Small Fractions of Muscular Dystrophy Embryonic Stem Cells Yield Severe Cardiac and Skeletal Muscle Defects in Adult Mouse Chimeras

Abstract

Duchenne muscular dystrophy (DMD) is characterized by the loss of the protein dystrophin, leading to muscle fragility, progressive weakening, and susceptibility to mechanical stress. Although dystrophin-negative mdx mouse models have classically been used to study DMD, phenotypes appear mild compared to patients. As a result, characterization of muscle pathology, especially in the heart, has proven difficult. We report that injection of mdx embryonic stem cells (ESCs) into Wild Type blastocysts produces adult mouse chimeras with severe DMD phenotypes in the heart and skeletal muscle. Inflammation, regeneration and fibrosis are observed at the whole organ level, both in dystrophin-negative and dystrophin-positive portions of the chimeric tissues. Skeletal and cardiac muscle function are also decreased to mdx levels. In contrast to mdx heterozygous carriers, which show no significant phenotypes, these effects are even observed in chimeras with low levels of mdx ESC incorporation (10%-30%). Chimeric mice lack typical compensatory utrophin upregulation, and show pathological remodeling of Connexin-43. In addition, dystrophin-negative and dystrophin-positive isolated cardiomyocytes show augmented calcium response to mechanical stress, similar to mdx cells. These global effects highlight a novel role of mdx ESCs in triggering muscular dystrophy even when only low amounts are present. Stem Cells 2017;35:597-610.

Connexin-43 reduction prevents muscle defects in a mouse model of manifesting Duchenne muscular dystrophy female carriers

“…WT (C57BL/6J), mdx (C57BL/10ScSn-Dmdmdx/J), and DsRed (B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J), mice were purchased from Jackson Laboratories (Bar Harbor, ME, https:// www.jax.org). Chimeras: Mouse and embryonic stem cell derivation and maintenance, and mouse chimera generation have been previously described in Gonzalez et al 14 . We generated mdx/WT and mdx/ WT-CX43(+/−) chimeras by injecting mdx ESCs into WT:Cx43(+/−) blastocysts.…”

“…We generated mdx/WT and mdx/ WT-CX43(+/−) chimeras by injecting mdx ESCs into WT:Cx43(+/−) blastocysts. Quantification of ESC incorporation by genotyping of inserted genomic DsRed gene in mdx ESCs using primers fwd: 5′-GGTGATGTCCAGCTTGGAGT-3′ and rev: 5′-CCCCGTAATGCAGAAGAAGA-3′ (DsRed Jax) or previously published sequences 58 and protocols 14 . Internal positive control primers provided by Jax Labs were used: fwd: 5′-CTAGGCCACAGAATTGAAAGATCT and rev: 5′-GTAGGTGGAAATTCTAGCATCATCC-3′.…”

“…A few chimeras were also sacrificed at 6 months of age. Chimeras were characterized and matched in pairs according to similar chimeric signatures determined by DsRed fluorescence from tail tip sections collected postnatally 14 and DsRed genomic PCR of tissues collected at sacrifice (Figs. 1A and 2A, Supplementrary Fig.…”

“…WT and mdx/WT-Cx43(+/−) chimeras by injecting mdx (dystrophin−) murine embryonic stem cells (ESCs) into WT:Cx43(+/+) and WT:Cx43(+/−) (dystrophin+) blastocysts 14 . Seven day-old pups were genotyped for the presence of a heterozygous mutation in the Cx43 gene using DNA from tail tips.…”

“…Half of the littermates contained the mutation and half did not (data not shown). Mdx ESCs contain a reporter DsRed transgene inserted into their genome whose ratio of band intensities compared to internal control, supplied by Jax labs, was used to determine the degree of ESC-derived cells (degree of chimerism) 14 . The presence of the DsRed transgene was assessed in pups tail tips (data not shown) 14 and then confirmed at time of sacrifice using heart ( Fig.…”