Cmah-dystrophin deficient mdx mice display an accelerated cardiac phenotype that is improved following peptide-PMO exon skipping treatment.

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids-the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah-/-;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah-/-;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah-/-;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah-/-;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah-/-;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics.

Implications for Cardiac Function Following Rescue of the Dystrophic Diaphragm in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by absence of the integral structural protein, dystrophin, which renders muscle fibres susceptible to injury and degeneration. This ultimately results in cardiorespiratory dysfunction, which is the predominant cause of death in DMD patients, and highlights the importance of therapeutic targeting of the cardiorespiratory system. While there is some evidence to suggest that restoring dystrophin in the diaphragm improves both respiratory and cardiac function, the role of the diaphragm is not well understood. Here using exon skipping oligonucleotides we predominantly restored dystrophin in the diaphragm and assessed cardiac function by MRI. This approach reduced diaphragmatic pathophysiology and markedly improved diaphragm function but did not improve cardiac function or pathophysiology, with or without exercise. Interestingly, exercise resulted in a reduction of dystrophin protein and exon skipping in the diaphragm. This suggests that treatment regimens may require modification in more active patients. In conclusion, whilst the diaphragm is an important respiratory muscle, it is likely that dystrophin needs to be restored in other tissues, including multiple accessory respiratory muscles, and of course the heart itself for appropriate therapeutic outcomes. This supports the requirement of a body-wide therapy to treat DMD.

The transgenic expression of LARGE exacerbates the muscle phenotype of dystroglycanopathy mice.

Mutations in fukutin-related protein (FKRP) underlie a group of muscular dystrophies associated with the hypoglycosylation of α-dystroglycan (α-DG), a proportion of which show central nervous system involvement. Our original FKRP knock-down mouse (FKRP(KD)) replicated many of the characteristics seen in patients at the severe end of the dystroglycanopathy spectrum but died perinatally precluding its full phenotyping and use in testing potential therapies. We have now overcome this by crossing FKRP(KD) mice with those expressing Cre recombinase under the Sox1 promoter. Owing to our original targeting strategy, this has resulted in the restoration of Fkrp levels in the central nervous system but not the muscle, thereby generating a new model (FKRP(MD)) which develops a progressive muscular dystrophy resembling what is observed in limb girdle muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) is a bifunctional glycosyltransferase previously shown to hyperglycosylate α-DG. To investigate the therapeutic potential of LARGE up-regulation, we have now crossed the FKRP(MD) line with one overexpressing LARGE and show that, contrary to expectation, this results in a worsening of the muscle pathology implying that any future strategies based upon LARGE up-regulation require careful management.

The neuroprotective effects of heat shock protein 27 overexpression in transgenic animals against kainate-induced seizures and hippocampal cell death.

The 27-kDa heat shock protein (HSP27) has a potent ability to increase cell survival in response to a wide range of cellular challenges. In order to investigate the mode of action of HSP27 in vivo, we have developed transgenic lines, which express human HSP27 at high levels throughout the brain, spinal cord, and other tissues. In view of the particular property of HSP27 compared with other HSPs to protect neurons against apoptosis, we have tested these transgenic lines in a well established in vivo model of neurotoxicity produced by kainic acid, where apoptotic cell death occurs. Our results demonstrate for the first time the marked protective effects of HSP27 overexpression in vivo, which significantly reduces kainate-induced seizure severity and mortality rate (>50%) in two independent lines and markedly reduces neuronal cell death in the CA3 region of hippocampus. This reduced seizure severity in HSP27 transgenic animals was associated with a marked attenuation of caspase 3 induction and apoptotic features. These studies clearly demonstrate that HSP27 has a major neuroprotective effect in the central nervous system in keeping with its properties demonstrated in culture and highlight an early stage in the cell death pathway that is affected by HSP27.