Download The onset of left ventricular diastolic dysfunction in SHR - AJP

Am J Physiol Heart Circ Physiol 302: H1524–H1532, 2012.
First published January 27, 2012; doi:10.1152/ajpheart.00955.2010.
The onset of left ventricular diastolic dysfunction in SHR rats is not related
to hypertrophy or hypertension
S. Dupont,1,2 J. Maizel,1,2 R. Mentaverri,1,2 J.-M. Chillon,1,2 I. Six,1,2 P. Giummelly,3 M. Brazier,1,2
G. Choukroun,1,2 C. Tribouilloy,1,2 Z. A. Massy,1,2 and M. Slama1,2
1
INSERM U 1088; 2Jules Verne University of Picardy and Amiens University Medical Center, Amiens; and 3Cardiovascular
Pharmacology Laboratory (EA 3452), Nancy, France
Submitted 23 September 2010; accepted in final form 4 January 2012
diastole; ultrasonic; calcium; spontaneously hypertensive rats
LEFT VENTRICULAR (LV) DIASTOLIC dysfunction is responsible for
more than one half of all hospitalizations for congestive heart
failure or acute pulmonary oedema, which has been shown to
be the consequence of multiple clinical factors, mostly hypertension (15). LV diastolic dysfunction, particularly relaxation
abnormalities, is usually recognized to be closely associated
with the development of left ventricular hypertrophy (LVH) in
hypertensive humans and spontaneously hypertensive rats
(SHR), a model of human genetic hypertension (35). Nevertheless, in a previous study, we demonstrated LV relaxation
Address for reprint requests and other correspondence: M. Slama, INSERM
U 1088, Univ. of Picardy and Amiens Univ. Medical Center, Dept. of
Nephrology-Medical Intensive Care Unit, Ave. René Laennec, F-80054
Amiens, France (e-mail: [email protected]).
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impairment in young SHR without LVH (37), subsequently
confirmed by Aeschbacher et al (1). Di Bello et al. (13) have
also demonstrated early LV diastolic dysfunction before the
onset of hypertension and LVH. In view of these data, LV
diastolic dysfunction may be considered to be an early event in
the cascade of processes leading to the development of hypertension and LVH. However, to date, no clear information is
available concerning the kinetics of development of diastolic
dysfunction.
Noteworthy, LVH is associated with structural and functional abnormalities of cardiomyocytes. Under physiological conditions, following the increase in intracytosolic Ca2⫹
concentration ([Ca2⫹]i) during contraction, myocyte relaxation occurs when Ca2⫹ is transported back into the sarcoplasmic reticulum (SR) by the SR Ca2⫹ ATPase [sarco(endo)plasmic Ca2⫹-ATPase isoform (SERCA)] and, to a lesser
extent, when Ca2⫹ is pumped out of the cytosol by the
Na⫹/Ca2⫹ exchange (NCX) and sarcolemmal Ca2⫹ ATPase.
SERCA is regulated by its inhibitor phospholamban (PLB).
Phosphorylation of PLB at Ser16 (p16-PLB) by protein
kinase A and/or Thr17 by Ca2⫹/calmodulin-dependent kinase II reduces its inhibitory effects on SERCA, resulting in
an increased affinity of SERCA for Ca2⫹, increasing SR
Ca2⫹ uptake, whereas dephosphorylation of PLB by SRassociated phosphatase (PP1 and PP2a) restores the inhibitory effect of PLB on SERCA. The present study mainly
concerned p16-PLB, since it is considered to be the major
regulator of PLB activity during ␤-adrenergic stimulation
(10). Under pathological conditions, several studies have
reported decreased expression of SERCA 2a, the predominant isoform in the heart (43), in failing human myocardium
(2, 34) and in animal models of pressure overload-induced
hypertrophy, especially in SHR (3). This abnormality of
SERCA 2a would lead to abnormalities in Ca2⫹ handling,
especially significant alteration of [Ca2⫹]i (4, 5), which
appears to be involved in hypertrophic cell growth (28).
Chen-izu et al. (9) recently showed that alterations in Ca2⫹
signaling already occur at the onset of hypertension and
precede the development of hypertrophy.
We therefore hypothesized that LV diastolic dysfunction and
abnormal [Ca2⫹]i in genetic hypertension may precede not
only LVH but also the onset of hypertension. We decided 1) to
analyze LV myocardial function and particularly diastolic
function (by invasive and noninvasive methods) in 3-weekold SHR rats before the onset of hypertension and in
5-week-old SHR rats after the onset of hypertension and
2) to investigate variations in [Ca2⫹]i as well as SERCA 2a
RNA and protein expression level in these animals.
0363-6135/12 Copyright © 2012 the American Physiological Society
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Dupont S, Maizel J, Mentaverri R, Chillon JM, Six I, Giummelly P, Brazier M, Choukroun G, Tribouilloy C, Massy ZA,
Slama M. The onset of left ventricular diastolic dysfunction in SHR
rats is not related to hypertrophy or hypertension. Am J Physiol Heart
Circ Physiol 302: H1524 –H1532, 2012. First published January 27,
2012; doi:10.1152/ajpheart.00955.2010.—Left ventricular (LV) diastolic dysfunction, particularly relaxation abnormalities, are known to
be associated with the development of LV hypertrophy (LVH).
