A novel protective effect of erythropoietin in the infarcted heart (original) (raw)
EPO signaling and proliferation in H9c2 myoblasts. EPO is known to stimulate three common cell survival pathways, including PI3K/Akt, ERK1/2 MAPK, and Jak-STAT in hematopoietic (25–27) and endothelial (28) cell lines. ERK and Akt activation (via phosphorylation) were assessed in H9c2 cardiomyoblasts after EPO addition and quantified by Western blot analysis and densitometry at serial time points (1–60 minutes) to determine signaling intermediates (Figure 1, a and b). EPO induced significant Akt activation within 15 minutes and lasted up to 60 minutes (1.5-fold ± 0.1-fold over control; P = 0.008). ERK was activated 2.96-fold ± 0.4-fold after 60 minutes versus control (P = 0.0016). Significant Jak1 phosphorylation occurs at 15 minutes (1.39-fold ± 0.18-fold over control, P = 0.027) and continues to serially increase up to 60 minutes (2.41-fold ± 0.54-fold, P = 0.014) (Figure 1c). One of the downstream intermediates, STAT3, indicates peak activation at 10 minutes (2.07-fold ± 0.16-fold versus control, P = 0.0004) with progressive mitigation of phosphorylation over serial time points (Figure 1d). Akin to its effect in erythroblasts (5, 7), EPO could stimulate proliferation in cardiac myoblasts. H9c2 cellular proliferation, as measured by [3H]-thymidine incorporation, mildly increased 9.3% ± 1.4% using EPO (4.0 U/ml) compared with control (P < 0.05). As a reference, the positive mitogen PDGF induced 12.2% ± 2.1% proliferation in H9c2 cells (P < 0.05 versus control).
EPO-mediated activation of critical intracellular kinase cascades including cell survival pathways. Shown are representative Western blots for ERK (a), Akt (b), Jak1 (c), and STAT3 (d) over serial time points. Activated (p-) signaling intermediates were assessed using anti-phospho Ab’s, and activated forms were normalized to total protein content of the corresponding molecule (lower panel of all blots). Data are representative of n = 3–4 experiments.
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 2). As shown in Figure 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).
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 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 2b). Importantly, wortmanin or PD98059 compound did not significantly alter survival of H9c2 cells in the absence of EPO. Figure 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 2c) compared with H9c2 cells treated with EPO (Figure 2d).
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 1, and cardiac contractility and relaxation in response to βAR stimulation are shown in Figure 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 3c) and LVEDP (Figure 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.
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 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).
Summary of in vivo baseline hemodynamic data in rabbits
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 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 5a). Representative staining of area at risk and infarct size can be seen in Figure 5, b and c, for saline- and EPO-treated MI hearts, respectively, demonstrating significantly less infarction (nonstained tissue) after EPO treatment.
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 6a). No TUNEL-positive cells were seen in sham (no MI, no EPO) animals. Representative TUNEL-stained sections are shown in Figure 6, b and c, demonstrating fewer apoptotic positive cells in the EPO-treated MI rabbit heart (Figure 6c), which is consistent with lower apoptosis in cardiac myoblasts in vitro following oxidative stress and hypoxia.
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.
In vivo myocardial Akt and ERK activation induced by EPO. Since the PI3K/Akt pathway as well as the ERK pathway was shown to be activated by EPO in H9c2 cells and implicated in protection against hypoxic injury and perhaps mitogenesis, we examined whether these critical kinase cascades could be activated by EPO in the intact adult rabbit heart. Normal rabbits were treated with either saline (controls) or EPO (1,000 U/kg) and sacrificed 12 hours later. Phosphorylated (i.e., activated) Akt and ERK were then measured in lysates prepared from control and EPO-treated rabbit hearts by protein immunoblotting as carried out above in H9c2 cells (Figure 7). As shown in Figure 7, both Akt and ERK were found to be significantly activated in vivo in the rabbit LV after EPO administration. Akt was activated a modest 50% over control values (Figure 7a), while ERK activity was robustly stimulated at more than sevenfold over controls (Figure 7b). The activation of the cell survival kinase Akt by EPO treatment in the adult rabbit heart in vivo is consistent with lower apoptosis seen when EPO is given at the time of MI (Figure 6).
In vivo activation of signaling kinases in the intact adult rabbit heart after EPO treatment. Shown in graphic representation is the activation of (a) Akt and (b) ERK assessed in LV lysates prepared 12 hours after the administration of EPO (1,000 U/kg) or saline (Control). Activated Akt and ERK were assessed using anti-phospho Ab’s, and activated forms were normalized to total protein content of the corresponding molecule as in Figure 1. *P < 0.05 versus control values (n = 4 each).