Research ArticleCell Biology

The β4 subunit of Cav1.2 channels is required for an optimal interferon response in cardiac muscle cells

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Science Signaling  11 Dec 2018:
Vol. 11, Issue 560, eaaj1676
DOI: 10.1126/scisignal.aaj1676
  • Fig. 1 Analysis of the localization of the β4 subunit in H9c2 cells.

    (A) H9c2 cells transfected with empty plasmid or with plasmid encoding the β4 subunit were processed to generate cytosolic and nuclear fractions that were analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of three experiments. The anti-β4 antibody was obtained from Abcam. (B) H9c2 cells transfected with plasmid encoding the β4 subunit were lysed, and the indicated amounts of cell lysate were analyzed by Western blotting with antibodies against β4 (StressMark) and actin, which served as a loading control. Blots are representative of three independent experiments. (C) H9c2 cells that were left untransfected (top) or were transfected with plasmid expressing the β4 subunit (bottom) were analyzed by confocal microscopy to determine the cellular localization of β4. The β4 subunit was detected with the monoclonal antibody from Abcam (1:100 dilution, top; 1:200 dilution, bottom) and Alexa Fluor 488–conjugated secondary antibody (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) counterstained (blue). Images are representative of three independent experiments.

  • Fig. 2 Effects of β4 on antiviral factors in H9c2 cells.

    (A) H9c2 cells transfected with empty plasmid (open bars) or with plasmid expressing the β4 subunit (filled bars) were analyzed by qRT-PCR to determine the relative abundances of mRNAs for the indicated antiviral factors and of Cacnb4 mRNA. Data are means ± SEM of 10 to 14 experiments, and values are shown relative to those in control cells, which were set at 1. (B) Whole-cell lysates of H9c2 cells transfected with empty plasmid (left lanes) or with plasmid expressing the β4 subunit were analyzed by Western blotting with antibodies against the indicated proteins. Actin was used as a loading control. Blots are representative of three to five experiments. (C) Analysis of the relative abundances of DDX58, IRF7, IFITM3, and STAT2 proteins, normalized to that of actin, in H9c2 cells transfected with empty plasmid (empty bars) or with plasmid expressing the β4 subunit (filled bars) from the experiments shown in (B). Data are means ± SEM of three to seven experiments. *P < 0.05, **P < 0.01 compared to control.

  • Fig. 3 Effect of IFN-β and DENV2 infection on β4 abundance and its nuclear localization in H9c2 cells.

    (A) H9c2 cells were left untreated (top) or were treated with IFN-β (500 U/ml) for 48 hours (bottom) before being analyzed by confocal microscopy. Images show the localization of the β4 subunit (green) and DAPI-stained nuclei (blue). Scale bar, 20 μm. Images are representative of three independent experiments. (B) H9c2 cells were left untreated or were treated with IFN-β (500 U/ml) for 48 hours before whole-cell lysates were analyzed by Western blotting with antibodies specific for the β4 subunit or actin, as indicated (inset). The bar graph shows the relative abundance of the β4 subunit normalized to that of actin in the indicated cells. Data are means ± SEM of three experiments. *P < 0.05 compared to control, untreated cells. (C) H9c2 cells were left uninfected (0) or were infected with DENV2 at an MOI of 5 or 10, as indicated, for 48 hours. The cells were then separated into nuclear (left) and cytosolic (right) fractions, which were analyzed by Western blotting with antibodies against the β4 subunit or actin (insets). Bar graphs show the relative abundance of the β4 subunit normalized to that of actin in the indicated cells. Data are means ± SEM of three experiments. *P < 0.05 compared to control, uninfected cells. (D) H9c2 cells that were uninfected or infected with DENV2 at an MOI of 5 were analyzed by qRT-PCR to determine the relative abundance of Cacnb4 mRNA. Data are means ± SEM of three experiments. *P < 0.05 compared to control, uninfected cells. (E) H9c2 cells were left uninfected (top) or were infected with DENV2 at an MOI of 5 (middle) or 10 (bottom) for 48 hours. The cells were then analyzed by confocal microscopy to visualize the localization of the β4 subunit (green) and the NS3 viral protein (red), with DAPI-labeled nuclei (blue). Images are representative of three experiments. Scale bars, 30 μm. Mean fluorescence intensity data and analysis of β4 subunit localization in the cells labeled 1 to 4 are presented in table S2.

  • Fig. 4 Knockdown of the β4 subunit results in the decreased abundance of IRF7, STAT2, IFITM3, and IFN-β mRNA and protein.

