title: PDE5A suppression of acute beta-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation.
abstract: Phosphodiesterase type 5A (PDE5A) inhibitors acutely suppress beta-adrenergic receptor (beta-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 >or=50% by the PDE5A inhibitor sildenafil (SIL, 1 microM), 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 beta3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in beta3-AR knockout (beta3-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 beta3-AR(-/-) myocytes. These data support a cascade involving beta3-AR stimulation, and subsequent PKG-dependent TnI S23, S24 phosphorylation as primary factors underlying the capacity of acute PDE5A inhibition to blunt myocardial beta-adrenergic stimulation.
authors.affiliation: Ross 858, Division of Cardiology, Department of Medicine, Johns Hopkins University Medical Institutions, Baltimore, MD, 21205, USA.
authors.forename: Dong I
authors.initials: DI
authors.lastname: Lee
authors.forename: Susan
authors.initials: S
authors.lastname: Vahebi
authors.forename: Carlo Gabriele
authors.initials: CG
authors.lastname: Tocchetti
authors.forename: Lili A
authors.initials: LA
authors.lastname: Barouch
authors.forename: R John
authors.initials: RJ
authors.lastname: Solaro
authors.forename: Eiki
authors.initials: E
authors.lastname: Takimoto
authors.forename: David A
authors.initials: DA
authors.lastname: Kass
mesh_terms: D000818:Animals; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2 / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D023421:Models, Animal; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D058986:Phosphodiesterase 5 Inhibitors; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D015363:Quinolones / Q000494:pharmacology; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D000068677:Sildenafil Citrate; D013450:Sulfones / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology; D019210:Troponin I / Q000378:metabolism*
chemical_list: C539142:2-(3,4-dimethoxybenzyl)-7-(1-(1-hydroxyethyl)-4-phenylbutyl)-5-methylimidazo(5,1-f)(1,2,4)triazin-4 (3H)-one; D007093:Imidazoles; D058986:Phosphodiesterase 5 Inhibitors; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D015363:Quinolones; D011943:Receptors, Adrenergic, beta; D022702:Receptors, Adrenergic, beta-3; D013450:Sulfones; D014227:Triazines; D019210:Troponin I; C021294:cilostamide; D000068677:Sildenafil Citrate; D017869:Cyclic GMP-Dependent Protein Kinases; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517289:Pde3a protein, mouse; C517293:Pde3b protein, mouse; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517382:Pde5a protein, mouse