Western blot analysis.

Western blot analysis was performed for ERK, Akt, Jak1, and STAT3 activation using specific antiphosphorylated Ab’s. H9c2 cells were serum starved in DMEM with 0.1% BSA for 24 hours. Serum-starved cells in six-well plates were stimulated with 4 U/ml of EPO for the indicated time at 37°C. After stimulation, monolayers were washed once with ice-cold PBS and lysed in buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin. After lysis, the same amount of proteins was applied for 4–20% polyacrylamide/Tris-glycine gel. Phosphorylated protein was then visualized by enzyme-linked chemiluminescence (Amersham Biosciences) and quantified by scanning laser densitometry, normalizing to total amounts of the corresponding proteins.

DNA synthesis.

DNA synthesis was determined as incorporation of [³H]-thymidine into trichloroacetic acid–insoluble (TCA-insoluble) material. Plates (n = 6) were seeded at an initial density of 2 × 10⁴ cells/well in 12 wells and grown to subconfluence. Cells were rendered quiescent by 48 hours in serum-free DMEM supplemented with 0.1% BSA and stimulated by the addition of fresh medium containing the agonists. [³H]-thymidine (1 μCi/ml) was added to the medium at the time of stimulation. Cells were washed twice with PBS and three times with 0.5% TCA, and then cells were lysed with 1N NaOH. Extent of cell growth was determined by counting incorporation of [³H]-thymidine into nascent DNA strands at 48 hours.

Apoptosis quantification after H₂0₂ treatment and hypoxia.

H9c2 cells were plated at 3 × 10⁵/well in six-well tissue culture plates and pretreated with EPO (0.4 or 10 U/ml) or vehicle for 24 hours prior to beginning the H₂O₂ (200 μM) treatment in DMEM without glucose and FBS. Following H₂O₂ treatment for 22 hours, adherent cells were fixed with 4% paraformaldehyde, and nuclei were stained with Hoechst 33258 dye to visualize morphological changes by fluorescence microscopy. Alternatively, similar H9c2 cell densities were plated into 12-well plates at 75% confluence. Experimental wells received EPO (8 U/ml), PD98059 (5 μg/ml), wortmannin (500 nM), and combinations thereof in the culture media. All plates were subjected to 12 hours of anoxia (95% N₂, 5% CO₂) and subsequently fixed and stained as detailed above. Apoptotic cell death was determined by bright staining on condensed nuclei or discrete fragmented nuclei (21).

Induction of MI.

Animals used in this in vivo study were adult male New Zealand White rabbits (2.5–3.5 kg), and all procedures were performed in accordance with the regulations adopted by the NIH and approved by the Animal Care and Use Committee of Duke University. MI was induced in anesthetized rabbits by a left thoracotomy and ligation of a branch of the left circumflex coronary artery (LCx) as described previously (22). Rabbits were sedated with a mixture of ketamine (30 mg/kg) and acepromazine (0.05 mg/kg), intubated, and mechanically ventilated. Inhalation anesthesia (isoflurane) was titrated to maintain the absence of pedal and corneal reflexes. Animals were preoperatively randomized to receive a single dose of either EPO (5,000 U/kg; Amgen Inc., Thousand Oaks, California, USA) (n = 12) or normal saline (3 ml) (n = 11) by the animal technician at the time of MI. Sham animals (n = 5) underwent thoracotomies in which the LCx was circled with a prolene suture, but was not occluded. Rabbits were sacrificed on postoperative day 4, and their hearts were harvested for infarction quantification. Infarct size, as a percentage of the left ventricle (LV) and septal mass, was quantified using triphenyltetrazolium chloride (TTC) staining as described previously (23). Fixed heart sections were placed on a camera mount to standardize camera-to-specimen distance, and digital photographs were taken. Using enlarged projections, the boundary of the stained areas on each slice was traced in a blinded fashion, and Adobe Photoshop 6.0 (Adobe Systems Inc., San Jose, California, USA) was used to quantify infarcted versus normal myocardium.

Assessment of area at risk.

