Compartmentation of β2 -adrenoceptor stimulated cAMP responses by phosphodiesterase types 2 and 3 in cardiac ventricular myocytes

Abstract

Background and purpose: In cardiac myocytes, cyclic AMP (cAMP) produced by both β₁ - and β₂ -adrenoceptors increases L-type Ca²⁺ channel activity and myocyte contraction. However, only cAMP produced by β₁ -adrenoceptors enhances myocyte relaxation through phospholamban-dependent regulation of the sarco/endoplasmic reticulum Ca²⁺ ATPase 2 (SERCA2). Here we have tested the hypothesis that stimulation of β₂ -adrenoceptors produces a cAMP signal that is unable to reach SERCA2 and determine what role, if any, phosphodiesterase (PDE) activity plays in this compartmentation.

Experimental approach: The cAMP responses produced by β₁ -and β₂ -adrenoceptor stimulation were studied in adult rat ventricular myocytes using two different fluorescence resonance energy transfer (FRET)-based biosensors, the Epac2-camps, which is expressed uniformly throughout the cytoplasm of the entire cell and the Epac2-αKAP, which is targeted to the SERCA2 signalling complex.

Key results: Selective activation of β₁ - or β₂ -adrenoceptors produced cAMP responses detected by Epac2-camps. However, only stimulation of β₁ -adrenoceptors produced a cAMP response detected by Epac2-αKAP. Yet, stimulation of β₂ -adrenoceptors was able to produce a cAMP signal detected by Epac2-αKAP in the presence of selective inhibitors of PDE2 or PDE3, but not PDE4.

Conclusion and implications: These results support the conclusion that cAMP produced by β₂ -adrenoceptor stimulation was not able to reach subcellular locations where the SERCA2 pump is located. Furthermore, this compartmentalized response is due at least in part to PDE2 and PDE3 activity. This discovery could lead to novel PDE-based therapeutic treatments aimed at correcting cardiac relaxation defects associated with certain forms of heart failure.

Keywords: cAMP; compartmentation; phosphodiesterase; ventricular myocyte; β-adrenergic receptors.

Figure 1.

FRET biosensor expression and localization in adult rat ventricular myocytes (ARVMs). Schematic representation of the Epac2-αKAP (A) and Epac2-camps (C) biosensors and representative confocal images of myocytes expressing each probe. Representative super-resolution images of fixed ARVMs expressing Epac2-αKAP (B) and Epac2-camps (D) (n/N = 12/3 for each probe). Left hand panels are of each probe labeled with a GFP antibody. Center panels are the same cells co-labeled with SERCA2 antibody. Right hand panels represent the merged images, with the ROI magnified 4x (insets). White scale bar: 3.0 μm. See Methods for details.

Figure 2.

Characterization of Epac2-αKAP cAMP detection in vivo. (A) Maximum Epac2-αKAP response in a ventricular myocyte exposed to Iso (1 μM) plus the non-selective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 μM). (B) Minimum Epac2-αKAP response in a ventricular myocyte exposed to the non-selective adenylyl cyclase inhibitor MDL 12330A (MDL, 100 μM). (C) Average maximum (positive) and minimum (negative) Epac2-αKAP responses measured in ventricular myocytes. (D) Representative time course of Epac2-αKAP response following exposure to Iso (10 nM) and subsequent saturation of the probe with Iso (1 μM) plus IBMX (100 μM) (n/N = 10/3). (E) Pseudocolor images of FRET-ratio recorded at time-points indicated in panel D. Yellow scale bar: 10 μm. (F) Concentration dependence of Epac2-αKAP response in ventricular myocytes exposed to various concentrations of Iso (EC_50, 3.8 nM; Hill Coefficient, 0.9) (n/N = 10–14/3–4).

Figure 3.

