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European Journal of Heart Failure 2008 10(1):22-29; doi:10.1016/j.ejheart.2007.10.008
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© 2008 European Society of Cardiology

Low-dose erythropoietin improves cardiac function in experimental heart failure without increasing haematocrit

Erik Lipsic, B. Daan Westenbrink*, Peter van der Meer, Pim van der Harst, Adriaan A. Voors, Dirk J. van Veldhuisen, Regien G. Schoemaker and Wiek H. van Gilst

Department of Cardiology, University Medical Center Groningen, University of Groningen The Netherlands

* Corresponding author. Department of Cardiology, Thoraxcenter, University Medical Center Groningen, Hanzeplein 1, P.O. Box 30001, 9700 RB Groningen, The Netherlands. Tel.: +31 50 3613876; fax: +31 50 3614391. E-mail address: b.d.westenbrink{at}thorax.umcg.nl (B. D. Westenbrink)


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Conflict of interests
 References
 
Background: Erythropoietin (EPO) may improve cardiac function and induce neovascularisation in experimental models of chronic heart failure (CHF). However, the increased haematocrit associated with EPO treatment might exert concomitant deleterious effects.

Aim: To investigate the haematocrit independent effects of EPO on cardiac function.

Methods and results: Rats underwent permanent coronary artery ligation to induce myocardial infarction (MI) or sham surgery. Three weeks after MI, rats were randomly allocated to treatment with vehicle (MI) or the long-acting EPO analogue darbepoetin alfa administered in a high (40 µ/kg/3 weeks, MI-EPO-high) or a low-dose (0.4 µ/kg/3 weeks, MI-EPO-low). After 9 weeks, haemodynamic parameters, myocardial histology and Myosin Heavy Chain (MHC) isoforms were determined. High-dose EPO resulted in a significant increase in haematocrit (p<0.01) while low-dose EPO had no effect on haematocrit levels. EPO significantly improved cardiac function in both EPO groups, reflected by increased left ventricular (LV)-developed pressure and improved contractility (dP/dtmax) and relaxation (dP/dtmin) indices of the LV at 9-weeks (all p<0.05 compared to MI). The improved cardiac function was associated with increased capillary growth (38% in MI-EPO-high (p<0.01) and 27% in MI-EPO-low (p<0.05)) and an attenuated switch to slow β-MHC isoforms in both EPO groups.

Conclusions: EPO improves cardiac function and induces neovascularisation at a dose that does not increase haematocrit, thereby circumventing the possible deleterious effects of increased erythropoiesis.

Key Words: Capillaries • Heart failure • Ventricular function

Received May 24, 2007; Revised August 31, 2007; Accepted October 16, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Conflict of interests
 References
 
The classical role of erythropoietin (EPO) is related to its haematopoietic effects. EPO is produced in the kidneys and acts as a major regulator of erythropoiesis, by increasing survival and promoting proliferation of erythroid progenitor cells.

However, EPO has recently been shown to render organ protection in various experimental models of acute ischaemia, including stroke and myocardial infarction, mainly through a reduction of apoptotic cell death [1-4]. In addition, EPO improves cardiac function in experimental models of chronic myocardial dysfunction, which is consistently associated with improved microvascularisation of the myocardium [5-10]. The neovascularisation by EPO is associated with marked mobilisation and vascular incorporation of endothelial progenitor cells (EPC) [11-14]. In addition, EPO stimulates neovascularisation by inducing direct mitogenic effects on endothelial cells through local upregulation of vascular endothelial growth factor (VEGF) [14-16]. Therefore, the ancillary properties of EPO seem independent of erythropoiesis.

