Erythropoietin Protects Cardiomyocytes from Cell Death during Hypoxia/Reperfusion Injury through Activation of Survival Signaling Pathways

<div><p>Hypoxia/Reoxygenation (H/R) cardiac injury is of great importance in understanding Myocardial Infarctions, which affect a major part of the working population causing debilitating side effects and often-premature mortality. H/R injury primarily consists of apoptotic and necrotic death of cardiomyocytes due to a compromise in the integrity of the mitochondrial membrane. Major factors associated in the deregulation of the membrane include fluctuating reactive oxygen species (ROS), deregulation of mitochondrial permeability transport pore (MPTP), uncontrolled calcium (Ca²⁺) fluxes, and abnormal caspase-3 activity. Erythropoietin (EPO) is strongly inferred to be cardioprotective and acts by inhibiting the above-mentioned processes. Surprisingly, the underlying mechanism of EPO's action and H/R injury is yet to be fully investigated and elucidated. This study examined whether EPO maintains Ca²⁺ homeostasis and the mitochondrial membrane potential (ΔΨ_m) in cardiomyocytes when subjected to H/R injury and further explored the underlying mechanisms involved. H9C2 cells were exposed to different concentrations of EPO post-H/R, and 20 U/ml EPO was found to significantly increase cell viability by inhibiting the intracellular production of ROS and caspase-3 activity. The protective effect of EPO was abolished when H/R-induced H9C2 cells were treated with Wortmannin, an inhibitor of Akt, suggesting the mechanism of action through the activation Akt, a major survival pathway.</p></div

Pre-treatment of EPO maintains mitochondrial membrane potential in H/R induced H9C2 cells.

<p>The effect of EPO on Membrane potential was determined by colocalization studies using Rhodamine-123 and DCFH-DA. H9C2 cells were subjected to H/R with or without pre-treatment with 20 U/ml EPO for 24 hrs. H9C2 cells pretreated with EPO and control cells showed Rhodamine-123 fluorescence only in the perinuclear region about 58% and 52% colocalzation respectively whereas H9C2 cells subjected to H/R showed Rhodamine-123 fluorescence both in perinuclear region and cytosol about 94.7% co localization in Figure 5 A. Data are presented as means ± SEM of the ratios from three independent experiments * denotes p<0.001 for analyses compared to H/R.</p

Pre-treatment of EPO in H/R induced H9C2 cells had uniformly green nuclei with intact plasma and nuclear membranes.

<p>The effect of EPO on apoptosis and necrosis was determined using Acridine/Orange (Ao/EtBr) double staining assay. H9C2 cells were subjected to H/R with or without pre-treatment with 20 U/ml EPO for 24 hrs. (A) EPO stabilizes plasma membrane and nuclear membrane showed byuniform green nuclei. In H/R induced cells, early apoptotic cells were indicated by intact membrane but nuclei was condensed and green. Late-stage apoptotic cells were indicated by bright orange-stained nuclei. Necrotic cells were indicated by red stained nuclei. (B) Represents the quantification of the above image. Data are presented as means ± SEM of the ratios from three independent ratios *p<0.001 for analyses compared to H/R.</p

Characterization of H9C2 cells by Anti α – Sarcomeric actin.

<p>Panel A shows DAPI, Panel B shows α-sarcomeric actinin and Panel C shows merged image of DAPI and α-sarcomeric actinin.</p

Pre-treatment of EPO decreases caspase-3 activity in H/R induced H9C2 cells.

<p>The effect of EPO on caspase-3 activity was determined using caspase-3 colorimetric assay. H9C2 cells were subjected to H/R with or without pre-treatment with 20 U/ml EPO for 24 hrs and incubated with 1 µM of Wortmannin 30 mins before EPO treatment. EPO decrease caspase-3 activity after H/R. H9C2 cells pretreated with wortmannin showed increase in caspase-3 activity. Data are presented as means ± SEM of the ratios from three independent experiments * denotes p<0.001 for analyses compared to H/R.</p

Western blot analysis demonstrating the effect of EPO on AKT.

<p>The effect of EPO on p-Akt was determined using Western blot. H9C2 cells were ⁱsubjected to H/R with or without pre-treatment with 20 U/ml EPO for 24 hrs and incubated with 1 µM of Wortmannin 30 mins before EPO treatment. (A) Samples treated with EPO showed increase in phosphorylation of Akt (p-Akt). Akt remain unaltered and demonstrate equal protein loading in all lanes. (B) Represent the quantization of western blot, which indicates the increase in p-Akt levels Data are presented as means ± SEM of the ratios from three independent experiments * denotes p<0.001 for analyses compared to H/R.</p

Pre-treatment of EPO increases intracellular p-Akt in H/R induced H9C2 cells.

<p>The effect of EPO on p-Akt was determined. H9C2 cells (A) were subjected to 8 hrs hypoxia and 30 mins of reperfusion with or without pre-treatment with 20 U/ml EPO and 1 µM Wortmannin before 30 mins for 24 hrs and stained with p-AKT antibody. EPO increase p-Akt. (B) Represents the quantification green florescence, which indicates the increase in levels of p-Akt. Data are presented as means ± SEM of the ratios from three independent experiments * denotes p<0.001 for analyses compared to H/R.</p

Pre-treatment of EPO increases cell viability in H/R induced H9C2 cells.

<p>The effect of EPO on cell viability was determined using MTT assay. H9C2 cells were subjected to H/R with or without pre-treatment with (10 U/ml, 15 U/ml and 20 U/ml) EPO for 24 hrs. 20 U/ml EPO significantly increases cell viability after H/R. Data are presented as means ± SEM of the ratios from five independent experiments. ^$denotes p<0.05, ^& denotes p<0.01, * denotes p<0.001 for analyses compared to H/R.</p

Pre-treatment of EPO maintains intracellular calcium and prevents MPTP opening in H/R induced H9C2 cells.

<p>The effect of EPO on intracellular calcium and MPTP opening was determined using time–lapse imaging in confocal microscope. (A) H9C2 cells were subjected to 8 hrs hypoxia and 15 mins reperfusion with or without pre-treatment with 20 U/ml EPO for 24 hrs and stained with Fluo-4 AM and performed live cell imaging. H9C2 cells pretreated with EPO accumulate Ca²⁺ inside the mitochondrial membrane even after reperfusion and showed increase Fluo-4 AM fluorescence. H9C2 cells subjected to H/R showed release of Ca²⁺ outside the mitochondria due to overload of Ca²⁺ during reperfusion rupture of mitochondrial membrane occurs. (B) Represents the quantification of Fluo-4 AM fluorescence. Data are presented as means ± SEM of the ratios from three independent experiments * denotes p<0.001 for analyses compared to H/R.</p