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PDE5A suppression of acute β-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation

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Abstract

Phosphodiesterase type 5A (PDE5A) inhibitors acutely suppress beta-adrenergic receptor (β-AR) stimulation in left ventricular myocytes and hearts. This modulation requires cyclic GMP synthesis via nitric oxide synthase (NOS)-NO stimulation, but upstream and downstream mechanisms remain un-defined. To determine this, adult cardiac myocytes from genetically engineered mice and controls were studied by video microscopy to assess sarcomere shortening (SS) and fura2-AM fluorescence to measure calcium transients (CaT). Enhanced SS from isoproterenol (ISO, 10 nM) was suppressed ≥50% by the PDE5A inhibitor sildenafil (SIL, 1 μM), without altering CaT. This regulation was unaltered despite co-inhibition of either the cGMP-stimulated cAMP-esterase PDE2 (Bay 60-7550), or cGMP-inhibited cAMP-esterase PDE3 (cilostamide). Thus, the SIL response could not be ascribed to cGMP interaction with alternative PDEs. However, genetic deletion (or pharmacologic blockade) of β3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in β3-AR knockout (β3-AR−/−) myocytes as in controls. Downstream, cGMP stimulates protein kinase G (PKG), and we found contractile modulation by SIL required PKG activation and enhanced TnI phosphorylation at S23, S24. Myocytes expressing the slow skeletal TnI isoform which lacks these sites displayed no modulation of ISO responses by SIL. Non-equilibrium isoelectric focusing gel electrophoresis showed SIL increased TnI phosphorylation above that from concomitant ISO in control but not β3-AR−/− myocytes. These data support a cascade involving β3-AR stimulation, and subsequent PKG-dependent TnI S23, S24 phosphorylation as primary factors underlying the capacity of acute PDE5A inhibition to blunt myocardial β-adrenergic stimulation.

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References

  1. Borlaug BA, Melenovsky V, Marhin T, Fitzgerald P, Kass DA (2005) Sildenafil inhibits beta-adrenergic-stimulated cardiac contractility in humans. Circulation 112:2642–2649

    Article  CAS  PubMed  Google Scholar 

  2. Bremer YA, Salloum F, Ockaili R, Chou E, Moskowitz WB, Kukreja RC (2005) Sildenafil citrate (viagra) induces cardioprotective effects after ischemia/reperfusion injury in infant rabbits. Pediatr Res 57:22–27

    Article  CAS  PubMed  Google Scholar 

  3. Brixius K, Bloch W, Ziskoven C, Bolck B, Napp A, Pott C, Steinritz D, Jiminez M, Addicks K, Giacobino JP, Schwinger RH (2006) Beta3-adrenergic eNOS stimulation in left ventricular murine myocardium. Can J Physiol Pharmacol 84:1051–1060

    Article  CAS  PubMed  Google Scholar 

  4. Castro LR, Verde I, Cooper DM, Fischmeister R (2006) Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes. Circulation 113:2221–2228

    Article  CAS  PubMed  Google Scholar 

  5. Ding B, Abe J, Wei H, Huang Q, Walsh RA, Molina CA, Zhao A, Sadoshima J, Blaxall BC, Berk BC, Yan C (2005) Functional role of phosphodiesterase 3 in cardiomyocyte apoptosis: implication in heart failure. Circulation 111:2469–2476

    Article  CAS  PubMed  Google Scholar 

  6. Dostmann WR, Taylor MS, Nickl CK, Brayden JE, Frank R, Tegge WJ (2000) Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Ialpha inhibit NO-induced cerebral dilation. Proc Natl Acad Sci USA 97:14772–14777

    Article  CAS  PubMed  Google Scholar 

  7. Fischmeister R, Castro LR, Abi-Gerges A, Rochais F, Jurevicius J, Leroy J, Vandecasteele G (2006) Compartmentation of cyclic nucleotide signaling in the heart: the role of cyclic nucleotide phosphodiesterases. Circ Res 99:816–828

    Article  CAS  PubMed  Google Scholar 

  8. Fisher PW, Salloum F, Das A, Hyder H, Kukreja RC (2005) Phosphodiesterase-5 inhibition with sildenafil attenuates cardiomyocyte apoptosis and left ventricular dysfunction in a chronic model of doxorubicin cardiotoxicity. Circulation 111:1601–1610

    Article  CAS  PubMed  Google Scholar 

  9. Gauthier C, Leblais V, Kobzik L, Trochu JN, Khandoudi N, Bril A, Balligand JL, Le Marec H (1998) The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle. J Clin Invest 102:1377–1384

