From the Division of Clinical Pharmacology,
Departments of Medicine and
Pharmacology,
Georgetown University Medical Center, Washington, D. C. 20007, § Cardiovascular Research Institute, Washington Hospital
Center, Washington, D. C. 20010, the ** Department of Anesthesiology,
Mayo Foundation, Rochester, Minnesota 55905, and the
¶ Departments of Molecular and Cellular Pharmacology and
Pathology, University of Miami School of Medicine,
Miami, Florida 33136
Received for publication, July 27, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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The cardiac troponin T (TnT) I79N mutation has
been linked to familial hypertrophic cardiomyopathy and a high
incidence of sudden death, despite causing little or no cardiac
hypertrophy. In skinned fibers, I79N increased myofilamental calcium
sensitivity (Miller, T., Szczesna, D., Housmans, P. R., Zhao, J.,
deFreitas, F., Gomes, A. V., Culbreath, L., McCue, J., Wang, Y.,
Xu, Y., Kerrick, W. G., and Potter, J. D. (2001)
J. Biol. Chem. 276, 3743-3755). To further study the
functional consequences of this mutation, we compared the cardiac
performance of transgenic mice expressing either human TnT-I79N or
human wild-type TnT. In isolated hearts, cardiac function was different
depending on the Ca2+ concentration of the perfusate;
systolic function was significantly increased in Tg-I79N hearts at 0.5 and 1 mmol/liter. At higher Ca2+ concentrations, systolic
function was not different, but diastolic dysfunction became manifest
as increased end-diastolic pressure and time to 90% relaxation.
In vivo measurements by echocardiography and Doppler
confirmed that base-line systolic function was significantly higher in
Tg-I79N mice without evidence for diastolic dysfunction. Inotropic
stimulation with isoproterenol resulted only in a modest contractile
response but caused significant mortality in Tg-I79N mice. Doppler
studies ruled out aortic outflow obstruction and were consistent with
increased chamber stiffness. We conclude that in vivo, the
increased myofilament Ca2+ sensitivity due to the I79N
mutation enhances base-line contractility but leads to cardiac
dysfunction during inotropic stimulation.
Mutations in cardiac troponin T
(TnT)1 have been implicated
in familial hypertrophic cardiomyopathy (FHC) (1-5). Individuals with
cardiac TnT mutations appear to have a high incidence of sudden cardiac
death at a young age, although heterozygote individuals have either
little or no cardiac hypertrophy (1, 3, 4). At present, there is
no clear understanding as to why TnT mutations in particular pose a
high risk for sudden death, as opposed to, for example, mutations in
the myosin heavy chain, which usually cause a much greater degree of
cardiac hypertrophy. Sudden cardiac death of FHC patients is often
caused by ventricular tachyarrhythmias (6), but its cause remains
unknown for patients with TnT mutations. In fact, the clinical features
of hypertrophic cardiomyopathy have been established mostly without
knowledge of the genotype and may not apply to patients carrying
specific TnT mutations. Given the paucity of clinical information, a
transgenic mouse model provides the opportunity to study the functional
consequences of a TnT mutation in an in vivo system.
To investigate the mechanisms of how a TnT mutation alters cardiac
function and lead to sudden cardiac death, we have generated transgenic
mice expressing the human cardiac TnT-I79N mutant (Tg-I79N). Similar to
humans carrying this mutation, Tg-I79N mice show no cardiac hypertrophy
(7). We found a large increase in Ca2+ sensitivity of the
skinned cardiac fibers from Tg-I79N mice compared with fibers from
transgenic mice expressing human wild-type TnT (Tg-WT), but maximal
developed force was significantly lower in cardiac fibers from Tg-I79N
mice (7).
In this study, we examined the effect of the I79N mutation on cardiac
performance and electrophysiological properties of the whole heart,
in vitro and in vivo. We found that the effect of the mutant protein was dependent on the inotropic state of the heart;
under base-line conditions, systolic function was higher in Tg-I79N
mice, with little effect on diastolic function. However, during
inotropic stimulation with high extracellular Ca2+ or with
isoproterenol, diastolic function was impaired, both in the isolated
heart and in vivo. Based on these results, we conclude that
the increased myofilament Ca2+ sensitivity due to the I79N
mutation enhances base-line contractility but leads to cardiac
dysfunction during inotropic stimulation. In humans carrying the
TnT-I79N mutation, this mechanism may contribute to excess mortality
during exercise or in pathophysiological states with high levels of
endogenous catecholamines, even in the absence of significant cardiac hypertrophy.
Experimental Protocol--
All studies were carried out
according to National Institutes of Health guidelines and approved by
the institutional animal care and use committee. To examine the effect
of the I79N mutation on cardiac performance and electrophysiology, we
compared 3-4-month-old mice: transgenic mice expressing either human
wild-type (Tg-WT, line 3) or mutant cardiac TnT (Tg-I79N, lines 8 and
9) and nontransgenic littermates (non-Tg). The generation and in
vitro characterization of this transgenic model has been described
(7). All three transgenic lines had high expression levels of the
transgenic protein, with 70, 52, and 34% replacement of endogenous
mouse TnT (line 3, 8, and 9, respectively).
