Skip Navigation

Cardiovascular Research 2004 61(1):105-114; doi:10.1016/j.cardiores.2003.10.026
© 2004 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Deten, A.
Right arrow Articles by Zimmer, H.-G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deten, A.
Right arrow Articles by Zimmer, H.-G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2004, European Society of Cardiology

Norepinephrine-induced acute heart failure in transgenic mice overexpressing erythropoietin

Alexander Deten*,a, Junpei Shibatab, Dimitri Scholzc, Wilfried Briesta, Klaus F Wagnerb, Roland H Wengera and Heinz-Gerd Zimmera

aCarl-Ludwig-Institute of Physiology, Leipzig University, Liebigstraße 27, D-04103 Leipzig, Germany
bDepartment of Anesthesiology, Lübeck University, Ratzeburger Allee 160, D-23538 Lübeck, Germany
cDepartment of Experimental Cardiology, Max-Planck-Institute, Benekestraße 2, D-61231 Bad Nauheim, Germany

* Corresponding author. Tel.: +49-341-971-5500; fax: +49-341-971-5509. deta{at}medizin.uni-leipzig.de

Received 2 May 2003; revised 3 October 2003; accepted 27 October 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Overexpression of erythropoietin (Epo) in mice (Epo-tg6) leads to an increase in hematocrit and blood volume, and strongly reduces endurance upon exercise. It was the aim of this study to characterize the mechanisms underlying the reduced cardiac performance. Methods: Left (LV) and right (RV) ventricular function was measured with and without norepinephrine (NE) stimulation in 12 anaesthetized Epo-tg6 and in 13 wild-type (WT) control mice. Results: There were no differences in heart function under baseline resting conditions. Stimulation with NE (10 µl bolus injections of 1–100 ng per mouse) in WT mice led to a dose-dependent increase in heart rate (HR), LV developed pressure (LVDP) and rate of rise in LV pressure (LV dP/dtmax), while LV end-diastolic pressure (LVEDP) was unchanged. Except for HR, these parameters increased to a lesser extent in EPO-tg6 mice. Strikingly, LVEDP strongly increased in Epo-tg6 mice after NE (up to >20 mmHg). Eleven out of 13 Epo-tg6, but none of the WT mice died or required resuscitation after high-doses of NE. In these cases severe diastolic dysfunction became overt since the relative myocardial relaxation time was significantly prolonged and the duration of diastole was shortened. Moreover, the ECG showed a marked ST segment depression as well as deep negative T-waves. The NE-induced reduction in myocardial adenosin-triphosphate (ATP) content was more pronounced in Epo-tg6 mice after 10 min of continuous NE infusion (50 ng/min per mouse). Conclusion: NE-induced stress in Epo-tg6 mice led to acute heart failure associated with diastolic dysfunction and myocardial ischemia.

KEYWORDS Adrenergic agonists; Heart failure; Ischemia


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The 30 kDa glycoprotein hormone erythropoietin (Epo) is produced primarily in the kidney upon hypoxic stimulation and regulates the production of erythrocytes in the bone marrow. Recently, a transgenic mouse line (Epo-tg6) was generated that constitutively overexpresses the human Epo cDNA in an oxygen-independent manner [1]. These Epo-tg6 mice exhibit a more than 12-fold elevation in plasma Epo levels. This results in excessive erythrocytosis with hematocrit levels of 0.80 and above, which gradually develops within the first 2 months of life [2]. Interestingly, the Epo transgenic mice appear to be well adapted to the high hematocrit and do not have arterial hypertension, despite an increase in blood viscosity and an increase in blood volume of 75% [2,3]. Neither heart rate (HR) nor cardiac output, differ from wild-type (WT) control animals [1,2]. There are also no signs of thrombosis or embolizations [4]. Enhanced bioavailability of NO by induction and activation of the endothelial NO synthase (eNOS) seems to be part of the mechanism to compensate for the high hematocrit. Indeed, eNOS levels and NO-mediated endothelium-dependent vasorelaxation are significantly increased in the Epo-tg6 mice despite concomitant expression of the vasoconstrictor endothelin-1 (ET-1) [1,5,6].