Rabbits were anesthetized and underwent a thoracotomy as described above. Rabbits were anticoagulated with intravenous heparin (70 U/kg), followed by 30 minutes of LCx occlusion. In a randomized fashion, rabbits received EPO (5,000 U/kg) (n = 5) or an equivalent volume of normal saline (n = 5) following LCx occlusion via a peripheral ear vein. At the start of reperfusion, a marking stitch was left at the site of occlusion, and on day 3 the animals were sacrificed and their hearts harvested. The area of the LV at risk for ischemic injury was stained as described (23, 24) using TTC staining, followed by counterstaining with pthalo blue after occlusion at the previous ligation site using the marking stitch. The heart was then dissected, cut into five or six 2-mm-thick transverse slices, fixed, and photographed as described (23). Total weights of area at risk and area of necrosis were then blindly calculated and expressed as percentage of total LV weight.

TUNEL.

In the subset of animals chosen for in vivo TUNEL assessment, animals were pretreated with 1,000 U/kg EPO (n = 4) or normal saline (n = 4) 24 hours prior to LCx ligation and survived for 6 hours. Histologic heart samples were frozen in cryomolds, and TUNEL labeling was performed on 5-μm sections per the manufacturer’s protocol (In Situ Cell Death Detection kit; Roche Diagnostics, Indianapolis, Indiana, USA). A notable exception was that the cell membrane–permeablization step was extended to 15 minutes. Digital photographs were taken under fluorescence microscopy at ×200 magnification. Twenty-five random high-power fields from each heart sample were chosen and blindly quantified.

In vivo kinase activity.

For assessment of Akt and ERK activation, normal rabbits received either 1,000 U/kg (n = 4) or saline (control, n = 4) intravenously and were sacrificed 12 hours later. LV samples were then homogenized in the buffer described above for the cell experiments, and activated Akt and ERK were assessed by protein immunoblotting using Ab’s raised against the phosphorylated kinases and quantified as described above.

In vivo hemodynamic measurements.

All animals underwent hemodynamic assessment 24 hours prior to MI or sham operation, and a repeat assessment was done on postoperative day 3, as we have described previously (22). LV pressure and heart rate were measured by a 2.5 French micromanometer (Millar Instruments Inc., Houston, Texas, USA) advanced into the LV cavity, and data were recorded on a PowerLab System (ADInstruments Pty Ltd., Mountain View, California, USA) as described (22).

Hematocrit measurements.

Blood samples were obtained from a peripheral ear vein of sedated rabbits at serial time points: day 0, 1, 3, and 4. Samples were analyzed using a blood gas/electrolyte analyzer (Model 5500; Instrumentation Laboratory, Ann Arbor, Michigan, USA).

EPO’s antiapoptotic effect in H9c2 myoblasts.

The H9c2 cell line is known to undergo apoptotic cell death with H₂O₂ treatment (29) and was thus used to assess cardiovascular cell protective properties of EPO. Tissue ischemia is associated with respiratory uncoupling and reactive oxygen species (ROS) production (30, 31), and thus, H₂O₂ represents an in vitro model of ischemic injury due to oxidative stress (19). H9c2 cells were treated with 200 μM H₂O₂ for 22 hours to see the effect of pretreatment with EPO to protect cells from apoptosis. H9c2 cells pretreated with EPO exhibited increased survival when compared with cells that did not receive EPO treatment (Figure ​(Figure2).2). As shown in Figure ​Figure2a,2a, this protection appears to be due to inhibition of apoptosis; after exposure to H₂O₂, cells pretreated with EPO (either 0.4 or 10 U/ml) exhibited a significant (~50%) decrease in apoptotic nuclear morphological change (P < 0.05).

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Figure 2

Apoptotic cell death in H9c2 myoblasts exposed to oxidative stress and hypoxia. (a) The ratio of apoptotic cells to total adherent cells in the dish following oxidative stress (H₂O₂ exposure). Cells were treated with H₂O₂ (200 μM) following the treatment with EPO (0.4 or 10 U/ml, n = 8 each). Cells were stained with Hoechst 33258 dye, and nuclear morphology was revealed by fluorescent microscopy (described in Methods). Data shown are the mean ± SEM. *P < 0.05 versus untreated cells. (b) The ratio of apoptotic cells to total adherent cells in the dish after hypoxic injury. Cells were exposed to anoxia (12 hours) and percentage of nuclear fragmentation quantified under the following conditions: vehicle (control), DMSO (n = 4), white bars; wortmanin (n = 4), black bars; and PD98059 (n = 4), gray bars. Presence (8 U/ml) or absence of EPO is indicated by + and –, respectively. Cells were stained as above. *P < 0.05 versus vehicle-treated (DMSO), ^†P < 0.05 versus EPO alone. (c and d) Representative sample of H9c2 cells treated with H₂O₂ (200 μM) without EPO pretreatment (c) or with EPO (10 U/ml) for 24 hours (d). Arrows indicate fragmented nuclei under both conditions.

