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. 2022 Sep 26;23(19):11315.
doi: 10.3390/ijms231911315.

Carvedilol Selectively Stimulates βArrestin2-Dependent SERCA2a Activity in Cardiomyocytes to Augment Contractility

Jennifer Maning  1 , Victoria L Desimine  1 , Celina M Pollard  1 , Jennifer Ghandour  1 , Anastasios Lymperopoulos  1
Affiliations

Carvedilol Selectively Stimulates βArrestin2-Dependent SERCA2a Activity in Cardiomyocytes to Augment Contractility

Jennifer Maning et al. Int J Mol Sci. .

Abstract

Heart failure (HF) carries the highest mortality in the western world and β-blockers [β-adrenergic receptor (AR) antagonists] are part of the cornerstone pharmacotherapy for post-myocardial infarction (MI) chronic HF. Cardiac β1AR-activated βarrestin2, a G protein-coupled receptor (GPCR) adapter protein, promotes Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a SUMO (small ubiquitin-like modifier)-ylation and activity, thereby directly increasing cardiac contractility. Given that certain β-blockers, such as carvedilol and metoprolol, can activate βarrestins and/or SERCA2a in the heart, we investigated the effects of these two agents on cardiac βarrestin2-dependent SERCA2a SUMOylation and activity. We found that carvedilol, but not metoprolol, acutely induces βarrestin2 interaction with SERCA2a in H9c2 cardiomyocytes and in neonatal rat ventricular myocytes (NRVMs), resulting in enhanced SERCA2a SUMOylation. However, this translates into enhanced SERCA2a activity only in the presence of the β2AR-selective inverse agonist ICI 118,551 (ICI), indicating an opposing effect of carvedilol-occupied β2AR subtype on carvedilol-occupied β1AR-stimulated, βarrestin2-dependent SERCA2a activation. In addition, the amplitude of fractional shortening of NRVMs, transfected to overexpress βarrestin2, is acutely enhanced by carvedilol, again in the presence of ICI only. In contrast, metoprolol was without effect on NRVMs' shortening amplitude irrespective of ICI co-treatment. Importantly, the pro-contractile effect of carvedilol was also observed in human induced pluripotent stem cell (hIPSC)-derived cardiac myocytes (CMs) overexpressing βarrestin2, and, in fact, it was present even without concomitant ICI treatment of human CMs. Metoprolol with or without concomitant ICI did not affect contractility of human CMs, either. In conclusion, carvedilol, but not metoprolol, stimulates βarrestin2-mediated SERCA2a SUMOylation and activity through the β1AR in cardiac myocytes, translating into direct positive inotropy. However, this unique βarrestin2-dependent pro-contractile effect of carvedilol may be opposed or masked by carvedilol-bound β2AR subtype signaling.