The most obvious difference regarding heart and circulatory function between the Epo transgenic and the WT control mice are enlarged hearts as well as increased preload in the transgenics [2,3]. Furthermore, the Epo-tg6 mice become exhausted earlier during exercise [2]. We have examined the hypothesis that the cardiovascular system is the limiting factor for the reduced exercise performance. Therefore, heart function of Epo transgenic mice was characterized using ultraminiature catheter pressure transducers for catheterization of the left and right heart under basal resting conditions [7]. Furthermore we have applied stress to the cardiovascular system by i.v. injection and infusion of norepinephrine (NE).


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Animals
The transgenic mouse line termed TgN(PDGFBEPO)321Zbz (short: tg6) carries the human Epo cDNA driven by the PDGF-β promoter as described earlier [1]. Hemizygous transgenic males were mated to WT C57Bl/6 females. Their offspring were controlled for heterozygous mice and WT littermates, the latter serving as controls. Twenty-two male WT and 23 male Epo-tg6 mice (32±1.6 and 31±1.4 weeks of age, respectively, and 35.0±1.4 and 32.6±1.0 g of body weight, respectively) were used. The animals were kept in conventional housing conditions and allowed to move freely in their cages with unrestricted access to standard rodent chow and tap water. All experimental procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the appropriate State agency of Saxony.

2.2 Hemodynamic measurements
Heart and circulatory function was measured with ultraminiature catheter pressure transducers in closed-chest spontaneously breathing animals anaesthetized with thiopental sodium (Trapanal® 80 mg/kg i.p., Byk Gulden, Konstanz, Germany) as previously described [7]. Briefly, the right ventricular (RV) catheter (2F model SPR-612, Millar Instruments Inc., Houston, TX, USA) was inserted into the right jugular vein and advanced into the RV via the right atrium. After collection of the RV data, the left ventricular (LV) catheter (1.4F model SPR-671, Millar Instruments Inc.) was placed in the right carotid artery and advanced upstream to the aorta and into the LV. HR, RVP and LVP (pressure processor amplifier 13-4615-52, Gould-Nicolet, Erlensee, Germany), the rates in rise and fall of ventricular pressure (LV and RV dP/dt, differentiator amplifier 13-4615-71, Gould-Nicolet), and the electrocardiogram (ECG, standard limb leads with needle-electrodes, Biotach amplifier 20-4615-65, Gould-Nicolet) were recorded continuously on a PC at a sampling rate of 2 kHz using the PCI-6023E multifunction analogue and digital conversion card and DASYLab V7.00 software (National Instruments, Mönchengladbach, Germany). Hemodynamic parameters were analyzed using IOX V1.7.0.18 software (EMKA Technologies, Paris, France). The time constant of early relaxation tau ({tau}) was calculated as the time of pressure decay from ventricular pressure at dP/dtmin to 1/e of that pressure. To correct for HR, {tau} was also expressed as percent of total heart cycle length ({tau}rel). To further assess the impact of {tau} on myocardial performance a relaxation time index was calculated by dividing the available time for myocardial relaxation (measured from dP/dtmin till the time of the EDP of the following beat) by 3.5 times {tau}, which is the time required to complete myocardial relaxation by 97% [8].

For NE application, an i.v. cannula (22 G, Vasocan® Braunüle®, B|Braun, Melsungen, Germany) was placed in the right jugular vein of anesthetized mice. NE was diluted appropriately in 0.9% NaCl, containing 100 mg/l ascorbic acid to prevent oxidation, and 1, 5, 10, 25, 50, and 100 ng NE per mouse were injected each in a 10 µl bolus. Injections were done using a precision syringe (Hamilton 702N 25 µl, Hamilton), additionally introducing a spacer so that the tip of the needle was placed at the tip of the cannula in the jugular vein. For continuous NE infusion (50 ng/min per mouse), the cannula in the jugular vein was replaced by an infusion catheter (PE50 tubing, Becton Dickinson) and connected to an external pump (B0|Braun). The pump rate was adjusted to 10 µl/min. NE infusion was started by inserting the catheter and stopped by removing it.

