RESUMO
AIM: To test a hypothesis attributing arrhythmia in Brugada Syndrome to right ventricular (RV) outflow tract (RVOT) conduction abnormalities arising from Nav 1.5 insufficiency and fibrotic change. METHODS: Arrhythmic properties of Langendorff-perfused Scn5a+/- and wild-type mouse hearts were correlated with ventricular effective refractory periods (VERPs), multi-electrode array (MEA) measurements of action potential (AP) conduction velocities and dispersions in conduction direction (CD), Nav 1.5 expression levels, and fibrotic change, as measured at the RVOT and RV. Two-way anova was used to test for both independent and interacting effects of anatomical region and genotype on these parameters. RESULTS: Scn5a+/- hearts showed greater arrhythmic frequencies during programmed electrical stimulation at the RVOT but not the RV. The Scn5a+/- genotype caused an independent increase of VERP regardless of whether the recording site was the RVOT or RV. Effective AP conduction velocities (CVs), derived from fitting regression planes to arrays of observed local activation times were reduced in Scn5a+/- hearts and at the RVOT independently. AP conduction velocity magnitudes derived by averaging MEA results from local vector analyses, CV*, were reduced by the Scn5a+/- genotype alone. In contrast, dispersions in conduction direction, were greater in the RVOT than the RV, when the atrioventricular node was used as the pacing site. The observed reductions in Nav 1.5 expression were attributable to Scn5a+/-, whereas increased levels of fibrosis were associated with the RVOT. CONCLUSIONS: The Scn5a+/- RVOT recapitulates clinical findings of increased arrhythmogenicity through reduced CV reflecting reduced CV* attributable to reduced Nav 1.5 expression and increased CD attributable to fibrosis.
Assuntos
Arritmias Cardíacas/fisiopatologia , Síndrome de Brugada/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Coração/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Western Blotting , Modelos Animais de Doenças , Eletrofisiologia , Feminino , Masculino , Camundongos , Camundongos Mutantes , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Técnicas de Cultura de ÓrgãosRESUMO
AIM: clinical observations suggest that alternans in action potential (AP) characteristics presages breakdown of normal ordered cardiac electrical activity culminating in ventricular arrhythmogenesis. We compared such temporal nonuniformities in monophasic action potential (MAP) waveforms in left (LV) and right ventricular (RV) epicardia and endocardia of Langendorff-perfused murine wild-type (WT), and Scn5a(+/-) hearts modelling Brugada syndrome (BrS) for the first time. METHODS: a dynamic pacing protocol imposed successively incremented steady pacing rates between 5.5 and 33 Hz. A signal analysis algorithm detected sequences of >10 beats showing alternans. Results were compared before and following the introduction of flecainide (10 microm) and quinidine (5 microm) known to exert pro- and anti-arrhythmic effects in BrS. RESULTS: sustained and transient amplitude and duration alternans were both frequently followed by ventricular ectopic beats and ventricular tachycardia or fibrillation. Diastolic intervals (DIs) that coincided with onsets of transient (tr) or sustained (ss) alternans in MAP duration (DI*) and amplitude (DI') were determined. Kruskal-Wallis tests followed by Bonferroni-corrected Mann-Whitney U-tests were applied to these DI results sorted by recording site, pharmacological conditions or experimental populations. WT hearts showed no significant heterogeneities in any DI. Untreated Scn5a (+/-) hearts showed earlier onsets of transient but not sustained duration alternans in LV endocardium compared with RV endocardium or LV epicardium. Flecainide administration caused earlier onsets of both transient and sustained duration alternans selectively in the RV epicardium in the Scn5a (+/-) hearts. CONCLUSION: these findings in a genetic model thus implicate RV epicardial changes in the arrhythmogenicity produced by flecainide challenge in previously asymptomatic clinical BrS.
Assuntos
Potenciais de Ação/fisiologia , Coração/fisiopatologia , Canais de Sódio/genética , Animais , Síndrome de Brugada/genética , Síndrome de Brugada/fisiopatologia , Estimulação Cardíaca Artificial , Eletrofisiologia , Camundongos , Camundongos Mutantes , Canal de Sódio Disparado por Voltagem NAV1.5 , Técnicas de Cultura de ÓrgãosRESUMO
The ovine NRAMP1 and cervine NRAMP1 cDNAs were cloned by RT PCR of RNA derived from macrophage enriched leukocyte preparations. The complete coding and 3' regions were sequenced. Both sheep and deer NRAMP1 proteins are 548 amino acids long. There are 77 and 73 amino acid differences, respectively, compared to the mouse Nramp1 sequence. Dinucleotide repeats were found in both the ovine and cervine 3' non-coding sequence. Amplification of these regions in individual sheep and deer showed them to be polymorphic microsatellites. They have polymorphism information content values of 0.76 and 0.84 in sheep and deer, respectively. Using these microsatellites, the ovine NRAMP1 gene was mapped in a linkage group on ovine chromosome 2q and cervine NRAMP1 was mapped in a linkage group syntenic with human chromosome 2, mouse chromosome 1 and sheep chromosome 2.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte de Cátions , Cervos/genética , Imunidade Inata/genética , Proteínas de Membrana/genética , Ovinos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/química , Bovinos , Mapeamento Cromossômico/veterinária , Clonagem Molecular , Sequência Conservada , Primers do DNA , DNA Complementar/química , DNA Complementar/genética , Repetições de Dinucleotídeos/genética , Eletroforese em Gel de Poliacrilamida/veterinária , Humanos , Macrófagos/química , Macrófagos/fisiologia , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Técnica de Amplificação ao Acaso de DNA Polimórfico/veterinária , Análise de Sequência de DNA/veterinária , Transcrição GênicaRESUMO
Retrotransposons are mobile genetic elements. They can transpose via the reverse transcription of mRNA into double-stranded DNA (dsDNA) followed by the insertion of this dsDNA into new sites within the host genome. The unintegrated, linear, dsDNA form of retrotransposons is usually very rare. We report here the isolation of a retrotransposon from Candida albicans which is unusual in this respect. This element, which we have named pCal, was first identified as a distinct band when uncut C. albicans DNA was examined on an agarose gel. Sequence analysis of the cloned element revealed that it is a retrotransposon belonging to the Ty1/copia group. It is estimated that pCal produces 50 to 100 free, linear, dsDNA copies of itself per cell. This is a much higher level of expression than even that of the system in which Ty1 is expressed behind the highly active GAL1 promoter on a high-copy-number plasmid (about 10 copies per cell). Another unusual feature of pCal is that its Pol enzymes are likely to be expressed via the pseudoknot-assisted suppression of an upstream, in-phase stop codon, as has been shown for Moloney murine leukemia virus.