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Am J Physiol Heart Circ Physiol 293: H60-H68, 2007. First published March 23, 2007; doi:10.1152/ajpheart.00227.2007
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TRANSLATIONAL PHYSIOLOGY

Darbepoetin alfa, a long-acting erythropoietin analog, offers novel and delayed cardioprotection for the ischemic heart

Erhe Gao,1 Matthieu Boucher,1 J. Kurt Chuprun,1 Rui-Hai Zhou,2 Andrea D. Eckhart,2 and Walter J. Koch1

1George Zallie and Family Laboratory for Cardiovascular Gene Therapy and 2Eugene Feiner Laboratory for Vascular Biology and Thrombosis, Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania

Submitted 21 February 2007 ; accepted in final form 20 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies from our lab and others have shown that the hematopoietic cytokine erythropoietin (EPO) can protect the heart from ischemic damage in a red blood cell-independent manner. Here we examined any protective effects of the long-acting EPO analog darbepoetin alfa (DA) in a rat model of ischemia-reperfusion (I/R) injury. Rats were subjected to 30-min ischemia followed by 72-h reperfusion. In a dose-response study, DA (2, 7, 11, and 30 µg/kg) or vehicle was administered as a single bolus at the start of ischemia. To determine the time window of potential cardioprotection, a single high dose of DA (30 µg/kg) was given at either the initiation or the end of ischemia or at 1 or 24 h after reperfusion. After 3 days, cardiac function and infarct size were assessed. Acute myocyte apoptosis was quantified by TUNEL staining on myocardial sections and by caspase-3 activity assays. DA significantly reduced infarct size from 32.8 ± 3.5% (vehicle) to 11.0 ± 3.3% in a dose-dependent manner, while there was no difference in ischemic area between groups. Treatment with DA as late as 24 h after the beginning of reperfusion still demonstrated a significant reduction in infarct size (17.0 ± 1.6%). Consistent with infarction data, DA improved in vivo cardiac reserve compared with vehicle. Finally, DA significantly decreased myocyte apoptosis and caspase-3 activity after I/R. These data indicate that DA protects the heart against I/R injury and improves cardiac function, apparently through a reduction of myocyte apoptosis. Of clinical importance pointing toward a relevant therapeutic utility, we report that even if given 24 h after I/R injury, DA can significantly protect the myocardium.

myocardial ischemia; ischemia-reperfusion injury; apoptosis

CARDIOVASCULAR DISEASE REPRESENTS the number one cause of mortality and hospitalization in developed countries, and ischemic heart disease greatly contributes to this worldwide health concern. Despite effective therapy for coronary artery disease, outcomes in congestive heart failure have not improved in the past two decades (35). Protection of the myocardium during myocardial ischemia resulting from coronary heart disease and myocardial infarction (MI) has been a target of investigation for the development of innovative cardioprotective therapies (14). One such approach that may have clinical utility is to protect the ischemic heart against cell death, including apoptosis, which can be robustly stimulated by ischemic/hypoxic conditions (14). Of importance, limiting infarct size after MI may prevent or significantly delay ventricular dysfunction and thus decrease the future occurrence of chronic heart failure.