2.3 Myocardial ATP content
NE or saline was infused in subgroups of anesthetized WT and Epo-tg6 mice (50 ng NE/min per mouse at 10 µl/min). After 10 min of infusion, the thorax was quickly opened, and the apical two thirds of the heart were rapidly cut and immediately frozen in liquid nitrogen. Tissue pieces (about 30 mg) were weighed and homogenized with nine parts 10% trichloroacetic acid with an Ultra-Turrax T8 (IKA, Staufen, Germany). Five volumes of water saturated ether were added to the supernatant cleared by centrifugation, and the aqueous layer was extracted. This extraction was repeated twice. The samples were diluted 1:5 (v/v) in Tris–EDTA buffer, and the content in adenosin-triphosphate (ATP) was determined luminometrically using the ATP Bioluminescence Assay Kit CLS II (Roche Diagnostics, Boehringer Mannheim Biochemicals, Mannheim, Germany). Measurements were performed in triplicate (1, 3 and 10 s detection time) in two further dilutions (1:500 and 1:5000). ATP concentration was calculated according to the standard curve and normalized to the weight of tissue used.

2.4 Perfusion fixation, ultrastructural analysis and morphometry
The aorta was retrogradely cannulated, and the coronary arteries were rinsed free of blood for 2 min with a modified tyrode buffer under a pressure of 100 cmH2O. This was followed by infusion of freshly prepared 1% formaldehyde for 10 min. Collateral vessels were dissected and cubes of about 1 mm3 were dissected from the middle part of left ventricle free wall. Myocardial tissue was postfixed in 3% glutaraldehyde, then in 1% OsO4, dehydrated in alcohols and embedded in Epon using the Lynx Microscopy Tissue Processor (Reichert-Jung, Bensheim, Germany). Semithin sections were cut with the Ultracut microtome (Reichert-Jung) to a thickness of 0.5 µm, stained with toluidin blue, and analyzed with a Leica DAM (Leica, Bensheim, Germany) microscope.

High resolution pictures were obtained with a Leica 63 x 1.40 oil immersion objective and Leica DM-RB microscope combined with a Leica DC200 digital camera. Diameters and areas taken by cardiomyocytes on cross-section were measured using the NIH Image 1.62 software (for Apple Macintosh). The whole section was photographed but only cells with nucleus profile and no cross-striation were evaluated.

2.5 Statistical analysis
The data are expressed as mean±S.E.M. The Mann–Whitney U-test (SigmaStat 2.0, SPSS Inc., Chicago, IL) was used for two-group comparison. One way repeated measures ANOVA, subsequently utilizing Dunnett's multiple comparison procedure was used to test for differences in the dose–response to NE stimulation. A value of P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Hemodynamic measurements
LV and RV function was comparable in Epo-tg6 and WT littermate control mice under baseline conditions (Table 1). LV end-diastolic (LVEDP) and developed pressure were not different in Epo-tg6 mice when compared with WT controls. The maximal rates in rise and fall of ventricular pressure (LV dP/dtmax and LV dP/dtmin, respectively) as well as the time constant for early relaxation {tau} were not significantly different in Epo-tg6 mice from WT littermate control mice. Additionally, the pressure-rate-product multiplied by LV dP/dtmax, indicative for myocardial oxygen consumption, was comparable in WT and Epo-tg6 mice. The myocardial relaxation time index indicated for both, WT and Epo-tg6 mice; that the time available for relaxation (measured from dP/dtmin till the time of the EDP of the following beat) was more than two times that required for nearly complete relaxation (estimated as 3.5 x {tau}). Therefore, the statistically significant difference in the myocardial relaxation time index between Epo-tg6 and WT mice under baseline resting conditions (Table 1) has no important functional relevance. The amplitude of aortic pressure, however, was reduced in Epo-tg6 mice when compared with WT controls.


