Skip to main content
SpringerLink
Log in

Pepducin ICL1-9-Mediated β2-Adrenergic Receptor-Dependent Cardiomyocyte Contractility Occurs in a Gi Protein/ROCK/PKD-Sensitive Manner

  • Original Article
  • Published:
Cardiovascular Drugs and Therapy Aims and scope Submit manuscript

Abstract

Purpose

β-Adrenergic receptors (βAR) are essential targets for the treatment of heart failure (HF); however, chronic use of βAR agonists as positive inotropes to increase contractility in a Gs protein-dependent manner is associated with increased mortality. Alternatively, we previously reported that allosteric modulation of β2AR with the pepducin intracellular loop (ICL)1-9 increased cardiomyocyte contractility in a β-arrestin (βarr)-dependent manner, and subsequently showed that ICL1-9 activates the Ras homolog family member A (RhoA). Here, we aimed to elucidate both the proximal and downstream signaling mediators involved in the promotion of cardiomyocyte contractility in response to ICL1-9.

Methods

We measured adult mouse cardiomyocyte contractility in response to ICL1-9 or isoproterenol (ISO, as a positive control) alone or in the presence of inhibitors of various potential components of βarr- or RhoA-dependent signaling. We also assessed the contractile effects of ICL1-9 on cardiomyocytes lacking G protein-coupled receptor (GPCR) kinase 2 (GRK2) or 5 (GRK5).

Results

Consistent with RhoA activation by ICL1-9, both Rho-associated protein kinase (ROCK) and protein kinase D (PKD) inhibition were able to attenuate ICL1-9-mediated contractility, as was inhibition of myosin light chain kinase (MLCK). While neither GRK2 nor GRK5 deletion impacted ICL1-9-mediated contractility, pertussis toxin attenuated the response, suggesting that ICL1-9 promotes downstream RhoA-dependent signaling in a Gi protein-dependent manner.

Conclusion

Altogether, our study highlights a novel signaling modality that may offer a new approach to the promotion, or preservation, of cardiac contractility during HF via the allosteric regulation of β2AR to promote Gi protein/βarr-dependent activation of RhoA/ROCK/PKD signaling.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Francis GS, Bartos JA, Adatya S. Inotropes. J Am Coll Cardiol. 2014;63:2069–78.

    Article  PubMed  Google Scholar 

  2. Bristow MR. Treatment of chronic heart failure with beta-adrenergic receptor antagonists: a convergence of receptor pharmacology and clinical cardiology. Circ Res. 2011;109:1176–94.

    Article  CAS  PubMed  Google Scholar 

  3. Abdel-Magid AF. Allosteric modulators: an emerging concept in drug discovery. ACS Med Chem Lett. 2015;6:104–7.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Ahn S, Pani B, Kahsai AW, Olsen EK, Husemoen G, Vestergaard M, et al. Small-molecule positive allosteric modulators of the beta2-adrenoceptor isolated from DNA-encoded libraries. Mol Pharmacol. 2018;94:850–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Okyere AD, Tilley DG. Self-made allostery: endogenous COMP antagonizes pathologic AT1AR signaling. Cell Res. 2021. https://doi.org/10.1038/s41422-021-00493-x.

  6. Wang J, Gareri C, Rockman HA. G-protein-coupled receptors in heart disease. Circ Res. 2018;123:716–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhang P, Leger AJ, Baleja JD, Rana R, Corlin T, Nguyen N, et al. Allosteric activation of a G protein-coupled receptor with cell-penetrating receptor mimetics. J Biol Chem. 2015;290:15785–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Carr R 3rd, Benovic JL. From biased signalling to polypharmacology: unlocking unique intracellular signalling using pepducins. Biochem Soc Trans. 2016;44:555–61.

    Article  CAS  PubMed  Google Scholar 

  9. Carr R 3rd, Schilling J, Song J, Carter RL, Du Y, Yoo SM, et al. beta-Arrestin-biased signaling through the beta2-adrenergic receptor promotes cardiomyocyte contraction. Proc Natl Acad Sci USA. 2016;113:E4107–16.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Wang J, Hanada K, Staus DP, Makara MA, Dahal GR, Chen Q, et al. Galphai is required for carvedilol-induced beta1 adrenergic receptor beta-arrestin biased signaling. Nat Commun. 2017;8:1706.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Grisanti LA, Thomas TP, Carter RL, de Lucia C, Gao E, Koch WJ, et al. Pepducin-mediated cardioprotection via beta-arrestin-biased beta2-adrenergic receptor-specific signaling. Theranostics. 2018;8:4664–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lauriol J, Keith K, Jaffre F, Couvillon A, Saci A, Goonasekera SA, et al. RhoA signaling in cardiomyocytes protects against stress-induced heart failure but facilitates cardiac fibrosis. Sci Signal. 2014;7:ra100.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Raake PW, Vinge LE, Gao E, Boucher M, Rengo G, Chen X, et al. G protein-coupled receptor kinase 2 ablation in cardiac myocytes before or after myocardial infarction prevents heart failure. Circ Res. 2008;103:413–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Gainetdinov RR, Bohn LM, Walker JK, Laporte SA, Macrae AD, Caron MG, et al. Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice. Neuron. 1999;24:1029–36.

