Targeting myocardial equilibrative nucleoside transporter ENT1 provides cardioprotection by enhancing myeloid Adora2b signaling

Abstract

Previous studies implicate extracellular adenosine signaling in attenuating myocardial ischemia and reperfusion injury (IRI). This extracellular adenosine signaling is terminated by its uptake into cells by equilibrative nucleoside transporters (ENTs). Thus, we hypothesized that targeting ENTs would function to increase cardiac adenosine signaling and concomitant cardioprotection against IRI. Mice were exposed to myocardial ischemia and reperfusion injury. Myocardial injury was attenuated in mice treated with the nonspecific ENT inhibitor dipyridamole. A comparison of mice with global Ent1 or Ent2 deletion showed cardioprotection only in Ent1-/- mice. Moreover, studies with tissue-specific Ent deletion revealed that mice with myocyte-specific Ent1 deletion (Ent1loxP/loxP Myosin Cre+ mice) experienced smaller infarct sizes. Measurements of cardiac adenosine levels demonstrated that postischemic elevations of adenosine persisted during reperfusion after targeting ENTs. Finally, studies in mice with global or myeloid-specific deletion of the Adora2b adenosine receptor (Adora2bloxP/loxP LysM Cre+ mice) implied that Adora2b signaling on myeloid-inflammatory cells in cardioprotection provided by ENT inhibition. These studies reveal a previously unrecognized role for myocyte-specific ENT1 in cardioprotection by enhancing myeloid-dependent Adora2b signaling during reperfusion. Extension of these findings implicates adenosine transporter inhibitors in cardioprotection against ischemia and reperfusion injury.

Keywords: Cardiology; Cardiovascular disease; Hypoxia; Inflammation.

Figure 1. Treatment with ENT inhibitor dipyridamole provides cardioprotection.

(A) Experimental strategy. C57BL/6J mice were given either PBS (WT + vehicle group) or dipyridamole (5 mg/kg, i.p.) (WT + dipyridamole group) 1 hour prior to in situ myocardial ischemia (60 minutes) and reperfusion (2 hours). (B) Infarct sizes in vehicle- or dipyridamole-treated mice (n = 7; 2-tailed unpaired t test, **P < 0.01). (C) Representative images after Evans blue injection and subsequent TTC staining. The infarct area is outlined by a green line; AAR is outlined by a blue line. Scale bar: 1 mm. (D) Troponin I (cTnI) levels (n = 7; Mann-Whitney U test, ***P < 0.001). Data are shown as mean ± SEM. Each dot represents 1 mouse.

Figure 2. Global deletion of Ent1, but not Ent2, confers ENT-dependent cardioprotection.

(A and B) Ent1 (A) or Ent2 (B) transcript levels in the heart of WT, Ent1^–/–, or Ent2^–/– mice (n = 3–5; 1-way ANOVA, **P < 0.01, ***P < 0.001 in Bonferroni’s multiple-comparison test). (C) Cardiac ENT1 protein by Western blot analysis. (n = 5–7; 1-way ANOVA, ***P < 0.001 in Bonferroni’s multiple-comparison test). (D) Cardiac ENT2 protein by Western blot analysis (n = 3; 1-way ANOVA, **P < 0.01 in Bonferroni’s multiple-comparison test). (E) Experimental strategy for the murine myocardial IRI in WT, Ent1^–/–, or Ent2^–/– mice. (F) Myocardial infarct sizes in WT, Ent1^–/–, or Ent2^–/– mice (n = 8 for WT, n = 5 for Ent1^–/–, n = 7 for Ent2^–/–, 1-way ANOVA, ***P < 0.001 in Bonferroni’s multiple-comparison test). (G) Representative images of left ventricles stained by Evans blue and TTC. The infarct area is outlined by a green line; AAR is outlined by a blue line. Scale bar: 1 mm. (H) cTnI levels after myocardial injury (n = 8 for WT, n = 5 for Ent1^–/–, n = 7 for Ent2^–/–, 1-way ANOVA, *P < 0.05 in Bonferroni’s multiple-comparison test). Data are shown as mean ± SEM. Each dot represents 1 mouse.

Figure 3. Tissue-specific deletion of Ent1 identifies cardiac myocytes as an important source for Ent1-dependent cardioprotection.

(A) Experimental approach to study myocardial ischemia (60 minutes) and reperfusion (2 hours) injury in control (Myosin Cre⁺) or Ent1^loxP/loxP Myosin Cre⁺ mice. (B) Ent1 or Ent2 transcript levels in isolated cardiomyocytes in Myosin Cre⁺ or Ent1^loxP/loxP Myosin Cre⁺ mice (n = 3; 2-way ANOVA, *P < 0.05 in Bonferroni’s multiple-comparison test). (C) ENT1 or ENT2 protein levels by Western blot analysis. (D) Quantification of C (n = 7; 2-way ANOVA, ***P < 0.001 in Bonferroni’s multiple-comparison test). (E) Infarct sizes in Myosin Cre⁺ or Ent1^loxP/loxP Myosin Cre⁺ mice (n = 5 for Myosin Cre⁺, n = 6 for Ent1^loxP/loxP Myosin Cre⁺, 2-tailed unpaired t test, **P < 0.01). (F) Representative TTC staining. The infarct area is outlined by a green line; AAR is outlined by a blue line. Scale bar: 1 mm. (G) cTnI levels (n = 5 for Myosin Cre⁺, n = 6 for Ent1^loxP/loxP Myosin Cre⁺, Welch’s t test, *P < 0.05). Data are shown as mean ± SEM. Each dot represents 1 mouse.

