EphA2-receptor deficiency exacerbates myocardial infarction and reduces survival in hyperglycemic mice

Background We have previously shown that EphrinA1/EphA expression profile changes in response to myocardial infarction (MI), exogenous EphrinA1-Fc administration following MI positively influences wound healing, and that deletion of the EphA2 Receptor (EphA2-R) exacerbates injury and remodeling. To determine whether or not ephrinA1-Fc would be of therapeutic value in the hyperglycemic infarcted heart, it is critical to evaluate how ephrinA1/EphA signaling changes in the hyperglycemic myocardium in response to MI. Methods Streptozotocin (STZ)-induced hyperglycemia in wild type (WT) and EphA2-receptor mutant (EphA2-R-M) mice was initiated by an intraperitoneal injection of STZ (150 mg/kg) 10 days before surgery. MI was induced by permanent ligation of the left anterior descending coronary artery and analyses were performed at 4 days post-MI. ANOVAs with Student-Newman Keuls multiple comparison post-hoc analysis illustrated which groups were significantly different, with significance of at least p < 0.05. Results Both WT and EphA2-R-M mice responded adversely to STZ, but only hyperglycemic EphA2-R-M mice had lower ejection fraction (EF) and fractional shortening (FS). At 4 days post-MI, we observed greater post-MI mortality in EphA2-R-M mice compared with WT and this was greater still in the EphA2-R-M hyperglycemic mice. Although infarct size was greater in hyperglycemic WT mice vs normoglycemic mice, there was no difference between hyperglycemic EphA2-R-M mice and normoglycemic EphA2-R-M mice. The hypertrophic response that normally occurs in viable myocardium remote to the infarct was noticeably absent in epicardial cardiomyocytes and cardiac dysfunction worsened in hyperglycemic EphA2-R-M hearts post-MI. The characteristic interstitial fibrotic response in the compensating myocardium remote to the infarct also did not occur in hyperglycemic EphA2-R-M mouse hearts to the same extent as that observed in the hyperglycemic WT mouse hearts. Differences in neutrophil and pan-leukocyte infiltration and serum cytokines implicate EphA2-R in modulation of injury and the differences in ephrinA1 and EphA6-R expression in governing this are discussed. Conclusions We conclude that EphA2-mutant mice are more prone to hyperglycemia-induced increased injury, decreased survival, and worsened LV remodeling due to impaired wound healing

EphA2-receptor deficiency exacerbates myocardial infarction and reduces survival in hyperglycemic mice

Background We have previously shown that EphrinA1/EphA expression profile changes in response to myocardial infarction (MI), exogenous EphrinA1-Fc administration following MI positively influences wound healing, and that deletion of the EphA2 Receptor (EphA2-R) exacerbates injury and remodeling. To determine whether or not ephrinA1-Fc would be of therapeutic value in the hyperglycemic infarcted heart, it is critical to evaluate how ephrinA1/EphA signaling changes in the hyperglycemic myocardium in response to MI. Methods Streptozotocin (STZ)-induced hyperglycemia in wild type (WT) and EphA2-receptor mutant (EphA2-R-M) mice was initiated by an intraperitoneal injection of STZ (150 mg/kg) 10 days before surgery. MI was induced by permanent ligation of the left anterior descending coronary artery and analyses were performed at 4 days post-MI. ANOVAs with Student-Newman Keuls multiple comparison post-hoc analysis illustrated which groups were significantly different, with significance of at least p?&lt;?0.05. Results Both WT and EphA2-R-M mice responded adversely to STZ, but only hyperglycemic EphA2-R-M mice had lower ejection fraction (EF) and fractional shortening (FS). At 4 days post-MI, we observed greater post-MI mortality in EphA2-R-M mice compared with WT and this was greater still in the EphA2-R-M hyperglycemic mice. Although infarct size was greater in hyperglycemic WT mice vs normoglycemic mice, there was no difference between hyperglycemic EphA2-R-M mice and normoglycemic EphA2-R-M mice. The hypertrophic response that normally occurs in viable myocardium remote to the infarct was noticeably absent in epicardial cardiomyocytes and cardiac dysfunction worsened in hyperglycemic EphA2-R-M hearts post-MI. The characteristic interstitial fibrotic response in the compensating myocardium remote to the infarct also did not occur in hyperglycemic EphA2-R-M mouse hearts to the same extent as that observed in the hyperglycemic WT mouse hearts. Differences in neutrophil and pan-leukocyte infiltration and serum cytokines implicate EphA2-R in modulation of injury and the differences in ephrinA1 and EphA6-R expression in governing this are discussed. Conclusions We conclude that EphA2-mutant mice are more prone to hyperglycemia-induced increased injury, decreased survival, and worsened LV remodeling due to impaired wound healing