However, the effects of EPO in chronic myocardial dysfunction have been established with repetitive dosing regimens that significantly increased haematocrit levels. Therefore, these effects might at least to some extent, be related to the increased oxygen-carrying capacity of blood. Moreover, EPO treatment in patients could lead to unwanted elevation of haematocrit, associated with higher risk for thrombosis and hypertension [17]. Indeed, the recently published CREATE and CHOIR studies revealed that overcorrection of anaemia with recombinant human EPO in patients with chronic kidney disease (CKD) was associated with increased cardiovascular events [18,19]. In Chronic Heart Failure (CHF) patients, appropriately powered phase-3 studies are lacking, although safety and feasibility studies have demonstrated promising beneficial effects without additional harm [20]. Nevertheless, treatment aimed at improving cardiac function in non-anaemic CHF patients at already elevated cardiovascular risk necessitates treatment strategies that exert the beneficial effects of EPO without increasing haematocrit levels.

The erythropoietin receptor in the heart is structurally and functionally distinct from its haematopoietic counterpart, and can be specifically targeted [21]. Moreover, chronic EPO administration in a dose that was insufficient to increase haematocrit, was shown to improve survival, ameliorate endothelial damage and preserve renal function in a rat remnant kidney model, suggesting a different dose-response relationship for the erythropoietic and pleiotropic effects [22]. Low-dose EPO might therefore exert similar beneficial effects on the myocardium, without the deleterious effects on haematocrit.

We hypothesized that the ancillary properties of EPO in CHF are independent of the effects on erythropoiesis, and can be induced with an EPO dose that does not increase haematocrit. We therefore studied the effects of high- and low-dose EPO treatment on cardiac function, neovascularisation and myosin heavy chain (MHC) isoform expression in an experimental model of post-MI heart failure.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Conflict of interests
 References
 
2.1. Animals
We used male sprague dawely rats weighing 270-330 g (Harlan, Zeist, The Netherlands). Animals were fed ad libitum, and housed in groups of four to five rats, according to institutional rules with 12:12 hour light-dark cycles. The experimental protocol was approved by the Animal Ethics Committee of the University Medical Center Groningen.

2.2. Design of the study
Rats were either subjected to left coronary artery ligation to induce myocardial infarction (MI, n=63) or sham surgery (n=11). Rats with MI were randomly allocated to 3 groups: control (MI) and two EPO treatment groups with different dosages of the long-acting EPO analogue darbepoetin: 40 µg/kg (MI-EPO-high) or 0.4 µg/kg (MI-EPO-low). Darbepoetin-alfa (Aranesp, Amgen Inc., Thousands Oaks, CA, USA) was administered intra-peritoneally, once every three weeks, starting three weeks after the coronary artery ligation, hence after the healing phase of MI. Control (MI) and SHAM rats received corresponding injections of saline. The high-dose of darbepoetin was based on our previous study [10], demonstrating increased neovascularisation in this model, together with significant elevation of haematocrit levels. To avoid the effect of EPO treatment on haematocrit we included a low-dose EPO group, with 100-times lower darbepoetin dosage (0.4 µg/kg/3 weeks), which in a pilot experiment did not cause elevation of haematocrit (data not shown). Haematocrit was measured at baseline and 3, 4, 6 and 9 weeks after surgery.

2.3. Myocardial infarction model
This model has been described previously [23]. Briefly, rats were anesthetized with 2.5% isoflurane and placed on a heating pad (37 °C). Animals were intubated and mechanically ventilated using room air enriched with 1.0 l/min oxygen. After left-side thoracotomy, MI was induced by ligating the proximal portion of the left coronary artery, beneath the left atrial appendage. In sham operated rats, the same surgery was performed, without ligating the suture.

2.4. Haemodynamic measurements
After nine weeks, rats were anesthetized as described above. A Microtip pressure transducer (Millar Instr. Inc., Houston, Texas, USA) was inserted into the left ventricular cavity via the right carotid artery. After a 3-min period of stabilization, heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and developed left ventricular pressure (dLVP=LVSP-LVEDP) were measured. As indices of contractility and relaxation, the maximal rates of increase and decrease in LVP (dP/dtmax and dP/dtmin) were determined. The catheter was retracted into the aortic arch and arterial systolic and diastolic blood pressures (SBP, DBP) were recorded.

