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Wallenberg and Lundberg Laboratories (E.B., E.O., B.M., B.S., M.L.X.F., F.W.), Research Center for Endocrinology and Metabolism (O.I., G.M., J.I.), Department of Internal Medicine, Department of Clinical Physiology (V.K.), Sahlgrenska University Hospital, 413 45 Goteborg, Sweden; Department of Physiology (M.B., J.T.), Goteborg University, Medicinaregatan 7B, 413 90, Goteborg, Sweden; Max-Delbrück-Centrum fur Molekulare Medizin (W.S.), MDC, Robert-Rossle-Strasse 10, Berlin, D-13125, Germany
Address all correspondence and requests for reprints to: Jorgen Isgaard, M.D., Ph.D., Research Center for Endocrinology and Metabolism, Sahlgrenska University Hospital, 413 45 Goteborg, Sweden. E-mail: jorgen.isgaard{at}ss.gu.se
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Abstract |
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Female transgenic mice expressing bGH gene (n = 11) 8 months old and aged matched controls (n = 11) were used. They were studied with two-dimensional guided M-mode and Doppler echocardiography. The animals (n = 6) for each group were examined with 31P magnetic resonance spectroscopy to determine the cardiac energy status. Transgenic mice had a significantly higher body weight (BW), 53.2 ± 2.4 vs. 34.6 ± 3.7 g (P < 0.0001) and hypertrophy of left ventricle (LV) compared with normal controls: LV mass/BW 5.6 ± 1.6 vs. 2.7 ± 0.2 mg/g, P < 0.01. Several indexes of systolic function were depressed in transgenic animals compared with controls mice such as shortening fraction 25 ± 3.0% vs. 39.9 ± 3.1%; ejection fraction, 57 ± 9 vs. 77 ± 5; mean velocity of circumferential shortening, 4.5 ± 0.8 vs. 7.0 ± 1.1 circ/sec, p < 0.01. Creatine phosphate-to-ATP ratio was significantly lower in bGH overexpressing mice (1.3 ± 0.08 vs. 2.1 ± 0.23 in controls, P < 0.05). Ultrastructural examination of the hearts from transgenic mice revealed substantial changes of mitochondria.
This study provides new insight into possible mechanisms behind the deteriorating effects of long exposure to high level of GH on heart function.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic technology has provided the research community with different lines of transgenic mice (TG) that have integrated rat, human, or bovine GH gene under the control of an independent inducible system into their genome. They express very high level of the foreign GH and have been extensively used to investigate the process of growth and the effect of excess GH in many organs and tissues (19). There is a lack of information regarding the cardiovascular physiology of these transgenic mice. Therefore, using adult transgenic mice overexpressing bovine GH (bGH) gene we aimed to assess in vivo, the effects of long-term exposure to high levels of circulating concentrations of GH on cardiac structure, function and energy status. Our hypothesis was that deterioration of cardiac performance could be linked to alterations in energy metabolism. To test that we combined two noninvasive tools echocardiography and in vivo volume-selective 31P magnetic resonance spectroscopy (31P MRS).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After performing echocardiography and/or 31PMRS, the experiment was terminated with an overdose of ip chloral hydrate; the blood samples were collected; hearts were excised and the atria and vessels dissected. Free biventricular tissue was weighed and frozen in liquid nitrogen and stored at -80 C. Liver and kidneys were also weighed.
The study protocol was approved by the Animal Ethics Committee of the Gothenburg’s University and conducted in accordance with NIH guidelines for use of experimental animals.
Echocardiography
Twenty offspring of Mt-bGH transgenic male mice derived from one
founder animal were investigated, 10 transgenic and 10 eight-month-old
nontransgenic female mice. Mice were anesthetized with isoflurane
(0.4–0.6%) and mixture of O2 and
N2O 2:1, using a nose mask. The anterior chest
was shaved and ECG leads were placed on extremities. A warming pad was
used to maintain body temperature. Cardiac ultrasound studies were
performed using a commercially available ultrasonograph (GE Vingmed
Ultrasonograph, West Milwaukee, WI) by methods previously validated
(21, 22). A 10 MHz linear transducer was used for obtaining of
two-dimensional parasternal short axis imaging closed to the papillary
muscles. This served as a guide for M-mode tracing. For pulsed-wave
Doppler recordings, the minimum sample size was used and the pulse
frequency of 5 MHz to record the estimated peak left ventricle (LV)
outflow tract velocity and the mitral inflow velocities. All tracings
were recorded at a sweep of 200 mm/s and were stored in magnetic
optical discs for off-line measurements. Off-line measurements were
done using image analysis system (Echo Pac 5.4, Ving Med) by one
observer without prior knowledge of the type of mice.
