Galphas-biased beta2-adrenergic receptor signaling from restoring synchronous contraction in the failing heart

Abstract

Cardiac resynchronization therapy (CRT), in which both ventricles are paced to recoordinate contraction in hearts that are dyssynchronous from conduction delay, is the only heart failure (HF) therapy to date to clinically improve acute and chronic function while also lowering mortality. CRT acutely enhances chamber mechanical efficiency but chronically alters myocyte signaling, including improving β-adrenergic receptor reserve. We speculated that the latter would identify unique CRT effects that might themselves be effective for HF more generally. HF was induced in dogs by 6 weeks of atrial rapid pacing with (HFdys, left bundle ablated) or without (HFsyn) dyssynchrony. We used dyssynchronous followed by resynchronized tachypacing (each 3 weeks) for CRT. Both HFdys and HFsyn myocytes had similarly depressed rest and β-adrenergic receptor sarcomere and calcium responses, particularly the β2-adrenergic response, whereas cells subjected to CRT behaved similarly to those from healthy controls. CRT myocytes exhibited suppressed Gαi signaling linked to increased regulator of G protein (heterotrimeric guanine nucleotide-binding protein) signaling (RGS2, RGS3), yielding Gαs-biased β2-adrenergic responses. This included increased adenosine cyclic AMP responsiveness and activation of sarcoplasmic reticulum-localized protein kinase A. Human CRT responders also showed up-regulated myocardial RGS2 and RGS3. Inhibition of Gαi (with pertussis toxin, RGS3, or RGS2 transfection), stimulation with a Gαs-biased β2 agonist (fenoterol), or transient (2-week) exposure to dyssynchrony restored β-adrenergic receptor responses in HFsyn to the values obtained after CRT. These results identify a key pathway that is triggered by restoring contractile synchrony and that may represent a new therapeutic approach for a broad population of HF patients.

Fig. 1

Impact of synchronous, dyssynchronous, and resynchronized HF on regional left ventricular wall motion, and myocyte β-adrenergic responsiveness. (A) Regional longitudinal strain (derived from speckle tracking) for septal/anterior wall (solid line) and lateral wall (dotted line) of left ventricles in control and HF models. Normal controls have similar, simultaneous strains in both regions. In HF_dys, septal shortening precedes the lateral wall, with reciprocal septal stretch when the latter wall contracts. Restoration of synchrony is observed in the CRT model. Synchronous HF (HF_syn) displays concurrent strain in both regions. For V3A3, dyssynchrony is observed during the initial 3-week RV pacing period (RVP) and this is reverted to synchronous in the latter atrial pacing period (AP:V3A3). (B) Sarcomere shortening and whole-cell calcium transients from isolated myocytes in each model. Data are from cells isolated from the lateral wall, although, as reported, HF_dys and CRT models show similar behavior in both anterior and lateral walls (11). Basal function (baseline) and peak calcium transient, as well as their stimulation by isoproterenol (ISO), were equally depressed in HF_dys and HF_syn models, but enhanced in both resynchronized models (V3A3 and CRT) back to near-control levels. (C) Summary results for peak rest and isoproterenol-stimulated shortening, cell relengthening velocity, peak calcium, and rate of calcium decline (n = 12 to 20 cells per heart, n = 4 to 5 hearts per group). All of the properties were depressed only in HF_dys and HF_syn models. *P < 0.01 versus all other groups; ^†P < 0.001 versus baseline.

Fig. 2

Resynchronization enhances β-receptor density and shifts signaling away from Gα_i coupling to generate a Gα_s-biased response. (A) β-Receptor maximal affinity binding (Bmax) reflecting surface membrane receptor abundance for combined and individual β₁-AR and β₂-AR subtypes. n = 4 for each group. *P < 0.05 versus other HF groups and control (within respective receptor group); ^†P < 0.01 versus β₂-AR response. (B) Sarcomere shortening (%SS) and whole-cell calcium transient in cells from different models when stimulated with isoproterenol without or with pretreatment by pertussis toxin (PTX). PTX treatment greatly enhanced both behaviors in HF_dys and HF_syn, but had no impact in resynchronized models. (C) %SS and peak Ca²⁺ transient in cells exposed to β₂-AR–selective agonist zinterol (ZIN), zinterol + PTX, or the G_s-biased β₂ agonist fenoterol. n = 12 to 20 cells per heart, and 3 to 4 hearts per group. *P < 0.01 versus other groups; ^†P < 0.001 versus zinterol; ^‡P < 0.001 versus fenoterol.