Erythropoietin (EPO), a hematopoietic cytokine that is primarily synthesized in kidney peritubular cells, has long been used as a clinical treatment for conditions that produce anemia. The physiological effects of EPO in hematopoietic cells are mediated through its interaction with its specific cellular receptor (EPO-R), a member of the type I superfamily of single-transmembrane cytokine receptors (8). Recently, EPO has also been found to be synthesized in a variety of other tissues including liver, peripheral endothelial cells, vascular smooth muscle cells, and cardiomyocytes (23). EPO-Rs have also been reported in those cells (1, 2, 5, 43) although recent studies have suggested that caution should be used in interpreting some of that data, given the lack of specificity of anti-EPO-R antibodies used (5a, 9a, 27a). Recombinant human (rh)EPO administration has been shown to exert a broad tissue-protective effect in a variety of experimental ischemic injury models in brain, retina, and kidney (10, 19, 36). Furthermore, EPO has been found to exhibit significant therapeutic value in rodent models of diabetic neuropathy (21), autoimmune encephalomyelitis (37), and stroke (42). In the cardiovascular system, through the binding to the EPO-R, EPO induces endothelial cell proliferation and angiogenesis (34, 40, 44). Moreover, rhEPO has been shown to exert marked myocardial protective effects against ischemia-reperfusion (I/R) injury in rats, rabbits, and dogs when administrated at early time points (18, 28, 30). Studies from our lab as well as others showed that rhEPO administration reduced left ventricular (LV) infarct size, enhanced recovery of LV function, reduced the number of myocytes undergoing apoptotic cell death, and activated phosphatidylinositol 3-kinase (PI3-kinase) and Akt (28, 30, 33, 41). These protective effects of EPO were all demonstrated to be independent of a rise in hematocrit. The acute benefits of EPO on the ischemic heart are quite clear, but it is still debatable whether there are long-term protective benefits of EPO or whether increased hematopoiesis may negate these benefits in chronic ischemic heart disease (15, 40).

In the present study, we examined the cardioprotective effects of darbepoetin alfa (DA), an erythropoietic agent with a threefold longer plasma half-life than rhEPO (22), on the rat heart, including its effect on apoptosis following I/R injury. Our results show that DA displays robust dose-dependent myocardial protection after ischemic injury and that this protection, due to limiting myocardial apoptosis, is evident even when a single dose is administered as late as 24 h after the beginning of reperfusion. These results suggest that DA has a rather wide therapeutic window of cardioprotective opportunity that increases its potential clinical utility in cases of myocardial ischemia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental protocol. Experiments were carried out according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Thomas Jefferson University Committee on Animal Care. For the rat model of I/R injury, a previously described method was used with some modification (12). Male Sprague-Dawley rats (200–300 g) were anesthetized with 2% isoflurane inhalation. A skin incision was made over the left thorax, and the pectoral muscles were retracted to expose the ribs. The heart was exposed through a left thoracotomy at the level of the fifth intercostal space. A slipknot was made around the left anterior descending coronary artery (LAD) 2–3 mm from its origin with a 6-0 silk suture. After the slipknot was tied, the heart was immediately placed back into the intrathoracic space, followed by manual evacuation of pneumothoraces and closure of muscle and the skin suture by means of the previously placed purse-string suture. Sham-operated animals were subjected to the same surgical procedures except that the suture was passed under the LAD but was not tied. Animals were recovered from anesthesia within 5 min after the completion of surgery and received appropriate postsurgery analgesia. After 30 min of ischemia, the slipknot was released and the myocardium was reperfused for 3 h to determine caspase-3 activity, 24–48 h to determine myocardial apoptosis [terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)], or 72 h to assess cardiovascular function and myocardial infarct size. The overall study protocol is illustrated in Fig. 1. In the DA dose-response study, rats were subjected to sham surgery (n = 10) or I/R and randomized to receive DA (Amgen, Thousand Oaks, CA) at the initiation of ischemia at a dose of 2 (n = 8), 7 (n = 8), 11 (n = 8) or 30 µg/kg (n = 10), or saline as the control vehicle (n = 10), intravenously (via tail vein). In the time course study, rats were subjected to sham surgery (n = 9) or I/R. DA (30 µg/kg) was given at the initiation (T0; n = 9) or end (T0.5; n = 9) of ischemia or at 1 (T1.5; n = 6) or 24 (T24; n = 19) h after the beginning of reperfusion by intravenous injection. Drug vehicle (n = 9) was given at T0.


