Evaluation of skeletal and cardiac muscle function after chronic administration of thymosin beta-4 in the dystrophin deficient mouse

Abstract

Thymosin beta-4 (Tbeta4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tbeta4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8-10 weeks old, were treated with 150 microg of Tbeta4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tbeta4 and amount of fibrosis were quantified using immunohistochemistry and Gomori's tri-chrome staining, respectively. Mdx mice treated with Tbeta4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tbeta4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tbeta4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.

Figure 1. Significantly decreased cardiac function (mean ± SEM) measured as percent shortening fraction (%SF) in mdx mice is seen after 6 months of treatment with thymosin-beta 4 compared to wild type mice.

There is no significant difference between treated and untreated mdx mice.

Figure 2. Significantly increased percent collagen (mean ± SEM) for cardiac (n = 3 for treated and untreated mdx mice and untreated wild type, n = 4 for treated wild type; panel A), diaphragm (n = 5 for all groups; panel B), and gastrocnemius (n = 3 for all groups; panel C) is seen in mdx mice compared to wild type.

There was no significant difference between treated and untreated mdx mice.

Figure 3. Gomori's tri-chrome stained slides of cardiac tissue showing increased fibrosis in mdx mice.

A) Untreated wild type cardiac tissue showing minimal collagen staining (light blue color) corresponding to a percent collagen of 1.82±0.5%. B) Untreated mdx cardiac tissue showing diffuse fibrosis in the LV and RV ventricular walls corresponding to a percent collagen of 3.83±0.5%. C) Tβ4 treated wild type mice showing few increased areas of collagen staining corresponding to a percent collagen of 1.6±0.5%. D) Tβ4 treated mdx cardiac tissue showing large areas of collagen staining in the LV and RV walls corresponding to a percent collagen of 4.39±0.7%. (LV – left ventricle, RV – right ventricle).

Figure 4. Peroxidase staining of regenerating fibers using anti-TB4 antibodies in skeletal muscle (gastrocnemius).

A) Mdx muscle treated with anti-TB4 antibody shows peroxidase staining of regenerating fibers (*); B) Mdx muscle treated with anti-desmin antibody shows staining in regenerating fibers (#), the same as in plate A; C) Wild type muscle treated with anti-TB4 antibodies shows no staining; D) Wild type muscle treated with anti-desmin antibodies shows no staining.