Preliminary human and animal studies suggested that early LV diastolic dysfunction may be revealed independently of LVH. However,
whether LV diastolic dysfunction is compromised before the onset of
hypertension and LVH remains unknown. We therefore evaluated LV
diastolic function in spontaneously hypertensive rats (SHR) at different ages and tested whether LV diastolic dysfunction is associated
with abnormal intracellular calcium homeostasis. LV systolic and
diastolic functions were evaluated by invasive and echocardiographic
methods in 3-week-old (without hypertension) and 5-week-old (with
hypertension) SHR and Wistar-Kyoto control rats. Basal intracytoplasmic calcium and sarcoplasmic reticulum (SR) Ca2⫹ contents were
measured in cardiomyocytes using fura-2 AM. Sarco(endo)plasmic
Ca2⫹-ATPase isoform 2a (SERCA 2a) and phospholamban (PLB)
expressions were quantified by Western blot and quantitative RT-PCR
techniques. LV relaxation dysfunction was observed in 3-week-old
SHR rats before onset of hypertension and LVH. An increase in basal
intracytoplasmic Ca2⫹ and a decrease in SR Ca2⫹ release were
demonstrated in SHR. Decreased expression of SERCA 2a and Ser16
PLB (p16-PLB) protein levels was also observed in SHR rats, whereas
mRNA expression was not decreased. For the first time, we have
shown that LV myocardial dysfunction precedes hypertension in
3-week-old SHR rats. This LV myocardial dysfunction was associated
with high diastolic [Ca2⫹]i possibly due to decreased SERCA 2a and
p16-PLB protein levels. Diastolic dysfunction may be a potential
predictive marker of arterial hypertension in genetic hypertension
syndromes.
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EARLY DIASTOLIC DYSFUNCTION IN SHR RATS
MATERIALS AND METHODS
Table 1. Invasive hemodynamic parameters in anesthetized
WKY and SHR
3 Wk
n
SAP, mmHg
DAP, mmHg
MAP, mmHg
PP, mmHg
HR, beats/min
␶, ms
5 Wk
WKY
SHR
WKY
SHR
12
69 ⫾ 4
52 ⫾ 2
58 ⫾ 3
17 ⫾ 3
329 ⫾ 11
9.3 ⫾ 0.4
10
75 ⫾ 3
53 ⫾ 3
60 ⫾ 2
22 ⫾ 2
288 ⫾ 8*
10.7 ⫾ 0.4*
6
86 ⫾ 5†
67 ⫾ 6†
74 ⫾ 5†
19 ⫾ 2
386 ⫾ 12†
8.1 ⫾ 0.2†
8
105 ⫾ 2*†
76 ⫾ 3†
86 ⫾ 2*†
29 ⫾ 2*
332 ⫾ 6*†
9.0 ⫾ 0.2*†
Values are means ⫾ SE. WKY, Wistar-Kyoto rats; SHR, spontaneously
hypertensive rats; SAP, systolic arterial pressure; DAP, diastolic arterial
pressure; MAP, mean arterial pressure; PP, pulse pressure; HR, heart rate; and
␶, time constant of isovolumic LV relaxation. *P ⬍ 0.05 vs. age-matched
WKY; †P ⬍ 0.05 vs. the same strain at 3 wk of age.
3 Wk
n
SAP, mmHg
DAP, mmHg
MAP, mmHg
PP, mmHg
HR, beats/min
5 Wk
WKY
SHR
WKY
SHR
5
106 ⫾ 4
70 ⫾ 5
82 ⫾ 3
37 ⫾ 7
332 ⫾ 17
5
95 ⫾ 3
63 ⫾ 4
73 ⫾ 3
32 ⫾ 5
344 ⫾ 12
5
101 ⫾ 3
80 ⫾ 3
87 ⫾ 3
21 ⫾ 1
308 ⫾ 15
5
129 ⫾ 4*†
84 ⫾ 5†
99 ⫾ 4*
45 ⫾ 3*†
312 ⫾ 20
Values are means ⫾ SE. *P ⬍ 0.05 vs. age-matched WKY; †P ⬍ 0.05 vs.
the same strain at 3 wk of age.
to 2% isoflurane. Transthoracic echocardiographic measurements
were performed in the left lateral decubitus position with a commercially available echocardiography system (Phillips IE33 with a 15MHz transducer, France) using two-dimensional and Doppler imaging, as described previously (36). Echocardiographic evaluation was
carried out in blind fashion. The transthoracic echocardiographic
transducer was placed to obtain short- and long-axis, four-chamber
(ventricles and atria) and five-chamber (ventricles, atria, and aorta)
apical cardiac views. M-mode LV imaging was obtained in the cardiac
long-axis and LV end-diastolic diameter (LVEDD), LV end-systolic
diameter (LVESD), and posterior and septal diastolic wall thickness
(PW and IVS) were measured according to the American Society of
Echocardiography guidelines (33). LV mass (LVM) was calculated as
follows: LVM ⫽ 1.04 ⫻ [(LVEDD ⫹ PW ⫹ IVS)3 ⫺ LVEDD3].
Shortening fraction (SF) was calculated according to the following
equation: SF ⫽ (LVEDD ⫺ LVESD)/LVEDD. From a five-chamber
apical view, aortic flow was recorded using pulsed Doppler imaging.
