The β2-Subunit of Voltage-Gated Calcium Channels Regulates Cardiomyocyte Hypertrophy

Figures

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  Figure 1. Cavβ2 downregulation enhances PE-induced hypertrophy of NRCs. (A) RT-PCR analysis of the expression of the four Cavβ isoforms in NRCs and adult brain. The lower panel shows the expression of the different Cavβ2 splice variants in NRCs. (B) Scheme of the domain organization of Cavβ2, showing the regions where the designed shRNAs bind: NT (N-terminal), SH3 (Src-3 homology domain), GK (guanylate kinase domain), CT (C-terminal). (C) Representative western blot analysis of Cavβ2 expression in homogenates from NRCs transduced with the different shRNAs for Cavβ2-silencing and the scrambled shRNA (shRNAsc) used as negative control. Cells were transduced with each shRNA at an adenoviral MOI of 5 or 20. Per lane, 50 μg of protein were loaded. An anti-GAPDH western blot was used as loading control. (D) Bar plot of Cavβ2 expression normalized vs. GAPDH in homogenates from NRCs transduced with the different shRNAs for Cavβ2-silencing and the scrambled shRNA (shRNAsc.) at an adenoviral MOI of 5 or 20. Mean ± SEM from 3 replicated experiments; ##p < 0.05 vs. shRNAsc treated at the same MOI (two-way ANOVA with Holm–Sidak's method). (E) Representative fluorescent images of control and…

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  Figure 2. Analyses of calcium transients in control and Cavβ2-downregulated NRCs after vehicle or PE treatments. (A) Representative calcium transients of control (shRNAsc) and Cavβ2-downregulated NRCs (shRNA892) treated with vehicle or 50 μM phenylephrine (PE) and stimulated at 1.0 Hz. (B–E) Bar plots of the mean values of the effect of vehicle or PE treatments on the diastolic calcium, amplitude, peak time, and decay time constant of the calcium transients in control or Cavβ2-downregulated NRCs. Mean ± SEM; n = 11–14 cells per group from 3 different NRC isolations; §§p < 0.05 vs. vehicle-treated cells in the same group (two-way ANOVA with Holm–Sidak's method).

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  Figure 3. A fraction of Cavβ2 locates in the nucleus in neonatal and adult cardiomyocytes. (A) Confocal fluorescence images of representative NRCs showing a fraction of Cavβ2 located in the nucleus (indicated with white arrows). Cells were fixed and stained for Cavβ2 (red), α-actinin (green) and nucleus (DAPI, blue). Scale bar represents 15 μm. (B) Bar plot of the Manders coefficient colocalization analyses between Cavβ2 and DAPI nuclear staining in NRCs after treatment with vehicle or 50 μM phenylephrine (PE). Mean ± SEM; 150–200 cells from 20 randomly chosen fields and from 3 replicated experiments were measured; §§p < 0.01 (two-tailed unpaired t-test). (C) Confocal fluorescence images of a representative adult mouse cardiomyocyte showing a fraction of Cavβ2 located in the nucleus (indicated with white arrows). Cells were stained for Cavβ2 (green), ryanodine receptor 2 (RyR2, red) and nucleus (DAPI, blue). Scale bar represents 15 μm. (D) Western blots of subcellular fractions from adult mouse cardiomyocytes. Cytosolic fraction (Cyt.), membrane fraction (Mem.), soluble nuclear fraction (So.NF.) and chromatin bound nuclear fraction (Chr.NF.). Per lane, 50 μg of total proteins were loaded.…

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  Figure 4. In vivo induction of left ventriclulare hypertrophy produces a decrease in nucleus-targeted Cavβ2. Western blot analyses (left panels) and bar plots of mean values of densitometry analyses from western blots (right panels) of: (A) left ventricle (LV) nuclear fractions, (B) LV membrane fractions and (C) right ventricle (RV) nuclear fractions. Per well, 50 μg of total proteins from sham-operated (N = 3) or TAC-operated (N = 3) mice were loaded and detected using an anti-Cavβ2 antibody. Immunoblots to confirm the presence of the compartment-specific protein markers histone H3 (nucleus), Na+/K+ ATPase (membrane) and GAPDH (cytosol) were also performed. Cavβ2 expression was normalized to histone-H3 (in A and C) or to Na+/K+ ATPase expression (in B). §§p < 0.05 vs. sham (two-tailed unpaired t-test).

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  Figure 5. Nuclear overexpression of Cavβ2 abolishes in vitro PE-induced cardiomyocyte hypertrophy. (A) Schematic representations of the three evaluated constructs. To achieve a complete Cavβ2 nuclear-targeting, the nuclear localization signal (NLS) of the SV40 large T-antigen (PPKKKRKV) was inserted at the N-terminus of the Cavβ2b-YFP construct (NLS-Cavβ2b-YFP). YFP and a construct with a mutated NLS (NLSK4T-Cavβ2b-YFP, red star) were used as negative controls. (B) Fluorescent images of NRCs expressing the controls (YFP or NLSK4T-Cavβ2b-YFP) or nuclear overexpressed Cavβ2b (NLS-Cavβ2b-YFP) and treated with vehicle or 50 μM phenylephrine (PE). Cells were stained for α-actinin (red) and nucleus (DAPI, blue), while the YFP fluorescence was directly monitored. Scale bar represents 25 μm. (C) Bar plot of the cell area analyses of NRCs expressing YFP, NLS-Cavβ2b-YFP or NLSK4T-Cavβ2b-YFP after the treatment with vehicle or PE. Mean ± SEM; 150–200 cells per group from 20 randomly chosen fields from 3 replicated experiments were measured; §§p < 0.01 vs. vehicle-treated cells in the same group; ##p < 0.05 vs. YFP expressing cells treated similarly (two-way ANOVA with Holm–Sidak's method).

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  Figure 6. Quantitative proteomic analysis of control and Cavβ2-downregulated NRCs. Subset of upregulated (red) or downregulated (green) proteins identified by quantitative mass-spectrometry-based analyses after Cavβ2 downregulation in NRCs. A complete list of proteins is provided in Supplementary Table 1.

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  Figure 7. Inhibition of calpain activity abolishes the enhanced PE-induced cardiomyocyte hypertrophy in Cavβ2-downregulated NRCs. (A) Representative western blot analysis of calpastatin expression in homogenates from control and Cavβ2 downregulated NRCs. Per well, 50 μg of protein were loaded. Anti-GAPDH western blot was used as loading control. (B) Bar plot of calpastatin expression normalized vs. GAPDH in homogenates from control and Cavβ2 downregulated NRCs. Mean ± SEM of 3 different experiments; ##p < 0.05 (two-tailed unpaired t-test). (C) Bar plot of the qRT-PCR analyses of calpastatin expression in control and Cavβ2 downregulated NRCs; ##p < 0.05 (two-tailed unpaired t-test). (D) Bar plot of calpain activity analyses of control and Cavβ2-downregulated NRCs after the treatment with vehicle or 50 μM phenylephrine (PE). Mean ± SEM of 6 different experiments; §§p < 0.05 vs. vehicle-treated cells in the same group; ##p < 0.01 vs. control cells treated similarly (two-way ANOVA with Holm–Sidak's method). (E) Bar plot of the mean values of the cell area of the control and Cavβ2-downregulated NRCs after vehicle and 25 μM of calpeptin or PE and calpeptin treatments. Mean ± SEM; 150–200 cells…