Basic Research in Cardiology

, Volume 105, Issue 3, pp 337–347 | Cite as

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

  • Dong I. Lee
  • Susan Vahebi
  • Carlo Gabriele Tocchetti
  • Lili A. Barouch
  • R. John Solaro
  • Eiki Takimoto
  • David A. KassEmail author
Original Contribution


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.


Phosphodiesterase Sildenafil Myocytes Adrenergic Contractility Calcium Troponin I Beta-3 adrenergic receptor Protein kinase G Cyclic GMP 



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).

Conflict of interest statement


Supplementary material

395_2010_84_MOESM1_ESM.ppt (234 kb)
(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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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Dong I. Lee
    • 1
  • Susan Vahebi
    • 1
  • Carlo Gabriele Tocchetti
    • 1
  • Lili A. Barouch
    • 1
  • R. John Solaro
    • 2
  • Eiki Takimoto
    • 1
  • David A. Kass
    • 1
    Email author
  1. 1.Ross 858, Division of Cardiology, Department of MedicineJohns Hopkins University Medical InstitutionsBaltimoreUSA
  2. 2.Department of Physiology and BiophysicsUniversity of Illinois at ChicagoChicagoUSA

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