Figure 4. Tissue-specific deletion of Ent2 is not associated with cardioprotection.

(A) Experimental set-up to study myocardial ischemia (60 minutes) and reperfusion (2 hours) injury in control (Myosin Cre⁺) or Ent2^loxP/loxP Myosin Cre⁺ mice. (B) Ent1 or Ent2 transcript levels were measured from isolated cardiomyocytes from Myosin Cre⁺ or Ent2^loxP/loxP Myosin Cre⁺ mice (n = 3; 2-way ANOVA, **P < 0.01 in Bonferroni’s multiple-comparison test). (C) ENT1 or ENT2 protein levels by Western blot analysis. (D) Quantification of C (n = 4–6; 2-way ANOVA, ***P < 0.001 in Bonferroni’s multiple-comparison test). (E) Infarct sizes in Myosin Cre⁺ or Ent2^loxP/loxP Myosin Cre⁺ mice (n = 6 for Myosin Cre⁺, n = 7 for Ent2^loxP/loxP Myosin Cre⁺, Mann-Whitney U test). (F) Representative images of infarct staining. The infarct area is outlined by a green line; AAR is outlined by a blue line. Scale bar: 1 mm. (G) cTnI levels after surgery (n = 6 for Myosin Cre⁺, n = 7 for Ent1^loxP/loxP Myosin Cre⁺, 2-tailed unpaired t test). Data are shown as mean ± SEM. Each dot represents 1 mouse.

Figure 5. ENT inhibition extends cardiac adenosine elevations throughout reperfusion.

(A) C57BL/6J mice received either PBS or dipyridamole (5 mg/kg, i.p.) 1 hour before being exposed to 1 of the 3 conditions: sham, 60 minutes of ischemia (I group), or 60 minutes of ischemia followed by 2 hours of reperfusion (IR group). (B) Cardiac adenosine levels were measured from the area at risk by high-performance liquid chromatography (HPLC) (n = 3–5, 2-way ANOVA, *P < 0.05, ***P < 0.001, ****P < 0.0001 in Bonferroni’s multiple-comparison test). (C) Representative cardiac adenosine levels in the IR group in WT mice treated with PBS or dipyridamole. (D) WT or Ent1^–/– mice were exposed to sham, 60 minutes of ischemia (I group), or 60 minutes of ischemia and 2 hours of reperfusion (IR group). (E) Cardiac adenosine levels were measured from the area at risk by HPLC (n = 3–4, 2-way ANOVA, *P < 0.05, ***P < 0.001 in Bonferroni’s multiple-comparison test). (F) Representative cardiac adenosine levels in the IR group in WT or Ent1^–/– mouse hearts. Data are shown as mean ± SEM. Each dot represents 1 mouse.

Figure 6. Global Adora2b whole-body KO dampens the cardioprotective effects of dipyridamole treatment.

(A) WT control and Adora2b^–/– mice treated with PBS or dipyridamole (5 mg/kg, i.p.) were exposed to in situ myocardial IRI. (B) Infarct sizes in WT control and Adora2b^–/– mice (n = 7; 2-way ANOVA, **P < 0.01, ****P < 0.0001 in Bonferroni’s multiple-comparison test). (C) Representative images of infarct staining. The infarct area is outlined by a green line; AAR is outlined by a blue line. Scale bar: 1 mm. (D) cTnI levels after surgery (n = 7; 2-way ANOVA, **P < 0.01, ***P < 0.001 in Bonferroni’s multiple-comparison test). Data are shown as mean ± SEM. Each dot represents 1 mouse.

Figure 7. Myeloid Adora2b deletion dampens the cardioprotective effects of dipyridamole treatment.

(A) LysM Cre⁺ and Adora2b^loxP/loxP LysM Cre⁺ mice treated with PBS or dipyridamole (5 mg/kg, i.p.) were exposed to in situ myocardial IRI. (B) Infarct sizes in LysM Cre⁺ and Adora2b^loxP/loxP LysM Cre⁺ mice (n = 7–10; 2-way ANOVA, ****P < 0.0001 in Bonferroni’s multiple-comparison test). (C) Representative images of infarct staining. The infarct area is outlined by a green line; AAR is outlined by a blue line. Scale bar: 1 mm. (D) cTnI levels after surgery (n = 7–8; 2-way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001 in Bonferroni’s multiple-comparison test). Data are shown as mean ± SEM. Each dot represents 1 mouse.

Figure 8. Targeting myocardial ENT1 provides cardioprotection by enhancing myeloid Adora2b signaling.

Previous studies implicate adenosine signaling in attenuating myocardial ischemia and reperfusion injury. Here, we show that pharmacologic inhibition or genetic deletion of Ent1 enhances extracellular adenosine signaling during myocardial IRI, ultimately promoting cardioprotection through myeloid Adora2b adenosine receptors. Gray arrow indicates the direction of the adenosine flow. A2B, Adora2b adenosine receptor.