Simultaneous Dystrophin and Dysferlin Deficiencies Associated with High-Level Expression of the Coxsackie and Adenovirus Receptor in Transgenic Mice

Abstract

The Coxsackie and adenovirus receptor (CAR) , a cell adhesion molecule of the immunoglobulin superfamily , is usually confined to the sarcolemma at the neuromuscular junction in mature skeletal muscle fibers. Previously , we reported that adenovirusmediated gene transfer is greatly facilitated in hemizygous transgenic mice with extrasynaptic CAR expression driven by a muscle-specific promoter. However , in the present study , when these mice were bred to homozygosity , they developed a severe myopathic phenotype and died prematurely. Large numbers of necrotic and regenerating fibers were present in the skeletal muscle of the homozygous CAR transgenics. The myopathy was further characterized by increased levels of caveolin-3 and ␤-dystroglycan and decreased levels of dystrophin , dysferlin , and neuronal nitric-oxide synthase. Even the hemizygotes manifested a subtle phenotype , displaying deficits in isometric force generation and perturbed mitogen-activated protein kinase (MAPK-erk1/2) activation during contraction. There are few naturally occurring or engineered mouse lines showing as severe a skeletal myopathy as observed with ectopic expression of CAR in the homozygotes. Taken together , these findings suggest that substantial overexpression of CAR may lead to physiological dysfunction by disturbing sarcolemmal integrity (through dystrophin deficiency) , impairing sarcolemmal repair (through dysferlin deficiency) , and interfering with normal signaling (through alterations in caveolin-3 and neuronal nitric-oxide synthase levels). (Am J Pathol

Modulation of coxsackie and adenovirus receptor expression for gene transfer to normal and dystrophic skeletal muscle

“…To determine whether the levels of CAR could be limiting even within the context of regenerating skeletal muscle, transgenic mice expressing CAR were bred into the mdx background. The immunofluorescent localization of CAR in these mdx /CAR mice was reminiscent of the pattern that we described previously for the CAR hemizygous transgenics 17. Examination of frozen cryostat sections of TA muscle immunostained with an antibody raised against the extracellular domain of CAR (ab 2240) revealed plasma membrane (sarcolemmal) expression in all fibers (Figure 1A); CAR also accumulated ectopically within the cytoplasm (Figure 1A), as reported previously for the CAR transgenics 17.…”

“…From the transgenesis experiments, it was not clear what degree of increase of CAR expression was required to improve transducibility. Although much higher levels of CAR are present in the skeletal muscle of the mdx /CAR mice as determined by western blot analysis (Figure 1C), most of the CAR protein accumulates intracellularly, with lesser amounts being present on the sarcolemmal membrane (Figure 1A) as reported previously 17. However, even in normal skeletal muscle, in which CAR is confined to the neuromuscular junction, a local increase of CAR expression through the administration of recombinant AAV2‐CAR resulted in a significant increase in subsequent AdV transducibility (Figure 4).…”

“…The immunofluorescent localization of CAR in these mdx /CAR mice was reminiscent of the pattern that we described previously for the CAR hemizygous transgenics 17. Examination of frozen cryostat sections of TA muscle immunostained with an antibody raised against the extracellular domain of CAR (ab 2240) revealed plasma membrane (sarcolemmal) expression in all fibers (Figure 1A); CAR also accumulated ectopically within the cytoplasm (Figure 1A), as reported previously for the CAR transgenics 17. By contrast, in the regenerating fibers of mdx muscle, the sarcolemmal expression of CAR was patchier (Figure 1B), rarely occurring in a continuous pattern as observed in the mdx /CAR mice.…”

The IgCAMs CAR, BT-IgSF, and CLMP: Structure, Function, and Diseases

“…In adult skeletal muscle fi bers CAR is confi ned to the sarcolemma at the neuromuscular junction (Nalbantoglu et al 1999 ;Shaw et al 2004 ) whereas in diseased muscle with necrosis and regeneration (polymyositis and Duchenne muscular dystrophy, DMD) extrasynaptic sarcolemmal and cytoplasmic CAR is found to be co-expressed with regeneration markers such as desmin and utrophin (Sinnreich et al 2005 ). Additionally, similar to cardiac overexpression of CAR, homozygous transgenic mice, in which CAR is regulated by the muscle creatine kinase (MCK) promoter, showed a severe myopathy with a large numbers of necrotic and regenerating fi bers and premature death that was associated with an upregulation of caveolin-3 levels and defi ciencies in dystrophin and dysferlin (Shaw et al 2006 ). In summary, the general view that emerges from these studies on cardiac and skeletal muscles is that CAR serves as a factor that is transiently expressed during development to establish cell-cell contact-mediated signaling.…”

A Method of Utrophin Up-Regulation through RNAi-Mediated Knockdown of the Transcription Factor EN1

“…18 Additionally, utrophin is up-regulated during periods where there is lack of necrosis in dystrophindeficient muscle. 19 To identify additional transcription factors that may regulate UTRN gene expression, the role of transcription factors was examined in the present study. Homeobox protein engrailed-1 (EN1) is an attractive candidate because it has two potential binding sites present in the UTRN promoter, one of which lies in close proximity to the N-box site (Fig.…”