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Am J Physiol Heart Circ Physiol 284: H464–H474, 2003;
10.1152/ajpheart.00540.2002.
Increasing myocardial contraction and blood pressure
in C57BL/6 mice during early postnatal development
KLAUS TIEMANN,3 DIRK WEYER,1 P. CHRYSO DJOUFACK,1 ALEXANDER GHANEM,3
THORSTEN LEWALTER,3 ULRIKE DREINER,2 RAINER MEYER,2
CHRISTIAN GROHÉ,3 AND KLAUS B. FINK1
1
Department of Pharmacology, Universitätsklinik Bonn, 53113 Bonn;
2
Department of Physiology II, Universitätsklinik Bonn, 53115 Bonn; and 3Medizinische Klinik
und Poliklinik II, Universitätsklinik Bonn, University of Bonn, 53105 Bonn, Germany
Submitted 28 June 2002; accepted in final form 3 October 2002
cardiovascular disease models; electrocardiography; echocardiography; sarcomere shortening
as specific cardiovascular
disease models have become invaluable instruments
for understanding cardiac pathophysiology (7). Techniques such as transverse aortic constriction (33), induction of ischemia-reperfusion, and myocardial in-
GENETICALLY ENGINEERED MICE
Address for reprint requests and other correspondence: K. B. Fink,
Dept. of Pharmacology, Univ. of Bonn, Medical School, Reuterstrasse
2b, 53113 Bonn, Germany (E-mail: [email protected]).
H464
farction (19) have been adapted to the smaller
dimensions in the mouse and are now readily employed
in this species. However, the physiological cardiovascular phenotype of the mouse strain C57BL/6, which is
most abundantly used as the background strain of
genetically engineered mice, is still incompletely characterized. Most notably, developmental changes in left
ventricular (LV) dimensions and mass, heart rate
(HR), blood pressure, and contractility of ventricular
myocytes need to be further evaluated. It was the
primary aim of this study to check for age- and genderdependent changes of cardiovascular parameters in
C57BL/6 mice and for the underlying mechanisms.
These data are of particular interest for comparison of
homozygously bred mutant mice with C57BL/6 as the
wild type rather than for comparison of mutant mice
(⫺/⫺ or ⫹/⫺) bred from heterozygous parents with
their homozygous ⫹/⫹ littermates. Littermates would
unequivocally provide the closest genetic background
and best control but are not always available in sufficient numbers.
Because transgenic mice or mice mutated by targeted gene disruption are usually rare early after generation, it is desirable to analyze cardiac size and
functional parameters without invasive procedures
(10). High-resolution echocardiography (EC) allows
noninvasive assessment of LV dimensions and mass
(18, 31). However, the reproducibility of this technique
is somewhat limited by the small size of the mouse and
the high HR. Although LV myocardial mass in EC
correlates with necropsy heart weight (5), the correlation of LV myocardial mass in the developing heart
determined by EC with that determined by extremely
reliable histomorphometry (HM) has not been systematically studied. Therefore, the second aim of this study
was to evaluate the accuracy of EC to study cardiac
dimensions in mice at defined ages and compare three
common calculation algorithms for LV myocardial
mass based on one- or two-dimensional EC recordings
with the corresponding HM reference measurements.
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0363-6135/03 $5.00 Copyright © 2003 the American Physiological Society
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Tiemann, Klaus, Dirk Weyer, P. Chryso Djoufack,
Alexander Ghanem, Thorsten Lewalter, Ulrike Dreiner,
Rainer Meyer, Christian Grohé, and Klaus B. Fink.
Increasing myocardial contraction and blood pressure in
C57BL/6 mice during early postnatal development. Am J
Physiol Heart Circ Physiol 284: H464–H474, 2003;10.1152/
ajpheart.00540.2002.—Knowledge of the developmental changes
of cardiovascular parameters in the genetic background of a
mouse strain is important for understanding phenotypic
changes in transgenic or knockout mouse models for heart
disease. We studied arterial blood pressure and myocardial
contractility in mice of the common background strain
C57BL/6, aged 21 days [postnatal day 21 (P21)] to 580 days.
Heart rate increased during maturation from 396 beats/min
at P21 to 551 beats/min at postnatal day 50 (P50), and mean
arterial blood pressure increased in parallel from 86 to 110
mmHg and remained constant afterward. Echocardiographically determined left ventricular myocardial wall dimensions
(R ⫽ 0.79, P ⬍ 0.0001) and left ventricular mass calculated
using the area-length algorithm correlated strongly with
histomorphometrical measurements (R ⫽ 0.93, P ⬍ 0.001).
Sarcomere shortening records from isolated ventricular myocytes used as a measure for myocardial contractility revealed
a negative shortening-frequency relation under a pacing frequency of 2 Hz and a positive relation above 2 Hz. Shortening
amplitudes recorded from P21 myocytes were smaller, and
the shortening-frequency relation was less steep than in
adult myocytes. A stimulation pause was followed by a negative “staircase” at pacing frequency of ⱕ6 Hz and a positive
staircase at ⱖ6 Hz. P21 myocytes developed positive staircases at 8 and 10 Hz, and adult myocytes also developed
them at 6 Hz. Blood pressure increase during maturation
until P50 may originate from increasing single cardiomyocyte contractility.
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MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
METHODS
AJP-Heart Circ Physiol • VOL
Fig. 1. Representative lead II surface electrocardiogram (ECG) from
an adult C57Bl/6 mouse at postnatal day 111 (P111) with an explanation of the ECG parameter definition. The heart rate (HR) in the
example shown is 580 beats/min. The T-wave is attached to the
S-wave and is often bipolar.
edge). End-diastolic measurements were obtained at the
peak of the R-wave, whereas end-systolic measurements (Mmode and B-mode) were obtained at the time of minimum
internal chamber dimensions.
For the assessment of LVM, three different algorithms
were applied: one based on B-mode-guided M-mode measurements and two based on B-mode measurements. The first
algorithm is based on M-mode data (PENN convention) (9)
LVMPENN
⫽ 1.05[(IVST ⫹ LVEDD ⫹ PWT)3 ⫺ (LVEDD)3]
(1)
where the factor 1.05 represents the specific density of the
myocardium. IVST and PWT are LV wall dimensions (interventricular septum thickness and posterior wall thickness,
respectively).
The second algorithm is the area-length method, based on
short- and long-axis B-mode data
冋
LVMArea-Length ⫽ 1.05
册
5
5
A1共L ⫹ T兲 ⫺ A2 共L兲
6
6
(2)
where A1 and A2 are the epicardial and endocardial shortaxis areas, L is the length in the long-axis view, and T is the
mean wall thickness calculated from A1 and A2 (5).
