No abstract

The blocking efficacy of 4,9-anhydro-TTX (4,9-ah-TTX) and TTX on several isoforms of voltage-dependent sodium channels, expressed in Xenopus laevis oocytes, was tested (Na(v1.2), Na(v1.3), Na(v1.4), Na(v1.5), Na(v1.6), Na(v1.7), and Na(v1.8)). Generally, TTX was 40-231 times more effective, when compared with 4,9-ah-TTX, on a given isoform. An exception was Na(v1.6), where 4,9-ah-TTX in nanomole per liter concentrations sufficed to result in substantial block, indicating that 4,9-ah-TTX acts specifically at this peculiar isoform. The IC(50) values for TTX/4,9-ah-TTX were as follows (in nmol/l): 7.8 +/- 1.3/1,260 +/- 121 (Na(v1.2)), 2.8 +/- 2.3/341 +/- 36 (Na(v1.3)), 4.5 +/- 1.0/988 +/- 62 (Na(v1.4)), 1,970 +/- 565/78,500 +/- 11,600 (Na(v1.5)), 3.8 +/- 1.5/7.8 +/- 2.3 (Na(v1.6)), 5.5 +/- 1.4/1,270 +/- 251 (Na(v1.7)), and 1,330 +/- 459/>30,000 (Na(v1.8)). Analysis of approximal half-maximal doses of both compounds revealed minor effects on voltage-dependent activation only, whereas steady-state inactivation was shifted to more negative potentials by both TTX and 4,9-ah-TTX in the case of the Na(v1.6) subunit, but not in the case of other TTX-sensitive ones. TTX shifted steady-state inactivation also to more negative potentials in case of the TTX-insensitive Na(v1.5) subunit, where it also exerted profound effects on the time course of recovery from inactivation. Isoform-specific interaction of toxins with ion channels is frequently observed in the case of proteinaceous toxins. Although the sensitivity of Na(v1.1) to 4,9-ah-TTX is not known, here we report evidence on a highly isoform-specific TTX analog that may well turn out to be an invaluable tool in research for the identification of Na(v1.6)-mediated function, but also for therapeutic intervention.

Objective: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca 2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na + /Ca 2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes. Methods: Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein-protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy. Results: TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Coimmunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GSTpulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca 2+ entry, which was dependent on TRPC3-mediated Na + loading of myocytes. This NCX-mediated Ca 2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1. Conclusion: These results suggest a PLC-dependent recruitment of a TRPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca 2+ homeostasis.

Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.

Rationale: Myofibroblasts typically appear in the myocardium after insults to the heart like mechanical overload and infarction. Apart from contributing to fibrotic remodeling, myofibroblasts induce arrhythmogenic slow conduction and ectopic activity in cardiomyocytes after establishment of heterocellular electrotonic coupling in vitro. So far, it is not known whether ␣-smooth muscle actin (␣-SMA) containing stress fibers, the cytoskeletal components that set myofibroblasts apart from resident fibroblasts, are essential for myofibroblasts to develop arrhythmogenic interactions with cardiomyocytes.Objective: We investigated whether pharmacological ablation of ␣-SMA containing stress fibers by actin-targeting drugs affects arrhythmogenic myofibroblast-cardiomyocyte cross-talk. Methods and Results:Experiments were performed with patterned growth cell cultures of neonatal rat ventricular cardiomyocytes coated with cardiac myofibroblasts. The preparations exhibited slow conduction and ectopic activity under control conditions. Exposure to actin-targeting drugs (Cytochalasin D, Latrunculin B, Jasplakinolide) for 24 hours led to disruption of ␣-SMA containing stress fibers. In parallel, conduction velocities increased dose-dependently to values indistinguishable from cardiomyocyte-only preparations and ectopic activity measured continuously over 24 hours was completely suppressed. Mechanistically, antiarrhythmic effects were due to myofibroblast hyperpolarization (Cytochalasin D, Latrunculin B) and disruption of heterocellular gap junctional coupling (Jasplakinolide), which caused normalization of membrane polarization of adjacent cardiomyocytes. Conclusions:The results suggest that ␣-SMA containing stress fibers importantly contribute to myofibro- Key Words: arrhythmia Ⅲ conduction Ⅲ fibroblast Ⅲ gap junction Ⅲ myocardial structure F ibrotic remodeling of the working myocardium is a common consequence of various insults to the heart like pressure/volume overload, infarction, genetic predisposition, and old age. 1 Apart from compromising mechanical function, fibrotic remodeling is a major cause of cardiac arrhythmias. Arrhythmogenesis in fibrotically remodeled hearts is thought to be due to the disruption of the normally dense and orderly three-dimensional cytoarchitecture of parenchymal cells by excessive amounts of extracellular matrix (ECM) produced by stromal cells. The resulting disorganization of the electrically excitable substrate causes slow conduction (discontinuous and/or "zigzag" conduction) and conduction blocks that contribute to the precipitation of arrhythmias. 2,3 Stromal cells responsible for fibrotic remodeling include so-called myofibroblasts (MFBs), which contribute to fibrosis by hypersecretion of ECM proteins. 4 MFBs, which are not normally present in healthy myocardia, typically appear in the context of the pathologies mentioned and tend to locally persist for extended periods of time.Apart from contributing to fibrotic remodeling, MFBs recently came into focus as a cell type that might direct...