Figure 4

Delayed expression of adult RyR1 mRNA splice variant in muscles from mouse models of SMA. (A) RT-PCR on RNA from hindlimb muscle from wild type mice with primers directed against ASII (+) and ASII (-). GAPDH served as a loading control to confirm equivalence of starting cDNA levels . Note that relative ratio of ASII (+) to ASII (-) increases from P2 to P21. (B) RT-PCR results demonstrated no change in the expression of ASI (+) and ASI (-) variants in control and Smn^-/-;SMN2 samples at P5 (upper panel). However, there was decreased expression of ASII (+) and sustained expression of ASII (-) in muscle samples from P5 Smn^-/-;SMN2 compared with controls (middle panel). GAPDH served as a loading control. N = 5 for each genotype. (C) In control P21 mice, we observed increased expression of ASI (+) transcripts relative to ASI (-) transcripts. However in Smn^2B/- mice, the relative ratio of ASI (+) to ASI (-) transcripts was decreased (upper panel). Furthermore, for the ASII variant, we observed the presence of a single transcript [ASII (+)] in P21 control samples, while in Smn^2B/- samples, we observed a decrease in ASII (+) transcripts compared with controls. The ASII (-) variant was also now apparent (middle panel). GAPDH served as a loading control. N = 5 for each variant. (D) Quantification of RT-PCR data show significant changes in the ASII+/ASII - ratio in Smn^-/-;SMN2 samples compared with controls. The relative levels of adult and neonatal RYR1 isoforms was significantly altered for both the ASI and ASII variants in Smn^2B/- animals compared with controls. (E,F) The relative levels of adult and neonatal ASII RyR1 transcript variants are not altered in P14 mice one (E) and seven (F) days post-denervation compared with sham operated mice. N = 3.