Skip to main content

Phenolic Metabolites Modulate Cardiomyocyte Beating in Response to Isoproterenol

Abstract

Cardiovascular disease (CVD) is a public health concern, and the third cause of death worldwide. Several epidemiological studies and experimental approaches have demonstrated that consumption of polyphenol-enriched fruits and vegetables can promote cardioprotection. Thus, diet plays a key role in CVD development and/or prevention. Physiological β-adrenergic stimulation promotes beneficial inotropic effects by increasing heart rate, contractility and relaxation speed of cardiomyocytes. Nevertheless, chronic activation of β-adrenergic receptors can cause arrhythmias, oxidative stress and cell death. Herein the cardioprotective effect of human metabolites derived from polyphenols present in berries was assessed in cardiomyocytes, in response to chronic β-adrenergic stimulation, to disclose some of the underlying molecular mechanisms. Ventricular cardiomyocytes derived from neonate rats were treated with three human bioavailable phenolic metabolites found in circulating human plasma, following berries’ ingestion (catechol-O-sulphate, pyrogallol-O-sulphate, and 1-methylpyrogallol-O-sulphate). The experimental conditions mimic the physiological concentrations and circulating time of these metabolites in the human plasma (2 h). Cardiomyocytes were then challenged with the β-adrenergic agonist isoproterenol (ISO) for 24 h. The presence of phenolic metabolites limited ISO-induced mitochondrial oxidative stress. Likewise, phenolic metabolites increased cell beating rate and synchronized cardiomyocyte beating population, following prolonged β-adrenergic receptor activation. Finally, phenolic metabolites also prevented ISO-increased activation of PKA–cAMP pathway, modulating Ca2+ signalling and rescuing cells from an arrhythmogenic Ca2+ transients’ phenotype. Unexpected cardioprotective properties of the recently identified human-circulating berry-derived polyphenol metabolites were identified. These metabolites modulate cardiomyocyte beating and Ca2+ transients following β-adrenergic prolonged stimulation.

This is a preview of subscription content, access via your institution.

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

Abbreviations

β-AR:

β-Adrenergic receptors

BDP:

Berry-derived polyphenols

CaMKII:

Calcium calmodulin-dependent kinase II

cAMP:

Cyclic adenosine monophosphate

CVD:

Cardiovascular diseases

ISO:

Isoproterenol

PKA:

cAMP-dependent protein kinase A

ROS:

Reactive oxidative species

RyR:

Ryanodine receptors

SR:

Sarcoplasmic reticulum

SERCA:

Sarcoplasmic reticulum calcium-ATPase

GPCRs:

G protein-coupled receptors

References

  1. Baker, A. J. (2014). Adrenergic signaling in heart failure: A balance of toxic and protective effects. Pflugers Archiv European Journal of Physiology, 466(6), 1139–1150. https://doi.org/10.1007/s00424-014-1491-5.

    Article  CAS  PubMed  Google Scholar 

  2. Bers, D. M. (2008). Calcium cycling and signaling in cardiac myocytes. Annual Review of Physiology, 70, 23–49. https://doi.org/10.1146/annurev.physiol.70.113006.100455.

    Article  CAS  PubMed  Google Scholar 

  3. Najafi, A., Sequeira, V., Kuster, D. W., & van der Velden, J. (2016). β-Adrenergic receptor signalling and its functional consequences in the diseased heart. European Journal of Clinical Investigation, 46(4), 362–374. https://doi.org/10.1111/eci.12598.

    Article  CAS  PubMed  Google Scholar 

  4. El-Armouche, A., & Eschenhagen, T. (2009). β-Adrenergic stimulation and myocardial function in the failing heart. Heart Failure Reviews, 14(4), 225–241. https://doi.org/10.1007/s10741-008-9132-8.

    Article  CAS  PubMed  Google Scholar 

  5. Bers, D. M. (2002). Cardiac excitation-contraction coupling. Nature, 415(6868), 198–205. https://doi.org/10.1038/415198a.

