Jewell A. Jessup

Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function

Angiotensin-converting enzyme 2 (ACE2) is the first human homologue of ACE to be described. ACE2 is a type I integral membrane protein that functions as a carboxypeptidase, cleaving a single hydrophobic/basic residue from the COOH-terminus of its substrates. Because ACE2 efficiently hydrolyzes the potent vasoconstrictor angiotensin II to angiotensin (1–7), this has changed our overall perspective about the classical view of the renin angiotensin system in the regulation of hypertension and heart and renal function, because it represents the first example of a feedforward mechanism directed toward mitigation of the actions of angiotensin II. This paper reviews the new data regarding the biochemistry of angiotensin-(1–7)-forming enzymes and discusses key findings such as the elucidation of the regulatory mechanisms participating in the expression of ACE2 and angiotensin-(1–7) in the control of the circulation.

Effect of Angiotensin-Converting Enzyme Inhibition and Angiotensin II Receptor Blockers on Cardiac Angiotensin-Converting Enzyme 2

Background—Angiotensin-converting enzyme 2 (ACE2) has emerged as a novel regulator of cardiac function and arterial pressure by converting angiotensin II (Ang II) into the vasodilator and antitrophic heptapeptide, angiotensin-(1–7) [Ang-(1–7)]. As the only known human homolog of ACE, the demonstration that ACE2 is insensitive to blockade by ACE inhibitors prompted us to define the effect of ACE inhibition on the ACE2 gene. Methods and Results—Blood pressure, cardiac rate, and plasma and cardiac tissue levels of Ang II and Ang-(1–7), together with cardiac ACE2, neprilysin, Ang II type 1 receptor (AT1), and mas receptor mRNAs, were measured in Lewis rats 12 days after continuous administration of vehicle, lisinopril, losartan, or both drugs combined in their drinking water. Equivalent decreases in blood pressure were obtained in rats given lisinopril or losartan alone or in combination. ACE inhibitor therapy caused a 1.8-fold increase in plasma Ang-(1–7), decreased plasma Ang II, and increased cardiac ACE2 mRNA but not cardiac ACE2 activity. Losartan increased plasma levels of both Ang II and Ang-(1–7), as well as cardiac ACE2 mRNA and cardiac ACE2 activity. Combination therapy duplicated the effects found in rats medicated with lisinopril, except that cardiac ACE2 mRNA fell to values found in vehicle-treated rats. Losartan treatment but not lisinopril increased cardiac tissue levels of Ang II and Ang-(1–7), whereas none of the treatments had an effect on cardiac neprilysin mRNA. Conclusions—Selective blockade of either Ang II synthesis or activity induced increases in cardiac ACE2 gene expression and cardiac ACE2 activity, whereas the combination of losartan and lisinopril was associated with elevated cardiac ACE2 activity but not cardiac ACE2 mRNA. Although the predominant effect of ACE inhibition may result from the combined effect of reduced Ang II formation and Ang-(1–7) metabolism, the antihypertensive action of AT1 antagonists may in part be due to increased Ang II metabolism by ACE2.

Angiotensin-(1-12) is an alternate substrate for angiotensin peptide production in the heart

Identification of angiotensin-(1-12) as an intermediate precursor derived directly from angiotensinogen led us to explore whether the heart has the capacity to process angiotensin-(1-12) into biologically active angiotensin peptides. The generation of angiotensin I, angiotensin II, and angiotensin-(1-7) from exogenous angiotensin-(1-12) was evaluated in the effluent of isolated perfused hearts mounted on a Langendorff apparatus in three normotensive and two hypertensive strains: Sprague-Dawley, Lewis, congenic mRen2.Lewis, Wistar-Kyoto, and spontaneously hypertensive rats. Hearts were perfused with Krebs solution for 60 min before and after the addition of angiotensin-(1-12) (10 nmol/l). Angiotensin-(1-12) caused the rapid appearance of both angiotensin I and angiotensin II in the perfusate that peaked between 30 and 60 min of recirculation. Production of angiotensin-(1-7) from exogenous angiotensin-(1-12) rose steadily over the course of the 60-min experiment. These data directly demonstrate that angiotensin-(1-12) is a substrate for the formation of angiotensin peptides in cardiac tissue. This finding further suggests that this angiotensinogen-derived product is a previously unrecognized important precursor peptide to the renin-angiotensin system cascade.

