RESUMEN
Muscle and bone are cooperatively preserved in Daurian ground squirrels (Spermophilus dauricus) during hibernation. As such, we hypothesized that IGF-1 and myostatin may contribute to musculoskeletal maintenance during this period. Thus, we systematically assessed changes in the protein expression levels of IGF-1 and myostatin, as well as their corresponding downstream targets, in the vastus medialis (VM) muscle and femur in Daurian ground squirrels during different stages. Group differences were determined using one-way analysis of variance (ANOVA). Results indicated that the co-localization levels of IGF-1 and its receptor (IGF-1R) increased by 50% during the pre-hibernation period (PRE) and by 35% during re-entry into torpor (RET) compared to the summer active period (SA). The phosphorylation level of FOXO1 in the VM muscle increased by 50% in the torpor (TOR) group and by 82% in the inter-bout arousal (IBA) group compared to the PRE group. The phosphorylation level of SGK-1 increased by 54% in the IBA group and by 62% in the RET group compared to the SA group. In contrast, the protein expression of IGF-1 and phosphorylation levels of PI3K, Akt, mTOR, and GSK3ß in the VM muscle showed no obvious differences among the different groups. ß-catenin protein expression was up-regulated by 84% in the RET group compared to the SA group, while the content of IGF-1 protein, correlation coefficients of IGF-1 and IGF-1R, and phosphorylation levels of PI3K, Akt, and GSK3ß in the femur showed no significant differences among groups. Regarding myostatin and its downstream targets, myostatin protein expression decreased by 70% in the RET group compared to the SA group, whereas ActRIIB protein expression and Smad2/3 phosphorylation in the VM muscle showed no obvious differences among groups. Furthermore, Smad2/3 phosphorylation decreased by 58% in the TOR group and 53% in the RET group compared to the SA group, whereas ActRIIB protein expression in the femur showed no obvious differences among groups. Overall, the observed changes in IGF-1 and myostatin expression and their downstream targets may be involved in musculoskeletal preservation during hibernation in Daurian ground squirrels.
RESUMEN
Disuse-induced muscular atrophy is frequently accompanied by iron overload. Hibernating animals are a natural animal model for resistance to disuse muscle atrophy. In this paper, we explored changes in skeletal muscle iron content of Daurian ground squirrels (Spermophilus dauricus) during different periods of hibernation as well as the regulatory mechanisms involved. The results revealed that compared with the summer active group (SA), iron content in the soleus muscle (SOL) decreased (- 65%) in the torpor group (TOR), but returned to normal levels in the inter-bout arousal (IBA); splenic iron content increased in the TOR group (vs. SA, + 67%), decreased in the IBA group (vs. TOR, - 37%). Expression of serum hepcidin decreased in the TOR group (vs. SA, - 22%) and returned to normal levels in the IBA groups; serum ferritin increased in the TOR group (vs. SA, + 31%), then recovered in the IBA groups. Soleus muscle transferrin receptor 1 (TfR1) expression increased in the TOR group (vs. SA, + 83%), decreased in the IBA group (vs. TOR, - 30%); ferroportin 1 increased in the IBA group (vs. SA, + 55%); ferritin increased in the IBA group (vs. SA, + 42%). No significant differences in extensor digitorum longus in iron content or iron metabolism-related protein expression were observed among the groups. Significantly, all increased or decreased indicators in this study returned to normal levels after the post-hibernation group, showing remarkable plasticity. In summary, avoiding iron overload may be a potential mechanism for hibernating Daurian ground squirrels to avoid disuse induced muscular atrophy. In addition, the different skeletal muscle types exhibited unique strategies for regulating iron homeostasis.
