Mast cell chymase limits the cardiac efficacy of Ang I-converting enzyme inhibitor therapy in rodents

Abstract

Ang I-converting enzyme (ACE) inhibitors are widely believed to suppress the deleterious cardiac effects of Ang II by inhibiting locally generated Ang II. However, the recent demonstration that chymase, an Ang II-forming enzyme stored in mast cell granules, is present in the heart has added uncertainty to this view. As discussed here, using microdialysis probes tethered to the heart of conscious mice, we have shown that chronic ACE inhibitor treatment did not suppress Ang II levels in the LV interstitial fluid (ISF) despite marked inhibition of ACE. However, chronic ACE inhibition caused a marked bradykinin/B2 receptor-mediated increase in LV ISF chymase activity that was not observed in mast cell-deficient KitW/KitW-v mice. In chronic ACE inhibitor-treated mast cell-sufficient littermates, chymase inhibition decreased LV ISF Ang II levels substantially, indicating the importance of mast cell chymase in regulating cardiac Ang II levels. Chymase-dependent processing of other regulatory peptides also promotes inflammation and tissue remodeling. We found that combined chymase and ACE inhibition, relative to ACE inhibition alone, improved LV function, decreased adverse cardiac remodeling, and improved survival after myocardial infarction in hamsters. These results suggest that chymase inhibitors could be a useful addition to ACE inhibitor therapy in the treatment of heart failure.

Figure 1. ACE and non-ACE Ang II–forming activities and ACE immunoreactivity in WT and W/W^v LV homogenates (A).

Values are mean ± SEM. In each group, n = 6. All measurements were made in 7-week-old mice. Ang II–forming activity: total, which was determined in the absence of any inhibitors; ACE, that level of the total that was inhibited by the ACE inhibitor lisinopril (10 μM); and non-ACE, the total minus the ACE activity. ***P < 0.001. Photomicrographs showing ACE immunoreactivity in (B) WT and (C) W/W^v LV tissue sections. ACE (red); CD31, which identifies ECs (green); and DAPI, which identifies nuclei (blue). Note ACE immunoreactivity in clusters of cardiomyocytes in the W/W^v LV tissue section (C) but not in WT LV section (B). ACE immunoreactivity was only weakly associated with CD31-positive ECs. Scale bar: 20 μm.

Figure 2. Basal Ang II (A), Ang I (B), ACE activity (C), and chymase activity (D) in the LV ISF of conscious WT and W/W^v mice.

In vivo microdialysis was used to collect samples for these measurements. In addition, using this procedure, [Pro¹⁰]Ang I and [Pro¹¹,DAla¹²]Ang I conversion to Ang II in the LV ISF was used to measure ACE- and chymase-like activities, respectively. Values are mean ± SEM. In each group, values in parentheses represent n. **P < 0.01.

Figure 3. Effect of chronic ACE inhibitor treatment on angiotensins and Ang II–forming enzyme activities in conscious mice.

Effect of chronic captopril treatment (150 mg captopril/kg/d; for 2 weeks) on (A) LV ISF ACE activity, (B) LV ISF, (C) plasma Ang II, (E) plasma Ang I, and (G) MC density in WT mice. Effect of chronic captopril treatment on (D) LV ISF Ang I and (F) LV ISF chymase activity in WT and W/W^v mice. Values are mean ± SEM. In each group, values in parentheses represent n. *P < 0.05; **P < 0.01; ***P < 0.001. Veh, vehicle; Cap, captopril.

Figure 4. Bradykinin B2 receptors in LV MMCP4⁺ MCs.

Photomicrographs of a LV section from an 8-week-old WT mouse stained for (A) MMCP4 to identify MC, (B) DAPI to detect nuclei, (C) and the B2 kinin receptor as well as the (D) composite image. Arrows show the location of the MMCP4⁺ MC. Scale bar: 20 μm.

Figure 5. Effect of chronic (2 weeks) ACE inhibition (Captopril, 150 mg/kg/d) on (A) chymase activity and (B) Ang II in the LV ISF on conscious WT mice concurrently treated with vehicle, chymase inhibitor (CI-A, 200 mg/kg, b.i.d.) (CI), or bradykinin B2 receptor antagonist (Hoe-140, 0.5 mg/kg/d).

Values are mean ± SEM. In each group, values in parentheses represent n. *P < 0.05; ***P < 0.001.

Figure 6. β-actin–normalized MMCP1, MMCP2, MMCP4, and MMCP5 mRNA levels in LV tissues from WT and W/W^v mice.

Values are mean ± SEM; n = 7 in each group. **P < 0.01; ***P < 0.001.

Figure 7. Effect of ACE inhibitor, chymase inhibitor, or combination therapy on LV function, cardiomyocyte size, interstitial fibrosis, infarct size, and survival in hamsters after MI.

EF (A), FS (B), average cardiomyocyte diameter (C), LV interstitial fibrosis (D), infarct size (E), and LV end-diastolic dimension (F) in hamsters with MI treated with vehicle (MI) ACE inhibitor (temocapril, 10 mg/kg/d) (ACEI), chymase inhibitor (CI-B, 100 mg/kg/d) (CI), or combination therapy (10 mg temocapril/kg/d + 100 mg CI-B/kg/d) (ACEI + CI) started 24 hours after MI for 34 days or after a sham operation. (G) Actuarial survival after MI in hamsters following treatment with vehicle, ACE inhibitor, chymase inhibitor, or combination therapy or after a sham operation. Values are mean ± SEM. In each group, values in parentheses represent n. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8. ACE-dependent bradykinin degradation limits baseline secretion of MC chymase in the LV.

Chronic inhibition of ACE reduces its direct effects on Ang I to Ang II conversion, but it increases bradykinin/B2 receptor–dependent chymase release from MCs, which counteracts the direct effect of ACE inhibition on Ang II formation through the chymase pathway of Ang II formation. In this scheme, we suggest that the effect of bradykinin on MC chymase release is direct; however, it is also possible that the bradykinin/B2 receptor mechanism indirectly causes chymase release. Chymase release also has a myriad of effects on factors that promote the inflammatory response and tissue remodeling. ET-1, endothelin-1; TIMP-1, tissue inhibitor-1 of metalloproteinase.