
PS1dE9 mice were developed by co-injection of the two transgene
constructs [mouse/human (Mo/Hu) chimeric APP695 harboring
the Swedish (K594M/N595L) mutation and exon-9-deleted PS1]
into pronuclei with a single genomic insertion site resulting in the
two transgenes being transmitted as a single mendelian locus. To
identify the transgenic founder mice, genomic DNA was isolated
from 1-cm tail clips from 10-month-old mice and genotyped by
polymerase chain reaction (PCR) technique using the PCR
conditions suggested by Jackson Laboratory. All primers were
synthesized by Integrated DNA Technologies Inc. (Coralville, IA,
USA) with the following sequences. APPswe: 59-GAC TGA CCA
CTC GAC CAG GTT CTG -39 and 59- CTT GTA AGT TGG
ATT CTC ATA TCC G-39; PSEN1dE9: 59-AAT AGA GAA
CGGCAGGAGCA-39and59-GCC ATGAGGGCACTA ATC
AT-39). Levels of fasting blood glucose and systolic blood pressure
were measured using a glucose monitor (Accu-ChekII, model 792,
Boehringer-Mannheim Diagnostics, Indianapolis, IN, USA) and a
CODA non-invasive blood pressure system (Kent Scientific Co.,
Torrington, CT, USA), respectively. At the time of sacrifice, left
tibial length was measured with a micrometer.
Histological examination
Following anesthesia, hearts WT and APP/PSEN mice were
excised and immediately placed in 10% neutral-buffered formalin
at room temperature for 24 hrs after a brief rinse with PBS.
Thereafter, the tissues were dehydrated through serial alcohols
and cleared in xylenes. The specimen were then embedded in
paraffin, cut in 5 mm sections and stained with hematoxylin and
eosin (H&E) or Masson’s trichrome (to detect fibrosis) for
histological observation [20].
Morris water maze test
Mice aged 8–10 months from WT and APP/PS1 groups were
subjected to behavioral testing. The Morris water maze tests were
conducted in a 120-cm (diameter) tank filled with opaque water.
The water was kept at 25uC and surrounded by dark walls
containing geometric designs that served as distal visual cues. In
the first phase of the training, mice were required to find a
platform (15615 cm) marked with a colored pole, placed in
different quadrants of the pool. Each animal underwent four trials
per day for 4 days, with a maximum of 60 s to find the submerged
platform. If the mice failed to find the platform within 60 s, they
were physically guided to it and allowed to remain on the platform
for 20 s. After visible platform training, two consecutive days of
hidden platform training was conducted in which the platform is
submerged in the opaque water 1 cm deep, with 4 trials per day. A
probe trial was performed at the end of training, in which the
platform was removed. Performance in all tasks (latency to
platform, time spent in target quadrant) was recorded by a
computer-based video tracking system [21].
ECG measurement
Mice were anesthetized with an intraperitoneal injection of
ketamine HCl/xylazine HCl solution (1 ml/kg) and were placed
in a supine position on a warm pad (37uC) for acclimatization.
Afterwards, three surface probes were inserted subcutaneously and
the ECG signal was obtained for 1 minute using a PowerLab
(ML866) and Animal Bio Amp (ML136; AD Instruments,
Colorado Springs, CO, USA). The signal was analyzed with
Chart 5.0 software.
Cardiomyocyte isolation and in vitro drug treatment
Murine cardiomyocytes were isolated as described [20]. After
ketamine/xylazine sedation, hearts were removed and perfused
with Ca2+-free Tyrode’s solution containing (in mM): NaCl 135,
KCl 4.0, MgCl21.0, HEPES 10, NaH2PO40.33, glucose 10,
butanedione monoxime 10, and the solution was gassed with 5%
CO2/95% O2. Hearts were digested with Liberase Blendzyme 4
(Hoffmann-La Roche Inc., Indianapolis, IN, USA) for 20 min. Left
ventricles were removed and minced before being filtered. Tissue
pieces were gently agitated and pellet of cells was resuspended.
Table 1. General characteristics and ECG properties of WT
and APP/PS1) mice.