    Article  CAS  PubMed  Google Scholar 

  15. Grisanti LA, Schumacher SM, Tilley DG, Koch WJ. Designer approaches for G protein-coupled receptor modulation for cardiovascular disease. JACC Basic Transl Sci. 2018;3:550–62.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Pfleger J, Gresham K, Koch WJ. G protein-coupled receptor kinases as therapeutic targets in the heart. Nat Rev Cardiol. 2019;16:612–22.

    Article  PubMed  Google Scholar 

  17. Daaka Y, Luttrell LM, Lefkowitz RJ. Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A. Nature. 1997;390:88–91.

    Article  CAS  PubMed  Google Scholar 

  18. Anthony DF, Sin YY, Vadrevu S, Advant N, Day JP, Byrne AM, et al. beta-Arrestin 1 inhibits the GTPase-activating protein function of ARHGAP21, promoting activation of RhoA following angiotensin II type 1A receptor stimulation. Mol Cell Biol. 2011;31:1066–75.

    Article  CAS  PubMed  Google Scholar 

  19. Barnes WG, Reiter E, Violin JD, Ren XR, Milligan G, Lefkowitz RJ. beta-Arrestin 1 and Galphaq/11 coordinately activate RhoA and stress fiber formation following receptor stimulation. J Biol Chem. 2005;280:8041–50.

    Article  CAS  PubMed  Google Scholar 

  20. Godin CM, Ferguson SS. The angiotensin II type 1 receptor induces membrane blebbing by coupling to Rho A, Rho kinase, and myosin light chain kinase. Mol Pharmacol. 2010;77:903–11.

    Article  CAS  PubMed  Google Scholar 

  21. Tocci P, Cianfrocca R, Di Castro V, Rosano L, Sacconi A, Donzelli S, et al. beta-arrestin1/YAP/mutant p53 complexes orchestrate the endothelin A receptor signaling in high-grade serous ovarian cancer. Nat Commun. 2019;10:3196.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Xiang SY, Vanhoutte D, Del Re DP, Purcell NH, Ling H, Banerjee I, et al. RhoA protects the mouse heart against ischemia/reperfusion injury. J Clin Invest. 2011;121:3269–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yung BS, Brand CS, Xiang SY, Gray CB, Means CK, Rosen H, et al. Selective coupling of the S1P3 receptor subtype to S1P-mediated RhoA activation and cardioprotection. J Mol Cell Cardiol. 2017;103:1–10.

    Article  CAS  PubMed  Google Scholar 

  24. Martin-Garrido A, Biesiadecki BJ, Salhi HE, Shaifta Y, Dos Remedios CG, Ayaz-Guner S, et al. Monophosphorylation of cardiac troponin-I at Ser-23/24 is sufficient to regulate cardiac myofibrillar Ca(2+) sensitivity and calpain-induced proteolysis. J Biol Chem. 2018;293:8588–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ryba DM, Li J, Cowan CL, Russell B, Wolska BM, Solaro RJ. Long-term biased beta-arrestin signaling improves cardiac structure and function in dilated cardiomyopathy. Circulation. 2017;135:1056–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tarigopula M, Davis RT 3rd, Mungai PT, Ryba DM, Wieczorek DF, Cowan CL, et al. Cardiac myosin light chain phosphorylation and inotropic effects of a biased ligand, TRV120023, in a dilated cardiomyopathy model. Cardiovasc Res. 2015;107:226–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Thomsen ARB, Plouffe B, Cahill TJ 3rd, Shukla AK, Tarrasch JT, Dosey AM, et al. GPCR-G protein-beta-arrestin super-complex mediates sustained G protein signaling. Cell. 2016;166:907–19.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nguyen AH, Thomsen ARB, Cahill TJ 3rd, Huang R, Huang LY, Marcink T, et al. Structure of an endosomal signaling GPCR-G protein-beta-arrestin megacomplex. Nat Struct Mol Biol. 2019;26:1123–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Song J, Li J, Lulla A, Evers BM, Chung DH. Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-delta pathway activation. Am J Physiol Cell Physiol. 2006;290:C1469–76.