Isolated Perfused Heart Preparation--
Mice were
anesthetized with 20 ml/kg 2% tribromoethanol (Avertin) via
intraperitoneal injection. After a surgical level of anesthesia was
confirmed, a thoracotomy performed, the heart was removed, and the
animal was killed by exsanguination. Hearts of four non-Tg, five Tg-WT,
and five Tg-I79N mice (line 9) were isolated and perfused in the
Langendorff mode as described previously for mouse hearts (8). In
brief, the chest was opened, the heart was rapidly excised, and the
aorta was cannulated. Retrograde perfusion via the aorta was carried
out at a constant perfusion pressure of 68 mm Hg at 37 °C. The flow
of thebesian veins was drained via a thin polyethylene tube (PE-10)
pierced through the apex of the left ventricle. Krebs-Henseleit buffer
containing 118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4,
0.5 mM Na-EDTA, 25 mM NaHCO3, 1.2 mM KH2PO4, and 11 mM
glucose was prepared at the time of the experiment. To maintain a
constant heart rate, the atrioventricular node was ablated, and the
hearts were epicardially paced at 420 min
Hearts were allowed to equilibrate for 15 min before base-line left
ventricular pressure recordings were obtained. To examine the effects
of different inotropic states, the heart was then perfused with KH
buffer containing free [Ca2+] of 0.5, 1, 2, 3, and 4 mM. Free [Ca2+]o was calculated using
Fabiato's program (9). Contractile measurements were obtained at the
end of each 5 min at the respective [Ca2+]o. All
perfusion buffers were equilibrated with 95% O2 plus 5%
CO2 for at least 1 h prior to the experiment, yielding a pH of 7.4.
Measurement of Isovolumic Cardiac Performance--
A
water-filled balloon custom-made of polyvinylchloride film was inserted
through the mitral valve into the left ventricle via an incision in the
left atrium as described (8). A 2.0-French microtip transducer
(Millar, Inc, Texas) was guided through the polyethylene tube and
positioned inside the balloon. The balloon was inflated to adjust the
end-diastolic pressure (EDP) at 6-8 mm Hg, and the balloon volume was
held constant for the duration of the experiment. The left ventricular
pressure was digitized at 1000 samples/s with the use of a commercially
available data acquisition system (PowerLab, ADInstruments, Inc.). The
fast sampling rate coupled with the use of a pressure catheter with a
flat frequency response up to 10 kHz allowed us to do the detailed
kinetic analysis of the pressure recordings outlined below.
The following indices of cardiac performance were measured off-line
using custom built software (National Instruments, Inc.) and averaged
from three consecutive beats at base line and at the end of each
Ca2+ concentration step: left ventricular systolic
pressure, EDP, developed pressure (the difference
between systolic pressure and EDP), the minimum and maximum values of
the first derivative of left ventricular pressure
(+dP/dt and Surface Electrocardiogram (ECG) and Systolic Blood Pressure
Measurements--
For the ECG measurements, mice were anesthetized
either with ketamine (100 mg/kg) and xylazine (5 mg/kg) or with 20 ml/kg of 2% tribromoethanol (Avertin) via intraperitoneal injection. Mice were positioned prone in a shielded box, with all four extremities immersed in 3 M KCl-filled wells to reduce skin resistance.
The bottom of the shielded box was heated to 37 °C using a
circulating water bath to maintain a stable body temperature during the
experiment. To accurately capture the ECG signals from mice, which have
the high heart rates (500-600 beats) and a very short QRS duration, a
custom-built ECG amplifier (Vibraspec, Bear Island, ME 04662) was used.
This amplifier provided a high frequency response and large gain (1000 Hz and 10,000-fold gain) and was used to record bipolar limb leads in
standard fashion as described (10). Signals were digitized at 1 kHz
using a National Instruments, Inc. data acquisition system with custom
built software. For each animal, PR and QRS intervals were measured
from three consecutive beats in leads I and II and averaged. For QT
interval measurements, the recordings were signal-averaged over
a 30-s time interval using the peak of the R-wave as the fiduciary
point as described (11). This procedure reduced the effect of
respiratory and motion artifacts on QT measurements. The heart rate was
measured as the average over the 30-s interval, and the QT interval was
measured in the lead with the longest and most prominent T-wave.
Systolic blood pressure and heart rate were measured in conscious and
anesthetized mice using a commercially available, noninvasive computerized tail-cuff system (BP-2000, Visitech, Apex, NC).
Echocardiography and Doppler Measurements--
Mice were
anesthetized as for the ECG measurements and positioned supine on a
heated surface (37 °C) to maintain a stable body temperature during
the experiment. Echocardiography and Doppler measurements were
performed with a 1-cm gel standoff in a left lateral position similar
to the technique used by Hoit et al. (12). An Acuson Sequoia
C256 system with a linear transducer in 13-MHz mode was used to obtain
high resolution two-dimensional and M-mode measurements (17). A
two-dimensional guided M-mode of the LV was performed in short axis at
the tip of the mitral leaflets and through the center of the LV cavity.
The proper probe position was confirmed by rotating the probe 90° and
obtaining two-dimensional guided long axis M-mode measurements in the
same animal. Two-dimensional guided Doppler flow measurements of aortic outflow and mitral inflow were obtained. Mitral inflow velocities were
recorded only after extensive scanning from multiple vantage points to
ensure that the maximal velocity was obtained. In most situations, this
was an apical window that corresponds to an "off-axis" apical
window (displaced toward the parasternal window). A limb-lead ECG was
simultaneously recorded during echocardiography and stored both on
computer, for off-line analysis, and on the echocardiogram videotape.