Fig. 3

Resynchronization enhances myocyte cAMP levels and PKA activation in the SR in response to β₂ stimulation. (A) Domain structure of ICUE3 FRET probe for analysis of membrane-localized cAMP generation. Cells were studied in the presence of IBMX to inhibit PDEs and stimulated with either zinterol or fenoterol. (B) Example of cell fluorescence ratio–encoded images from myocytes in each group, showing enhanced cAMP generated in CRT models stimulated by zinterol. (C) Upper panels show time tracings for FRET ratio in cells stimulated with zinterol or fenoterol, and lower panels summarize data from all experiments (n = 8 to 15 per group). Zinterol induced a greater response in CRT models, whereas fenoterol led to a similarly elevated response in all groups. (D) Domain structure of SR-AKAR3 FRET probe used for sarcoplasmic reticulum (SR)-localized PKA activity. (E) Myocyte expressing SR-AKAR3 shows fluorescence in tubular SR throughout the cell. (F) Example time tracings of SR-AKAR3 FRET in cells treated with zinterol. Only myocytes from CRT models showed PKA activation in the SR. Forskolin (Fsk) is used as a positive control showing functionality of the probe after direct AC stimulation (n = 4 to 5 per group).

Fig. 4

Protein expression of Gα_i and GRK2 but not Gα_s increases in each HF model over control. (A and B) Western blots (A) and summary densitometry (B) for protein levels of Gα_i(1,2,3), Gα_s, and GRK2 (n = 4 to 5 per group). Analysis is normalized to GAPDH as a loading control. *P < 0.05 versus control.

Fig. 5

Up-regulation of RGS2 and RGS3 in canine models of resynchronization and human responders to chronic CRT. (A and B) Western blots (A) and summary densitometry (B) for protein expression of RGS2, RGS3, and RGS4 (n = 4 to 5 per group). Analysis is normalized to GAPDH as a loading control. *P < 0.001 versus control (Con); P < 0.01 versus HF_dys and HF_syn; ^†P < 0.01 versus control; P < 0.001 versus HF_dys and HF_syn; ^#P < 0.001 versus all other groups. (C) Enhanced RGS2 and RGS3 mRNA expression is present in human LV biopsy samples from responder (R) patients, whereas it is absent in nonresponders (NR). Responders also demonstrated a significant decline in myocardial B-type natriuretic peptide (BNP). Differential response in EF is also displayed for the two groups.

Fig. 6

Up-regulation of RGS2 or RGS3 in HF_dys myocytes is sufficient to convert the β₂-adrenergic stimulation phenotype to that of CRT (or V3A3) responses. (A) Isolated myocytes from HF_dys hearts were exposed to adenovirus with either GFP (control) or RGS2 or RGS3 vectors. Up-regulation of protein was confirmed in the myocytes and was in the range of 50 to 60% over controls. Adenovirus-GFP controls were similar to noninfected HF_dys cells. *P < 0.05 versus control and HF_dys; ^†P < 0.05 versus other groups. (B) Summary data showing sarcomere shortening response to zinterol in myocytes from HF_dys, HF_syn, or CRT hearts after 24-hour infection with adenovirus containing GFP or full-length RGS2 or RGS3. Data are also shown with or without concomitant PTX treatment. n = 12 to 18 cells per heart, n = 2 to 3 hearts per group. *P < 0.01 versus CRT; ^†P < 0.001 versus CRT.

Fig. 7

Enhanced β-AR responsiveness in myocytes from hearts exposed to RV (dyssynchrony) pacing during the middle 2 weeks of otherwise 6-week atrial tachypacing (AVA). (A) %SS and peak Ca²⁺ transients are similar at baseline and after isoproterenol stimulation in myocytes from an AVA heart to those observed after CRT. *P < 0.001 versus baseline (nonstimulated). (B) Minimal augmentation of functional or Ca²⁺ response to zinterol in AVA myocytes by addition of PTX. Data are compared with the HF_syn model, which displayed marked augmentation. *P < 0.05 versus zinterol alone. n = 12 to 20 cells per heart, n = 4 hearts for each group. (C) Protein expression for RGS3 and RGS2 in AVA model, shown in comparison with the HF_syn model. There was no up-regulation in this model, unlike CRT (n = 4 to 5 per model). (D) Protein expression (left) and summary densitometry (right) for β-arrestin2, shown in comparison to HF_syn data. Unlike HF_syn, which had no change in expression over control or the other models (fig. S4), AVA myocytes had very low levels. *P < 0.001.