Igor L. Baptista

LEUCINE ATTENUATES SKELETAL MUSCLE WASTING VIA INHIBITION OF UBIQUITIN LIGASES

The aim of this study was to assess the effect of leucine supplementation on elements of the ubiquitin–proteasome system (UPS) in rat skeletal muscle during immobilization. This effect was evaluated by submitting the animals to a leucine supplementation protocol during hindlimb immobilization, after which different parameters were determined, including: muscle mass; cross‐sectional area (CSA); gene expression of E3 ligases/deubiquitinating enzymes; content of ubiquitinated proteins; and rate of protein synthesis. Our results show that leucine supplementation attenuates soleus muscle mass loss driven by immobilization. In addition, the marked decrease in the CSA in soleus muscle type I fibers, but not type II fibers, induced by immobilization was minimized by leucine feeding. Interestingly, leucine supplementation severely minimized the early transient increase in E3 ligase [muscle ring finger 1 (MuRF1) and muscle atrophy F‐box (MAFbx)/atrogin‐1] gene expression observed during immobilization. The reduced peak of E3 ligase gene expression was paralleled by a decreased content of ubiquitinated proteins during leucine feeding. The protein synthesis rate decreased by immobilization and was not affected by leucine supplementation. Our results strongly suggest that leucine supplementation attenuates muscle wasting induced by immobilization via minimizing gene expression of E3 ligases, which consequently could downregulate UPS‐driven protein degradation. It is notable that leucine supplementation does not restore decreased protein synthesis driven by immobilization. Muscle Nerve, 2010

MuRF1 is a muscle fiber-type II associated factor and together with MuRF2 regulates type-II fiber trophicity and maintenance.

MuRF1 is a member of the RBCC (RING, B-box, coiled-coil) superfamily that has been proposed to act as an atrogin during muscle wasting. Here, we show that MuRF1 is preferentially induced in type-II muscle fibers after denervation. Fourteen days after denervation, MuRF1 protein was further elevated but remained preferentially expressed in type-II muscle fibers. Consistent with a fiber-type dependent function of MuRF1, the tibialis anterior muscle (rich in type-II muscle fibers) was considerably more protected in MuRF1-KO mice from muscle wasting when compared to soleus muscle with mixed fiber-types. We also determined fiber-type distributions in MuRF1/MuRF2 double-deficient KO (dKO) mice, because MuRF2 is a close homolog of MuRF1. MuRF1/MuRF2 dKO mice showed a profound loss of type-II fibers in soleus muscle. As a potential mechanism we identified the interaction of MuRF1/MuRF2 with myozenin-1, a calcineurin/NFAT regulator and a factor required for maintenance of type-II muscle fibers. MuRF1/MuRF2 dKO mice had lost myozenin-1 expression in tibialis anterior muscle, implicating MuRF1/MuRF2 as regulators of the calcineurin/NFAT pathway. In summary, our data suggest that expression of MuRF1 is required for remodeling of type-II fibers under pathophysiological stress states, whereas MuRF1 and MuRF2 together are required for maintenance of type-II fibers, possibly via the regulation of myozenin-1.

Leucine Supplementation Improves Skeletal Muscle Regeneration after Cryolesion in Rats

This study was undertaken in order to provide further insight into the role of leucine supplementation in the skeletal muscle regeneration process, focusing on myofiber size and strength recovery. Young (2-month-old) rats were subjected or not to leucine supplementation (1.35 g/kg per day) started 3 days prior to cryolesion. Then, soleus muscles were cryolesioned and continued receiving leucine supplementation until 1, 3 and 10 days later. Soleus muscles from leucine-supplemented animals displayed an increase in myofiber size and a reduction in collagen type III expression on post-cryolesion day 10. Leucine was also effective in reducing FOXO3a activation and ubiquitinated protein accumulation in muscles at post-cryolesion days 3 and 10. In addition, leucine supplementation minimized the cryolesion-induced decrease in tetanic strength and increase in fatigue in regenerating muscles at post-cryolesion day 10. These beneficial effects of leucine were not accompanied by activation of any elements of the phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin signalling pathway in the regenerating muscles. Our results show that leucine improves myofiber size gain and strength recovery in regenerating soleus muscles through attenuation of protein ubiquitination. In addition, leucine might have therapeutic effects for muscle recovery following injury and in some muscle diseases.

