Abstract
Purpose:
Duchenne and Becker Muscular Dystophy (DMD/BMD) patients with mutations that overlap dystrophin isoform Dp260 have a scotopic ERG with a reduced b-wave amplitude. The mdx3cv mouse model of DMD in which no known dystrophin isoforms are expressed also demonstrates a negative scotopic ERG. We previously found that in the mdx3cv , the b-wave component of the ERG was normal but a superimposed abnormal slow PIII component resulted in the ERG defect. This finding is consistent with the observation that dystrophin isoform Dp71 is required to localize the Kir4.1 channel to the Müller cell end-feet. Absence of Dp-71 results in altered distribution of Kir4.1 channels which affects potassium homeostasis. Mice that lack only Dp71, however, have a normal ERG (Dalloz et al., 2003). In this study, we aimed to determine how over-expression of Dp71 would affect the ERG.
Methods:
The Dp71-TG mouse has a Dp71 transgene including the alternatively spliced exon 78 on a C57BL/6 background, leading to over-expression of Dp71. Genotyping was confirmed by PCR. ERGs were performed using the HMsERG (Ocuscience, Kansas, USA) adapted for isofluorane anesthesia. Scotopic ERGs were performed at half log unit intervals from an intensity of 0.03 to 30 mcd.s/m2. Amplitudes and implicit times for both a- and b-waves were measured and averaged. The experiments were performed using 4-6 week old Dp71-TG mice and results were compared using age and sex-matched C57Bl/6 mice. The Student t-test was used for statistical comparison and a value of p < 0.05 was deemed to be significant.
Results:
The PCR product length was approx. 580 bp, as expected. Sequencing of the PCR product confirmed the presence of dystrophin Dp 71. Average a-wave amplitude and implicit times were 191 ± 16 µV and 26 ± 2 mS respectively for the C57 mouse vs. 178 ± 19 µV and 24 ± 1 mS for Dp71-TG mice using a 3 mcd.s/m2 flash. The corresponding b-wave values were 422 ± 45 µV and 42 ± 3 mS for C57 compared to 420 ± 78 and 42 ± 3 mS for Dp71-TG mice. The results between the two groups were not significantly different.
Conclusions:
Mice expressing excess Dp71 have no b-wave abnormality. Specifically, these mice did not show a supernormal ERG b-wave. These findings, in concert with the previous work by Dalloz and colleagues (2003) showing a normal ERG b-wave in a Dp71 null mouse, raise questions regarding the proposed roles of Dp71 in the normal mouse ERG that merit further consideration.
Keywords: 510 electroretinography: non-clinical •
538 gene transfer/gene therapy •
533 gene/expression