View this table:
[in this window]
[in a new window]

  		Table 1 Heart function at baseline and after NE-infusion for 10 min (50 ng/min)

 
The parameters of LV function increased in a dose-dependent manner in WT mice after stimulation with NE (Fig. 1). HR, LVSP, LV dP/dtmax, and LV dP/dtmin were maximally elevated by 44%, 83%, 186%, and 114%, respectively, after NE application. After high-dose NE, LVEDP increased slightly but significantly in WT mice (Fig. 1D). In Epo-tg6 mice, there was a dose-dependent response to NE stimulation, too, but it was significantly attenuated compared to WT mice. The maximum response was limited to about 75% of that seen in WT mice with the exception that the increase in HR was comparable in WT and Epo-tg6 mice (Fig. 1). The increase in LVEDP, however, occurred in Epo-tg6 mice already after moderate NE dosages and was significantly pronounced when compared to WT controls. After 100 ng NE, LVEDP increased to 20 mmHg and above, indicating severely impaired heart function.


Figure 1
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 LV hemodynamic data after NE bolus injections. Dose-dependent changes in HR (A), triple product of LVSP x HR x LV dP/dtmax (B), LVSP (C), LVEDP (D), LV dP/dtmax (E) and –LV dP/dtmin (F) in WT (closed circles) and Epo-tg6 mice (open circles) after NE bolus injections as indicated (ng per mouse). Data are mean±S.E.M.; n = 12 for WT and n = 13 for Epo-tg6 mice except n = 9 for Epo-tg6 after 100 ng NE; {dagger}P<0.05 vs. baseline; *P<0.05 vs. WT (same treatment).

 
In a subgroup of animals (n = 4 for each group), also the parameters of RV function increased in a dose-dependent manner after stimulation with NE (Fig. 2). The increases in RVSP, RV dP/dtmax and RV dP/dtmin were similar in WT and Epo-tg6 mice. Significant differences were observed for RV dP/dtmin after moderate NE dosages as well as for the maximal increase in RVSP. RVEDP was unchanged in WT mice after NE stimulation, but significantly increased in Epo-tg6 mice already after 5 ng NE (Fig. 2B).


Figure 2
View larger version (20K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 RV hemodynamic data after NE bolus injections. Dose-dependent changes in RVSP (A), RVEDP (B), RV dP/dtmax (C) and –RV dP/dtmin (D) in WT (closed circles) and Epo-tg6 mice (open circles) after NE bolus injections as indicated (ng per mouse). Data are mean±S.E.M.; n = 4 for each group; {dagger}P<0.05 vs. baseline; *P<0.05 vs. WT (same treatment).

 
Heart function attained a new steady state at elevated levels nearly immediately after the initiation of continuous NE infusion. As for single bolus injections, the increase in LVSP and both LV dP/dtmax and LV dP/dtmin was attenuated in Epo-tg6 mice after continuous NE infusion for 10 min, whereas there were no differences in HR (Table 1). The time constant of early relaxation {tau} significantly decreased in WT mice after NE stimulation. The relation of {tau} to total heart cycle length ({tau}rel) remained constant. In Epo-tg6 mice, {tau} was not changed after NE stimulation resulting in a significant increase in {tau}rel (Table 1). Importantly, the relaxation time index dropped below values of 1 in Epo-tg6 mice within a minute after starting NE infusion and remained close to that for the duration of NE stimulation (Fig. 3). In WT mice the relaxation time index also decreased, but slightly and remained above values of 2. Furthermore, LVEDP strongly increased in Epo-tg6 mice after 10 min NE, while it was unchanged in WT controls. All parameters returned to baseline levels within a few minutes after removing the infusion catheter (Fig. 4).


Figure 3
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Myocardial relaxation time index after continuous NE application. Plot of the relaxation time index over time in a WT (left) and Epo-tg6 mouse (right) under baseline resting conditions as well as after continuous NE infusion (50 ng/min, start and stop marked by the arrows at the bottom). The relaxation time index was calculated by dividing the time available for myocardial relaxation (measured from dP/dtmin till time of EDP of the next beat) by the time needed for a 97% myocardial relaxation (estimated as 3.5 x {tau}).

