Western blot and RTPCR analysis of phosphodiesterase (PDE4) expression in mouse leg muscle

Duchenne muscular dystrophy, an X‐linked recessive disorder, is a progressive skeletal muscle disease caused by absence of the protein dystrophin. Previous work in this lab showed elevated cAMP phosphodiesterase (PDE) activity in muscle from 5 week‐old dystrophic mdx mice versus control 10SnJ mice, followed by a decline in PDE4 activity at fifteen weeks in mdx muscle. We hypothesize that PDE4 expression is specifically and differentially regulated in mdx muscle. Specific PDE4 isoforms in muscle were identified using western blots with commercial affinity‐purified antibodies. A custom made antibody against a conserved peptide in all PDE4 families was used to confirm the other antibodies. PDE4A and PDE4D were identified in 7 week and 8‐week‐old male 10SnJ hind leg muscles, respectively. The presence of PDE4C and PDE4B was not detected in 6 week and 7‐week‐old male 10SnJ hind leg muscles, respectively. To more specifically identify expressed isoforms, the presence of specific PDE4 mRNAs was assessed using RT‐PCR. This approach found expression of mRNA from all four PDE4 families, including PDE4A5, PDE4B2, and at least 2 isoforms of PDE4D. Primers for conserved PDE4C sequences also produced a sequence‐verified fragment. These results lay the groundwork for examining differences in the expression of PDE4 in mdx versus 10SnJ tissue. The project described was supported by NIH Grant Number 1R15AR050408‐01A2 from NIAMS.