    Article  CAS  PubMed  Google Scholar 

  10. Gauthier C, Seze-Goismier C, Rozec B (2007) Beta 3-adrenoceptors in the cardiovascular system. Clin Hemorheol Microcirc 37:193–204

    CAS  PubMed  Google Scholar 

  11. Kobayashi T, Jin L, de Tombe PP (2008) Cardiac thin filament regulation. Pflugers Arch 457:37–46

    Article  CAS  PubMed  Google Scholar 

  12. Kobayashi T, Yang X, Walker LA, Van Breemen RB, Solaro RJ (2005) A non-equilibrium isoelectric focusing method to determine states of phosphorylation of cardiac troponin I: identification of Ser-23 and Ser-24 as significant sites of phosphorylation by protein kinase C. J Mol Cell Cardiol 38:213–218

    Article  CAS  PubMed  Google Scholar 

  13. Kooij V, Boontje N, Zaremba R, Jaquet K, Dos RC, Stienen GJ, van der Velden (2009) Protein kinase C alpha and epsilon phosphorylation of troponin and myosin binding protein C reduce Ca(2+) sensitivity in human myocardium. Basic Res Cardiol. doi:10.1007/s00395-009-0053-z

  14. Layland J, Li JM, Shah AM (2002) Role of cyclic GMP-dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes. J Physiol 540:457–467

    Article  CAS  PubMed  Google Scholar 

  15. Leroy J, Abi-Gerges A, Nikolaev VO, Richter W, Lechene P, Mazet JL, Conti M, Fischmeister R, Vandecasteele G (2008) Spatiotemporal dynamics of beta-adrenergic cAMP signals and L-type Ca2+ channel regulation in adult rat ventricular myocytes: role of phosphodiesterases. Circ Res 102:1091–1100

    Article  CAS  PubMed  Google Scholar 

  16. Marston SB, de Tombe PP (2008) Troponin phosphorylation and myofilament Ca2+-sensitivity in heart failure: increased or decreased? J Mol Cell Cardiol 45:603–607

    Article  CAS  PubMed  Google Scholar 

  17. Martinez SE, Wu AY, Glavas NA, Tang XB, Turley S, Hol WG, Beavo JA (2002) The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding. Proc Natl Acad Sci USA 99:13260–13265

    Article  CAS  PubMed  Google Scholar 

  18. Massion PB, Dessy C, Desjardins F, Pelat M, Havaux X, Belge C, Moulin P, Guiot Y, Feron O, Janssens S, Balligand JL (2004) Cardiomyocyte-restricted overexpression of endothelial nitric oxide synthase (NOS3) attenuates beta-adrenergic stimulation and reinforces vagal inhibition of cardiac contraction. Circulation 110:2666–2672

    Article  CAS  PubMed  Google Scholar 

  19. Moens AL, Leyton-Mange JS, Niu X, Yang R, Cingolani O, Arkenbout EK, Champion HC, Bedja D, Gabrielson KL, Chen J, Xia Y, Hale AB, Channon KM, Halushka MK, Barker N, Wuyts FL, Kaminski PM, Wolin MS, Kass DA, Barouch LA (2009) Adverse ventricular remodeling and exacerbated NOS uncoupling from pressure-overload in mice lacking the beta3-adrenoreceptor. J Mol Cell Cardiol 47:576–585

    Article  CAS  PubMed  Google Scholar 

  20. Mongillo M, Tocchetti CG, Terrin A, Lissandron V, Cheung YF, Dostmann WR, Pozzan T, Kass DA, Paolocci N, Houslay MD, Zaccolo M (2006) Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway. Circ Res 98:226–234

    Article  CAS  PubMed  Google Scholar 

  21. Moniotte S, Kobzik L, Feron O, Trochu JN, Gauthier C, Balligand JL (2001) Upregulation of beta(3)-adrenoceptors and altered contractile response to inotropic amines in human failing myocardium. Circulation 103:1649–1655

    CAS  PubMed  Google Scholar 

  22. Nagayama T, Hsu S, Zhang M, Koitabashi N, Bedja D, Gabrielson K, Takimoto E, Kass DA (2009) Sildenafil stops progressive chamber, cellular, and molecular remodeling and improves calcium handling and function in hearts with pre-existing advanced hypertrophy due to pressure-overload. J Am Coll Cardiol 53:207–215

    Article  CAS  PubMed  Google Scholar 

  23. Nagayama T, Zhang M, Hsu S, Takimoto E, Kass DA (2008) Sustained soluble guanylate cyclase stimulation offsets nitric-oxide synthase inhibition to restore acute cardiac modulation by sildenafil. J Pharmacol Exp Ther 326:380–387