Asunto(s)
Antígenos CD , Ferritinas , Hepcidinas , Hibernación , Homeostasis , Hierro , Músculo Esquelético , Atrofia Muscular , Receptores de Transferrina , Sciuridae , Animales , Sciuridae/fisiología , Hibernación/fisiología , Hierro/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Hepcidinas/metabolismo , Receptores de Transferrina/metabolismo , Ferritinas/metabolismo , Masculino , Bazo/metabolismo , Proteínas de Transporte de Catión/metabolismoRESUMEN
Hibernating Spermophilus dauricus experiences minor muscle atrophy, which is an attractive anti-disuse muscle atrophy model. Integrated metabolomics and proteomics analysis was performed on the hibernating S. dauricus during the pre-hibernation (PRE) stage, torpor (TOR) stage, interbout arousal (IBA) stage, and post-hibernation (POST) stage. Time course stage transition-based (TOR vs. PRE, IBA vs. TOR, POST vs. IBA) differential expression analysis was performed based on the R limma package. A total of 14 co-differential metabolites were detected. Among these, l-cystathionine, l-proline, ketoleucine, serine, and 1-Hydroxy-3,6,7-Trimethoxy-2, 8-Diprenylxanthone demonstrated the highest levels in the TOR stage; Beta-Nicotinamide adenine dinucleotide, Dihydrozeatin, Pannaric acid, and Propionylcarnitine demonstrated the highest levels in the IBA stage; Adrenosterone, PS (18:0/14,15-EpETE), S-Carboxymethylcysteine, TxB2, and 3-Phenoxybenzylalcohol demonstrated the highest levels in the POST stage. Kyoto Encyclopedia of Genes and Genomes pathways annotation analysis indicated that biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism were co-differential metabolism pathways during the different stages of hibernation. The stage-specific metabolism processes and integrated enzyme-centered metabolism networks in the different stages were also deciphered. Overall, our findings suggest that (1) the periodic change of proline, ketoleucine, and serine contributes to the hindlimb lean tissue preservation; and (2) key metabolites related to the biosynthesis of amino acids, ATP-binding cassette transporters, and cysteine and methionine metabolism may be associated with muscle atrophy resistance. In conclusion, our co-differential metabolites, co-differential metabolism pathways, stage-specific metabolism pathways, and integrated enzyme-centered metabolism networks are informative for biologists to generate hypotheses for functional analyses to perturb disuse-induced muscle atrophy.
Asunto(s)
Hibernación , Cetoácidos , Músculo Esquelético , Animales , Músculo Esquelético/metabolismo , Sciuridae/metabolismo , Proteómica , Cisteína/metabolismo , Criopreservación/métodos , Atrofia Muscular/metabolismo , Hibernación/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Serina/metabolismo , Metionina/metabolismoRESUMEN
Disuse atrophy of skeletal muscle is associated with a severe imbalance in cellular Ca2+ homeostasis and marked increase in nuclear apoptosis. Nuclear Ca2+ is involved in the regulation of cellular Ca2+ homeostasis. However, it remains unclear whether nuclear Ca2+ levels change under skeletal muscle disuse conditions, and whether changes in nuclear Ca2+ levels are associated with nuclear apoptosis. In this study, changes in Ca2+ levels, Ca2+ transporters, and regulatory factors in the nucleus of hindlimb unloaded rat soleus muscle were examined to investigate the effects of disuse on nuclear Ca2+ homeostasis and apoptosis. Results showed that, after hindlimb unloading, the nuclear envelope Ca2+ levels ([Ca2+]NE) and nucleocytoplasmic Ca2+ levels ([Ca2+]NC) increased by 78% (p < 0.01) and 106% (p < 0.01), respectively. The levels of Ca2+-ATPase type 2 (Ca2+-ATPase2), Ryanodine receptor 1 (RyR1), Inositol 1,4,5-tetrakisphosphate receptor 1 (IP3R1), Cyclic ADP ribose hydrolase (CD38) and Inositol 1,4,5-tetrakisphosphate (IP3) increased by 470% (p < 0.001), 94% (p < 0.05), 170% (p < 0.001), 640% (p < 0.001) and 12% (p < 0.05), respectively, and the levels of Na+/Ca2+ exchanger 3 (NCX3), Ca2+/calmodulin dependent protein kinase II (CaMK II) and Protein kinase A (PKA) decreased by 54% (p < 0.001), 33% (p < 0.05) and 5% (p > 0.05), respectively. In addition, DNase X is mainly localized in the myonucleus and its activity is elevated after hindlimb unloading. Overall, our results suggest that enhanced Ca2+ uptake from cytoplasm is involved in the increase in [Ca2+]NE after hindlimb unloading. Moreover, the increase in [Ca2+]NC is attributed to increased Ca2+ release into nucleocytoplasm and weakened Ca2+ uptake from nucleocytoplasm. DNase X is activated due to elevated [Ca2+]NC, leading to DNA fragmentation in myonucleus, ultimately initiating myonuclear apoptosis. Nucleocytoplasmic Ca2+ overload may contribute to the increased incidence of myonuclear apoptosis in disused skeletal muscle.
