G.P.125: Manganese enhanced muscle MRI as a sensitive outcome measure of dystrophin restoration in the mdx mouse

Abstract

A central component of dystrophic pathology in DMD patients as well as mdx mice is a persistent increase in steady-state calcium levels in muscle. Increased calcium load leads to loss of muscle strength, activation of calcium-dependent proteases and may increase muscle cell necrosis. As a key primary event in dystrophic pathology, measurement of calcium levels may offer a sensitive marker of the efficacy of therapeutic agents. Manganese is a divalent cation with very similar chemical properties to calcium and is thought to be taken up by cardiac and skeletal muscle by the same receptors as calcium ions. It is also a contrast agent in T1 weighted MRI, enabling calcium levels in muscle to be measured non-invasively. We have previously shown that manganese-enhanced MRI (MEMRI) contrast is elevated in cardiac pathology in mdx and can track the dynamics of calcium channel blockade in dystrophic models. Here, we have extended this analysis by infusing manganese intravenously and observing the uptake by skeletal muscle using MRI, showing that the mdx mouse has an increased steady-state level of MEMRI contrast in both chest wall and triceps. Furthermore, we treated a cohort ( n = 6) of 12-week old mdx mice with an increasing dosage (64 mg/kg or 133 mg/kg) of phosphorothioate Morpholino oligonucleotides (PMOs) to induce exon skipping. Animals were treated with a series of three i.v. injections spaced two weeks apart, scanned after a further two weeks, and muscle (heart, chest wall and triceps) harvested for dystrophin quantification by IF and Western blotting, as well as for histology. We show that inducing exon skipping, even at very low levels, can be detected by changes in the MEMRI signal in both cardiac and skeletal muscle, even in the absence of discernable changes in histology. These data suggest that muscle MEMRI may be a valuable tool for longitudinal studies on dystrophin restoration in mdx and may have increased sensitivity when compared to more established methods.