Upregulation of the creatine synthetic pathway in skeletal muscles of mature mdx mice

Abstract

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular human disease caused by dystrophin deficiency. The mdx mouse lacks dystrophin protein, yet does not exhibit the debilitating DMD phenotype. Investigating compensatory mechanisms in the mdx mouse may shed new insights into modifying DMD pathogenesis. This study targets two metabolic genes, guanidinoacetate methyltransferase (GAMT) and arginine:glycine amidinotransferase (AGAT) which are required for creatine synthesis. We show that GAMT and AGAT mRNA are up-regulated 5.4- and 1.9-fold respectively in adult mdx muscle compared to C57. In addition, GAMT protein expression is up-regulated at least 2.5-fold in five different muscles of mdx vs. control. Furthermore, we find GAMT immunoreactivity in up to 80% of mature mdx muscle fibers in addition to small regenerating fibers and rare revertants; while GAMT immunoreactivity is equal to background levels in all muscle fibers of mature C57 mice. The up-regulation of the creatine synthetic pathway may help maintain muscle creatine levels and limit cellular energy failure in leaky mdx skeletal muscles. These results may help better understand the mild phenotype of the mdx mouse and may offer new treatment horizons for DMD.

Figure 1

Average distances run in a 24 hour period were measured daily over a six week interval. In panel A, C57 mice (n=10) are denoted with a solid black box and mdx mice (n=12) are denoted with an open diamond. In panel B, bar graph summarizes the average daily voluntary wheel run distances of mature mdx and C57 mice. No statistical difference is seen.

Figure 2

Quantitative real time Taq-man RT-PCR in gastrocnemius muscles of 16 week old mdx vs C57 mice. In mdx, both GAMT and AGAT mRNAs are significantly elevated, with p = 0.003 and p = 0.047, respectively. Mdx mice (n = 5) are denoted by black and white diagonal bars while C57 (n = 5) are denoted by black and gray diagonal bars.

Figure 3

Panel A shows GAMT immunoblot analysis of five different muscle groups of 16 week old mdx and C57 mice. Lanes 1–4 are mdx and lanes 5–8 are C57. Lane 9 is liver used as a positive control tissue for GAMT protein. GAMT protein has a calculated MW= 26kD. GAST is gastrocnemius; RF is rectus femoris; TA is tibialis anterior; HAM is hamstring; and Dia is diaphragm. N=8–12 in all groups have been tested with this representative blot showing n=4 per group. Two GAMT bands seen in the TA and HAM of C57 have unclear significance. Two alternative transcripts been reported in NCBI database and a human case report from lymphoblastoid tissue suggested the smaller GAMT species lacks a terminal exon and may be non-enzymatic [79]. The predominate GAMT band in the mdx mice is the larger 26 kd species. The function of this smaller GAMT species in C57 is not known but does not undermine the observation that the mdx mouse highly upregulates 26 kD GAMT unlike C57. Panel B shows the upper half of blot membrane in Panel A probed with desmin antibody to verify relative equal loading and internal expression control. Expected molecular weight of desmin = 53 kD. (C) Gastrocnemius immunoblot analysis for GAMT in mdx and C57 mice ages postnatal age 5 weeks, 4 weeks and 2 weeks. Desmin immunoblotting demonstrated equivalent protein loading and internal expression control for each sample.

Figure 4

Panel A. Indirect immunofluorescence of GAMT expression in transverse gastrocnemius cryosections of C57 and mdx mice. Sections were treated identically on the same slide and images were captured using identical exposure parameters. Panel B. Fiber cross-sectional area plotted against relative GAMT expression in 100 fibers of each C57 (O) and mdx gastrocnemius fibers grouped into central (▲) or non-central nucleated fibers (△).

Figure 5

Indirect immunofluorescence on serial cryosections to show both GAMT and EMyHC protein expression in gastrocnemius muscles of 16 weeks old mdx mice. Panel A is immunostained for GAMT (green) and the area within the yellow line is a pathologic area. Panel B is a serial section of panel A, which shows the same region with fibers staining for EMyHC in red, circled with blue. Yellow stars denote fibers that are positive for GAMT and negative for EMyHC. White Xs denote fibers of a similar size that are negative for both GAMT and EMyHC. Adjacent cryosections incubated with a polyclonal antibody against green fluorescent protein in Panel C for non-specific antigenicity labeling and Panel D for secondary antibody only control for non-specific labeling. 16 week old C57 gastrocnemius cryosections are not immunoreactive to GAMT nor EMyHC antibodies (data not shown). Bar = 50 microns.

Figure 6

Panel A – D are serial sections showing an isolated hyper-contracted (pathologic) fiber, denoted with an X, from mature mdx rectus femoris. Panel A. H&E staining. Bar = 40 microns. Panel B. Indirect immunoflourescence of GAMT protein appears green with Alexa 488 plus nuclear DNA (blue) with Hoechst stain. Panel C is the same section as panel B using different filter set to show Evans Blue Dye (red) is leaking into a subset of cells. Panel D shows succinate dehydrogenase (SDH) enzymatic activity with the greatest activity in the darkest fibers. White and black arrows denote fibers that are leaking (EBD positive), expressing GAMT, have high SDH activity but do not appear pathologic on H&E features. Images were captured using a 40X objective lens.

Figure 7

We propose that leaky mdx muscle fibers are able to maintain near normal creatine concentrations by synthesizing creatine de novo through up-regulation of both GAMT and AGAT. Our data shows that creatine levels are similar in the serum and muscle homogenates of both mdx and control mice. The up-regulation of GAMT and AGAT for de novo synthesis of creatine would allow for discrete mdx metabolic adaptations to offset leaky membranes and excess loss of intracellular creatine. Limiting energy failure in muscle cells, mdx mice may be divergent in a unique metabolic adaptation compared to muscles from boys with DMD. Further studies including human analysis are warranted.