Angiotensin Peptides As Neurotransmitters/ Neuromodulators In The Dorsomedial Medulla

1. The present review provides an update on evidence of the neurotransmitter pathways and location of receptors within the nucleus tractus solitarii (NTS) mediating the baroreflex and other haemodynamic actions of angiotensin (Ang) II.

Role of estrogen in diastolic dysfunction

The prevalence of left ventricular diastolic dysfunction (LVDD) sharply increases in women after menopause and may lead to heart failure. While evidence suggests that estrogens protect the premenopausal heart from hypertension and ventricular remodeling, the specific mechanisms involved remain elusive. Moreover, whether there is a protective role of estrogens against cardiovascular disease, and specifically LVDD, continues to be controversial. Clinical and basic science have implicated activation of the renin-angiotensin-aldosterone system (RAAS), linked to the loss of ovarian estrogens, in the pathogenesis of postmenopausal diastolic dysfunction. As a consequence of increased tissue ANG II and low estrogen, a maladaptive nitric oxide synthase (NOS) system produces ROS that contribute to female sex-specific hypertensive heart disease. Recent insights from rodent models that mimic the cardiac phenotype of an estrogen-insufficient or -deficient woman (e.g., premature ovarian failure or postmenopausal), including the ovariectomized congenic mRen2.Lewis female rat, provide evidence showing that estrogen modulates the tissue RAAS and NOS system and related intracellular signaling pathways, in part via the membrane G protein-coupled receptor 30 (GPR30; also called G protein-coupled estrogen receptor 1). Complementing the cardiovascular research in this field, the echocardiographic correlates of LVDD as well as inherent limitations to its use in preclinical rodent studies will be briefly presented. Understanding the roles of estrogen and GPR30, their interactions with the local RAAS and NOS system, and the relationship of each of these to LVDD is necessary to identify new therapeutic targets and alternative treatments for diastolic heart failure that achieve the cardiovascular benefits of estrogen replacement without its side effects and contraindications.

Attenuation of Salt-Induced Cardiac Remodeling and Diastolic Dysfunction by the GPER Agonist G-1 in Female mRen2.Lewis Rats

Introduction The G protein-coupled estrogen receptor (GPER) is expressed in various tissues including the heart. Since the mRen2.Lewis strain exhibits salt-dependent hypertension and early diastolic dysfunction, we assessed the effects of the GPER agonist (G-1, 40 nmol/kg/hr for 14 days) or vehicle (VEH, DMSO/EtOH) on cardiac function and structure. Methods Intact female mRen2.Lewis rats were fed a normal salt (0.5% sodium; NS) diet or a high salt (4% sodium; HS) diet for 10 weeks beginning at 5 weeks of age. Results Prolonged intake of HS in mRen2.Lewis females resulted in significantly increased blood pressure, mildly reduced systolic function, and left ventricular (LV) diastolic compliance (as signified by a reduced E deceleration time and E deceleration slope), increased relative wall thickness, myocyte size, and mid-myocardial interstitial and perivascular fibrosis. G-1 administration attenuated wall thickness and myocyte hypertrophy, with nominal effects on blood pressure, LV systolic function, LV compliance and cardiac fibrosis in the HS group. G-1 treatment significantly increased LV lusitropy [early mitral annular descent (e′)] independent of prevailing salt, and improved the e′/a′ ratio in HS versus NS rats (P<0.05) as determined by tissue Doppler. Conclusion Activation of GPER improved myocardial relaxation in the hypertensive female mRen2.Lewis rat and reduced cardiac myocyte hypertrophy and wall thickness in those rats fed a high salt diet. Moreover, these advantageous effects of the GPER agonist on ventricular lusitropy and remodeling do not appear to be associated with overt changes in blood pressure.