The echo images were digitized from videotape and measurements performed using a digital image analysis package (Nova
Microsonics/Eastman Kodak Co.). LV end-systolic and end-diastolic
internal dimensions (LVESO and LVEDD, respectively) and LV wall
thickness were measured for each animal from the M-mode image. Aortic
outflow velocity, aortic ejection time, aortic acceleration time to
peak outflow, early and late mitral inflow velocity (E-wave and A-wave,
respectively), and E-wave deceleration time were measured from the
Doppler recordings in standard fashion, and the E to A ratio was
calculated. To assess overall left ventricular systolic performance, LV
shortening fraction (SF = (LVEDD Isoproterenol Application--
Isoproterenol was given as
a bolus intraperitoneal injection after stable base-line ECG and
echocardiography recordings had been obtained (5-10 min). Mice were
initially challenged with a dose of 1.5 mg/kg (high dose) based on
reports in the literature (12). After deaths occurred in the Tg-I79N
group, we examined the effects of a lower dose (0.1 mg/kg), which was
sufficient to produce a significant increase in heart rate in all four
groups of mice. All ECG and echocardiographic assessments described
above were repeated at 3-5 min after injection, when a significant
increase in heart rate was present.
Mice Exercise Protocol--
A swimming protocol described by
Geisterfer-Lowrance et al. (13) was utilized to examine the
effects of chronic aerobic exercise. Groups of four animals, 2 months
old, representing the different lines, were exercised by swimming. Mice
were adapted to the swimming program by beginning with 10-min sessions
two times a day separated by 4 h. These were incremented by 10 min per day until reaching 90 min per session. The program was completed in
4 weeks. During each session, they were monitored for inability to
sustain the exercise and/or sudden death.
Autopsy and Histopathology of Tg Mice--
Every mouse
that died during the isoproterenol challenge was autopsied to rule out
other causes of death such as hemorrhage. This is especially important
because drugs were administered via intraperitoneal injection. One
death (a non-Tg mouse) was indeed due to a hemorrhage from the right
iliac artery close to the injection site. This animal was excluded from
further analysis. We did not find any evidence for bleeding in any of
the other deaths.
Three separate groups of mice (four non-Tg, four Tg-WT, six
Tg-I79N line 9) were specifically sacrificed to examine the heart morphology and histology. Two mice of each group had undergone chronic
exercise. After euthanasia, each mouse was autopsied with dissection of
the heart. The heart was weighed, and any gross abnormalities (chamber
enlargement, mural thrombi, etc.) were recorded. The 10% buffered
formalin-fixed heart was sectioned along its transverse plane and
embedded in paraffin. Microscopic sections were cut from the transected
surface at 4-6 µm and stained with hematoxylin and eosin for overall
morphology and Mason's trichrome for collagen. The histologic slides
were examined for the presence of asymmetric hypertrophy, fibrosis, or
myocyte disarray without knowledge of genotype, degree of exercise
training, or in vivo test results.
Computer Simulations--
Force generation in Tg-WT and Tg-I79N
myocardium was simulated by computer based on a modification of the
model of Robertson et al. (14) and a two-state cross-bridge
model (15) utilizing an exponential dependence of the TnC off-rate for
Ca2+ (Ca2+-specific site II) on force (16).
These simulations have been extensively described previously (7).
Statistical Analysis--
All experiments (left ventricular
pressure, ECG, and echocardiography) were done in random sequence in
respect to the genotype, and measurements were taken by a single
observer who was blinded to the genotype. Differences between groups
were assessed using a one-way analysis of variance. If statistically
significant differences were found, individual groups were compared
with Student's two-sided t test without correction for
multiple comparisons. Incidence rates were compared using the
Cardiac Performance of the Isolated Heart
Base-line Measurements--
To examine the functional consequences
of the I79N mutation in the whole heart, we measured indices of cardiac
performance in the isolated perfused, isovolumically contracting heart.
This experimental preparation permits exact control of the loading conditions and has been used to examine both systolic and diastolic function in other murine models (8, 17-19). Systolic function was
assessed from systolic pressure, time to peak pressure, and maximum
rate of pressure rise (+dP/dt). Diastolic
function was assessed from the maximum rate of pressure decay
(
Force traces were simulated as would be observed during isometric
twitch contractions of Tg-WT and Tg-I79N ventricular myocardium using
variables (off-rate of Ca2+ from troponin C and
cross-bridge dissociation constant) derived previously (7). Transgenic
I79N myocardium relaxed more slowly than Tg-WT, which is consistent
with the experimental observations in isovolumically beating heart.
Ca2+ Dependence of Cardiac Performance--
We also
examined the changes in cardiac performance in response to changes in
[Ca2+]o (Fig. 2).
At [Ca2+]o of 0.5 and 1 mM, developed
pressure was significantly higher in Tg-I79N than in Tg-WT hearts (Fig.
2A). At higher [Ca2+]o, however,
developed pressure was not significantly different between the two
groups. Thus, there was a leftward shift of the
[Ca2+]o-pressure relationship in Tg-I79N hearts.
Indices of diastolic function demonstrated a different
[Ca2+]o dependence; at
[Ca2+]o of 0.5 and 1 mM, EDP was
significantly lower in I79N hearts (Fig. 2B), while time to
90% relaxation was not significantly different from Tg-WT (Fig.
