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. 2017 Dec 13;12(12):e0189307.
doi: 10.1371/journal.pone.0189307. eCollection 2017.

EphrinA1-Fc Attenuates Myocardial Ischemia/Reperfusion Injury in Mice

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Free PMC article

EphrinA1-Fc Attenuates Myocardial Ischemia/Reperfusion Injury in Mice

Augustin DuSablon et al. PLoS One. .
Free PMC article

Abstract

EphrinA1, a membrane-bound receptor tyrosine kinase ligand expressed in healthy cardiomyocytes, 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 ischemia 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 myocardium. 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 homogenates from mice that underwent 30min ischemia and 24hr of reperfusion (I/R) indicates that ephrinA1-Fc administration alters several regulators of signaling pathways that attenuate apoptosis, promote autophagy, and shift from FA metabolism in favor of increased glycolysis 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
Echocardiographic data and representative M-mode traces for uninjured controls, IgG-Fc-treated, and ephrinA1-Fc-treated mice that have undergone with 30 min of ischemia followed by 24 h reperfusion (1a) or 30 min ischemic followed by 4 d of reperfusion (1b). Fractional shortening (FS) (57.58 ± 2.99% p < 0.01) and ejection fraction (EF) (87.92 ± 2.35%; *p < 0.05) was significantly impaired in IgG-Fc-treated mice and systolic volume and diameter were increased. In contrast, mice treated with ephrinA1-Fc showed no change in FS (69.46 ± 1.45%; p > 0.05) or EF (95.27 ± 0.64%; p > 0.05). After four days of reperfusion, infarcted IgG-Fc-treated animals display further impairment in FS (48.75 ± 7.32%; p < 0.01) and EF (81.08 ± 7.17%; p < 0.01) and both systolic and diastolic diameters and volumes were significantly different from uninured control and ephrinA1-Fc-treated mice whereas ephrinA1-Fc-treated animals showed no significant difference in FS (88.2 ± 5.5%; p > 0.05) or EF (96.6 ± 1.9%; p > 0.05). There were no differences in heart rate observed between the groups (control: 598 ± 64, IgG-Fc: 638 ± 69, ephrinA1-Fc: 630 ± 36) at 24hrs or 4days post-injury (control: 577 ± 53, IgG-Fc: 586 ± 57, ephrinA1-Fc: 563 ± 44). From left to right in the sham heart, blue lines denote anterior wall thickness, red lines denote chamber diameter, and green lines denote posterior wall thickness at diastole and systole, respectively.
Fig 2
Fig 2. Area at risk and infarct size.
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.
Fig 3
Fig 3. Inflammatory infiltrate in the infarct zone of IgG-Fc and ephrinA1-Fc treated mouse hearts and serum CXCL1 at 24hrs.
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.
Fig 4
Fig 4. Western blots of EphA1, EphA4, and EphA7 receptor expression in left ventricular homogenates of control, IgG-Fc-treated, and ephrinA1-Fc-treated mouse hearts at 24hrs.
(a) EphA1-R expression increased by 16% in ephrinA1-Fc-treated hearts compared to control hearts but were not different from IgG-Fc-treated hearts. (b) EphA4-R protein expression decreased in ephrinA1-Fc-treated hearts compared to IgG-Fc-treated hearts which were not difference from controls. (c) EphA7-R expression decreased 33% in IgG-Fc-treated hearts compared to controls and was 26% higher in ephrinA1-Fc-treatred hearts compared to IgG-Fc-treated hearts but not significantly different from control hearts. *p<0.05 compared to control, p<0.05 compared to IgG-Fc, ‡ p<0.01 compared to control.
Fig 5
Fig 5. 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.
(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.

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