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. 2019 Jun 7;14(6):e0218110.
doi: 10.1371/journal.pone.0218110. eCollection 2019.

Constitutive inhibitory G protein activity upon adenylyl cyclase-dependent cardiac contractility is limited to adenylyl cyclase type 6

Caroline Bull Melsom  1 , Marie-Victoire Cosson  1 , Øivind Ørstavik  1 , Ngai Chin Lai  2   3 , H Kirk Hammond  2   3 , Jan-Bjørn Osnes  1 , Tor Skomedal  1 , Viacheslav Nikolaev  4 , Finn Olav Levy  1 , Kurt Allen Krobert  1
Affiliations

Constitutive inhibitory G protein activity upon adenylyl cyclase-dependent cardiac contractility is limited to adenylyl cyclase type 6

Caroline Bull Melsom et al. PLoS One. .

Abstract

Purpose: We previously reported that inhibitory G protein (Gi) exerts intrinsic receptor-independent inhibitory activity upon adenylyl cyclase (AC) that regulates contractile force in rat ventricle. The two major subtypes of AC in the heart are AC5 and AC6. The aim of this study was to determine if this intrinsic Gi inhibition regulating contractile force is AC subtype selective.

Methods: Wild-type (WT), AC5 knockout (AC5KO) and AC6 knockout (AC6KO) mice were injected with pertussis toxin (PTX) to inactivate Gi or saline (control).Three days after injection, we evaluated the effect of simultaneous inhibition of phosphodiesterases (PDE) 3 and 4 with cilostamide and rolipram respectively upon in vivo and ex vivo left ventricular (LV) contractile function. Also, changes in the level of cAMP were measured in left ventricular homogenates and at the membrane surface in cardiomyocytes obtained from the same mouse strains expressing the cAMP sensor pmEPAC1 using fluorescence resonance energy transfer (FRET).

Results: Simultaneous PDE3 and PDE4 inhibition increased in vivo and ex vivo rate of LV contractility only in PTX-treated WT and AC5KO mice but not in saline-treated controls. Likewise, Simultaneous PDE3 and PDE4 inhibition elevated total cAMP levels in PTX-treated WT and AC5KO mice compared to saline-treated controls. In contrast, simultaneous PDE3 and PDE4 inhibition did not increase in vivo or ex vivo rate of LV contractility or cAMP levels in PTX-treated AC6KO mice compared to saline-treated controls. Using FRET analysis, an increase of cAMP level was detected at the membrane of cardiomyocytes after simultaneous PDE3 and PDE4 inhibition in WT and AC5KO but not AC6KO. These FRET data are consistent with the functional data indicating that AC6 activity and PTX inhibition of Gi is necessary for simultaneous inhibition of PDE3 and PDE4 to elicit an increase in contractility.

