Generation and phenotypic changes in BMD mice carrying d45–47, d45–48 and d45–49 in-frame mutation in mouse DMD.

(A) Schematic representation of mouse DMD from exon 44 to exon 50, and the location of guide RNAs. We established d45–47, d45–48 and d45–49 BMD mouse models, using guide RNA corresponding to the sequences of intron 44 and 47, intron 44 and 48, intron 44 and 49 respectively. All BMD mouse models were confirmed having desired mutations by RT-PCR. (B) Body weight (g) in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1, 3, 6 and 12 months (n = 10 at 1 and 3 months, n = 4 at 6 and 12 months). (C) Relative (to body weight (g)) forelimb grip strength in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1, 3, 6 and 12 months (n = 10 at 1 and 3 months, n = 4 at 6 and 12 months). Bar: mean ± S.D.; *P < 0.05, **P < 0.01.

(A) The intronic deletion breakpoint of BMD mouse models having d45–47, d45–48 and d45–49. DNA sequences that is enclosed by a box indicates protospacer adjacent motif (PAM), and that is marked yellow of blue label corresponds guide RNA. (B) The holding time of hanging wire test (sec, maximum limitation = 600sec) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3), at the age of 3 months. Bar: mean ± S.E.; *P < 0.05, **P < 0.01.

Histopathological changes in BMD mice carrying d45–47, d45–48 and d45–49.

(A) Hematoxylin-Eosin stain of tibialis anterior muscles (TA) in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1, 3, 6 and 12 months. Yellow asterisk: mean opaque-fibers. Arrow head: mean necrotic fibers. (B) Sirius-Red stain of TA in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1, 3, 6 and 12 months.(C) Percent opaque fibers at TA in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1 and 3 months, (n = 3). (D) Percent necrotic fibers at TA in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1 and 3 months, (n = 3). (E) Percent centronuclear-fibers at TA in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1 and 3 months, (n = 3). (F) Percent fibrosis area at TA in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1, 3, 6 and 12 months, (n = 3). Bar: mean ± S.D.; *P < 0.05, **P < 0.01. Scale bar means 100 µm.

(A) Hematoxylin-Eosin and Sirius Red stain of diaphragm muscles in WT, d45–48, d45–47, d45–49 and mdx mice, at the age of 12 months. Scale bar means 100 µm. (B) Hematoxylin-Eosin and Sirius Red stain of cardiac muscles in WT, d45–48, d45–47, d45–49 and mdx mice, at the age of 12 months. Scale bar means 100 µm. (C) Schematic representation of time-coursed histopathological changes about muscle degeneration and fibrosis in WT, d45–48, d45–47, d45–49 and mdx mice. d45–49 and mdx mice showed muscle degeneration at an earlier age compared with d45–48 and d45–47 mice.

All BMD mice showed truncated-dystrophin expression and decreased sarcolemmal neuronal nitric oxide synthetase (nNOS) expression.

(A) Dystrophin, nNOS and alpha-Sarcoglycan immunohistochemistry of TA in WT, d45–48, d45–47, d45–49 and mdx mice, at the age of 1 and 3 months. (B) Western blot analysis for dystrophin in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1 and 3 months. (C) Western blot analysis for dystrophin-homolog utrophin in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1 and 3 months. (D) Western blot analysis for nNOS in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1 and 3 months. (E) Relative Dp427 protein levels (normalized by total protein band intensity) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3). (F) Relative Up395 protein levels (normalized by total protein band intensity) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3). (G) Relative nNOS protein levels (normalized by total protein band intensity) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3). Bar: mean ± S.D.; *P < 0.05, **P < 0.01. Scale bar means 100 µm.

(A) Relative mRNA levels of dystrophin exons 75–77 (normalized by 18S rRNA levels) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3). (B) Relative mRNA levels of utrophin exons 70–72 (normalized by 18S rRNA levels) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3). (C) Relative mRNA levels of neuronal nitric oxide synthetase (nNOS) (normalized by 18S rRNA levels) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3). Bar: mean ± S.D.; *P < 0.05, **P < 0.01.

All BMD mice showed site-specific muscle degeneration especially in inner part of TA with type IIa fiber reduction.