 

Figure 4
View larger version (48K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Effect of continuous NE application. (A) Representative original recordings of LVP (upper tracing), LV dP/dt (middle tracing) and HR (lower tracing) in a WT (left) and an Epo-tg6 mouse (right). NE infusion (10 µl/min of 5 ng/µl) was stopped as indicated by the arrows at the bottom of the panels. (B) Representative original recordings of LVP (upper tracing), LV dP/dt (middle tracing) and ECG (standard limb lead I, lower tracing) in a WT (ECG 1) and an Epo-tg6 mouse (ECG 2 and ECG 3). The tracings were taken from the animals of (A) at the time points indicated there and shown at a higher resolution.

 
Strikingly, 14 out of the 18 Epo-tg6 mice stimulated with NE died or required resuscitation after either high-dose or continuous NE application. Contrarily, NE was well tolerated in all WT littermate control mice even after 500 ng bolus injections (data not shown). Additionally, there were deep ST-segment depressions and negative T-waves in the ECG (Fig. 4B, ECG 2) starting after about 3–5 min of high-dose or continuous NE application and preceding acute heart failure. This was highly reproducible, but occurred in none of the WT mice (Fig. 4B, ECG 1). When the NE infusion was stopped during the periods with prominent changes in the ECG, the mouse could be rescued, and ECG as well as hemodynamic parameters normalized (Fig. 4B, ECG 3).

3.2 Myocardial ATP content
Myocardial ATP content was slightly but significantly lower in Epo-tg6 mice at baseline when compared to WT controls (Fig. 5, left). NE treatment for 10 min decreased myocardial ATP content in both, WT and Epo-tg6 mice. This decrease, however, was significantly pronounced in Epo-tg6 mice in comparison to WT mice (32.8 vs. 18.6%, respectively, Fig. 5, right).


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Myocardial ATP content after 10 min of NE infusion. Myocardial ATP content was determined luminometrically in WT (light columns) and Epo-tg6 mice (dark columns) after 10 min of saline (CTRL) or NE infusion. Data are mean±S.E.M.; n = 5 per group except for n = 3 for Epo-tg6 CTRL; {dagger}P<0.05 vs. CTRL; *P<0.05 vs. WT (same treatment).

 
3.3 Morphology and ultrastructure
The most prominent difference in the myocardial tissue of Epo-tg6 mice to WT control mice were large euchromatic nuclei with prominent nucleoli (Fig. 6) suggesting activated transcription. The myocytes of Epo-tg6 mice also showed disarray of myofibers (Fig. 6C) as well as significant hypertrophy (Fig. 6B and D). Myocyte cross sectional area was 495±32 and 644±49 µm2 for WT and Epo-tg6 mice, respectively (P<0.05), and myocyte diameter was 18.3±0.9 and 22.3±0.9 µm, respectively (P<0.05).


Figure 6
View larger version (145K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Ultrastructural analysis of hearts from WT and Epo overexpressing mice. Original microphotographs of cross (A and B) and longitudinal sections (C) of WT (A) and Epo-tg6 mice (B and C) demonstrating large euchromatic nuclei with prominent nucleoli, enlarged myocytes and disarray of myofibers. Scale bar provided in A represents 10 µm, same magnification for all microphotographs. (D) Box-Blot of myocyte diameters of WT (n = 6) and Epo-tg6 mice (n = 7); — median, -··-mean, *P<0.05 vs. WT.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
In this study, heart and circulatory function of Epo overexpressing (Epo-tg6) and WT mice was stimulated by the application of NE. There was a normal HR response, however, the increase in both LV pressure as well as in contractility was significantly attenuated in Epo-tg6 mice even at low dosages of NE (Fig. 1). After higher NE concentrations or continuous infusion, also LVEDP markedly increased and, most strikingly, about 80% of the Epo-tg6 but none of the WT mice died or required resuscitation due to severe diastolic dysfunction, myocardial ischemia and ultimately acute heart failure. The drop in the myocardial relaxation time index below a value of 1 indicating incomplete myocardial relaxation occurred within a minute after NE infusion. Myocardial ischemia became overt after 3–5 min of NE infusion preceding acute heart failure after 5–10 min. This is in accordance with our previous observation that Epo-tg6 mice suffered from exhaustion in treadmill test after 6 min [2].

