G protein βγ subunits regulate cardiomyocyte hypertrophy through a perinuclear Golgi phosphatidylinositol 4-phosphate hydrolysis pathway

Abstract

We recently identified a novel GPCR-dependent pathway for regulation of cardiac hypertrophy that depends on Golgi phosphatidylinositol 4-phosphate (PI4P) hydrolysis by a specific isoform of phospholipase C (PLC), PLCε, at the nuclear envelope. How stimuli are transmitted from cell surface GPCRs to activation of perinuclear PLCε is not clear. Here we tested the role of G protein βγ subunits. Gβγ inhibition blocked ET-1-stimulated Golgi PI4P depletion in neonatal and adult ventricular myocytes. Blocking Gβγ at the Golgi inhibited ET-1-dependent PI4P depletion and nuclear PKD activation. Translocation of Gβγ to the Golgi stimulated perinuclear Golgi PI4P depletion and nuclear PKD activation. Finally, blocking Gβγ at the Golgi or PM blocked ET-1-dependent cardiomyocyte hypertrophy. These data indicate that Gβγ regulation of the perinuclear Golgi PI4P pathway and a separate pathway at the PM is required for ET-1-stimulated hypertrophy, and the efficacy of Gβγ inhibition in preventing heart failure maybe due in part to its blocking both these pathways.

FIGURE 1:

PI4P-associated GFP-FAPP-PH fluorescence colocalizes with a Golgi marker and is closely associated with nuclear envelope–localized mAKAP. (A) NRVMs were transduced with adenovirus expressing GFP-FAPP-PH. Cells were fixed and stained for Golgi-specific 58K protein (Alexa Fluor 546 secondary antibody) and imaged for GFP and red fluorescence. (B) Same as A, except that cells were stained for mAKAP instead of Golgi and treated with and without 10 μM brefeldin A for 20 min as indicated. (C) Cells were treated for the indicated times with brefeldin A, fixed, and costained for mAKAP and Golgi-specific 58K protein. Scale bars, 10 μm.

FIGURE 2:

Gβγ inhibitors block ET-1–stimulated perinuclear PI4P depletion in NRVMs. (A) NRVMs transfected with GFP-FAPP-PH were pretreated with 10 μM gallein or PBS vehicle control, followed by stimulation with 100 nM ET-1. Perinuclear FAPP fluorescence intensity was measured over time by confocal microscopy. (B) NRVMs were treated as in A, except that 10 μM cpTOME was added to stimulate PI4P hydrolysis. (C) NRVMs were cotransfected with GFP-FAPP-PH and GRK2ct or vehicle control, followed by ET-1 stimulation as in A. (D) NRVMs were transfected as in A were pretreated for 18 h with 100 ng/ml pertussis toxin. All traces represent pooled data from at least three cells from three separate myocyte preparations ± SEM. The last five values from each time course were averaged and tested for significance using Student's t test. ***p < 0.005; ns, not significant.

FIGURE 3:

Gβγ signaling at the Golgi apparatus stimulates perinuclear PI4P depletion in NRVMs and nuclear PKD activation. (A) NRVMs were transfected with Golgi-GRK2ct. GRK2ct localization was analyzed by immunocytochemistry with a GRK2 antibody with an Alexa Fluor 546–conjugated secondary antibody and imaged in the red channel. The Golgi-specific antibody (anti-Golgi 58K protein) was used to identify the Golgi apparatus, with Alexa Fluor 488 secondary antibody imaged in the green channel. (B) Cells cotransfected with PM-GRK2ct, and myristoylated YFP to mark the PM, were analyzed for YFP fluorescence and GRK2ct localization as in A. (C) Cells were cotransfected with GFP-FAPP-PH and either LacZ or PM-GRK2ct constructs. Perinuclear GFP-FAPP-PH fluorescence was monitored with time after ET-1 addition. (D) Same as C, except that Golgi-targeted GRK2ct was used. (E) Quantitation of data in C and D. Curves in C and D were fitted with single-exponential decay curves using GraphPad Prism 6 and analyzed for statistical significance. *p < 0.05 and ****p < 0.001 relative to ET-1 control. (F) NRVMs were cotransfected with nDKAR and LacZ, PM-GRK2ct, or Golgi-GRK2ct, and the nDKAR YFP/CFP ratio in the nucleus was monitored over time after addition of ET-1. (G) Changes in YFP/CFP ratio, normalized to time 0, ± SEM for each trace in F were pooled from 35 to 40 min, averaged, and analyzed by a one-way analysis of variance (ANOVA). (H) Curves from F were fitted and analyzed as in E. All traces (C, D, F, G) are pooled data from at least three cells from three separate myocyte preparations ± SEM. Scale bars, 10 μm.

