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

Abstract

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.

Figure 1

Cardiac function and pathology of mdx and Cmah−/−;mdx hearts compared to C57BL10 at 12 and 24 weeks of age. (A) Representative cine MRI images showing LV and RVs for heart during diastole and systole. (B) Cardiac function parameters, RV EF (%), RV SV, LV EDV and LV SV. (B) Quantitative real time (qRT)-PCR for the expression of fibrotic and injury markers namely Ctgf, Nppa and Nox4 in heart tissue normalized to 12 week C57BL10. Data displayed as mean ± SEM. For cardiac function parameters N = 5–7, and qRT-PCR data n = 3–6. Significance calculated using two-way ANOVA, Tukey post-hoc test (^***P < 0.001, ^**P < 0.01, ^*P < 0.05).

Figure 2

Metabolic profile of C57BL10, mdx and Cmah−/−;mdx hearts at 12 and 24 weeks of age-hyperpolarized MRS and gene expression analysis. (A) Bicarbonate and lactate production normalized to maximum pyruvate signal. Data displayed as mean ± SEM. N = 5–7. (B) Quantitative real-time (qRT)-PCR for the expression of Pdk1, Pdk4, Ucp3, Cpt1, CD36 and Ppara in heart tissue normalized to 12-week C57BL10. Data displayed as mean ± SEM. N = 3–6. Statistical significance was determined using two-way ANOVA, Tukey post-hoc test (^****P < 0.0001, ^***P < 0.001, ^**P < 0.01. ^*P < 0.05).

Figure 3

Biochemical data from plasma samples of C57BL10, mdx and Cmah−/−;mdx mice at 12 and 24 weeks. Graphs showing total cholesterol, FFA, HDL and LDL measurements. Data displayed as mean ± SEM. N = 4–6. Statistical significance was determined using two-way ANOVA, Tukey post-hoc test (^****P < 0.0001, ^***P < 0.001, ^**P < 0.01, ^*P < 0.05).

Figure 4

PPMO treatment restores dystrophin and improves RV function and pathology in Cmah−/−;mdx hearts at 8 months of age. (A) Quantification of exon 23-skipped transcripts (RT-qPCR) and dystrophin protein restoration in TA, diaphragm and heart following Pip6A-PMO treatment (n = 4). (B) Cardiac function parameters were measured by cine-MRI. Graphs indicating RV ESV, RV EF and HR shown. (C) RT-qPCR for the expression of injury and metabolic markers namely Nox4, Nppa, Ucp3, Pdk1 and Pdk4 in heart tissue normalized to C57BL10. Data displayed as mean ± SEM. For cardiac function parameters significance calculated using one-way ANOVA, Tukey post-hoc test. For RT-qPCR data significance was determined using Student’s t-test (^***P < 0.001, ^**P < 0.01^*P < 0.05).

Figure 5

PPMO treatment improves muscle function in untreated and treated Cmah−/−;mdx mice. (A) Specific force–frequency relationship for untreated Cmah−/−;mdx (n = 6), chronically Pip6a-PMO treated Cmah−/−;mdx (n = 8), and C57BL10 mice (n = 12). Treatment with Pip6a-PMO significantly increased specific force in the Cmah−/−;mdx mice (P < 0.001). (B) Force drop associated with eccentric exercise in untreated Cmah−/−;mdx and chronically Pip6a-PMO treated Cmah−/−;mdx. C57BL10 mice not included as their plot overlays the result for the chronically Pip6a-PMO treated Cmah−/−;mdx mice. The Cmah−/−;mdx mice show a significant force drop with eccentric exercise that is prevented by Pip6a-PMO treatment (P < 0.001). Pip6a-PMO treated Cmah−/−;mdx and C57BL10 mice are not significantly different.