Proteomic Profiling of Hindlimb Skeletal Muscle Disuse in a Murine Model of Sepsis.

CONTEXT: Sepsis leads to multiple organ dysfunction and negatively impacts patient outcomes. Skeletal muscle disuse is a significant comorbidity in septic patients during their ICU stay due to prolonged immobilization. HYPOTHESIS: Combination of sepsis and muscle disuse will promote a unique proteomic signature in skeletal muscle in comparison to disuse and sepsis separately. METHODS AND MODELS: Following cecal ligation and puncture (CLP) or Sham surgeries, mice were subjected to hindlimb suspension (HLS) or maintained normal ambulation (NA). Tibialis anterior muscles from 24 C57BL6/J male mice were harvested for proteomic analysis. Proteomic profiles were assessed using nano-liquid chromatography with tandem mass spectrometry, followed by data analysis including Partial Least Squares Discriminant Analysis (PLS-DA), to compare the differential protein expression across groups. RESULTS: A total of 2876 differentially expressed proteins were identified, with marked differences between groups. In mice subjected to CLP and HLS combined, there was a distinctive proteomic signature characterized by a significant decrease in the expression of proteins involved in mitochondrial function and muscle metabolism, alongside a marked increase in proteins related to muscle degradation pathways. The PLS-DA demonstrated a clear separation among experimental groups, highlighting the unique profile of the CLP/HLS group. This suggests an important interaction between sepsis-induced inflammation and disuse atrophy mechanisms in sepsis-induced myopathy. INTERPRETATIONS AND CONCLUSIONS: Our findings reveal a complex proteomic landscape in skeletal muscle exposed to sepsis and disuse, consistent with an exacerbation of muscle protein degradation under these combined stressors. The identified proteins and their roles in cellular stress responses and muscle pathology provide potential targets for intervention to mitigate muscle dysfunction in septic conditions, highlighting the importance of addressing both sepsis and disuse concurrently in clinical and experimental settings.

[Buyang Huanwu Decoction promotes arteriogenesis after hindlimb ischemia in mice by activating PDGF signaling pathway].

This study aims to investigate the effect of Buyang Huanwu Decoction on blood flow recovery and arteriogenesis after hindlimb ischemia in mice via the platelet-derived growth factor(PDGF) signaling pathway. Forty C57BL/6 mice were randomized into model(clean water, 10 mL·kg~(-1)·d~(-1)), beraprost sodium(positive control, 18 µg·kg~(-1)·d~(-1)), and low-, medium-, and high-dose(10, 20, and 40 g·kg~(-1)·d~(-1), respectively) Buyang Huanwu Decoction groups(n=8). The hindlimb ischemia model was established by femoral artery ligation. The mice were administrated with corresponding agents by gavage daily for 14 days after ligation. For laser Doppler perfusion imaging, the mice were anesthetized and measured under a Periscan PSI imager. The density of capillary and arterio-le in the ischemic gastrocnemius was measured using immunofluorescence staining of the frozen tissue sections. Western blot was employed to determine the expression of PDGF subunit B(PDGFB), phosphorylated mitogen extracellular kinase(p-MEK), MEK, phosphorylated extracellular signal-regulated kinase(p-ERK), and ERK. Real-time PCR was employed to determine the mRNA level of PDGFB. The Buyang Huanwu Decoction-containing serum was used to treat the vascular smooth muscle cells(VSMCs) in hypoxia at doses of 10% and 20%. The proliferation and migration of VSMCs was assessed in vitro. The results showed that compared with the model group, beraprost sodium and Buyang Huanwu Decoction enhanced the blood flow recovery, increased the capillary and arteriole density, and up-regulated the protein levels of PDGFB, p-MEK, p-ERK, and mRNA levels of PDGFB, with the medium-dose Buyang Huanwu Decoction demonstrating the most significant effect. The 10% Buyang Huanwu Decoction-containing serum enhanced the proliferation and migration of VSMCs. Our findings demonstrate that Buyang Huanwu Decoction up-regulates PDGFB transcription and activates PDGF signaling pathway to promote arteriogenesis and blood flow recovery in ischemic gastrocnemius.