Keywords: G protein-coupled receptor; SERACA2a; SUMOylation; cardiomyocyte; contractility; signal transduction; β-adrenergic receptor; βarrestin2.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
β-blockers and βarrestin2-dependent SERCA2 SUMOylation in H9c2 cardiomyocytes. (A) Immunoblotting for βarrestins in control (infected with adenovirus encoding for green fluorescent protein, AdGFP) or in H9c2 cardiomyocytes infected with adenovirus encoding for βarrestin2 (Adβarr2) to confirm βarrestin2 overexpression in the latter cells. H9c2 cardiomyocytes express only βarrestin1 (βarr1) endogenously at detectable levels (see AdGFP lanes and Refs. [7,20]). Shown is a representative blot of three independent experiments. (B,C) Immunoblotting for βarrestins, SUMO-1 and SERCA2a in SERCA2a immunoprecipitates (IPs) prepared from these cells and treated with vehicle (0.1% DMSO), 1 μM carvedilol (Carv), or 1 μM metoprolol (Meto) for 20 min. Representative blots are shown in (B) and SERCA2a SUMOylation quantitation, as measured by densitometry, is shown in (C). IB: Immunoblotting; IP: Immunoprecipitation; S-SERCA2a: SUMOylated SERCA2a; A.U.: Arbitrary (densitometric) units. Only βarrestin2 (not βarrestin1) could be detected in the SERCA2a IPs and only in Adβarr2-infected cells treated with carvedilol. *, p < 0.05, vs. any other treatment/cell clone; n = 3 independent experiments.
Figure 2
Figure 2
β-blockers and SERCA2a activity in βarrestin2-overexpessing H9c2 cardiomyocytes. Maximal SERCA activity in microsomes isolated from βarrestin2-expressing (Adβarr2) or control (AdGFP) H9c2 cardiomyocytes and treated with 1 μM metoprolol (Meto), 1 μM carvedilol (Carv), or 1 μM carvedilol in the presence of 10 μM ICI 118,551 (Carv + ICI) for 30 min. *, p < 0.05, vs. any other group; n = 3 independent measurements/transfection group/treatment.
Figure 3
Figure 3
β-blockers and βarrestin2-dependent SERCA2a function and contractility in NRVMs. (A) Immunoblotting for βarrestins in control (AdGFP) or in NRVMs expressing βarrestin2 (Adβarr2) to confirm βarrestin2 transgene induction. Like H9c2 cardiomyocytes, NRVMs also appear to express only βarrestin1 endogenously at detectable levels (see AdGFP lanes). Shown is a representative blot from three independent experiments. (B,C) Immunoblotting for βarrestins, SUMO1 and SERCA2a in SERCA2a IPs prepared from these cells and treated with vehicle (0.1% DMSO), 1 μM carvedilol (Carv), or 1 μM metoprolol (Meto) for 20 min. Representative blots are shown in (B) and SERCA2a SUMOylation quantitation, as measured by densitometry, is shown in (C). S-SERCA2a: SUMOylated SERCA2a; A.U.: Arbitrary (densitometric) units. As in H9c2 cardiomyocytes, only βarrestin2 could be detected in SERCA2a IPs and only in Adβarr2-infected cells treated with carvedilol. *, p < 0.05, vs. any other treatment/cell clone; n = 3 independent experiments. (D) Maximal SERCA activity potentiation in microsomes isolated from βarrestin2-expressing (Adβarr2) or control (AdGFP) NRVMs. ICI: 10 μM ICI 118,551; Meto: 1 μM metoprolol; Carv: 1 μM carvedilol, for 30 min. *, p < 0.05, vs. any other group; n = 3 independent measurements/transfection group/treatment. (E) Cell shortening response of βarrestin2-overexpressing NRVMs treated as in (D). *, p < 0.05, vs. any other treatment; NS: Not significant vs. basal (0.1% DMSO) at p = 0.05; n = 3 independent measurements (in triplicate)/treatment group.
Figure 4
Figure 4
Cell shortening response of βarrestin2-overexpressing hIPSC-CMs. (A) Co-immunofluorescence performed on culture day 16 (48 h post-adenoviral infection) for NKX2.5 (cardiac myocyte marker, red) and for GFP (green) to confirm βarrestin2 overexpression (the adenovirus encoding for βarrestin2 also encoded for GFP, see Section 4.2 for details). (B) ICI: 10 μM ICI 118,551; Meto: 1 μM metoprolol; Carv: 1 μM carvedilol; Dob: 1 μM dobutamine. *, p < 0.05, vs. baseline (0.1% DMSO); #, p < 0.05, vs. Carv alone; ^, p < 0.05, vs. any other treatment; NS: Not significant vs. basal (0.1% DMSO) at p = 0.05; n = 3 independent measurements in 6–8 different myocytes per drug tested.
Figure 5
Figure 5
Schematic illustration of carvedilol’s positive inotropic effect via stimulation of the β1AR-βarrestin2-SERCA2a SUMOylation axis in cardiac myocytes. See text for details. SR: Sarcoplasmic reticulum; SUMO1: Small ubiquitin-like modifier-1; ???: Effect/signaling pathway unknown.
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