2C). At higher [Ca2+]o, EDP was
significantly higher (Fig. 2B), and time to 90% relaxation
was significantly longer in I79N hearts than in Tg-WT hearts (Fig.
2C). Therefore, I79N hearts demonstrate increased systolic
performance at low [Ca2+]o but performance
comparable with that of Tg-WT at a [Ca2+]o
of 2 mM or above. On the other hand, diastolic function is
comparable with that of Tg-WT at low [Ca2+]o but
significantly impaired at high [Ca2+]o.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
via a bipolar Ag-AgCl contact electrode placed on the base of the right ventricle.
dP/dt), ratio
of +dP/dt and
dP/dt, time
to reach peak systolic pressure, time to reach peak
+dP/dt, and time from peak systolic pressure to
reach 90% relaxation. To determine the time constant of isovolumic
pressure decay, the left ventricle (LV) pressure curve was fitted from the time of minimum dP/dt to a level 5 mm Hg
above the EDP of the next beat using an monoexponential function as
described (8).
LVESD)/LVEDD) and heart
rate-corrected velocity of circumferential fiber shortening (VCFc = (SF)(RR0.5)/ejection time) were calculated.
2 test statistic. Results were considered statistically
significant if the p value was less than 0.05. Unless
otherwise indicated, results are expressed as means and S.E.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
dP/dt) and from time to 90% relaxation and
the time constant of isovolumic relaxation. When hearts were paced at
420 min
1 and end-diastolic pressure was set
at 6-8 mm Hg, systolic performance was not statistically different
between Tg-I79N, Tg-WT, and non-Tg hearts (Table
I). However, the initial rise of pressure
was faster in Tg-I79N hearts (Fig. 1,
arrow). Thus, time to maximum +dP/dt was significantly shorter in the Tg-I79N hearts (Table I). On the other
hand, time to 90% relaxation and the relaxation time constant were
significantly longer in the Tg-I79N hearts compared with both Tg-WT and
non-Tg hearts (Table I). The pressure tracings of Fig. 1 illustrate the
slower relaxation of the Tg-I79N hearts. The ratio of
+dP/dt to
dP/dt, which
can be used to assess diastolic function relative to systolic function
(20), was significantly increased in Tg-I79N mice (Table I).
Furthermore, the LV balloon volume at an end-diastolic pressure of 8 mm
Hg was lower in Tg-I79N mice (Tg-I79N: 13.0 ± 0.3 µl; Tg-WT:
15.1 ± 0.8 µl, p < 0.05). Taken together,
these results indicate that Tg-I79N hearts had significantly impaired
diastolic function.
Base-line contractile parameters of isovolumetrically contracting mouse
hearts (mean ± S.E.)
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Fig. 1.
Left ventricular isovolumic pressure tracings
from Tg-WT and Tg-I79N (line 9) mice. Representative examples are
shown of isovolumic pressure tracings recorded at 1 kHz from a Tg-WT
and a Tg-I79N heart. Note the faster onset of pressure rise
(arrow) and the slower pressure decay in the Tg-I79N
heart.
View larger version (12K):
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Fig. 2.
Relationship between Ca2+
concentration and isovolumic contractile performance.
A, developed pressure; B, EDP; C, time
to 90% relaxation as a function of perfusate Ca2+
concentrations in hearts from Tg-I79N mice (line 9, filled
squares) and from Tg-WT mice (open
squares). Data are expressed as means ± S.E. *,
p < 0.05.
In Vivo Cardiac Performance and Cardiac Electrophysiology of Tg Mice
Base-line Echocardiography and Doppler
Measurements--
Echocardiography and Doppler measurements have been
used extensively to characterize the cardiac phenotype of transgenic
mouse models (21). We measured cardiac dimensions, indices of
systolic function, and diastolic
ventricular filling by two-dimensional guided M-mode (Fig.
3A) and Doppler (Fig. 3B). Table
II summarizes the results. Indices of
systolic contractile function (LV shortening fraction and VCFc) were
significantly higher in both Tg-I79N lines compared with Tg-WT.
Evidence for increased contractility was also present on flow
measurements by Doppler. Tg-I79N mice had a significantly shorter
time to peak outflow velocity (Fig. 3B). Base-line diastolic
filling parameters measured by Doppler (E-wave, A-wave, E-wave
deceleration time, E/A ratio) were not statistically different between
all four lines. Thus, unlike the isolated perfused heart, systolic
function was increased in Tg-I79N mice, but diastolic function appeared
to be normal. One reason for this apparent discrepancy may be related
to the slow heart rates (~250 min1) induced
by the ketamine/xylazine anesthesia. We therefore repeated the
echocardiography and Doppler measurements using a different anesthetic,
Avertin, which produces much less bradycardia. Since there were no
large differences in the cardiac parameters between Tg-I79N lines 8 and
9, we compared Tg-I79N line 9 with Tg-WT mice and non-Tg mice. Heart
rates indeed increased to a similar extent in all groups (Table II).
