Neuronal nitric oxide synthase expression is increased, but contraction‐induced phosphorylation is impaired in hindlimb muscles of rats with femoral artery ligation
Authors: Kuczmarski J. Matthew , Harms Jonathan E. , Thomas Gail D. 
Published: June 2021

Patients with peripheral artery disease experience repeated bouts of leg ischemia and reperfusion, promoting oxidative stress and inflammation in the affected muscles. While these conditions can reduce nitric oxide (NO) bioactivity, little is known about the effect of intermittent ischemia on neuronal NO synthase (nNOS), which is the main source of NO in contracting muscle. Reactive oxygen species (ROS) can disrupt nNOS signaling in muscle by reducing sarcolemmal nNOS, uncoupling nNOS, and impairing nNOS phosphorylation. We recently reported that oxidative stress occurs in the contracting hindlimbs of rats with femoral arteries that were ligated (LIG) for 72 h compared to rats with freely perfused (FP) femoral arteries. Therefore, in this study, we sought to determine if nNOS expression and regulation are disrupted in the hindlimb muscles of rats after femoral artery ligation. In skeletal muscles from FP or LIG hindlimbs of male Sprague Dawley rats, we measured protein expression of the NADPH oxidase subunit p67phox, cytosolic and mitochondrial superoxide dismutase (SOD1, SOD2), total nNOS, and nNOS dimerization. We also assessed nNOS phosphorylation (S1416) in resting muscle and after 10 min of rhythmic contractions. Consistent with our recent findings, p67phox was increased in muscles from LIG vs FP hindlimbs (plantaris, +160±55%, p<0.05; soleus, +418±236%, p=0.06), suggesting that NADPH oxidase is a significant source of ROS in LIG skeletal muscle. We also found that SOD1 was decreased (−13±2%, p<0.05) and SOD2 was increased (+52±18%, p=0.05) in the soleus but not plantaris muscles of LIG vs FP hindlimbs. This suggests that mitochondria may contribute to the increased ROS in oxidative muscle of the LIG hindlimbs. Surprisingly, nNOS was increased in soleus (+48±10%, p<0.05) and plantaris (+28±12%, p<0.05) muscles of LIG vs FP hindlimbs. Immunohistochemistry of the plantaris muscles confirmed that sarcolemmal nNOS localization was retained in the LIG hindlimbs. Although nNOS monomer levels increased in LIG vs FP muscles (soleus, +53±19%; plantaris, +36±12%; both p<0.05), similar increases in dimer levels resulted in unchanged nNOS dimer to monomer ratios, suggesting that nNOS remained coupled. Contraction of FP hindlimbs increased nNOS phosphorylation (+199±106%, p<0.05 vs rest), but this post‐transcriptional regulation was disrupted in muscle from LIG hindlimbs (+44±50%, p>0.05 vs rest). We then assessed signaling by the metabolic regulator AMP‐activated protein kinase (AMPK), which is known to phosphorylate nNOS. Total AMPK was unchanged in muscle from LIG vs FP hindlimbs, but the increased AMPK T172 phosphorylation seen in contracting muscle from FP hindlimbs (+21±6%, p<0.05 vs rest) was absent in muscle from LIG hindlimbs. Impaired AMPK regulation was confirmed by evaluating another known target, acetyl‐CoA carboxylase S79 phosphorylation (FP, +153±52%, p<0.05 vs rest; LIG, +49±25%, p>0.05 vs rest). Our findings suggest that impaired nNOS phosphorylation during contraction may contribute to reduced NO bioactivity in the muscles of the LIG hindlimb and raise the possibility that nNOS signaling might be restored by treatment with AMPK activators.Support or Funding InformationNIH P01 HL134609This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.