Mouse groupWT APP/PS1
Body Weight (g)27.061.0 27.961.0
Heart Weight (mg)16366 16966
Heart weight/Body weight (mg/g) 5.5160.69 5.5360.69
Tibial length (mm) 16.260.917.060.9
Heart weight /Tibia length (mg/mm)8.8360.429.1960.63
Liver Weight (g)1.5860.041.5360.04
Kidney Weight (g) 0.4260.03 0.3860.03
Fasting Blood Glucose (mg/dl)110.665.0 116.6610.5
Systolic Blood Pressure (mmHg)122.968.3126.9610.2
Heart Rate (bpm) 3236738264*
R-R interval (msec)22964 16462*
P-R interval (msec)39.265.233.660.4
Q-T interval (msec)25.260.322.160.1
QRS Width (msec)12.660.110.260.6
R Wave Amplitude0.9860.220.3760.03*
QRS Complex Amplitude1.1260.170.8060.02*
Mean6SEM,*p,0.05 vs. wild-type (WT) group, n=6–11 mice per group.
doi:10.1371/journal.pone.0006033.t001
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Extracellular Ca2+was added incrementally back to 1.20 mM over
a period of 30 min. Isolated myocytes were used within 8 hrs of
isolation. Normally, a yield of 50–60% viable rod-shaped
cardiomyocytes with clear sarcomere striations was achieved. Only
rod-shaped myocytes with clear edges were selected for mechanical
study. To directly assess the role of oxidative stress in AD-associated
cardiomyocyte contractile function, a cohort of cardiomyocytes
from WT and APP/PS1 mice were incubated at 37uC for 2 hrs in
the absence or presence of the antioxidant N-acetylcysteine (NAC,
500 mM) [22] prior to mechanical function assessment.
Figure 1. A: The Morris water maze learning curves of mean latency time for WT and APP/PS1 mice; B: Percentage of time spent in the target
quadrant for WT and APP/PS1 mice during the probe trial. Data for both panels were obtained from 4 consecutive days of training (4 trials per day); C:
Representative ECG traces obtained from WT and APP/PS1 mice; and D: Representative microphotographs of the H&E and Masson’s trichrome stained
left ventricular tissue sections from WT and APP/PS1 mice (magnification=4006). Note that Masson’s trichrome stains collagen blue to detect
interstitial fibrosis. Mean6SEM, n=5–6 mice for panels A and B, *p,0.05 vs. WT group.
doi:10.1371/journal.pone.0006033.g001
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Cell shortening/relengthening
Mechanical properties of cardiomyocytes were assessed using a
video-based edge-detection system (IonOptix Corporation, Mil-
ton, MA, USA) [20]. In brief, cells were placed in a Warner
chamber mounted on the stage of an inverted microscope
(Olympus, IX-70) with a buffer containing (in mM): 131 NaCl,
4 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, at pH 7.4. The
cells were field stimulated with suprathreshold voltage at a
frequency of 0.5 Hz, 3 msec duration, using a pair of platinum
wires placed on opposite sides of the chamber connected to a FHC
stimulator (Brunswick, NE, USA). The polarity of stimulation
electrodes was reversed frequently to avoid possible build up of
electrolyte by-products. The myocyte being studied was displayed
on the computer monitor using an IonOptix MyuC am camera,
which rapidly scans the image area at every 8.3 msec such that the
amplitude and velocity of shortening/relengthening is recorded
with good fidelity. The soft-edge software (IonOptix) was used to
capture changes in cell length during shortening and relengthening.
Cell shortening and relengthening were assessed using the following
indices: peak shortening (PS), which indicates peak ventricular
Figure 2. Cardiomyocyte contractile properties in WT, APP/PS1, APP and PS1 mouse hearts. A: Resting cell length (CL); B: Peak shortening
(PS, normalized to CL); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening (TPS50and
TPS90); and F: Time-to-relengthening (TR50and TR90). Mean6SEM, n=132–135 cells from four mice per group, * p,0.05 vs. WT group.