Systolic function was significantly different between all three groups
(LV shortening fraction: Tg-I79N 43 ± 1.2%, Tg-WT 27 ± 1.2%, non-Tg 33 ± 1.2%, p < 0.00001 by
analysis of variance). Interestingly, at these faster heart rates, the end-diastolic dimension was significantly smaller in Tg-I79N mice (3.2 ± 0.1 mm, p < 0.0001 against both WT and
non-Tg) than in Tg-WT mice (3.8 ± 0.1 mm) or in non-Tg mice
(3.6 ± 0.2 mm). Nevertheless, Doppler indices of diastolic
filling were not significantly different. Unfortunately, the A-wave
could only be measured in less than 30% of the examined animals.
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To exclude the possibility that the differences in LV shortening fraction were merely due to a difference in afterload, we also measured the blood pressure in anesthetized mice (Avertin). Systolic blood pressure was not significantly different (Tg-I79N mice 95 ± 4 mm Hg, n = 10, versus Tg-WT 89 ± 4 mm Hg, n = 8, p = not significant). Taken together, these data demonstrate that anesthetized Tg-I79N mice have increased systolic cardiac performance in vivo. A likely explanation for the difference between the systolic function in vivo and in the isolated heart is the relatively low free [Ca2+] of mouse blood; average free [Ca2+] measured in four nontransgenic littermates after 15 min of ketamine/xylazine anesthesia was 1.26 ± 0.03 mM, which is significantly lower than the perfusate [Ca2+] for the base-line isolated heart recordings.
In another series of experiments, we examined whether the increased systolic function can be demonstrated in conscious Tg-I79N mice. Systolic blood pressure was not significantly different between Tg-I79N (112 ± 2 mm Hg, n = 32) and Tg-WT mice (114 ± 2 mm Hg, n = 23, p = not significant). Heart rate, on the other hand, was significantly lower in Tg-I79N mice (521 beats/min versus 604 beats/min, p < 0.00001). It remains to be determined whether the slow heart rate of Tg-I79N mice represents a vagal response to the increased cardiac contractility.
ECG Measurements--
Cardiac arrhythmias are a potential cause of
sudden death. Limb-lead ECGs were recorded from anesthetized non-Tg,
Tg-WT, and Tg-I79N mice (Fig.
4A). The most significant
finding was that Tg-I79N had a consistently prolonged PR interval
representing the conduction time from the atria to the ventricle. The
PR prolongation was independent of the anesthetic used (data not shown)
and was more pronounced in chronically exercised mice (Fig.
4B). As shown in Fig. 4C, the degree of PR
prolongation appeared to be related to the expression level of the
mutant TnT protein; line 8 with higher levels of Tg-I79N had more PR
prolongation than line 9 with lower protein levels. Although PR
prolongation is not a common ECG finding in FHC patients, left atrial
enlargement is often observed. Left atrial hypertrophy in Tg-I79N mice
was documented on autopsy. QRS duration and heart rate were not
statistically different, but the QT interval was significantly shorter
in Tg-I79N mice (line 9: 43 ± 2.6 ms, n = 8; line
8: 51 ± 6.0 ms, n = 5; Tg-WT: 67 ± 4.5 ms,
n = 13; non-Tg: 67 ± 2.5 ms, n = 13, p < 0.05 between groups). Arrhythmias were not
observed in Tg-I79N mice at base line.
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In Vivo Cardiac Performance under Inotropic Stimulation--
Based
on the results from the isolated heart studies and the simulation
studies, raising [Ca2+]o predominantly affects
diastolic function (Figs. 1 and 2). To test this hypothesis in
vivo, we examined the effect of raising intracellular
[Ca2+] via inotropic stimulation with isoproterenol,
because it is difficult to raise plasma-free
[Ca2+]o in vivo without causing
significant toxicity. To detect abnormalities in diastolic function,
ketamine/xylazine anesthesia was used for echocardiography and Doppler
studies, because, based on our experience from the base-line studies,
it allowed better Doppler recordings of mitral inflow compared with
Avertin anesthesia. When mice were injected with 1.5 mg/kg
isoproterenol, several Tg-I79N mice died shortly after injection.
Therefore, echocardiography and Doppler studies were performed with a
lower dose of 0.1 mg/kg isoproterenol intraperitoneally (Table II).
Whereas isoproterenol produced a dramatic increase in contractility in
non-TG and Tg-WT mice (LV shortening fraction increased by 26 ± 3% in Tg-WT, n = 12), Tg-I79N mice showed a smaller
inotropic response (LV shortening fraction increased only by 9 ± 3% and 8 ± 3% in lines 9 and 8, n = 7 and 6, p < 0.01 against WT). In fact, four out of 13 Tg-I79N mice, but none of the 12 Tg-WT mice, later developed global LV hypokinesis and died. Doppler measurements demonstrated that this impaired inotropic response was not caused by an outflow obstruction, since both WT and I79N had similar aortic outflow velocities (see Table
II). Impaired ventricular relaxation and increased diastolic chamber
stiffness, as evidenced by the shorter E-wave deceleration time, loss
of A-wave, and increased E/A ratio on Doppler measurements in I79N mice
(Fig. 5) probably contributed to the
isoproterenol-induced cardiac dysfunction.
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To further investigate the isoproterenol-induced deaths of TgI79N mice,
we repeated the isoproterenol experiments with continuous ECG
monitoring. At the lower dose (0.1 mg/kg intraperitoneally), 75% of
the examined Tg-I79N mice exhibited loss in R-wave amplitude, QRS
widening, and T-wave inversion, and 25% died (Fig.