Mammalian target of rapamycin complex 1 is involved in differentiation of regenerating myofibers in vivo

This work was undertaken to provide further insight into the role of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle regeneration, focusing on myofiber size recovery. Rats were treated or not with rapamycin, an mTORC1 inhibitor. Soleus muscles were then subjected to cryolesion and analyzed 1, 10, and 21 days later. A decrease in soleus myofiber cross‐section area on post‐cryolesion days 10 and 21 was accentuated by rapamycin, which was also effective in reducing protein synthesis in these freeze‐injured muscles. The incidence of proliferating satellite cells during regeneration was unaltered by rapamycin, although immunolabeling for neonatal myosin heavy chain (MHC) was weaker in cryolesion+rapamycin muscles than in cryolesion‐only muscles. In addition, the decline in tetanic contraction of freeze‐injured muscles was accentuated by rapamycin. This study indicates that mTORC1 plays a key role in the recovery of muscle mass and the differentiation of regenerating myofibers, independently of necrosis and satellite cell proliferation mechanisms. Muscle Nerve 42: 778–787, 2010

Expression of genes related to myostatin signaling during rat skeletal muscle longitudinal growth.

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin‐like 3 protein (FLST‐L3), growth and differentiation factor–associated protein‐1 (GASP‐1), activin IIB receptor (ActIIB), and SMAD‐7 were determined by real‐time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12–24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST‐L3, and GASP‐1 mRNA levels increased significantly over all time‐points. ActIIB gene expression decreased quickly at 12–24 hours. SMAD‐7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching. Muscle Nerve, 2009

Leucine and HMB Differentially Modulate Proteasome System in Skeletal Muscle under Different Sarcopenic Conditions

In the present study we have compared the effects of leucine supplementation and its metabolite β-hydroxy-β-methyl butyrate (HMB) on the ubiquitin-proteasome system and the PI3K/Akt pathway during two distinct atrophic conditions, hindlimb immobilization and dexamethasone treatment. Leucine supplementation was able to minimize the reduction in rat soleus mass driven by immobilization. On the other hand, leucine supplementation was unable to provide protection against soleus mass loss in dexamethasone treated rats. Interestingly, HMB supplementation was unable to provide protection against mass loss in all treatments. While solely fiber type I cross sectional area (CSA) was protected in immobilized soleus of leucine-supplemented rats, none of the fiber types were protected by leucine supplementation in rats under dexamethasone treatment. In addition and in line with muscle mass results, HMB treatment did not attenuate CSA decrease in all fiber types against either immobilization or dexamethasone treatment. While leucine supplementation was able to minimize increased expression of both Mafbx/Atrogin and MuRF1 in immobilized rats, leucine was only able to minimize Mafbx/Atrogin in dexamethasone treated rats. In contrast, HMB was unable to restrain the increase in those atrogenes in immobilized rats, but in dexamethasone treated rats, HMB minimized increased expression of Mafbx/Atrogin. The amount of ubiquitinated proteins, as expected, was increased in immobilized and dexamethasone treated rats and only leucine was able to block this increase in immobilized rats but not in dexamethasone treated rats. Leucine supplementation maintained soleus tetanic peak force in immobilized rats at normal level. On the other hand, HMB treatment failed to maintain tetanic peak force regardless of treatment. The present data suggested that the anti-atrophic effects of leucine are not mediated by its metabolite HMB.

Involvement of eukaryotic translation initiation factor 5A (eIF5A) in skeletal muscle stem cell differentiation

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post‐translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non‐differentiated satellite cells and skeletal muscle. The treatment with N1‐guanyl‐1,7‐diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L‐Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration. J. Cell. Physiol. 218: 480–489, 2009.