Figure 1
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  		Fig. 1. Schematic illustration of experimental protocols. 1) Testing the dose dependence for the cardioprotective properties of darbepoetin alfa (DA) (dose response). Doses were 2, 7, 11, and 30 µg/kg administered at the beginning of the ischemia period. 2) Testing the time course of this cardioprotection. In this study, the dose of 30 µg/kg DA was used. 3) Determining DA's effects on myocardial apoptosis (caspase-3 activity and TUNEL staining) after ischemia-reperfusion (I/R) (see text for details). TTC, triphenyltetrazolium chloride.

 
In vivo hemodynamic measurements. We assessed in vivo cardiac hemodynamic function 72 h after I/R. Rats were anesthetized with a 2% isoflurane, and the right common carotid artery was isolated and cannulated with a 2-Fr micromanometer (Millar Instruments, Houston, TX) (26, 31). LV pressure, LV end-diastolic pressure (LVEDP), and heart rate (HR) were measured by this catheter advanced into the LV cavity, and data were recorded and analyzed on a PowerLab System (AD Instruments, Mountain View, CA) as we have previously described (26, 31). These parameters as well as LV maximum positive change in pressure over time (+dP/dtmax), as a measure of cardiac contractility, and LV minimum negative change in pressure over time (–dP/dtmin), as a measure of relaxation, were recorded at baseline and after administration of the beta-adrenergic receptor (beta-AR) agonist isoproterenol (Iso, 0.1–1,000 ng) as described elsewhere (26, 31).

Determination of LV area at risk and infarct size. At the end of the 72-h reperfusion period, the ligature around the LAD was retied through the previous ligation, and 0.2 ml of 2% Evans blue dye was injected. The dye was circulated uniformly and distributed in the heart to areas perfused by the open coronary arteries. The heart was quickly excised, and atria, RV, and fatty tissues were removed. The LV was then sliced into six 1.2-mm-thick sections perpendicular to the long axis of the heart. The sections were then incubated in PBS containing 2% triphenyltetrazolium chloride (TTC; Sigma) at room temperature for 15 min and digitally photographed. The Evans blue-stained area (area not at risk, ANAR), TTC-stained area, and TTC-negative staining area [infarcted myocardium; area at risk (AAR), including both TTC-negative and -positive areas] were measured with the computer-based image analyzer SigmaScan Pro 5.0 (SPSS Science, Chicago, IL). Myocardial infarct size was expressed as a percentage of the AAR (I/AAR), and the AAR was expressed as the percentage of total LV (AAR/AAR+ANAR).

Determination of myocardial apoptosis. Myocardial apoptosis was analyzed by TUNEL staining and caspase-3 activity assay. For TUNEL staining, at the end of 24 (where DA was administered at T0) or 48 h (where DA was administered at T24) of reperfusion, rats (n = 4 for sham-operated group, n = 5 for vehicle group, and n = 5 for DA group) were anesthetized. Hearts were removed, perfused and fixed with 4% paraformaldehyde, and then stored at 4°C. The hearts were then embedded in paraffin and cut into 6-µm-thickness sections. The sections were treated as instructed with an in situ cell death detection kit (Roche, 1684817). Slides were covered with a glass cover slide applied with mounting medium containing DAPI. The entire population was visualized under a fluorescence microscope with the DAPI filter (330–380 nm); the same population was also examined with a FITC filter (465–495 nm), and apoptotic cells with green fluorescence were counted under a high-power field (>5 fields in >3 different sections/animal in the infarction border zone).

Cardiac caspase-3 activity was measured with a caspase colorimetric assay kit (Chemicon International, Temecula, CA) according to the manufacturer's instructions. At the end of the 3-h reperfusion period, a subgroup of rats (n = 7 for sham-operated group, n = 8 for vehicle group, and n = 8 for DA group) were anesthetized; hearts were removed and stored at –80°C until analysis. The myocardial tissue was homogenized in ice-cold lysis buffer for 30 s with a tissue homogenizer. The homogenates were centrifuged for 5 min at 10,000 g and 4°C, supernatants were collected, and protein concentrations were measured by the bicinchoninic acid method (Pierce Chemical, Rockford, IL). To each well of a 96-well plate, supernatant containing 200 µg of protein was loaded and incubated with 25 µg of N-acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) as colorimetric-specific substrate at 37°C for 1.5 h. The pNA was cleaved from DEVD by caspase-3. This free pNA was quantified with a SpectraMax-Plus microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) at 405 nm. Changes in caspase activity in the I/R tissue samples were calculated against the mean value of caspase activity in the sham-operated tissue and expressed as picomoles of pNA per milligram of protein.