Conclusions: Together, these data suggest that AC6 is tightly regulated by intrinsic receptor-independent Gi activity, thus providing a mechanism for maintaining low basal cAMP levels in the functional compartment that regulates contractility.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Representative autoradiogram showing ADP-ribosylated Gαi protein in WT, AC5KO and AC6KO mouse left ventricle pre-treated with saline or pertussis toxin (PTX).
Fig 2
Fig 2. Experimental protocol and analysis of in vivo experiments.
(A,B) Representative screenshots from wild-type mice showing left ventricular pressure (mm Hg) over time during baseline, followed by 1 μM timolol, followed by the response to simultaneous addition of 1 μM cilostamide (Cil) and 10 μM rolipram (Rol) and lastly a second administration of timolol to verify responses to cilostamide and rolipram did not result from βAR activation. Displayed is the continuous recording of left ventricular pressure over the entire experimental period. The mice were pre-treated with either saline (A,C,E) or PTX (B,D,F; 120 μg/kg i.p. three days prior to study). Note that cilostamide and rolipram evoked an increase in pressure only in PTX treated mice in the presence of timolol. (C-F) Shown are individual contraction-relaxation cycles of changes in pressure from before and after addition of each respective drug administration expressed in mm Hg (C,D) or as a percentage of each individual maximal pressure (E,F; normalized to 100%) to illustrate the time base of the contraction-relaxation cycle. Cilostamide and rolipram increased pressure in AC5KO similar to WT but not in AC6KO (see Fig 3).
Fig 3
Fig 3. PDE3 and PDE4 inhibition after PTX treatment increased in vivo rate of left ventricle contractility in wild type (WT) and AC5KO mice but not in AC6KO mice.
Left ventricular (LV) +(dP/dt)max (A-C), -(dP/dt)max (D-F) and heart rate (G-I) recorded in WT (A, D, G), AC5KO (B, E, H) and AC6KO (C, F, I) mice treated first with timolol (2.5 mg/kg i.p.) followed by combined PDE3 inhibitor cilostamide (3 mg/kg i.p.) and PDE4 inhibitor rolipram (10 mg/kg i.p.). To verify continuous and complete βAR blockade, timolol (2.5 mg/kg i.p.) was administered a second time after the PDE inhibitors. All mice received either saline or PTX injection i.p. three days prior to study. Experiments were conducted after vagotomy. Data are mean±SEM. *P<0.05, two-way repeated measures ANOVA with Tukey’s multiple comparisons test; **P<0.05, two-way ANOVA with Sidak’s multiple comparisons test; #P<0.05 PTX-treated AC6KO vs. PTX-treated WT and AC5KO, two-way ANOVA with Sidak’s multiple comparisons test. ##P<0.05 vs. baseline, Cil/Rol and the second timolol in respective saline and PTX treated groups, two-way repeated measures ANOVA with Tukey’s multiple comparisons test.
Fig 4
Fig 4. Ex vivo experimental timeline and contraction-relaxation cycles.
(A-C) Representative traces showing inotropic responses (mN) evoked by simultaneous addition of 1 μM cilostamide (Cil) and 10 μM rolipram (Rol) and the subsequent effect of 10 μM carbachol and reversal of carbachol effects by 1 μM atropine followed by 1 μM timolol in left ventricular strips of WT mice. The mice were pre-treated with either saline (A,B) or PTX (C; 120 μg/kg i.p. three days prior to study). Displayed is the Fmax of each contraction–relaxation cycle (CRC) in 1 ms intervals (each point) over the entire experimental period (A,B,C). Note that timolol blockade of βARs in animals pre-treated with saline was sufficient to prevent an inotropic response by cilostamide and rolipram. In addition, cilostamide or rolipram evoked an inotropic and lusitropic response only in PTX treated mice in the presence of timolol. (D-F) Averaged CRCs (mean data of 10–30 consecutive cycles) from before and after each drug administration expressed in mN or as (G-I) a percentage of each individual maximum Fmax to illustrate lusitropic effects as reduction of relaxation time. Similar effects were obtained for AC5KO as WT, but cilostamide and rolipram elicited no inotropic or lusitropic effect in AC6KO (see Fig 5).
Fig 5
Fig 5. Knockout of AC6 prevented the inotropic and lusitropic response to combined PDE3 and PDE4 inhibition in isolated ventricular strips from mice treated with pertussis toxin (PTX).
(A) Maximal inotropic response (reported as (dF/dt)max in percent above basal) and (B) lusitropic response (reported as Δ relaxation time (RT) in ms) elicited by simultaneous addition of PDE3 (cilostamide, 1 μM) and PDE4 (rolipram, 10 μM) inhibitors in left ventricular strips from saline-treated or PTX-treated mice (120 μg/kg i.p. three days prior to study). All experiments were conducted in the presence of the βAR blocker timolol (1 μM), α1-adrenergic receptor antagonist prazosin (0.1 μM) followed by addition of cilostamide and rolipram. Data are mean±SEM. *P<0.05 vs. respective PTX treated group, two-way ANOVA with Sidak’s multiple comparisons test. **P<0.05 vs. PTX-treated AC6KO, two-way ANOVA with Tukey’s multiple comparison’s test.
Fig 6
Fig 6. PTX treatment increased cAMP levels evoked by simultaneous inhibition of PDE3 and PDE4 is dependent on AC6.
The effect of simultaneous inhibition of PDE3 (cilostamide, 1μM) and PDE4 (rolipram, 10 μM) upon cAMP levels in left ventricular homogenates after saline or PTX pre-treatment (120 μg/kg i.p. three days prior to study). The experiments were conducted in the presence of the βAR inverse agonist timolol (1 μM) and α1-adrenergic receptor antagonist prazosin (0.1 μM). The strips were flash frozen in liquid nitrogen after ~20 minutes of incubation with the PDE inhibitors. Data are mean±SEM and reported as pmol cAMP/mg protein (A,B,C) or pmol cAMP/mg protein above control (no cilostamide or rolipram) (D). *P<0.05, unpaired t-test.
Fig 7
Fig 7. Increase in cAMP at the plasma membrane evoked by simultaneous inhibition of PDE3 and PDE4 only in WT and AC5KO.
(A) Representative cardiomyocytes excited at 500±10 nm expressing (top) and not expressing (bottom) the pmEPAC1 sensor. Note the stronger signal on the plasma membrane of the top cell. (B) Representative traces displaying FRET detection of cAMP at the plasma membrane of PTX-treated cardiomyocytes after simultaneous inhibition of PDE3 (cilostamide, 1μM) and PDE4 (rolipram, 10 μM) in wild type (top), AC5KO (middle) and AC6KO (bottom). Isolated cardiomyocytes were incubated for 3 hours with 1 μg/ml PTX or saline (data not shown) of equal volume in the incubation media (1.2 ml reaction volume). FRET experiments were conducted in the presence of 1 μM timolol to block βARs.
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