Data analysis
M-mode measurements of LV internal diameters and wall thickness
in diastole and systole were made by using the leading-edge convention
of the American Society of Echocardiography. End diastole was taken at
the onset of the QRS complex, and end systole was taken at the peak
inward of interventricular septum (IVS) motion. Four or more beats were
averaged for each measurement.
LV fraction shortening (FS) was calculated as follows. (LVIDd-LVIDs)/LVIDd x 100 where LVIDd and LVIDs are respectively LV internal diameters in diastole and systole. Ejection fraction (EF), relative wall thickness (RWT), velocity of circumferential shortening (Vcf) were calculated by formulas described elsewhere (23).
IVS thickening (IVS %) = [(IVSsystole - IVSdiastole)/IVSdiastole] x 100
Posterior wall thickening (PW %) = [(PWsystole - PWdiastole)/ PWdiastole] x 100.
31P Magnetic resonance spectroscopy
Twelve mice (6 TG and 6 nontransgenic), randomly chosen were
investigated noninvasively with 31P-MRS for
evaluation of cardiac energy metabolism.
Mice were given induction anesthesia with fentanyl/fluanizon (Hypnorm, Janssen Pharmaceuticals, Beerse, Belgium) 0.5 mg/kg and diazepam (Stesolid) 2.5 mg/kg ip and kept anesthetized by continued anesthesia with isoflurane (0.4–0.6%) in the mixture of O2/N2O 2:1, at the flow rate 1–2 liters/min). Body temperature was maintained constant (37 C) during the investigation. Cardiac gated MR imaging and spectroscopy were performed on a 2.35 T horizontal magnet with a 20 cm bore (Bruker BioSpec Products, Inc., Faellanden, Switzerland, 24/30). A double tuned (1H and 31P) Helmholtz coil of 2 cm was used for transmission and reception of radiofrequencies. Anesthetized mouse was placed between Helmholtz coil in a prone position in such a way that chest region was lying in the B1 field. More technical details about the localization strategy were given in our earlier investigations on the skeletal muscle (24). Cardiac-gated gradient echo method was used for imaging of the thoraco-abdominal region in transversal and sagittal plane (5 slices each with thickness of 1.5 mm). The volume of interest (VOI) was 8 x 4 x 4 mm (128 µl) and included as much of the LV ventricle as possible. The position of the VOI was defined both in sagittal and transversal plane. Care was taken not to include parts of diaphragm, chest skeletal muscles, and liver into VOI. After selecting the VOI, the magnetic field homogeneity was further optimized by localized shimming using STEAM localization method. Cardiac-gated Image Selected In vivo Spectroscopy (ISIS) was then employed for acquisition of 31P MR spectra of the heart. Acquisition parameters were 4096 scans with repetition time 2.5 sec, giving a total scan time of approximately 3 h.
To correct for blood contamination of the ATP signal, 31P MRS of the mouse blood was performed. The correction of ATP was then calculated according to the formula: ATP = calculated ATP - [0.5 x (ATP/2.3 DPG)blood x 2,3-DPGheart)] (25, 26).
Blood pressure measurement
Animals were anesthetized with chloral hydrate (459 µg/g ip).
The left carotid artery was cannulated and connected to a Statham P23
DC transducer. Mean arterial blood pressure was recorded on a Grass
Model 7D polygraph. Blood pressure values were calculated as means of
30 samples.
Serum IGF-I
Serum was prepared and frozen at -20 C until assayed for
measurements of IGF-I level. The serum concentration of IGF-I was
determined by a hydrochlorid acid-ethanol extraction RIA using human
IGF-I for labeling (Nichols Institute Diagnostics, San
Juan Capistrano, CA). The assay was performed according to the
manufacturer’s protocol after centrifugation of precipitated serum
proteins at +4 C followed by neutralization with Tris base and another
centrifugation at +4 C.
Histologic examination
The whole hearts were cut serially from apex to the base (n
= 6). Every second slice was paraffin embedded. From each paraffin
block a section was cut, stained with Masson’s trichrome and mounted.
The measurements were carried out on an IBAS system and all images used
for measuring were captured with high-resolution digital camera
(Progress).
Briefly, in 9–10 systematically randomly selected high power fields, the colourmetric spectrum of blue stained fibrous tissue from sections of left ventricle was defined and its area fraction was calculated in the IBAS. Vascular profiles and subendocardial fibrous zone were excluded in the sampling procedures. The area fraction of diffuse fibrosis was expressed as percent of sampled myocardium.
Electron microscopy
Small pieces of the hearts were fixed in 2% glutaraldehyde in
0.1 M cacodylate buffer for 2 h at 4 C. After being
washed in PBS (0.1 phosphate buffer) for 2 h, the tissues were
postfixed with 1% osmium tetroxide for 1 h. After the dehydration
with a graded series of ethanol concentration, the samples were
embedded in Epon resin. Ultrathin sections were stained with uranyl
acetate and examined with Tesla EM BS 500.
