Direct inhibition of the pacemaker (I-f) current in rabbit sinoatrial node cells by genistein

1 Genistein is a tyrosine kinase inhibitor which interferes with the activity of several ionic channels either by altering modulatory phosphorylating processes or by direct binding. In whole-cell conditions, genistein induces a partial inhibition of the pacemaker (If) current recorded in cardiac sinoatrial and ventricular myocytes. 2 We investigated the mechanism of action of genistein (50 mM) on the If current in whole-cell, cellattached, and inside-out configurations, and the measured fractional inhibitions were similar: 26.6, 27.2, and 33.6%, respectively. 3 When ATP was removed from the whole-cell pipette solution no differences were revealed in the effect of the drug when compared to metabolically active cells. Genistein fully maintained its blocking ability even when herbimycin, a tyrosine kinase inhibitor, was added to the whole-cell ATP-free pipette solution. 4 Genistein-induced block was independent of the gating state of the channel and did not display voltage or current dependence; this independence distinguishes genistein from all other f-channel blockers. 5 When inside-out experiments were performed to test for a direct interaction with the channel, genistein, superfused on the intracellular side of the membrane, decreased the maximal If conductance, and slightly shifted the current\u2013activation curve to the left. Furthermore, the effect of genistein was independent of cAMP modulation. 6 We conclude that, in addition to its tyrosine kinase-inhibitory properties, genistein also blocks If by directly interacting with the channel, and thus cannot be considered a valuable pharmacological tool to investigate phosphorylation-dependent modulatory pathways of the If current and of cardiac rhythm

Il canale Nav1.1 nel nodo senoatriale di coniglio : struttura primaria, identificazione di varianti di splicing e loro possibile significato funzionale : Knock-down del canale HCN4 nel nodo senoatriale di coniglio neonato mediante la tecnica dell\u2019RNA Interference

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Properties of ivabradine-induced block of HCN1 and HCN4 pacemaker channels

Ivabradine is a 'heart rate-reducing' agent able to slow heart rate, without complicating side-effects. Its action results from a selective and specific block of pacemaker f-channels of the cardiac sinoatrial node (SAN). Investigation has shown that block by ivabradine requires open f-channels, is use dependent, and is affected by the direction of current flow. The constitutive elements of native pacemaker channels are the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, of which four isoforms (HCN1-4) are known; in rabbit SAN tissue HCN4 is expressed strongly, and HCN1 weakly. In this study we have investigated the blocking action of ivabradine on mouse (m) HCN1 and human (h) HCN4 channels heterologously expressed in HEK 293 cells. Ivabradine blocked both channels in a dose-dependent way with half-block concentrations of 0.94 μ for mHCNI and 2.0 μM for hHCN4.Properties of block changed substantially for the two channels. Block of hHCN4 required open channels, was strengthened by depolarization and was relieved by hyperpolarization. Block of mHCN1 did not occur, nor was it relieved, when channels were in the open state during hyperpolarization; block required channels to be either closed, or in a transitional state between open and closed configurations. The dependence of block upon current flow was limited for hHCN4, and not significant for mHCN1 channels. In summary our results indicate that ivabradine is an 'open-channel' blocker of hHCN4, and a 'closed-channel' blocker of mHCN1 channels. The mode of action of ivabradine on the two channels is discussed by implementing a simplified version of a previously developed model of f-channel kinetics

siRNA-mediated knock-down of the HCN4 gene in cultured rabbit neonatal SAN cells

siRNA-mediated knock-down of the HCN4 gene in cultured rabbit neonatal SAN cell

Mechanism of HCN1 and HCN4 block by ivabradine

Mechanism of HCN1 and HCN4 block by ivabradin

Cardiac-derived mesoangioblasts can differentiate into autorhythmic myocytes

Cardiac-derived mesoangioblasts can differentiate into autorhythmic myocyte

Molecular characterization of adult cardiac stem cells during differentiation towards a pacemaker phenotype

Several cardiac diseases cause disturbances in rhythm propagation, requiring a pharmacological treatment, and/or the implantation of electronic pacemakers. An alternative approach to electronic devices is the \u201cbiological\u201d pacemaker. Possible biological pacemakers consist of cells with pacemaker-like properties, able to pace spontaneously and rhythmically. This work aims to characterize a cellular substrate derived from adult murine stem cells. Self-renewing clones of mesoangioblasts (MABS), obtained from ventricle biopsies, were analyzed for the presence of common stem cell markers. Flow cytometry experiments indicated that these cells are positive for the CD34, c-kit, Sca-1 and CD44 antigens, and for the endothelial antigen CD31. When grown in differentiating medium (low serum, 2%) about 5 days, a fraction of cells started to contract spontaneously. Electrophysiological analysis showed that these cells are able to fire action potentials spontaneously and express the pacemaker current with characteristics similar to the native If (Half-activation at -72.5\ub12.07 mV, n=16). Immunofluorescence was used to investigate the expression of HCN channels, the molecular components of native f-channels. HCN4 was the most expressed isoform; a signal for HCN3 could be detected in a few cells, while the HCN1 and HCN2 isoforms were not detected. As expected, only cells presenting an organized cytoskeleton showed some degree of HCN labeling. Moreover, cells with an organized sarcomeric structure expressed the connexin 43 protein, one of the major components of atrial gap junctions, in vicinity of contacts between cells. In conclusion our data show that MABS could be a potential substrate for the development of a biological pacemaker, although their properties must be further investigated