(A) Hematoxylin-Eosin stain (upper panels), MYH-2, -4, -7 immunohistochemistry (middle panels: type IIa, IIb, I fibers, respectively) and MYH-1 immunohistochemistry (lower panels: type IIx fibers) at outer- or inner- part of TA in WT, d45–48, d45–47, d45–49 and mdx mice, at the age of 3 months. (B) The proportion of muscle fiber types at outer- or inner-part of TA about type I, IIa, IIb, and others, in WT, d45–48, d45–47, d45–49 and mdx mice, at the age of 3 months, (n = 3). (C) The proportion of muscle fiber types at outer- or inner-part of TA about type IIx and others, in WT, d45–48, d45–47, d45–49 and mdx mice, at the age of 3 months, (n = 3). (D) Hematoxylin-Eosin stain at four component muscles of quadriceps muscle: vastus lateralis (VL), rectus femoris (RF), vastus medialis (VM) and vastus intermedius (VI), in WT, d45–49 and mdx mice, at the age of 3 months. (E) MYH-2, -4, -7 immunohistochemistry (type IIa, IIb, I fibers (blue, red, green, respectively)) at VL, RF, VM and VI in WT, d45–49 and mdx mice, at the age of 3 months. (F) The proportion of muscle fiber types at VL, RF, VM and VI in WT, d45–49 and mdx mice, at the age of 3 months, (average of n = 3). (G) Hematoxylin-Eosin stain at three component muscles of erector spinae muscle: multifidus (MF), longissimus (LG) and iliocostalis (IC), in WT, d45–49 and mdx, at the age of 3 months. (H) MYH-2, -4, -7 immunohistochemistry immunohistochemistry (type IIa, IIb, I fibers (blue, red, green, respectively)) at MF, LG and IC in WT, d45–49 and mdx mice, at the age of 3 months. (I) The proportion of muscle fiber types at MF, LG and IC in WT, d45–49 and mdx mice, at the age of 3 months, (average of n = 3). Scale bar means 100 µm.

(A) Serum CK levels (IU/L) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3), at the ages of 1, 3, 6 and 12 months. Bar: mean ± S.E.; *P < 0.05, **P < 0.01. (B) Cross-sectional area (CSA) (mm2) at the middle section of TA muscles in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3). (C) Relative mRNA levels of muscle ring finger protein-1 (Murf1) (normalized by 18S rRNA levels) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3). (D) Relative mRNA levels of atrogin-1 (normalized by 18S rRNA levels) in WT, d45–48, d45–47, d45–49 and mdx mice (n = 3). Bar: mean ± S.D.; *P < 0.05, **P < 0.01.

Type IIa fiber decrement started from the age of 3 months in BMD mice.

(A) MYH-2, -4, -7 immunohistochemistry (type IIa, IIb, I fibers, respectively) at TA under low-magnification in WT, d45–48, d45–47, d45–49 and mdx mice, at the age of 1, 3, 6 and 12 months. (B) The count of Type IIa fibers involved in one TA section (at the middle part of TA) in WT, d45–48, d45–47, d45–49 and mdx mice, at the ages of 1, 3, 6 and 12 months, (n = 3). (C) Schematic representation about the character of type I, IIa, IIx and IIb fibers and its proportional changes in BMD mice compared with WT mice. Bar: mean ± S.D.; *P < 0.05, **P < 0.01. Scale bar means 100 µm.

Hematoxylin-Eosin stain (left panels), MYH-2, -4, -7 (type IIa, IIb, I fibers respectively) immunohistochemistry (middle panels), and MYH-1 (type IIx fibers) immunohistochemistry (right panels) of 6 weeks TA in WT mice at 1, 3, 5, 7, 14 days after cardiotoxin (CTX) injection. The muscle fiber recovering in type IIb, IIa and IIx fibers were shown at day 5, 14 and 28 respectively. Scale bar means 100 µm.

Capillaries contacting type IIa fiber were decreased and altered in morphology in BMD mice.

(A) MYH-2, -4, -7 and PECAM-1 immunohistochemistry (upper panels: type IIa, IIb fibers and capillaries, respectively) and MYH-1 and PECAM-1 immunohistochemistry (lower panels: type IIx and capillaries, respectively) at the inner- and the outer-part of TA in WT, d45–49 and mdx mice, at the age of 3 months. < A >: mean type IIa fibers, < B >: mean type IIb fibers, < X >: mean type IIx fibers. (B) High-magnification images of PECAM-1 immunohistochemistry (capillaries) around type IIa, IIx and IIb fibers at the inner-part of TA, and around type I fibers at vastus intermedius in WT, d45–49 and mdx mice, at the age of 3 months. < A >: mean type IIa fibers, < B >: mean type IIb fibers, < X >: mean type IIx fibers, < I >: mean type I fibers. (C) The Endothelial area (µm2) of capillaries contacting one type IIa, IIx, IIb and I fiber at the inner-part of TA in WT, d45–49 and mdx mice, at the ages of 3 months, (n = 3). (D) Schematic representation of an expected mechanisms of morphological changes in capillaries contacting to type IIa fibers from ‘ring-pattern’ to ‘dot-pattern’ in BMD mice. (E) PECAM-1 immunohistochemistry (capillaries) of the longitudinal sections at the inner-part of TA in WT, d45–49 and mdx mice, at the age of 3 months (upper panels), and the interconnected branches and capillary loops around a representative type IIa fiber (lower panels). < A >: mean type IIa fibers. Bar: mean ± S.D.; *P < 0.05, **P < 0.01. Scale bar means 100 µm.