cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes

Abstract

Rationale: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs).

Objective: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments.

Methods and results: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility.

Conclusions: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

Fig. 1. Activation of sGC or pGC generates different cGMP signals in the PKA-RI and PKA-RII compartments

(A) Cardiac myocytes co-expressing the targeted cGMP FRET sensor RI_cygnet-2.1 (upper left panel) or RII_cygnet-2.1 (lower left panel) and protein ZASP_RFP (middle panels). The overlay between sensor localization and ZASP-RFP is shown in the right panels. Reported on the right is the intensity profile of the probe signal (in blue) and of the ZASP_RFP signal (in red) in the region indicated by the white line. RI_cygnet-2.1 and RII_cygnet-2.1 show the same localization previously described for RI_Epac and RII_Epac, respectively. Scale bars are 10 μm. (B) Average FRET changes upon treatment with 100 μmol/L SNAP or 100 nmol/L ANP, as indicated. n≥9. Throughout, error bars indicate SEM, unless otherwise stated. *0.01<p<0.05.

Fig. 2. Effects of cGMP signals on cAMP levels

(A) Representative kinetics of cAMP changes detected in NRVMs by the targeted cAMP sensors RI_epac (left panels) or RII_epac (right panels) upon stimulation with 10 nmol/L ISO and subsequent addition of 100 μmol/L IBMX. Where indicated cells were pre-incubated for 10 min with SNAP (100 μmol/L) or SNAP and ODQ (10 μmol/L). (B) Summary of all experiments performed as in A. n≥13 (C) cAMP changes detected in ARVMs expressing RI_epac and RII_epac upon stimulation with 100 nmol/L ISO with or without 100 μmol/L SNAP, as indicated. n≥7. *0.01<p<0.05, **0.001<p<0.01, ***<0.001.

Fig. 3. ANP reduces the cAMP response to ISO selectively in the PKA-RII compartment

(A) Representative kinetics of cAMP changes detected by the targeted cAMP reporters RI_epac or RII_epac in NRVMs upon stimulation with 10 nmol/L ISO and 100 μmol/L IBMX. Where indicated cells were pre-treated for 10 min with 100 nmol/L ANP. (B) Summary of all the experiments performed as in A. n≥15. ***p<0.001.

Fig. 4. Effect of selective PDE2 and PDE3 inhibition

(A) cAMP changes induced by ISO in NRVMs expressing the targeted cAMP sensors RI_epac or RII_epac. Where indicated myocytes were pre-treated for 10 min with 100 μmol/L SNAP, 10 μmol/L BAY or 10 μmol/L CILO. n≥15. (B) cAMP changes induced by ISO in NRVMs expressing RI_epac or RII_epac. Where indicated myocytes were pre-treated for 10 min with 100 nmol/L ANP, 10 μmol/L BAY or 10 μmol/L CILO. n≥7. *0.01<p<0.05, **0.001<p<0.01, ***<0.001.

Fig. 5. Effects of displacing endogenous PDE2A

cAMP changes induced by 10 nmol/L ISO in NRVMs expressing RI_epac or RII_epac with or without dnPDE2A-mRFP or PDE2A_D485A, as indicated. Where indicated cells were pre-treated for 10 min with 100 μmol/L SNAP (A) or 100 nmol/L ANP (B). n≥9, *0.01<p<0.05, **0.001<p<0.01, ***<0.001.

Fig. 6. Effect of SNAP on PKA isoforms activity

(A) Cardiac myocytes expressing the targeted PKA activity reporter RI_AKAR3 or RII_AKAR3 (left panels) and the marker ZASP_RFP (middle panels). The overlay between probe localization and ZASP-RFP is shown in the right panels. On the right the intensity profile of the probe signal (in blue) and of the ZASP_RFP signal (in red) in the region indicated by the white line is reported. RI_AKAR3 and RII_AKAR3 show the same localization as RI_cygnet-2.1 and RII_cygnet-2.1, respectively. Scale bars are 10 μm. (B) cAMP changes detected by RI_AKAR3 or RII_AKAR3 upon application of 0.5 nmol/L ISO. Where indicated cells were pre-incubated with 100 μmol/L SNAP or SNAP and 10 μmol/L ODQ. n≥7, *0.01<p<0.05, **0.001<p<0.01, ***<0.001.

Fig. 7. Functional effects of cAMP/cGMP interplay

(A) Example tracing showing the fractional shortening recorded in isolated ventriculocytes obtained from adult hearts in the absence or in the presence of 10 nmol/L ISO in control cells and cells treated with 100 μmol/L SNAP. (B) Summary of the results obtained from three independent experiments performed as in (A). (C) Summary of the results obtained in three independent experiments in which cells were treated as in A but pretreated with BAY 10 μmol/L. n≥11. *=p<0.05, ns = non significant.

Fig. 8. Model of cGMP-mediated modulation of local cAMP signals

(A) β-AR stimulation generates a spatially restricted pool of cAMP that preferentially activates PKA-RII over PKA-RI. (B) Activation of sGC generates a uniform increase in cGMP in both PKA-RI and PKA-RII compartments. Increased cGMP levels inhibit PDE3, which is mainly confined to the PKA-RI compartment, and at the same time activate PDE2 associated with the PKA-RII compartment, thereby leading to an inversion of the cAMP gradients in response to catecholamines. (C) Activation of pGC generates a local pool of cGMP that specifically affects the PKA-RII compartment and blunts the cAMP response to catecholamines via PDE2 activation selectively in this locale.