    Article  CAS  PubMed  Google Scholar 

  6. Andersson, D. C., Fauconnier, J., Yamada, T., Lacampagne, A., Zhang, S.-J., Katz, A., et al. (2011). Mitochondrial production of reactive oxygen species contributes to the β-adrenergic stimulation of mouse cardiomycytes. The Journal of Physiology, 589(7), 1791–1801. https://doi.org/10.1113/jphysiol.2010.202838.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Curran, J., Hinton, M. J., Rı, E., Bers, D. M., & Shannon, T. R. (2007). Beta-adrenergic enhancement of sarcoplasmic reticulum calcium leak in cardiac myocytes is mediated by calcium/calmodulin-dependent protein kinase. Circulation Research, 100(3), 391–398. https://doi.org/10.1161/01.RES.0000258172.74570.e6.

    Article  CAS  PubMed  Google Scholar 

  8. Bovo, E., Lipsius, S. L., & Zima, A. V. (2012). Reactive oxygen species contribute to the development of arrhythmogenic Ca2+ waves during β-adrenergic receptor stimulation in rabbit cardiomyocytes. The Journal of Physiology, 590(14), 3291–3304. https://doi.org/10.1113/jphysiol.2012.230748.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bovo, E., Mazurek, S. R., De Tombe, P. P., & Zima, A. V. (2015). Increased energy demand during adrenergic receptor stimulation contributes to Ca2+ wave generation. Biophysical Journal, 109(8), 1583–1591. https://doi.org/10.1016/j.bpj.2015.09.002.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Branco, A. F., Sampaio, S. F., Wieckowski, M. R., Sardão, V. A., & Oliveira, P. J. (2013). Mitochondrial disruption occurs downstream from β-adrenergic overactivation by isoproterenol in differentiated, but not undifferentiated H9c2 cardiomyoblasts: Differential activation of stress and survival pathways. International Journal of Biochemistry and Cell Biology, 45(11), 2379–2391. https://doi.org/10.1016/j.biocel.2013.08.006.

    Article  CAS  PubMed  Google Scholar 

  11. Branco, A. F., Pereira, S. L., & Oliveira, P. J. (2011). Isoproterenol cytotoxicity is dependent on the differentiation state of the cardiomyoblast H9c2 cell line. Cardiovascular Toxicology, 11, 191–203. https://doi.org/10.1007/s12012-011-9111-5.

    Article  CAS  PubMed  Google Scholar 

  12. Mendis, S., Puska, P., Norrving, B. (Eds.). (2011). Global Atlas on cardiovascular disease prevention and control. Geneva: World Health Organization in collaboration with the World Heart Federation and the World Stroke Organization.

    Google Scholar 

  13. Arts, I. C. W., & Hollman, P. C. H. (2005). Polyphenols and disease risk in epidemiologic studies 1–4. The American Journal of Clinical Nutrition, 81(1), 317S–325S.

    Article  CAS  PubMed  Google Scholar 

  14. Vauzour, D., Rodriguez-Mateos, A., Corona, G., Oruna-Concha, M. J., & Spencer, J. P. E. (2010). Polyphenols and human health: Prevention of disease and mechanisms of action. Nutrients, 2(11), 1106–1131. https://doi.org/10.3390/nu2111106.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Johnson, S. A., Figueroa, A., Navaei, N., Wong, A., Kalfon, R., Ormsbee, L. T., et al. (2015). Daily blueberry consumption improves blood pressure and arterial stiffness in postmenopausal women with pre- and stage 1-hypertension: A randomized, double-blind, placebo-controlled clinical trial. Journal of the Academy of Nutrition and Dietetics, 115(3), 369–377. https://doi.org/10.1016/j.jand.2014.11.001.

    Article  PubMed  Google Scholar 

  16. Watson, R. R., Victor, P., & Zibadi, S. (2014). Polyphenols in human health and disease. San Diego: Academic Press.

    Google Scholar 

  17. Du, G., Sun, L., Zhao, R., Du, L., Song, J., Zhang, L., et al. (2016). Polyphenols: Potential source of drugs for the treatment of ischaemic heart disease. Pharmacology & Therapeutics, 162, 23–34. https://doi.org/10.1016/j.pharmthera.2016.04.008.

