A Morpholino Oligomer Therapy Regime That Restores Mitochondrial Function and Prevents mdx Cardiomyopathy

Figures

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  Figure 1. Short-Term, High-Dose Treatment of 4- to 5-Day-Old mdx Mice With PMO Results in Exon Skipping of Dystrophin, and Partial Restoration of Ψm and Flavoprotein Oxidation in Response to Activation of ICa-L (A) Reverse transcriptase polymerase chain reaction on cardiac ribonucleic acid from phosphorodiamidate morpholino oligomer (PMO)-treated murine model of Duchenne muscular dystrophy (mdx) mice demonstrating exon 23 skipping (Δ 23 687 base pairs [bp]), as indicated by asterisks, and untreated mdx and wild-type (wt) control mice. (B) Representative ratiometric 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) fluorescence recorded in myocytes from PMO-treated mdx mice, and an untreated mdx mouse before and after exposure to 10 μM BayK(–) under calcium-free conditions (0 mM Ca2+). (C) Mean ± SEM of JC-1 fluorescence for all myocytes exposed to drugs as indicated. (D) Representative traces of flavoprotein fluorescence recorded in myocytes from PMO-treated mdx mice and an untreated mdx mouse before and after exposure to 10 μM BayK(–). (E) Mean ± SEM of flavoprotein fluorescence for all myocytes exposed to drugs as indicated. PMO treatments: 40 mg/kg…

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  Figure 2. Long-Term, High-Dose Treatment of 24-Week-Old mdx Mice With PMO Results in Exon Skipping and Expression of Dystrophin (A) Reverse transcriptase polymerase chain reaction performed on cardiac muscle ribonucleic acid from PMO-treated mdx mice demonstrating exon 23 skipping (Δ 23 687 bp) (indicated by asterisk) and age-matched untreated mdx and wt control mice. The 22/23 541 bp amplicon represents an out-of-frame transcript missing exons 22 and 23 and has been previously reported 37, 38. (B) Immunoblot performed on cardiac muscle from untreated wt (5%, 2%, and 1% dilutions), untreated mdx mice (undiluted), and PMO-treated mdx mice (undiluted) demonstrating presence of dystrophin (indicated by asterisk). (C) Immunostaining of heart cryosections from untreated wt and mdx mice and PMO-treated mdx mice demonstrating presence of dystrophin. Bars = 100 μm. PMO treatment: 40 mg/kg PMO 3× per week for 19 weeks; other abbreviations as in Figure 1.

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  Figure 3. Long-Term, High-Dose Treatment of 24-Week-Old mdx Mice With PMO Completely Restores Ψm and Flavoprotein Oxidation in Response to Activation of ICa-L (A) Representative ratiometric JC-1 fluorescence recorded in myocytes from a PMO-treated mouse and an untreated mdx mouse before and after exposure to 10 μM BayK(–) ± 15 μM nisoldipine (Nisol) under calcium-free conditions (0 mM Ca2+). Arrow indicates addition of drugs. (B) Mean ± SEM of JC-1 fluorescence for all myocytes exposed to drugs as indicated. (C) Representative traces of flavoprotein fluorescence recorded in myocytes from a PMO-treated mouse and an untreated mdx mouse before and after exposure to 10 μM BayK(–) ± 15 μM nisoldipine (Nisol). Arrow indicates addition of drugs. (D) Mean ± SEM of flavoprotein fluorescence for all myocytes exposed to drugs as indicated. BayK(+) = 10 μM; FCCP = 50 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; NaCN = 40 mM sodium cyanide; PMO treatment = 40 mg/kg PMO 3× per week for 19 weeks; Oligo = 20 μM oligomycin; other abbreviations as in Figure 1.

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  Figure 4. Long-Term, High-Dose Treatment of 24-Week-Old mdx Mice With PMO Completely Restores Metabolic Activity in Response to Activation of ICa-L (A) Formation of formazan measured as change in absorbance in myocytes from untreated wt and mdx mice and PMO-treated mice (40 mg/kg PMO 3× per week for 19 weeks), after addition of 10 μM BayK(+) or BayK(–). (B) Mean ± SEM of increases in absorbance for all myocytes exposed to drugs as indicated. Dant = 20 μM dantrolene; Nisol = 15 μM nisoldipine; Oligo = 20 μM oligomycin; Ru360 = 15 μM; other abbreviations as in Figure 1.

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  Figure 5. Long-Term, High-Dose Treatment of 24-Week-Old mdx Mice With PMO Prevents Development of mdx Cardiomyopathy and Is Not Toxic (A) Representative images of serial echocardiographic measurements from PMO-treated mdx mice and untreated wt and mdx mice pre- (24 weeks) and post-treatment (Rx; 43 weeks). (B) Mean ± SEM of all echocardiographic measurements. *p < 0.05 compared with untreated age-matched wt; #p < 0.05 compared with untreated age-matched mdx. (C) Mean ± SEM of urea, creatinine, and alanine transaminase (ALT) concentrations in PMO-treated mice compared to untreated mdx mice. bpm = beats/min; FS = fractional shortening; HR = heart rate; IVDS = intraventricular septum in diastole; IVSS = intraventricular septum in systole; LVEDD = left ventricular end-diastolic diameter; LVESD = left ventricular end-systolic diameter; LVDPW = left ventricular posterior wall in diastole; LVSPW = left ventricular posterior wall in systole; PMO Rx = 40 mg/kg PMO 3× per week for 19 weeks; other abbreviations as in Figure 1.