LV ejection time (ET) was measured as the time from the beginning
to the end of the aortic flow wave. Velocity of circumferential fiber
shortening was also calculated as VCF ⫽ SF/ET. Mitral flow was
recorded at the tip of the mitral valve from an apical view using
Doppler imaging. Isovolumic relaxation time (IVRT) was measured
as the interval between the aortic closure click and the start of mitral
flow. The Tei index was calculated on the same apical view as follows
(39): Tei index ⫽ (DD ⫺ MD)/ET where DD is diastole duration and
Table 3. Echocardiographic parameters in SHR
and WKY rats
3 Wk
n
BW, g
HR, beat/min
LVEDD, mm
LVESD, mm
PW, mm
IVS, mm
LVM, g
LVM/BW mg/g
ESAo, %
VTIao, cm
VTImi, cm
VE, cm/s
ETao, ms
5 Wk
WKY
SHR
WKY
SHR
31
59 ⫾ 2
375 ⫾ 1
4.5 ⫾ 0.1
2.2 ⫾ 0.1
1.3 ⫾ 0.1
1.1 ⫾ 0.1
0.25 ⫾ 0.01
4.2 ⫾ 0.1
8.3 ⫾ 0.8
4.0 ⫾ 0.1
3.3 ⫾ 0.1
78.3 ⫾ 1.4
76.2 ⫾ 0.8
31
51 ⫾ 1*
372 ⫾ 2
4.3 ⫾ 0.1
2.5 ⫾ 0.1
1.2 ⫾ 0.1
1.1 ⫾ 0.1
0.23 ⫾ 0.01
4.5 ⫾ 0.1
9.7 ⫾ 1.3
4.0 ⫾ 0.1
2.9 ⫾ 0.1*
76.0 ⫾ 1.5
77.5 ⫾ 1.0
27
145 ⫾ 5†
383 ⫾ 2
6.2 ⫾ 0.1†
3.1 ⫾ 0.1†
1.4 ⫾ 0.1
1.2 ⫾ 0.1
0.48 ⫾ 0.01†
3.3 ⫾ 0.1†
9.5 ⫾ 1.3
4.8 ⫾ 0.2†
4.2 ⫾ 0.1†
93.5 ⫾ 2.2†
73.7 ⫾ 0.9†
27
111 ⫾ 2*†
378 ⫾ 2
6.0 ⫾ 0.1†
3.4 ⫾ 0.1*†
1.2 ⫾ 0.1*
1.1 ⫾ 0.1
0.40 ⫾ 0.01†
3.3 ⫾ 0.2†
10.9 ⫾ 0.8
4.4 ⫾ 0.1*†
3.8 ⫾ 0.1*†
91.5 ⫾ 1.9†
72.9 ⫾ 1.1†
Values are means ⫾ SE. BW, body weight; LVEDD, left ventricular (LV)
end-diastolic diameter; LVESD, LV end-systolic diameter; PW, posterior wall
end-diastolic diameter; IVS, interventricular septum end-diastolic diameter;
LVM, LV mass; LVM/BW and ESAo, systolic expansion rate of aorta; VTIao
and VTImi, aortic and mitral velocity time integral; VE, early wave velocity;
ETao, aortic ejection time. *P ⬍ 0.05 vs. age-matched WKY; †P ⬍ 0.05 vs.
the same strain at 3 wk of age.
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Materials. All chemicals were obtained from Sigma-Aldrich. Primers were obtained from Invitrogen (France). Primary mouse antiSERCA 2a and rabbit anti-Ser16 phospho-phospholamban (p16-PLB)
antibodies were purchased from Affinity Bioreagent. Rabbit anticalnexin, goat anti-PLB, secondary anti-rabbit, anti-goat, and antimouse peroxidase IgG antibodies were purchased from Santa Cruz.
Pentobarbital sodium was obtained from Ceva Santé, France.
Animals. Three-week-old and 5-week-old male SHR and WistarKyoto (WKY) rats were purchased from Janvier Breeding Laboratories (Le Genest-Saint-Isle, France). They were maintained in temperature- and humidity-controlled rooms with a 12-h:12-h light/dark
cycle and were given standard chow. All rats were handled in
accordance with French legislation, and the protocol was approved by
an institutional animal care committee.
Blood and LV pressure measurements. At 3 and 5 weeks of age,
respectively, five WKY, five SHR, five WKY, and five SHR rats were
anesthetized with ketamine-zylazine solution. A polyethylene catheter
was introduced into the right carotid artery and tunneled subcutaneously to exit behind the head. The surgical procedure was performed
in the morning. Animals awoke 1 h after the procedure, and the
catheter was then connected to a pressure transducer to record arterial
blood pressure in conscious animals after resting for 8 h. Systolic
arterial pressure (SAP), diastolic arterial pressure, mean arterial blood
pressure (MAP), pulse pressures (PP), and heart rate (HR) were
measured by the data acquisition system (IOX v2.4.2.6; EMKA
technologies, Paris, France) during a stable period.
In parallel, at 3 and 5 weeks of age, respectively, these same
parameters were measured in 12 WKY, 10 SHR, 6 WKY, and 8 SHR
anesthetized animals. The catheter was then carefully advanced into
the left ventricle through the aortic valve. Time constants of isovolumic LV relaxation (␶) were calculated automatically from the LV
pressure curve (IOX v2.4.2.6; EMKA technologies) during a stable
period.