It has been demonstrated previously that the plasma levels of Epo which are about 10–12-fold increased in Epo-tg6 mice resulting in a hematocrit of 0.75–0.80 [1,2,6]. In the present study, heart function was comparable in Epo-tg6 and WT mice under baseline resting conditions (Table 1). Additionally, there were no clinical signs of manifest heart failure, i.e. shortness of breath, edema, pleural effusions or ascites. This is in agreement with the results of previous studies in which blood pressure and cardiac output were not different between WT and Epo-tg6 mice [1,2]. Normal heart function is surprising since hematocrit is a major regulator of whole blood viscosity, determining peripheral vascular resistance. The tremendous increase in hematocrit should, therefore, lead to a reduction in blood pressure or cardiac output or both. That this does not occur under baseline conditions may be due to one or several compensatory mechanism(s). Recently, it was shown that this adaptation to high hematocrit involves regulated elevation of blood viscosity by increasing erythrocyte flexibility [3]. Moreover, increased eNOS expression in the vessels of Epo-tg6 mice as well as increased NO bioavailability has been observed [1]. This may be induced by Epo directly [9,10] or as a result of the increased shear stress. The importance of increased NO bioavailability as compensatory mechanism in the Epo-tg6 mice was further emphasized by the severely increased mortality in Epo-tg6 mice that were treated with L-NAME to inhibit endothelial NO formation [1,5]. It has, however, also been reported that the Epo-tg6 mice show an activation of the ET-1 system, contributing to the deleterious effects of L-NAME treatment in Epo-tg6 mice [5]. A limited number of studies on isolated erythrocytosis have been carried out in dogs, rats and mice so far. Despite differences between species or in the experimental setup, it is obvious that the mechanisms to compensate for the high hematocrit are time-dependent. In an early study in dogs, cardiac output was reduced by about 50% when hematocrit was raised to a mean of 47% above normal by bleeding and reinjecting freshly spun red blood cells within only 3 min [11]. In contrast, no cardiovascular alterations were observed in rats when hematocrit was increased to 0.63 over 3 weeks by the administration of 500 units Epo thrice weekly [12]. Although the hematocrit increased in Epo-tg6 mice to much higher values, the gradual increase probably enabled the cardiovascular system to adapt.

Besides an increase in whole blood viscosity [3] the increase in hematocrit is also accompanied by an increase in blood volume [2,3]. This, in turn, leads to ventricular dilation [2] and hypertrophy [1,2]. However, no cardiac hypertrophy was observed in rats with a hematocrit of 0.63 by Epo administration [12,13]. In contrast, moderate hypertrophy of the right ventricle or of both the right and left ventricle occurred in mice after intraperitoneal injection of packed red cells into mice resulting in a hematocrit of 0.67–0.80 [14,15]. Since erythrocytosis developed gradually in Epo-tg6 mice over several weeks while growing up [2] the cardiovascular system attains enough time to adapt. At the age of 8-month the hearts are dilated, but also show hypertrophic cardiomyopathy.

Secondly, the hearts of Epo-tg6 mice suffer from severe diastolic dysfunction, typically associated with hypertrophic cardiomyopathy, leading to coronary insufficiency and acute heart failure after NE stimulation. Stimulation with NE-induced a significant increase in HR, LVSP, LV dP/dtmax and LV dP/dtmin in WT mice. These effects except for the increase in HR were attenuated in Epo-tg6 mice (Fig. 4A) indicating abnormal systolic performance and afterload mismatch. Importantly, the time constant of relaxation {tau} was not changed in Epo-tg6 mice after NE, but decreased in WT mice (Table 1). Since especially the diastole is being shortened when HR increases, the time required for complete myocardial relaxation (estimated as 3.5 x {tau}) [8] equaled the time available (estimated as time from LV dP/dtmin till the time of EDP of the following beat) as indicated by the drop in the relaxation time index to values of 1 in Epo-tg6 mice (Fig. 3, Table 1). This effect may even be underestimated, since only data of those animals were included in Table 1 that survived for at least 7 min of continuous NE stimulation. Incomplete myocardial relaxation and afterload mismatch are capable to induce an upward shift of the diastolic pressure–volume relation as well as a rightward shift along that relation [16–18]. This in turn leads to a (further) dilation of the LV cavity as well as to (further) increased wall tension. As a result, myocardial ischemia and ultimately acute heart failure occur.