Localization of the novel angiotensin peptide, angiotensin-(1-12), in heart and kidney of hypertensive and normotensive rats

A low expression of angiotensinogen in the heart has been construed as indicating a circulating uptake mechanism to explain the local effects of angiotensin II on tissues. The recent identification of angiotensin-(1-12) in an array of rat organs suggests this propeptide may be an alternate substrate for local angiotensin production. To test this hypothesis, tissues from 11-wk-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (n = 14) were stained with purified antibodies directed to the COOH terminus of angiotensin-(1-12). Robust angiotensin-(1-12) staining was predominantly found in ventricular myocytes with less staining found in the medial layer of intracoronary arteries and vascular endothelium. In addition, angiotensin-(1-12) immunoreactivity was present in the proximal, distal, and collecting renal tubules within the deep cortical and outer medullary zones in both strains. Preadsorption of the antibody with angiotensin-(1-12) abolished staining in both tissues. Corresponding tissue measurements by radioimmunoassay showed 47% higher levels of angiotensin-(1-12) in the heart of SHR compared with WKY rats (P < 0.05). In contrast, renal angiotensin-(1-12) levels were 16.5% lower in SHR compared with the WKY rats (P < 0.05). This study shows for first time the localization of angiotensin-(1-12) in both cardiac myocytes and renal tubular components of WKY and SHR. In addition, we show that increased cardiac angiotensin-(1-12) concentrations in SHR is associated with a small, but statistically significant, reduction in renal angiotensin-(1-12) levels.

Activation of GPR30 attenuates diastolic dysfunction and left ventricle remodelling in oophorectomized mRen2.Lewis rats

AIMS GPR30 is a novel oestrogen receptor expressed in various tissues, including the heart. We determined the role of GPR30 in the maintenance of left ventricular (LV) structure and diastolic function after the surgical loss of ovarian hormones in the female mRen2.Lewis rat, a model emulating the cardiac phenotype of the post-menopausal woman. METHODS AND RESULTS Bilateral oophorectomy (OVX) or sham surgery was performed in study rats; the selective GPR30 agonist, G-1 (50 µg/kg/day), or vehicle was given subcutaneously to OVX rats from 13-15 weeks of age. Similar to the cardiac phenotype of sham rats, G-1 preserved diastolic function and structure relative to vehicle-treated OVX littermates independent of changes in blood pressure. G-1 limited the OVX-induced increase in LV filling pressure, LV mass, wall thickness, interstitial collagen deposition, atrial natriuretic factor and brain natriuretic peptide mRNA levels, and cardiac NAD(P)H oxidase 4 (NOX4) expression. In vitro studies showed that G-1 inhibited angiotensin II-induced hypertrophy in H9c2 cardiomyocytes, evidenced by reductions in cell size, protein content per cell, and atrial natriuretic factor mRNA levels. The GPR30 antagonist, G15, inhibited the protective effects of both oestradiol and G-1 on this hypertrophy. CONCLUSION These data show that the GPR30 agonist G-1 mitigates the adverse effects of oestrogen loss on LV remodelling and the development of diastolic dysfunction in the study rats. This expands our knowledge of the sex-specific mechanisms underlying diastolic dysfunction and provides a potential therapeutic target for reducing the progression of this cardiovascular disease process in post-menopausal women.

