Functional Deficits in nNOSμ-Deficient Skeletal Muscle: Myopathy in nNOS Knockout Mice

Skeletal muscle nNOSμ (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSμ; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSμ. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSμ signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSμ expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention

SOD AND GSH INHIBIT THE HIGH GLUCOSE-INDUCED OXIDATIVE DAMAGE AND THE PDGF INCREASED SECRETION IN CULTURED HUMAN ENDOTHELIAL-CELLS

Poor control of blood glucose has been established as a key pathogenetic mechanism in the vascular complications of diabetes. It has been reported that glucose may autooxidize generating free radicals which have been suggested to delay proliferation, to modify mobility, to influence platelet-derived growth factor and other secretory protein production in a variety of cell systems. Platelet-derived growth factor, in turn, may induce proliferation and migration of vascular smooth muscle cells and thus play a role in atherogenesis. In the present study the effects of antioxidants on the high glucose-dependent oxidative cell damage and increased platelet-derived growth factor secretion have been investigated using cultured human endothelial cells. Our findings show that rising the glucose concentration in the culture medium from 5 mM to 20 mM, increased the production of free radicals cell damage markers, such as malondialdehyde and conjugated dienes, as well as the production of platelet-derived growth factor. The addition of super oxide dismutase or glutathione prevents both such effects. These results suggest that antioxidants may be a helpful therapeutic adjuvant to reduce the vascular complications of diabetes

Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin results in reduced sarcolemmal integrity and increased susceptibility to muscle damage. The α7β1-integrin is a laminin-binding protein up-regulated in the skeletal muscle of DMD patients and in the mdx mouse model. Transgenic overexpression of the α7-integrin alleviates muscle disease in dystrophic mice, making this gene a target for pharmacological intervention. Studies suggest laminin may regulate α7-integrin expression. To test this hypothesis, mouse and human myoblasts were treated with laminin and assayed for α7-integrin expression. We show that laminin-111 (α1, β1, γ1), which is expressed during embryonic development but absent in normal or dystrophic skeletal muscle, increased α7-integrin expression in mouse and DMD patient myoblasts. Injection of laminin-111 protein into the mdx mouse model of DMD increased expression of α7-integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscle from exercised-induced damage. These findings demonstrate that laminin-111 is a highly potent therapeutic agent for the mdx mouse model of DMD and represents a paradigm for the systemic delivery of extracellular matrix proteins as therapies for genetic diseases

In vivo acceleration of heart relaxation performance by parvalbumin gene delivery

Defective cardiac muscle relaxation plays a causal role in heart failure. Shown here is the new in vivo application of parvalbumin, a calcium-binding protein that facilitates ultrafast relaxation of specialized skeletal muscles. Parvalbumin is not naturally expressed in the heart. We show that parvalbumin gene transfer to the heart in vivo produces levels of parvalbumin characteristic of fast skeletal muscles, causes a physiologically relevant acceleration of heart relaxation performance in normal hearts, and enhances relaxation performance in an animal model of slowed cardiac muscle relaxation. Parvalbumin may offer the unique potential to correct defective relaxation in energetically compromised failing hearts because the relaxation-enhancement effect of parvalbumin arises from an ATP-independent mechanism. Additionally, parvalbumin gene transfer may provide a new therapeutic approach to correct cellular disturbances in calcium signaling pathways that cause abnormal growth or damage in the heart or other organs

Inaugural Article: Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of alpha-dystroglycan

Skeletal muscle basal lamina is linked to the sarcolemma through transmembrane receptors, including integrins and dystroglycan. The function of dystroglycan relies critically on posttranslational glycosylation, a common target shared by a genetically heterogeneous group of muscular dystrophies characterized by α-dystroglycan hypoglycosylation. Here we show that both dystroglycan and integrin α7 contribute to force-production of muscles, but that only disruption of dystroglycan causes detachment of the basal lamina from the sarcolemma and renders muscle prone to contraction-induced injury. These phenotypes of dystroglycan-null muscles are recapitulated by Largemyd muscles, which have an intact dystrophin–glycoprotein complex and lack only the laminin globular domain-binding motif on α-dystroglycan. Compromised sarcolemmal integrity is directly shown in Largemyd muscles and similarly in normal muscles when arenaviruses compete with matrix proteins for binding α-dystroglycan. These data provide direct mechanistic insight into how the dystroglycan-linked basal lamina contributes to the maintenance of sarcolemmal integrity and protects muscles from damage