Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Aug;121:51-59.
doi: 10.1016/j.yjmcc.2018.06.002. Epub 2018 Jun 7.

Compartmentalized Cyclic Nucleotides Have Opposing Effects on Regulation of Hypertrophic Phospholipase Cε Signaling in Cardiac Myocytes

Affiliations
Free PMC article

Compartmentalized Cyclic Nucleotides Have Opposing Effects on Regulation of Hypertrophic Phospholipase Cε Signaling in Cardiac Myocytes

Craig A Nash et al. J Mol Cell Cardiol. .
Free PMC article

Abstract

In cardiac myocytes activation of an exchange factor activated by cAMP (Epac) leads to activation of phospholipase Cε (PLCε)-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) in the Golgi apparatus a process critical for development of cardiac hypertrophy. Here we show that β-adrenergic receptor (βAR) stimulation does not stimulate this pathway in the presence of the broad spectrum phosphodiesterase (PDE) inhibitor IBMX, but selective PDE3 inhibition revealed βAR-dependent PI4P depletion. On the other hand, selective inhibition of PDE2 or PDE9A blocked endothelin-1 (ET-1) and cAMP-dependent PI4P hydrolysis by PLCε. Direct activation of protein kinase A (PKA), protein kinase G (PKG), or the atrial natriuretic factor (ANF) receptor abolished PI4P hydrolysis in response to multiple upstream stimuli. These results reveal distinct pools of cyclic nucleotides that either inhibit PLCε at the Golgi through PKA/PKG, or activate PLCε at the Golgi through Epac. These data together reveal a new mechanism by which ANF and selective PDE inhibitors can protect against cardiac hypertrophy.

Keywords: A kinase anchoring protein; Cardiac hypertrophy; Cyclic AMP; Epac; Golgi apparatus; Phosphatidylinositol 4-phosphate; Phosphodiesterase; Phospholipase C.