Sall genes regulate hindlimb initiation in mouse embryos.

Vertebrate limbs start to develop as paired protrusions from the lateral plate mesoderm at specific locations of the body with forelimb buds developing anteriorly and hindlimb buds posteriorly. During the initiation process, limb progenitor cells maintain active proliferation to form protrusions and start to express Fgf10, which triggers molecular processes for outgrowth and patterning. Although both processes occur in both types of limbs, forelimbs (Tbx5), and hindlimbs (Isl1) utilize distinct transcriptional systems to trigger their development. Here, we report that Sall1 and Sall4, zinc finger transcription factor genes, regulate hindlimb initiation in mouse embryos. Compared to the 100% frequency loss of hindlimb buds in TCre; Isl1 conditional knockouts, Hoxb6Cre; Isl1 conditional knockout causes a hypomorphic phenotype with only approximately 5% of mutants lacking the hindlimb. Our previous study of SALL4 ChIP-seq showed SALL4 enrichment in an Isl1 enhancer, suggesting that SALL4 acts upstream of Isl1. Removing 1 allele of Sall4 from the hypomorphic Hoxb6Cre; Isl1 mutant background caused loss of hindlimbs, but removing both alleles caused an even higher frequency of loss of hindlimbs, suggesting a genetic interaction between Sall4 and Isl1. Furthermore, TCre-mediated conditional double knockouts of Sall1 and Sall4 displayed a loss of expression of hindlimb progenitor markers (Isl1, Pitx1, Tbx4) and failed to develop hindlimbs, demonstrating functional redundancy between Sall1 and Sall4. Our data provides genetic evidence that Sall1 and Sall4 act as master regulators of hindlimb initiation.

Amplified P2X3 pathway activity in muscle afferent dorsal root ganglion neurons and exercise pressor reflex regulation in hindlimb ischaemia-reperfusion.

Hindlimb ischaemia-reperfusion (IR) is among the most prominent pathophysiological conditions observed in peripheral artery disease (PAD). An exaggerated arterial blood pressure (BP) response during exercise is associated with an elevated risk of cardiovascular events in individuals with PAD. However, the precise mechanisms leading to this exaggerated BP response are poorly elucidated. The P2X3 signalling pathway, which plays a key role in modifying the exercise pressor reflex (EPR), is the focus of the present study. We determined the regulatory role of P2X3 on the EPR in a rat model of hindlimb IR. In vivo and in vitro approaches were used to determine the expression and functions of P2X3 in muscle afferent nerves and EPR in IR rats. We found that in IR rats there was (1) upregulation of P2X3 protein expression in the L4-6 dorsal root ganglia (DRG); (2) amplified P2X currents in isolated isolectin B4 (IB4)-positive muscle DRG neurons; and (3) amplification of the P2X-mediated BP response. We further verified that both A-317491 and siRNA knockdown of P2X3 significantly decreased the activity of P2X currents in isolated muscle DRG neurons. Moreover, inhibition of muscle afferents' P2X3 receptor using A-317491 was observed to alleviate the exaggerated BP response induced by static muscle contraction and P2X-induced BP response by α,ß-methylene ATP injection. P2X3 signalling pathway activity is amplified in muscle afferent DRG neurons in regulating the EPR following hindlimb IR.

A non-invasive mouse model that recapitulates disuse-induced muscle atrophy in immobilized patients.