doi:10.1371/journal.pone.0006033.g002
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contractility; time-to250% and 90% PS (TPS50and TPS90), which
indicate rapid and entire phase of myocyte shortening, respectively;
time-to250% and 90% relengthening (TR50and TR90), which
indicates early and entire phase of myocyte relengthening,
respectively; as well as maximal velocity of shortening/relengthen-
ing (6 dL/dt), which depicts maximal velocity of cardiomyocyte
contraction and relaxation. For duration of myocyte shortening and
relengthening, 90% rather 100% of the entire duration was used in
an effort to avoid noisy signal near baseline. In the case of altering
stimulus frequency (from 0.1 Hz to 5.0 Hz), the steady-state
contraction of myocyte was achieved (usually after the first five to
six beats) before PS amplitude was recorded. The grid-crossing
methodwasused forrandomcellselectiontominimizethe influence
of heterogeneous cell contraction in response to field stimuli.
Myocytes with obvious sarcolemmal blebs or spontaneous contrac-
tions were not used for mechanical recording. All measurements
were performed at 25–27uC.
Intracellular Ca2+fluorescence measurement
A separate cohort of myocytes was loaded with fura-2/AM
(0.5 mM) for 15 min and fluorescence measurements were
recorded with a dual-excitation fluorescence photomultiplier
system (Ionoptix) as previously described [20]. In brief, myocytes
were placed on an Olympus IX-70 inverted microscope equipped
with a temperature-controlled (25uC) Warner chamber and
imaged through a Fluor640 oil objective. Myocytes were exposed
to light emitted by a 75W lamp and passed through either a 360 or
a 380 nm filter (bandwidths were615 nm), while being electrically
stimulated to contract at 0.5 Hz. Fluorescence emissions were
detected between 480–520 nm by a photomultiplier tube after first
illuminating the cardiomyocytes at 360 nm for 500 msec then at
380 nm for the duration of the recording protocol (9 sec at a
sampling rate of 333 Hz). The 360 nm excitation scan was
repeated for another 500 msec at the end of the protocol and
qualitative changes in intracellular Ca2+concentration ([Ca2+]i)
were inferred from the ratio of the fluorescence intensity at two
wavelengths (360/380). This ‘‘interpolated’’ technique was used
since 360 nm is the isobestic point for fura-2 at which the
numerator is independent of intracellular Ca2+concentration. The
majority of the recording was done using the 380 nm filter (except
the first and last 500 msec) since the strongest signal is the 380 nm-
excited emission. The time course of the fluorescence signal decay
Figure 3. Intracellular Ca2+transient properties in cardiomyocytes from WT and APP/PS1 mouse hearts. A: Representative intracellular
Ca2+transients in cardiomyocytes from WT (solid line) and APP/PS1 (dotted line) mice; B: Baseline intracellular Ca2+levels; C: Peak intracellular Ca2+
levels; D: Increase of intracellular Ca2+in response to electrical stimuli; and E: Intracellular Ca2+transient decay rate (t). Mean6SEM, n=62–64 cells
from four mice per group, * p,0.05 vs. WT group.
doi:10.1371/journal.pone.0006033.g003
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was fit to a single exponential equation, and the time constant (t)
was used as a measure of the rate of intracellular Ca2+decay.
Western blot analysis
The total protein was prepared as described [20]. In brief, tissue
samples from mouse ventricles were removed and homogenized in
a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl,
1 mM EDTA, 1 mM EGTA, 1% Triton, 0.1% SDS, and protease
inhibitor cocktail. Samples were then sonicated for 15 sec and
centrifuged at 13,0006 g for 20 min at 4uC. The protein
concentration of the supernatant was determined using Protein
Assay Reagent (Bio-Rad Laboratories, Richmond, CA, USA).