Quadriceps Muscle Atrophy After Anterior Cruciate Ligament Transection Involves Increased mRNA Levels of Atrogin-1, Muscle Ring Finger 1, and Myostatin

ObjectiveThe aim of this study was to assess the mRNA levels of atrogin-1, muscle ring finger 1, and myostatin in rat quadriceps after anterior cruciate ligament (ACL) transection. DesignWistar rats were randomized into three different groups: ACL (surgery and ACL transection), sham (surgery without ACL transection), and control. Vastus medialis, rectus femoris, and vastus lateralis muscles were harvested at 1, 2, 3, 7, and 15 days after ACL transection. The mRNA levels of atrogin-1, muscle ring finger 1, and myostatin, as well as the ubiquitinated protein content, muscle mass, and cross-sectional area of the muscle fibers, were evaluated. ResultsElevated levels of atrogin-1, muscle ring finger 1, and myostatin mRNA were detected in all tested muscles at most time points. The ubiquitinated protein content was increased at 3 days in the ACL and sham groups. The muscle mass of the ACL group was reduced at 3, 7, and 15 days (vastus lateralis and vastus medialis) and at 7 and 15 days (rectus femoris), whereas it was reduced in the sham group at 3 and 7 days (vastus lateralis and vastus medialis) and at 7 days (rectus femoris). The cross-sectional area of vastus medialis was reduced at 3, 7, and 15 days in the ACL group and at 3 and 7 days in the sham group. The cross-sectional area of the vastus lateralis was reduced at 7 and 15 days in the ACL group and at 7 days in the sham group. Whereas muscle mass and cross-sectional area recovery was noted in the sham group, no recovery was observed in the ACL group. ConclusionsQuadriceps atrophy after ACL transection involves increased levels of myostatin, atrogin-1, and muscle ring finger 1 mRNA and the accumulation of ubiquitinated protein.

Radicicol improves regeneration of skeletal muscle previously damaged by crotoxin in mice.

This work investigates the influence of heat shock proteins (HSPs) on necrosis and subsequent skeletal muscle regeneration induced by crotoxin (CTX), the major component of Crotalus durissus terrificus venom. Mice were treated with radicicol, a HSP inductor, followed by an intramuscular injection of CTX into the gastrocnemius muscle. Treated groups were sacrificed 1, 10 and 21 days after CTX injection. Muscle histological sections were stained with toluidine blue and assayed for acid phosphatase or immunostained with either neuronal cell adhesion molecule (NCAM) or neonatal myosin heavy chain (MHCn). Muscle samples were also submitted to Western blotting analysis. The results show that CTX alone and CTX combined with radicicol induced a similar degree of myofiber necrosis. CTX-injured muscles treated with radicicol had increased cross-sectional areas at 10 and 21 days post-lesion compared with untreated CTX-injured muscles. Additionally, radicicol significantly increased the number of NCAM-positive satellite cells in the gastrocnemius at one day post-CTX injury. CTX-injured muscles treated with radicicol contained more MHCn-positive regenerating myofibers compared with untreated CTX-injured muscles. These results suggest that HSPs contribute to the regeneration of myofibers damaged by CTX. Additionally, further studies should investigate the potential therapeutic effects of radicicol in skeletal muscles affected by Crotalus venom.

FoxO3a suppression and VPS34 activity are essential to anti-atrophic effects of leucine in skeletal muscle

Our aim is to gain insight into the mechanisms underlying the anti-atrophic effects of leucine, namely, the way that this amino acid can restrain the up-regulation of MuRF1 and Mafbx/Atrogin-1 in muscle atrophy. Male rats received dietary leucine supplementation for 1–3 days, during which time their hind limbs were immobilized. Our results showed that leucine inhibited Forkhead Box O3 (FoxO3a) translocation to cell nuclei. In addition, leucine was able to reverse the expected reduction of FoXO3a ubiquitination caused by immobilization. Unexpectedly, leucine promoted these effects independently of the Class I PI3K/Akt pathway. Vacuolar protein sorting 34 (VPS34; a Class III PI3K) was strongly localized in nuclei after immobilization and leucine supplementation was able to prevent this effect. In experiments on cultured primary myotubes, dexamethasone led to the localization of VPS34 in the nucleus. In addition, the pharmacological inhibition of VPS34 blocked VPS34 nuclear localization and impaired the protective effect of leucine upon myotube trophicity. Finally, the pharmacological inhibition of VPS34 in primary myotubes prevented the protective effects of leucine upon MuRF1 and Mafbx/Atrogin-1 gene expression. Autophagy-related target genes were not responsive to leucine. Thus, we demonstrate that the anti-atrophic effect of leucine is dependent upon FoxO3a suppression and VPS34 activity.