    Article  CAS  Google Scholar 

  18. Dolinsky, V. W., Chakrabarti, S., Pereira, T. J., Oka, T., Levasseur, J., Beker, D., et al. (2013). Resveratrol prevents hypertension and cardiac hypertrophy in hypertensive rats and mice. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1832(10), 1723–1733. https://doi.org/10.1016/j.bbadis.2013.05.018.

    Article  CAS  Google Scholar 

  19. Manach, C. (2004). Polyphenols: Food sources and bioavailability. American Journal of Clinical Nutrition, 79(5), 727–747.

    Article  CAS  PubMed  Google Scholar 

  20. Rodriguez-Mateos, A., Heiss, C., Borges, G., & Crozier, A. (2014). Berry (poly)phenols and cardiovascular health. Journal of Agricultural and Food Chemistry, 62(18), 3842–3851. https://doi.org/10.1021/jf403757g.

    Article  CAS  PubMed  Google Scholar 

  21. Pimpão, R. C., Dew, T., Figueira, M. E., Mcdougall, G. J., Stewart, D., Ferreira, R. B., et al. (2014). Urinary metabolite profiling identifies novel colonic metabolites and conjugates of phenolics in healthy volunteers. Molecular Nutrition and Food Research, 58(7), 1414–1425. https://doi.org/10.1002/mnfr.201300822.

    Article  CAS  PubMed  Google Scholar 

  22. Pimpão, R. C., Ventura, M. R., Ferreira, R. B., Williamson, G., & Santos, C. N. (2015). Phenolic sulfates as new and highly abundant metabolites in human plasma after ingestion of a mixed berry fruit purée. The British journal of nutrition, 113(3), 454–463. https://doi.org/10.1017/S0007114514003511.

    Article  CAS  PubMed  Google Scholar 

  23. Nicklas, W., Baneux, P., Boot, R., Decelle, T., Deeny, A. A., Fumanelli, M., et al. (2002). Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units. Laboratory Animals, 36(1), 20–42. https://doi.org/10.1258/0023677021911740.

    Article  CAS  PubMed  Google Scholar 

  24. Louch, W. E., Sheehan, K. A., & Wolska, B. M. (2011). Methods in cardiomyocyte isolation, culture, and gene transfer. Journal of Molecular and Cellular Cardiology, 51(3), 288–298. https://doi.org/10.1016/j.yjmcc.2011.06.012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., et al. (2012). Fiji: An open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. https://doi.org/10.1038/nmeth.2019.

    Article  CAS  PubMed  Google Scholar 

  26. Willis, B. C., Salazar-Cantú, A., Silva-Platas, C., Fernández-Sada, E., Villegas, C., Rios-Argaiz, E., et al. (2015). Impaired oxidative metabolism and calcium mishandling underlie cardiac dysfunction in a rat model of post-acute isoproterenol-induced cardiomyopathy. American Journal of Physiology-Heart and Circulatory Physiology, 308(5), H467–H477. https://doi.org/10.1152/ajpheart.00734.2013.

    Article  CAS  PubMed  Google Scholar 

  27. Moore, M. J., Kanter, J. R., Jones, K. C., & Taylor, S. S. (2002). Phosphorylation of the catalytic subunit of protein kinase A. Journal of Biological Chemistry, 277(49), 47878–47884. https://doi.org/10.1074/jbc.M204970200.

    Article  CAS  PubMed  Google Scholar 

  28. Song, Y.-H., Choi, E., Park, S.-H., Lee, S.-H., Cho, H., Ho, W.-K., et al. (2011). Sustained CaMKII activity mediates transient oxidative stress-induced long-term facilitation of L-type Ca2+ current in cardiomyocytes. Free Radical Biology and Medicine, 51(9), 1708–1716. https://doi.org/10.1016/j.freeradbiomed.2011.07.022.