    Article  CAS  PubMed  Google Scholar 

  24. Nagendran J, Archer SL, Soliman D, Gurtu V, Moudgil R, Haromy A, St Aubin C, Webster L, Rebeyka IM, Ross DB, Light PE, Dyck JR, Michelakis ED (2007) Phosphodiesterase type 5 is highly expressed in the hypertrophied human right ventricle, and acute inhibition of phosphodiesterase type 5 improves contractility. Circulation 116:238–248

    Article  CAS  PubMed  Google Scholar 

  25. Piggott LA, Hassell KA, Berkova Z, Morris AP, Silberbach M, Rich TC (2006) Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments. J Gen Physiol 128:3–14

    Article  CAS  PubMed  Google Scholar 

  26. Pokreisz P, Vandenwijngaert S, Bito V, Van den BA, Lenaerts I, Busch C, Marsboom G, Gheysens O, Vermeersch P, Biesmans L, Liu X, Gillijns H, Pellens M, Van Lommel A, Buys E, Schoonjans L, Vanhaecke J, Verbeken E, Sipido K, Herijgers P, Bloch KD, Janssens SP (2009) Ventricular phosphodiesterase-5 expression is increased in patients with advanced heart failure and contributes to adverse ventricular remodeling after myocardial infarction in mice. Circulation 119:408–416

  27. Recchia FA, McConnell PI, Bernstein RD, Vogel TR, Xu X, Hintze TH (1998) Reduced nitric oxide production and altered myocardial metabolism during the decompensation of pacing-induced heart failure in the conscious dog. Circ Res 83:969–979

    CAS  PubMed  Google Scholar 

  28. Rozec B, Gauthier C (2006) beta3-adrenoceptors in the cardiovascular system: putative roles in human pathologies. Pharmacol Ther 111:652–673

    Article  CAS  PubMed  Google Scholar 

  29. Salloum FN, Abbate A, Das A, Houser JE, Mudrick CA, Qureshi I, Hoke NN, Roy SK, Brown WR, Prabhakar S (2008) Kukreja RC (2008) Sildenafil (Viagra) Attenuates Ischemic Cardiomyopathy and Improves Left VentricularFunction in Mice. Am J Physiol Heart Circ Physiol 294:H1398–H1406

    Article  CAS  PubMed  Google Scholar 

  30. Satoh S, Makino N (2001) Intracellular mechanisms of cGMP-mediated regulation of myocardial contraction. Basic Res Cardiol 96:652–658

    Article  CAS  PubMed  Google Scholar 

  31. Senzaki H, Smith CJ, Juang GJ, Isoda T, Mayer SP, Ohler A, Paolocci N, Tomaselli GF, Hare JM, Kass DA (2001) Cardiac phosphodiesterase 5 (cGMP-specific) modulates beta-adrenergic signaling in vivo and is down-regulated in heart failure. FASEB J 15:1718–1726

    Article  CAS  PubMed  Google Scholar 

  32. Shah AM, Spurgeon HA, Sollott SJ, Talo A, Lakatta EG (1994) 8-bromo-cGMP reduces the myofilament response to Ca2+ in intact cardiac myocytes. Circ Res 74:970–978

    CAS  PubMed  Google Scholar 

  33. Surapisitchat J, Jeon KI, Yan C, Beavo JA (2007) Differential regulation of endothelial cell permeability by cGMP via phosphodiesterases 2 and 3. Circ Res 101:811–818

    Article  CAS  PubMed  Google Scholar 

  34. Takimoto E, Belardi D, Tocchetti CG, Vahebi S, Cormaci G, Ketner EA, Moens AL, Champion HC, Kass DA (2007) Compartmentalization of cardiac beta-adrenergic inotropy modulation by phosphodiesterase type 5. Circulation 115:2159–2167

    Article  CAS  PubMed  Google Scholar 

  35. Takimoto E, Champion HC, Belardi D, Moslehi J, Mongillo M, Mergia E, Montrose DC, Isoda T, Aufiero K, Zaccolo M, Dostmann WR, Smith CJ, Kass DA (2005) cGMP catabolism by phosphodiesterase 5A regulates cardiac adrenergic stimulation by NOS3-dependent mechanism. Circ Res 96:100–109

    Article  CAS  PubMed  Google Scholar 

  36. Takimoto E, Champion HC, Li M, Belardi D, Ren S, Rodriguez ER, Bedja D, Gabrielson KL, Wang Y, Kass DA (2005) Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nat Med 11:214–222

    Article  CAS  PubMed  Google Scholar 

  37. Tsai EJ, Kass DA (2009) Cyclic GMP signaling in cardiovascular pathophysiology and therapeutics. Pharmacol Ther 122:216–238