    (A and B) H9c2 cells transfected with empty plasmid (open bars) or with plasmid encoding β4-specific small interfering RNA (siRNA; filled bars) were subjected to qRT-PCR analysis of the relative abundance of Cacnb4 mRNA (A) and to Western blotting analysis of the relative abundance of β4 subunit protein (B). Data are means ± SEM of three to five experiments. **P < 0.01 compared to control cells. (C) H9c2 cells transfected with empty plasmid (open bars) or with plasmid encoding β4-specific siRNA (filled bars) were subjected to qRT-PCR analysis of the relative abundances of the indicated mRNAs. Data are means ± SEM of three to five experiments. **P < 0.01 compared to control cells. (D to F) Top: H9c2 cells were transfected with empty plasmid (left lanes) or with plasmid encoding β4-specific siRNA (right lanes). Thereafter, the cells were treated with IFN-β (D), infected with DENV2 (E), or treated with poly(I:C) (F). Whole-cell lysates were then analyzed by Western blotting with antibodies against IRF7, STAT2, or IFITM3. Blots are representative of three to five experiments. Bottom: The relative abundances of the appropriate bands were determined by densitometry. Data are means ± SEM of three to five experiments. *P < 0.05, **P < 0.01.

  • Fig. 5 β4 and IRF7 interactions.

    (A) H9c2 cells transfected with empty plasmid (−) or with plasmid expressing the β4 subunit (+) were lysed, separated into nuclear and cytosolic fractions, and then subjected to immunoprecipitation (IP) with antibody against IRF7 and analyzed by Western blotting (IB) with antibody against the β4 subunit. (B) H9c2 cells transfected with empty plasmid (−) or with plasmid expressing the β4 subunit (+) were lysed, separated into nuclear and cytosolic fractions, and then subjected to immunoprecipitation with antibody against the β4 subunit and analyzed by Western blotting with antibody against IRF7. (C) H9c2 cells transfected with empty plasmid (−) or with plasmid expressing the β4 subunit (+) were lysed, separated into nuclear and cytosolic fractions, and then subjected to immunoprecipitation with antibody against the β4 subunit and analyzed by Western blotting with antibody against β4. Western blots in (A) to (C) are representative of four experiments. (D) H9c2 cells were transfected with the pGL2 vector expressing the Irf7 promoter and the pSG5 plasmid alone (empty bar) or in combination with plasmid expressing the β4 subunit (filled bar). The cells were then subjected to a luciferase reporter assay as described in Materials and Methods. Data are means ± SEM of eight experiments. *P < 0.05.

  • Fig. 6 β4 and β4-regulated antiviral factors abundance is mediated by JAK1.

    (A to D) H9c2 cells transfected with empty plasmid or with plasmid expressing the β4 subunit were left untreated or were treated with 1 μM filgotinib (GLPG0634) for 48 hours, as indicated. The cells were then lysed and subjected to Western blotting analysis with antibodies against IFITM3 (A) or STAT2 (C). Blots are representative of four to six experiments. The relative abundances of IFITM3 (B) and STAT2 (D) in the indicated samples were determined by densitometry. Data are means ± SEM of five to nine experiments. *P < 0.05, **P < 0.01. (E to G) H9c2 cells were left untreated or were treated with 1 μM filgotinib (GLPG0634) for 24 hours, as indicated. (E) The cells were then lysed and subjected to Western blotting analysis with antibodies against β4. Actin was used as a loading control. Blots are representative of six experiments. (F) The relative abundances of β4 in the indicated samples represented in (E) were determined by densitometry. Data are means ± SEM of five to nine experiments. *P < 0.05. (G) The same cells described in (E) were analyzed by qRT-PCR to determine the relative abundance of Cacnb4 mRNA. Data are means ± SEM of six experiments. **P < 0.01.

  • Fig. 7 β4 reduces DENV infection.