FIGURE 4:

Targeting Gβγ to the Golgi apparatus stimulates PI4P hydrolysis and nuclear PKD activation. (A) Cells were transfected with mCherry-Gβ₁, Gγ₂FRB, and CFP-Golgi-FKBP. mCherry fluorescence (top two) and CFP fluorescence (bottom two) before and 10 min after rapamycin addition. (B) NRVMs were transfected with GFP-FAPP-PH, Gβ₁, Gγ₂FRB, and either CFP-Golgi-FKBP or PM-FKBP. At time 0, 24 h after transfection, 10 μM rapamycin was added, and perinuclear PI4P fluorescence was monitored with time. (C) Cells were transfected with Gβ₁, Gγ₂FRB, and nDKAR and either PM-FKBP or Golgi FKBP. At time 0, 24 h after transfection, 10 μM rapamycin was added, and nDKAR YFP/CFP fluorescence in the nucleus was monitored. All traces represent pooled data from at least three cells from three separate myocyte preparations ± SEM. Scale bars, 10 μm.

FIGURE 5:

Blocking Gβγ signaling in the Golgi or PM prevents ET-1–stimulated hypertrophic gene expression in NRVMs. (A) NRVMs were cotransfected with YFP and GRK2ct, followed by treatment with 100 nM ET-1 or vehicle. After 24 h, cells were fixed and stained for ANF (red). With ET-1 treatment, the intensity of red staining increases in untransfected cells but not in cells expressing YFP and GRK2ct. Scale bar, 40 μm. (B) Cells were transfected with YFP alone or YFP plus GRK2ct, Golgi GRK2ct, or PM-GRK2ct. Cells were treated as in A. Scale bar, 20 μm. (C) The intensity of ANF staining was quantitated with and without ET-1 only in transfected cells identified by YFP fluorescence. Quantitation is based on combined data (mean ±SEM) from four separate experiments. ****p < 0.0001, statistically different from YFP control. None of the other samples is statistically different from YFP control (one-way ANOVA).

FIGURE 6:

Blocking Gβγ signaling in the Golgi or PM prevents ET-1–stimulated cardiomyocyte hypertrophic cell growth. (A) NRVMs were infected with adenoviruses expressing YFP or YFP and the indicated targeted GRK2ct constructs. Cells were treated with 100 nM ET-1 or vehicle. After 48 h, cells were visualized in the YFP channel. Scale bar, 40 μm. (B) Cells treated as in A quantitated for cell area using ImageJ (National Institutes of Health, Bethesda, MD). Quantitation is based on combined data (mean ±SEM) from three separate experiments; 50 cells each experiment. Data were analyzed by one-way ANOVA; ***p < 0.005.

FIGURE 7:

Blocking Gβγ signaling in the Golgi or PM prevents ET-1–stimulated ANF expression in AVMs. (A) AVMs were infected with adenoviruses expressing either Tubby-GFP (top) or GFP-FAPP-PH (bottom). (B) AVMs were infected with GFP-FAPP-PH and stimulated with 100 nM ET-1, and perinuclear GFP-FAPP-PH was monitored over time as indicated. Each trace represents pooled data from at least five cells from five separate myocyte preparations ± SEM. (C) AVMs were infected with viruses (50 MOI) expressing YFP, Golgi-GRK2ct, or PM-GRK2ct (only cells expressing YFP or Golgi-GRK2ct are shown). Viruses expressing Golgi-GRK2ct and PM-GRK2ct also expressed YFP from a separate cassette. Cells were stimulated for 24 h with 100 nM ET-1 and fixed and stained for ANF expression. (D) ANF expression was quantitated as mean ± SEM for all treatments as described in Materials and Methods. ****p < 0.0001, statistically different from YFP control. None of the other samples is statistically different from YFP control (one-way ANOVA). Scale bars, 10 μm.