Protein samples were then mixed 1:2 with Laemmli sample buffer
with 5% 2-mercaptoethanol and heated at 95uC for 5 min. Equal
amounts (50 mg protein/lane) of the protein mixture, or the
SeeBlue Plus2 PreStained markers (Invitrogen, Carlsbad, CA,
USA) were separated on 10% or 15% SDS-polyacrylamide gels in
a minigel apparatus (Mini-PROTEAN II, Bio-Rad); then were
transferred electrophoreticallyto Nitrocellulosemembranes
(0.2 mm pore size, Bio-Rad Laboratories, Inc, Hercules, CA,
USA). Membranes were incubated for 1 hr at room temperature
in a blocking solution containing 5% milk in Tris-buffered saline
(TBS). After TBS washed, membranes were incubated overnight at
4uC with primary antibody including rabbit anti-SERCA2a
(1:1,000), rabbit anti-Na+-Ca2+exchanger (NCX, 1:1,000) and
mouse anti-phospholamban(PLB,
(1:1,000), goat anti-calregulin (calreticulin) (1:1,000), rabbit anti-
IRE1a (1:500), rabbit anti-phospho-IRE1a (1:1,000), rabbit anti-b1
adrenergic receptor antibody (1:500), rabbit anti-a tubulin (1:1,000
as loading control) and anti-a smooth muscle actin (1:1,200 as
loading control) antibodies. After three washes with TBS-T to
remove excessive primary antibody binding, blots were incubated
with horseradish peroxidase (HRP)–conjugated secondary antibody
(1:5,000) for 1 hr at room temperature. The antigens were detected
by the luminescence method. Quantification of band density was
determined using Quantity One software (Bio-Rad, version 4.4.0)
and reported in optical density per square millimeter.
1:2,000), rabbit anti-BiP
Protein carbonyl assay
Proteins were extracted and minced to prevent proteolytic
degradation. Nucleic acids were eliminated by treating the samples
with 1% streptomycin sulphate for 15 min, followed by a 10 min
centrifugation (11,0006g). Protein was precipitated by adding an
equal volume of 20% trichloroacetic acid (TCA) to protein
(0.5 mg) and centrifuged for 1 min. The TCA solution was
removed and the sample resuspended in 10 mmol/L 2, 4-
dinitrophenylhydrazine(2,4-DNPH)
incubated at room temperature for 15–30 min. After adding
500 mL of 20% TCA, samples were centrifuged for 3 min. The
supernatant was discarded, the pellet washed in ethanol: ethyl
acetate and allowed to incubate at room temperature for 10 min.
The samples were centrifuged again for 3 min and the ethanol:
ethyl acetate steps repeated twice or more times. The precipitate
was resuspended in 6 mol/L guanidine solution, centrifuged for
3 min and any insoluble debris removed. The maximum
absorbance (360–390 nm) of the supernatant was read against
appropriate blanks (water, 2 mol/L HCl) and the carbonyl content
was calculated using the molar absorption coefficient of 22
000 mol/L21cm21[23].
solution. Sampleswere
Statistical analysis
Data are presented as Mean6SEM. Statistical significance
(p,0.05) was estimated by ANOVA or t-test, where appropriate.
Results
General features of animals and ECG analysis
Little difference was identified in tibial length, body and organ
(heart, liver and kidney) weights between the APP/PS1 mice and
their wild-type (WT) littermates. Both heart-to-body weight and
heart weight-to-tibial length ratios were comparable between the
two mouse groups. Fasting glucose levels and systolic blood
pressure were similar between WT and APP/PS1 mice, excluding
the potential contribution of diabetes and hypertension. Surface
ECG examination showed that heart rate was significantly faster in
the APP/PS1 mice compared to WT mice. Correspondingly, R-R
interval was shorter in APP/PS1 mice. P-R interval, Q-T interval
and QRS width were all normal in APP/PS1 mice compared with
WT. Interestingly, both R wave and QRS complex amplitudes,
which are reliable indicatives of myocardial contractility [24], were
significantly diminished in APP/PS1 group compared with those
from WT control (Table 1). Representative ECG traces from
APP/PS1 and WT mice were depicted in Fig. 1.
Figure 4. A: Peak shortening amplitude of cardiomyocytes
isolated from WT and APP/PS1 mouse hearts at different
stimulus frequencies (0.1–5.0 Hz). Peak shortening amplitude is
normalized to value obtained at 0.1 Hz of the same cell. B: Adrenergic
response in cardiomyocytes from WT and APP/PS1 mice. Data are
shown as percent change from baseline in the absence of adrenergic
agonists, norepinephrine (NE, 1 mM) or isoproterenol (ISO, 0.1 and
1 mM). Mean6SEM, n=13–26 cells from four to six mice per data point,
* p,0.05 vs. WT group.
doi:10.1371/journal.pone.0006033.g004
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Behavioral evaluation
The standard Morris water maze test was used to evaluate the
spatial memory of mice from both groups. Results shown in Fig. 1A
indicated that the performance of APP/PS1 mice tended to be
poorer (although not significant) than that of WT mice when the
platform was hidden. Following 4 days of pre-training, all mice
were subjected to a probe trial with the platform taken away. Data
from Fig. 1B showed that APP/PS1 mice spent significantly lesser
time in the target quadrant than the WT control, indicating spatial
memory impairment in the APP/PS1 mice.