Pulse wave velocity measurements. Rats were anesthetized with
ketamine-xylazine solution. A polyethylene catheter was introduced
into the right carotid artery via the aortic arch. Another catheter was
inserted into the abdominal aorta via the left femoral artery. When
blood pressure and heart rate were stable, the two pressure waves were
recorded simultaneously for at least 3 min. The propagation time for
the pulse wave moving from the aortic arch to the abdominal aorta
was measured by the time interval between the upstroke (foot) of each
pressure wave front. After euthanasia, the distance between the two
catheters was determined with a cotton thread. The pulse wave
velocity (PWV) was obtained by dividing this distance by the time
interval between the two pressure wave fronts.
Echocardiography. Thirty-one SHR and WKY rats (3 week old)
and 27 SHR and WKY rats (5 week old) were anesthetized with 1%
Table 2. Arterial blood pressure in conscious SHR
and WKY rats
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EARLY DIASTOLIC DYSFUNCTION IN SHR RATS
Ca2⫹-free buffer supplemented with fetal bovine serum (10%), and
filtered through a nylon mesh (100 ␮m). Cells were finally resuspended successively in buffers containing increasing extracellular
Ca2⫹ concentrations to reach a final Ca2⫹ concentration of 1.2 mM.
An average of 70 – 80% of rod-shaped cardiomyocytes was observed
on light microscopy. Cells were then plated on laminin-coated 96-well
plates at a density of 20,000 cells/well and maintained at 37°C in a
humidified atmosphere containing 5% CO2 for least 1 h, before
intracellular calcium measurements.
Measurement of [Ca2⫹]i. Fresh cardiomyocytes were incubated
with 5 ␮M fura-2 AM for 30 min, as described previously (45).
Excitation wavelengths were 340 and 380 nm (calcium bound and
unbound to fura-2 AM, respectively). Fluorescence emission was
monitored at 510 nm by Envision 2103 Multilabel Reader (Perkin
Elmer). In view of the recognized uncertainties inherent to the
measurement of absolute [Ca2⫹]i, the results are expressed as the
(340/380 nm) fluorescence ratio throughout this study, which provides
an index of [Ca2⫹]i. To assess the sarcoplasmic reticulum (SR) Ca2⫹
release, various ATP concentrations (0.5, 5, 50 ␮M) were used in the
presence of 1 mM Ca2⫹. Values are expressed as variations of the
340-to-380 nm ratio calculated as: 340-to-380 nm ratio after stimulation with ATP normalized to 340-to-380 nm ratio observed at steady
state (i.e., before stimulation of cardiomyocytes).
The maximum (Rmax) and minimum (Rmin) fluorescence intensities
were compared in both WKY and SHR rats, at 3 and 5 weeks of age.
Rmax was assessed after addition of Triton X-100 and 5 mM Ca2⫹,
and Rmin was determined after addition of 25 mM EGTA.
Fig. 1. A–D: Doppler echocardiography systolic and diastolic indexes in 3-week-old and
5-week-old Wistar-Kyoto rats (WKY) and
spontaneously hypertensive rats (SHR).
IVRT, isovolumic relaxation time; SF, shortening fraction; VCF, velocity circumferential
fiber shortening. Three-week-old WKY and
SHR (n ⫽ 31) 5-week-old WKY and SHR
(n ⫽ 27) are shown. *P ⬍ 0.05 vs. agematched WKY.
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MD is mitral duration. The systolic expansion rate of the aorta (ESAo)
was calculated as described previously (24). HR must be identical to
compare echocardiographic parameters between SHR and WKY rats,
particularly diastolic function (IVRT), which is highly dependent
on HR.
Aortic and left ventricle collagen and desmosine contents. Twenty
rats (5 rats in each of the 4 groups: 3- and 5-week-old SHR and WKY
animals) were assigned for the analyses of aortic composition in
collagen and desmosine. Samples of frozen aorta and left ventricle
were removed, weighed, and hydrolyzed in 6 M hydrochloric acid for
24 h at 105°C. Protein content was determined with the dinitrofluorobenzene method, taking 92 kDa as the molecular weight of an amino
acid. Collagen content was determined with the chloramine T/paradimethylaminobenzaldehyde reaction as [(hydroxyproline content ⫻
7.46/protein content) ⫻ 100] (25). Elastin and the elastin-specific,
cross-linking amino acids desmosine and isodesmosine were assayed
by capillary zone electrophoresis and UV detection (16).
