NOX2 Inhibition Restores Contractility, Intracellular Calcium Handling and Reduces Arrhythmogenicity in Dystrophic Cardiomyopathy

Adriana Treuer1, Raul Dulce2, Daniel Gonzalez1. 1Universidad de Talca, Talca, Chile, 2University of Miami, Miami, Florida, USA

Dystrophic cardiomyopathy is the cardiac manifestation of Duchenne muscular dystrophy (DMD. Intracellular calcium (Ca2+) handling is abnormal in the cardiomyocytes of the mdx mouse, a model of DMD (1). We tested the hypothesis that NADPH oxidase (NOX2)-derived oxidative stress may cause disturbances in Ca2+ handling and contractility in mdx mice heart cells.

Mdx mice (19 months old, n= 35) and background controls mice (C57BL/10SnJ, n=45) were used for the study. To assess superoxide production, isolated cardiac myocytes from wild type and mdx myocytes were loaded with 2′,7′-dichlorofluorescein diacetate (DCF), and treated with vehicle (DMSO) or the NOX inhibitors apocynin (100 µmol/L) or VAS2870 (20 µmol/L) (60 cells each group, from 4 hearts) and visualized using confocal microscopy. Contractility, assessed as sarcomere shortening and calcium was evaluated using fura-2 (2). NOX2 expression was evaluated by Western Blotting.

Fig 1.Effect of NOX inhibition superoxide production in mdx myocytes. ** p<0.001 vs. wt control, ***p<0.0001 vs. wt control, ††, p<0.001 vs. mdx control, †††, p<0.0001 vs. mdx control (ANOVA).

NOX2 expression was increased fivefold in the mdx hearts compared to wild type (n= 6 each group, p<0.005, t test) and NOX2 inhibition with apocyinin and VAS2870 decreased superoxide production in mdx myocytes (Fig. 1). Next, we studied the impact of NOX2 inhibition on contractility and Ca2+ handling in mdx cardiomyocytes. Contractility was decreased in mdx myocytes (n=25) compared to wild type (n= 31, p<0.05, ANOVA). Pre-treatment with apocynin restored this response towards normal. In addition, the amplitude of evoked Ca2+ transients that was diminished in mdx myocytes (n= 21) compared to wild type (n=35), was also restored upon NOX2 inhibition (p<0.05, ANOVA). Total sarcoplasmic reticulum (SR) Ca2+ content was reduced in mdx myocytes (n=12 wild type and mdx, p<0.05, ANOVA). This content was normalized by apocynin treatment. At the same time, NOX2 inhibition decreased dramatically the production of spontaneous diastolic Ca2+ release events (p<0.05, ANOVA) and decreased the SR Ca2+ leak in mdx myocytes (p<0.05, ANOVA).

These results indicate that in mdx hearts, NOX2 inhibition increases contractility by improving the SR calcium handling. NOX2 inhibition reduced SR Ca2+ leak, probably reducing the sensitivity of the ryanodine receptor. Targeting NOX2 in dystrophic cardiomyopathies may help to restore heart function.

(1) Fauconnier J., et al. (2010) Proc Natl Acad Sci U S A ;107:1559-1564

(2) Gonzalez DR et al (2010 J Biol Chem ;285:28938-28945