The third formula represents the truncated-ellipsoid
method
LVMTruncated-Ellipsoid ⫽ 1.05 ␲
冤 冦
冧 冢
冣冥
2
2
共a ⫹ T兲 ⫹ d ⫺ d3
a ⫹ d ⫺ d3
3
3
2
2
⫻ 共b ⫹ T兲
⫺b
关3共a ⫹ T兲2兴
3a2
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Animal housing and body weight. All animal procedures
were approved by the local committee for animal care and
conducted in conformity with the “Guiding Principles for
Research Involving Animals and Human Beings” established
by the American Physiological Society. C57BL/6 mice of either sex (Charles River; Sulzfeld, Germany) were housed at
24°C with a 12:12-h light-dark cycle. C57BL/6 mice (35 male
and 34 female) were prospectively included in this study and
euthanized on postnatal days 21, 30, 50, 70, and 111 (P21,
P30, P50, P70, and P111, respectively). A senescence group (6
male and 4 female) was investigated on postnatal day 580
(P580). Arterial blood pressure measurements, electrocardiography (ECG) recordings, LV muscle mass (LVM) evaluation, and ECs were performed on P21, P30, P50, P70, P111,
and P580. Body weight was determined from P21 to P50 each
3rd day, from P50 to P75 each 5th day, and from P75 to P111
each 14th day. The senescence group was weighed on P580.
Arterial blood pressure. Mice were anesthetized with enflurane (3% for induction or surgical procedures and 0.8–1%
for blood pressure measurements) in 70% nitrous oxide and
30% oxygen by face mask. The degree of anesthesia was
adapted to minimize cardiodepressive effects. Thus the HR
during anesthesia was maintained in the physiological range
of conscious mice under resting conditions (21, 27). The core
temperature was maintained at 37°C using a feedback-controlled heating pad. Mean arterial blood pressure (MABP),
systolic blood pressure (SBP), and diastolic blood pressure
(DBP) were recorded in the left carotid artery with an 8-cmlong fluid-filled polyethylene-10 catheter (pressure transducer, Braun; Melsungen, Germany; and Servomed blood
pressure monitor, Hellige; Freiburg, Germany).
Electrocardiography. Lead II surface ECG was recorded
simultaneously to arterial blood pressure recordings with a
Servomed ECG monitor (Hellige). HR, sinus cycle length
(SCL), PQ, QRS, and QT periods were evaluated as previously described (16). QRS was measured from the Q-wave
onset to the return of the S-wave to the isoelectric line, and
the QT interval was defined from the Q-wave onset to the
final return of the uni- or bipolar T-wave to the isoelectric
line (Fig. 1). The QT was rate corrected (QTc) according to
Mitchell (28).
Echocardiography. For comparison, EC and HM were performed on identical mice on the same day. EC was performed
using a commercially available ultrasound device equipped
with a new linear array transducer operating at an emission
frequency of 15 MHz with frame rates up to 280 Hz (HDI5000, Philips Medical Systems; Bothell, WA). Animals were
anesthetized, and the HR was monitored as described above.
Parasternal short- and long-axis views were obtained in
two-dimensional B-mode. At least 20 cardiac cycles were
obtained in B-mode imaging for each view, and each imaging
plane was acquired three times to assess reproducibility.
One-dimensional M-mode imaging was performed two-dimensionally guided in the parasternal short-axis view at the
level of the papillary muscle. Parasternal short-axis views
were divided into six segments, and long-axis views were
divided into seven segments (4). Imaging was considered
adequate if the endocardial and epicardial borders could be
properly visualized in five or more segments. The endocardial
borders were manually traced on the innermost endocardial
edge while the epicardial borders were defined by tracing
along the first bright pixel adjacent to myocardial tissue (5).
LV end-diastolic and end-systolic internal chamber diameters (LVEDD and LVESD, respectively) and wall thickness
was assessed from M-mode traces (leading edge to trailing
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MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
enzymatically as previously described for guinea pigs, rats,
and rabbits (24). Briefly, animals were euthanized and the
hearts excised. Hearts were prepared in Tyrode solution with
EGTA instead of CaCl2 (in mM: 135 NaCl, 4 KCl, 1 MgCl2, 2
HEPES, 2.6 EGTA, and 10 glucose and 1 mg/ml BSA; pH 7.4)
and mounted in a Langendorff perfusion system. Pressure
was adjusted to 0.05 bar and temperature to 36°C. Hearts
were perfused with Tyrode solution with EGTA for 5 min
and, subsequently, with high-K⫹ solution for 5 min [in mM:
4 NaCl, 10 KCl, 130 K-glutamate, 1 MgCl2, 0.05 CaCl2, 2
HEPES, and 10 glucose and 1 mg/ml BSA; pH 7.4 (KOH)]
before enzymes were added to the high-K⫹ solution. The
hearts were perfused first for 8–10 min with trypsin (1,000
Bae units/40 ml, Roche Molecular Biochemicals; Mannheim,
Germany) and second for 10–13 min with collagenase (type
L, 25 mg in 40 ml, Sigma; St. Louis, MO) and finally sectioned in small parts, which were directly transferred into
normal Tyrode solution (composition as described above) but
with 1.8 mM CaCl2 instead of EGTA plus trypsin inhibitor
(0.167 mg/ml, Sigma). In this solution, the pieces were disintegrated completely by stirring with glass rods. The solution was passed through a filter (125 ␮m mesh width) and
centrifuged gently. The cells were taken from the pellet. For
the isolation of myocytes from the hearts of P21 mice, enzyme
concentrations and perfusion times were reduced by one-half.
Isolated cells were kept in oxygenated standard Tyrode solution (in mM: 135 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 2
HEPES, and 11 glucose and 1 mg/ml BSA; pH 7.4) at 22°C up
to 6 h until use. Sarcomere shortening of ventricular myo-
Fig. 2. A: representative image of hematoxylin-eosin-stained heart section
stack viewed from the ventricle apex
toward valves. The frontal section represents the position of in vivo echocardiography (EC) 4.5 mm from the apex,
whereas the more apical sections of
this three-dimensional file are omitted.
