Expression of aldosterone synthase gene in failing human heart: quantitative analysis using modified real-time polymerase chain reaction.
We have shown that aldosterone, previously thought to be synthesized solely in the adrenal cortex, is also produced in the failing human heart. One way to induce aldosterone synthesis in the heart would be to increase the expression of CYP11B2, the enzyme catalyzing the terminal step in aldosterone synthesis. However, CYP11B2 expression has never been examined in cardiac tissue from patients with heart failure. We assayed CYP11B2 expression in left ventricular tissue obtained at autopsy from seven patients. Total RNA was extracted from frozen samples. CYP11B2 gene expression was then quantitatively analyzed using a modified real-time PCR method that enabled assay of samples containing very small amounts of template DNA. The template DNA was initially amplified 1024-fold by subjecting it to 10 PCR cycles in the absence of the TaqMan probe. Thereafter, conventional real-time PCR was simultaneously performed on both target and standard samples. We measured the small quantities of CYP11B2 gene transcript and found the levels to be significantly higher in samples from heart failure patients than in those from cardiovascular disease-free patients. Our modified real-time PCR method enables quantitative analysis of gene expression using very small amounts of template DNA. CYP11B2 expression is up-regulated in the failing human heart.
Yoshimura
Michihiro
M
Department of Cardiovascular Medicine, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan. bnp@kumamoto-u.ac.jp
Nakamura
Shota
S
Ito
Teruhiko
T
Nakayama
Masafumi
M
Harada
Eisaku
E
Mizuno
Yuji
Y
Sakamoto
Tomohiro
T
Yamamuro
Megumi
M
Saito
Yoshihiko
Y
Nakao
Kazuwa
K
Yasue
Hirofumi
H
Ogawa
Hisao
H
D019405:Cytochrome P-450 CYP11B2 / Q000235:genetics*; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D006333:Heart Failure / Q000201:enzymology* / Q000503:physiopathology; D006801:Humans; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015854:Up-Regulation
D019405:Cytochrome P-450 CYP11B2