Angiotensin-Converting Enzyme in the Human Heart
Effect of the Deletion/Insertion Polymorphism
Background An insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been associated with differences in the plasma levels of ACE as well as with myocardial infarction, cardiomyopathy, left ventricular hypertrophy, and coronary artery disease.
Methods and Results We determined the cardiac ACE activity and the ACE genotype in 71 subjects who died of noncardiac disorders. Cardiac ACE activity was significantly higher (P<.01) in subjects with the ACE DD genotype (12.7±1.9 mU/g wet wt) compared with subjects with the ID (8.7±0.8 mU/g) and the II (9.1±1.0 mU/g) genotypes. This difference was independent of sex, age, and the time required for tissue collection.
Conclusions Cardiac ACE activity is highest in subjects with the DD genotype. Elevated cardiac ACE activity in these subjects may result in increased cardiac angiotensin II levels, and this may be a mechanism underlying the reported association between the ACE deletion polymorphism and the increased risk for several cardiovascular disorders.
A deletion polymorphism in the gene encoding angiotensin-converting enzyme (ACE) has been associated with an increased risk of myocardial infarction,1 cardiomyopathy,2 3 left ventricular hypertrophy,4 and coronary artery disease.5 However, these associations could not be confirmed in all studies,6 7 and the mechanism underlying this increased genetic risk remains unclear.
Plasma ACE levels are known to be elevated in subjects homozygous for the deletion allele (DD) compared with heterozygotes (ID) or subjects homozygous for the insertion (II) allele.8 ACE levels in human T lymphocytes are genetically determined in a similar way.9
At present, no information is available on the ACE activity of cardiac tissue in relation to the deletion/insertion ACE genotype. We determined ACE activity and genotype in human cardiac tissue so as to study whether the cardiac ACE level is, in part, genetically determined as well.
Left ventricular cardiac tissue was obtained from 71 organ donors (38 men and 33 women 1 to 54 years old; median, 35 years) who died of noncardiac disorders (29 cerebrovascular accident, 32 polytrauma, 7 cerebral hypoxia, 1 encephalitis, 1 meningitis, and 1 hydrocephalus). The donor hearts were provided by the Rotterdam Heart Valve Bank (Bio Implant Services Foundation/Eurotransplant Foundation). Immediately after circulatory arrest, the hearts were stored at 0°C to 4°C in a sterile organ-protecting solution (University of Wisconsin, EuroCollins, or HTK-Bretschneider solution) for a maximum of 24 hours (cold ischemia time). After the aortic and pulmonary valves had been excised for homograft valve transplantation, ≈1 to 3 g of left ventricular free wall was removed and stored at −70°C.
Frozen tissue (100 to 200 mg) was homogenized with a Polytron PT10/35 (Kinematica) in 1 mL 0.01 mol/L phosphate buffer, pH 7.4, containing 0.15 mol/L NaCl. ACE activity in the homogenate was measured in duplicate with a commercial kit (ACEcolor, Fujirebio) containing p-hydroxybenzoyl-glycyl-l-histidyl-l-leucine as synthetic substrate.10 Multiple dilutions (50- or 100-μL aliquots containing ≈1 and ≈2 mg protein, respectively) of the homogenate were incubated for 2 hours with 10 mmol/L substrate (total incubation volume, 300 μL) at 37°C and pH 8.3. Incubations of homogenate with substrate in the presence of 3.0 mmol/L disodium-EDTA served as blanks. After 2 hours, the reaction of ACE on the synthetic substrate was stopped by addition of 750 μL stopper/developer solution containing 3 mmol/L disodium-EDTA. The tubes were mixed, incubated at 37°C for 10 minutes to allow formation of index color from the converted substrate, and centrifuged at 3000g for 5 minutes at room temperature. Absorbance was then read at 505 nm.
Generation of p-hydroxybenzoic acid over time was linear, and ACE activity, expressed as nanomoles of p-hydroxybenzoic acid per minute per gram of tissue (or milliunits per gram of tissue), was not different for the samples containing ≈1 and ≈2 mg protein. The lower limit of detection was 1.5 mU/g tissue.
To extract genomic DNA, 50 to 100 mg of left ventricular tissue was homogenized in a buffer containing 155 mmol/L ammonium chloride, 10 mmol/L potassium carbonate, and 0.1 mmol/L EDTA (pH 8.0). The homogenate was then centrifuged at 6000 rpm for 15 minutes at 4°C, and the resulting supernatant (0.5 mL) was diluted in 4.5 mL of the same buffer, incubated at 0°C for 10 minutes, and centrifuged at 13 000 rpm for 15 minutes at 4°C. The pellet was resuspended in 100 μL 10 mmol/L Tris-HCl buffer, pH 8.0, containing 1 mmol/L disodium-EDTA, subjected to proteinase K digestion, and ethanol-precipitated as described previously.4 The genotype was then determined by the polymerase chain reaction (PCR) and subsequent gel electrophoresis of the PCR products.4 The laboratory responsible for genotyping was blinded for values of cardiac ACE activity.
The distribution of the ACE genotypes in the present population was not different from that reported previously for normal populations.1 3 4 The frequency of the D allele was 0.53. Cardiac ACE activity (expressed per gram wet weight) was in the same range as plasma ACE activity measured normally in our laboratory (7 to 20 mU/mL), which indicates that the presence of ACE in cardiac tissue cannot be attributed to trapped blood plasma. Cardiac ACE activity (expressed per gram wet weight or per gram protein) was significantly higher in subjects with the DD genotype (Table⇓; P<.02 versus ID, P<.01 versus ID+II). The difference remained statistically significant (P<.01) after correction for sex, age, and cold ischemia time. No difference in cardiac ACE activity was found between subjects with the ID and the II genotypes.
Our results show that in subjects with the DD genotype in whom plasma ACE is known to be elevated, cardiac ACE activity is elevated as well. Previous studies in experimental animals have demonstrated that cardiac ACE activity is closely correlated with cardiac ACE mRNA levels.8 ACE, an ectoenzyme anchored in the cell membrane, is released into the circulation upon cleavage from its anchor. Both increased synthesis and accelerated release into the circulation would lead to elevated plasma ACE activity. Our finding that, besides plasma ACE activity, cardiac ACE activity is also increased in subjects with the DD genotype would favor increased synthesis as an explanation for the increased plasma ACE levels.
ACE activity in subjects with the II genotype was not different from that in subjects with the ID genotype. This contrasts with earlier findings in plasma, in which ACE activity was lowest in subjects with the II genotype.8 It resembles, however, the findings of Costerousse et al9 in human T lymphocytes. The absence of a gene dosage effect in cardiac tissue may be due to unknown genetic or environmental effects. Furthermore, the number of subjects in our study may have been too small to detect a statistical difference between II and ID subjects.
Elevated ACE levels in cardiac tissue may, through increased conversion of angiotensin I to angiotensin II,11 12 lead to higher cardiac angiotensin II levels.13 Since angiotensin II affects cardiac function as well as cardiovascular growth and remodeling, our findings may help to explain the associations between the ACE deletion polymorphism and cardiac disease.1 2 3 4 5
This work was supported in part by a grant from the Netherlands Heart Foundation (NHS 93.004), the Deutsche Forschungsgemeinschaft (Schu 672/3), and the Bundesministerium für Forschung und Technologie (KBF 01GB9403). We wish to acknowledge the excellent technical support by Susanne Kürzinger, as well as advice for statistical analysis by Dr M. Gliese.
- Received April 26, 1995.
- Revision received June 13, 1995.
- Accepted July 5, 1995.
- Copyright © 1995 by American Heart Association
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