Measurement of plasma DA concentration. At the end of the 72-h reperfusion period, ~1 ml of blood was collected and centrifuged for 1 min at 10,000 g. The resultant plasma was decanted and kept at –80°C until analysis. The samples were thawed, and DA levels were measured spectrophotometrically at 450 nm with the Quantikine In Vitro Diagnosis kit for human EPO (R&D System, Minneapolis, MN). Data are expressed as milli-international units per milliliter.

Measurement of hematocrit. Hematocrit was measured at the end of 72-h reperfusion as described previously (28, 30).

Statistical analysis. All values are presented as means ± SE of independent experiments from given n sizes. Statistical significance of multiple treatments was determined by one-way or two-way ANOVA followed by the Bonferroni post hoc test when appropriate. P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Dose-dependent effects of DA on in vivo cardiac function. To examine potential cardioprotective effects of DA in vivo, we chose a rat model of I/R injury via LAD ligation for 30 min followed by up to 72 h of reperfusion (12). This model generally produces LV infarcts of >30% and results in significant cardiac dysfunction. First, rats were randomly divided into six groups: sham operated, I/R + vehicle (saline), and I/R + DA (2, 7, 11 or 30 µg/kg). Treatments were randomly and blindly administered at the time of LAD ligation (Fig. 1). After 72 h of reperfusion, we measured in vivo LV contractile function via Millar catheterization and hemodynamic measurements. Parameters were studied both at baseline and in response to the beta-AR agonist Iso and included peak LV pressure, LVEDP, HR, and LV +dP/dtmax and LV –dP/dtmin as measures of ventricular contractility and relaxation, respectively (Fig. 2). Rats undergoing I/R had significantly impaired LV contractile function compared with sham-operated animals (Fig. 2, A and B). Treatment with DA at the onset of ischemia significantly preserved impaired LV contractility and relaxation in a dose-dependent manner, with the most beneficial effect seen at the dosages of 11 and 30 µg/kg under inotropic stimulation (Iso; Fig. 2, A and B). Minor improvements in LV +dP/dtmax and LV –dP/dtmin were also seen in animals treated with 7 µg/kg of DA after Iso challenge. There were no treatment effects at the lowest dose of DA (2 µg/kg). Finally, there were no significant differences in LVEDP (Fig. 2C) and HR (Fig. 2D) as well as mean arterial blood pressure (MABP; data not shown) among all groups.


Figure 2
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  		Fig. 2. Dose-dependent effects of DA on postischemic cardiac hemodynamic function. Rats were subjected to sham operation (Sham) or 30-min ischemia followed by 72 h of reperfusion treated with saline (Vehicle), 2 µg/kg DA, 7 µg/kg DA, 11 µg/kg DA, or 30 µg/kg DA (DA 30 µg) at the onset of ischemia. Data shown were recorded 72 h after I/R: left ventricular (LV) maximum positive change in pressure over time (LV +dP/dtmax, A), LV minimum negative change in pressure over time (LV –dP/dtmin, B), LV end-diastolic pressure (LVEDP, C), and heart rate (HR, D). These hemodynamic measurements were recorded at baseline (B) and on isoproterenol (Iso, 0.1–1,000 ng) administration (n = 8–10/group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle.