Myocardial histology
To assess the impact of APP/PS1 mutation on myocardial
histology, histological studies were performed using the H&E and
Masson’s trichrome staining techniques. No overt difference was
found under the light microscope with regards to the appearance
of isolated cardiomyocytes from APP/PS1 and WT groups (data
not shown), which was supported H&E and Masson’s trichrome
staining in myocardial tissue sections from WT and APP/PS1
mice. Little difference was identified for either cardiomyocyte size
(H&E staining) or interstitial fibrosis (Masson’s trichrome staining)
between the two mouse groups (Fig. 1D).
Cardiomyocyte mechanics in myocytes from APP, PS1
and APP/PS1 transgenic murine model
The APP/PS1 mouse model used in our study possesses
concurrent mutated forms of APP and Presenilin 1 (PS1), both
identified separately in human Alzheimer diseases [1]. To examine
if individual mutation in APP or PS1 is capable of eliciting
myocardial contractile dysfunction independently, cardiomyocyte
mechanical function was assessed in the gender- and weight-
matched mice with mutation of APP or PS1 (3 mice each, body
weight=27–29 g), or both (APP/PS1). The resting cell lengths
(CL) were comparable in WT, APP/PS1, APP and PS1 transgenic
mice. At the pacing frequency of 0.5 Hz, peak shortening
amplitude (PS) and maximal velocity of shortening/relengthening
(6dL/dt) were significantly reduced in cardiomyocytes from APP/
PS1 but not APP or PS1 mouse hearts. The time-to-peak
shortening (TPS50and TPS90) and time-to290% relengthening
(TR50and TR90) were comparable between the WT and APP/
PS1 mice (Fig. 2). Similarly, TPS50, TPS90, TR50and TR90were
unaffected by single mutation of APP or PS1 at the stimulus
frequency of 0.5 Hz (data not shown). Given the higher heart rate
in rodents, cell mechanical indices were also examined in WT and
APP/PS1 mouse cardiomyocytes paced at 5 Hz. PS and6dL/dt
Figure 5. Expression of intracellular Ca2+regulatory proteins in myocardium from WT and APP/PS1 mice. A: Representative gel blots
depicting the expression of SERCA2a, NCX and PLB using specific antibodies. a-tubulin was used as an internal loading control; B: SERCA2a; Panel C:
Phospholamban (PLB); and D: Na+-Ca2+exchanger (NCX). Mean6SEM, n=4, * p,0.05 vs. WT group.
doi:10.1371/journal.pone.0006033.g005
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were significantly lowered in APP/PS1 cardiomyocytes (PS:
1.6760.30%; + dL/dt: 66.4611.8 mm/sec; - dL/dt: 258.06
13.5 mm/sec, n=18 cells) compared with those from WT group
(PS: 2.8860.40%; + dL/dt: 91.4613.8 mm/sec; - dL/dt:
276.5614.7 mm/sec, n=21 cells, p,0.05 for all parameters
between the two groups). TPS90 and TR90 were comparable
between WT (7263 and 8364 msec, respectively) and APP/PS1
(6864 and 7865 msec, respectively) mice at the stimulus
frequency of 5 Hz.
Intracellular Ca2+transient properties
To further explore the possible mechanism of action underlying
APP/PS1-associated cardiomyocyte mechanical dysfunction, the
membrane permeable intracellular Ca2+fluorescent dye fura-2
was used to evaluate intracellular Ca2+homeostasis in cardiomy-
ocytes. Our results depicted unchanged peak and resting
intracellular Ca2+, as well as intracellular Ca2+decay rate (t)
associated with a slight although significant decrease in intracel-
lular Ca2+rise (D[Ca2+]i) in cardiomyocytes from APP/PS1 mice
compared with those from WT mice (Fig. 3). These data indicated
the presence of intracellular Ca2+handling defect in cardiomyo-
cytes from this APP/PS1 transgenic mouse model of AD.
