Pathological alterations in the gastrointestinal tract of a porcine model of DMD

Abstract

Background: Patients with Duchenne muscular dystrophy (DMD) develop severe skeletal and cardiac muscle pathologies, which result in premature death. Therefore, the current therapeutic efforts are mainly targeted to correct dystrophin expression in skeletal muscle and heart. However, it was reported that DMD patients may also exhibit gastrointestinal and nutritional problems. How the pathological alterations in gastrointestinal tissues contribute to the disease are not fully explored.

Results: Here we employed the CRISPR/Cas9 system combined with somatic nuclear transfer technology (SCNT) to establish a porcine model of DMD and explored their pathological alterations. We found that genetic disruption of dystrophin expression led to morphological gastrointestinal tract alterations, weakened the gastrointestinal tract digestion and absorption capacity, and eventually led to malnutrition and gastric dysfunction in the DMD pigs.

Conclusions: This work provides important insights into the pathogenesis of DMD and highlights the need to consider the gastrointestinal dysfunction as an additional therapeutic target for DMD patients.

Keywords: CRISPR; Duchenne muscular dystrophy; Gastrointestinal tract; Genome editing; Pig; Porcine; Swine.

Fig. 1

PFFs with DMD exon 51 deficiency showed impaired cell membrane integrity and early cell apoptosis. A Sanger sequencing of PFFs showed different mutations induced by Cas9/sgRNA electrotransfection. WT sequence is shown at the top of the targeting sequence. PAM sequences are highlighted in red. B NRD uptake assay of PFF clones carrying DMD exon 51 mutations at different cell densities. ***P < 0.001, **P < 0.01 and *P < 0.05. C LDH activities in culture medium at different time points were measured by the LDH-kit. ***P < 0.001, **P < 0.01 and *P < 0.05. D Cell apoptosis was analyzed by flow cytometry. *P < 0.05. E Western blotting analysis of autophagy in PFFs did not detect significant difference between WT and DMD-mutant PFFs

Fig. 2

Generation and identification of DMD exon 51 defective pigs. A The construction flowchart of Bama miniature pigs with edited DMD exon 51. ①② Isolation and culture of miniature PFFs; ③ Transfection of miniature PFFs with Cas9/sgRNA; ④ Single cell clone picking and culturing; ⑤ Acquisition of oocytes; ⑥ Enucleation of oocytes; ⑦ Somatic cell nuclear transfer; ⑧ Embryo transfer; ⑨ Delivery and identification of DMD-delE51 pigs. B PCR analysis of DMD exon 51 in all piglets. C Mutation analysis by T-cloning and Sanger sequencing in all piglets. WT sequence is shown at the top. PAM sites are highlighted in red; target sequences are shown in green. D The relative expression of DMD mRNA in DMD-delE51 pigs and the age-matched wild-type pigs. DIA diaphragm, GAST gastrocnemius; ***P < 0.001. E Western blotting showed the disrupted expression of dystrophin in DMD-delE51 pigs. F IHC analysis of dystrophin expression in heart, diaphragm and gastrocnemius muscles. Scale bars: 50 µm and 100 µm

Fig. 3

Development of muscular dystrophy and cardiomyopathy in DMD-delE51 pigs. A Piglets carrying DMD exon 51 mutations showed abnormal posture. B Survival curves of DMD-delE51 pigs and WT-pigs. C–E H&E staining of heart, diaphragm and gastrocnemius muscle sections from WT and DMD-delE51 pigs at the age of 12 weeks. Scale bars: 100 µm. Fiber fracture (green arrows) and hypertrophic fiber (black arrows) were seen in heart sections. Excessive fiber size variation (red arrows), central nucleated fibers (yellow arrows), fiber fracture (green arrows) and inflammatory cell infiltration (blue arrows) were readily visible in muscle sections. F Serum biochemical profiles of WT and DMD-delE51 pigs. ***P < 0.001, **P < 0.01 and *P < 0.05. G, I Quantification of cross-sectional area (CSA) of diaphragm and gastrocnemius muscle fibers in WT and DMD-delE51 pigs. H, J Quantification of centrally nucleated fiber (CNF) percentage in WT and DMD-delE51 pigs. K, L Size distribution of diaphragm and gastrocnemius muscle in WT and DMD-delE51 pigs. Scale bars: 100 µm. ***P < 0.001, **P < 0.01 and *P < 0.05; n = 5

Fig. 4

DMD-delE51 pigs exhibited symptoms of malnutrition. A Photograph of DMD-delE51 pigs and WT control at the age of 1 week. B Body mass of DMD-delE51 and WT pigs from birth to 12 weeks of age. C, D Serum ALB and PA levels of WT and DMD-delE51 pigs. E, F The contents of glycogen and triglyceride in diaphragm and gastrocnemius muscle. G H&E staining of the small intestine sections of WT and DMD-delE51 pigs. Red dotted lines indicate the thickness of the intestinal wall, black arrows indicate the height of the small intestine villi and blue arrows indicate the depth of the small intestine crypt. H The thickness of small intestinal wall in WT and DMD-delE51 pigs. I The relative ratio of small intestine villus height/crypt depth of WT and DMD-delE51 pigs. Scale bars: 50 µm and 100 µm. ***P < 0.001, **P < 0.01 and *P < 0.05; n = 5

Fig. 5

DMD-delE51 pigs suffered from atrophic gastritis. A H&E staining of stomach sections from WT and DMD-delE51 pigs at the age of 12 weeks. The thickness of gastric fundus glands (red rectangle) was reduced in DMD-delE51 pigs. B Quantification of the thickness of gastric fundus glands in WT and DMD-delE51 pigs. C Measurements of serum G-17 in WT and DMD-delE51 pigs. D Measurement of serum PGI in WT and DMD-delE51 pigs. E Measurement of serum PGII in WT and DMD-delE51 pigs. F The ratio of PGI/PGII in WT and DMD-delE51 pigs. Scale bars: 50 µm and 100 µm. ***P < 0.001, **P < 0.01 and *P < 0.05; n = 5