Lawrence S. Zisman

Angiotensin-converting enzyme DD genotype in patients with ischaemic or idiopathic dilated cardiomyopathy

Polymorphism in the angiotensin-converting enzyme (ACE) gene has been shown to correlate with circulating ACE concentrations, and also to be an independent risk factor for the development of myocardial infarction, particularly in men thought to be at low risk by standard criteria. We determined the genotypes of individuals with end-stage heart failure due to either ischaemic dilated cardiomyopathy (102) or idiopathic dilated cardiomyopathy (112) and compared these to organ donors with normally functioning hearts (79). Genotypes were determined by the polymerase chain reaction with oligonucleotide primers flanking the polymorphic region in intron 16 of the ACE gene to amplify template DNA isolated from patients. Compared with the DD frequency in the control population, the frequency of the ACE DD genotype was 48% higher in individuals with idiopathic dilated cardiomyopathy (p = 0.008) and 63% higher in subjects with ischaemic cardiomyopathy (p = 0.008), suggesting that an ACE gene variant may contribute to the pathogenesis of both types of cardiomyopathy.

Selective Downregulation of the Angiotensin II AT1-Receptor Subtype in Failing Human Ventricular Myocardium

BACKGROUND The regulation of angiotensin II receptors and the two major subtypes (AT1 and AT2) in chronically failing human ventricular myocardium has not been previously examined. METHODS AND RESULTS Angiotensin II receptors were measured by saturation binding of 125I-[Sar1,Ile8]angiotensin II in crude membranes from nonfailing (n = 19) and failing human left ventricles with idiopathic dilated cardiomyopathy (IDC; n = 31) or ischemic cardiomyopathy (ISC; n = 21) and membranes from a limited number of right ventricles in each category. The AT1 and AT2 fractions were determined by use of an AT1-selective antagonist, losartan. beta-Adrenergic receptors were also measured by binding of 125I-iodocyanopindolol with the beta 1 and beta 2 fractions determined by use of a beta 1-selective antagonist, CGP20712A, AT1 but not AT2 density was significantly decreased in the combined (IDC + ISC) failing left ventricles (nonfailing: AT1 4.66 +/- 0.48, AT2 2.73 +/- 0.39; failing: AT1 3.20 +/- 0.29, AT2 2.70 +/- 0.33 fmol/mg protein; mean +/- SE). The decrease in AT1 density was greater in the IDC than in the ISC left ventricles (IDC: 2.73 +/- 0.40, P < .01; ISC: 3.89 +/- 0.39 fmol/mg protein, P = NS versus nonfailing). beta 1 but not beta 2 density was decreased in the failing left ventricles. AT1 density was correlated with beta 1 density in all left ventricles (r = .43). AT1 density was also decreased in IDC right ventricles. In situ reverse transcription-polymerase chain reaction in sections of nonfailing and failing ventricles indicated that AT1 mRNA was present in both myocytes and nonmyocytes. CONCLUSIONS AT1 receptors are selectively downregulated in failing human ventricles, similar to the selective downregulation of beta 1 receptors. The relative lack of AT1 downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.

Angiotensin-(1-7) Formation in the Intact Human Heart In Vivo Dependence on Angiotensin II as Substrate