2.5. Infarct size and myocyte hypertrophy
After haemodynamic measurements, hearts were rapidly excised and weighed. Mid-papillary slices were fixed in 4% paraformaldehyde and paraffin-embedded. Infarct size was determined by planimeter in transverse slices on picrosirius red/fast green-stained sections. Infarct size was expressed as the percentage of scar length to total left ventricular circumference, as previously described [24]. Deparaffinised 5-µm thick sections were stained with a Gomori's silver staining. Using image analysis (Zeiss KS 400, Germany), concentric myocyte hypertrophy in the viable LV wall, remote from the infarcted area, was measured as the cross-sectional area of transversally cut myocytes showing a nucleus [23]. Myocyte density was calculated as the average number of myocytes per tissue area (mm2). In each stained section, measurements were averaged from three different counting fields (±75 myocytes per heart).

2.6. Capillary density
To visualize the capillaries in the myocardium of the LV free wall, endothelial cells were stained with biotin-labelled Lectin GSL (1:100; Sigma-Aldrich, St. Louis, Missouri, USA), as previously described. Since lectins stain not only capillaries but other vessels as well, a size criterion of 10 µm was used to exclude small arterioles and venules. Image analysis (Image-Pro Plus for Windows, version 4.5.0.29) was used to measure capillary density, calculated as the number of capillaries per tissue area (mm2). As a measure of neovascularisation, capillary-to-myocyte ratio was calculated dividing capillary with myocyte density, as previously described [23].

2.7. Myosin heavy chain (MHC) isoform analysis
Myosin heavy chain (MHC) isoform analysis was performed as a molecular marker of changes in myocardial contractility. Samples of the non-infarcted left ventricular free wall were snap frozen and stored at –80 °C until analysis. Gel electrophoresis was performed on tissue lysates as described previously [25]. In brief, samples were run at a constant current (24 mA) for 5 h. Silver staining was then performed and the percentage of fast {alpha}-MHC vs. slow β-MHC was determined with laser scanning densitometry. Increased expression of slow β-MHC results in impaired contractility of cardiomyocytes and represents a molecular marker of impaired contractility.

2.8. Apoptosis analysis
To visualize apoptotic cells in the myocardium, deparaffinised sections were stained with a monoclonal anti-cleaved caspase-3 antibody (ASP175, Cell Signaling Technologies, MA, USA) and visualized with the ENVISION kit (DAKO Cytomation, Glostrup, Denmark) according to the guidelines provided by the supplier. Cleaved caspase-3 positive cells were considered apoptotic and were expressed per 105 cells.

2.9. Statistical analysis
Data are presented as mean±SEM, or as median±IQR (25th and 75th percentile) depending on their distribution. Differences among groups were tested using one-way analysis of variance, followed by LSD post-hoc analysis if normally distributed, and by Kruskal-Wallis test if the distribution was skewed. Correlation analysis was performed with Spearman's correlation test. All reported probability values were 2-tailed, and a p-value<0.05 was considered statistically significant. All statistical analyses were performed with SPSS version 11.0.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Conflict of interests
 References
 
3.1. Mortality and general characteristics
Overall 24-hour mortality following MI was 41%. Five additional MI rats died during follow-up (2 in MI group, 1 in MI-EPO-high and 2 in MI-EPO-low group).

Two rats (1 in MI and 1 in MI-EPO-high group) had an infarct size <25% and were excluded from further analysis. General characteristics after nine weeks are shown in Table 1. LV-infarct size (% of LV) was comparable between all MI groups (Table 1). Body weight (BW) was significantly higher only in the MI-EPO-low group (Table 1). The heart weight to BW ratio was significantly increased in the rats with MI compared to the sham rats (all p<0.05; Table 1). A lower heart weight to BW compared to MI group was observed in MI-EPO-high and MI-EPO-low groups (both p<0.05).


