Overexpression of Human Soluble Epoxide Hydrolase Exacerbates Coronary Reactive Hyperemia Reduction in Angiotensin-II-Treated Mouse Hearts

Abstract

Coronary reactive hyperemia (CRH) is impaired in cardiovascular diseases, and angiotensin-II (Ang-II) exacerbates it. However, it is unknown how Ang-II affects CRH in Tie2-sEH Tr (human-sEH-overexpressed) versus wild-type (WT) mice. sEH-overexpression resulted in CRH reduction in Tie2-sEH Tr versus WT. We hypothesized that Ang-II exacerbates CRH reduction in Tie2-sEH Tr versus WT. The Langendorff system measured coronary flow in Tie2-sEH Tr and WT. The hearts were exposed to 15-second ischemia, and CRH was assessed in 10 mice each. Repayment volume was reduced by 40.50% in WT treated with Ang-II versus WT (7.42 ± 0.8 to 4.49 ± 0.8 mL/g) and 48% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (5.18 ± 0.4 to 2.68 ± 0.3 mL/g). Ang-II decreased repayment duration by 50% in WT-treated with Ang-II versus WT (2.46 ± 0.5 to 1.24 ± 0.4 minutes) and 54% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (1.66 ± 0.4 to 0.76 ± 0.2 minutes). Peak repayment flow was reduced by 11.2% in WT treated with Ang-II versus WT (35.98 ± 0.7 to 32.11 ± 1.4 mL/g) and 4% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (32.18 ± 0.6 to 30.89 ± 1.5 mL/g). Furthermore, coronary flow was reduced by 43% in WT treated with Ang-II versus WT (14.2 ± 0.5 to 8.15 ± 0.8 mL/min/g) and 32% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (12.1 ± 0.8 to 8.3 ± 1.2 mL/min/g). Moreover, the Ang-II-AT 1 -receptor and CYP4A were increased in Tie2-sEHTr. Our results demonstrate that Ang-II exacerbates CRH reduction in Tie2-sEH Tr mice.

Fig. 1.

Schematic picture showing epoxyeicosatrienoic acids (EETs), epoxy-fatty acids (anti-inflammatory oxylipins) formation in the presence of CYP-epoxygenases and degradation to inactive diols called dihydroxyeicosatrienoic acids (DHETs), dihydroxy-fatty acids (proinflammatory oxylipins) in the presence of soluble epoxide hydrolase (sEH).

Fig. 2.

Typical tracing of coronary flow showing the different components of reactive hyperemia (RH), typical tracing of coronary flow (CF) showing the different components of coronary reactive hyperemic (CRH) response, tracing depicted CF at the baseline before and after 15-seconds no-flow ischemia, and the Repayment Volume ml/g heart wt.=CRH.

Fig. 3.

Representative Western blots and densitometric analysis for Angiotensin-II AT₁ receptor (AT₁R) proteins in aortas of Tie2-sEH Tr (sEH-overexpressed) and C57Bl/6 (WT) mice, n = six per group (*p<0.05, A); representative Western blots and densitometric analysis for cytochrome P450–4A (CYP4A) proteins in aortas of Tie2-sEH Tr and C57Bl/6 (WT) mice, n = six per group (*p<0.05, B).

Fig. 4:

Effect of angiotensin-II (Ang-II, 1 nM) on CRH response in WT and Tie2-sEH Tr mouse hearts. Administration of Ang-II decreased CRH in both WT and Tie2-sEH Tr. Repayment Volume (ml/g) was reduced by 31% in non-Ang-II treated Tie2-sEH Tr compared to non-Ang-II treated WT mouse hearts (*p < 0.05, n=10); Repayment Volume (ml/g) was decreased by 40.5% in WT-treated with Ang-II vs. non-treated WT mouse hearts (*p < 0.05, n=10); similarly, Repayment Volume (ml/g) was reduced by 48% in Tie2-sEH Tr-treated with Ang-II vs. non-treated Tie2-sEH Tr mouse hearts (^#p < 0.05, n=10). The Repayment Volume (ml/g) was reduced by 40% in Tie2-sEH Tr-treated with Ang-II vs. Ang-II treated WT mouse hearts (^ψp < 0.05, n=10).

Fig. 5:

Repayment Duration (min) was reduced by 32.5% in non-Ang-II treated Tie2-sEH Tr compared to non-Ang-II treated WT mouse hearts (*p < 0.05, n=10); Repayment Duration (min) was reduced by 50% in WT-treated with Ang-II vs. non-treated WT mouse hearts (*p < 0.05, n=10); similarly, Repayment Duration (min) was reduced by 54% in Tie2-sEH Tr-treated with Ang-II vs. non-treated Tie2-sEH Tr mouse hearts (^#p < 0.05, n=10), and the Repayment Duration (min) was reduced by 39% in Tie2-sEH Tr-treated with Ang-II vs. Ang-II treated WT mouse hearts (^#p < 0.05, n=10).

