NITRIC OXIDE AND DMD

Relation of Nitric Oxide Production Is Associated With DMD

Table of Contents

Abstract3

Introduction4

Materials and methods5

Muscle biopsies, patients5

Immunohistochemistry6

Cytophotometry7

Visualization and image processing8

Western blotting8

Results and discussion9

References16

Abstract

Nitric oxide mediates fundamental physiological actions on skeletal muscle. The neuronal NO synthase isoform (NOS1) was reported to be located exclusively in the sarcolemma. Its loss from the sarcolemma was associated with development of Duchenne muscular dystrophy . However, new studies evidence that all three NOS isoforms—NOS1, NOS2, and NOS3—are co-expressed in the sarcoplasm both in normal and in DMD skeletal muscles. To address this controversy, we assayed NOS expression in DMD myofibers in situ cytophotometrically and found NOS expression in DMD myofibers up-regulated. These results support the hypothesis that NO deficiency with consequent muscle degeneration in DMD results from NO scavenging by superoxides rather than from reduced NOS expression.

Relation of Nitric Oxide Production Is Associated With DMD

Introduction

In skeletal musculature, the neuronal NOS (nNOS, designated also as NOS1) was originally reported to be localized around the border of some muscle fibers identified as type II (fast) fibers . In the following decade, the circumferential NOS1 immunostaining pattern of muscle fibers, albeit without discriminating between fast and slow myofiber types, was reproduced by various groups. Positive NOS1 immunolabeling and and NADPH-diaphorase staining surrounding the myofibers were interpreted by the above-quoted authors as a proof for localization of NOS1 in the sarcolemma. The loss of NOS1 from skeletal muscle sarcolemma was declared to underlie the development of the clinical phenotype of DMD . This concept cannot be, however, well reconciled with the fact that the correction of some pathology in mdx mice (a mouse model of Duchenne myopathy) by dystrophin expression or by utrophin over-expression was independent of the presence of NOS1 . Interestingly, mice lacking NOS1 did not develop a musculardystrophy in spite of the originally reported association of DMD with the loss of NOS1 from skeletal muscle sarcolemma .

With the advent of more powerful immunohistochemical technique employing antigen retrieval in combination with signal amplification and , it was shown that NOS1 in skeletal muscles was not restricted to the sarcolemma and co-existed with NOS2 and NOS3 also in the intracellular compartments such as mitochondria, sarcoplasmic reticulum, and along contractile filaments . Recently, it was reported that, in contrast to the commonly accepted view, skeletal muscles of DMD patients retained all three NOS isoforms . To address this controversy, we have undertaken in this study a quantitative in situ determination of NOS expression in muscular fibers of DMD patients using tyramide signal amplification with following cytophotomentrical assessment of absorbance values for NOS1-3 immunostaining. In view of the NO involvement in muscle repair and , a better understanding of the NO pathways in dystrophic skeletal muscles may provide further insight into fundamental mechanisms underlying the development of a clinical phenotype in DMD patients and might have implications in the interception of the NO signaling for designing new adjunctive therapies for muscular dystrophies.

Materials and methods

Muscle biopsies, patients

Muscle biopsies obtained during routine diagnostic procedures from five DMD patients (aged 2-4 years) ...