The left ventricular (LV) cavum is colored red, and the right ventricular (RV)
cavum is colored blue. B: EC M-mode
sweep obtained in the corresponding
imaging plane of the same heart in
vivo. C: short-axis view in the EC Bmode; D: long-axis view in the EC Bmode. E and F: position of the measured areas and distances. A1 and A2,
epicardial and endocardial short-axis
areas; L, length in the long-axis view;
a, full major radius; b, minor axis radius of the internal diameter of the LV
at the tip of the papillary muscle (as
assessed in the long-axis view); d, truncated major radius.
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where b is the minor axis radius of the internal diameter of
the LV at the tip of the papillary muscle (as assessed in the
long-axis view). b subdivides the length (L) into the full major
radius (a) and the truncated major radius (d). The average
thickness T was calculated from the epicardial and endocardial areas (5).
LVM histomorphometry. Hearts were excised after blood
pressure and ECG recordings and perfused in a Langendorff
apparatus with Ca2⫹-free Tyrode solution (in mM: 135 NaCl,
4 KCl, 1 MgCl2, 2 HEPES, and 2.6 EGTA; pH 7.4 and
perfusion pressure 5 kPa) to yield myocardial relaxation and
to maintain the heart under reproducible conditions for freezing in liquid N2. Transversal cryostat sections (20 ␮m thick)
were taken in 1-mm steps parallel to the atrioventricular
groove starting 0.5 mm from the apex. After the sections were
stained with hematoxylin and eosin (H-E), magnified and
calibrated images of the sections were generated with a
charge-coupled device camera. Images were analyzed morphometrically 1) for myocardial wall thickness at the same
position as that used for in vivo EC and 2) for LV mass using
three-dimensional reconstruction (Fig. 2) with image analysis software (Scion Image 4; Frederick, MD). Tibia length was
measured to calculate individual heart weight-to-tibia length
ratios.
Sarcomere shortening. For the recording of sarcomere
shortening, isolated ventricular myocytes were prepared
from female mice of four age groups (⫾SD; group 1: P21 ⫾ 1,
n ⫽ 4; group 2: P93 ⫾ 4, n ⫽ 4; group 3: P169 ⫾ 4, n ⫽ 7; and
group 4: P362 ⫾ 2, n ⫽ 4). Ventricular myocytes were isolated
MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
Y ⫽ [SS 1 ⫺ (SS 1 ⫺ PostRest)e ⫺ x/␶1]
⫹ [(SS1 ⫺ SS2)e ⫺ x/␶2] ⫺ (SS1 ⫺ SS2)
(4)
PostRest is the sarcomere length at the peak of the postrest
shortening. In the case of a negative “staircase,” SS1 represents the sarcomere length in the steady state. If the postrest
shortening is followed by a positive staircase, SS1 represents
a sarcomere length near the diastolic level. During a positive
staircase, SS2 represents the sarcomere length during the
steady state. The resulting time constants ␶1 and ␶2 were
investigated as a function of stimulation frequency. In the
case of negative staircases, SS1 and SS2 are identical, and
thus only the first part of the equation was fitted to the data.
Statistical analysis. Values are presented as means ⫾ SE.
The SE of the estimate (SEE) is given for linear regression.
Statistical comparisons were performed by unpaired Student’s t-test (two-tail criterion) or two-way ANOVA, followed
by post hoc Student’s t-tests for multiple comparisons. P ⬍
0.05 was considered significant. Bland-Altman analysis (bias
plot) was performed to assess the agreement between EC and
HM (bias ⫾ 2SD) (1). Linear regression and ANOVA were
calculated with GraphPad Prism 3.03 (GraphPad Software;
San Diego, CA).
RESULTS
Developmental changes of body weight and tibia
length. The body weights of male and female mice
increased continuously from 10.2 ⫾ 0.3 and 9.8 ⫾ 0.3 g
at P21 to 33.2 ⫾ 1.2 and 29.4 ⫾ 1.8 g at P580, respectively. In female mice, the body weight increased
slower until P60. Between P60 and P580, the body
weights of adult male and female mice increased at a
comparable rate by 40.1% and 44.1%, respectively. The
body weight of male mice was higher at any given time
(Fig. 3A). Tibia length developed without gender differences, but growth slowed down after P70 (Fig. 3B).
The heart weight-to-body weight ratio remained constant but was higher in female mice except for P580
(5.00 ⫾ 0.19 in females vs. 4.25 ⫾ 0.09 in males, P ⬍
0.006; Fig. 3C), whereas the heart weight-to-tibia
length ratio increased until P50 and did not show
gender differences (Fig. 3D).
Fig. 3. A: development of body weight
(BW) in male and female C57BL/6
mice. B: development of tibia length in
male and female mice. C: calculated
heart weight (HW)-to-BW ratio (HW/
BW). D: calculated HW-to-tibia length
ratio. Values are means ⫾ SE of 8–15
mice.
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cytes was recorded with a video imaging system and SarcLen
software (IonOptix; Milton, MA). The regular striation pattern of the sarcomeres is analyzed by fast Fourier transformation. Sarcomere shortening shifts the power spectrum
peak, which corresponds to the absolute sarcomere length
(23). The video system was mounted to an inverted microscope (Zeiss IM 35; Jena, Germany; lens, Neofluar ⫻40, 0.75)
equipped with an experimental chamber with permanent
perfusion of Tyrode solution heated to 36°C. Contractions
were induced by bipolar external stimuli (0.4 ms, 30 V, SD9,
Grass; Quincy, MA). Stimuli were applied in 20-s pulse trains
interrupted by a 30-s stimulation pause. The stimulation
protocol was as follows: 0.5, 10, 1, 8, 2, 6, and 4 Hz.
The shortening records were evaluated in different ways.
First, resting sarcomere length was calculated. Second, to
obtain a representative of the shortening-frequency relationship, the five last shortening signals of each train were
averaged at each stimulation frequency. The resulting signal
was evaluated for resting sarcomere length, amplitude, duration, and relaxation time. Within age groups, the mean
values of these parameters were plotted as function of frequency. Finally, to characterize the postrest behavior of the
shortening, the answers to the different pulse trains were
investigated. The series of contractions during a pulse train
at a defined frequency was averaged within each age group.
The contraction peaks were fitted by a double-exponential
function
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MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
Developmental changes of arterial blood pressure.
SBP, DBP, MABP, and HR increased until P50 in
either sex and then remained constant until P580 (Fig.
4 and Table 1). Arterial blood pressure did not differ
between male and female mice at any age.