    (A) H9c2 cells transfected with empty plasmid (control) or plasmid expressing the β4 subunit were subjected to infection with the indicated viruses for 48 hours before the amount of NS1 secreted by the cells was determined by ELISA. (B) H9c2 cells transfected with empty plasmid (control) or plasmid expressing the β4 subunit were subjected to infection with DENV4 for 48 hours before the relative abundance of the viral genome was determined by qRT-PCR. (C and D) H9c2 cells transfected with empty plasmid (control) or plasmid expressing the β4 subunit were subjected to infection with DENV2 for 48 hours before the relative abundance of the NS3 protein (C) and viral E-protein (D) was determined by Western blotting (insets). Actin was used as a loading control. Bar graphs show the relative abundance of the indicated proteins as determined by densitometry. (E) Top: Scheme outlining the infection of Vero cells with culture medium from infected H9c2 cells. Bottom: Analysis of the number of focus-forming units of Vero cells 24 hours after the addition of culture medium from control or β4-expressing H9c2 cells. Data are means ± SEM of three to six experiments. *P < 0.05, **P < 0.01. (F and G) H9c2 cells transfected with empty vector plasmid (control) or plasmid expressing the β4 subunit were infected 24 hours later with DENV2 or DENV4 (F and G) at an MOI of 5. (F) Infected cells were examined by microscopy at the indicated magnifications. Images are representative of three experiments. (G) The numbers of DENV2-infected and DENV4-infected cells were then counted. Data are means ± SEM of three experiments. **P < 0.01.

  • Fig. 8 β4 reduces infection in differentiated H9c2 cells.

    (A and B) Differentiated H9c2 cells were transfected with empty plasmid (left lanes and bars) or plasmid expressing β4-specific siRNA (right lanes and bars) and then were treated with 500 U/ml IFN-β for 42 hours. (A) Whole-cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. (B) The relative abundances of the indicated proteins from the experiments represented in (A) were determined by densitometry. Data are means ± SEM of three or four experiments. *P < 0.05, **P < 0.01. (C and D) Differentiated H9c2 cells were transfected with empty plasmid (left lanes and bars) or plasmid expressing β4 (right lanes and bars). (C) Whole-cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. (D) The relative abundances of the indicated proteins from the experiments represented in (C) were determined by densitometry. Data are means ± SEM of three or four experiments. *P < 0.05, **P < 0.01. (E and F) Differentiated H9c2 cells transfected with empty plasmid (left lanes and empty bar) or plasmid expressing β4 (right lanes and filled bar) were infected with DENV2 at an MOI of 5. (E) Whole-cell lysates were then analyzed by Western blotting to detect viral E-protein. Actin was used as a loading control. (F) The relative amounts of E-protein in the cells represented in (E) were determined by densitometry. Data are means ± SEM of four experiments. **P < 0.01. (G) Differentiated H9c2 cells transfected with empty plasmid (left) or plasmid expressing β4 (right) were infected with DENV2 at an MOI of 5 for 24 hours, and the relative amounts of NS1 secreted by the cells were determined by ELISA. Data are means ± SEM of four experiments. **P < 0.01. (H) Mean values of three experiments (± SEM) of focus-forming units in Vero cells were incubated with cell culture taken from DENV2-infected, differentiated H9c2 cells transfected with empty vector (empty bar) or with plasmid expressing the β4 subunit. The number of focus-forming units were then determined as described in Fig. 7E. **P < 0.01.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/560/eaaj1676/DC1

    Fig. S1. Linearity range of Western blots.

    Fig. S2. IFN-β increases the abundance of antiviral factors in H9c2 cells.

    Fig. S3. The increase in β4 abundance and its nuclear localization in response to poly(I:C).

    Fig. S4. Differentiation of H9c2 cells toward a cardiac phenotype.

    Fig. S5. STAT2 expression and localization in β4-silenced differentiated and undifferentiated H9c2 cells.

    Fig. S6. Effects of DENV2 infection and poly(I:C) transfection on cytosolic Ca2+.

    Table S1. Effects of transfection with empty plasmid or β4-encoding plasmid on the expression of viral infection response–related genes.

    Table S2. Summary of effects of DENV or mock infection on quantitated β4 localization.

  • This PDF file includes:

    • Fig. S1. Linearity range of Western blots.
    • Fig. S2. IFN-β increases the abundance of antiviral factors in H9c2 cells.
    • Fig. S3. The increase in β4 abundance and its nuclear localization in response to poly(I:C).
    • Fig. S4. Differentiation of H9c2 cells toward a cardiac phenotype.
    • Fig. S5. STAT2 expression and localization in β4-silenced differentiated and undifferentiated H9c2 cells.
    • Fig. S6. Effects of DENV2 infection and poly(I:C) transfection on cytosolic Ca2+.
    • Table S1. Effects of transfection with empty plasmid or β4-encoding plasmid on the expression of viral infection response–related genes.
    • Table S2. Summary of effects of DENV or mock infection on quantitated β4 localization.

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