6A). Heart rate peaked 3 min
after injection of isoproterenol before significant ECG changes
developed in the Tg-I79N mice. Despite a similar maximal heart rate in
all four groups, the significant PR prolongation in the Tg-I79N mice
was consistently observed (Tg-I79N line 8, n = 5:
43 ± 3.2 ms; line 9, n = 8: 44 ± 1.6 ms;
Tg-WT, n = 12: 35 ± 1.2 ms; non-Tg,
n = 13: 34 ± 0.8 ms, p < 0.05 Tg-79 versus Tg-WT). QRS duration and QT interval were not
statistically different between the four groups. At a higher dose of
isoproterenol (1.5 mg/kg intraperitoneally), all Tg-I79N mice exhibited
loss in R-wave amplitude, QRS widening, and T-wave inversion, and 75%
of the animals died within 20 min of injection (Fig. 6B).
Compared with the sustained acceleration in heart rate of Tg-WT mice,
the same isoproterenol injection resulted only in a very transient
increase in heart rate in Tg-I79N mice, followed by progressive
conduction blocks and eventually death (Fig.
7). The insets in Fig. 7
illustrate the profound changes in QRS morphology (progressive loss in
R-wave amplitude, QRS widening, and T-wave inversion) that developed after isoproterenol injection. Ventricular tachyarrhythmias were observed only in a few mice and were never sustained more than a few
seconds. Tg-I79N mice died with progressive conduction blocks present
on ECG. Autopsy examination demonstrated dilated atria with pulmonary
and hepatic congestion and no evidence for bleeding. Taken together,
these data indicate that the isoproterenol-induced deaths were not
caused by ventricular tachyarrhythmias. It remains to be determined
whether the bradyarrhythmias were the primary cause of the deaths or
whether they were a secondary phenomenon (e.g. as a result
of pulmonary edema and respiratory failure that developed because of
the isoproterenol-induced cardiac dysfunction).
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Effect of Chronic Exercise-- Eight Tg-WT, eight Tg-I79N, and eight non-Tg mice underwent 4 weeks of chronic swimming exercise. Two deaths occurred, one each in the Tg-WT and the Tg-I79N group. No apparent difference in exercise tolerance was observed between the groups. Except for the increase in PR interval in Tg-I79N mice (Fig. 4B), there were no other ECG abnormalities or arrhythmias found in ECG studies obtained after the completion of the exercise program.
Histopathology of Tg Mice
Hearts of Tg-I79N were smaller than age- and sex-matched Tg-WT and
non-Tg mice, with a significantly decreased heart weight/body weight
ratio, as already reported in the preceding article (7). However, we
found evidence for left atrial hypertrophy (left atrial weight Tg-I79N,
line 9: 10 ± 2 mg versus Tg-WT: 5 ± 1 mg,
n = 6 each, p = 0.08), which was
significantly different when the values were corrected for the lower
ventricular weight of Tg-I79N mice (left atrial weight/heart weight
ratio Tg-I79N: 0.048 ± 0.009 versus Tg-WT: 0.023 ± 0.003, n = 6 each, p = 0.025).
Histological examinations of hematoxylin and eosin
(H+E)-stained ventricular sections from non-Tg, Tg-WT
(line 3), and Tg-I79N (line
9) revealed normal myocyte architecture and no major
differences between the three lines (Fig.
8, upper panels).
Tg-WT (line 3) and Tg-I79N (line 9) may have somewhat more nuclear
pleomorphism (greater variation in the size and appearance of the
nuclei) than the non-Tg mice. On trichrome (TRIC)-stained
sections, where muscle and red blood cells are stained red and
areas containing fibrosis or collagen are stained blue, no differences
between the three lines were observed. Chronic exercise had no effect
on cardiac histology in either group.
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DISCUSSION |
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The major finding of this study is that significant alterations in cardiac contractile and electrical properties occur in transgenic mice carrying the TnT-I79N mutation. In the isolated heart, the effect of the I79N mutation on cardiac performance depended on the Ca2+ concentration of the perfusate; systolic function was significantly increased in Tg-I79N hearts only at 0.5 and 1 mmol/liter [Ca2+]o, with no difference in diastolic function. At higher [Ca2+]o, diastolic dysfunction became manifest as both an increase in end-diastolic pressure and in time to 90% relaxation (Table I and Figs. 1 and 2). In vivo measurements with echocardiography and Doppler confirmed that, at base-line, systolic function was significantly higher in Tg-I79N mice without evidence for diastolic dysfunction (Table II and Fig. 3). However, inotropic stimulation with isoproterenol resulted only in a modest initial contractile response but later caused significant mortality only in Tg-I79N mice (Fig. 6). Doppler studies ruled out aortic outflow obstruction and were consistent with impaired diastolic filling (Table II and Fig. 5). Continuous ECG monitoring ruled out ventricular tachyarrhythmias as a cause of death and showed changes in ECG morphology and bradyarrhythmias (Fig. 7). Morphological and histological studies ruled out significant ventricular hypertrophy, fibrosis, or myocyte disarray (Fig. 8). Our results are consistent with the hypothesis that the increased myofilament Ca2+ sensitivity due to the I79N mutation enhances base-line contractility but leads to cardiac dysfunction under inotropic stimulation. This mechanism may contribute to excess mortality during exercise or other states with high levels of endogenous catecholamines, even in the absence of significant cardiac hypertrophy.