    Article  CAS  PubMed  Google Scholar 

  29. Mustroph, J., Neef, S., & Maier, L. S. (2017). CaMKII as a target for arrhythmia suppression. Pharmacology and Therapeutics. https://doi.org/10.1016/j.pharmthera.2016.10.006.

    Article  PubMed  Google Scholar 

  30. Dewenter, M., Neef, S., Vettel, C., Lämmle, S., Beushausen, C., Zelarayan, L. C., et al. (2017). Calcium/calmodulin-dependent protein kinase II activity persists during chronic β-adrenoceptor blockade in experimental and human heart failure. Circulation: Heart Failure, 10(5), e003840. https://doi.org/10.1161/CIRCHEARTFAILURE.117.003840.

    CAS  Article  Google Scholar 

  31. Aratyn-Schaus, Y., Pasqualini, F. S., Yuan, H., McCain, M. L., Ye, G. J. C., Sheehy, S. P., et al. (2016). Coupling primary and stem cell-derived cardiomyocytes in an in vitro model of cardiac cell therapy. The Journal of Cell Biology, 212(4), 389–397. https://doi.org/10.1083/jcb.201508026.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bito, V., Sipido, K. R., & Macquaide, N. (2015). Basic methods for monitoring intracellular Ca2+ in cardiac myocytes using Fluo-3. Cold Spring Harbor Protocols, 2015(4), 392–397. https://doi.org/10.1101/pdb.prot076950.

    Article  PubMed  Google Scholar 

  33. Dries, E., Santiago, D. J., Johnson, D. M., Gilbert, G., Holemans, P., Korte, S. M., et al. (2016). Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1 dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft. Journal of Chemical Information and Modeling, 53(9), 1689–1699. https://doi.org/10.1017/CBO9781107415324.004.

    Article  Google Scholar 

  34. Vanni, S., Neri, M., Tavernelli, I., & Rothlisberger, U. (2011). Predicting novel binding modes of agonists to β adrenergic receptors using all-atom molecular dynamics simulations. PLoS Computational Biology, 7(1), e1001053. https://doi.org/10.1371/journal.pcbi.1001053.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lefkowitz, R. J., & Williams, L. T. (1977). Catecholamine binding to the beta-adrenergic receptor. Proceedings of the National Academy of Sciences of the United States of America, 74(2), 515–519.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Deng, H., & Fang, Y. (2013). The three catecholics benserazide, catechol and pyrogallol are GPR35 agonists. Pharmaceuticals, 6(12), 500–509. https://doi.org/10.3390/ph6040500.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ambrosio, C., Molinari, P., Cotecchia, S., & Costa, T. (2000). Catechol-binding serines of beta(2)-adrenergic receptors control the equilibrium between active and inactive receptor states. Molecular Pharmacology, 57(1), 198–210

    CAS  PubMed  Google Scholar 

  38. Moniotte, S., Kobzik, L., Feron, O., Trochu, J.-N., Gauthier, C., & Balligand, J.-L. (2001). Upregulation of 3-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium. Circulation, 103(12), 1649–1655. https://doi.org/10.1161/01.CIR.103.12.1649.

    Article  CAS  PubMed  Google Scholar 

  39. Ufer, C., & Germack, R. (2009). Cross-regulation between β 1- and β 3-adrenoceptors following chronic β-adrenergic stimulation in neonatal rat cardiomyocytes. British Journal of Pharmacology, 158(1), 300–313. https://doi.org/10.1111/j.1476-5381.2009.00328.x.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lohse, M. J., Engelhardt, S., Danner, S., & Böhm, M. (1996). Mechanisms of b-adrenergic receptor desensitization: From molecular biology to heart failure. Basic Research in Cardiology, 91(S1), 29–34. https://doi.org/10.1007/BF00795359.

    Article  CAS  PubMed  Google Scholar 

  41. Hausdorff, W. P., Caron, M. G., & Lefkowitz, R. J. (1990). Turning off the signal: Desensitization of beta-adrenergic receptor function. The FASEB Journal, 4(11), 2881–2889. https://doi.org/10.1096/fasebj.4.11.2165947.