Effect of increased stimulation frequency on
cardiomyocyte shortening
Mouse hearts normally contract at very high frequencies
(400 beat/min), whereas our baseline mechanical assessment was
conducted at 0.5 Hz. To investigate the possible role of
sarcoplasmic reticulum Ca2+storage and release in APP/PS1
mouse cardiomyocytes, a frequency-response was constructed.
Cardiomyocytes were initially stimulated to contract at 0.5 Hz for
5 min to ensure steady-state before commencing the frequency
sequence. While increased stimulating frequency (0.1–5.0 Hz)
triggered a negative staircase in peak shortening amplitude in both
WT and APP/PS1 groups. The degree of decrease was more
pronounced in APP/PS1 mice at frequencies greater than 1 Hz
(Fig. 4A), indicating existence of compromised SR storage and
release of intracellular Ca2+in APP/PS1 mouse hearts.
Figure 6. Expression of ER stress markers in myocardium from WT and APP/PS1 mice. A: Representative gel blots depicting the ER stress
markers IRE1a, phosphorylated IRE1a, BiP and calregulin (calreticulin) using specific antibodies. a-tubulin was used as an loading control; B:
phosphorylated IRE 1a/IRE 1a ratio; C: calregulin; and D: Bip. Mean6SEM, n=4.
doi:10.1371/journal.pone.0006033.g006
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Effect of norepinephrine and isoproterenol on
cardiomyocyte shortening
To explore the alterations in cardiac excitation-contraction
coupling, cardiomyocytes from APP/PS1 and WT mice were
exposed to norepinephrine (NE, 1 mM) and isoproterenol (ISO,
0.1 and 1 mM) and myocyte shortening was examined. Results
from Fig. 4B displayed a reduced responsiveness to both NE and
ISO in cardiomyocytes from APP/PS1 mice compared with those
from WT group.
Protein expression of intracellular Ca2+regulatory
proteins, ER stress markers and b-adrenergic receptor
Given that intracellular Ca2+homeostasis is tightly regulated by
a cascade of intracellular Ca2+regulatory proteins including
SERCA, phospholamban (PLB) and NCX [18], Western blot
analysis was performed to evaluate the expression of SERCA2a,
PLB and NCX. Our results shown in Fig. 5 revealed a significantly
reduced expression of PLB associated with unchanged SERCA2a
and NCX in myocardium from APP/PS1 mice compared with the
WT group. To explore if alteration in protein trafficking and
folding was involved in myocardial dysfunction in APP/PS1 mice,
protein expression of the ER stress markers IRE-1a, calregulin
and Bip was examined. Results shown in Fig. 6 revealed that IRE-
1a, IRE-1a phosphorylation, calregulin and Bip were all
comparable between the APP/PS1 and WT groups, not favoring
a potential involvement of protein misfolding in APP/PS1
mutation-associated cardiac dysfunction. Our further immuno-
blotting analysis revealed similar levels of b1-adrenergic receptor in
myocardium between the APP/PS1 and WT groups (Fig. 7).
Protein carbonyl formation and effect of antioxidant on
cardiomyocyte mechanics in APP/PS1 mice
Oxidative stress and subsequent protein oxidation have been
reported to play an important role in the pathogenesis of AD brain
[25–28]. To assess the cardiac oxidative protein damage in the
APP/PS1 AD model, protein carbonyl formation was determined.
