Rapid induction of aldosterone synthesis in cultured neonatal rat cardiomyocytes under high glucose conditions

Abstract

In addition to classical adrenal cortical biosynthetic pathway, there is increasing evidence that aldosterone is produced in extra-adrenal tissues. Although we previously reported aldosterone production in the heart, the concept of cardiac aldosterone synthesis remains controversial. This is partly due to lack of established experimental models representing aldosterone synthase (CYP11B2) expression in robustly reproducible fashion. We herein investigated suitable conditions in neonatal rat cardiomyocytes (NRCMs) culture system producing CYP11B2 with considerable efficacy. NRCMs were cultured with various glucose doses for 2-24 hours. CYP11B2 mRNA expression and aldosterone concentrations secreted from NRCMs were determined using real-time PCR and enzyme immunoassay, respectively. We found that suitable conditions for CYP11B2 induction included four-hour incubation with high glucose conditions. Under these particular conditions, CYP11B2 expression, in accordance with aldosterone secretion, was significantly increased compared to those observed in the cells cultured under standard-glucose condition. Angiotensin II receptor blocker partially inhibited this CYP11B2 induction, suggesting that there is local renin-angiotensin-aldosterone system activation under high glucose conditions. The suitable conditions for CYP11B2 induction in NRCMs culture system are now clarified: high-glucose conditions with relatively brief period of culture promote CYP11B2 expression in cardiomyocytes. The current system will help to accelerate further progress in research on cardiac tissue aldosterone synthesis.

Figure 1

Quantification of the CYP11B2 transcript levels in NRCMs after four hours of incubation with the indicated glucose concentrations (5.5 mM, n = 15; 15 mM, n = 4; 25 mM, n = 15; 40 mM, n = 4). The qPCR data were normalized to 18S rRNA. The data are shown as the fold changes normalized to the levels found in NRCMs incubated with 5.5 mM glucose. **P < 0.01 and *P < 0.03 versus 5.5 mM glucose.

Figure 2

Time course of the relative expression of CYP11B2 mRNA in NRCMs. NRCMs were grown in culture medium containing either 5.5 mM (gray) or 25 mM (black) glucose for the indicated periods of time (2 hours, n = 6 each; 4 hours, n = 15 each; 8 hours, n = 5 each; 12 hours, n = 6 each; 24 hours, n = 5 each). The qPCR data were normalized to 18S rRNA. The results obtained from cells cultured under normal glucose conditions (gray bars) are shown as the fold changes normalized to the levels after 5.5 mM exposure for four hours, whereas the significance of the differences in the high glucose concentration (black bars) was assessed by comparison of the data with those from the corresponding normal glucose controls (**P < 0.01).

Figure 3

Time course of high glucose-stimulated aldosterone secretion from cardiomyocytes into the culture medium. NRCMs were grown in culture medium containing either 5.5 mM (gray) or 25 mM (black) glucose for the indicated periods of time (4 and 8 hours, n = 14 each). The white bar indicates the aldosterone concentration in the culture medium at baseline after primary myocyte culture, which was followed by an 18-hour serum-free incubation, as described in Section 2 (n = 12). The aldosterone concentration secreted into the culture medium was measured using an enzyme immunoassay. **P < 0.01 versus baseline, ^# P < 0.05 versus 5.5 mM glucose at four hours.

Figure 4

The inhibitory effects of ARB, losartan, on the high glucose-induced CYP11B2 expression in NRCMs. NRCMs were grown in culture medium containing either 5.5 mM (gray) or 25 mM (black) glucose for four hours, with or without 10⁻⁷ M of losartan (n = 11 each). The qPCR data were normalized to 18S rRNA expression. The data are shown as the fold changes normalized to the levels found in NRCMs incubated with 25 mM glucose alone for four hours. **P < 0.01 and ^# P < 0.02 versus 25 mM glucose at four hours.