Isolation of cardiomyocytes. Following echocardiographic measurements, animals were euthanized by pentobarbital sodium. The
heart was removed, and cardiomyocytes were isolated from the LV
wall, as described previously (32). Briefly, the hearts were rapidly
excised, mounted on a Langendorff apparatus, and retrogradely perfused for 5 min with Ca2⫹-free buffer containing (in mM) 120.4 NaCl,
5.4 KCl, 0.6 KH2PO4, 0.6 Na2PO4, 1.2 MgSO4, 10 HEPES, 4.6
NaHCO3, 30 taurine, 10 2,3 butanedione monoxime, and 5.5 glucose
at pH 7.2. The hearts were then perfused with a Ca2⫹-free buffer
containing 1 mg/ml collagenase II (Worthington) until they started to
become flaccid. The LV were removed, minced into small pieces in
EARLY DIASTOLIC DYSFUNCTION IN SHR RATS
Table 4. Collagen, desmosine, and isodesmosine contents in
the aorta and the left ventricle in SHR and WKY rats
3 Wk
n
Aorta ww, mg
Desmosine, ␮g/mg
aorta ww
Isodesmosime, ␮g/mg
aorta ww
Collagen, ␮g/mg
aorta ww
LV ww, mg
Collagen, ␮g/mg
LV ww
5 Wk
WKY
SHR
WKY
SHR
5
6.9 ⫾ 0.2
5
6.5 ⫾ 0.2
5
6.9 ⫾ 0.4
5
6.6 ⫾ 0.3
0.7 ⫾ 0.1
0.5 ⫾ 0.1
0.6 ⫾ 0.1
0.6 ⫾ 0.1
0.8 ⫾ 0.1
0.7 ⫾ 0.1
0.7 ⫾ 0.1
0.8 ⫾ 0.1
59.1 ⫾ 2.6
9.1 ⫾ 0.4
65.7 ⫾ 3.4
9.4 ⫾ 0.5
79.2 ⫾ 5.1†
9.3 ⫾ 0.3
83.1 ⫾ 5.7†
9.6 ⫾ 0.5
3.6 ⫾ 0.3
4.0 ⫾ 0.3
3.5 ⫾ 0.2
3.7 ⫾ 0.2
Quantitative RT-PCR. Total RNA was extracted from left ventricles using Tri-reagent, and total RNA (1 ␮g) was transcribed into
complementary DNA using the high capacity cDNA reverse transcription kit according to the manufacturer’s protocol (Applied Biosystems). The following primers were used: SERCA 2a (forward, TGA
ATC TGA CCC AGT GGC TGA; reverse, ACT CCA GTA TTG
CAG GCT CCA), SERCA 2b (forward, CCT TTG CCA CTC ATT
TTC CAG A; reverse, GCA CAC TCT TTA CCA GGC TCC A),
PLB (forward, TGA GCT CCC AGA CTT CAC ACA; reverse, TTG
ACA GCA GGC AGC CAA A), NCX 1 (forward, TCA TTG GAG
ATC TGG CTT CCC; reverse, TGG ACG CAT CTG CAT ACT
GGT), and ␤-actin (forward, CAT TGC TGA CAG GAT GCA;
reverse, AGA GCC ACC AAT CCA CAC AGA). SYBR Green
chemistry was used to perform quantitative determinations of the
mRNAs encoding for SERCA 2a, SERCA 2b, PLB, NCX 1, and a
housekeeping gene, ␤-actin. cDNAs were amplified using an ABI
PRISM 7900 sequence detection system (Applied Biosystems). ␤-Actin was used to normalize differences in RNA isolation, RNA degradation, and the efficiency of the reverse transcription reaction.
Changes in mRNA expression were determined using the comparative
threshold method (23). RNA expression was also expressed in relation
to the levels observed in WKY rats.
Preparation of SR-enriched microsomes. After rats were euthanized, the hearts were removed and left ventricles were isolated to
obtain SR-enriched microsomes, as described previously (19). The
protein concentration was determined by Bradford assay (Bio-Rad).
Aliquots were frozen at ⫺80°C until Western blot and SERCA 2a
activity analysis.
Western blot. Proteins of interest were quantified using Western
blot analysis. Proteins from SR-enriched microsomes were denatured
in Laemmli buffer containing 125 mM Tris·HCl, 2% SDS, 1%
␤-mercaptoethanol, 10% glycerol, and 0.005% bromophenol blue (pH
6.8). Fifty micrograms of proteins per sample were separated on 8%
(SERCA 2a, calnexin) or 15% (PLB, Ser16 PLB, or p16-PLB)
acrylamide/bisacrylamide SDS-PAGE under constant current. Proteins were then transferred to polyvinylidene difluoride (0.45 ␮m
membrane; Millipore) in a transfer buffer, at 14 V overnight at 4°C.
Ponceau red staining technique was used to confirm the amount of
protein loaded on the membrane. Membranes were then blocked with
5% non-fat milk in Tris-buffered saline 0.1% Tween 20 for 1 to 2 h
at room temperature and immunoblotted with the desired primary
antibody in 0.5% non-fat milk for 90 min at room temperature
(anti-SERCA 2a; 1:5,000) or overnight (anti-p16-PLB, anti-PLB, and
anti-calnexin; 1:5,000). After several washings in Tris-buffered saline
0.1% Tween 20, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (SERCA 2a;
1:5,000), HRP-conjugated goat anti-rabbit antibodies (p16-PLB and
calnexin; 1:5,000), or HRP-conjugated rabbit anti-goat antibodies
(PLB; 1:5,000). Each secondary antibody was incubated for 1 h at
room temperature before development using the enhanced chemiluminescence detection kit (Amersham). Bands were visualized and
quantified using the GeneGenius Bio Imaging System (Syngene, UK).
SERCA 2a protein levels were normalized to endogenous calnexin
levels. The p16-PLB-to-PLB ratio was also determined.
