In vivo expression of angiotensin-(1-7) lowers blood pressure and improves baroreflex function in transgenic (mRen2)27 rats

Abstract

Transgenic (mRen2)27 rats are hypertensive with impaired baroreflex sensitivity for control of heart rate compared with Hannover Sprague-Dawley rats. We assessed blood pressure and baroreflex function in male hemizygous (mRen2)27 rats (30-40 weeks of age) instrumented for arterial pressure recordings and receiving into the cisterna magna either an Ang-(1-7) fusion protein or a control fusion protein (CTL-FP). The maximum reduction in mean arterial pressure achieved was -38 ± 7 mm Hg on day 3, accompanied by a 55% enhancement in baroreflex sensitivity in Ang-(1-7) fusion protein-treated rats. Both the high-frequency alpha index (HF-α) and heart rate variability increased, suggesting increased parasympathetic tone for cardiac control. The mRNA levels of several components of the renin-angiotensin system in the dorsal medulla were markedly reduced including renin (-80%), neprilysin (-40%), and the AT1a receptor (-40%). However, there was a 2-fold to 3-fold increase in the mRNA levels of the phosphatases PTP-1b and dual-specificity phosphatase 1 in the medulla of Ang-(1-7) fusion protein-treated rats. Our finding that replacement of Ang-(1-7) in the brain of (mRen2)27 rats reverses in part the hypertension and baroreflex impairment is consistent with a functional deficit of Ang-(1-7) in this hypertensive strain. We conclude that the increased mRNA expression of phosphatases known to counteract the phosphoinositol 3 kinase and mitogen-activated protein kinases, and the reduction of renin and AT1a receptor mRNA levels may contribute to the reduction in arterial pressure and improvement in baroreflex sensitivity in response to Ang-(1-7).

Figure 1

Changes in mean arterial pressure (MAP) and heart rate (HR) in (mRen2)27 rats after injection into the cisterna magna of Ang-(1 7)-FP (solid bars) and CTL-FP (open bars). The MAP was monitored for 3 days following injection of the Ang-(1-7)-FP (n = 9) or CTL-FP (n = 4). * P < 0.05 vs CTL-FP.

Figure 2

Frequency domain and sequence analysis of spontaneous baroreflex function, low frequency alpha (LFα), high frequency alpha (HFα), sequence up (Seq Up), sequence down (Seq Down), sequence all (Seq ALL) parameters calculated for CTL-FP and Ang-(1-7)-FP before (A7-FP pre) and after (A7-FP post) injection into the cisterna magna. CTL- FP pre (white bars), CTL- post (diagonal bars) correspond to parameters before and day 2 after intracisternal injection the CTL-FP, whereas A7-FP pre (black bar) and A7-FP post (grey bar) correspond to before and day 2 after intracisternal injection of the A7-FP. * p < 0.05 vs CTL-FP post, # p < 0.05 vs pre.

Figure 3

Standard deviation of the beat to beat interval (SDRR, panel A) and rMSSD (panel B) calculated for CTL-FP and Ang-(1-7)-FP (A7-FP) before and after (post) injection into the cisterna magna. CTL-pre (white bar), CTL-post (diagonal bars) correspond to parameters before and after intracisternal injection the CTL-FP, whereas A7-FP pre (black bar) and A7-FP post (grey bar) correspond to before and after intracisternal injection of the A7-FP. *p < 0.05 vs. CTL-FP post.

Figure 4

Western blot showing the expression of the fusion protein in hypothalamus and dorsal medulla. The IgG2b protein generated from the construct has a molecular weight of 34 kiloDaltons (kDa). Lanes: IgG2b standard alone (1 ng); hypothalamic and medullary tissue from control rats (cH and cM, respectively); control medulla spiked with IgG2b (cM +IgG2b, 1 ng); hypothalamus and medulla from the CTL-FP administration (CTL-FP H and CTL-FP M, respectively). Tissues were collected at day 3 following CTL-FP administration when the maximal hemodynamic effect was observed Data are representative of three independent experiments for each treatment group.

Figure 5

Dorsal medulla oblongata mRNA levels of renin, angiotensinogen (Aogen), ACE and ACE2 following 3 days of Ang-(1-7)-FP treatment. The values for control (CTL-FP) and Ang-(1-7) treated animals (A7-FP) are expressed as relative target gene expression (n=4, each group). *P<0.05 and ***P<0.001 vs. CTL-FP group.

Figure 6

Dorsal medulla oblongata mRNA levels of neprilysin (NEP), Mas receptor, AT_1a and AT_1b receptor isoforms following 3 days of Ang-(1-7)-FP treatment. The values for control (CTL-FP) and Ang-(1-7) treated animals (A7-FP) are expressed as relative target gene expression, (n=4, each group). *P<0.05 vs CTL-FP group.

Figure 7

Hypothalamic mRNA levels of renin, neprilysin (NEP), Mas receptor, AT_1a and AT_1b receptor isoforms following 3 days of Ang-(1-7)-FP treatment. The values for control (CTL-FP) and Ang-(1-7) treated animals (A7-FP) are expressed as relative target gene expression, (n=3-4 per group). *P<0.05 vs. CTL-FP group.

Figure 8

Dorsal medulla oblongata and hypothalamic mRNA levels of the phosphatases DUSP1 and PTP-1b following 3 days of Ang-(1-7)-FP treatment. The values for control (CTL-FP) and Ang-(1-7) treated animals (A7-FP) are expressed as relative target gene expression, (n=4, each group). *P<0.05 and **P<0.01 vs. CTL-FP group.