Developmental changes of HR, atrioventricular conduction, and repolarization. HR increased from 396 ⫾
16 beats/min in P21 to 570 ⫾ 31 beats/min in P70 mice,
whereas the SCL was shorter in young mice, accordingly. PQ decreased from P21 to P50 and remained
constant from then on. QRS remained constant during
the life span (Table 1). QT decreased from P21 to P50,
consistent with the parallel increase in HR, whereas
the QTc values, which were calculated using a modified
Bazett equation to account for the high resting HR in
mice (28), remained constant (Table 1).
Developmental changes of LV wall dimensions determined by EC and HM. LV wall thickness determined
by B-mode-guided M-mode acquisition correlated
strongly with HM (y ⫽ 0.89x ⫹ 0.052, R ⫽ 0.79, P ⬍
0.0001, SEE: 0.078 mm; Fig. 5, A–C). No significant
differences were found between anteroseptal and posterior wall dimensions in EC or HM. Wall dimensions
could be accurately assessed at all ages, even at P5
(data not shown).
Table 1. Blood pressure and ECG parameters of C57BL/6 mice at defined ages
Age, days
n
Heart rate, beats/min
SBP, mmHg
DBP, mmHg
MABP, mmHg
SCL, ms
PQ, ms
QRS, ms
QT, ms
QTc
21
30
50
70
111
580
14
396 ⫾ 16*
98.5 ⫾ 1.7*
74.3 ⫾ 2.3*
86.3 ⫾ 1.7*
191.2 ⫾ 10.5*
56.2 ⫾ 2.0*
18.5 ⫾ 0.4
48.2 ⫾ 2.3*
35.0 ⫾ 1.5
15
460 ⫾ 16*
113.2 ⫾ 2.8*
84.7 ⫾ 2.5*
97.5 ⫾ 2.7*
151.7 ⫾ 8.8*
52.7 ⫾ 1.8*
17.5 ⫾ 0.3
45.9 ⫾ 1.9*
37.9 ⫾ 2.0
8
551 ⫾ 12
127.5 ⫾ 3.1
93.4 ⫾ 2.3
110.4 ⫾ 2.4
124.8 ⫾ 6.3
47.5 ⫾ 1.3
17.1 ⫾ 0.5
39.2 ⫾ 2.2
35.1 ⫾ 1.7
8
570 ⫾ 31
128.4 ⫾ 1.2
100.0 ⫾ 2.5
113.4 ⫾ 1.6
106.8 ⫾ 2.0
47.5 ⫾ 2.4
17.5 ⫾ 0.2
39.7 ⫾ 1.2
38.4 ⫾ 0.9
14
580 ⫾ 10
130.2 ⫾ 3.0
102.4 ⫾ 2.0
115.7 ⫾ 2.1
107.7 ⫾ 3.3
45.0 ⫾ 1.4
17.7 ⫾ 0.3
35.9 ⫾ 2.2
34.7 ⫾ 2.2
10
570 ⫾ 19
134.3 ⫾ 2.5
101.4 ⫾ 2.4
117.1 ⫾ 2.9
110.0 ⫾ 3.3
44.6 ⫾ 2.4
17.2 ⫾ 0.2
34.3 ⫾ 1.3
32.7 ⫾ 1.0
Values are means ⫾ SE; n ⫽ no. of mice. PQ, QRS, and QT were taken from ECG. Rate-corrected QT (QTc) was calculated according to
Mitchell et al. (28). SBP, systolic blood pressure; DBP, diastolic blood pressure; MABP, mean arterial blood pressure; SCL, sinus cycle length.
* P ⬍ 0.05 compared with the corresponding values at postnatal days 50, 70, 111, or 580.
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Fig. 4. Mean arterial blood pressure (BP) of male and female
C57BL/6 mice over the life span from P21 to P580. BP was measured
using a liquid-filled catheter in the left common carotid artery
connected to a pressure transducer. Values are means ⫾ SE of 8–15
mice.
Developmental changes of LVM calculated from EC
and HM. Linear regression analysis revealed the
strongest correlation between LVMHM and diastolic
LVMArea-Length (y ⫽ 1.11x ⫺ 6.45, R ⫽ 0.93, P ⬍ 0.001;
Fig. 5E). The SEE was calculated as 7.01 mg for LVMArea-Length. LVMPENN underestimated LVM substantially; thus correlation between LVMHM and LVMPENN
was weaker (y ⫽ 0.84x ⫺ 10.05, R ⫽ 0.84, P ⬍ 0.001).
Although the truncated-ellipsoid method overestimated LVM systematically compared with the HM
reference, a strong positive linear correlation was also
found for this algorithm (y ⫽ 1.41x ⫺ 3.70, R ⫽ 0.86,
P ⬍ 0.001). All systolic measurements correlated less
with LVMHM, with the highest correlation between
LVMArea-Length and LVMHM (y ⫽ 1.42x ⫺ 4.90, R ⫽
0.87, P ⬍ 0.001).
Bland-Altman analysis was performed to compare
the estimates of LVM by EC with HM. The bias (mean
difference) between the EC-based algorithms and
LVMHM was lowest for the LVMArea-Length algorithm
(⫺1.9 mg; Fig. 5F). However, LVM assessment in
smaller animals resulted in slightly, albeit not significantly, higher LVM values by HM (Fig. 5F). As indicated above, Bland-Altman analysis revealed a significant overestimation of LVM by the truncated-ellipsoid
algorithm, but no proportional error (data not shown).
For LVMPENN, Bland-Altman analysis revealed a substantial proportional error with significant underestimation of smaller hearts and overestimation of bigger
hearts (P ⬍ 0.001; not shown). In particular, smaller
hearts (20–60 mg) could not be analyzed adequately
using M-mode-based algorithms.
Developmental changes of sarcomere shortening. Sarcomere shortening of isolated ventricular cardiac myocytes was monitored to compare the detected age dependency of MABP with potential changes in the
myocardium. In age groups 1–4, resting sarcomere
length ranged from 1.84 to 1.89 ␮m (group 1: 1.862 ⫾
0.006 ␮m, n ⫽ 9; group 2: 1.867 ⫾ 0.008 ␮m, n ⫽ 20;
group 3: 1.890 ⫾ 0.008 ␮m, n ⫽ 13; and group 4:
1.843 ⫾ 0.005 ␮m, n ⫽ 12; means ⫾ SE). Significant
differences could be shown between most groups (between groups 1 and 3, 1 and 4, 2 and 4, and 3 and 4),
MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
H469
indicating an increase of sarcomere length with age in
young animals, followed by a decrease in advanced age.
