RESUMEN
AIMS: In atrial fibrillation, increased function of the Na+/Ca2+-exchanger (NCX) is one among several electrical remodeling mechanisms. METHODS/RESULTS: Using the patch-clamp- and Ca2+ imaging-methods, we investigated atrial myocytes from NCX-homozygous-overexpressor (OE)- and heterozygous-knockout (KO)-mice and their corresponding wildtypes (WTOE; WTKO). NCX mediated Ca2+ extrusion capacity was reduced in KO and increased in OE. There was no evidence for structural or molecular remodeling. During a proarrhythmic pacing-protocol, the number of low amplitude delayed afterdepolarizations (DADs) was unaltered in OE vs. WTOE and KO vs. WTKO. However, DADs triggered full spontaneous action potentials (sAP) significantly more often in OE vs. WTOE (ratio sAP/DAD: OE:0.18±0.05; WTOE:0.02±0.02; p<0.001). Using the same protocol, a DAD triggered an sAP by tendency less often in KO vs. WTKO (p=0.06) and significantly less often under a more aggressive proarrhythmic protocol (ratio sAP/DAD: KO:0.01±0.003; WT KO: 0.12±0.05; p=0.007). The DAD amplitude was increased in OE vs. WTOE and decreased in KO vs. WTKO. There were no differences in SR-Ca2+-load, the number of spontaneous Ca2+-release-events or IKACh/IK1. CONCLUSIONS: Atrial myocytes with increased NCX expression exhibited increased vulnerability towards sAPs while atriomyocytes with reduced NCX expression were protected. The underlying mechanism consists of a modification of the DAD-amplitude by the level of NCX-activity. Thus, although the number of spontaneous Ca2+-releases and therefore DADs is unaltered, the higher DAD-amplitude in OE made a transgression of the voltage-threshold of an sAP more likely. These findings indicate that the level of NCX activity could influence triggered activity in atrial myocytes independent of possible remodeling processes.
Asunto(s)
Atrios Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Potenciales de Acción/genética , Animales , Calcio/metabolismo , Señalización del Calcio , Femenino , Expresión Génica , Masculino , Potenciales de la Membrana/genética , Ratones , Ratones Transgénicos , Contracción Miocárdica/genética , Miocardio/metabolismo , Retículo Sarcoplasmático/metabolismo , Intercambiador de Sodio-Calcio/genéticaRESUMEN
The expression of the tobacco (Nicotiana tabacum) retrotransposon Tnt1 has previously been shown to be strongly regulated and driven from the 5' long terminal repeat (LTR). We report here that the Tnt1 LTR can promote activity of the beta-glucuronidase (GUS) reporter gene in two heterologous species of the Brassicaceae family, namely rapeseed (Brassica napus) and Arabidopsis thaliana. The translational LTR-GUS fusion was active in transient expression studies performed with tobacco and rapeseed protoplasts, indicating that the LTR sequences are recognized in heterologous species. Our results also showed that Tnt1 LTR-promoted GUS expression in transgenic Arabidopsis is strongly regulated, and that, in contrast to tobacco, hormonal activation plays a significant role in the expression of the Tnt1 LTR in Arabidopsis. LTR sequences were shown to be more effective than the CaMV 35S enhancer region in transient expression studies performed with tobacco or rapeseed protoplasts, and substitution of the LTR sequences upstream from the major transcriptional start with the CaMV 35S enhancer region gave high levels of expression in transgenic tobacco and Arabidopsis leaves, suggesting that a Tnt1 element with similar substitutions in its 5' LTR might be suited for gene-tagging experiments in heterologous species.