Fig. 6:

Baseline Coronary Flow or Coronary Flow (CF ml/min/gm) was reduced by 15% in non-Ang-II treated Tie2-sEH Tr compared to non-Ang-II treated WT mouse hearts (*p < 0.05, n=10); CF (ml/min/gm) was reduced by 42.6% in WT-treated with Ang-II vs. non-treated WT mouse hearts (*p < 0.05, n=10); moreover, CF (ml/min/gm) was reduced by 31% in Tie2-sEH Tr-treated with Ang-II vs. non-treated Tie2-sEH Tr mouse hearts (^#p < 0.05, n=10), and d. CF was not changed in Tie2-sEH Tr-treated with Ang-II vs. Ang-II treated WT mouse hearts (p > 0.05, n=10).

Fig. 7:

Peak Hyperemic Flow (PHF ml/gm) was reduced by 10.6% in non-Ang-II treated Tie2-sEH Tr compared to non-Ang-II treated WT mouse hearts (barely significant, *p < 0.05, n=10); PHF (ml/gm) was reduced by 10.8% in WT-treated with Ang-II vs. non-treated WT mouse hearts (barely significant, *p < 0.05, n=10); moreover, PHF (ml/gm) was reduced by 4% in Tie2-sEH Tr-treated with Ang-II vs. non-treated Tie2-sEH Tr mouse hearts (p >0.05, n=10), and the PHF (ml/gm) was reduced by 4% in Tie2-sEH Tr-treated with Ang-II vs. Ang-II treated WT mouse hearts (p > 0.05, n=10).

Fig. 8:

Left Ventricular Developed Pressure (LVDP mmHg) was reduced by 21% in non-Ang-II treated Tie2-sEH Tr compared to non-Ang-II treated WT mouse hearts (p > 0.05, n=10); LVDP (mmHg) was reduced by 17% in WT-treated with Ang-II vs. non-treated WT mouse hearts (*p < 0.05, n=10); moreover, LVDP (mmHg) was decreased by 4% in Tie2-sEH Tr-treated with Ang-II vs. non-treated Tie2-sEH Tr mouse hearts (p >0.05, n=10), and the LVDP (mmHg) was reduced by 10% in Tie2-sEH Tr-treated with Ang-II vs. Ang-II treated WT mouse hearts (p > 0.05, n=10).

Fig. 9:

Repayment/Debt ratio was reduced by 16.5% in non-Ang-II treated Tie2-sEH Tr compared to non-Ang-II treated WT mouse hearts (p > 0.05, n=10); Repayment /Debt ratio was not changed between the WT-treated with Ang-II vs. non-treated WT mouse hearts (p > 0.05, n=10); moreover, Repayment /Debt ratio was reduced by 10% in Tie2-sEH Tr-treated with Ang-II vs. non-treated Tie2-sEH Tr mouse hearts (p > 0.05, n=10), and the Repayment /Debt ratio was decreased by 24% in Tie2-sEH Tr-treated with Ang-II vs. Ang-II treated WT mouse hearts (p > 0.05, n=10).

Fig. 10:

Heart Rate (HR beat/min) was not changed between non-Ang-II treated Tie2-sEH Tr compared to non-Ang-II treated WT mouse hearts (p > 0.05, n=10); HR (beat/min) was reduced by 6% in WT-treated with Ang-II vs. non-treated WT mouse hearts (p > 0.05, n=10); moreover, HR (beat/min) was reduced by 5% in Tie2-sEH Tr-treated with Ang-II vs. non-treated Tie2-sEH Tr mouse hearts (p >0.05, n=10), and the HR was not changed between Tie2-sEH Tr-treated with Ang-II vs. Ang-II treated WT mouse hearts (p > 0.05, n=10).

Fig. 11:

Summary Diagram: Comparison between Tie2-sEH Tr (sEH-overexpressed) vs. C57Bl/6 (WT) mice with differences in sEH overexpression, AT₁R and CYP4A upregulation, and abnormal CRH response without Ang-II infusion in Tie2-sEH Tr compared to WT mice. After Ang-II infusion, both Tie2-sEH Tr and WT mice had abnormal CRH response, but in Tie2-sEH Tr, the severity of CRH reduction response was immense compared to WT mice. This shows that sEH overexpression increases the sensitivity to Ang-II in reducing CRH response.