Cardiac Performance of Tg-I79N Mice-- In the isolated heart of I79N mice, systolic function was higher, and relaxation was impaired. Although no data from humans or other transgenic models carrying the I79N mutation exist, similar findings have been reported in a mouse model expressing the FHC-linked TnT-R92Q mutation (17-19), whereas impaired systolic function has been reported in an independently created transgenic mouse model of the same mutations (22). Data presented here may provide explanation for the disparate results; at low [Ca2+]o, the positive inotropic effect of the leftward shift in Ca2+ sensitivity of skinned fibers predominates (Fig. 2A). At higher [Ca2+]o or during inotropic stimulation, peak intracellular [Ca2+] may reach levels closer to the flat portion of the calcium-force relationship in I79N mice, and little additional force is produced. Under those conditions, the impaired relaxation due to the increased myofilamental Ca2+ sensitivity predominates. Thus, the measured cardiac performance depends critically on the inotropic state of the preparation and may differ depending on the experimental conditions.
What other factors besides the level of free Ca2+ could have contributed to the difference in systolic function measured under base-line conditions (i.e. increased in vivo, but no difference in the isolated heart)? One explanation is that end-diastolic pressure was set at 8 mm Hg for the isolated heart measurements. This experimental strategy allows one to compare contractile performance of hearts of different sizes but underestimates developed pressure when diastolic compliance is impaired (31). However, the lower LV balloon volume of I79N hearts would also be consistent with a decreased diastolic compliance. Thus, if diastolic compliance was impaired in the I79N hearts at a perfusate [Ca2+]o of 2 mM, their systolic performance may actually have been underestimated in our isolated heart experiments.
Contractile indices obtained by echocardiography are significantly influenced by changes in preload and afterload. There are several reasons to believe that the reported in vivo results represent true differences in myocardial systolic performance; afterload, as estimated by systolic blood pressure, was not significantly different between Tg-I79N and Tg-WT. Although we did not directly measure the end-diastolic pressure (preload) in vivo, the end-diastolic LV dimensions can be used as a correlate if heart rate and diastolic compliance are the same. End-diastolic LV dimensions were significantly smaller in the Tg-I79N mice, suggesting that either EDP was lower, or compliance was decreased in Tg-I79N mice. Thus, changes in preload are unlikely to be the explanation for the profound increase of the shortening fraction in the I79N mice.
In the Tg-I79N mice, ventricular relaxation was predicted to be slowed under basal conditions, based on our simulation studies and at a [Ca2+]o of 2 mM or above based on the isolated heart studies (Table I). Yet, we did not find evidence for impaired filling on Doppler under base-line conditions. One possibility is that the Doppler technique is not sensitive enough to detect subtle increases in diastolic chamber stiffness. Another possible explanation is the relatively low free [Ca2+]o (1.25 mM) in mouse blood. In the isolated heart experiments, diastolic relaxation times were not significantly different at [Ca2+]o of 0.5 or 1 mM, and EDP was significantly higher in the Tg-WT compared with Tg-I79N hearts (Fig. 2B). This increase of EDP at low [Ca2+]o is somewhat surprising but has been observed previously in the isolated heart preparation and is thought to be related to the turgor effect, when the perfusion pressure exceeds the systolic pressure during continuous retrograde perfusion (18).
Cardiac Electrophysiology and Arrhythmias--
Unlike in the
MHC403+/ mouse model of FHC (23), ventricular arrhythmias were not
observed in sedentary or exercised I79N mice at base line. However,
this may not be expected during the short period of ECG recordings
employed in our study, since even in FHC patients ventricular
arrhythmias are relatively rare and occur on average less than once per
month (6).
Surprisingly, transgenic mice expressing TnT-I79N had a consistently prolonged PR interval on ECG recordings. The PR prolongation was independent of the anesthetic used, was correlated to gene dosage (Fig. 4C), and was more pronounced in chronically exercised (Fig. 4B). The duration of the PR interval is determined by conduction from the atria to the ventricle and is controlled by the autonomic nervous system. An increase in vagal tone therefore could account for the PR prolongation and could represent a reflex mechanism to increased cardiac contractility of I79N mice (e.g. via the baroreceptor reflex). The slower heart rate of conscious I79N mice is consistent with this hypothesis. Alternatively, atrial hypertrophy has been associated with PR prolongation in other mouse models of cardiac hypertrophy (10). We found significant atrial hypertrophy in I79N mice, and this may have contributed to the PR prolongation. The atrial hypertrophy could result from atrial I79N overexpression driven by the MHC promoter, which occurs earlier in the atria than in the ventricle (24), or might be a compensatory response to impaired diastolic relaxation of the ventricle, or both. Atrial hypertrophy has also been described in mice expressing the TnT-R92Q mutation (17).