Measurement of SERCA 2a activity. SERCA 2a activity was
determined from the rate of inorganic phosphate release, as described
previously (18). The Ca2⫹-ATPase activity was calculated as the
difference between Ca2⫹, Mg2⫹-ATPase activity (in the presence of
calcium) and Mg2⫹-ATPase activity measured in the absence of
calcium (3 mM EGTA).
Statistical analysis. The results are expressed as the mean ⫾ SE of
the indicated number of experiments. All variables were tested for a
normal distribution (Kolmogorov-Smirnov test). Statistical analysis
was performed using Student’s t-test in the case of a normal distribution and a Mann-Whitney test in the case of an abnormal distribution or number of samples is ⬍10. A value of P ⬍ 0.05 was
considered to be significant.
RESULTS
Arterial and LV pressure. Data on arterial pressure are
shown in Tables 1 and 2. Body weight was significantly lower
in 3-week-old and 5-week-old SHR than in age-matched WKY
rats. No difference in SAP, diastolic arterial pressure, MAP,
PP, and HR values were observed between 3-week-old WKY
and SHR rats or between anesthetized and conscious animals.
At the age of 5 weeks, a higher blood pressure (SAP, MAP,
Fig. 2. Measurement of basal intracytosolic calcium levels. Before stimulation
with ATP, basal intracellular calcium was measured in cardiomyocytes isolated from 3-week-old and 5-week-old WKY and SHR rats and was expressed
as the 340-to-380 nm ratio. In our experiments, the minimum and maximum
fluorescence intensities (Rmin and Rmax) were not significantly different between WKY and SHR. Three-week-old SHR and WKY rats (n ⫽ 15) and
5-week-old SHR and WKY rats (n ⫽ 8). *P ⬍ 0.05 vs. age-matched WKY.
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Values are means ⫾ SE. ww, Wet weight. †P ⬍ 0.05 vs. the same strain at
3 wk of age.
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EARLY DIASTOLIC DYSFUNCTION IN SHR RATS
Fig. 3. ATP dose-response. Cardiomyocytes isolated from WKY and SHR rats
were stimulated with ATP concentrations ranging from 0.5 to 50 ␮M. ATPinduced SR Ca2⫹ release was measured as: 340-to-380 nm ratio after stimulation with ATP (t ⫽ 10 s) normalized to the steady-state 340-to-380 nm ratio.
A: 3-week-old SHR and WKY rats (n ⫽ 15). B: 5-week-old SHR and WKY
rats (n ⫽ 8). Data were analyzed by t-test. WKY, *P ⬍ 0.05 vs. age-matched
WKY.
and PP) was observed in SHR rats compared with WKY
control rats in both anesthetized and conscious animals. LV
catheterization showed a higher ␶ value in both 3-week-old and
5-week-old SHR rats compared with WKY rats (Table 1).
PP amplification and PWV. At 3 and 5 weeks, the strains did
not differ in terms of PP amplification (expressed either in
mmHg or percentage). In contrast, the PWV was significantly
higher in the SHR groups (440 ⫾ 40, n ⫽ 6; P ⫽ 0.026 and
500 ⫾ 30 cm/s, n ⫽ 5; P ⫽ 0.015 at 3 and 5 weeks,
respectively) than in the WKY groups (320 ⫾ 30, n ⫽ 6 and
380 ⫾ 20 cm/s, n ⫽ 5, respectively) at both time points.
Doppler echocardiography findings. M-mode echocardiography data are shown in Table 3. LVEDD and IVS were similar
in 3-week-old and 5-week-old SHR compared with agematched WKY rats. Echocardiography also revealed a significant increase of LVESD in 5-week-old SHR compared with
WKY control rats, whereas no difference was observed at 3
weeks of age.
These parameters were used to calculate LVM. No difference in LVM was observed in 3-week-old and 5-week-old
SHR compared with age-matched WKY control rats. This
Fig. 4. Quantitative RT-PCR analysis of calcium regulatory gene in the left
ventricle from 3-week-old and 5-week-old WKY and SHR. Graphs depicting
relative sarco(endo)plasmic Ca2⫹-ATPase isoform (SERCA) 2a and 2b, phospholamban (PLB), and Na⫹/Ca2⫹ exchange (NCX1) expression normalized to
␤-actin in 3-week-old (A) and 5-week-old (B) SHR rats compared with WKY
(control or ct). 3-week-old WKY and SHR rats: n ⫽ 10, 5-week-old WKY; and
SHR rats: n ⫽ 7. The error bar for WKY refers to SERCA 2a.
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result was confirmed by weighing left ventricles after euthanasia (WKY: 0.19 ⫾ 0.01 and SHR: 0.18 ⫾ 0.01 g for
3-week-old rats; WKY: 0.38 ⫾ 0.02 and SHR: 0.32 ⫾ 0.03 g
for 5-week-old rats) and after normalizing to body weight
(WKY: 3.2 ⫾ 0.5 and SHR: 3.4 ⫾ 0.5 mg/g for 3-week-old
rats; WKY: 2.6 ⫾ 0.1 and SHR: 2.7 ⫾ 0.1 mg/g for 5-week-old
rats).
ESAo was similar in 3-week-old and 5-week-old SHR compared with age-matched WKY control rats.
