Fig 1.
The inotropic response to OR-1896 is enhanced by PDE4 inhibition and absent in the presence of PDE3 inhibition.
The figure shows representative original tracings of experiments showing the effect of increasing concentrations of OR-1896 (OR) alone and in the presence of PDE4 (A) and PDE3 (B) inhibitors. A) Effect of OR-1896 in increasing concentrations (OR-7: 100 nM, OR-6: 1 μM and OR-5: 10 μM, n = 6) and the effect of OR-1896 in the presence of the PDE4 inhibitor rolipram (Rol, 10 μM, n = 6). B) Effect of OR-1896 in the presence of a PDE3 inhibitor, either cilostamide (Cil, 1 μM, n = 6) or milrinone (Mil, 1 μM). A, B) EMD57033 (EMD, 3 μM) was added after OR-5. Isoprenaline (Iso, 100 μM) administered at the end gave the maximum inotropic response achievable in the strip. C) Bar graph showing the inotropic response to different concentrations of OR-1896 in the presence and absence (Ctr) of PDE inhibitors in rat myocardial strips (n = 6). D) Bar graph comparing the inotropic response of OR-1896 (1 μM) to the inotropic response of different PDE inhibitors in rat ventricular strips (n = 6). All experiments were conducted on rat ventricular muscle strips in the absence of timolol and in the presence of α1-AR (prazosin, 100 nM) and muscarinic receptor blockade (atropine, 1 μM). Basal force values for each group (mN): OR-1896: 4.3±0.7; Rol: 3.5±0.5; Cil: 3.2±0.5; Mil: 2.6±0.3.
Fig 2.
The OR-1896-evoked inotropic response is associated with a lusitropic response with similar characteristics of cAMP-dependence.
A) Lusitropic response, shown as the change in relaxation time (RT) compared to basal elicited by OR-1896 and known PDE inhibitors in rat ventricular strips in the absence of timolol (n = 6). B&C) Bar graphs showing the effect of carbachol (CCh, 20 μM) on the inotropic and lusitropic response to combined OR-1896 (1 μM) and rolipram stimulation in rat ventricular strips (n = 6 strips from 3 rats). The results were compared to a group which received timolol (Tim) prior to OR-1896 and rolipram (n = 6 strips from 3 rats). Basal force values (mN): Ctr: 5.0±1.1 vs Tim: 3.7±1.2 A, B, C) PDE4 inhibitor: Rolipram (Rol, 10 μM), A) PDE3 inhibitors: cilostamide (Cil, 1 μM), milrinone (Mil, 1 μM). All data are mean ± SEM, * = p<0.05.
Fig 3.
A) Lack of Ca2+ sensitization by OR-1896.
The effect of EMD57033 (EMD, 3 μM, n = 7) or OR-1896 (1 μM, n = 7)) on the Ca2+ concentration-response relationship. Basal force values (mN): Ctr: 2.5±0.3 vs. OR-1896: 2.4±0.3. Mean ± SEM, * p<0.05. B) PDE3 inhibition by OR-1896. Effect of OR-1896 (1 μM) and PDE inhibitors (Cilostamide, Cil 1 μM; Rolipram, Rol 10 μM) upon PDE activity in homogenates of normal rat heart ventricle. Mean ± SEM, *** = p<0.005, ns = non-significant.
Fig 4.
OR-1896 causes cAMP increase by PDE3 inhibition.
A, C, E) Experimental readouts of cAMP measured as the FRET signal from the RII_epac sensor in isolated cardiomyocytes. All cells were primed with a small concentration of isoprenaline (5 nM) prior to PDE inhibition. Forskolin (Fsk, 3 μM) was administered at the end as a measure of the maximum achievable FRET response, indicating that the sensors were not fully saturated during measurements. The readouts are normalized to the maximal response to Fsk. B) Bar graph showing the FRET response to OR-1896 (OR, 1 μM), followed by rolipram (Rol, 10 μM) and the attenuated response to cilostamide (Cil, 1 μM) following OR-1896 + Rol compared to Rol alone (n = 38 cells from 3 animals) The FRET responses are compared to a set of cells that did not receive OR-1896 (Ctr). D) Bar graph showing the FRET response to OR-1896 and the attenuated response to Cil after OR-1896 (n = 26 cells from 2 animals). F) Bar graph showing the FRET response to Cil and the attenuated response to OR-1896 after Cil (n = 24 cells from 2 animals). Data are mean ± SEM, * = p<0.05, ** = p<0.05 compared to OR-1896 (Fig. 4D), *** = p<0.05 compared to Cil (Fig. 4F), # = p<0.005.
Fig 5.
Effect of OR-1896, levosimendan and the Ca2+ sensitizer EMD57033 on concentration-response curves to β- and α1-adrenoceptor stimulation of inotropic response in rat ventricular strips.
A) The effect of OR-1896 (1 μM, n = 7) and EMD57033 (EMD, 3 μM, n = 7) alone and in combination (n = 7) on the concentration-response curves of isoprenaline in the presence of α1-adrenergic (prazosin, 100 nM) and muscarinic (atropine, 1 μM) receptor blockade. Basal force values (mN): Ctr: 3.9±0.3, OR-1896: 3.1±0.4, EMD: 3.4±0.4, EMD+OR-1896: 3.4±0.3. B) The effect of levosimendan (Lev, 1 μM, n = 14) and EMD alone (n = 12) and in combination (n = 7) on the concentration-response curves of isoprenaline in the presence of α1-adrenergic and muscarinic receptor blockade. Basal force values (mN): Ctr: 3.9±0.4, Lev: 3.7±0.4, EMD: 3.7±0.3, EMD+Lev: 3.2±0.4. C) The effect of levosimendan (1 μM, n = 7), OR-1896 (1 μM, n = 15) and EMD (3 μM, n = 14) on the concentration-response curves of phenylephrine in the presence of β-adrenergic (timolol, 1 μM) and muscarinic receptor blockade. Basal force values (mN): Ctr: 4.2±0.5, Lev: 4.0±0.3, OR-1896: 4.5±0.3, EMD: 3.5±0.4. Data are mean ± SEM, * = p<0.05.