Abstract-To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43 Ϫ/Ϫ mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, g j , was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye. Compared with wild-type (Cx43 ϩ/ϩ ) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43 ϩ/Ϫ strands and was absent in Cx43 Ϫ/Ϫ strands. Cx45 signal was reduced by 46% in Cx43 ϩ/Ϫ strands and to the limit of detection in Cx43 Ϫ/Ϫ strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in Ϸ 2% of myocytes. Intercellular conductance, g j , was reduced by 32% in Cx43 ϩ/Ϫ cell pairs and by 96% in Cx43 Ϫ/Ϫ cell pairs. The symmetrical dependence of g j on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43Ϫ/Ϫ cells. Propagation in Cx43 Ϫ/Ϫ strands was very slow (2.1 cm/s versus 52 cm/s in Cx43 ϩ/ϩ ) and highly discontinuous, with simultaneous excitation within and long conduction delays (2 to 3 ms) between individual cells. Propagation was abolished by 1 mmol/L heptanol, indicating residual junctional coupling. In summary, knockout of Cx43 in ventricular myocytes leads to very slow conduction dependent on the presence of Cx45. Electrical field effect transmission does not contribute to propagation in synthetic strands. Key Words: Cx43 Ⅲ Cx45 Ⅲ very slow electrical propagation Ⅲ discontinuous propagation Ⅲ heptanol C onnexin (Cx) proteins form intercellular channels enabling the intercellular exchange of ions and small molecules. 1 In the heart, they facilitate rapid, coordinated electrical excitation, a prerequisite for normal rhythmic contraction. Three different connexins, Cx43, Cx40, and Cx45, are expressed in heart. 2,3 Cx43, the most abundant connexin, is found in ventricular and atrial myocardium. Cx40 is expressed in atrial tissue and the cardiac conduction system. Although Cx45 expression in working ventricular myocardium is modest compared with Cx43, Cx45 is vital for early embryogenesis and cardiac development. 4,5 Cx45 is also expressed in the sinoatrial and atrioventricular nodes. It colocalizes with Cx40 in the conduction system, 6 and with Cx43 in ventricular myocardium. 7 The three cardiac connexins form channels with unique properties. 8 Although multiple connexins are coexpressed in cardiac tissues, their interactions and contributions to electrical or metabolic function are not understood completely. Cx43 and Cx45 likely form heteromeric/heterotypic channels, 9 -12 which may fulfill distinct functions. Cardiac diseases that lead to arrhythmias are associated with gap junction remodeling. [13][14][15] Therefore, know...
Primary cultured human coronary myocytes (HCMs) derived from ischemic human hearts express an atypical voltage-gated tetrodotoxin (TTX)-sensitive sodium current (I(Na)). The whole-cell patch-clamp technique was used to study the properties of I(Na) in HCMs. The variations of intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were monitored in non-voltage-clamped cells loaded with Fura-2 or benzofuran isophthalate, respectively, using microspectrofluorimetry. The activation and steady-state inactivation properties of I(Na) determined a "window" current between -50 and -10 mV suggestive of a steady-state Na+ influx at the cell resting membrane potential. Consistent with this hypothesis, the resting [Na+]i was decreased by TTX (1 micromol/L). In contrast, it was increased by Na+ channel agonists that also promoted a large rise in [Ca2+]i. Veratridine (10 micromol/L), toxin V from Anemonia sulcata (0.1 micromol/L), and N-bromoacetamide (300 micromol/L) increased [Ca2+]i by 7- to 15-fold. This increase was prevented by prior application of TTX or lidocaine (10 micromol/L) and by the use of Na(+)-free or Ca(2+)-free external solutions. The Ca(2+)-channel antagonist nicardipine (5 micromol/L) blocked the effect of veratridine on [Ca2+]i only partially. The residual component disappeared when external Na+ was replaced by Li+ known to block the Na+/Ca2+ exchanger. The resting [Ca2+]i was decreased by TTX in some cells. In conclusion, I(Na) regulates [Ca2+]i in primary cultured HCMs. This regulation, effective at baseline, involves a tonic control of Ca2+ influx via depolarization-gated Ca2+ channels and, to a lesser extent, via a Na+/Ca2+ exchanger working in the reverse mode.
Although the long-lasting effects of neurotrophins have been extensively studied, less data are available on their rapid effects, especially on peptide release. In the present report, we investigated rapid effects of neurotrophins on somatostatin release and on intracellular calcium concentration ([Ca2+]i) in primary cultures of hypothalamic neurons. RT-PCR experiments revealed mRNA expression of the three high-affinity neurotrophin receptors tyrosine kinase (Trk) TrkA, TrkB and TrkC, indicating potential responses to their preferential ligands: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3), respectively. We demonstrated that BDNF, and to a lesser extent NT-3, induced significant time- and concentration-dependent somatostatin release, while NGF was devoid of any effect. BDNF or NT-3 induction of somatostatin release was inhibited by the Trk inhibitors K-252a and genistein, whereas K-252b, a less effective inhibitor, had no effect. BDNF- and NT-3-induced somatostatin release depended upon extra- and intracellular Ca2+ since it was completely abolished in the presence of the Ca2+ chelators BAPTA (bis-(α-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid) or BAPTA-AM (bis-(α-aminophenoxy)-ethane-N,N,N′,N′-tetraacetoxymethylester), respectively. In addition, BDNF and NT-3 induced a sustained and rapid increase in [Ca2+]i which depended on the extracellular Ca2+ concentration. MK-801 (dizocilpine) and tetrodotoxin (TTX) entirely blocked neurotrophin-evoked somatostatin release and [Ca2+]i rise in response to BDNF and NT-3 application in most neurons. Neurotrophin-induced [Ca2+]i rise was completely blocked by K-252a. The present results are consistent with: (1) an indirect effect of neurotrophins on somatostatin release via endogenous glutamate release and subsequent NMDA receptor activation, (2) a major indirect effect of neurotrophins on Ca2+ rise in hypothalamic neurons which very likely occurs through NMDA receptor activation. Taken altogether, these results indicate that BDNF and NT-3 can rapidly affect the activity of hypothalamic neurons.
Primary cultured human coronary myocytes express a tetrodotoxin-sensitive sodium current (I(Na)). Here, we have investigated whether I(Na) is expressed in vascular smooth muscles cells (VSMCs) isolated from other large arteries, and other mammals. VSMCs were enzymatically dissociated, kept in primary culture, and macroscopic I(Na) was recorded using the whole-cell patch-clamp technique. We found that I(Na) is expressed in VSMCs grown from human aortic (90%; n=50) and pulmonary (44%; n=19) arteries, and in the human aortic myocyte cell line HAVSMC (94%; n=27). I(Na) was also detected in pig coronary (60%; n=33), and rabbit aortic (47%; n=15), but not in rat aortic VSMCs (n=20). These different I(Na) had similar voltage thresholds for activation (approximately equal to -50 mV), and were highly sensitive to extracellularly applied tetrodotoxin. We conclude that I(Na) is expressed in VSMCs grown from various types of large arteries in humans, pig and rabbit.
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