 
DA reduces post-I/R myocardial injury. MI represents the total myocardial injury caused by myocyte necrosis and apoptosis. To determine the effect of DA on myocardial injury and total LV infarction in our model of I/R, we dissected hearts after 72 h of reperfusion and stained them with TTC to determine the LV infarct size (I/AAR) and total AAR (AAR/LV). Our data show that control rats, which had I/R but were treated with saline, had infarction size of 32.8 ± 3.5% within the total AAR (Fig. 3). Consistent with the in vivo functional data described above, DA significantly reduced LV infarct size in a dose-dependent manner (Fig. 3, A and B). There was no difference in the value of AAR/LV between groups (including the sham-operated animals that only underwent occlusion of the LAD for AAR measurement purposes), indicating that a comparable degree of ischemia was induced in those groups (Fig. 3C). To determine whether the dose-dependent myocardial protective effects of DA correlated with circulating DA concentration, blood was drawn after 72 h of reperfusion and plasma DA levels were measured. Importantly, plasma concentrations of DA were significantly increased in a dose-dependent manner (Fig. 4A). Previous results by our group (26, 28) with EPO in MI and I/R demonstrated cardioprotection independent of hematopoiesis. Thus we determined hematocrit levels in our animals at the end of the 72-h reperfusion period treated with a single dose of DA. The hematocrit was increased by <12% with the two higher doses of DA (Fig. 4B). Previously with EPO we found (28, 20) that hematocrit was similarly elevated at this time point but not raised earlier when actual cardioprotection was seen. Thus we also believe DA's protection was hematocrit independent.


Figure 3
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  		Fig. 3. LV infarct sizes in vehicle and DA-treated post-I/R rat hearts. A: representative photographs of TTC-stained rat heart sections obtained from different treatment groups after I/R injury. B: graphic representation of LV infarct size expressed as % of total ischemic area (AAR) in each group (n = 7–9/group). *P < 0.05, **P < 0.01 vs. vehicle (V). C: % of LV AAR for each group.

 

Figure 4
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  		Fig. 4. A: serum concentration of DA measured in the various I/R rats treated with increasing doses of DA and between groups (n = 7–9/group). *P < 0.01 vs. vehicle. B: blood hematocrit measured in rats subjected to I/R and treated with vehicle or increasing doses of DA (n = 4–8/group). *P < 0.05 vs. vehicle. For both DA measurements and hematocrit, blood was taken at the end of 72 h of reperfusion.

 
Time-dependent effects of DA on I/R injury. To determine the time dependence of DA supplementation on postischemic myocardial injury as well as its therapeutic window, a single dose of DA (30 µg/kg) was given by intravenous injection at the initiation (T0) or end (T0.5) of ischemia or at 1 (T1.5) or 24 (T24) h after the beginning of reperfusion. As above, in the vehicle group myocardial I/R resulted in significant in vivo hemodynamic depression after 72 h of reperfusion when animals were challenged with Iso (Fig. 5, A and B). Importantly, treatment with DA at T0, T0.5, and T1.5 resulted in significantly improved Iso-stimulated cardiac contractility and relaxation (Fig. 5, A and B), with no differences in HR, LVEDP, and MABP among groups (data not shown). Remarkably, late administration of DA at 24 h after beginning of reperfusion (T24) still had significant effects on LV function when under inotropic stimulation (Fig. 5, A and B). Consistent with the above infarction data, I/R in these rats resulted in an average LV infarct size of 31.2 ± 3.9% when they were treated with vehicle (Fig. 5, C and D). After administration of DA at T0, T0.5, and T1.5, infarct size was reduced significantly to 9.1 ± 0.8%, 9.0 ± 2.3%, and 10.1 ± 2.1%, respectively (Fig. 5D). Interestingly, treating animals with DA as late as 24 h after the beginning of reperfusion still resulted in significant cardiac protection, because the myocardial infarct size was 17.0 ± 1.6%, a significant 45% reduction from the vehicle-treated control I/R group (Fig. 5, C and D). All infarct size measurements were performed in a double-blinded manner for the late treatment group (n = 19). No difference was noted in AAR size (data not shown).