Impairment of the balance between myocardial oxygen demand and delivery are demonstrated by deep ST-segment depressions and negative T-waves in the ECG preceding acute heart failure (Fig. 4B, ECG 2). Additionally, the pressure-rate-product multiplied by LV dP/dtmax, indicative for myocardial oxygen consumption, reached maximum in the Epo-tg6 mice already at 25–50 ng NE (Fig. 1B), while it still increased in WT mice after 500 ng NE (not shown). That myocardial ischemia occurs, is further strengthened by the more pronounced decrease in myocardial ATP content in the Epo-tg6 mice after 10 min of NE infusion compared to their WT littermates (Fig. 5). In addition to the incomplete myocardial relaxation, the mechanism(s) underlying NE-induced myocardial ischemia in the Epo-tg6 mice likely also include increased oxygen diffusion distances due to myocyte hypertrophy which further impairs oxygen delivery. Moreover, the Epo-tg6 mice seem to be well adapted to increased blood volume and viscosity as discussed earlier [1,5,6]. Although not analyzed in this study, it seems reasonable to suggest that any vasoconstriction or increase in oxygen and metabolic requirements of the myocardium induced by NE may exceed the capacity of these compensatory mechanisms thereby contributing to myocardial ischemia. However, myocardial ischemia is reversible within a few minutes after stopping NE infusion (Fig. 4B, ECG 3).

The main difference between the Epo-tg6 mice and the usual pathophysiological conditions of elevated hematocrit is that it is hypoxia-independent such as in Epo-doped athletes. Beside hypoxia, most erythrocytotic conditions involve all three blood cell lines (polycythemia vera) or are secondary to others disorders (i.e. cyanotic congenital heart disease, malignancies or Cushing disease) to which symptoms and complications have to be attributed to. That erythrocytosis in the Epo-tg6 mice occurs isolated from other cell lines or hormones may, in part, also explain why there is no sign of thrombosis or embolization. Obviously there are additional protective mechanisms regarding coagulation [4]. The relationship between hematocrit and blood pressure, however, remains unclear. Both aspects may be associated, [19,20] but erythrocytosis does not necessarily lead to hypertension [21,22]. However, the Epo-tg6 mice show that behind normal resting values a severe cardio-vascular disease had developed. Once challenged by NE, as in this study, stress-induced heart failure became overt. Taken into account that Epo-tg6 mice seem to have a reduced life span, [2] further studies need to investigate whether or not this stress (NE)-induced heart failure may turn into manifest heart failure and which mechanisms may lead to failure of a well-adapted heart.


   Acknowledgements
 
This work was supported by the Deutsche Forschungsgemeinschaft (ZI 199/10-3, 4), the formel.1 program of the medical faculty of the Leipzig University (formel.1–19) and by grants of BMBF (NBL-3-Förderung; Kennzeichen 01ZZ0106).