Background—Several enzymes that hydrolyze angiotensin I (Ang I) and Ang II to Ang-(1-7) have been identified, but their relative importance in the intact human heart is not known. Methods and Results—Intracoronary (IC) 123I-Ang I was administered to 4 heart transplantation recipients. Arterial and coronary sinus (CS) samples were taken before and after coadministration of IC enalaprilat. 123I-Ang metabolites were separated by high-pressure liquid chromatography, and 123I-Ang-(1-7) and 123I-Ang II were quantified across the myocardial circulation. 123I-Ang II formation (as measured by fractional conversion) at steady state was 0.43±0.05 and was reduced to 0.042±0.02 after IC enalaprilat (P <0.01). The fractional conversion of 123I-Ang-(1-7) was 0.198±0.032 but was reduced to 0.06±0.01 during IC enalaprilat (P <0.01). Net Ang II production at steady state was 2720±704 pg/min. Ang-(1-7) production was 3489±768 pg/min. After IC enalaprilat, Ang II production fell to 436±66.8 pg/min (P <0.05 versus Ang II production). After suppression of Ang II production with enalaprilat, there was net uptake of Ang-(1-7): −289±144 pg/min (P <0.05). Conclusions—Ang-(1-7) was formed in the intact human myocardial circulation and was decreased when Ang II formation was suppressed. These data indicate that the major pathway for Ang-(1-7) generation in the intact human heart was dependent on substrate availability of Ang II. Ang-(1-7)–forming enzymes that demonstrate substrate preference for Ang II are likely to play an important role in the regulation of Ang-(1-7) formation in the intact human heart.

Differential Regulation of Cardiac Angiotensin Converting Enzyme Binding Sites and AT1 Receptor Density in the Failing Human Heart

BACKGROUND The regulation and interaction of ACE and the angiotensin II (Ang II) type I (AT1) receptor in the failing human heart are not understood. METHODS AND RESULTS Radioligand binding with 3H-ramiprilat was used to measure ACE protein in membrane preparations of hearts obtained from 36 subjects with idiopathic dilated cardiomyopathy (IDC), 8 subjects with primary pulmonary hypertension (PPH), and 32 organ donors with normal cardiac function (NF hearts). 125I-Ang II formation was measured in a subset of hearts. Saralasin (125I-(Sar1,Ile8)-Ang II) was used to measure total Ang II receptor density. AT1 and AT2 receptor binding were determined with the AT1 receptor antagonist losartan. Maximal ACE binding (Bmax) was 578+/-47 fmol/mg in IDC left ventricle (LV), 713+/-97 fmol/mg in PPH LV, and 325+/-27 fmol/mg in NF LV (P<0.001, IDC or PPH versus NF). In IDC, PPH, and NF right ventricles (RV), ACE Bmax was 737+/-78, 638+/-137, and 422+/-49 fmol/mg, respectively (P=0.02, IDC versus NF; P=0.08, PPH versus NF). 125I-Ang II formation correlated with ACE binding sites (r=0.60, P=0.00005). There was selective downregulation of the AT1 receptor subtype in failing PPH ventricles: 6.41+/-1.23 fmol/mg in PPH LV, 2.37+/-0.50 fmol/mg in PPH RV, 5.38+/-0.53 fmol/mg in NF LV, and 7.30+/-1.10 fmol/mg in NF RV (P=0.01, PPH RV versus PPH LV; P=0.0006, PPH RV versus NF RV). CONCLUSIONS ACE binding sites are increased in both failing IDC and nonfailing PPH ventricles. In PPH hearts, the AT1 receptor is downregulated only in the failing RV.

Inhibiting tissue angiotensin-converting enzyme: a pound of flesh without the blood?

“This bond doth give thee here no jot of blood. The words expressly are ‘a pound of flesh.’ ” Portia, in Shakespeare’s The Merchant of Venice Angiotensin-converting enzyme catalyzes the formation of angiotensin II (Ang II) from Ang I but also degrades bradykinin (BK). Ang II, acting through the AT1 receptor, is a potent vasoconstrictor, stimulates norepinephrine release from sympathetic nerve terminals in the heart, and causes hypertrophy of cardiac myocytes. BK, via activation of the BK2 receptor, stimulates the release of NO and prostaglandins and may counteract Ang II–mediated effects. ACE inhibition may exert beneficial effects both by interrupting Ang II–mediated AT1 receptor signal transduction and by augmenting BK2 receptor activation. Because of the proven survival benefit from ACE-inhibitor therapy in patients with heart failure, great attention has been given to understanding the structure and function of ACE and to the design of optimally effective ACE inhibitors. In this issue of Circulation , Hornig and colleagues1 compare the effects of 2 such ACE inhibitors, enalaprilat and quinaprilat, on the peripheral circulation in patients with heart failure. To understand the implications of their work, a discussion of the ACE molecule itself, the chemical structure of ACE inhibitors, and their interaction with ACE is required. The major issue to be examined is the proposed dichotomy between high- and low-affinity tissue ACE inhibitors. In somatic tissues, ACE is a glycoprotein of ≈140 kDa; in testicular cells, it is synthesized from the same gene but at an alternative transcription start site that results in a protein of 100 kDa.2 The somatic form of ACE has 2 homologous domains (the amino, or N-terminal, and carboxy, or C-terminal, sites) that are both catalytically active and that both require the presence of Zn2+ for activity.3 Testicular ACE contains …