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  		Table 1 Characteristics of the experimental groups at sacrifice (9 weeks)

 
3.2. Effects of EPO on haematocrit
The changes in haematocrit throughout the study are shown in Fig. 1. Only treatment with high-dose EPO led to a significant increase in haematocrit levels, which persisted throughout the experiment. Importantly, haematocrit levels in the MI-EPO-low group were similar to those of the MI and sham groups.


Figure 01
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  		Fig. 1 Effect of EPO treatment on longitudinal changes in haematocrit. *p<0.01 vs. sham.

 
3.3. Effects of EPO on cardiac function
Invasive pressure measurements were performed 9 weeks after the surgery immediately before the rats were sacrificed. Myocardial contractility (dP/dtmax) and myocardial relaxation (dP/dtmin) were both impaired in all MI groups compared to the sham group (all p<0.05). Both low- and high-dose EPO treatments resulted in improved contractility and relaxation compared to MI (both p<0.05; Fig. 2). LVSP and developed LVP (dLVP) were both decreased in all MI groups compared to sham operated rats (p<0.05 for all). MI-EPO-high showed a significantly higher LVSP and dLVP (Table 1, Fig. 2), compared to MI (both p<0.01). Low-dose EPO resulted in a 17% higher dLVP (p<0.05), and a trend towards elevation of LVSP, compared to the MI group (p=0.07; Table 1). LVEDP was elevated in the MI group compared to the sham operated rats (p<0.01; Table 1). Compared to the MI group, LVEDP was 34% (p<0.05) lower in the MI-EPO-high group and 20% lower in the MI-EPO-low group (p=NS). SBP and DBP were higher only in the MI-EPO-high group, compared to MI (both p<0.05).


Figure 02
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  		Fig. 2 Effects of myocardial infarction and different doses of EPO treatment on haemodynamic parameters. dP/dtmax and dP/dtmin, maximal rate of increase and decrease of ventricular pressure, respectively. * p<0.05 vs. MI, ** p<0.01 vs. MI. dLVP, developed left ventricular pressure.

 
3.4. Effects of EPO on neovascularisation
Fig. 3C shows representative photomicrographs of the four different groups. Capillary density was significantly reduced in MI compared to sham group (p<0.01). High-dose EPO treatment prevented the decrease in capillary density after induction of MI and restored it to sham values, as shown in Fig. 3A (p=NS vs. sham). In the MI-EPO-high group we observed a 33% increase in capillary density compared to MI group (p<0.01). Treatment with low-dose EPO resulted in a 20% higher capillary density (p<0.05). The cross-sectional area of cardiomyocytes increased in all MI groups compared to sham, although EPO treatment had no effect on cardiomyocyte hypertrophy. Compared to the MI group, the capillary-to-myocyte ratio increased by 39% in MI-EPO-high (p<0.01) and by 27% in MI-EPO-low (p<0.05) (Fig. 3B). The differences between MI-EPO-high and MI-EPO-low group were not statistically significant.


Figure 03
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  		Fig. 3 Effect of EPO treatment on neovascularisation. A, Actual measurements of capillary density in number of capillaries per mm2. B, Bar graph representing the capillary-to-myocyte ratio in different groups. C, Tissue sections with lectin in the viable free wall of the four different groups, showing individual capillaries. *p<0.05 vs. MI, ** p<0.01 vs. MI.

 
3.5. Effects of EPO on MHC isoforms
Induction of heart failure resulted in a 2.5 fold increase in the expression of β-MHC in the MI group (24±5% vs. 58±5% in sham vs. MI, Fig. 4). EPO significantly attenuated the shift from {alpha}-MHC to β-MHC (Fig. 4, p<0.01) by 31% in MI-EPO high (40±3%) and by 28% in the MI-EPO low group (42±4%).


