Phosphodiesterase inhibitor KMUP-3 displays cardioprotection via protein kinase G and increases cardiac output via G-protein-coupled receptor agonist activity and Ca2+ sensitization

Abstract KMUP-3 (7-{2-[4-(4-nitrobenzene) piperazinyl]ethyl}-1, 3-dimethylxanthine) displays cardioprotection and increases cardiac output, and is suggested to increase cardiac performance and improve myocardial infarction. To determine whether KMUP-3 improves outcomes in hypoperfused myocardium by inducing Ca 2+ sensitization to oppose protein kinase (PK)G-mediated Ca 2+ blockade, we measured left ventricular systolic blood pressure, maximal rates of pressure development, mean arterial pressure and heart rate in rats, and measured contractility and expression of PKs/RhoA/Rho kinase (ROCK)II in beating guinea pig left atria. Hemodynamic changes induced by KMUP-3 (0.5–3.0 mg/kg, intravenously) were inhibited by Y27632 [( R )-(+)- trans -4-1-aminoethyl)-N-(4-Pyridyl) cyclohexane carboxamide] and ketanserin (1 mg/kg, intravenously). In electrically stimulated left guinea pig atria, positive inotropy induced by KMUP-3 (0.1–100μM) was inhibited by the endothelial NO synthase (eNOS) inhibitors N -nitro- l -arginine methyl ester (L-NAME) and 7-nitroindazole, cyclic AMP antagonist SQ22536 [9-(terahydro-2-furanyl)-9 H -purin-6-amine], soluble guanylyl cyclase (sGC) antagonist ODQ (1 H -[1,2,4] oxadiazolo[4,3- a ] quinoxalin-1-one), RhoA inhibitor C3 exoenzyme, β-blocker propranolol, 5-hydroxytryptamine 2A antagonist ketanserin, ROCK inhibitor Y27632 and KMUP-1 (7-{2-[4-(2-chlorobenzene) piperazinyl]ethyl}-1, 3-dimethylxanthine) at 10μM. Western blotting assays indicated that KMUP-3 (0.1–10μM) increased PKA, RhoA/ROCKII, and PKC translocation and CIP-17 (an endogenous 17-kDa inhibitory protein) activation. In spontaneous right atria, KMUP-3 induced negative chronotropy that was blunted by 7-nitroindazole and atropine. In neonatal myocytes, L-NAME inhibited KMUP-3-induced eNOS phosphorylation and RhoA/ROCK activation. In H9c2 cells, Y-27632 (50μM) and PKG antagonist KT5823 [2,3,9,10,11,12-hexahydro-10R- methoxy-2,9-dimethyl-1-oxo-9S,12R-epoxy-1H-diindolo(1,2,3-fg:3′,2′,1′-kl) pyrrolo(3,4-i)(1,6)benzodiazocine-10-carboxylic acid, methyl ester] (3μM) reversed KMUP-3 (1–100μM)-induced Ca 2+ -entry blockade. GPCR agonist activity of KMUP-3 appeared opposed to KMUP-1, and increased cardiac output via Ca 2+ sensitization, and displayed cardioprotection via cyclic GMP/PKG-mediated myocardial preconditioning in animal studies.