onist. Quantification of Ca2 spike frequency of co-stimulated CMs with a constant concentration of nonanoic acid and a rising concentration of the antagonist in Ca2 imaging experiments. The data are shown as the mean SEM. Significance was calculated by Student’s t test 123 Basic Res Cardiol 112:13 Page 11 of 20 13 Fig. 5 OR51E1 signaling in CMs. a Detection of transcripts of ORsignaling pathway components, including Gaolf, adenylyl cyclase III and CNG channel subunits in 169939-93-9 web ventricle and septum of explanted human heart and iCell-CMs by RT-PCR. b Verification of Gaolf protein and AC-III expression in human heart tissue by western blot. The molecular size of the two splice variants of Gas/olf are 45 kDa and 52 kDa. The 39 kDa band may represent the proteolytic fragment of Gas/olf. c Co-immunoprecipitations of different G protein alpha-subunits with OR51E1 protein. The human heart lysate was immunoprecipitated with one of the indicated G proteins and detected with antibodies against the OR51E1 protein. d Representative recordings of Fura-2 loaded iCell-CMs. Co-application of nonanoic acid and inhibitor gallein. e Quantification of nonanoic acid-induced negative chronotropic effects by pretreating with or without gallein in Ca2 imaging experiments. The data are shown as the mean SEM. Significance was calculated Student’s t test or MannWhitney U test represent a population of ventricular, atrial, and nodal cells, we observed the RNA expression of three members of the canonical olfactory pathway, GNAL, ADCY3 and CNGA2, whereas olfactory CNGA4 and CNGB1 mRNA expression was not observed. By comparing expression profiles, we concluded that the OR51E1-initiated signal transduction mechanism in cardiomyocytes differs from the canonical olfactory signaling pathway. However, we aimed to elucidate which G protein couples to OR51E1 in the human heart and could show that Gas/olf interacts with OR51E1 by performing co-immunoprecipitations. The antibodies against different Ga subunits pulled down protein complexes from lysates of the isolated human heart tissue. The western blots revealed that OR51E1 protein was co-precipitated with Gas/ olf protein. A weak band for OR51E1 is also visible in the Gai sample. We next aimed to pharmacologically characterize in vitro the nonanoic acid-induced signaling that triggers the observed negative 
chronotropic effect. In pacemaker cells of the human heart, stimulation of the muscarinic acetylcholine receptor M2 by acetylcholine or carbachol mediates negative chronotropic and inotropic effects by coupling to a PTX-sensitive G protein, which results in the inhibition of adenylyl cyclase and thereby a decrease in cAMP level and protein kinase A phosphorylation. Furthermore, the Gbc subunit of the G protein activates G protein regulated inward-rectifier potassium channels, which cause a hyperpolarization of the cell membrane and move the sinoatrial node further from depolarization. Thus, it takes longer for HCN channels to depolarize the cell, resulting in a reduced heart rate. Using the stem cell-derived cardiomyocyte model, we excluded the notion that nonanoic acid activates the muscarinic receptor pathway because the Gi/Go protein inhibitor pertussis toxin showed no effect on the nonanoic acid-induced negative chronotropic effect in Ca2 imaging experiments. However, gallein, an inhibitor of G protein bc subunit-dependent signaling, significantly abolished the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19793655 123 13 Page 12 of 20 Basic Res Cardiol 112:13 nonanoic acid-ind
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