Figure 04
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  		Fig. 4 Effect of EPO treatment on β-myosin heavy chain (MHC) expression. Graphic representation of the expression of β-MHC as a percentage of total MHC. *p<0.001 vs. sham {ddagger} p<0.01 vs. MI.

 
3.6. Effects of EPO on myocardial apoptosis
The number of cleaved caspase-3 positive cells was significantly higher in rats with heart failure, compared to shams (6.6±2, 40±3.4, 29±3, 32±8 in sham, MI, MI-EPO-high and MI-EPO-low respectively, p<0.01, Fig. 5). However, the numbers of cleaved caspase-3 positive cells did not differ significantly between the MI groups.


Figure 05
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  		Fig. 5 Effects of EPO on myocardial apoptosis. A. Immunohistochemical staining of a cleaved caspase-3 positive cardiomyocyte under high power magnification showing a nucleus (blue) and cytoplasmatic presence of cleaved caspase 3 (brown, upper panel). Rabbit immunoglobulin IgG was used as a negative control (lower panel). B. Graphic representation of the number of cleaved caspase-3 positive cells in the viable myocardium. *p<0.01 vs. sham.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Conflict of interests
 References
 
Our study demonstrates that the beneficial effects of EPO on cardiac function in post-MI heart failure are at least in part independent of an increased haematocrit. EPO treatment in a dose that was insufficient to raise haematocrit levels, markedly improved cardiac function. The functional improvement was associated with induction of neovascularisation, and attenuated expression of slow β-MHC isoforms. Most of the beneficial effects were slightly less pronounced in the low-dose group, which might indicate that some of the beneficial effects are related to an increased haematocrit. Alternatively, it might reflect the dose dependent nature of the pleiotropic effects of EPO on the heart, which are completely distinct from erythropoiesis.

In the clinical setting, current therapy after MI is focused on prevention of ventricular remodelling and development of heart failure. Myocardial regeneration may offer possibilities that could improve cardiac function in these patients [26]. Although cardiomyocyte proliferation after ischaemic injury seems limited, the formation of new vessels in the non-infarcted part of the ventricle could lead to an improvement of function and attenuation of ventricular remodelling [27,28]. Evidence is accumulating to suggest that EPO exerts potent pleiotropic effects on the myocardium in the setting of acute myocardial infarction and chronic heart failure as well [2,5-10,29]. Therefore, in addition to its acute protective effects, EPO might evolve as a standard "cardioregenerative" therapy in the setting of chronic myocardial dysfunction.

The dosing regimens used in previous studies, all resulted in supra-physiological haematocrit levels. When applied to the clinical situation, this could lead to hypertension, seizures, vascular thrombosis and death, possibly related to abruptly increased haematocrit values [17,19,30]. This could be of potential concern in patients at already elevated cardiovascular risk. Therefore, we investigated the effects of a non-erythropoietic EPO dose to avoid elevation of haematocrit, and consequently the changes in rheology and oxygen-binding capacity of the blood. We compared these effects to a high EPO dose, with previously established effects on cardiac function in post-MI heart failure [10]. Moreover, similar to the high-dose treatment, low-dose treatment resulted in noticeably improved cardiac function, as reflected by a significantly enhanced developed LVP, together with improved contractility and relaxation indices of the LV. Similar to high-dose EPO, low-dose EPO induced neovascularisation and attenuated the unfavourable switch to slow β-MHC isoforms. However, in contrast to high-dose EPO, LVEDP was not significantly attenuated in the low-dose group compared to the untreated MI. Thus, in spite of increased filling pressures, low-dose EPO improves the contractile properties of the non-infarcted part of the myocardium. Although the (non-significant) larger infarct size in the low-dose EPO group could have partially averted the beneficial effects of EPO, neovascularisation was also less pronounced in the MI-EPO-low group. This might indicate that some of the beneficial effects are related to an increased haematocrit and consequently increased oxygen delivery. However, the low-dose EPO group was treated with a dose 100 times lower than the high-dose group. Therefore, the slightly less pronounced effects in the MI-EPO-low group might also reflect a reduced magnitude of the non-erythropoietic effects. The anti-apoptotic effects of EPO in the myocardium have been well described in the setting of acute ischaemia, yet EPO did not reduce apoptotic cells in our study. Although these results might seem to contradict previous reports, apoptosis was determined 3 weeks after the last EPO dose, when from our previous study we know that EPO levels will be comparable between the EPO and saline groups [14]. Since the anti-apoptotic effects of EPO are dependent on EPO receptor signalling, comparable EPO levels will have a comparable anti-apoptotic effect.