Systolic and diastolic functions were then evaluated by
Doppler echocardiography. Assessment of diastolic function at
constant HR showed that IVRT and Tei index, two parameters
of LV relaxation, were significantly increased in both 3-weekold and 5-week-old SHR compared with control WKY rats
(Fig. 1, A and B). Conversely, no significant increase in IVRT
and Tei index was observed between 3-week-old and 5-weekold rats of either strain. Mitral velocity time integral was also
significantly lower in 3-week-old and 5-week-old SHR compared with control WKY rats, whereas aortic velocity time was
significantly decreased only in 5-week-old rats (Table 3).
Assessment of systolic function revealed a significant decrease in SF and VCF in both 3-week-old and 5-week-old SHR
compared with control WKY rats (Fig. 1, C and D). In contrast,
no significant increase in SF and VCF was observed between
3-week-old and 5-week-old rats of either strain.
Aortic and left ventricle collagen and desmosine contents.
At both 3 and 5 weeks of age, the SHR and control WKY
EARLY DIASTOLIC DYSFUNCTION IN SHR RATS
increase of the 340-to-380 nm ratio in both SHR and WKY
cardiomyocytes. ATP-induced release of SR Ca2⫹ was significantly lower in 3-week-old SHR than in age-matched control
WKY rats, at ATP concentrations of 5 and 50 ␮M (Fig. 3A). At
5 weeks of age, similar data were obtained with a significant
difference between the two strains starting at an ATP concentration of 0.5 ␮M (Fig. 3B).
mRNA expression by quantitative RT-PCR. Gene expression
was determined to further assess the mechanisms underlying
the alterations in calcium cycling observed in left ventricles
isolated from 3- and 5-week-old rats. No significant difference
in mRNA expression, normalized to ␤-actin encoding for
SERCA 2a and SERCA 2b, PLB, and NCX 1, was observed
between 3-week-old and 5-week-old SHR and their control
WKY rats (Fig. 4, A and B). Similar results were obtained
when hypoxanthine-guanine phosphoribosyltransferase was
used as housekeeping gene instead of ␤-actin (data not shown).
Calcium regulatory protein abundance. The abundance of
key calcium regulatory proteins, particularly SERCA 2a, PLB,
and p16-PLB, was measured in SR-enriched microsomes isolated from left ventricles of 3-week-old and 5-week-old rats.
As shown in Fig. 5, A and C, a decrease of SERCA 2a protein
expression was observed in both 3-week-old and 5-week-old
SHR rats compared with age-matched control WKY rats.
SERCA 2a/calnexin, assessed by the GeneGenius Bio Imaging
System, was significantly lower in both 3-week-old and
5-week-old SHR compared with WKY control rats (Fig. 5, B
Fig. 5. Western blot analysis of SERCA 2a in the left ventricle from 3-week-old and 5-week-old WKY and SHR. A and B: representative Western blot using
anti-SERCA 2a antibodies in 3-week-old rats (WKY, n ⫽ 11; SHR, n ⫽ 13) and 5-week-old rats (WKY, n ⫽ 9; SHR: n ⫽ 7), respectively. Calnexin protein
expression was used as internal control. C and D: graphs depicting quantification of SERCA 2a normalized to calnexin. *P ⬍ 0.05 vs. age-matched WKY.
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groups did not differ in terms of the aortic desmosine, isodesmosine, and collagen contents. However, the collagen contents
were higher at 5 weeks than at 3 weeks. Left ventricle collagen
contents were similar in SHR and age-matched WKY control
rats at both 3 and 5 weeks of age (Table 4).
Variation of [Ca2⫹]i in SHR and WKY rats. Calcium is the
main mediator in cardiac excitation-contraction coupling and
relaxation and abnormalities of calcium currents may therefore
explain the disorders observed. We therefore investigated
[Ca2⫹]i in cardiomyocytes isolated from the left ventricles of
SHR and WKY rats. No significant difference in Rmin and Rmax
values was observed between cardiomyocytes isolated from
3-week-old and 5-week-old SHR and WKY rats (data not
shown). However, basal [Ca2⫹]i expressed as the 340-to-380
nm ratio was higher in cardiomyocytes isolated from SHR
compared with age-matched WKY rats at steady state (before
stimulation) and independently of age (3 weeks or 5 weeks old)
(Fig. 2). A less marked, but nonsignificant, difference in
340-to-380 nm ratio was observed between cardiomyocytes
isolated from 5-week-old rats of the two strains, suggesting
that an adaptive mechanism affecting intracellular calcium
homeostasis becomes effective between the ages of 3 weeks
and 5 weeks in SHR rats.
ATP, which triggers release of Ca2⫹ from the SR via
activation of metabotropic (P2Y) and ionotropic (P2X) receptors, was used to evaluate SR Ca2⫹ load. ATP, introduced at
concentrations of 0.1 to 50 ␮M, induced a dose-dependent
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EARLY DIASTOLIC DYSFUNCTION IN SHR RATS
and D). Although no difference in PLB was observed between
SHR and WKY rats, p16-PLB and p16-PLB/PLB were significantly lower in 3-week-old SHR than in age-matched WKY
control rats (Fig. 6, A and B). Assessment of the p16-PLB-toPLB ratio did not reveal any significant difference in 5-weekold rats (Fig. 6, C and D).