Beneficial versus harmful effects of Angiotensin (1-7) on impulse propagation and cardiac arrhythmias in the failing heart

Introduction. The presence of Angiotensin (1-7) (Ang 1-7) and ACE 2 in the ventricle of cardiomyopathic hamsters as well as the influence of Ang (1-7) on membrane potential, impulse propagation and cardiac excitability were investigated. Methods. Histology and immunochemistry were used to demonstrate the presence of Ang (1-7) and ACE 2 in the ventricle of cardiomyopathic hamsters. Measurements of transmembrane potentials, conduction velocity and refractoriness were made using conventional intracellular microelectrodes. The influence of Ang (1-7) on sodium pump current was investigated in voltageclamped myocytes isolated from the ventricle. Results. The results indicated the presence of Ang (1-7) and ACE 2 in myocytes of cardiomyopathic hamsters. Moreover, Ang (1-7) (10-8 M) hyperpolarised the heart cell, increased the conduction velocity, and I reduced transiently the action potential duration. The cardiac refractoriness was also increased by the heptapeptide, an effect in part reduced by an inhibitor of mas receptor. These findings indicate that Ang (1-7) has important antiarrhythmic properties. However, the beneficial effects of Ang (1-7) are dose-dependent because at higher concentration (10-7 M) the heptapeptide elicited an appreciable increase of action potential duration and early-after depolarisations. Since losartan (10-7 M) did not counteract this effect of the high dose of the heptapeptide, it is possible to conclude that activation of AT1-receptors is not involved in this effect of Ang (1-7).To investigate the mechanism of the hyperpolarising action of Ang (1-7) the influence of the heptapeptide on the sodium potassium pump current was studied in myocytes isolated from the ventricle of cardiomyopathic hamsters. The peak pump current density was measured under voltage clamp using the whole cell configuration. The results indicated that Ang (1-7) (10—8 M) enhanced the electrogenic sodium pump, an effect suppressed by ouabain (10—7 M). Conclusions. Ang (1-7) has beneficial effects on the failing heart by activating the sodium pump, hyperpolarising the cell membrane and increasing the conduction velocity. These effects as well as the increment of refractoriness indicate that Ang (1-7) has antiarrhythmic properties. At higher concentrations (10—7 M), however, the heptapeptide induced early-after depolarisations which leads to the conclusion that an optimal generation of Ang (1-7) must be achieved to permit a protective role of Ang (1-7) on cardiac arrhythmias.

Characterization of the cardiac renin angiotensin system in oophorectomized and estrogen-replete mRen2.Lewis rats.

The cardioprotective effects of estrogen are well recognized, but the mechanisms remain poorly understood. Accumulating evidence suggests that the local cardiac renin-angiotensin system (RAS) is involved in the development and progression of cardiac hypertrophy, remodeling, and heart failure. Estrogen attenuates the effects of an activated circulating RAS; however, its role in regulating the cardiac RAS is unclear. Bilateral oophorectomy (OVX; n = 17) or sham-operation (Sham; n = 13) was performed in 4-week-old, female mRen2.Lewis rats. At 11 weeks of age, the rats were randomized and received either 17 β-estradiol (E2, 36 µg/pellet, 60-day release, n = 8) or vehicle (OVX-V, n = 9) for 4 weeks. The rats were sacrificed, and blood and hearts were used to determine protein and/or gene expression of circulating and tissue RAS components. E2 treatment minimized the rise in circulating angiotensin (Ang) II and aldosterone produced by loss of ovarian estrogens. Chronic E2 also attenuated OVX-associated increases in cardiac Ang II, Ang-(1–7) content, chymase gene expression, and mast cell number. Neither OVX nor OVX+E2 altered cardiac expression or activity of renin, angiotensinogen, angiotensin-converting enzyme (ACE), and Ang II type 1 receptor (AT1R). E2 treatment in OVX rats significantly decreased gene expression of MMP-9, ACE2, and Ang-(1–7) mas receptor, in comparison to sham-operated and OVX littermates. E2 treatment appears to inhibit upsurges in cardiac Ang II expression in the OVX-mRen2 rat, possibly by reducing chymase-dependent Ang II formation. Further studies are warranted to determine whether an E2-mediated reduction in cardiac chymase directly contributes to this response in OVX rats.