Results from the present study revealed that myocardial protein
carbonylformationwas significantly
(3.13960.716 nmol/mg protein)
0.215 nmol/mg protein, p,0.05 between the two groups). To
further examine the role of oxidative stress in APP/PS1-induced
cardiac contractile defects, freshly isolated cardiomyocytes from
WT and APP/PS1 mice were incubated for 2 hrs in the absence
or presence of the antioxidant NAC (500 mM). Our data revealed
that NAC effectively ablated APP/PS1-induced mechanical
defects (depressed PS and6dL/dt) without eliciting any effects
on cardiomyocyte mechanics itself (Fig. 8). These data provided
direct evidence for a likely role of oxidative stress in AD-induced
cardiac contractile dysfunction, consistent with elevated protein
carbonyl formation.
higher
WT
inAPP/PS1
(0.9936
thanmice
Discussion
Data from our current study have clearly depicted cardiomy-
ocyte contractile dysfunction in the APPswe/PS1dE9 (APP/PS1)
transgenic model of AD. The major mechanical defects observed
in cardiomyocytes from APP/PS1 mice include decreased cell
shortening, reduced velocities of shortening/relengthening at the
stimulus of both 0.5 Hz and 5 Hz and exaggerated frequency
response. Our results indicated that individual mutation in either
APP or PS1 alone is insufficient to trigger overtly compromised
cardiomyocyte contractile function compared with concurrent
presence of both. These findings are consistent with literature that
the expression of APPswe alone results in less Ab production than
APPswe/PS1dE9 and PS1dE9 transgene enhances the production
of Ab in APPswe mice while little effect exerted by itself [29,30].
Our further study revealed the possible contribution of decreased
adrenergic response, dysregulated intracellular Ca2+homeostasis,
protein damage and oxidative stress to impaired cardiomyocyte
mechanical function in APP/PS1 mice.
In our study, the APP/PS1 mice were euglycemic and
normotensive, excluding potential contribution of diabetes and
hypertension to the compromised cardiac function. Our data
revealed overt mechanical changes associated with exacerbated
spatial learning and memory impairment in APP/PS1 mice.
Approximately 2% of populations in developed countries are
afflicted with AD [31]. In 2004, AD was ranked the 7th among the
10 leading causes of death in the US [32]. Although there has been
an intensive effort to understand AD, the main focus thus far is to
elucidate the pathogenesis of neurodegeneration in AD brain with
rather limited data for the organ-specific complications of AD.
During the past two decades, a growing body of evidence has
revealed many risk factors of cardiac diseases which are also
Figure 7. Expression of b1-adrenergic receptor (b1-AR) in
myocardium from WT and APP/PS1 mice. Inset: Representative
gel blots of b1-AR and a-smooth muscle actin (used as an internal
loading control). Mean6SEM, n=4.
doi:10.1371/journal.pone.0006033.g007
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detrimental to AD, indicating a close link between cardiac diseases
and AD [33]. To the best of our knowledge, our current study is
the first report to provide direct evidence of cardiac dysfunction in
AD using this transgenic line.
In our study, the heart rate was higher in AD mice. This may be
associated with cardiac autonomic dysfunction in AD although
conflicting data were seen. Elevated catecholamine levels and
ablated vagal tone were reported in AD patients [34,35],
supporting the higher heart rate observed in our current study.
Impaired vagal parasympathetic function was reported in AD
patients evaluated by R-R interval variation (RRIV) and
sympathetic skin response and orthostatic cardiovascular reflexes
[36]. In addition, QT dispersion was found to be tightly correlated
with the degree of cognitive impairment in AD patients. As shown
Figure 8. Cardiomyocyte contractile properties in myocytes from WT and APP/PS1 mice incubated for 2 hrs in the presence or
absence of the antioxidant N-acetylcysteine (NAC, 500 mM). A: Resting cell length; B: Peak shortening (normalized to cell length); C: Maximal
velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening (TPS); and F: Time-to290% relengthening
(TR90). Mean6SEM, n=50 cells from 3 mice per group, * p,0.05 vs. WT group, # p,0.05 vs.APP/PS1 group.
doi:10.1371/journal.pone.0006033.g008
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in our current study, surface ECG revealed for the first time
increased heart rate, reduced RR interval and diminished QRS
amplitude in the APP/PS1 mice compared with WT mice.
The most prominent cardiac dysfunctions in the APP/PS1 AD
mouse model observed in our study were reduced cardiomyocyte
contraction and diminished maximal velocity of contraction/
relaxation (6dL/dt) at both low and high stimulus frequencies
associated with unchanged duration of contraction and relaxation.
