Sarcolemmal loss of active nNOS (Nos1) is an oxidative stress‐dependent, early event driving disuse atrophy

Abstract

Skeletal muscle atrophy following unloading or immobilization represents a major invalidating event in bedridden patients. Among mechanisms involved in atrophy development, a controversial role is played by neuronal NOS (nNOS; NOS1), whose dysregulation at the protein level and/or subcellular distribution also characterizes other neuromuscular disorders. This study aimed to investigate unloading-induced changes in nNOS before any evidence of myofiber atrophy, using vastus lateralis biopsies obtained from young healthy subjects after a short bed-rest and rat soleus muscles after exposure to short unloading periods. Our results showed that (1) changes in nNOS subcellular distribution using NADPH-diaphorase histochemistry to detect enzyme activity were observed earlier than using immunofluorescence to visualize the protein; (2) loss of active nNOS from the physiological subsarcolemmal localization occurred before myofiber atrophy, i.e. in 8-day bed-rest biopsies and in 6 h-unloaded rat soleus, and was accompanied by increased nNOS activity in the sarcoplasm; (3) nNOS (Nos1) transcript and protein levels decreased significantly in the rat soleus after 6 h and 1 day unloading, respectively, to return to ambulatory levels after 4 and 7 days of unloading, respectively; (4) unloading-induced nNOS redistribution appeared dependent on mitochondrial-derived oxidant species, indirectly measured by tropomyosin disulfide bonds which had increased significantly in the rat soleus already after a 6 h-unloading bout; (5) activity of displaced nNOS molecules is required for translocation of the FoxO3 transcription factor to myofiber nuclei. FoxO3 nuclear localization in rat soleus increased after 6 h unloading (about four-fold the ambulatory level), whereas it did not when nNOS expression and activity were inhibited in vivo before and during 6 h unloading. In conclusion, this study demonstrates that the redistribution of active nNOS molecules from sarcolemma to sarcoplasm not only is ahead of the atrophy of unloaded myofibers, and is induced by increased production of mitochondrial superoxide anion, but also drives FoxO3 activation to initiate muscle atrophy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Expression of SUMO enzymes is fiber type dependent in skeletal muscles and is dysregulated in muscle disuse

“…Surprising, in this model that mimic a condition of not physiological muscle position, along the treatment, the observed enrichment in more glycolytic fiber type phenotype was followed by an increasing of total SUMO‐1 and SUMO‐2 fluorescence (Figure H), that did not correlate with the fluorescence observed in the same fibers from ambulatory rats (Figure B). To prove the hindlimb‐unloading treatment worked on the soleus muscle samples, histograms and statistics of mean MW/BW ratio (Supplementary Figure 9), and increased localization of FoxO3 protein in myonuclei in the unloaded soleus muscle sections, compared to ambulatory one (Supplementary Figure 8), were performed according to Lechado i Terradas et al…”

“…Hindlimb muscles were unloaded using the tail‐suspension rat model, by wrapping the tail root with tape under general anesthesia induced with intraperitoneal administration of 20 mg/kg of Zolazepam chlorhydrate and tiletamine chlorhydrate (Zoletil; Virbac SRL, Milano, Italy). All animals were euthanized after inducing anesthesia with isofluorane; soleus muscles were excised and frozen in liquid nitrogen . The experimental protocol was performed following the recommendations provided by the European Convention for the protection of Vertebrate Animals used for Experimental and Scientific purposes (Council of Europe number 123, Strasbourg, 1985) and authorized by the Animal Ethics Committee of the University of Padova, Italy and the Italian Health Ministry (103/2007B).…”

“…All animals were euthanized after inducing anesthesia with isofluorane; soleus muscles were excised and frozen in liquid nitrogen. 27…”

“…We employed soleus muscles from ambulatory and after 1, 2, and 4 days of tailed-suspended Wistar-Han rats. 27,28 To evaluate the changes in the total SUMOylated proteins during unloading, the muscles lysates were separated in a SDS-PAGE gel and membranes probed with anti-SUMO-1, anti-SUMO-2, and anti-Ubiquitin antibodies, (Figure 5A,C,E). A significant (P < .05) increase of total SUMOylated proteins was detected with both anti-SUMO-1 and anti-SUMO-2 antibodies immediately after 1 day of unloading treatment ( Figure 5B,D), while on the contrary, no modification in the total polyubiquitinated proteins levels was detected along the same period ( Figure 5E,F), suggesting an intervention of the SUMO machinery immediately after change in the muscle activity and before the activation of protein degradation pathway.…”

“…Surprising, in this model that mimic a condition of not physiological muscle position, along the treatment, the observed enrichment in more glycolytic fiber type phenotype was followed by an increasing of total SUMO-1 and SUMO-2 fluorescence ( Figure 5H), that did not correlate with the fluorescence observed in the same fibers from ambulatory rats ( Figure 2B). To prove the hindlimb-unloading treatment worked on the soleus muscle samples, histograms and statistics of mean MW/BW ratio (Supplementary Figure 9), and increased localization of FoxO3 protein in myonuclei in the unloaded soleus muscle sections, compared to ambulatory one ( Supplementary Figure 8), were performed according to Lechado i Terradas et al 27 To understand the molecular mechanisms behind this unexpected alteration in the SUMO conjugation reaction during muscle unloading, we performed a time-series analysis of SUMO component transcript levels on soleus muscle after different times of unloading ( Figure 6A). Notably, the unloading treatment altered the transcription of almost all the SUMO machinery components immediately after 1 day of treatment as also clearly represented in the correspondent PCA obtained from the SUMO transcriptional signature of unloaded soleus ( Figure 6B).…”