AAV-microdystrophin therapy improves cardiac performance in aged female mdx mice

Abstract

Dystrophin deficiency leads to lethal dilated Duchenne cardiomyopathy. A promising therapy is to deliver a highly abbreviated microdystrophin gene to the heart using adeno-associated virus (AAV). Microdystrophin has been shown to mitigate dystrophin-deficient skeletal muscle disease. However, it is not clear whether microdystrophin is equally effective in treating Duchenne cardiomyopathy. To evaluate microdystrophin therapy in the heart, we injected 5 × 10(12) viral genome particles/mouse of AAV-9 ΔR4-23/ΔC microdystrophin vector via tail vein to ~16-20-month-old (average 18.7-month-old) female mdx mice, a manifesting model of Duchenne cardiomyopathy. Cardiac transduction and heart function were examined at 2-8 months after gene transfer. We observed robust myocardial microdystrophin expression. Electrocardiography (ECG) and left ventricular catheter hemodynamic assays also revealed significant improvement. Furthermore, AAV-microdystrophin therapy prevented dobutamine-stress induced acute cardiac death. We demonstrate for the first time that AAV microdystrophin therapy significantly ameliorates functional deficiency in a phenotypic model of Duchenne cardiomyopathy. Our results support further exploration of microdystrophin therapy to treat Duchenne cardiomyopathy.

Figure 1

Adeno-associated virus serotype-9 (AAV-9) microdystrophin vector efficiently transduced the myocardium of aged mdx mice. (a) Representative Dys-3 immunofluorescence staining of whole mdx heart infected with AAV-9 microdystrophin vector. (b) Representative high-power images of immunofluorescence staining from untreated mdx and AAV-9 microdystrophin vector-treated mdx. Top panel: Dys-3 antibody immunostaining. Dys-3 is an antibody that specifically recognizes human dystrophin (Hum Dys). Dys-3 immunostaining reveals the expression of human-derived microdystrophin. Middle panel: Mandys-8 antibody (R11) immunostaining. Mandys-8 antibody specifically reacts with spectrin-like repeat 11, a repeat deleted in microdystrophin. Bottom panel: Utrophin immunostaining. (c) Representative western blot analysis of dystrophin expression in BL10, untreated mdx and AAV-infected mdx mice. Top panel: western blot with the DysB antibody; bottom panel: Rapid blue staining of a duplicated gel. µ-Dys, microdystrophin. Arrowhead, full-length mouse dystrophin; *Human ΔR4-23/ΔC microdystrophin.

Figure 2

Evaluation of cardiac fibrosis and cardiomyocyte size in adeno-associated virus serotype-9 (AAV-9) microdystrophin-treated mdx mice. Representative Masson trichrome staining photomicrographs from the hearts of BL10, untreated mdx, and AAV microdystrophin-treated mdx mice. Blue staining shows fibrosis. (a) Age-matched BL10 control. (b) Untreated mdx. (c) A 16-month-old mdx mouse treated for 4 months. (d) A 16-month-old mdx mouse treated for 8 months. (e) A 20-month-old mdx mouse treated for 3 months. (f) Another 20-month-old mdx mouse treated for 3 months. (g) Quantification of heart hydroxyproline content in untreated and AAV-treated mdx mice. *P ≤ 0.016. (h) Quantification of cardiomyocyte size in BL10, untreated mdx, and AAV microdystrophin-treated mdx mice. AU, arbitrary unit. *Significantly different from other groups. BL10, age and gender-matched control; mdx, untreated mdx; AAV, AAV microdystrophin-treated mdx.

Figure 3

Adeno-associated virus serotype-9 (AAV-9) microdystrophin therapy partially improved the electrocardiography (ECG) profile. Quantitative evaluation of ECG in BL10, mdx, and AAV-treated mdx. HR, heart rate; bpm, beats/min; Q Amp, Q wave amplitude; C. Index, cardiomyopathy index. *Significantly different from the other two groups; cross, significantly different from BL10 only.

Figure 4

Left ventricular hemodynamics was enhanced by adeno-associated virus serotype-9 (AAV-9) microdystrophin therapy. (a) Pressure–volume (PV) loops from eight AAV-treated mdx mice. The age of AAV injection and the duration of therapy are indicated at the bottom of each panel. Representative PV loops from BL10 and mdx mice are depicted in each panel for comparison. Vertical axis, pressure (mm Hg); horizontal axis, volume (µl). (b) Systolic parameters. (c) Diastolic parameters. (d) Overall cardiac function. *Significantly different from the other two groups; cross, significantly different from BL10 only. (e) Kaplan–Meier survival curve within 15 minutes of dobutamine administration. Neither BL10 nor AAV-treated mdx mice died during this period. The survival rate of untreated mdx is significantly lower than that of BL10 and AAV microdystrophin-treated mdx mice (P ≤ 0.0079). N = 7 for BL10, N = 7 for untreated mdx, N = 8 for AAV-treated mdx.