Genetic silencing of Nrf2 enhances X-ROS in dysferlin-deficient muscle

Abstract

Oxidative stress is a critical disease modifier in the muscular dystrophies. Recently, we discovered a pathway by which mechanical stretch activates NADPH Oxidase 2 (Nox2) dependent ROS generation (X-ROS). Our work in dystrophic skeletal muscle revealed that X-ROS is excessive in dystrophin-deficient (mdx) skeletal muscle and contributes to muscle injury susceptibility, a hallmark of the dystrophic process. We also observed widespread alterations in the expression of genes associated with the X-ROS pathway and redox homeostasis in muscles from both Duchenne muscular dystrophy patients and mdx mice. As nuclear factor erythroid 2-related factor 2 (Nrf2) plays an essential role in the transcriptional regulation of genes involved in redox homeostasis, we hypothesized that Nrf2 deficiency may contribute to enhanced X-ROS signaling by reducing redox buffering. To directly test the effect of diminished Nrf2 activity, Nrf2 was genetically silenced in the A/J model of dysferlinopathy-a model with a mild histopathologic and functional phenotype. Nrf2-deficient A/J mice exhibited significant muscle-specific functional deficits, histopathologic abnormalities, and dramatically enhanced X-ROS compared to control A/J and WT mice, both with functional Nrf2. Having identified that reduced Nrf2 activity is a negative disease modifier, we propose that strategies targeting Nrf2 activation may address the generalized reduction in redox homeostasis to halt or slow dystrophic progression.

Keywords: Nrf2; ROS; X-ROS; dysferlin; dystrophy.

Figure 1

X-ROS is enhanced in Nrf2 silenced dysferlin deficient muscle fibers. FDB fibers from WT (n = 3_animals, 8_fibers), dysferlin deficient A/J (n = 3_animals, 9_fibers), and Nrf2-silenced A/J (n = 3_animals, 11_fibers) were loaded with the fluorescent ROS probe DCF and attached with MyoTak between a high-sensitivity force transducer and length controller. The fibers were challenged with a brief 5 s passive stretch (~10% sarcomere length) and DCF fluorescence signals (A) were processed as previously described (Prosser et al., 2011). The DCF fluorescent rate, expressed as a % over the pre-stretch condition, revealed that WT stretch was not different from the pre-stretch condition, dysferlin deficient A/J was significantly elevated over WT, and Nrf2 silenced A/J were elevated compared to each other genotype. The population averages are represented in the bar graph in (B). ^*p < 0.05.

Figure 2

X-ROS protein expression is elevated in Nrf2 silenced dysferlin deficient muscle. Western blot analysis of gastrocnemius muscle from 1 year old mice identified that X-ROS protein content is significantly elevated in Nrf2 silenced A/J muscle (A) (n = 5) but not in Nrf2 silenced muscle (B) (n = 5). Percent changes in protein expression are demonstrated in the bar graphs to the right of both (A,B). ^*p < 0.05.

Figure 3

Nrf2 silenced A/J muscle exhibits a loss-of-function phenotype. (A) In vivo assessments of isomeric quadriceps torque and (B) in vitro assessments of EDL contractility via the force vs. stimulation frequency relationship both revealed significant loss-of-function phenotypes in the Nrf2 silenced dysferlin deficit A/J muscle. ^*p < 0.05.

Figure 4

Histological evidence of enhanced pathology in Nrf2 silenced A/J muscle. (A) Cryosections were H&E stained to evaluate myofiber morphology and immune cell infiltrate (top) and Picrosirius red stained for assaying collagen content (bottom). (B) The Nrf2 silenced A/J muscle exhibited a decrease in total myofiber area, an increase in collagen content, the number of CNFs, and fatty deposits. ^*p < 0.05.

Figure 5

Increased oxidative stress in of Nrf2 silenced A/J quadriceps muscle. Quadriceps muscles (n = 6) were isolated from A/J and A/J-Nrf2^‒/‒ mice at 1 year of age, and were stained with antibodies against oxPL. Sections from A/J-Nrf2^‒/‒ muscles (right panels) exhibit excess oxPL (dark brown staining), compared to sections derived from A/J mice (left panels).