lic
di
a,b,
are identified in experimental preeclampsia; they also reveal an adaptation in the use of energy substrates in pregnancy and its impairment by
enhanced volume load on the heart. Blood volume
expansion leads to adaptations of the myocardium that
affect mainly the left ventricle (LV), more susceptible to
cal conditions, these
Cardiovascular Research 69 (20* Corresponding author. Research Center of Sainte Justine Hospital, 3175
Cote Sainte Catherine, H3T 1C5 Montreal, Canada. Tel.: +1 514 345sodium supplementation.
D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: L-type calcium current; Cardiomyocytes; Metabolism; Pregnancy; Hypertension
1. Introduction
Pregnancy induces significant adaptations in the cardio-
vascular system, associated with hemodynamic and endo-
crine changes that contribute to maternal volume expansion
and are necessary for fetal homeostasis, development and
well-being [1]. Systemic arterial vasodilatation represents
one of the first detectable changes in hemodynamics that
initiates a cascade of compensations in the circulation and
volume homeostasis that also affect the heart: cardiac output
rises in response to increase in the heart rate and achieves its
greatest value in the final stages of delivery, placing anAvailable online 7 December 2005
Time for primary review 27 days
Abstract
Objectives: Pregnancy is an important physiological condition associated with hemodynamic and endocrine changes that affect the heart.
Nevertheless, very little is known about cardiomyocyte remodeling in this condition. Here, we studied the morphological, functional and
metabolic remodeling of rat left ventricular myocytes that occurs in late stages of normal pregnancy (P) and in experimental preeclampsia
induced by elevated (0.9%) sodium intake (P0.9).
Methods: We applied confocal microscopy to examine the morphology and the contractility of single cells, while the patch clamp technique
was used to assay ionic currents.
Results: Our results revealed a significant increase in the volume of single left ventricular cardiac myocytes in P, mainly resulting from cell
elongation. In P0.9, further increase in the cell length led to a significant rise in the length/width ratio. Cell contractility was significantly
decreased in glucose-based solutions in response to stimulation at 0.5 Hz and 6 Hz in P as well as in P0.9. The density of L-type calcium
current (ICaL) was not significantly altered in P or in P0.9. Metabolic substrates lactate and pyruvate, increased in the blood of P and P0.9
rats, enhanced contractility in P, without affecting ICaL. The same effect, present but blunted in P0.9, was associated with a significant
increase in ICaL.
Conclusion: Our results demonstrate that processes of adaptive remodeling take place in normal pregnancy, while maladaptive componentsReceived 10 May 2005; received in reviseda Research Center, Sainte-Justine Hospital, Canada
b Department of Pediatrics, University of Montreal, Canada
c Department of Nutrition, University of Montreal, Canada
form 24 October 2005; accepted 31 October 2005Structural, functional and metabo
myocytes in normal and in so
V. Bassien-Capsa a, J.-C. Fouron0008-6363/$ - see front matter D 2005 European Society of Cardiology. Publishe
doi:10.1016/j.cardiores.2005.10.017
4931x4366; fax
E-mail address: alzbeta.chorvatova@recherche-ste-justine.qc.ca
(A. Chorvatova).remodeling of rat left ventricular
um-supplemented pregnancy
B. Comte a,c, A. Chorvatova a,b,*
06) 423 – 431
www.elsevier.com/locate/cardioresincreased load. In contrast to pathologi: +1 514 345 4801.
alterations are associated with physiological reduction of
blood pressure (BP) and are reversible. The natural volume
d by Elsevier B.V. All rights reserved.

to preeclampsia was induced experimentally by sodium
supplementation, in regard to a recent work that showed that
asculan increase in the dietary NaCl in the last week of pregnancy
induced alterations similar to preeclampsia [14], including
higher BP and significantly and durably attenuated renin–
angiotensin–aldosterone system [15].
2. Materials and methods
2.1. Animals and cardiomyocyte isolation
Female Sprague–Dawley rats (13–14 weeks old, Charles
River, Canada) received a 0.9% NaCl supplement in tap
water for the last 7 days of pregnancy [14,15]. Two groups
of age-matched pregnant and never pregnant rats were
also used. Rats were killed at the 21st day of pregnancy by
decapitation. All procedures were performed in accordance
with the NIH and the Canadian Council for the Protection of
Animals (CCPA) guidelines. Left ventricular myocytes were
isolated following retrograde perfusion of the heart with
proteolytic enzymes [16]. Myocytes were maintained in a
storage solution at 4 -C until used. Only cells showing
clearly defined striations were used in up to 10 h following
isolation.
