Analysis by two-dimensional Blue Native/SDS-PAGE of membrane protein alterations in rat soleus muscle after hindlimb unloading

Abstract

Muscle atrophy occurring in several pathophysiological conditions determines decreases in muscle protein synthesis, increases in the rate of proteolysis and changes in muscle fiber composition. To determine the effect of muscle atrophy induced by hindlimb unloading (HU) on membrane proteins from rat soleus, a proteomic approach based on two-dimensional Blue Native/SDS-PAGE was performed. Proteomic analysis of normal and HU soleus muscle demonstrates statistically significant changes in the relative level of 36 proteins. Among the proteins identified by mass spectrometry, most are involved in pathways associated with muscle fuel utilization, indicating a shift in metabolism from oxidative to glycolytic. Moreover, immunoblotting analysis revealed an increase in aquaporin-4 (AQP4) water channel and an alteration of proteins belonging to the dystrophin-glycoprotein complex (DGC). AQP4 and DGC are regulated in soleus muscle subjected to simulated microgravity in response to compensatory mechanisms induced by muscle atrophy, and they parallel the slow-to-fast twitch conversion that occurs in soleus fibers during HU. In conclusion, the alterations of soleus muscle membrane proteome may play a pivotal role in the mechanisms involved in disuse-induced muscle atrophy.

Absence of Aquaporin-4 in Skeletal Muscle Alters Proteins Involved in Bioenergetic Pathways and Calcium Handling

“…With this method we are able to detect proteins with molecular masses between 10 kDa and up to 200 kDa. 2D BN/SDS-PAGE on skeletal muscle proteins was performed as previously described [6] with some modifications. Briefly, 250 µg of protein sample was applied on 4–15% polyacrylamide native gradient gels.…”

“…In a previous work we hypothesized that AQP4, together with the endothelial AQP1, allows high water exchange between the blood and the fibers in order to regulate volume changes occurring during muscle activity [2] which may be related to the substantial muscle swelling and intracellular osmolyte production occurring during exercise [3]–[5]. The physiological relevance of this water channel in the skeletal muscle is supported by the fact that muscle activity modulates AQP4 expression as it is evident during disuse [6], [7].…”

Long-term effects of simulated microgravity and/or chronic exposure to low-dose gamma radiation on behavior and blood–brain barrier integrity

“…It is associated with multiple types of skeletal muscle change and atrophy, 11 dysregulated gene expression that leads to altered protein synthesis and hemodynamics, 12 oxidative stress, 13 altered metabolism, 14 increased fatigability, 15 and vestibular issues. 16 In addition, aquaporin-4 (AQP4), a water channel protein involved in regulating water balance in the brain, 17,18 has been shown to be altered following hindlimb unloading, 19,20 a well-established Earth-bound model of microgravity that involves suspending a rodents’ hindlimbs off the ground for an extended period of time (see Materials and Methods for details). It has been suggested 19 that the increase in AQP4, which is prevalent in the neuromuscular system, 21 is a response to compensatory mechanisms initiated by muscle atrophy from extended periods of unloading.…”

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

“…In general, muscular atrophy is associated with a drastic decrease in protein synthesis and concomitant increase in the rates of protein breakdown, as well as slow-to-fast transitions in contractile kinetics and an oxidative-to-glycolytic shift in fiber metabolism [195]. A considerable number of gel-based studies have used animal models of denervation, immobilization or extended periods of muscle disuse to determine proteome-wide changes during muscular atrophy [196,197,198,199,200,201,202,203,204,205]. These studies have confirmed the general tendency of atrophying skeletal muscles to undergo a stepwise conversion to faster contractile properties and increased glycolytic metabolism.…”

“…The proteomic profiling of rat muscles following denervation [196], hindlimb suspension [197] or immobilization by the pin-heel method [198] showed increases in carbonic anhydrase CA3, enolase and fast MLC, as well as decreases in the fatty acid binding protein and slow MLC. As described in the proteomic studies listed in Table 2, neuromuscular unloading is clearly associated with a slow-to-fast transformation process [199,200,201,202,203] and complete denervation triggers extensive isoform switching in slow versus fast isoforms of the contractile proteins MyHC, MLC, TN and TM [204,205]. As already mentioned in above section on exercise proteomics, vibration training can be beneficial to counteract muscular atrophy triggered by extended periods of bed rest [174,175].…”