Similar results were obtained when H9c2 cells were subjected to isolated anoxic injury for 12 hours (Figure ​(Figure2b)2b) because EPO-mediated protection during hypoxia resulted in a mitigation of nuclear morphologic change as opposed to H9c2 cells exposed to anoxia alone (37.7% ± 3.9% versus 56.8% ± 6.7%; P < 0.01). Akt inhibition by the addition of the PI3K inhibitor wortmanin reversed EPO-mediated protection (54.2% ± 5.6%) after hypoxia, while ERK inhibitor (PD98059) did not (Figure ​(Figure2b).2b). Importantly, wortmanin or PD98059 compound did not significantly alter survival of H9c2 cells in the absence of EPO. Figure ​Figure2,2, c and d, contains representative cellular staining with Hoechst 33258 dye. Untreated H9c2 cells exposed to ischemic injury show more positive-stained apoptotic nuclei (Figure ​(Figure2c)2c) compared with H9c2 cells treated with EPO (Figure ​(Figure22d).

Effects of systemic EPO administration on in vivo cardiac function following MI.

To examine potential cardioprotective effects of EPO in vivo, we chose a rabbit model of MI caused by LCx ligation (22). This model generally produces LV infarcts of greater than 25% and results in significant LV dysfunction both acutely and chronically (22). In these experiments, 5,000 U/kg of human recombinant EPO was randomly and blindly administered at the time of LCx ligation. Control MI rabbits received saline; we also included a sham control group with no MI and no EPO. Blood was drawn for determination of hematocrit at the time of MI (day 0) and daily thereafter. At day 3 after MI, we measured in vivo cardiac function by Millar catheterization. Parameters were studied both at baseline and in response to the β-adrenergic receptor (βAR) agonist ISO and included peak LV pressure, LV end-diastolic pressure (LVEDP), heart rate, and LV +dP/dt_max and LV –dP/dt_min as measures of global cardiac contractility and relaxation, respectively. Complete baseline hemodynamic measurements are shown in Table ​Table1,1, and cardiac contractility and relaxation in response to βAR stimulation are shown in Figure ​Figure3,3, a and b, respectively. Overall, MI rabbits had impaired LV function compared with sham controls, as we have documented previously (22), and although EPO did not completely restore global cardiac function, there was significant improvement of inotropic reserve with EPO. Importantly, both heart rate (Figure ​(Figure3c)3c) and LVEDP (Figure ​(Figure3d),3d), which can influence global cardiac function, were not altered in either MI group. Thus, these data demonstrate a true enhancement of global cardiac function by EPO at this acute window of measurement.

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Figure 3

In vivo cardiac physiology in post-MI rabbits. Hemodynamic measurements at post-MI day 3 in the experimental groups MI + saline (n = 11), filled diamonds; MI + EPO (n = 12), filled squares; and normal sham rabbits (n = 5), filled triangles. Measurements were taken at baseline (0) (see Table ​Table1)1) and after progressive ISO stimulation. Data is the mean ± SEM. *P < 0.05 versus MI + saline; ^†P < 0.05 versus sham (ANOVA). (a) Global cardiac function measured by LV dP/dt_max. (b) LV relaxation as measured by LV dP/dt_min. (c) Heart rate. (d) LVEDP (EDP).

Table 1

Summary of in vivo baseline hemodynamic data in rabbits

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In regard to these positive functional effects of EPO in the post-MI heart, increased O₂ carrying capacity due to a higher hematocrit needs to be considered, especially since previous studies have described the beneficial effects of hematocrit augmentation on cardiac function, including the use of EPO (32, 33). Importantly, no discernable difference in hematocrit in rabbits was noted until day 4 after MI (Figure ​(Figure3),3), and all post-MI physiology data were obtained on day 3 after MI when hematocrits in the two groups were effectively identical.

