Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin

Abstract

SummaryDuchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin helps maintain sarcolemmal stability. Dystrophin also recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Failure to anchor nNOS to the membrane leads to functional ischemia and aggravates muscle disease in DMD. Over the past two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence, structural and functional similarity with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. Full-length utrophin cDNA was expressed in dystrophin-deficient mdx mice by gutted adenovirus or via transgenic overexpression. Subcellular nNOS localization was determined by immunofluorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supra-physiological utrophin expression, we did not detect nNOS at the sarcolemma. Furthermore, transgenic utrophin overexpression failed to protect mdx muscle from exercise-associated injury. Our results suggest that full-length utrophin cannot anchor nNOS to the sarcolemma. This finding might have important implications for the development of utrophin-based DMD therapies.

Microtubule binding distinguishes dystrophin from utrophin

“…Mice lacking sarcolemmal nNOS exhibit dramatically reduced cage activity after mild exercise (47). Although sarcolemmal nNOS localization is not rescued by the endogenous utrophin overexpression in mdx muscle or by transgenic utrophin overexpression in Fiona-mdx muscle (34), we show that postexercise activity in mdx mice is partially rescued by transgenic utrophin overexpression. Whereas mdx mice lose almost all physical activity after mild exercise, Fiona-mdx mice retain approximately half of WT physical activity levels after mild exercise.…”

“…Interestingly, the mini-Dys-TG-mdx mouse, which shows rescued microtubule morphology (Fig. 1E) but lacks the nNOS binding domain (34), exhibited postexercise activity that mirrored the activity of the Fiona-mdx mouse (P = 0.685), suggesting that microtubule lattice disorganization does not contribute to exercise-induced inactivity.…”

“…The transgenic utrophin overexpression mouse model does not restore the localization of nNOS to the sarcolemma, making this the only thus-far documented mdx phenotype not rescued in the Fiona-mdx mouse (9,34). The second possibility is that the amount of endogenous utrophin up-regulation in the mdx mouse is simply insufficient to restore microtubule lattice organization.…”

“…The mdx mouse shows greatly reduced physical activity after mild exercise that has been attributed to a lack of contraction-induced signaling from sarcolemmal-localized nNOS (37)(38)(39)(40)47). Although transgenic overexpression of utrophin in the Fiona-mdx mouse clearly fails to restore sarcolemmal nNOS localization (34), physical activity levels after mild exercise have not yet been measured. Similar to previously reported data (47), we found that mdx mice were significantly less active compared with WT mice after mild exercise ( Fig.…”

“…Although the mdx mouse expresses increased utrophin, these levels fail to restore nNOS to the sarcolemma or rescue the disorganized microtubule lattice that results owing to loss of dystrophin (16,19,34,35), indicating that utrophin cannot fully compensate for the lack of dystrophin. There are two main possibilities why endogenously up-regulated utrophin fails to retain microtubule lattice organization.…”