Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A

Abstract

MDC1A, the second most prevalent form of congenital muscular dystrophy, results from laminin-α2 chain deficiency. This disease is characterized by extensive muscle wasting that results in extremely weak skeletal muscles. A large percentage of children with MDC1A are faced with respiratory as well as ambulatory difficulties. We investigated the effects of overexpressing insulin-like growth factor-1 (IGF-1) as a potential therapeutic target for the disease in the Lama2(Dy-w) mouse, a model that closely resembles human MDC1A. IGF-1 transgenic Lama2(Dy-w) mice showed increased survivability, body weight and muscle weight. In addition, these mice showed better ability to stand up on their hind limbs: a typical exploratory behavior seen in healthy mice. Histology and immunohistochemistry analyses revealed increased regenerative capacity and proliferation in IGF-1 transgenic Lama2(Dy-w) muscles. Western blot analysis showed increased phosphorylation of Akt and ERK1/2, both known to enhance myogenesis. Additionally, we saw increases in the expression of the regeneration markers MyoD, myogenin and embryonic myosin (myosin heavy chain 3, MYH3). We conclude that overexpression of IGF-1 in Lama2(Dy-w) mice increases lifespan and improves their overall wellbeing mainly through the restoration of impaired muscle regeneration, as fibrosis or inflammation was not impacted by IGF-1 in this disease model. Our results demonstrate that IGF-1 has a promising therapeutic potential in the treatment of MDC1A.

Figure 1.

Increased survival and body weight observed in laminin-α2-deficient mice overexpressing IGF-1. (A) Survival curves showing that most Lama2^Dy-w mice died within 4 weeks of birth, whereas the lifespans of most Lama2^Dy-w+IGF-1tg mice were markedly increased to at least 21 weeks (n= 8 for both groups, P< 0.009). (B) At 3 weeks, the mean body weight of Lama2^Dy-w+IGF-1tg (n= 7) mice was twice that of Lama2^Dy-w mice (n= 6; P< 0.001). (C) Body weight curves showing a higher growth rate in Lama2^Dy-w+IGF-1tg mice than in Lama2^Dy-w mice. Furthermore, a greater divergence in body weight was seen over time as Lama2^Dy-w+IGF-1tg mice continued to grow, whereas Lama2^Dy-w mice showed poor growth overall.

Figure 2.

Overall functional activity and capacity were improved with IGF overexpression in Lama2^Dy-w mice. (A) Stand up test results showed a significant increase in exploratory behavior in Lama2^Dy-w+IGF-1tg mice than in Lama2^Dy-w mice (P< 0.005). (B) Pictures display leg extension and leg retraction in tail suspension tests performed to assess muscle weakness, with severity graded according to the average time to leg retraction (severe:<3 s; moderate: 3–6 s; mild: >6 s). Average muscle weakness was severe in Lama2^Dy-w mice and moderate in Lama2^Dy-w+IGF-1tg mice.

Figure 3.

Lama2^Dy-w+IGF-1tg mice showed increased hind limb muscle size and myofiber hypertrophy. (A) Average muscle weights of tibialis anterior (TA), gastrocnemius/soleus complex (GS) and quadriceps muscles (QD) were significantly greater in Lama2^Dy-w+IGF-1tg mice (black bars) than in its non-transgenic littermates (white bars) at 6 weeks (TA, P< 0.0001; GS, P= 0.0005; QD, P= 0.0002). (B) The mean cross-sectional area of the mid-belly portion of TA muscles in Lama2^Dy-w+IGF-1tg mice was 50% (n= 6) of its age- and sex-matched WT control, a 2-fold increase compared with that of Lama2^Dy-w littermates (25%, n= 4). (C) Lama2^Dy-w mice showed a significant increase in the number of fibers per square millimeter, whereas Lama2^Dy-w+IGF-1tg mice did not significantly vary in comparison with the WT (P= 0.0042, one-way ANOVA). (D) Minimum Feret histograms display a shift in fiber size distribution in Lama2^Dy-w+IGF-1tg muscles (black bars) towards the WT (gray bars) distribution in comparison with the Lama2^Dy-w (white bars) distribution. Although Lama2^Dy-w mice had more fibers per square millimeter, many of those fibers were of smaller caliber.

Figure 4.