The duration of myocyte shortening is frequency
dependent, similar to action potential duration, i.e.,
the higher the stimulation frequency, the shorter is the
duration of shortening. At 0.5 Hz, the shortening duration ranged from 92 to 101 ms and at 10 Hz from 65
to 72 ms. Significant differences between the age
groups could not be detected at any frequency. The
relaxation time exhibited comparable frequency dependency, again without significant differences between
age groups.
Like duration of shortening, shortening amplitude is
frequency dependent. In all age groups, shortening
amplitude was minimal around 1–2 Hz; above 2 Hz, a
positive shortening-frequency relationship was found;
and below 2 Hz, a negative shortening-frequency relation was found. Interestingly, within this frame, agerelated differences could be detected. In a plot of averaged shortening amplitude versus stimulation frequency,
all shortenings of the myocytes of age group 1 are below
those of the adult animals (age groups 2–4; Fig. 6A).
There were significant differences between values of
adult myocytes and those of young ones (Fig. 6A). No
significant differences between the shortening amplitudes of the three adult groups were detected at any
stimulation frequency. Thus shortening amplitudes of
the age groups 2–4 were summarized in one group (Fig.
6B). The shortening amplitude of adult myocytes is reAJP-Heart Circ Physiol • VOL
lated positively to stimulation frequency between 2 and 6
Hz and does not increase above 6 Hz. In young myocytes,
the shortening-frequency relation is less steep and shortening amplitude is significantly smaller at all frequencies
except 2 and 10 Hz.
Myocytes of C57BL/6 mice exhibit a stimulation frequency-dependent postrest shortening behavior, i.e.,
the first shortening after a stimulation rest has a
relatively high amplitude and the following shortenings form a negative or positive Bowditch staircase
(Fig. 7, A–C). At frequencies below 6 Hz, the postrest
shortening was followed by a negative staircase in all
age groups. Adult myocytes developed positive staircases at 6, 8, and 10 Hz (Fig. 7, B and C), whereas
young myocytes formed a positive staircase only at 8
and 10 Hz. To characterize the time course of the
staircases, the peaks of the shortening amplitudes
were fit by Eq. 4. In the case of negative staircases, a
convergent fit could be gained by applying only the first
part of the equation; thus the time course could be
described by one time constant. Time constants decreased with increasing frequency, e.g., in age group 2
from 9.9 s at 0.5 Hz to 0.04 s at 4 Hz. An influence of
age on the first time constant could not be detected, i.e.,
the time course of the negative staircase did not seem
to change in our age groups. Also, the second time
constants were not different with the exception of 6 Hz,
where only the juvenile myocytes exhibited a negative
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Fig. 5. Comparison of LV wall dimensions (A–C) and LV muscle mass (LVM; D–F) determined by or calculated
from EC or histomorphometry (HM). A and D: absolute LV wall thickness (A) or calculated LV myocardial mass
(D). Values are means ⫾ SE of 6–12 mice. B: correlation of EC measurements in B-mode-guided M-mode
acquisitions on the y-axis with HM measurements on the x-axis (y ⫽ 0.89x ⫺ 0.052, R ⫽ 0.79, P ⬍ 0.0001). C and
F: Bland-Altman blots comparing EC- and HM-based determinations of wall dimensions (C) or LVM calculations
(F). E: correlation of EC measurement-based LVM calculation (area-length algorithm) on the y-axis with HM
measurement-based LVM calculation on the x-axis (y ⫽ 1.11x ⫺ 6.45, R ⫽ 0.93, P ⬍ 0.001).
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MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
staircase. However, the steady-state values of shortening were different, as shown in Fig. 6.
DISCUSSION
Although pathophysiological processes found in cardiac disease have been studied extensively with the use
of cellular and molecular biology in mice, little is
known about early postnatal heart development.
Therefore, the aim of this study was to investigate the
developmental changes of morphological and functional cardiac parameters including the early postnatal
phase.
Determination of HR and blood pressure in mice is
difficult because either parameter responds instantly
to any kind of stressful situation. HR reported in adult
P70–P120 Swiss-Webster mice using a telemetric
blood pressure transponder ranged between 450 and
500 beats/min in awake resting mice to between 600
and 650 beats/min after light physical activity without
manipulation (21). Telemetric ECG recording in conscious adult Swiss mice at light activity revealed a
sinus rhythm of 630 beats/min (30). HR higher than
700 beats/min occurs only after manipulation such as
weighing, restraining, or cage changing (21). Thus our
HR recordings under mild enflurane anesthesia,
which, in mice of a similar age, ranged from 570 beats/
min in P70 to 581 beats/min in P111, are well consistent with those in awake resting animals. Deep anesthesia yielded by 3–4% enflurane or pentobarbital
(0.05–0.08 mg/g body wt) or ketamine plus xylazine
(0.08 mg/g plus 0.12 mg/g body wt, respectively) reduces HR substantially (⬍300 beats/min) due to negative chronotropic and inotropic effects (36). Mouse HR
increased during postnatal development until P50,
which is consistent with previous findings (34). We did
not observe gender differences in HR or any other ECG
parameter. PQ, PR, and QT were longer in young mice
(P21 and P30), whereas QRS remained constant over
the life span. For explanation of age-dependent decreases in atrioventricular conduction as well as in
AJP-Heart Circ Physiol • VOL
repolarization time, in vitro electrophysiological recordings are needed.
HM using image analysis of H-E-stained cryostat
sections as applied in this study has not been used so
far to determine myocardial mass. However, the analysis of cryostat sections is a common technique to
precisely calculate brain infarct volume in mice (11, 12)
and also allows precise determination of LV myocardial mass. Because this technique is neither affected by
blood and buffer remainders nor by inconsistent ventricle preparation, it allows more reliable and reproducible ventricular myocardial mass determinations
than wet heart weight.
We calculated heart weight-to-body weight ratios for
standardized myocardial mass assessment. However,
the index was always lower in male mice except for the
maximal age tested (P580). This gender difference results from higher body weights of male mice throughout life. Whereas the heart weight-to-body weight ratio
was clearly gender dependent, we also calculated heart
weight-to-tibia length ratios, which are supposed to be
less affected by gender. Heart weight-to-tibia length,
however, increases continuously with growth and remains constant after maturity, as shown in rats (37),
because the myocardial mass in growing mice of either
sex increases numerically faster than the tibia length.
Thus, for mice younger than P70, gender-specific heart
weight-to-body weight ratios appear to be most advantageous, whereas in adult mice beyond P70 heart
weight-to-tibia length appears to be a more reliable
index.