Another confounding variable in the physiological data after MI is infarct size, since smaller infarcts would naturally have less dysfunction. Rabbit hearts were harvested on day 4 after MI and stained with TTC (see Methods). Cross sections were captured as digital photographs and analyzed by computerized planimetry (22). Saline-treated MI rabbits exhibited an average infarct of 24.0% ± 3.1% of the LV free wall, while EPO-treated MI rabbits exhibited infarctions of 18.5% ± 2.7% of the LV. While not statistically significant (P = 0.1), there appears to be a strong trend for EPO to reduce infarct size. This data prompted us to examine more closely the ischemic area of risk in these infarcted rabbit hearts (see below).

Effect of EPO on LV area at risk after MI in rabbits.

We wanted to investigate further the apparent smaller infarct size induced by EPO by examining the total area of the LV at risk of ischemic injury following MI. In this protocol, rabbits underwent a 30-minute occlusion of the LCx followed by 3 days of reperfusion. Rabbits were randomly and blindly treated with EPO or saline at the time of reperfusion as described above, and 3 days later the LV ischemic area and infarcts were analyzed by standard TTC–pthalo blue staining (23, 24). Notably, while the potential ischemic zone was similar in both MI groups (EPO: 32.1% ± 3.9%, n = 5; saline: 31.1% ± 4.0%, n = 5), the percentage of infarcted tissue in this area at risk was significantly reduced in the EPO group (13.8% ± 4.7%) as compared with the saline group (35.1% ± 4.3%, P = 0.004) (Figure ​(Figure5a).5a). Representative staining of area at risk and infarct size can be seen in Figure ​Figure5,5, b and c, for saline- and EPO-treated MI hearts, respectively, demonstrating significantly less infarction (nonstained tissue) after EPO treatment.

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Figure 5

LV area at risk quantification after ischemia/reperfusion. (a) Graphic representation of the LV infarction size measured as the percentage of infarct of total ischemic area at risk in MI + saline (control) rabbits (black bar, n = 5) and MI + EPO rabbits (white bar, n = 5). The LV area at risk was virtually identical in both groups (see Results). Data are the mean ± SEM. *P < 0.005 versus MI + saline. (b) Representative LV cross sections from a MI + saline (control) rabbit and (c) from a MI + EPO rabbit. The dark blue–stained areas are nonischemic tissue, and red-stained areas represent the ischemic LV area at risk. Infarcted areas are indicated by blanched (yellow-white) areas within this area at risk, with much less seen in the MI + EPO heart (c).

TUNEL.

Since EPO-treated rabbits have smaller infarcts as a percentage of the LV ischemic area at risk and thus more viable myocardium — accounting for the enhanced post-MI function — we directly assessed whether EPO is protecting ischemic myocardium against apoptosis. Specifically, we carried out TUNEL labeling of LV sections 6 hours after MI in rabbits that were treated with either EPO or saline 24 hours previously. Six hours after LCx occlusion, TUNEL-positive cells were noted in both the saline- and EPO-treated rabbits, although significantly less staining was noted in the EPO group (13.8% ± 2.0% versus 29.3% ± 3.3%; P < 0.001) (Figure ​(Figure6a).6a). No TUNEL-positive cells were seen in sham (no MI, no EPO) animals. Representative TUNEL-stained sections are shown in Figure ​Figure6,6, b and c, demonstrating fewer apoptotic positive cells in the EPO-treated MI rabbit heart (Figure ​(Figure6c),6c), which is consistent with lower apoptosis in cardiac myoblasts in vitro following oxidative stress and hypoxia.

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Figure 6

TUNEL staining and quantification of apoptosis in vivo in postischemic rabbit hearts. (a) Graphic representation of TUNEL-positive nuclei in MI + saline (control) rabbit hearts (black bar, n = 4) and MI + EPO rabbits (white bar, n = 4). Data shown are the mean ± SEM of the number of TUNEL-positive cells (nuclei) per high-power field analyzed by microscopy. *P < 0.05 versus MI + saline (control) rabbits. (b) Representative TUNEL-stained LV section from an MI + saline (control) rabbit showing several positive-stained apoptotic nuclei. (c) Representative TUNEL-stained LV section from a MI + EPO rabbit with very few apoptotic nuclei.

The authors thank Elaine Parker, George Quick, and Andrea Koch for technical assistance and Damien Craig for technical and statistical assistance. This study was supported in part by NIH grants R01-HL59533 and R01-HL56205 (to W.J. Koch), R01-ES09206 (to J.S. Stamler), and an NIH/National Research Service Award Fellowship F32-HL70505 (to C.J. Parsa). J.S. Stamler is an Investigator of the Howard Hughes Medical Institute.