    Article  CAS  PubMed  Google Scholar 

  30. Okamoto R, Kato T, Mizoguchi A, Takahashi N, Nakakuki T, Mizutani H, et al. Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart. Cell Signal. 2006;18:1408–16.

    Article  CAS  PubMed  Google Scholar 

  31. Chang AN, Battiprolu PK, Cowley PM, Chen G, Gerard RD, Pinto JR, et al. Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo. J Biol Chem. 2015;290:10703–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chang AN, Kamm KE, Stull JT. Role of myosin light chain phosphatase in cardiac physiology and pathophysiology. J Mol Cell Cardiol. 2016;101:35–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Homan KT, Tesmer JJ. Molecular basis for small molecule inhibition of G protein-coupled receptor kinases. ACS Chem Biol. 2015;10:246–56.

    Article  CAS  PubMed  Google Scholar 

  34. Tressel SL, Koukos G, Tchernychev B, Jacques SL, Covic L, Kuliopulos A. Pharmacology, biodistribution, and efficacy of GPCR-based pepducins in disease models. Methods Mol Biol. 2011;683:259–75.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Gurbel PA, Bliden KP, Turner SE, Tantry US, Gesheff MG, Barr TP, et al. Cell-penetrating pepducin therapy targeting PAR1 in subjects with coronary artery disease. Arterioscler Thromb Vasc Biol. 2016;36:189–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Availability of Data and Materials

The data underlying this article will be shared upon reasonable request to the corresponding authors.

Funding

This work was supported by National Institutes of Health (R01 HL136219 to DGT and JLB; P01 HL147841 to WJK and DGT; F31 HL154814 to ADO; K99 HL132882 to SMS) and the American Heart Association (17POST33660942 to CdL).

Author information

Author notes

  1. Claudio de Lucia

    Present address: Instituti Clinici Scientifici Maugeri di Telese Terme, Telese Terme, Italy

  2. Sarah M. Schumacher

    Present address: Cleveland Clinic Lerner College of Medicine, Cleveland, OH, USA

Authors and Affiliations

  1. Center for Translational Medicine, Lewis Katz School of Medicine, Temple University, Room 945A MERB, 3500 N. Broad St, Philadelphia, PA, 19140, USA

    Ama Dedo Okyere, Jianliang Song, Viren Patwa, Rhonda L. Carter, Nitya Enjamuri, Anna Maria Lucchese, Jessica Ibetti, Claudio de Lucia, Sarah M. Schumacher, Walter J. Koch, Joseph Y. Cheung & Douglas G. Tilley

  2. Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, 19107, USA

    Jeffrey L. Benovic

Authors
  1. Ama Dedo Okyere
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianliang Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Viren Patwa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rhonda L. Carter
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nitya Enjamuri
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anna Maria Lucchese
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jessica Ibetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Claudio de Lucia
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sarah M. Schumacher
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Walter J. Koch
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Joseph Y. Cheung
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jeffrey L. Benovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Douglas G. Tilley
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the study conception and design. Material preparation and data collection and analysis were performed by Ama Okyere, Jianliang Song, Viren Patwa, Rhonda Carter, Nitya Enjamuri, Anna Maria Lucchese, Jessica Ibetti, Claudio de Lucia, and Sarah Schumacher. The first draft of the manuscript was written by Ama Okyere and Douglas Tilley and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Douglas G. Tilley.

Ethics declarations

Ethics Approval and Consent to Participate

All animal experiments were conducted under the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) at Temple University under Animal Care and Use Protocols #4902 and #5017.

Consent for Publication

Not applicable

Informed Consent

Not applicable

Conflict of Interest

The authors declare no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

ESM 1

(DOCX 15 kb)

ESM 2

(JPG 124 kb)

ESM 3

(JPG 61 kb)

ESM 4

(JPG 90 kb)

ESM 5

(JPG 103 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Okyere, A.D., Song, J., Patwa, V. et al. Pepducin ICL1-9-Mediated β2-Adrenergic Receptor-Dependent Cardiomyocyte Contractility Occurs in a Gi Protein/ROCK/PKD-Sensitive Manner. Cardiovasc Drugs Ther 37, 245–256 (2023). https://doi.org/10.1007/s10557-021-07299-4

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10557-021-07299-4

Keywords

Search

Navigation