    Article  CAS  PubMed  Google Scholar 

  42. Wallace, C. H. R., Baczkó, I., Jones, L., Fercho, M., & Light, P. E. (2006). Inhibition of cardiac voltage-gated sodium channels by grape polyphenols. British Journal of Pharmacology, 149(6), 657–665. https://doi.org/10.1038/sj.bjp.0706897.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Belevych, A. E. (2002). Genistein inhibits cardiac L-Type Ca2+ channel activity by a tyrosine kinase-independent mechanism. Molecular Pharmacology, 62(3), 554–565. https://doi.org/10.1124/mol.62.3.554.

    Article  CAS  PubMed  Google Scholar 

  44. Obayashi, K., Horie, M., Washizuka, T., Nishimoto, T., & Sasayama, S. (1999). On the mechanism of genistein-induced activation of protein kinase A-dependent Cl—Conductance in cardiac myocytes. Pflügers Archiv European Journal of Physiologygers Archiv European Journal of Physiology, 438(3), 269–277. https://doi.org/10.1007/s004240050909.

    Article  CAS  Google Scholar 

  45. Hool, L. C., Middleton, L. M., & Harvey, R. D. (1998). Genistein increases the sensitivity of cardiac ion channels to beta-adrenergic receptor stimulation. Circulation Research, 83(1), 33–42.

    Article  CAS  PubMed  Google Scholar 

  46. LIEW, R., Macleod, K. T., & COLLINS, P. (2003). Novel stimulatory actions of the phytoestrogen genistein: Effects on the gain of cardiac excitation-contraction coupling. The FASEB Journal, 17(10), 1307–1309. https://doi.org/10.1096/fj.02-0760fje.

    Article  CAS  PubMed  Google Scholar 

  47. Bode, A. M., & Dong, Z. (2015). Toxic phytochemicals and their potential risks for human cancer. Cancer Prevention Research, 8(1), 1–8. https://doi.org/10.1158/1940-6207.CAPR-14-0160.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Pedro Sampaio, from Cilia Regulation and Disease lab, CEDOC, for technical support regarding the cardiomyocyte beating measurement.

Funding

The present work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) [ANR-FCT/BEX-BCM/0001/2013], iNOVA4Health Unit (UID/Multi/04462/2013), the Agence National de la Recherche (France) [Grant ANR-13-ISV1-0001-01] and by the laBex LERMIT. Fundação para a Ciência e Tecnologia provided individual financial support to AG (SFRH/BD/103155/2014), HLAV (IF/00185/2012) and CNS (IF/01097/2013).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Helena L. A. Vieira.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Handling Editor: Shazina Saeed.

Electronic supplementary material

Below is the link to the electronic supplementary material.

12012_2018_9485_MOESM1_ESM.tif

Supplementary Figure 1 Schematic representation of the different phenolic metabolites used. The compounds were present in the human plasma in different concentrations: catechol-O-sulphate: 12 µM; pyrogallol-O-sulphate: 6 µM; and 1-methylpyrogallol-O-sulphate: 3 µM. (TIF 439 KB)

12012_2018_9485_MOESM2_ESM.tif

Supplementary Figure 2 Cell Viability. Cell viability was detected using propidium iodide, of differentiated H9c2 cells, treated with phenolic metabolites for 2 h and exposed to ISO for 48 h. Data are mean ± SD. ****P < 0.0001 versus Control and ####P < 0.0001 versus ISO (ISO: Isoproterenol). (TIF 482 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Dias-Pedroso, D., Guerra, J., Gomes, A. et al. Phenolic Metabolites Modulate Cardiomyocyte Beating in Response to Isoproterenol. Cardiovasc Toxicol 19, 156–167 (2019). https://doi.org/10.1007/s12012-018-9485-8

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12012-018-9485-8

Keywords

  • Cardiomyocytes
  • β-Adrenergic receptors
  • Isoproterenol
  • Human bioavailable phenolic metabolites
  • Polyphenols