The best agreement between HM three-dimensional
integration and EC-based mass calculation algorithms
was found for the area-length algorithm. Correlation
between EC data and HM data was even better than
for wall dimensions. This discrepancy could be expected because mass calculations that rely on measurements of areas rather than distances are not as sensitive to measurement artifacts caused by incorrect
placement of the measurement cursor (20).
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Fig. 6. Averaged sarcomere shortening amplitude of isolated ventricular myocytes of female mice plotted versus
the stimulation frequency. A: data points refer to the four age groups (AG) investigated. The measuring points of
age groups 2–4 (adult animals) were not significantly different. However, most shortening amplitudes of age group
1 differ from those of the older animals (0.5 Hz: group 1 to 3; 1 Hz: group 1 to 2 and 3; 4 Hz: group 1 to 3 and 4;
6 Hz: group 1 to 2, 3, and 4; and 8 Hz: group 1 to 2, 3, and 4). B: data of age groups 2–4 were joined and compared
with age group 1. The shortening amplitudes of group 1 were significantly smaller than those of the adult animals
at all frequencies except for 2 and 10 Hz. Values are means ⫾ SE of 6–20 cells. *P ⬍ 0.05.
MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
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We found an excellent correlation between wall dimensions by EC and HM. Best agreement was found
between end-diastolic EC data and HM data obtained
from hearts fixated in diastole. Repeated measurements delivered reproducible records. Epicardial and
endocardial structures could be clearly differentiated
both in M-mode and B-mode imaging. Papillary muscle
and the mitral valve could be easily identified. Endocardial border definition was possible in the majority of
all imaging sequences and allowed reliable measurements even in small hearts.
HM measurements in vitro revealed larger cavity
dimensions than in vivo EC. Hearts prepared for HM
were perfused with a Ca2⫹ chelating buffer at 5 kPa,
resulting in maximal dilatation, whereas in beating
hearts diastolic relaxation is always incomplete.
The data presented in this paper demonstrate the
feasibility of high-resolution EC in young, adult, and
elderly mice, allowing the assessment of developmenAJP-Heart Circ Physiol • VOL
tal changes in LV dimensions and mass. The highfrequency ultrasound transducer used in this study
allows vertical resolution of up to 90–100 ␮m in Bmode imaging. Because of HR between 400 and 600
beats/min in mice, high frame rates in EC are required
for measurements in defined phases of the cardiac
cycle. The ultrasound system used provides frame
rates up to 280 Hz, which provides sufficient temporal
resolution to study functional changes adequately (31).
Temporal and spatial resolution of EC are therefore
within the range of high-resolution MRI (35). EC as a
real-time imaging technique may be advantageous
over MRI in some scenarios because it does not require
proper image triggering by ECG, which frequently
hampers imaging by MRI.
Arterial blood pressure increased during development until P50 and remained constant after this age.
Increasing blood pressure during maturation is a novel
finding in mice, but a well-known phenomenon in hu-
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Fig. 7. Averaged sarcomere shortening
records after a 30-s stimulation rest.
Left, shortening trains of age group 1;
right, shortening trains of age group 2.
Each graph displays 20 shortenings;
therefore, the time scales are different.
The peaks of shortening ⫾ SE were
fitted by Eq. 4. A: stimulation frequency of 0.5 Hz; age group 1, average
of 10 cells; age group 2, average of 8
cells. B: stimulation frequency of 6 Hz;
age group 1, average of 5 cells; age
group 2, average of 4 cells. C: stimulation frequency of 10 Hz; age group 1,
average of 6 cells; age group 2, average
of 9 cells.
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MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
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in isolated murine cardiomyocytes without interference with their refractory time. We found a biphasic
shortening-frequency relation with a negative correlation below 2 Hz and positive correlation above. In adult
cardiomyocytes, the shortening amplitude saturated at
6 Hz. This agrees with the shortening-frequency relation of 4-mo-old C57BL/6 mice described by Lim et al.
(22). A biphasic force-frequency relation was also obtained in papillary muscle of 20-wk-old Black Swiss
mice (15). Maximal sarcomere shortening at 6 Hz was
4.4% here, and maximal cell shortening at 6 Hz was in
the same range in the study of Lim et al. (22). Thus the
values for adult cells recorded in this study appear to
be reliable. In young myocytes (age group 1), we obtained a smaller shortening amplitude in combination
with a less steep shortening-frequency relation. Because sarcomere shortening is independent of cell size,
the observed reduced shortening is an intrinsic characteristic of immature myocytes, which will result in a
reduced fractional shortening. Young myocytes proved
to be more sensitive to the enzymatic isolation procedure; therefore, we reduced both the enzyme concentration and application times (see METHODS). The
isolated young myocytes then exhibited healthy morphology, a sarcomere length above 1.84 ␮m, only a
small amount of spontaneously beating cells, and could
be paced up to 10 Hz. However, it cannot be excluded
that the reduced sarcomere shortening in these myocytes may be influenced by the isolation procedure.
Postrest behavior of murine cardiomyocytes is intriguing, because they develop a negative staircase at
low pacing frequencies that turns into a positive staircase at high frequencies. The negative staircase in
hearts and tissue strips of small rodents has been well
known for many decades. However, the change of a
negative staircase at low pacing frequency to a positive
one at high frequency has not yet been reported. The
reason for this change may be an increasing role of
calcium sequestration by the sarcoplasmic reticulum
at high pacing frequencies, because manipulations of
sarco(endo)plasmic reticulum Ca2⫹-ATPase 2 and
phospholamban change the force-frequency relation
(26). A frequency-dependent Ca2⫹ “sensitization” of the
myofilaments may also cause this effect (14). In this
context, it is interesting that positive staircases appeared only at pacing frequencies of 8 and 10 Hz in
cells of age group 1, whereas the staircase turned to a
positive one at 6 Hz in older animals. This may reflect
the less steep shortening-frequency relation in age
group 1.
In conclusion, EC allows precise noninvasive measurement of LV dimensions in P21–P580 mice and
yields reliable LV mass estimates in these mice if the
area-length algorithm is employed. The cardiac contribution to the arterial blood pressure increase during
postnatal maturation until P50 results from 1) an
increase in HR and, subsequently, cardiac output; 2)
an increase in shortening amplitude at most frequencies and a steeper shortening-frequency relation; and
3) an elevated shortening amplitude that is, due to the
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man cardiovascular development (32). Gender differences were not detected. SBP (127.5–130.2 mmHg),
MABP (110.4–115.7 mmHg), and DBP (93.4–102.4
mmHg) values found in adult C57BL/6 mice (P50–
P111) were consistent with telemetric measurements
reported for adult male Swiss-Webster mice during
light activity (110–124 mmHg) (21, 27). The 24-h average MABP reported for conscious C57Bl/6 at P38
(110 mmHg) (25) ranges between our MABP measurements in P30 (97.5 mmHg) and P50 (110.4 mmHg).
