Regulation of ACE2 in cardiac myocytes and fibroblasts

Abstract

Angiotensin-converting enzyme 2 (ACE2) preferentially forms angiotensin-(1-7) [ANG-(1-7)] from ANG II. We showed that cardiac ACE2 is elevated following treatment of coronary artery-ligated rats with AT1 receptor blockers (ARBs). Cardiac myocytes and fibroblasts were isolated from neonatal rats to determine the molecular mechanisms for the ACE2 upregulation by ARB treatment. ANG II significantly reduced ACE2 activity and downregulated ACE2 mRNA in cardiac myocytes, effects blocked by the ARB losartan, indicating that ANG II regulates ACE2. ANG II also reduced ACE2 mRNA in cardiac fibroblasts; however, no enzyme activity was detected, reflecting the limited expression of ACE2 in these cells. Endothelin-1 (ET-1) also significantly reduced myocyte ACE2 mRNA. The reduction in ACE2 mRNA by ANG II or ET-1 was blocked by inhibitors of mitogen-activated protein kinase kinase 1, suggesting that ANG II or ET-1 activates extracellular signal-regulated kinase (ERK) 1/ERK2 to reduce ACE2. Although ACE2 mRNA was not affected by ANG-(1-7), both the ANG II- and ET-1-mediated reductions in ACE2 mRNA were blocked by the heptapeptide. The ANG-(1-7) modulatory effect was prevented by the ANG-(1-7) receptor antagonist [D-Ala7]-ANG-(1-7), indicating that the ANG-(1-7) response was mediated by a specific AT(1-7) receptor. Myocyte treatment with atrial natriuretic peptide (ANP) also reversed the ACE2 mRNA downregulation by ANG II or ET-1, whereas treatment with ANP alone was ineffective. These results indicate that multiple hypertrophic and anti-hypertropic peptides regulate ACE2 production in myocytes, suggesting that ACE2 expression in the heart is dependent upon the compliment and concentration of regulatory molecules.

Fig. 1.

ANG II reduces angiotensin-converting enzyme (ACE2) activity in neonatal cardiac myocytes. Myocytes in serum-free media were incubated for 12 h with either 100 nM ANG II or 100 nM ANG II and 1 μM losartan (Los). EDTA (0.5 mM) was included to prevent the degradation of ANG II by ACE2, ACE, or neprilysin. ACE2 activity was measured using the fluorescent substrate 7-methoxycoumarin-4-acetyl-alanine-proline-lysine-(2,4dinitrophenyl)-OH; n = 3–4 rats. *P < 0.05 compared with Control (Con), in the absence of ANG II or losartan.

Fig. 2.

ACE2 mRNA in neonatal cardiac myocytes and fibroblasts. ACE2 mRNA was measured by RT real-time PCR in myocytes and fibroblasts isolated from neonatal rat heart; n = 4.

Fig. 3.

ANG II downregulates ACE2 mRNA in neonatal cardiac myocytes and fibroblasts. Myocytes (A) and cardiac fibroblasts (B) in serum-free media were incubated for 12 h with either 100 nM ANG II, in the presence or absence of 1 μM losartan (Los), with 1 μM losartan alone, or with 10 nM endothelin (ET)-1. ACE2 mRNA was measured by RT real-time PCR; n = 5. *P < 0.01 compared with Control (Con), in the absence of ANG II, losartan, or ET-1.

Fig. 4.

Mitogen/extracellular signal-regulated kinase (MEK) inhibitors prevent the downregulation of ACE2 by ANG II or ET-1. Myocytes in serum-free media were incubated for 12 h with 100 nM ANG II (AII, A) or 10 nM ET-1 (B) in the presence and absence of the MEK inhibitor PD-98059 (PD, 30 μM) or U-0126 (U, 5 μM), as indicated. ACE2 mRNA was measured by RT real-time PCR; n = 5–6. *P < 0.01 compared with the Control (Con), in the absence of ANG II or ET-1.

Fig. 5.

ANG-(1-7) prevents the downregulation of ACE2 mRNA by ANG II. Myocytes in serum-free media were incubated for 12 h with 100 nM ANG II, in the presence or absence of 1 μM ANG-(1-7) [A7], or with 1 μM ANG-(1-7) alone, as shown in A. B: myocytes in serum-free media were incubated for 12 h with 100 nM ANG II (AII) in the presence of increasing concentrations of ANG-(1-7) [A7]. For the studies shown in C, myocytes in serum-free media containing 1 μM [d-Ala⁷]-ANG-(1-7) were incubated for 12 h with 100 nM ANG II, in the presence or absence of 1 μM ANG-(1-7). ACE2 mRNA was measured by RT real-time PCR; n = 4–6. *P < 0.01 compared with the Control (Con), in the absence of ANG II or ANG-(1-7); δP < 0.05 compared with ANG II, in B.

Fig. 6.

Atrial natriuretic peptide (ANP) blocks the ANG II-mediated reduction in ACE2 mRNA. Myocytes in serum-free media containing 0.5 mM EDTA were incubated with 100 nM ANG II, in the presence or absence of 100 nM ANP. ACE2 mRNA was measured by RT real-time PCR; n = 5–6. *P < 0.01 compared with the Control, in the absence of ANG II or ANP.