Differential Regulation of Protective and Harmful Renin Transcripts by the cAMP/PKA/Ca2+-Pathway in Cardiac H9c2 Cells

Figures

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  Figure 1. Effect of 8Br-cAMP on renin mRNA levels in H9c2 cells. Analyses were performed using H9c2 cells exposed to control conditions or glucose starvation in combination with or without 5 mmol/L 8Br-cAMP supplementation. Expression pattern of (A) renin-a mRNA and (B) renin-b mRNA levels were normalised to the housekeeper gene YWHAZ. Data represent mean ± SEM values from six individual experiments. Statistical analyses were performed by two-way ANOVA followed by Tukey’s post hoc test with ** p < 0.01, *** p < 0.001.

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  Figure 2. Effects of adenylate cyclase activation or inhibition on renin mRNA levels in H9c2 cells. H9c2 cells were exposed to basal conditions or glucose starvation in combination with various adenylyl cyclase (AC) modulators. Renin-a mRNA and renin-b mRNA levels were normalised to the housekeeping gene YWHAZ. (A,B) Effects of the AC activator forskolin (10 µmol/L) and (C,D) Effects of the AC inhibitor SQ22536 (100 µmol/L). (E,F) Effect of glucose deprivation on cellular ATP and cAMP levels at the indicated time points. Data represent mean ± SEM values of 6 independent experiments. Statistical analyses of the renin transcript data (A–D) were performed by two-way ANOVA followed by Tukey’s post hoc test; the ATP and cAMP data (E,F) were analysed by one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 3. Effect of protein kinase A inhibition on renin expression in H9c2 cells. Analyses were performed using H9c2 cells exposed to basal conditions or glucose starvation in combination with the protein kinase A inhibitor KT5720 (5 µmol/L) and the AC activator forskolin (10 µmol/L), as indicated. The expression pattern of (A) renin-a and (B) renin-b mRNA were normalised to the housekeeping gene YWHAZ. Data represent mean ± SEM values of 7 independent experiments. Statistical analyses were performed by two-way ANOVA followed by Tukey’s post hoc test with * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 4. Renin expression in H9c2 cells is determined by the intracellular Ca2+ level. For analyses, H9c2 cells were exposed to basal conditions or glucose starvation alone or in combination with the calcium ionophore A23187 (1 µmol/L) or the Ca2+ chelator BAPTA (50 µmol/L) without or with forskolin (10 µmol/L) for 24 h. (A,D) The intracellular calcium content ([Ca2+]i) was monitored by flow cytometry using the Ca2+-sensitive fluorophore Fluo3AM (n = 7). The levels of (B,E) renin-a mRNA and (C,F) renin-b mRNA were normalised to the housekeeping gene YWHAZ. Data represent mean ± SEM values of 8 independent experiments. Statistical analyses were performed using two-way ANOVA followed by Tukey’s post hoc test with * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the respective controls, as indicated.

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  Figure 5. Influence of cAMP/PKA and Ca2+ signaling on the expression of renin-a and renin-b in cardiac H9c2 cells. In general, renin mRNA levels are regulated by the second messengers cAMP and Ca2+, which can affect both the renin expression itself and mRNA stabilization. The relevance of individual components of the signaling pathway was tested using specific inhibitors or stimulators of AC, PKA, and [Ca2+]i, as indicated. The results are summarised in the accompanying table with Ø without effects, ↑ significant increase, and ↓ significant decrease in renin mRNA levels.