ACE inhibitors and cardiac ACE mRNA in volume overload-induced cardiac hypertrophy.

Quinapril, an angiotensin-converting enzyme (ACE) inhibitor with high affinity for cardiac ACE, prevents increases in both plasma and cardiac angiotensin II (ANG II) and development of cardiac hypertrophy after aortocaval shunt in rats. In contrast, enalapril, an ACE inhibitor with low affinity for cardiac ACE, only prevents the increase in plasma ANG II. In the present study, we assessed whether these differences between enalapril and quinapril reflect different inhibition of cardiac tissue ACE and local ANG II by measuring their effects on cardiac ACE mRNA. Treatment with enalapril (250 mg/l) and quinapril (200 mg/l in drinking water) was started 3 days before the shunt and sham surgery. After 1 wk of aortocaval shunt, the hearts were excised and the left ventricle and right ventricle were weighed and used for reverse transcriptase-polymerase chain reaction (RT-PCR) assays for ACE and phosphoglycerate kinase-1 (internal standard). Quinapril, but not enalapril, inhibited the development of cardiac hypertrophy by aortocaval shunt. The shunt increased ACE mRNA in both left and right ventricles about twofold. In animals with aortocaval shunt, quinapril markedly further upregulated ACE mRNA in both ventricles, whereas enalapril did not cause significant changes. In sham rats, both ACE inhibitors increased ACE mRNA, but the increase was more pronounced by treatment with quinapril. These studies show that in vivo ACE inhibitors with low (enalapril) vs. high (quinapril) affinity for cardiac ACE differ in their effects on cardiac ACE mRNA. This difference is more pronounced in volume overload-induced cardiac hypertrophy, presumably reflecting their different effects on cardiac ANG II.

Rapid activation of the type B versus type A natriuretic factor gene by aortocaval shunt induced cardiac volume overload.

OBJECTIVE: The aim was to compare activities of the type A and type B natriuretic factor genes during development of cardiac hypertrophy by use of a non-radioactive method designed for assessment of stable atrial and brain natriuretic factor (ANF, BNF) transcript levels in biopsy sized tissue samples. METHODS: At 1 and 7 days after aortocaval shunt or sham surgery in rats, quantitative reverse transcriptase mediated polymerase chain reaction (Q-RT-PCR) was used to determine mRNA levels in cardiac tissues. Phosphoglycerate kinase-1 (PGK-1) mRNA levels served as an external standard for Q-RT-CR. RESULTS: The shunt increased left ventricular end diastolic pressure at days 1 and 7, and cardiac weight was increased by day 7. By day 1, left ventricular BNF mRNA levels were twice those of controls, whereas ANF mRNA levels were not changed. By day 7, left ventricular BNF mRNA levels were increased 15-fold, and those for ANF were increased fivefold; the BNF mRNAs were also increased in right atria and right ventricle, about fivefold in both cases. CONCLUSIONS: Both natriuretic factor genes were activated by cardiac volume overload, and the increase in the level of left ventricular BNF transcripts-observed for the first time-was in fact more rapid and exceeded that of ANF. The Q-RT-PCR assay will be of value to investigate the response to increased work load of cardiac muscle in vivo.

Random primer p(dN)6-digoxigenin labeling for quantitation of mRNA by Q-RT-PCR and ELISA.

The ability to accurately measure mRNA levels in samples of total RNA is essential for studies on control of gene expression. The mRNAs from the housekeeping gene for phosphoglycerate kinase (PGK-1) can serve as a quality control for RNA samples. We describe an enzyme-linked immunosorbent assay (ELISA) method for mRNA determination by Q-RT-PCR, a quantitative reverse transcriptase-mediated PCR assay with competitive internal standards. After PCR, two biotinylated capture primers, one specific for PGK-1 cDNA and another one for internal standard, are annealed in separate assays so that each can attach DNA to a streptavidin-coated microplate. The captured DNA is either internally labeled with digoxigenin (DIG) or is "developed" after annealing with DIG-labeled primers. Bound DNA is then quantitated by adding DIG-specific antibody with attached alkaline phosphatase and measuring phosphatase activity with a chromogenic substrate and a plate reader. We compared different capturing methods and various primers labeled with DIG at their 3' ends. We determined that amplified PGK-1 DNA specifically captured with biotinylated primers was efficiently assayed with random p(dN)6-DIG.

Stretch-mediated activation of cardiac renin gene.

This study was designed to quantitate cardiac mRNA levels encoding components of the local renin-angiotensin system during the development of volume overload-induced cardiac hypertrophy. Changes in cardiac renin mRNA levels were measured in relation to renin activity in the left ventricle (LV) and in plasma after acute passive stretch of the heart caused by an aortovenocaval shunt in the rat. A quantitative reverse-transcriptase polymerase chain reaction method with competitive internal standards was used to measure mRNA levels in total RNA derived from cardiac tissues after shunt. Seven days after shunt surgery, LV weight was increased by 23%. Renin activities were elevated four- and twofold in plasma and LV, respectively. LV angiotensinogen mRNAs were not significantly increased by shunt surgery; they were twofold higher than phosphoglycerate kinase mRNA from the housekeeping gene PGK-1. By day 7, LV levels for renin mRNA were significantly increased from well below 0.25% to approximately 1% of PGK-1 mRNA. Identity between renin polymerase chain reaction products from kidney and heart cDNAs and absence of "reninlike" amplification products were supported by Southern blotting. Volume overload caused increased expression of the renin gene in the stretched myocardium. This finding is consistent with the concept of a myocardial renin-angiotensin system that can be activated by locally produced renin and contributes to the hypertrophy of cardiac muscle.