Figure 5
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  		Fig. 5. Effect of time of DA treatment on myocardial protection and postischemic function. Rats were subjected to sham I/R or I/R, and vehicle or DA (30 µg/kg) was given either at the time of ischemia (T0), 0.5 h after beginning of ischemia (T0.5), 1.5 h after beginning of ischemia and 1 h after beginning of reperfusion (T1.5), or 24 h after beginning of reperfusion (T24). Shown are LV +dP/dtmax (A) and LV –dP/dtmin (B), where hemodynamic measurements were recorded with a Millar catheter after 72 h of reperfusion at baseline (B) and on Iso (0.1–1000 ng) administration (n = 6–9/group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle. C: representative photographs from sham-operated (S), I/R + vehicle (V), and I/R + DA rats treated at the various time points described above. See Fig. 3 for details of the staining. D: graphic representation of the LV infarct size expressed as % of total AAR; numbers inside bars represent the sample sizes in each group. **P < 0.01 vs. vehicle.

 
DA reduces myocardial apoptosis. A recent study from our lab (28, 30) demonstrated that EPO administration in a rabbit model of I/R could reduce myocardial apoptosis. However, direct evidence that DA also can limit infarct size by reducing apoptosis is lacking. Consistent with the results previously reported by our group and other investigators, 30 min of myocardial ischemia followed by 24 h of reperfusion resulted in significant myocardial apoptotic cell death manifested by a 15-fold increase in TUNEL-positive cell labeling (Fig. 6, A and B). In addition, a 2.4-fold increase in caspase-3 activity was observed after 3 h of reperfusion (Fig. 6C). Administration of 30 µg/kg of DA at the onset of ischemia significantly decreased myocardial apoptosis, as evidenced by ~37% reduction in TUNEL-positive cells (Fig. 6B) and a ~35% reduction in caspase-3 activity (Fig. 6C). To see whether a similar reduction of apoptosis was observed in the group treated with DA after 24 h of reperfusion, we did TUNEL staining at 48 h of reperfusion (24 h after this delayed DA treatment). As shown in Fig. 7, the number of TUNEL-positive cells was significantly reduced in the DA-treated group compared with vehicle, indicating reduction of apoptotic processes even with delayed administration. This attenuation of myocardial cell death was similar to what is seen when DA is given early in reperfusion (Fig. 6).


Figure 6
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  		Fig. 6. Apoptosis in rat hearts subjected to I/R and treated with DA before ischemia. A: representative photographs of in situ detection of apoptosis in heart tissue from rats subjected to sham operation or I/R and treated with vehicle or DA (30 µg/kg) at the time of ischemia. Total nuclei were labeled with DAPI (blue), and apoptotic nuclei were detected by TUNEL staining (green). B: average number of TUNEL-positive nuclei per high-power field (HPF) in tissue sections from each group (n = 4 or 5 per group). *P < 0.05 vs. vehicle. C: caspase-3 activity [expressed as pmol of p-nitroanilide (pNA) per mg of protein] in hearts from the same groups (n = 7 or 8 per group). *P < 0.05 vs. vehicle.

 

Figure 7
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  		Fig. 7. Apoptosis in rat hearts subjected to I/R and treated with DA at 24 h of reperfusion. A: representative photographs of in situ detection of apoptosis in heart tissue from rats subjected to sham operation or I/R and treated with saline (Vehicle) or DA (30 µg/kg) at 24 h of reperfusion. Total nuclei were labeled with DAPI (blue), and apoptotic nuclei were detected by TUNEL staining (green). B: average number of TUNEL-positive nuclei in tissue sections from each group (n = 5/group). *P < 0.05 vs. vehicle.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The novelty and significance of the present study begin with the demonstration that the long-acting EPO analog DA can limit in vivo myocardial damage following an infarction after a single intravenous administration. This protection of the ischemic heart was convincingly shown in a dose-dependent manner in this rat model of I/R injury. Perhaps more importantly, for the first time we demonstrate that a potent cardioprotective effect of DA can occur even if the drug is given up to 24 h after the beginning of reperfusion. The protective effects of DA, as we have previously shown for EPO (28, 30), appear independent of hematopoiesis since hematocrit was elevated at 3 days after administration while infarction size limitation is a process taking place early after the onset of reperfusion. Thus DA's effects on the heart appear to be direct, limiting apoptosis and reperfusion-induced myocyte death. This cardioprotective effect of DA leads to preserved and increased viable myocardium in the ischemic zone that translates to enhanced cardiac contractile function and increased inotropic reserve after I/R. Thus there appears to be a wide, clinically relevant therapeutic window for using a single dose of DA in patients experiencing myocardial I/R.