2.2. Whole heart echocardiography
Echocardiography was performed on isoflurane (2%/L of
O2/min)-anesthetized rats using a 128XP/10c instrument
from Acuston (Mountain View, U.S.A.) equipped with a 7overload in pregnancy is frequently associated with
increase in LV size and mass, leading to LV hypertrophy
(LVH) [2–4].
The most important pathology during pregnancy is
hypertension [5], in particular preeclampsia, affecting 6 to
10% of all pregnancies. In this condition, instead of
decreasing, BP is significantly increased, producing an
acute pressure overload on left ventricle [6,7]. For long,
duration of the disease was thought to be too brief to
induce structural cardiac changes. However, in recent
years, studies of gestational hypertension identified
changes in LV geometry as an important risk factor [8–
10], correlating with increase in fetal and maternal
mortality and morbidity. To date, very little information
is available on structure/function modifications at the single
cardiomyocyte level.
This study examined whether left ventricular cardiomyo-
cytes undergo structural, functional and metabolic remodel-
ing in normal pregnancy in rat and whether this remodeling
is altered by sodium supplementation. To investigate
cardiomyocyte remodeling during pregnancy, a rat model
was chosen due to close resemblances between pregnancies
in rats [11,12] and in humans [13]. A condition comparable
V. Bassien-Capsa et al. / Cardiov424MHz transducer. LV shortening fraction was calculated as
(diastolic
systolic) /diastolic dimensions.2.3. Confocal microscopy
To label sarcolemma and t-tubule system, myocytes were
stained with lipophilic membrane fluorescent probe di-8-
ANEPPS (5 Amol/L for 5 min at room temperature) [17]. 3D
images were taken by inverted laser scanning confocal
microscope LSM 510 (Zeiss, core lab of Sainte-Justine
Research Center; PlanNeofluar 63/1.3 oil, 488 nm Ar/ion,
LP 505) and recorded by averaging four lines using 1.2 Am
steps in the z-stack (pinhole opening of 417 Am, scaling 0.16
Am0.16 Am). Sarcomere lengthwas determined in each cell
as a mean length of at least three sarcomeres. For length and
width data, some measurements obtained from transmission
images were also added. Fluorescent probe Tetramethylrhod-
amine TMRM (10 nmol/L for 10 min at room temperature)
was used to image mitochondria (543 nm He/Ne, LP 560 nm,
pinhole opening 106 Am, scaling 0.2 Am0.2 Am). At least
three measurements were averaged in each cell.
Rapid line-scan confocal imaging was used to record
contractions. Cells were field-stimulated to contract using Pt
electrodes, operated by a pulse generator (DS-2A, Digitimer
Ltd., U.S.A.) and by a trigger generator (DG-2, Digitimer
Ltd., U.S.A.) under external perfusion at 35T1 -C.
Maximum contraction was calculated as a percentage of D
cell length=(length-maximal shortening) / length. Only cells
with two contracting edges were taken into consideration.
2.4. Electrophysiology
Myocytes were voltage clamped using the Axopatch
200B patch clamp amplifier linked to a Digidata 1200B A/D
interface. Currents were acquired using Clampex 9 and
analyzed with Clampfit 9 from pClamp package (Axon
Instruments, USA). Micropipettes were fabricated from
filamented borosilicate glass GC150TF (Harvard, Canada)
using pipette pooler (PP-830, Narishige, Japan) and polisher
(MF-830, Narishige, Japan) to give tip resistances of 1 MV
when filled with standard physiological solution (in mmol/L:
CsCl, 130.0; CaCl2, 1.0; NaCl, 10.0; HEPES, 10.0 adjusted
to pH 7.25 with CsOH). Cells were pre-incubated in the
presence of BAPTA-AM (10 Amol/L for 15 min) prior to
patch clamp experiments. Membrane currents were mea-
sured by the perforated patch clamp method using Ampho-
tericin B (240–480 Ag/mL) as the pore-forming agent. All
measurements were undertaken at 35T1 -C.
2.5. Blood analyses
Blood was collected immediately after decapitation. The
samples were processed within 24 h and determined by
standard enzymatic methods [18]. For pyruvate measure-
ment, blood sample was treated with 5% trichloroacetic acid
and immediately vortexed for 2 min. Each sample was then
centrifuged for 4 min at 10,000g, the supernatant collected
ar Research 69 (2006) 423–431and promptly frozen at
20 -C. Pyruvate was subsequently
determined by standard enzymatic dosage. The dosages