Angiotensin II Receptors in the Normal and Failing Heart

Mammalian myocardium expresses two major subtypes of angiotensin II receptors, namely, AT1 and AT2. These two subtypes mediate a diverse set of Ang II actions on the myocardium in multiple types of cells. AT1 receptors mediate most of the conventionally known angiotensin II actions such as vasoconstrictive, inotropic, chronotropic, and trophic–mitogenic effects. The function(s) of AT2 receptors remains obscure in the myocardium. Expression of the two receptor subtypes appears to be developmentally regulated, with high expression in the fetus followed by decreased expression in the early postnatal period. In end-stage failing human heart, expression of the two subtypes is differentially regulated, with selective downregulation of the AT1 subtype and unchanged or relative upregulation of the AT2 subtype. The selective AT1 receptor downregulation in failing human ventricular myocardium is similar to the behavior of β1-adrenergic receptors, and is “chamber-specific” or only present in failing chambers in the failing human heart. AT1 receptor downregulation in failing ventricular myocardium is likely caused directly or indirectly by activation of the cardiac renin–angiotensin system. Cardiac AT1 receptor downregulation is present in subjects treated with angiotensin-converting enzyme inhibitors, which suggests that blockade of AT1 receptors may be necessary to optimally inhibit adverse effects of angiotensin II. The overall effectiveness of selective AT1 receptor antagonist therapy will likely depend on the probable enhancement of unblocked AT2 receptor-mediated effects of Ang II in the failing human heart, the consequences of which are yet to be elucidated.

757-2 Angiotensin II Formation in the Intact Human Heart: Predominance of the Angiotensin Converting Enzyme Pathway

It has been proposed that the contribution of myocardial tissue angiotensin converting enzyme (ACE) to angiotensin II (Ang II) formation in the human heart is low compared with non-ACE pathways. However, little is known about the actual in vivo contribution of these pathways to Ang II formation in the human heart. To examine angiotensin II formation in the intact human heart, we administered intracoronary 123I-labeled angiotensin I (Ang I) with and without intracoronary enalaprilat to orthotopic heart transplant recipients. The fractional conversion of Ang I to Ang II, calculated after separation of angiotensin peptides by HPLC, was 0.415 +/- 0.104 (n = 5, mean +/- SD). Enalaprilat reduced fractional conversion by 89%, to a value of 0.044 +/- 0.053 (n = 4, P = 0.002). In a separate study of explanted hearts, a newly developed in vitro Ang II-forming assay was used to examine cardiac tissue ACE activity independent of circulating components. ACE activity in solubilized left ventricular membrane preparations from failing hearts was 49.6 +/- 5.3 fmol 125I-Ang II formed per minute per milligram of protein (n = 8, +/- SE), and 35.9 +/- 4.8 fmol/min/mg from nonfailing human hearts (n = 7, P = 0.08). In the presence of 1 microM enalaprilat, ACE activity was reduced by 85%, to 7.3 +/- 1.4 fmol/min/mg in the failing group and to 4.6 +/- 1.3 fmol/min/mg in the nonfailing group (P < 0.001). We conclude that the predominant pathway for angiotensin II formation in the human heart is through ACE.