Enflurane initially increases cytosolic Ca2⫹ concentration ([Ca2⫹]i) in vitro in isolated cardiac myocytes by
inhibition of the Na⫹/Ca2⫹ exchanger (15) or by increased Ca2⫹ release from the sarcoplasmic reticulum
(5). However, ⬎1.2% enflurane as well as isoflurane
reduce blood pressure and HR in mice concentration
dependently (unpublished data), indicating that in
vivo, during the steady state, anesthesia [Ca2⫹]i-decreasing effects, i.e., inhibition of Ca2⫹ influx through
L-type Ca2⫹ channels (2) or depletion of intracellular
Ca2⫹ stores, prevail over [Ca2⫹]i-increasing effects, i.e.,
release from intracellular stores and/or decreased Ca2⫹
outward transport.
Blood pressure is determined by both cardiac output
(HR ⫻ stroke volume) and total peripheral resistance.
Because HR was lower in P21 and P30 compared with
adult animals, we decided to also monitor myocardial
contractility. Cardiac contractility was recorded as sarcomere shortening in isolated myocytes during zero
load shortening. Sarcomere shortening is superior to
cell shortening, which is a more commonly recorded
parameter, because it is independent of cell size and
thus allows direct comparison between cells (29). Zero
load shortening of isolated myocytes is monitored
widely as assessment for cardiac contractility (8). Concerning the basic contractile characteristics like forcefrequency relation and postrest behavior, the results of
zero load shortenings are comparable to force recordings of cardiac muscle strips, e.g., changes in the shortening-frequency relation also appeared in the forcefrequency relation (3).
The sarcomere length found in this study (between
1.84 and 1.89 ␮m, depending on the age group) proves
that the cells used were well maintained. A clearly
shorter sarcomere length of ⬃1.72 ␮m in murine cardiomyocytes has been reported by Lim et al. (22) (1.72
␮m in 5-mo-old and 1.71 ␮m in 34-mo-old B6C3F1
hybrid mice), which may result from a different cell
isolation protocol. In rats, a decrease in resting sarcomere length with age has been reported when comparing three age groups, 2 mo (1.85 ␮m), 6–9 mo (1.83
␮m), and 24–25 mo (1.82 ␮m) (13). Such a distinct age
dependency was not detected here, likely due to the
younger age of the animals investigated in this study.
However, the sarcomere length in age group 4 (362
days) was also significantly shorter than that in the
younger animals.
HR from 550 to 600 beats/min, as reported here in
adult mice ⱖP50, corresponds to frequencies from 9 to
10 Hz. We therefore recorded shortening up to 10 Hz,
which is the highest contraction rate we could achieve
MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
positive shortening-frequency relation, induced by the
age-dependent HR increase.
The authors are grateful to Michael Hans for help with databases
and to Philips-Ultrasound, namely, Pat Rafter and Heinrich Beckermann, for technical support.
K. Tiemann, P. C. Djoufack, U. Dreiner, R. Meyer, C. Grohé, T.
Lewalter, and K. B. Fink were supported by Interdisciplinary Faculty Grant BONFOR O-708.0209 and A. Ghanem was supported by
the Studienstiftung des Deutschen Volkes.
REFERENCES
AJP-Heart Circ Physiol • VOL
17. Haworth RA and Goknur AB. Inhibition of sodium/calcium
exchange and calcium channels of heart cells by volatile anesthestics. Anesthesiology 82: 1255–1265, 1995.
18. Hoit BD. New approaches to phenotypic analysis in adult mice.
J Mol Cell Cardiol 33: 27–35, 2001.
19. Jones SP, Girod WG, Palazzo AJ, Granger DN, Grisham
MB, Jourd’Heuil D, Huang PL, and Lefer DJ. Myocardial
ischemia-reperfusion injury is exacerbated in absence of endothelial cell nitric oxide synthase. Am J Physiol Heart Circ
Physiol 276: H1567–H1573, 1999.
20. Kiatchoosakun S, Restivo J, Kirkpatrick D, and Hoit BD.
Assessment of left ventricular mass in mice: comparison between
two-dimensional and M-mode echocardiography. Echocardiography 19: 199–205, 2002.
21. Kramer K, Voss HP, Grimbergen JA, Mills PA, Huetteman
D, Zwiers L, and Brockway B. Telemetric monitoring of blood
pressure in freely moving mice: a preliminary study. Lab Anim
34: 272–280, 2000.
22. Lim CC, Apstein CS, Colucci WS, and Liao R. Impaired cell
shortening and relengthening with increased pacing frequency
are intrinsic to the senescent mouse cardiomyocyte. J Mol Cell
Cardiol 32: 2075–2082, 2000.
23. Lim CC, Helmes MH, Sawyer DB, Jain M, and Liao R.
High-throughput assessment of calcium sensitivity in skinned
cardiac myocytes. Am J Physiol Heart Circ Physiol 281: H969–
H974, 2001.
24. Linz KW and Meyer R. Profile and kinetics of L-type calcium
current during the cardiac ventricular action potential compared
in guinea-pigs, rats and rabbits. Pflügers Arch 439: 588–599,
2000.
25. Mattson DL. Comparison of arterial blood pressure in different
strains of mice. Am J Hypertens 14: 405–408, 2001.
26. Meyer M, Bluhm WF, He H, Post SR, Giordano FJ, Lew
WY, and Dillmann WH. Phospholamban-to-SERCA2 ratio controls the force-frequency relationship. Am J Physiol Heart Circ
Physiol 276: H779–H785, 1999.
27. Mills PA, Huetteman DA, Brockway BP, Zwiers LM,
Gelsema AJ, Schwartz RS, and Kramer K. A new method
for measurement of blood pressure, heart rate, and activity in
the mouse by radiotelemetry. J Appl Physiol 88: 1537–1544,
2000.
28. Mitchell GF, Jeron A, and Koren G. Measurement of heart
rate and Q-T interval in the conscious mouse. Am J Physiol
Heart Circ Physiol 274: H747–H751, 1998.
