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. 2010 May;105(3):337-47.
doi: 10.1007/s00395-010-0084-5. Epub 2010 Jan 27.

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

Dong I Lee  1 , Susan Vahebi, Carlo Gabriele Tocchetti, Lili A Barouch, R John Solaro, Eiki Takimoto, David A Kass
Affiliations

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

Dong I Lee et al. Basic Res Cardiol. 2010 May.

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.

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Conflict of interest statement

Conflict of interest statement None.

Figures

Fig. 1
Fig. 1
PDE2 modulation does not underlie suppression of isoproterenol stimulated contractility by the PDE5A inhibitor, sildenafil. a Example tracings (left) and summary results (right) for control C57BL/6 myocytes exposed to ISO (10 nM) followed by ISO + SIL (SIL, 1 µM). SIL suppressed the positive inotropic response to ISO without altering the Ca2+ transient. b Top cardiac myocytes were stimulated with ISO, then co-stimulated with ISO and PDE2 inhibitor (Bay 60-7550, 50 nM). The latter enhanced both contractile and Ca2+ responses to ISO, consistent with a rise in cAMP as PDE2 functions as a cGMP-stimulated cAMP-esterase [20]. Lower however, PDE2 inhibition did not block the suppressive effect of SIL on contraction stimulated by ISO. For all panels: *P < 0.01 versus control, #P < 0.02 versus control, † P < 0.01 versus middle condition (solid black bar). Data are mean ± SEM, n = 7–20 cells from at least four separate hearts in each group
Fig. 2
Fig. 2
PDE3 inhibition does not alter sildenafil-mediated suppression of acute β-adrenergic stimulation. a Cardiac myocytes stimulated with ISO followed by co-stimulation of ISO and the PDE3 inhibitor cilostamide (CIL, 1 µM). PDE3 inhibition did not alter the ISO response, supporting little basal esterase activity. b PDE3 inhibition had no effect on SIL suppression of ISO-stimulated contraction. Symbols and sample size for various studies are as in Fig. 1
Fig. 3
Fig. 3
Beta-3 adrenergic stimulation is required for effectiveness of SIL to suppress acute β-adrenergic contractile stimulation. a Sarcomere shortening and corresponding Ca2+ transients in myocytes genetically lacking the β3-AR receptor. ISO stimulation increased both, but SIL had no suppressive effect on myocyte shortening. b PDE5A protein expression in isolated myocytes from control β3-AR−/− cells was similar. c PDE5A activity measured by fluorescence polarization (FP) as previously described [36]. Consistent with protein expression, PDE5A activity was similar in control and β3-AR−/− myocytes. Thus, the lack of SIL effects was not due to less PDE expression or activity. d Sarcomere shortening and corresponding Ca2+ transients with pre-incubation (20 min) of selective β3-AR antagonist (L-748,337, 1 µM) in control myocytes. SIL did not suppress ISO-stimulated shortening, similar to results in β3-AR−/− cells. Symbols and sample size for various studies are as in Fig. 1
Fig. 4
Fig. 4
PKG-dependent signaling targeting troponin I phosphorylation is central to effect of PDE5A inhibition. a Inhibition of PKG by peptide antagonist DT2 blocks modulation of ISO (1 nM)- stimulated sarcomere shortening by SIL, while augmentation in the Ca2+ transient is unaltered. b Effect of SIL on ISO stimulation in control CD-1 myocytes (upper panels) and myocytes from transgenic hearts expressing the skeletal TnI isoform in myocytes (ssTnItg) (lower panels). Controls behaved as in other mouse strains, with SIL markedly suppressing shortening without altering the calcium transient. However, SIL had no impact on the ISO-stimulated changes in ssTnItg myocytes. Symbols and sample size similar to that for Fig. 1
Fig. 5
Fig. 5
TnI phosphorylation is augmented by SIL above that obtained with ISO in isolated control (C57BL/6), but not in β3-AR−/− cardiomyocytes. Cells isolated from C57BL/6 hearts were stimulated with ISO or ISO + SIL, and TnI phosphorylation was examined using a TnI phospho-S23/S24 antibody. Three primary phosphorylation states were identified, and summary data are shown normalized to the ISO response. a In control myocytes, resting cells displayed weak P1 and P2 states, and ISO stimulation mostly enhanced P2 and revealed an additional P3 state. SIL increased phosphorylation levels in all three states. *P < 0.03 versus ISO, P < 0.005 versus control, †P < 0.05 versus ISO. b In β3-AR−/− myocytes, there was greater resting phosphorylation, shown by a balance favoring P2 and appearance of P3. ISO stimulation resulted in largely a rise in the highest phosphorylation state—P3, and importantly, SIL did not impact this any further. *P < 0.0001 versus other groups, †P < 0.002 versus other two groups. ANOVAs performed using data adjusted for mean value per group
Fig. 6
Fig. 6
Schematic of proposed signaling underlying PDE5A inhibitory effects of acute β-adrenergic stimulation. Potential contributors are shown, with those identified by solid lines/arrows supported by prior and/or present results, while the dashed lines/arrows indicate pathways found unimportant to the SIL modulation in the current analysis. On the left side, activation of β1/β2 predominantly stimulates adenylate cyclase coupled to cAMP generation, which in turn activates PKA phosphorylating the L-type Ca2+ channel (LTCC), phospholamban (PLB), and troponin I (TnI) and enhancing contractility and Ca2+ transients. Co-stimulation of the β3-AR predominantly stimulates nitric oxide synthase (NOS) to activate soluble guanylate cyclase (sGC) generating cGMP. This in turn activates PKG with the dominant effect (related to suppression of contraction) being TnI phosphorylation. Potential regulation of cAMP levels by PDE2 or PDE3—activated or inhibited by cGMP respectively—are shown in the center, but do not appear to regulate SIL effects on β-stimulated contraction
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