Asunto(s)
Suspensión Trasera , Atrofia Muscular , Ratas , Animales , Suspensión Trasera/fisiología , Atrofia Muscular/patología , Músculo Esquelético/metabolismo , Daño del ADN , Desoxirribonucleasas/metabolismoRESUMEN
Hibernating Spermophilus dauricus is resistant to muscle atrophy. Comprehensive transcriptome and proteome time-course analyses based on Metascape can further reveal the underlying processes (pre-hibernation stage, PRE; torpor stage, TOR; interbout arousal stage, IBA; and post-hibernation stage, POST). Transcriptome analysis showed that the cellular responses to growth factor stimulus and discrete oxygen levels continuously changed during hibernation. Proteomic analysis showed that neutrophil degranulation, sulfur compound metabolic process, and generation of precursor metabolites and energy continuously changed during hibernation. Molecular complex detection (MCODE) analysis in both transcriptome and proteome indicated that smooth muscle contraction was involved in the POST versus IBA stage, and peroxisome proliferator-activated receptor delta (Ppard), Myc proto-oncogene (Myc), Sp1 transcription factor (Sp1), and nuclear factor Kappa B subunit 1 (NFκB1) are the common TFs during the hibernation process. Integrated transcriptome and proteome analyses found 18 molecules in the TOR versus PRE stage, 1 molecule in the IBA versus TOR stage, and 16 molecules in the POST versus IBA stage. Among these molecules, carnitine palmitoyltransferase 1A (Cpt1a), SET and MYND domain containing 2 (Smyd2), four and a half LIM domains 1(Fhl1), reactive oxygen species modulator 1 (Romo1), and translocase of the inner mitochondrial membrane 50 (Timm50) were testified by Western blot. In conclusion, novel muscle atrophy resistance mechanisms can be deciphered by time-course transcriptome and proteome analyses based on Metascape.
Asunto(s)
Hibernación , Sciuridae , Animales , Sciuridae/fisiología , Transcriptoma , Proteómica , Proteoma/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Hibernación/fisiologíaRESUMEN
Iron overload is an independent risk factor for disuse osteoporosis. Hibernating animals are natural models of anti-disuse osteoporosis; however, whether iron metabolism is involved in bone adaptation and maintenance during hibernation is unclear. To investigate this question, Daurian ground squirrels (Spermophilus dauricus) (n = 5-6/group) were used to study changes in bone iron metabolism and its possible role in anti-disuse osteoporosis during hibernation. Iron content in the femur and liver first decreased in the torpor group (vs. summer group, -66.8% and -25.8%, respectively), then recovered in the post-hibernation group, suggesting remarkable plasticity of bone iron content. The expression of ferritin in the femur and hepcidin in the liver also initially decreased in the torpor group (vs. summer group, -28.5% and -38.8%, respectively), then increased in the inter-bout arousal (vs. torpor group, 126.2% and 58.4%, respectively) and post-hibernation groups (vs. torpor group, 153.1% and 27.1%, respectively). In conclusion, bone iron metabolism in hibernating Daurian ground squirrels showed remarkable plasticity, which may be a potential mechanism to avoid disuse bone loss during extended periods of inactivity. However, the specific location of iron during low-iron hibernation and the source of iron in post-hibernation recovery need to be further explored.