Lower doses of EPO have also been shown to confer vascular and tissue protection in the kidney [22]. Low-dose darbepoetin treatment in a rat remnant kidney model improved the survival, ameliorated endothelial damage and preserved renal function, without an increase in haematocrit levels. In contrast, Prunier et al. recently reported that a weekly dose of 0.75 µg/kg darbepoetin alfa, initiated 1 week after myocardial ischaemia reperfusion injury, was insufficient to improve cardiac function and induce neovascularisation [9]. The dose used by Prunier et al. was clearly higher than in the present study and resulted in a significant elevation of haematocrit. The reason for the discrepancy between the study by Prunier et al. and our study is unclear. Possibly, the relatively limited infarct size associated with ischaemia reperfusion injury, and the consequently limited decline in cardiac function, might result in more equivocal results. Furthermore, since increased haematocrit levels might have deleterious effects, the balance between the negative effects of haematocrit elevation and the beneficial effects of EPO might become unfavourable.

Another option to circumvent the unwanted effects of EPO on haematocrit could be the use of recently discovered non-erythropoietic derivates of EPO, which retain the tissue protective properties, without the undesired effect on erythropoiesis [21]. The possibility of separating the erythropoietic and tissue protective effects of EPO could be explained through interaction with different receptors in the bone marrow and in "peripheral" tissues. Two independent studies have demonstrated that these non-erythropoietic EPO's retain their acute cardioprotective potential [31,32]. It is however uncertain whether these new EPO's will also improve cardiac function in CHF. Finally, a very recent study by Schneider et al. revealed that endocardial EPO injections improve the contractile function of hibernating myocardium, without affecting haematocrit levels [33]. One of the major shortcomings of the present study is the lack of proper evaluation of platelet activation and coagulability. Apart from differences in haematocrit values, the beneficial effect of low-dose EPO might include attenuated EPO-induced activation of platelets and coagulability. Although some reports have described enhanced coagulability in patients treated with EPO, a link between haematopoiesis-stimulating drugs and thrombosis has not been proven. Indeed, Lindenblatt et al. recently demonstrated that high-dose darbepoetin (10 µg/kg/week) did not affect coagulation and platelet aggregation in mice. Since we used an 8 times lower dose of EPO in the present study, similar results are expected, but need to be proven in future studies.

In summary, EPO treatment improves cardiac function and induces neovascularisation in post-MI heart failure, even at doses that do not increase haematocrit. Although time-limited treatment with high-dose EPO may be beneficial and safe during acute ischaemic injury, if prolonged therapy is required (heart failure), drug regimens using low-dose EPO may be more suitable to avoid the adverse effects of the treatment.


   Conflict of interests
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Conflict of interests
References
 
This study was sponsored by an unrestricted educational grant from Amgen Inc. Dr. Westenbrink, Dr. van der Meer, and Dr. van der Harst are supported by Netherlands Organisation of Scientific Research (NWO). Dr. Lipsic and H. Oeseburg are supported by GUIDE. Dr. van Veldhuisen and Dr. Voors are established investigators of the Netherlands Heart Foundation (grant D97-017 and grant 2006T037 respectively).


   Acknowledgements
 
We thank Azuwerus van Buiten, Maaike Goris, Liza Wong, Bianka Meijeringh and Richard van Veghel for their expert technical assistance.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Conflict of interests
 References
 

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