DA and apoptosis reduction. Accumulating evidence indicates that apoptosis, a special form of cell death that differs from necrosis, plays an essential role in cardiomyocyte death following induction of I/R injury (9, 13, 20). One of the most widely recognized biochemical features of apoptosis is the activation of a class of cysteine proteases known as caspases (39). Cells possess multiple caspases, which may work in a cascade fashion. Two pathways have been identified that activate caspases: one activated through a cell surface signal leading to caspase-8 activation and another more complicated pathway involving the mitochondria and resulting in caspase-9 activation (9, 13, 17, 39). Both pathways will activate the downstream caspase-3, leading to the cellular apoptosis and loss of myocardium. It is clear from our data that DA administration during or after the ischemic insult decreases apoptosis in the heart as determined by TUNEL staining and caspase-3 activity assay. This is consistent with previous studies from our lab (28, 30) and others (6, 7, 16, 18, 33, 38) with EPO in various animal models.

Although the exact mechanism of the protective effect of against myocardial ischemia and I/R injuries has not been completely clarified, several cell prosurvival pathways have been implicated. These include kinase cascades involving JAK-STAT (30, 33), PI3-kinase-Akt (7, 28), ERK1/2 (6), and PKC (16, 38). Previously, our results in rabbit models of myocardial ischemia (28, 30) demonstrated a critical role for PI3-kinase and Akt activation in the cardioprotection afforded by EPO delivery. In this study, we did not specifically address which of these kinase cascades might be involved mechanistically in DA's protection. However, it is clear that apoptosis limitation would be a mechanism for improved cardiac function. In the current study using DA, we show cardioprotection not only at the level of a reduction of infarct size but also in postischemic cardiac physiology that includes increased adrenergic-mediated cardiac contractile function in vivo.

DA-induced cardioprotection is dose dependent. DA is a drug used extensively in the clinic for hematopoiesis and has a threefold longer plasma half-life than rhEPO. Our data are the first to show a clear dose and plasma concentration dependence of the protection against myocardial I/R injury. Importantly, all doses that had apparent benefit did so independent of a major rise in hematocrit, as in our previous rabbit studies with EPO (28, 30). The cardioprotective effects of DA are distinct and suggest a direct tissue protective effect through myocardial EPO-Rs. Single higher doses of DA appear to eventually raise hematocrit slightly. Thus caution is warranted when giving a single high dose in patients with compromised circulatory systems such as in coronary artery disease, because increased blood viscosity could be harmful. Interestingly, recent reports have shown that EPO derivatives that do not stimulate hematopoiesis can still have tissue-protective effects including cardioprotection (11, 21, 25). These agents may be advantageous since there is a limited risk of increased thrombosis. However, a single intravenous dose of DA during the therapeutic window of the I/R injury, as demonstrated here, has minimal effects on hematocrit, and risks would be limited. Since DA is an approved drug with a proven safety profile, it could still be a choice for cardioprotection clinical trials.