Figures

Figure 1
Figure 1. cpTOME and Forskolin stimulate PI4P hydrolysis in NRVMS, but Isoproterenol does not
NRVMs were transduced with GFP-FAPP-PH adenovirus overnight before imaging in 1% FBS containing media. A) Representative images of Fsk (10μM)- and cpTOME (10μM) - induced PI4P hydrolysis. Bar-10μm B) Average data cpTOME (10μM), C) Average data Iso (1 μM), D) Average data Fsk (10μM), induced PI4P hydrolysis. Images taken from n=6 cells each from 6 separate preparations of NRVMs. All time course graphs are presented as mean ± standard error. Agonist treatments were compared to vehicle control performed on the same day and were added where indicated by the arrow. All data was analyzed by two way unpaired ANOVA with Sidak’s post-hoc test. ** p<0.001 *** p<0.0001 **** p<0.00001
Figure 2
Figure 2. Rolipram and Cilostamide stimulate PI4P hydrolysis in NRVMs, with Isoproterenol stimulation increasing the rate of Cilostamide-induced PI4P hydrolysis
NRVMs were transduced with GFP-FAPP-PH adenovirus overnight before imaging in 1% FBS containing media. A) Time course of IBMX (300μM) or Iso (1 μM) plus IBMX induced PI4P hydrolysis. Arrow indicates time of stimulant addition. B) cAMP accumulation by PDE inhibitors. NRVMs were grown for 48hrs before being serum starved overnight and stimulated with either Iso (10 μM), IBMX (300 μM) or Iso (1 μM) plus IBMX for 15 min. Cells were then lysed and ELISA for cAMP performed according to manufacturer’s instructions. Data are representative of experiments performed three times. C) Time course PDE4 inhibitor Rolipram (10μM) or PDE3 inhibitor Cilostamide (10μM) induced PI4P hydrolysis. Phosphodiesterase inhibitors were added as indicated by arrow. D) NRVMs were grown for 48hrs before being serum starved overnight and stimulated with either Cilostamide (10μM), Rolipram (10μM) or IBMX (300μM) for 15 min and assayed for cAMP accumulation as in B. E) Time course Rolipram (10μM) or Cilostamide (10μM) induced PI4P hydrolysis in the presence of Isoproterenol (1μM). Isoproterenol and phosphodiesterase inhibitors were added simultaneously as indicated by arrow. F) NRVMs were grown for 48hrs before being serum starved overnight and stimulated with either Cilostamide (10μM), Rolipram (10μM) or IBMX (300μM) for 15 min in combination with Isoproterenol (1μM) and assayed for cAMP accumulation as in B. G) Epac inhibition blocks PDE effects. The Epac inhibitor HJC0726 (1μM) was added for 15 min before imaging. Forskolin (10 μM), Rolipram (10μM) or Cilostamide (10μM) were added at the arrows and PI4P hydrolysis was measured. For all experiments Data taken from at least 3 cells from 3 separate preparations of NRVMs. All graphs are presented as mean ± standard error. All data was analyzed by two way unpaired ANOVA with Sidak’s post-hoc test. *p<0.05 ** p<0.001 *** p<0.0001 **** p<0.00001
Figure 3
Figure 3. Removal of PI4P or inhibiting PLCε inhibits NRVM hypertrophy in response to Iso, PDE3 or PDE4 inhibition
A) NRVMs were infected with adenoviruses expressing either random control shRNA (Ran shRNA) or PLCε shRNA prior to stimulation with the indicated drugs for 48h and cell area was measured. B) Experiments were performed as in A, except before fixation cells were stained with O-propargyl-puromycin for 2hrs. NRVMs from at least 3 separate preparations were fixed following stimulation with indicated compounds, as described in methods. Data was analyzed using ImageJ software and size of myocytes presented in pixels2. All graphs are presented as mean ± standard error. All data was analyzed by two way unpaired ANOVA with Sidak’s post-hoc test. *p<0.05 ** p<0.001 *** p<0.0001 **** p<0.00001. For the random siRNA transduced cells, Isoproterenol, cilostamide and Rolipram all significantly increased hypertrophy relative to the vehicle treated control for both cell area (P<0.01–0.005) and protein synthesis measurements (all P<0.001).
Figure 4
Figure 4. PKA or PKG activation inhibits PI4P hydrolysis by cAMP and Gβγ-dependent pathways
NRVMs were transduced with GFP-FAPP-PH adenovirus overnight before imaging. A) Time course of IBMX (300μM added at the arrow) induced PI4P hydrolysis in the presence of the PKA inhibitor myrPKI (1 μM) or B) PKG inhibitor, rp-cGMPS (10μM). C) Time course of cpTOME (10 μM) or D) ET-1 mediated PI4P hydrolysis in the presence of either vehicle DMSO or the PKA activator sp-cAMPs. E) Time course of cpTOME (10 μM) or F) ET-1 mediated PI4P hydrolysis in the presence of either vehicle DMSO or cGMP analogue, 8-Br-cGMP. PKI, rp-cGMPs, sp-cAMPs, or 8-Br-cGMP were added 15 mins before imaging. Data are from 4 cells from 4 separate preparations of NRVMs. All time course graphs are presented as mean ± standard error. All data was analyzed by two way unpaired ANOVA with Sidak’s post-hoc test. *p<0.05 ** p<0.001 *** p<0.0001 **** p<0.00001. Data is significantly different across complete time course or at each individual time point, as indicated.
Figure 5
Figure 5. BAY60-7550, PF04449613 and atrial natriuretic peptide (ANF) inhibit the ability of ET-1 and Forskolin to stimulate PI4P hydrolysis
A) Time course of BAY60-7550 (10 μM) or vehicle treated NRVMs after stimulation with either ET-1 (100 nM,) or B) Forskolin (10 μM) and measurement of PI4P hydrolysis. Images captured from at least n=4 cells from at least 4 separate preparations of NRVMs. C) Time course of PF-04449613 (10μM) or vehicle treated NRVMs after stimulation with either ET-1 (100nM, left) or D) Forskolin (10 μM, right) and measurement of PI4P hydrolysis. Images captured from at least n=3 cells from at least 3 separate preparations of NRVMs. E) Time course of either ET-1 (100 nM, left) or F) Forskolin (10 μM, right)–stimulated PI4P hydrolysis after incubation either ANF (10 nM) or vehicle control. Images and time courses are from at least 5 cells from 5 separate preparations of cells. NRVMs were transduced with GFP-FAPP-PH adenovirus overnight before imaging. BAY60-7550, PF04449613 and ANF were added 15 mins before samples were transferred to microscopy stage for imaging. All time course graphs are presented as mean ± standard error. All data was analyzed by two way unpaired ANOVA with Sidak’s post-hoc test. *p<0.05 ** p<0.001 *** p<0.0001 **** p<0.00001.
Figure 6
Figure 6. PKA can inhibit PLCε in COS-7 cells downstream of all activators, however, PKG only inhibits Rho-dependent activation
COS-7 cells were Cotransfected with PLCε and Gβγ (A and E), Rho (B and F), H-Ras G12V (C and G), Rap1 (D and H), and PKA catalytic subunit (A,B,C and D) or constitutively active PKG (E,F,G and H). Total IP production was measured 48 hours after transfection as indicated in Materials and Methods. Data was generated from at least 3 experiments analyzed by two way unpaired ANOVA with Sidak’s post-hoc test. *** p<0.0001 **** p<0.00001.
Figure 7
Figure 7. Model for control of PI4P hydrolysis by distinct inputs to PLCε signaling
In this model, β-adrenergic receptor stimulation of adenylyl cyclase (AC) generates pools of cAMP that are differentially regulated by PDE2 and PDE3. PDE3 limits the ability of cAMP to activate Epac-PLCε, while PDE2 limits cAMP dependent inhibition of PLCε through PKA. Similarly, cGMP generated by activation of guanylyl cyclase (GC) inhibits PLCε through PKG activation, but this pathway may be indirect, as indicated by the dashed lines. PDE9 and possible PDE2 limit the ability of cGMP to act on this inhibitory PKG-dependent pathway.

Similar articles

See all similar articles

Cited by 4 articles

Publication types

MeSH terms

Feedback