Specific Upregulation of TRPC1 and TRPC5 Channels by Mineralocorticoid Pathway in Adult Rat Ventricular Cardiomyocytes

Figures

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  Figure 1. A 24-h aldosterone treatment increases store-operated Ca2+ entry (SOCE) in adult rat ventricular cardiomyocytes (ARVMs). (A) Representative traces of fluorescence variation in Fluo-4/AM-loaded ARVMs pre-incubated in the absence (control (ctrl), gray trace) and in the presence of aldosterone (Aldo, 100 nM, dark-gray trace or 1 µM, red trace). Cells were exposed to 5 µM thapsigargin (Tg) plus 10 mM caffeine (caf) in the presence of 10 µM nifedipine (NIF) in Ca2+-free medium to deplete the SR and then to Ca2+-containing solution in the presence of 5 µM of KB-R7943 and NIF to evaluate the SOCE. (B) Averaged amplitude of SOCE (F/F0) in ARVMs incubated for 24 h in the absence (ctrl, white symbols) or in the presence of aldosterone (100 nM, gray symbols or 1 µM, red symbols) without or with 10 µM of the selective MR antagonist RU-28318 (RU, orange symbols in aldosterone condition and pink symbols in ctrl condition). Average results of 8–32 cells from three isolations. (C) Representative traces of [Ca2+]i variation (in nM) in Fura-2/AM-loaded ARVMs incubated for 24 h in the absence (ctrl, gray trace) or in the presence of aldosterone (100 nM, dark-gray trace; 1 µM, red trace). Fura-2/AM…

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  Figure 2. A 24-h aldosterone treatment increases SOC currents (ISOC) in ARVMs. (A,B) Representative current–voltage (I–V) relationships elicited by ramp voltage-clamp protocol in ARVMs incubated for 24 h without (A) or with aldosterone 1 μM (B), following SR Ca2+ depletion (black traces) and then in the presence of 5 µM BTP2 (orange traces). BTP2-sensitive (blue traces) represents the difference current. (C) Scatter plots of mean ± SEM of the BTP2-sensitive I–V relationships recorded in ARVMs incubated for 24 h without (gray trace, −0.19 ± 0.19 pA/pF at −90 mV and 0.30 ± 0.17 pA/pF at +60 mV, n = 7) or with Aldo 1 µM (red trace, −1.51 ± 0.20 pA/pF at −90 mV (p < 0.01) and 0.90 ± 0.15 pA/pF at +60 mV (p < 0.05), n = 9) from three cell isolations. Statistical significance was evaluated using Student’s t-test.

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  Figure 3. A 24-h aldosterone treatment increases transient receptor potential canonical channel 1 (TRPC1), TRPC5, and STIM1 protein expression in ARVMs. Representative Western blot (top panels) and pooled data (bottom bar graph) of TRPC1, TRPC5, STIM1, Orai1, TRPC3, TRPC4, TRPC6, STIM2, and Orai3 in ARVMs incubated for 24 h in the absence or in the presence of aldosterone (100 nM or 1 µM). Protein levels were normalized by β-actin and are expressed as fold changes of that determined in untreated ARVMs for each cell isolation. Results are presented as scatter plots with median and interquartile range. Statistical significance was evaluated using one-way ANOVA (Kruskal–Wallis) followed by post hoc Dunn’s test for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. ctrl.

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  Figure 4. Cellular localization of TRPC1, TRPC5, and STIM1 in ARVMs. Representative immunofluorescences images (top) and quantification of TRPC1, TRPC5, and STIM1 in ARVMs incubated for 24 h in the absence or in the presence of aldosterone 1 µM. Results are expressed as relative fluorescence/cellular area (RFA). Average results of 20–39 cells from three isolations. Results are presented as scatter plots with mean ± SEM. Statistical significance was evaluated using Student’s t-test. * p < 0.05, ** p < 0.01 vs. ctrl.

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  Figure 5. A 24-h aldosterone treatment increases TRPC1 and TRPC5 messenger RNA (mRNA) expression in ARVMs. Relative TRPC1, TRPC5, and STIM1 mRNA levels in ARVMs were incubated for 24 h in the absence or in the presence of aldosterone (Aldo 10 nM, 100 nM or 1 µM). The mRNA levels determined by qRT-PCR were normalized to housekeeping genes and are expressed as fold changes of that determined in ARVMs incubated in the absence of aldosterone for each cell isolation. Results are presented as scatter plots with median and interquartile range. Statistical significance was evaluated using one-way ANOVA (Kruskal–Wallis) followed by post hoc Dunn’s test for multiple comparisons. * p < 0.05, ** p < 0.01 vs. ctrl.