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 a parts per thousand yen50% by the PDE5A inhibitor sildenafil (SIL, 1 mu M), 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 beta 3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in beta 3-AR knockout (beta 3-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 beta 3-AR(-/-) myocytes. These data support a cascade involving beta 3-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.
PDE5A suppression of acute beta-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation / Lee Dong, I.; Vahebi, Susan; Tocchetti, CARLO GABRIELE; Barouch Lili, A.; Solaro R., John; Takimoto, Eiki; Kass David, A.. - In: BASIC RESEARCH IN CARDIOLOGY. - ISSN 0300-8428. - 105:3(2010), pp. 337-347. [10.1007/s00395-010-0084-5]
PDE5A suppression of acute beta-adrenergic activation requires modulation of myocyte beta-3 signaling coupled to PKG-mediated troponin I phosphorylation
TOCCHETTI, CARLO GABRIELE;
2010
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 a parts per thousand yen50% by the PDE5A inhibitor sildenafil (SIL, 1 mu M), 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 beta 3-ARs, which couple to NOS signaling, fully prevented SIL modulation of ISO-stimulated SS. Importantly, both PDE5A protein expression and activity were similar in beta 3-AR knockout (beta 3-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 beta 3-AR(-/-) myocytes. These data support a cascade involving beta 3-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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