EphrinA1-Fc attenuates myocardial ischemia/ reperfusion injury in mice

EphrinA1, a membrane-bound receptor tyrosine kinase ligand expressed in healthy car- diomyocytes, is lost in injured cells following myocardial infarction. Previously, we have reported that a single intramyocardial injection of chimeric ephrinA1-Fc at the time of ische- mia reduced injury in the nonreperfused myocardium by 50% at 4 days post-MI by reducing apoptosis and inflammatory cell infiltration. In a clinically relevant model of acute ischemia (30min)/reperfusion (24hr or 4 days) injury, we now demonstrate that ephrinA1-Fc reduces infarct size by 46% and completely preserves cardiac function (ejection fraction, fractional shortening, and chamber dimensions) in the short-term (24hrs post-MI) as well as long-term (4 days). At 24 hours post-MI, diminished serum inflammatory cell chemoattractants in ephrinA1-Fc-treated mice reduces recruitment of neutrophils and leukocytes into the myo- cardium. Differences in relative expression levels of EphA-Rs are described in the context of their putative role in mediating cardioprotection. Validation by Western blotting of selected targets from mass spectrometry analyses of pooled samples of left ventricular tissue ho- mogenates from mice that underwent 30min ischemia and 24hr of reperfusion (I/R) indicates that ephrinA1-Fc administration alters several regulators of signaling pathways that attenu- ate apoptosis, promote autophagy, and shift from FA metabolism in favor of increased gly- colysis to optimize anaerobic ATP production. Taken together, reduced injury is due a combination of adaptive metabolic reprogramming, improved cell survival, and decreased inflammatory cell recruitment, suggesting that ephrinA1-Fc enhances the capacity of the heart to withstand an ischemic insult

Area at risk and infarct size.

<p>Area at Risk (AAR) in IgG-Fc-treated hearts was not different from ephrinA1-Fc-treated hearts whereas infarct size was 46% smaller in the ephrinA1-Fc-treated group (*p< 0.05) compared to the IgG-Fc group.</p

Western blots of markers for apoptosis, autophagy, fatty acid metabolism, and glycolysis in left ventricular homogenates of control, IgG-Fc-treated, and ephrinA1-Fc-treated mouse hearts at 24hrs.

<p>(a)The ratio of bcl2/bax increased 2-fold and HSP20 increased by 35%, both of which are indicative of reduced apoptosis in ephrinA1-Fc treated mouse hearts compared to IgG-Fc. (b) The ratios of LC3II/LC3I increased 28% and pmTOR/mTOR decreased by 31%, indicating increased autophagy in ephrinA1-Fc relative to IgG-Fc-treated mouse hearts. (c) Decreased CD36 and MCD in ephrinA1-Fc-treated mice by 35% and 70% respectively compared to IgG-Fc treated mice is suggestive of reduced deleterious fatty acid accumulation and increased (d) PGAM2 and PDK2 by 26% and 33% respectively indicate altered glycolytic flux. *p<0.05 compared to control, ** p<0.05 compared to IgG-Fc, ^† p<0.01 compared to control, †† p<0.01 compared to IgG = Fc, ‡ p<0.001 compared to control, ‡‡ p<0.001 compared to IgG-Fc.</p

Inflammatory infiltrate in the infarct zone of IgG-Fc and ephrinA1-Fc treated mouse hearts and serum CXCL1 at 24hrs.

<p>The density of positively stained (A) Ly6G+ neutrophils and (B) CD45+ macrophages (methyl green stained nuclei completely surrounded by dark brown DAB staining) was decreased by 33% and 40% respectively in ephrinA1-Fc-treated compared to IgG-Fc-treated hearts. (C) CXCL1 in serum of ephrinA1-Fc-treated mice was significantly decreased by 39% compared to IgG-Fc-treated mice. *p<0.001 compared to control, ^†p<0.01 compared to control, ^‡ p<0.05 compared to IgG-Fc.</p

EphrinA1-Fc attenuates myocardial ischemia/ reperfusion injury in mice

EphrinA1, a membrane-bound receptor tyrosine kinase ligand expressed in healthy car- diomyocytes, is lost in injured cells following myocardial infarction. Previously, we have reported that a single intramyocardial injection of chimeric ephrinA1-Fc at the time of ische- mia reduced injury in the nonreperfused myocardium by 50% at 4 days post-MI by reducing apoptosis and inflammatory cell infiltration. In a clinically relevant model of acute ischemia (30min)/reperfusion (24hr or 4 days) injury, we now demonstrate that ephrinA1-Fc reduces infarct size by 46% and completely preserves cardiac function (ejection fraction, fractional shortening, and chamber dimensions) in the short-term (24hrs post-MI) as well as long-term (4 days). At 24 hours post-MI, diminished serum inflammatory cell chemoattractants in ephrinA1-Fc-treated mice reduces recruitment of neutrophils and leukocytes into the myo- cardium. Differences in relative expression levels of EphA-Rs are described in the context of their putative role in mediating cardioprotection. Validation by Western blotting of selected targets from mass spectrometry analyses of pooled samples of left ventricular tissue ho- mogenates from mice that underwent 30min ischemia and 24hr of reperfusion (I/R) indicates that ephrinA1-Fc administration alters several regulators of signaling pathways that attenu- ate apoptosis, promote autophagy, and shift from FA metabolism in favor of increased gly- colysis to optimize anaerobic ATP production. Taken together, reduced injury is due a combination of adaptive metabolic reprogramming, improved cell survival, and decreased inflammatory cell recruitment, suggesting that ephrinA1-Fc enhances the capacity of the heart to withstand an ischemic insult