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.

Keywords: AMPK; cardiomyocyte; dystrophin; nNOS; stretch.

Fig. 1.

Stretch-dependent NO signaling is impaired in dystrophin-deficient cardiomyocytes. (A) Experimental setup. Adult mouse cardiomyocytes loaded with DAF-FM are visualized under 10x magnification using an epifluorescence microscope throughout 1 h of 1-Hz stretch. (Scale bar: 100 µm.) DAF-FM fluorescence in each rod-shaped, nonfibrillatory cardiomyocyte is quantified throughout the protocol. (B) Validation of NO imaging assay. Stretch of WT cardiomyocytes induces a significant increase in DAF-FM fluorescence (*P < 0.05, **P < 0.01, ****P < 0.0001, stretch vs. no stretch + vehicle). Incubation with the NOS inhibitor L-NAME abolishes the stretch-induced increase in DAF-FM fluorescence (^P < 0.001, ^^P < 0.0001, stretch + vehicle vs. L-NAME). Treatment with the NO donor SNAP yields a significant increase in DAF-FM fluorescence (‡P < 0.01, ‡‡‡P < 0.0001, SNAP vs. no stretch + vehicle). (C) The increase in DAF-FM fluorescence during stretch is significantly lower in mdx cardiomyocytes compared with WT cardiomyocytes (*P < 0.05, **P < 0.01, WT vs. mdx stretch). (D) Treatment with SNAP after stretch yields similar increases in DAF-FM fluorescence in WT and mdx cardiomyocytes. (E) There is no significant difference in cGMP concentration between WT and mdx cardiomyocytes before stretch (n = 8 mice). (F) The change in cGMP concentration downstream of NO in response to stretch is significantly altered in mdx cardiomyocytes vs. WT cardiomyocytes (n = 8 mice) (*P < 0.05).

Fig. 2.

Stimulatory nNOS phosphorylation is impaired in dystrophin-deficient hearts in vivo. The ratio of phospho-Ser1412-nNOS to total nNOS is significantly reduced in mdx hearts vs. WT hearts (n = 3 mice) (*P < 0.05).

Fig. 3.

Stretch-induced nNOS and AMPK activation are impaired in dystrophin-deficient cardiomyocytes. (A) Acute stretch of WT cardiomyocytes elicits increased phosphorylation of nNOS at the stimulatory residue serine 1412, increased activating phosphorylation of AMPKα at threonine 172, and increased phosphorylation of ACC at serine 79 (*P < 0.05 vs. WT control). These effects are attenuated in mdx cardiomyocytes (^#P < 0.05 vs. WT stretch) (n = 6 mice). (B) The increase in DAF-FM fluorescence during stretch of WT cardiomyocytes is significantly reduced in cells incubated with the nNOS-selective inhibitor vinyl-l-NIO (*P < 0.05, **P < 0.01, stretch + vehicle vs. vinyl-l-NIO). (C) The increase in DAF-FM fluorescence during stretch of WT cardiomyocytes is significantly reduced in cells incubated with the AMPK inhibitor Compound C (*P < 0.05, stretch + vehicle vs. Compound C).

Fig. S1.

nNOS localization. WT and mdx cardiomyocytes exhibit cytoplasmic and nuclear localization of nNOS (green) at rest. nNOS is not specifically enriched at the sarcolemma and does not colocalize with dystrophin (red). Cyclic stretch (1-Hz) does not lead to a discernible change in nNOS localization in either WT or mdx cardiomyocytes. (Scale bar: 50 µm.)

Fig. S2.

AMPKα localization. WT and mdx cardiomyocytes exhibit cytoplasmic and nuclear localization of AMPKα (red) at rest. AMPKα is not specifically enriched at the sarcolemma and does not colocalize with dystrophin (green). Cyclic stretch (1-Hz) does not lead to a discernible change in AMPKα localization in either WT or mdx cardiomyocytes. (Scale bar: 50 µm.)

Fig. 4.

Pharmacologic AMPK activation increases nNOS activity in dystrophin-deficient cardiomyocytes. (A) Treatment with AICAR elicits a significant increase in DAF-FM fluorescence in both WT and mdx cardiomyocytes (*P < 0.01, WT AICAR vs. vehicle; ^P < 0.01, ^^P < 0.001, mdx AICAR vs. vehicle). (B) nNOS inhibition with vinyl-l-NIO attenuates the increase in DAF-FM fluorescence induced by AICAR in mdx cardiomyocytes (*P < 0.001, **P < 0.0001, AICAR + vehicle vs. AICAR + vinyl-l-NIO). (C) Treatment with salicylate elicits a significant increase in DAF-FM fluorescence in both WT and mdx cardiomyocytes (*P < 0.0001, WT salicylate vs. vehicle; ^P < 0.0001, mdx salicylate vs. vehicle). (D) nNOS inhibition with vinyl-l-NIO attenuates the increase in DAF-FM fluorescence induced by salicylate in mdx cardiomyocytes (*P < 0.0001, salicylate + vehicle vs. salicylate + vinyl-l-NIO).

Fig. 5.

Pharmacologic AMPK activation increases stimulatory nNOS phosphorylation in dystrophin-deficient cardiomyocytes. (A) Acute treatment of WT and mdx cardiomyocytes with the AMPK activator AICAR increases activating phosphorylation of AMPKα at threonine 172 and increases stimulatory phosphorylation of nNOS at serine 1412 (*P < 0.05, **P < 0.01 vs. control) (n = 6 mice). (B) Acute treatment of WT and mdx cardiomyocytes with the AMPK activator salicylate increases activating phosphorylation of AMPKα at threonine 172 and increases stimulatory phosphorylation of nNOS at serine 1412 (*P < 0.05, **P < 0.01 vs. control) (n = 7 mice).