TUNEL stain
Sections from heart tissue, 10 µm thick, were cut on a
cryostat and adhered to microscopic slides. The sections were then
fixed in 4% paraformaldehyde in PBS, pH 7.4. Endogenous peroxidase was
quenched with preincubation of the sections in 3%
H2O2 in PBS for 5 min. The
sections were then stained according to the TUNEL method using the
ApoTag plus peroxidase kit (Oncor) according to the manufacture
instructions. Thereafter the sections were counterstained with methyl
green and mounted. Sections preincubated with DNase I and sections of
ovary from 21-day-old rats served as positive controls.
Statistical analysis
All data are shown as mean ± SD, unless
otherwise stated. The significance of group differences was determined
with two-sided unpaired Student’s t test. A value of
P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Levels of IGF-I in serum were also
higher in bGH mice. The excess of bGH affected growth of various organs
to a different extent. Organ to body weight (presented as a percentage)
in transgenic mice also differed significantly from those seen in
controls (Table 1). In comparison to controls, the relative organ mass
in relation to BW was most pronounced for liver and kidneys followed by
the heart. Macroscopic appearance of the liver was characterized by
enlargement with nodular alterations. Heart weights in transgenic mice,
even when normalized to body weight, were increased compared with
normal controls. In four transgenics and five controls, blood pressure
was successfully measured showing no difference between the two groups
(mean ± SEM; bGH, 71 ± 9 mmHg; control, 84
± 4 mmHg).
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). In control heart, normal
geometry and orientation between muscle fibers and mitochondria are
observed. Myofibrils are separated by rows of mitochondria. The
mitochondria are pleomorphic and elongated in shape with well developed
cristae and outer membranes. Among them there are residual bodies with
electron dense matrix and myelin figures or lipids droplets (Fig. 1a). The ultrastructure of the transgenic
mouse heart exhibits the typical morphologic pattern with one important
exception. The mitochondria are swollen with dissolving of the regular
cristae arrangements and giant mitochondria are occasionally seen. The
outer membranes of the mitochondria are not homogeneously stained as
seen in the controls although identical contrasting procedures have
been used (Fig. 1b). Occasionally confluent mitochondria are seen; then
they are formed giant organelles over more than one sarcomere length.
The components of the sarcoplasmatic reticulum and T systems were
relatively unchanged.
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Left ventricle geometry and function (Fig. 2, Tables 2 and 3)
The mean heart rates were similar between the groups. Absolute LV
dimensions were significantly increased in bGH mice. The posterior wall
thickness was mildly increased, whereas the relative wall thickness was
not different between the two
groups. Together, these findings
indicate eccentric hypertrophy of left ventricle in transgenic
mice.
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Doppler measurement did not show any difference between the groups. Early (E-wave) and late-diastolic transmitral velocity with atrial contraction (A-wave) velocities were similar in control and transgenic animals (E wave: bGH, 6 ± 1 cm/sec; controls 6 ± 2 cm/sec); (A wave: bGH, 3 ± 1 cm/sec; controls 3 ± 1 cm/sec).
31P MRS
Representative spectra from a control and a transgenic mouse are
illustrated in Fig. 3. The amplitude and
the area of PCr relative to ATP are lower in the heart of bGH mouse.
The mean value ± SEM of PCr/ATP ratio are presented
in Fig. 4, showing a significant decrease
of this ratio in bGH mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cardiac enlargement is a consistent finding in acromegalic heart disease, presenting with varying degree of myocardial hypertrophy (4, 5, 27). In our animals, left ventricular mass and heart mass indexes were higher in transgenic mice compared with nontransgenic mice, marking a disproportionate growth of the heart compared with growth of the body. Echocardiographic results indicated presence of eccentric LV hypertrophy in the mice overexpressing bGH gene that was associated with increased interstitial fibrosis, a common finding in the hearts of acromegalic patients. Morphologic alterations were not only in the organ level but also in the cellular level. The ultrastructural alterations observed in the heart from bGH mice are mainly confined to the mitochondria. These include both changes in the size of the mitochondria, disarrangement of the cristae and outer membrane. Histologic data from other studies using bGH mice did not reveal pronounced pathological changes in cardiac tissue (28). The different age of animals might explain the discrepancy between the results.
Several clinical studies performed in patients with acromegaly at the early stages of the disease have demonstrated the presence of diastolic dysfunction at rest, while systolic function was normal whereas during exercise testing cardiac reserve is limited (4, 6, 27, 29).
