EphrinA1-Fc Attenuates Ventricular Remodeling and Dysfunction in Chronically Nonreperfused WT but not EphA2-R-M mice

Background: EphrinA1-Fc abolishes acute I/R injury and attenuates nonreperfused cardiac injury 4 days after permanent occlusion in mice. The goal of this study was to assess the capacity of a single intramyocardial administration of ephrinA1-Fc at the time of coronary artery ligation, to determine the degree to which early salvage effects translate to reduced adverse remodeling after 4 weeks of nonreperfused myocardial infarction (MI) in wild-type B6 and EphA2-R-M (EphA2 receptor null) mice. Methods: At 4 weeks post-MI, echocardiography, histologic and immunohistochemical analyses of B6 mouse hearts were performed. Primary mouse cardiac fibroblasts (FBs) isolated from B6 mice cultured in the presence of low and high dose ephrinA1-Fc, both with and without pro-fibrotic TGF-β stimulation and Western blots, were probed for relative expression of remodeling proteins MMP-2, MMP-9 and TIMP-1, in addition to DDR2 and (p)SMAD2/3/totalSMAD2/3. Results: EphrinA1-Fc preserved a significant degree of contractile function, decreased adverse left ventricular remodeling, attenuated excessive compensatory hypertrophy, and decreased interstitial fibrosis in wild-type (WT) B6 mouse hearts. In contrast, most of these parameters were poorer in ephrinA1-Fc-treated EphA2-R-M mice. Of note, fibrosis was proportionately decreased, implying that other EphA receptor(s) are more important in regulating the pro-fibrotic response. Primary FBs showed disparate alteration of MMP-2, MMP-9 and TIMP-1, as well as DDR2 and p-SMAD2/3/totalSMAD2/3, which indicates that matrix remodeling and cardiac fibrosis in the injured heart are influenced by ephrinA1-Fc. Conclusion: This study demonstrates the capacity of a single administration of ephrinA1-Fc at the onset of injury to attenuate long-term nonreperfused post-MI ventricular remodeling that results in progressive heart failure, and the important role of EphA2 in mitigating the deleterious effects

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

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

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

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