29. Mukherjee R, Crawford FA, Hewett KW, and Spinale FG.
Cell and sarcomere contractile performance from the same cardiocyte using video microscopy. J Appl Physiol 74: 2023–2033,
1993.
30. Nuyens D, Stengl M, Dugarmaa S, Rossenbacker T, Compernolle V, Rudy Y, Smits JF, Flameng W, Clancy CE,
Moons L, Vos MA, Dewerchin M, Benndorf K, Collen D,
Carmeliet E, and Carmeliet P. Abrupt rate accelerations or
premature beats cause life-threatening arrhythmias in mice
with long-QT3 syndrome. Nat Med 7: 1021–1027, 2001.
31. Roell W, Lu ZJ, Bloch W, Siedner S, Tiemann K, Xia Y,
Stoecker E, Fleischmann M, Bohlen H, Stehle R, Kolossov E, Brem G, Addicks K, Pfitzer G, Welz A, Hescheler J, and Fleischmann BK. Cellular cardiomyoplasty
improves survival after myocardial injury. Circulation 105:
2435–2441, 2002.
32. Rosner B, Prineas RJ, Loggie JM, and Daniels SR. Blood
pressure nomograms for children and adolescents, by height,
sex, and age, in the United States. J Pediatr 123: 871–886,
1993.
33. Van Eickels M, Grohe C, Cleutjens JP, Janssen BJ,
Wellens HJ, and Doevendans PA. 17␤-Estradiol attenuates
the development of pressure-overload hypertrophy. Circulation
104: 1419–1423, 2001.
34. Wang L, Swirp S, and Duff H. Age-dependent response of the
electrocardiogram to K⫹ channel blockers in mice. Am J Physiol
Cell Physiol 278: C73–C80, 2000.
284 • FEBRUARY 2003 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 5, 2017
1. Bland JM and Altman DJ. Regression analysis. Lancet 1:
908–909, 1986.
2. Bosnjak ZJ, Supan FD, and Rusch NJ. The effects of halothane, enflurane, and isoflurane on calcium current in isolated
canine ventricular cells. Anesthesiology 74: 340–345, 1991.
3. Brixius K, Hoischen S, Reuter H, Lasek K, and Schwinger
RH. Force/shortening-frequency relationship in multicellular
muscle strips and single cardiomyocytes of human failing and
nonfailing hearts. J Card Fail 7: 335–341, 2001.
4. Cerqueira MD, Weissman NJ, Dilsizian V, Jacobs AK, Kaul
S, Laskey WK, Pennell DJ, Rumberger JA, Ryan T, and
Verani MS. Standardized myocardial segmentation and nomenclature for tomographic imaging of the heart: a statement for
healthcare professionals from the Cardiac Imaging Committee of
the Council on Clinical Cardiology of the American Heart Association. J Nucl Cardiol 9: 240–245, 2002.
5. Collins KA, Korcarz CE, Shroff SG, Bednarz JE, Fentzke
RC, Lin H, Leiden JM, and Lang RM. Accuracy of echocardiographic estimates of left ventricular mass in mice. Am J
Physiol Heart Circ Physiol 280: H1954–H1962, 2001.
6. Connelly TJ and Coronado R. Activation of the Ca2⫹ release
channel of cardiac sarcoplasmic reticulum by volatile anesthetics. Anesthesiology 81: 459–469, 1994.
7. De Winther MP and Hofker MH. New mouse models for
lipoprotein metabolism and atherosclerosis. Curr Opin Lipidol
13: 191–197, 2002.
8. Delbridge LM and Roos KP. Optical methods to evaluate the
contractile function of unloaded isolated cardiac myocytes. J Mol
Cell Cardiol 29: 11–25, 1997.
9. Devereux RB and Reichek N. Echocardiographic determination of left ventricular mass in man. Anatomic validation of the
method. Circulation 55: 613–618, 1977.
10. Doevendans PA, Daemen MJ, de Muinck ED, and Smits
JF. Cardiovascular phenotyping in mice. Cardiovasc Res 39:
34–49, 1998.
11. Endres M, Namura S, Shimizu-Sasamata M, Waeber C,
Zhang L, Gomez-Isla T, Hyman BT, and Moskowitz MA.
Attenuation of delayed neuronal death after mild focal ischemia
in mice by inhibition of the caspase family. J Cereb Blood Flow
Metab 18: 238–247, 1998.
12. Fink K, Zhu J, Namura S, Shimizu-Sasamata M, Endres M,
Ma J, Dalkara T, Yuan J, and Moskowitz MA. Prolonged
therapeutic window for ischemic brain damage caused by delayed caspase activation. J Cereb Blood Flow Metab 18: 1071–
1076, 1998.
13. Fraticelli A, Josephson R, Danziger R, Lakatta E, and
Spurgeon H. Morphological and contractile characteristics of
rat cardiac myocytes from maturation to senescence. Am J
Physiol Heart Circ Physiol 257: H259–H265, 1989.
14. Gao WD, Perez NG, and Marban E. Calcium cycling and
contractile activation in intact mouse cardiac muscle. J Physiol
507: 175–184, 1998.
15. Gao WD, Perez NG, Seidman CE, Seidman JG, and Marban E. Altered cardiac excitation-contraction coupling in mutant mice with familial hypertrophic cardiomyopathy. J Clin
Invest 103: 661–666, 1999.
16. Hagendorff A, Schumacher B, Kirchhoff S, Luderitz B,
and Willecke K. Conduction disturbances and increased atrial
vulnerability in connexin40-deficient mice analyzed by transesophageal stimulation. Circulation 99: 1508–1515, 1999.
H473
H474
MYOCARDIAL CONTRACTION DEVELOPMENT IN C57BL/6 MICE
35. Wiesmann F, Ruff J, Hiller KH, Rommel E, Haase A, and
Neubauer S. Developmental changes of cardiac function and
mass assessed with MRI in neonatal, juvenile, and adult mice.
Am J Physiol Heart Circ Physiol 278: H652–H657, 2000.
36. Yang XP, Liu YH, Rhaleb NE, Kurihara N, Kim HE, and
Carretero OA. Echocardiographic assessment of cardiac func-
tion in conscious and anesthetized mice. Am J Physiol Heart Circ
Physiol 277: H1967–H1974, 1999.
37. Yin FC, Spurgeon HA, Rakusan K, Weisfeldt ML, and
Lakatta EG. Use of tibial length to quantify cardiac hypertrophy: application in the aging rat. Am J Physiol Heart Circ
Physiol 243: H941–H947, 1982.
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 5, 2017
AJP-Heart Circ Physiol • VOL
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