Isoproterenol-induced Contractile Abnormalities, ECG Changes, and Mortality-- Based on our echocardiography and Doppler measurements, isoproterenol had little effect on systolic indices but resulted in significantly altered measurements of diastolic filling in Tg-I79N mice. Although other factors such as pulmonary vein flow, end-diastolic pressure, and atrial compliance can contribute to the shape of Doppler signals during mitral inflow, Doppler indices can be used to detect diastolic dysfunction; Little et al. (25) have shown that in particular the E-wave deceleration time is well correlated with diastolic chamber stiffness independently of preload. Accordingly, changes in this parameter could detect even the transient increase of diastolic chamber stiffness that occurs after coronary artery bypass surgery (26, 27). Together with the data from the isolated heart and the modeling studies, the significantly shortened E-wave deceleration time (Table II and Fig. 5) in both transgenic lines suggests that isoproterenol induced an increased diastolic chamber stiffness in Tg-I79N mice. Obviously, an extensive cardiac catheterization study will have to confirm these observations in the future.
One surprising finding was the high mortality of Tg-I79N mice following the isoproterenol injection. Based on our echocardiography and Doppler findings, the deaths were not caused by an aortic outflow obstruction, which is a common cause of death in end-stage human FHC (28). Deaths were also not due to ventricular tachyarrhythmias, another major cause of death in FHC patients (6), or hemorrhage. Rather, Tg-I79N mice died after isoproterenol induced changes in ECG morphology (loss of R-wave, ST segment depression, and T-wave inversion) and heart block (Fig. 6). Although not diagnostic, similar ECG abnormalities can be observed with acute cardiac ischemia. Myocardial ischemia could be detected in all FHC patients that survived a cardiac arrest (29) and could play a role in the isoproterenol-induced mortality of Tg-I79N mice.
Several lines of evidence suggest that autonomic dysregulaton could have contributed to the isoproterenol induced mortality as follows. (i) Tg-I79N shows evidence of an increase in vagal output (lower basal heart rate, PR prolongation). (ii) Deaths occurred due to bradycardia and heart block. (iii) Isoproterenol challenge can be used to induce vasovagal syncope in humans. (iv) In FHC patients, autonomic dysfunction can cause an inappropriate decrease in systemic vascular resistance at high work loads (30) and has been reported in a patient carrying a TnT mutation (4). Somewhat against this hypothesis speaks the fact that both ECG changes and Doppler abnormalities occurred before significant bradycardia was present. Thus, vagally mediated bradycardia and vasodilation could have contributed to the Tg-I79N phenotype but are probably not the sole explanation of the isoproterenol-induced mortality.
It remains to be determined why excess mortality was restricted to the isoproterenol challenge under anesthesia and did not occur during the chronic swimming exercise, which should have produced a considerable endogenous catecholamine response. This suggests that the additional cardiovascular stress of anesthesia was needed to decompensate the Tg-I79N mice. On the other hand, sudden death in response to exercise is a rare event even in patients with FHC, and our chronic swimming exercise of 4 weeks in a limited number of mice might not have been sensitive enough to demonstrate significant differences in a rare event.
Thus, in an attempt to relate our previously published results obtained
from skinned fibers to the data presented here, we suggest the
following potential mechanisms for the isoproterenol-induced mortality.
(i) The increased Ca2+ sensitivity of myofilaments (7) will
enhance basal cardiac contractility (Table II, Figs. 2 and 3), but the
resulting slower decay of the Ca2+ transient and force
could prevent or hinder the increase in relaxation rate that normally
occurs after -adrenergic stimulation, causing diastolic dysfunction
(Figs. 2 and 5) (ii) The loss of the protective myofilamental pH
regulation (e.g. the loss of contractile uncoupling at low
pH previously reported (7)) could worsen any regional oxygen
supply-demand mismatch induced by impaired diastolic relaxation (Fig.
1). Thus, the isoproterenol-induced ECG changes (Fig. 7) may represent
evidence for regional myocardial ischemia or direct catecholamine
toxicity. (iii) Although I79N fibers may produce more force at low
[Ca2+]o (Fig. 2A), raising
[Ca2+] provides little increase in force, since the
fibers are already operating close to the flat part of the force/pCa
relationship. Thus, the lower maximal force per cross-sectional area
previously reported (7) might decrease the contractile reserve
available to increase cardiac output appropriately in response to
isoproterenol-induced vasodilatation. (iv) The cardiac
hypercontractility may activate ventricular mechanoreceptors and
thereby cause chronic alterations in vagal tone, which could contribute
to bradycardia and death in response to isoproterenol. In summary,
Tg-I79N mice may become a valuable model to explore potential
mechanisms of sudden death induced by the TnT-I79N mutation.
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ACKNOWLEDGEMENTS |
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We are very grateful for the help of Dr. Carl Apstein, who taught us the technique of isovolumic contractility measurements in the isolated mouse heart.
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FOOTNOTES |
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* This work was supported by American Heart Association fellowship 9920387U and Pharmaceutical Research and Manufacturers of America Foundation Faculty Development Award (to B. C. K.), and National Institutes of Health Grants AR 45391 and 2R01 HL42325 (to J. D. P.), R01 HL16152 (to M. M.), and R01 GM36365 (to P. R. H.).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.
§§ To whom correspondence should be addressed: Dept. of Molecular and Cellular Pharmacology, University of Miami School of Medicine, 1600 N.W. 10th Ave., Miami, FL 33136. Tel.: 305-243-5874; Fax: 305-243-6233; E-mail: jdpotter@miami.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M006745200
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ABBREVIATIONS |
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The abbreviations used are: TnT, cardiac troponin T; FHC, familial hypertrophic cardiomyopathy; WT, wild type; EDP, end-diastolic pressure; LV, left ventricle; ECG, electrocardiogram; Tg, transgenic.
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