    Article  CAS  PubMed  Google Scholar 

  38. Varghese P, Harrison RW, Lofthouse RA, Georgakopoulos D, Berkowitz DE, Hare JM (2000) beta(3)-adrenoceptor deficiency blocks nitric oxide-dependent inhibition of myocardial contractility. J Clin Invest 106:697–703

    Article  CAS  PubMed  Google Scholar 

  39. Vila-Petroff MG, Younes A, Egan J, Lakatta EG, Sollott SJ (1999) Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes. Circ Res 84:1020–1031

    CAS  PubMed  Google Scholar 

  40. Wang H, Kohr MJ, Traynham CJ, Ziolo MT (2009) Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca2+ current in cardiac myocytes. J Mol Cell Cardiol 47:304–314

    Article  CAS  PubMed  Google Scholar 

  41. Wattanapermpool J, Guo X, Solaro RJ (1995) The unique amino-terminal peptide of cardiac troponin I regulates myofibrillar activity only when it is phosphorylated. J Mol Cell Cardiol 27:1383–1391

    Article  CAS  PubMed  Google Scholar 

  42. Wolska BM, Vijayan K, Arteaga GM, Konhilas JP, Phillips RM, Kim R, Naya T, Leiden JM, Martin AF, de Tombe PP, Solaro RJ (2001) Expression of slow skeletal troponin I in adult transgenic mouse heart muscle reduces the force decline observed during acidic conditions. J Physiol 536:863–870

    Article  CAS  PubMed  Google Scholar 

  43. Wu AY, Tang XB, Martinez SE, Ikeda K, Beavo JA (2004) Molecular determinants for cyclic nucleotide binding to the regulatory domains of phosphodiesterase 2A. J Biol Chem 279:37928–37938

    Article  CAS  PubMed  Google Scholar 

  44. Yang L, Liu G, Zakharov SI, Bellinger AM, Mongillo M, Marx SO (2007) Protein kinase G phosphorylates Cav1.2 alpha1c and beta2 subunits. Circ Res 101:465–474

    Article  CAS  PubMed  Google Scholar 

  45. Yasuda S, Coutu P, Sadayappan S, Robbins J, Metzger JM (2007) Cardiac transgenic and gene transfer strategies converge to support an important role for troponin I in regulating relaxation in cardiac myocytes. Circ Res 101:377–386

    Article  CAS  PubMed  Google Scholar 

  46. Zaccolo M (2006) Phosphodiesterases and compartmentalized cAMP signalling in the heart. Eur J Cell Biol 85:693–697

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Chad M. Warren for his assistance in performing some of the isoelectric gel electrophoresis studies. This work was supported by Public Health Service NHLBI grants: HL-089297, HL-095408 (DAK), and T32 HL-07227 (DAK, SV), and RO1 HL-022231, and PO1-HL-062426 (RJS).

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Authors and Affiliations

  1. Ross 858, Division of Cardiology, Department of Medicine, Johns Hopkins University Medical Institutions, 720 Rutland Ave, Baltimore, MD, 21205, USA

    Dong I. Lee, Susan Vahebi, Carlo Gabriele Tocchetti, Lili A. Barouch, Eiki Takimoto & David A. Kass

  2. Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA

    R. John Solaro

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  1. Dong I. Lee
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  2. Susan Vahebi
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Correspondence to David A. Kass.

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D. I. Lee and S. Vahebi contributed equally to the work.

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395_2010_84_MOESM1_ESM.ppt

(A) Sarcomere shortening and corresponding Ca2+ transients in myocytes before and after treatment with the selective PDE2 inhibitor Bay 60-7550 (50 nM). Bay 60-7550, itself, had a no effect on either cell shortening or the Ca2+ transient in control (C57BL/6) myocytes. Example tracings are on left, summary data to the right. (B) Sarcomere shortening and corresponding Ca2+ transients in control myocytes before and after exposure to the PDE3 inhibitor cilostamide (CIL, 1 µM). CIL alone had no effect on either behavior in myocytes. Example tracings are on the left, summary data to the right. (C) (A) PDE3 inhibition alone (CIL) does not alter sarcomere shortening or calcium transients in myocytes lacking the β3-adrenergic receptor. (B) ISO stimulated contraction and calcium transients are unaltered by co-repression of PDE3 in β3 knockout cells. (C) SIL does not significantly alter contraction (nor calcium transients) in β3 knockout cells stimulated with ISO. See text for details (PPT 234 kb)

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Lee, D.I., Vahebi, S., Tocchetti, C.G. et al. PDE5A suppression of acute β-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation. Basic Res Cardiol 105, 337–347 (2010). https://doi.org/10.1007/s00395-010-0084-5

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