The systolic cardiac function evaluated in vivo was significantly depressed in the mice overexpressing bGH gene compared with controls. In present study, fraction shortening and velocity of circumferential shortening were significantly lower in transgenic mice, underlining a severe depression of systolic function. In the presence of hypertrophy and increased interstitial fibrosis, a normal Doppler diastolic inflow is unlikely. Therefore, the "normal" transmitral velocities seen in our study could be rather a pattern of pseudonormalization as result from counterbalancing influences from both abnormal relaxation and restrictive forces. Taken together, morphological and functional similarities between our observations and clinical data made this animal model potentially suitable for studying the chronic effects of excessive GH activity in the heart.
Using the rat bearing GH-secreting tumor, both an improvement (30) and deterioration (11, 31), of cardiac performance have been reported. The positive inotropic effect of GH is known, demonstrated in vivo in normal mice (32) treated with GH for a relatively short time and in more physiological doses and in animal model of myocardial infarction (33, 34). In vitro studies on isolated papillary muscle or papillary skinned fibers have shown improved contractile performance with significantly increment in calcium sensitivity of contractile proteins (35, 36), explaining the positive inotropic effect of GH seen in the early phase of acromegaly, where a hyperkinetic syndrome is present.
Several characteristics make the rats with transplanted GH-secreting tumors different from patients with acromegaly (7) and bGH transgenic mice. What predominates in this rat model is a functional rather than trophic component of GH action, which may be attributed to a relatively short exposure to GH excess. The bGH transgenic mice are exposed earlier in life to high levels of GH, and the exposure to these high GH levels are more prolonged than in rats with a GH producing tumor.
Our study provides new information about cardiovascular physiology and metabolism of bGH transgenic mice. The compromised cardiac performance in transgenic mice was accompanied with substantial alteration in myocardial energy metabolism. Timsit et al.(35) has reported increased contractility with low energy expenditure in rat model of GH hypersecretion. The time of exposure to GH excess was 18 weeks, whereas in our model, cardiac tissue was exposed twice longer. Marked changes observed in our study underlined the importance of duration of exposure to GH excess. Cardiac bioenergetic status was evaluated with 31P MRS. The validity of this method for studying the changes in high-energy phosphate metabolites in heart in normal and pathological conditions has been extensively demonstrated in animals (37, 38, 39) and in clinical studies (26, 40).
Our preliminary data showed the feasibility of performing in vivo volume selective 31P MRS for assessment of the cardiac energy status in the mouse heart (41) that has been confirmed from others (42). The marked reduction of PCr/ATP ratio observed in our study reflects an imbalance in energy demand and supply. Several studies performed in different models of LV hypertrophy have shown an association between increase in LV mass and decreased PCr/ATP ratio (43, 44). Quaife et al. (28) have shown that bGH transgenic mice are hyperinsulinemic with normal fasting glucose levels in blood. These findings are a reflection of insulin resistance that is often seen in acromegalic patients also. Disturbances in glucose metabolism, presence of hypertrophy, and increased interstitial fibrosis could affect the availability and pattern of energy substrate utilization in the myocardium. At the same time, we observed marked morphological changes of the mitochondria in the heart of transgenic mice. Increase in size and changes in the appearance of the mitochondria may be compensatory events to defective oxidative phosphorylation, as seen in mitochondria diseases (45). Knowing the direct effect of GH on Ca2+-handling, a long-term exposure to high circulatory levels of GH might have lead to calcium overload with negative consequences on mitochondria function and integrity (46). Nevertheless, characterization of the abnormalities of mitochondria function is needed to clarify the issue.
Apoptosis was recently reported to be increased in the heart of acromegalic patients (47). However, in the present study no signs of increased apoptosis could be detected. A possible explanation for the absence of apoptosis in our study might be that we only looked at one time-point, relatively late in the process of deterioration of cardiac structure and function and that apoptosis may indeed occur at an earlier stage.
It is important to point out that our data do not give us an exact information regarding the sequence of events in this study, i.e. whether the histologic and metabolic alterations into the myocardium are causative to or occur in parallel with the depression of cardiac function. Further studies with evaluation of cardiac morphology, performance, and myocardial energy status at different time points after exposure to GH excess are needed to clarify this issue. However, it is plausible to assume that while it cannot be proved that impaired energetics is the sole cause of decreased function, decreased myocardial energy stores can and does limit contraction. Therefore, this is likely to contribute to the depressed systolic function observed in our model.
Conclusions
Long-term exposure to high levels of GH increases cardiac mass and
deteriorates cardiac systolic function. These effects are in close
conjunction with bioenergetic abnormalities in the myocardium,
suggesting that impaired high-energy phosphate metabolism can be an
important mechanism behind the deterioration of cardiac function in
acromegaly.
Limitations
Technical limitations only allow a cautious interpretation of data
regarding Doppler recordings of mitral inflow with reference to
diastolic function.
We used relatively old animals where irreversible changes have been shown to occur in other organs (28, 48) that could to some extent have influenced our results.
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Footnotes |
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Received December 21, 1999.
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