SERCA 2a activity. A nonsignificant trend to a decrease in
SHR SERCA 2a activity was observed in both 3-week-old
(WKY 885 ⫾ 100; SHR 740 ⫾ 126 pmol Pi/mg ⫻ min n ⫽ 6)
and 5-week-old rats (WKY 816 ⫾ 63; SHR 696 ⫾ 95 pmol
Pi/mg ⫻ min n ⫽ 6).
DISCUSSION
Fig. 6. Western blot analysis of PLB and phosphorylation of PLB at Ser16 (p16-PLB) in the left ventricle from 3-week-old and 5-week-old WKY and SHR. A
and C: representative Western blot using anti-PLB and anti-p16-PLB antibodies in 3-week-old rats (WKY, n ⫽ 12; SHR, n ⫽ 10) and 5-week-old rats (WKY,
n ⫽ 9; SHR, n ⫽ 7), respectively. B and D: graphs depicting quantification of p16-PLB normalized to PLB. *P ⬍ 0.05 vs. age-matched WKY.
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This study demonstrates, for the first time, that 3-week-old
SHR rats develop marked myocardial dysfunction with impaired LV relaxation and alteration of LV systolic function,
before the onset of hypertension and LVH. Myocardial dysfunction is associated with decreased SR Ca2⫹ release and
increased steady-state intracellular calcium concentrations, at
least partly due to a decrease of SERCA 2a protein levels. This
deregulation was associated with a decrease in PLB phosphorylation, indicating possible functional inhibition of SERCA 2a,
in addition to decreased protein expression. The absence of
LVH is confirmed by the absence of difference for either LVM
or LVM/body weight despite the body weight difference be-
tween the two groups. In fact, young SHR rats were been
reported to have a lower body weight than age-matched WKY
rats, and this weight difference could be explained by the
underlying metabolic disorders observed in SHR (14).
LV diastolic dysfunction was assessed by IVRT, Tei index,
and ␶ in SHR rats. IVRT has been described as a reliable
marker of LV relaxation in clinical and experimental studies
(38). The Tei index is increased in the presence of systolic or
diastolic impairment (30). In the present study, IVRT and Tei
index were higher in 3-week-old SHR than in age-matched
WKY rats, confirming the presence of early LV diastolic
dysfunction. These results are consistent with those of a previous study conducted in our laboratory in older rats with LVH
and hypertension, confirming the accuracy of these echocardiographic parameters to detect systolic and diastolic dysfunction compared with invasive methods (36, 37). An alteration of
aortic stiffness leading to increased PWV has also been observed in 3-week-old SHR rats before the onset of hypertension. Similar results have been reported in 6-week-old SHR
(11, 41). This abnormality of aortic compliance has been
attributed to media hypertrophy due to an increased number of
smooth muscle cell layers rather than a modification in
intrinsic elastic properties, particularly collagen and elastin
contents (14). Our study results confirmed prior reports
because we did not find any difference in collagen and
EARLY DIASTOLIC DYSFUNCTION IN SHR RATS
Second, IVRT and myocardial index were used to analyze LV
relaxation. It would have been preferable to measure Doppler
imaging indexes, but these indexes are still difficult to record in
small animals weighing 40 – 60 g. In contrast, IVRT and
myocardial indexes have been validated in our laboratory and
have been shown to be very accurate to assess LV relaxation.
␶ was also measured and confirmed by our Doppler findings.
Finally, it would have been more appropriate to use a Millar
catheter to measure ␶. A polyethylene catheter was used in this
study, since it provides an optimal dynamic response with
negligible resonance (42). This study demonstrates for the first
time that diastolic dysfunction precedes hypertension. This
finding may completely change our understanding of the pathophysiology of hypertension. At least three hypotheses can be
proposed: 1) diastolic dysfunction may be the cause of hypertension by decreasing stroke volume and stimulating the sympathetic system resulting in hypertension and LV hypertrophy
that accentuates LV diastolic dysfunction; 2) diastolic dysfunction may be a part of the same disease, and two mechanisms
may be present related to two chromosomal alterations at the
same locus; and 3) arterial abnormalities and cardiac dysfunction share the same alterations. In the light of these results, LV
diastolic dysfunction could become a valuable predictive
marker for hypertension and/or LVH. In the light of these
findings, echocardiography may be useful to predict the onset
of hypertension in patients with a family history of hypertension.
In conclusion, this study demonstrates that early LV myocardial dysfunction precedes the onset of hypertension in SHR
rats. This myocardial dysfunction is associated with abnormal
Ca2⫹ homeostasis due to decreased SERCA 2a and p16-PLB
protein expression. These findings may facilitate the design of
appropriate and effective strategies for the treatment of cardiovascular diseases.
ACKNOWLEDGMENTS
We thank the French Society of Hypertension and the Picardie Regional
Council for valuable support.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: S.D., J.M., R.M., I.S., Z.A.M., and M.S. conception
and design of research; S.D., J.M., J.-M.C., and P.G. performed experiments;
S.D., J.M., P.G., and M.S. analyzed data; S.D., J.M., R.M., J.-M.C., M.B.,
Z.A.M., and M.S. interpreted results of experiments; S.D. and J.M. prepared
figures; S.D., J.M., and R.M. drafted manuscript; S.D., J.M., G.C., C.T., and
Z.A.M. edited and revised manuscript; S.D., J.M., R.M., J.-M.C., I.S., P.G.,
M.B., G.C., C.T., Z.A.M., and M.S. approved final version of manuscript.
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