Several factors may contribute to the impaired mechanical
function including defected contractile proteins (e.g., actin, myosin
heavy chain isoform), reduced intracellular Ca2+release, depressed
SR Ca2+load and altered myofilament Ca2+sensitivity. The
intracellular Ca2+measurement confirmed a decrease in intracel-
lular Ca2+rise associated with unchanged resting and peak
intracellular Ca2+levels in cardiomyocytes from APP/PS1 mice.
The unchanged intracellular Ca2+decay coincides with the
normal TR50 and TR90 in APP/PS1 mouse cardiomyocytes,
which is also supported by the normal protein levels of SERCA2a
and NCX, the main machineries to remove Ca2+from cytosolic
space for cardiac relaxation to occur, in APP/PS1 myocardium. In
addition, our data revealed exaggerated decline in the steady-state
myocyte peak shortening in response to increased stimulating
frequency in APP/PS1 mice, suggesting a possibly reduced
capacity in SR Ca2+storage and release in this murine model of
AD. The reduced cardiomyocyte shortening capacity received
convincing support from the reduced R-wave height in ECG
reading. Our data revealed down-regulation of phospholamban,
an endogenous inhibitor of SERCA, in APP/PS1 mouse hearts.
Although the link between reduced phospholamban expression
and altered cardiac contractile function in APP/PS1 hearts is
unclear, reduced phospholamban levels may trigger altered
intracellular Ca2+homeostasis en route to affect cardiomyocyte
contractile function [18]. Our study revealed enhanced protein
carbonyl formation in the absence of ER stress in myocardium
from APP/PS1 mice, suggesting a likely role of oxidative stress in
the altered cardiomyocyte contractile function in APP/PS1 mice.
This notion received further support from our in vitro study where
antioxidant NAC alleviates the cardiomyocyte mechanical dys-
function in APP/PS1 mice.
Catecholamine desensitization is a hallmark of heart failure
[37]. It has been shown that heart failure may trigger
catecholamine desensitization to both isoproterenol and norepi-
nephrine in the presence and absence of ganglionic blockade [37].
This is in line with our observation of reduced responsiveness to
norepinephrine and isoproterenol in APP/PS1 mouse cardiomy-
ocytes. However, the lack of overt change in myocardial histology
and b1-adrenergic receptor expression suggest the absence of any
advanced heart failure and adrenergic desensitization in APP/PS1
hearts. Although sympathetic denervation may lead to postsynap-
tic supersensitivity due to lack of neural uptake for norepinephrine
[37], to what extent adrenergic denervation or desensitization
contributes to myocardial complications in AD patients remains
controversial. Several reports have been seen to discriminate
changes in cardiac sympathetic innervation of AD patients from
other neurodegenerative disease such as dementia with Lewy
bodies and vascular dementia [38–40]. These studies measured
cardiac uptake of metaiodobenzylguanidine (MIBG), a physiologic
analogue of norepinephrine [41], as a mean of noninvasive index
of the integrity of sympathetic neurotransmission in the heart of
AD patients. It was found that cardiac MIBG uptake in AD
patients was essentially unaffected compared with other neour-
odegenrative diseases. Nevertheless, subtle differences may exist in
the diagnostic criteria of AD and different stages of disease process.
In summary, this study provides evidence for the first time of
altered cardiomyocyte contractile function in the APP/PS1
transgenic model of AD. Cardiomyocytes from APP/PS1 mice
exhibited decreased inotropic response and impaired intracellular
Ca2+homeostasis possibly due to mechanisms associated with
reduced intracellular Ca2+release, altered expression in Ca2+
regulatory proteins, reduced adrenergic function and oxidative
damage. However, other mechanisms should not be excluded at
this time such as susceptibility of myofilament Ca2+sensitivity and
altered expression of cardiac contractile proteins. Future study is
warranted to elucidate the subcellular signaling mechanisms
responsible for cardiac abnormalities in AD.
Acknowledgments
The authors would like to thank Drs. Susan Zhang and Ji Li from
University of Wyoming School of Pharmacy for technical assistance.
Author Contributions
Conceived and designed the experiments: ST JR. Performed the
experiments: ST RG AFH EMW. Analyzed the data: ST RG EMW BC
JR. Contributed reagents/materials/analysis tools: BC. Wrote the paper:
BC JR.
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