Increased muscle regeneration in Lama2^Dy-w+IGF-1tg mice. (A) H&E staining of Lama2^Dy-w+IGF-1tg TA muscle cross-section at 6 weeks. The image displays centrally nucleated fibers (arrow) indicating regeneration. The average percent of centrally nucleated fibers was significantly greater in Lama2^Dy-w+IGF-1tg mice (30%) than in Lama2^Dy-w mice (12%). (B) Transcript-level expression of embryonic myosin heavy chain (MYH3) increased 2-fold in muscle tissues of Lama2^Dy-w+IGF-1tg mice compared with Lama2^Dy-w mice. (C) Significant increases in relative RNA expression of MyoD (P= 0.002, one-way ANOVA) were found in Lama2^Dy-w+IGF-1tg muscles compared with non-transgenic littermates. (D) Top left panel: Immunohistochemistry shows a number of MyoD (red)-positive nuclei in the interstitial space between myofibers (white arrows). Top right panel: Nuclei stained with DAPI (blue). Bottom panel: A merged image showing dystrophin (green) and DAPI (blue) with MyoD (red).

Figure 5.

Increased muscle cell proliferation observed in Lama2^Dy-w mice with IGF overexpression. (A) 40× images of Lama2^Dy-w (left) and Lama2^Dy-w+IGF-1tg (right) TA muscles stained for Ki-67 (pink) (white arrow), dystrophin (green) and DAPI (blue). (B) The mean number of Ki67-positive cells per square millimeter was doubled in Lama2^Dy-w+IGF-1tg compared with Lama2^Dy-w TA muscles. (C) Immunohistochemical staining shows several Ki-67 (green)-positive nuclei stained positive for Pax7 (red), a satellite cell marker. Examples of these nuclei are indicated by white arrowheads in each panel. The last panel shows the merged image.

Figure 6.

Fibrosis and inflammation were not noticeably improved by IGF-1 overexpression in Lama2^Dy-w muscles. 20× images of Picro-sirius red staining for collagen (red) of (A) Lama2^Dy-w and (B) Lama2^Dy-w+IGF-1tg TA muscles revealed no noticeable difference in levels of fibrosis. Immunostaining for Mac-1/CD11B (green), a marker of monocytes/macrophages, showed no change in infiltrating inflammatory cells in (C) Lama2^Dy-w muscles and (D) Lama2^Dy-w+IGF-1tg muscles.

Figure 7.

Increased expression of downstream signaling targets of IGF-1 in Lama2^Dy-w+IGF-1tg muscle lysates. (A) Western blots of total Akt, phospho-Akt (Thr-308), phospho-Akt (Ser-473) and α-tubulin in muscles isolated from 6-week-old WT, Lama2^Dy-w and Lama2^Dy-w+IGF-1tg mice. (B) Relative optical density analysis of phospho-Thr-308-Akt levels in Lama2^Dy-w+IGF-1tg muscles (black bars) revealed a several-fold increase relative to Lama2^Dy-w (white bars) and WT (gray bars), whereas total Akt and phospho-Ser-473-Akt levels increased only marginally. (C) Western blots of phospho-ERK1/2, total ERK1/2 and α-tubulin in 6-week-old muscles isolated from Lama2^Dy-w and Lama2^Dy-w+IGF-1tg mice. (D) Relative optical density of total ERK1/2 was similar in Lama2^Dy-w and Lama2^Dy-w+IGF-1tg muscle lysates. However, an increase in relative phospho-ERK1/2 levels was seen in muscles of Lama2^Dy-w+IGF-1tg (black bars) compared with its non-transgenic littermates (white bars).

Figure 8.

IGF-1 overexpression leads to a decrease in Lama2^Dy-w-mediated apoptosis. (A) Caspase 3/7 activity was higher in Lama2^Dy-w compared with WT muscles (P< 0.0001). There was only a 12.8% decrease in caspase activity in the muscle lysates of Lama2^Dy-w+IGF-1tg mice compared with its non-transgenic littermates (n= 5, P< 0.05). (B) Representative muscle sections of Lama2^Dy-w (top panel) and Lama2^Dy-w+IGF-1tg (bottom panel) assayed for TUNEL-positive nuclei did not show any significant difference. TUNEL-positive nuclei (green) are indicated by white arrows. These sections were also stained with PAN laminin isoform (red) and DAPI (blue).