Dystrophin–glycoprotein complex regulates muscle nitric oxide production through mechanoregulation of AMPK signaling

Abstract

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. We used live-cell NO imaging and in vitro cyclic stretch of isolated adult mouse cardiomyocytes as a model system to investigate if and how the DGC directly regulates the mechanical activation of muscle NO signaling. Acute activation of NO synthesis by mechanical stretch was impaired in dystrophin-deficient mdx cardiomyocytes, accompanied by loss of stretch-induced neuronal NO synthase (nNOS) S1412 phosphorylation. Intriguingly, stretch induced the acute activation of AMP-activated protein kinase (AMPK) in normal cardiomyocytes but not in mdx cardiomyocytes, and specific inhibition of AMPK was sufficient to attenuate mechanoactivation of NO production. Therefore, we tested whether direct pharmacologic activation of AMPK could bypass defective mechanical signaling to restore nNOS activity in dystrophin-deficient cardiomyocytes. Indeed, activation of AMPK with 5-aminoimidazole-4-carboxamide riboside or salicylate increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC promotes the mechanical activation of cardiac nNOS by acting as a mechanosensor to regulate AMPK activity, and that pharmacologic AMPK activation may be a suitable therapeutic strategy for restoring nNOS activity in dystrophin-deficient hearts and muscle.T he muscular dystrophies are a group of muscle wasting disorders characterized by progressive weakening and degeneration of striated muscle. The most common form is Duchenne muscular dystrophy (DMD), an X-linked disorder caused by genetic disruption of dystrophin (1) that affects 1 in 3,500-5,000 males (2, 3). DMD and several other types of muscular dystrophy result from disruption of the dystrophin-glycoprotein complex (DGC), a structure that spans the sarcolemma and forms a mechanical linkage between the cytoskeleton and the extracellular matrix via the association of dystrophin with subsarcolemmal γ-actin and the binding of α-dystroglycan to laminin (4). The generally accepted role for this complex is to act as a molecular shock absorber and stabilize the plasma membrane during muscle contraction. Disruption of the DGC's linkage between the cytoskeleton and extracellular matrix, such as occurs in DMD (1, 5-7) or with the disruption of α-dystroglycan-laminin binding in glycosylation-deficient muscular dystrophies (8, 9), leads to destabilization of the plasma membrane, rendering skeletal muscle fibers and cardiomyocytes susceptible to stretch-or contraction-induced injury and cell death (10)(11)(12)(13)(14).In addition to this structural role, a signaling function for the DGC has been proposed based on its association with several signaling proteins including Grb2-Sos1 (15), MEK and ERK (16), heterotrimeric G protein subunits (17, 18), archvillin (19), and neuronal nitric oxide synthase (n...

Nitric oxide‐dependent attenuation of noradrenaline‐induced vasoconstriction is impaired in the canine model of Duchenne muscular dystrophy

“…; Hurt et al . ; Garbincius & Michele, ). We quantified the pnNOS level using a phospho‐S1412‐nNOS‐specific antibody by western blot.…”

The role of AMP‐activated protein kinase in the expression of the dystrophin‐associated protein complex in skeletal muscle

“…Indeed, recent investigations have revealed that pharmacological AMPK activation promotes translocation of the Na + /K + ‐ATPase a 1 subunit to the sarco‐lemma and increases activity of the ion pump (35). Furthermore, Garbincius and Michele (37) demonstrated that stretch‐induced production of NO in cardiomyocytes was dependent on AMPK signaling after mechano‐transduction from the DAPC. Thus, our findings support the concept that AMPK may be an important component of the structure and function of the sarcolemmal compartment.…”

“…Conversely, chronic pharmacological AMPK stimulation increases DAPC expression in skeletal muscle (11). Moreover, the presence and function of the kinase at the sarcolemma has been recently established (35, 37, 51). Based on this evidence, we hypothesized that removal of muscle AMPK would decrease DAPC gene expression and by extension would reduce its membrane localization.…”

“…Along these lines, an emerging area of research has focused on the role of AMPK in regulating sarcolemmal DAPC expression and function. Recent investigations have revealed that pharmacological activation of AMPK in models of DAPC deficiency leads to a rescue of DAPC expression and function and to increased levels of the dystrophin homolog utrophin in skeletal muscle (5, 10–13, 15, 36, 37). These adaptations occur concomitantly with a shift toward characteristics indicative of the slow, oxidative myogenic program.…”

Pharmacological therapeutics targeting the secondary defects and downstream pathology of Duchenne muscular dystrophy

“…The DAPC is thought to primarily serve a mechanical role, functioning as a physical link between the internal cytoskeleton and extracellular matrix (ECM) 4, 5 , though roles in mediating signaling are emerging 6, 7 . In DMD, loss of functional dystrophin and the consequential loss of the DAPC enhances sarcolemma susceptibility to contraction-induced damage resulting in repetitive cycles of muscle degeneration and regeneration 8 .…”