   Notes
 
Time for primary review 24 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Ruschitzka F.T., Wenger R.H., Stallmach T., et al. Nitric oxide prevents cardiovascular disease and determines survival in polyglobulic mice overexpressing erythropoietin. Proc. Natl. Acad. Sci. USA (2000) 97:11609–11613.[Abstract/Free Full Text]
  2. Wagner K.F., Katschinski D.M., Hasegawa J., et al. Chronic inborn erythrocytosis leads to cardiac dysfunction and premature death in mice overexpressing erythropoietin. Blood (2001) 97:536–542.[Abstract/Free Full Text]
  3. Vogel J., Kiessling I., Heinicke K., et al. Transgenic mice overexpressing erythropoietin adapt to excessive erythrocytosis by regulating blood viscosity. Blood (2003) 102:2278–2284.[Abstract/Free Full Text]
  4. Shibata J., Hasegawa J., Siemens H.J., et al. Hemostasis and coagulation at a hematocrit of 0.85: functional consequences of erythrocytosis. Blood (2003) 101:4416–4422.[Abstract/Free Full Text]
  5. Quaschning T., Ruschitzka F., Stallmach T., et al. Erythropoietin-induced excessive erythrocytosis activates the tissue endothelin system in mice. FASEB J. (2003) 17:259–261.[Abstract/Free Full Text]
  6. Slowinski T., Schulz N., Ruschitzka F.T., et al. Pattern of prepro-endothelin-1 expression revealed by reporter-gene activity in kidneys of erythropoietin-overexpressing mice. Clin. Sci. (2002) 103:44S–47S.[Web of Science][Medline]
  7. Deten A., Zimmer H.G. Heart function and cytokine expression is similar in mice and rats after myocardial infarction but differences occur in TNFalpha expression. Pflug. Arch. (2002) 445:289–296.[CrossRef][Web of Science][Medline]
  8. Weisfeldt M.L., Frederiksen J.W., Yin F.C., Weiss J.L. Evidence of incomplete left ventricular relaxation in the dog: prediction from the time constant for isovolumic pressure fall. J. Clin. Invest. (1978) 62:1296–1302.[Web of Science][Medline]
  9. Yamane Y., Murakami Y., Tsumori M., Koshimura K., Kato Y. Acute stimulatory effect of erythropoietin on nitric oxide production in human aortic endothelial cells. Biomed. Res. (1999) 20:281–285.[Web of Science]
  10. Banerjee D., Rodriguez M., Nag M., Adamson J. Exposure of endothelial cells to recombinant human erythropoietin induces nitric oxide synthase activity. Kidney Int. (2000) 57:1895–1904.[CrossRef][Web of Science][Medline]
  11. Richardson T.Q., Guyton A.C. Effects of polycythemia and anemia on cardiac output and other circulatory factors. Am. J. Physiol. (1959) 179:1167–1170.
  12. Petit R.D., Warburton R.R., Ou L.C., Hill N.S. Pulmonary vascular adaptations to augmented polycythemia during chronic hypoxia. J. Appl. Physiol. (1995) 79:229–235.[Abstract/Free Full Text]
  13. Petit R.D., Warburton R.R., Ou L.C., Brinck J.T., Hill N.S. Exogenous erythropoietin fails to augment hypoxic pulmonary hypertension in rats. Respir. Physiol. (1993) 91:271–282.[CrossRef][Web of Science][Medline]
  14. Naeye R.L. Polycythemia and hypoxia: individual effects on heart and pulmonary arteries. Am. J. Pathol. (1967) 50:1027–1033.[Web of Science][Medline]
  15. Swigart R.H. Polycythemia and right ventricular hypertrophy. Circ. Res. (1965) 17:30–38.[Abstract/Free Full Text]
  16. Leite-Moreira A.F., Correia-Pinto J., Gillebert T.C. Afterload induced changes in myocardial relaxation: a mechanism for diastolic dysfunction. Cardiovasc. Res. (1999) 43:344–353.[Abstract/Free Full Text]
  17. Sarnoff S.J., Mitchell J.H., Gilmore J.P., Remensmyder J.P. Homeometric autoregulation in the heart. Circ. Res. (1960) 8:1077–1091.[Abstract/Free Full Text]
  18. Glantz S.A., Parmley W.W. Factors which affect the diastolic pressure–volume curve. Circ. Res. (1978) 42:171–180.[Free Full Text]
  19. Cirillo M., Capasso G., DeSanto N.G. Relationship between hematocrit and blood pressure: implications for primary hypertension. Nephron (1993) 65:505–510.[Web of Science][Medline]
  20. Raine A.E. Seizures and hypertension events. Semin. Nephrol. (1990) 10:40–50.[Web of Science][Medline]
  21. Bilgrami S., Greenberg B.R. Polycythemia rubra vera. Semin. Oncol. (1995) 22:307–326.[Web of Science][Medline]
  22. de Wardener H.E., McSwiney R.R., Miles B.E. Renal haemodynamics in primary polycythemia. Lancet (1951) 2:204–206.[Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Deten, A.
Right arrow Articles by Zimmer, H.-G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deten, A.
Right arrow Articles by Zimmer, H.-G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?