DA offers novel cardioprotection with delayed, clinically relevant administration. One of the most striking results found in this study is that DA can protect the heart at several time points including up to 24 h after the beginning of reperfusion. This increases the clinical significance and potential applicability of this agent in the post-MI setting. It may be somewhat surprising that such a late administration can have a positive benefit for the ischemic and reperfused heart but a recent study has shown that after a brief period of ischemia, the majority of myocytes during early reperfusion died by a necrotic pathway, while the apoptotic cell death in perinecrotic myocardium was triggered during the late phase of reperfusion (46). In that study, Zhao et al. demonstrated that TUNEL-positive cells and DNA fragmentation from samples in the perinecrotic zone of the infarct progressively increased to up to 72 h after the beginning of reperfusion (46). Since apoptosis in the I/R heart is an active process (9, 13), inhibition of apoptosis is a better target than necrosis that occurs very early in the course of I/R injury generation. Our results with DA support these findings that it is possible to reduce/inhibit apoptosis in this later reperfusion period.

Of note, the ~45% reduction in LV infarct size seen with DA when given 24 h after beginning of reperfusion is in contrast with a recent report in which no protection was observed with EPO (given 24 h after permanent coronary occlusion model; Ref. 25). On the other hand, Prunier et al. (32) recently showed that DA administration led to reduced infarction size and LV remodeling after 8 wk (permanent occlusion model with DA administration after 1 wk of ischemia followed by a weekly treatment). Possible explanations for differences in results are the model used (permanent occlusion vs. I/R), the molecules themselves (EPO vs. DA), the doses used, as well as the treatment regimen. Importantly, permanent occlusion of the coronary artery will lead to almost complete to complete death of the area at risk, whereas I/R does not, principally because of the reperfusion. All these factors are known key modulators of infarction size and must be acknowledged to discriminate results between these studies. Nevertheless, our results clearly demonstrate that in our experimental setting of I/R injury, the therapeutic window of cardioprotection in regard to DA or EPO-related molecule is wider than expected. Of clinical interest is that our findings show that DA still retains protective characteristics when administered in late reperfusion (>1–24 h). This issue represents an extensive advantage over other protective pharmacological agents such as adenosine (3, 4, 45) or opioids (14), which must be administered in early reperfusion (≤5–10 min) to generate a significant reduction in infarct size (reviewed in Ref. 14).

Isoflurane has been shown to induce some level of cardioprotection with a mechanism similar to preconditioning (24, 27). In our study, isoflurane did not reduce infarct size. This discrepancy can be explained by the fact that preconditioning induction can be achieved by stopping administration of isoflurane a few minutes before the ischemic period begins, thereby achieving maximum cardioprotective effect. This was not the case in our study. Moreover, even if isoflurane can induce cardioprotection, all animals were anesthetized in the same manner. Thus the extent of infarct reduction would be the same in all animals, and infarction size comparison would still be valid.

The present study demonstrates that DA treatment in a rat model of I/R injury significantly reduces infarct size and improves cardiac functional responses to inotropic stimulation in a dose-dependent manner. This cardioprotection could be due at least in part to inhibition of apoptosis in the border zone of the previously myocardial ischemic region. Moreover, these results clearly show for the first time that a cardioprotective agent can have a beneficial effect when given as late as 24 h after the beginning of reperfusion. This demonstrates that a single dose of DA has a rather wide therapeutic window that should be translatable to the clinic. This bodes well for the planning of future clinical trials in this important pathological arena.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grant R01-HL-56205 (to W. J. Koch). M. Boucher is supported by an American Heart Association postdoctoral fellow award and a Fonds de la Recherche en Santé du Québec postdoctoral scholarship.


   ACKNOWLEDGMENTS
 
DA was provided by Amgen Corporation (Thousand Oaks, CA) as part of a Sponsored Research Agreement (to W. J. Koch). W. J. Koch is the W. W. Smith Professor of Medicine.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. J. Koch, Center for Translational Medicine, George Zallie and Family Laboratory for Cardiovascular Gene Therapy, Thomas Jefferson Univ., 1025 Walnut St., Rm. 317, Philadelphia, PA 19107 (e-mail: walter.koch{at}jefferson.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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