Microdomain specific cAMP responses to selective activation of β₁ or β₂ARs in adult ventricular myocytes. Time course of β₁AR responses elicited by exposure to 10 nM Iso in the presence of the selective β₂AR antagonist ICI 118,551 (ICI, 300 nM) in a myocyte expressing (A) Epac2-camps (red) or (B) Epac2-αKAP (blue). Time course of β₂AR responses elicited by exposure to 10 nM Iso in the presence of the selective β₂AR antagonist CGP 20712A (CGP, 100 nM) in a myocyte expressing (D) Epac2-camps or (E) Epac2-αKAP. Responses to β₁ or β₂AR activation were normalized to the magnitude of the maximal FRET response of the probe elicited by subsequent exposure to Iso (1 μM) plus IBMX (100 μM) in the same cell. (C) Average β₁AR responses in Epac2-camps or Epac2-αKAP expressing cardiac myocytes. (F) Average β₂AR responses in Epac2-camps or Epac2-αKAP expressing cardiac myocytes. Statistical significance between groups (#) was determined by unpaired two-tailed Student’s t-tests.

Figure 4.

Microdomain specific cAMP responses to selective inhibition of different phosphodiesterase (PDE) isoforms in adult ventricular myocytes. Time course of cAMP responses detected by Epac2-camps (A) or Epac2-αKAP (C) following selective inhibition of PDE2 with 10 μM EHNA, PDE3 with 10 μM cilostamide (cil), or PDE4 with 10 μM rolipram (rol). Responses to PDE inhibition were normalized to the magnitude of the maximal FRET response of each probe elicited by subsequent exposure to Iso (1 μM) plus IBMX (100 μM), in the same cell (not shown). Average responses to selective PDE inhibitors detected by Epac2-camps (B) or Epac2-αKAP (D). Statistically significant responses (#) were identified by one-way ANOVA, with post hoc comparison (Bonferroni t-test) between groups where appropriate.

Figure 5.

Microdomain specific effects of selective PDE isoform inhibition on the cAMP response produced by β₂AR activation. Time course of cAMP responses to selective activation of β₂ARs with 10 nM Iso in the presence of 100 nM CGP detected by Epac2-camps following inhibition of PDE2 with 10 μM EHNA (A), PDE3 with 10 μM cilostamide (B), or PDE4 with 10 μM rolipram (C). Time courses of cAMP responses to selective activation of β₂ARs with 10 nM Iso in the presence of 100 nM CGP detected by Epac2-αKAP following inhibition of PDE2 with 10 μM EHNA (E), PDE3 with 10 μM cilostamide (F), or PDE4 with 10 μM rolipram (G). Responses to β₂AR activation were normalized to the magnitude of the maximal FRET response of each probe elicited by subsequent exposure to Iso (1 μM) plus IBMX (100 μM), in the same cell. Bar graphs indicate responses to β₂AR stimulation detected by Epac2-camps (D) and Epac2-αKAP (H) in the presence of EHNA, cilostamide, or rolipram. Statistically significant responses (#) were identified by one-way ANOVA with post hoc comparison (Bonferroni t-test) of β₂AR responses measured in the presence of each PDE inhibitor to those measured in the absence of PDE inhibitor (see Figure 3).

Figure 6.

Schematic depiction of βAR compartmentation under varying conditions. (A) β₁ARs are located throughout the sarcolemma of ventricular myocytes. They stimulate production of cAMP that can be detected in dyadic cleft as well as free SR microdomains by Epac2-camps (cytosolic) and Epac2-αKAP (free SR) FRET probes. (B) β₂ARs are primarily located in the T-tubules of ventricular myocytes and produce a cAMP signal in dyadic clefts, which can be detected by Epac2-camps. Under normal conditions, cAMP produced by β₂ARs is prevented from reaching the SERCA2 microdomain due to hydrolysis by PDE2 and PDE3. (C) Upon selective inhibition of either PDE2 or PDE3, cAMP generated by β₂ARs is able to reach the SERCA2 microdomain, where it can be detected by Epac2-αKAP, indicating a loss of compartmentation.