Disuse muscle atrophy occurs consequent to prolonged limb immobility or bed rest, which represents an unmet medical need. As existing animal models of limb immobilization often cause skin erosion, edema, and other untoward effects, we here report an alternative method via thermoplastic immobilization of hindlimbs in mice. While significant decreases in the weight and fiber size were noted after 7 days of immobilization, no apparent skin erosion or edema was found. To shed light onto the molecular mechanism underlying this muscle wasting, we performed the next-generation sequencing analysis of gastrocnemius muscles from immobilized versus non-mobilized legs. Among a total of 55,487 genes analyzed, 787 genes were differentially expressed (> fourfold; 454 and 333 genes up- and down-regulated, respectively), which included genes associated with muscle tissue development, muscle system process, protein digestion and absorption, and inflammation-related signaling. From a clinical perspective, this model may help understand the molecular/cellular mechanism that drives muscle disuse and identify therapeutic strategies for this debilitating disease.

Neovascularization after ischemic injury: evaluation with 99mTc-HYNIC-RGD / Neovascularização após lesão isquêmica: avaliação com 99mTc-HYNIC-RGD

Purpose: Angiogenesis involves many mediators including integrins, and the tripeptide RGD is a target amino acid recognition sequence for many of them. Hindlimb ischemia is a simple and convenient animal model however standardization of the injection procedures in the devascularized and control limb is lacking, thus rendering difficult the interpretation of results. The aim of this investigations was to evaluate neovascularization in a hindlimb murine model by means of 99mTc-HYNIC-ß-Ala-RGD. Methods: 99mTc-HYNIC-RGD analog was prepared using coligands. Ischemia was induced in Wistar rats by double- ligation of the common femoral artery. Radiolabeled RGD was injected after 2h, as well as 1, 3, 5, 7, 10 and 14 days. Uptake was evaluated by planar imaging and biodistribution studies. Results: The highest ratio between ischemia and control was achieved at the 7th day (2.62 ± 0.95), with substantial decrease by the 14th day. For pertechnetate the 7th day ratio was 0.87 ± 0.23. Scintigraphic image confirmed different uptakes. Conclusion: 99mTc-HYNIC-RGD analog concentrated in ischemic tissue by the time of widespread angiogenesis and pertechnetate confirmed reduction in blood flow. In this sense, the protocol can be recommended for ischemic models.

Objetivo: A angiogênese em resposta a fenômenos isquêmicos envolve vários mediadores como as integrinas, sendo que o tripeptídeo RGD possui uma seqüência de aminoácidos com reconhecimento para este alvo. O modelo animal de isquemia de pata traseira é simples e conveniente, porém não há uma padronização do procedimento de injeção e controle radioisotópico em membro desvascularizado, dificultando, portanto a interpretação de resultados. O objetivo deste estudo foi avaliar a neovascularização em modelo murino de isquemia de pata traseira através do radiotraçador 99mTc-HYNIC-ß-Ala-RGD. Métodos: O análogo 99mTc-HYNIC-RGD foi preparado usando coligantes. A isquemia foi induzida em ratos Wistar por dupla-ligação da artéria femoral comum na prega inguinal. Peptídeo RGD radiomarcado foi injetado após 2h, assim como 1, 3, 5, 7, 10 e 14 dias. A captação foi avaliada por imagem planar e estudos de biodistribuição. Resultados: A maior diferença de captação entre isquemia e pata controle foi obtida no 7º dia (2,62 ± 0,95), com decréscimo acentuado no 14º dia. Para o pertecnetato a razão no 7º dia foi 0,87 ± 0,23. A imagem cintilográfica confirmou as diferentes captações. Conclusões: O análogo 99mTc-HYNIC-RGD concentrou-se no tecido isquêmico na etapa em que a angiogênese é mais acentuada, e o estudo do pertecnetato confirmou a redução no fluxo sanguíneo. Desta maneira, este protocolo diagnóstico pode ser recomendado para modelos isquêmicos.

Estimulação elétrica minimiza alterações metabólicas musculares durante e após a imobilização de membro posterior de ratos / Electrical stimulation minimizes muscle metabolic alterations during and after immobilization of rats hindlimb

O objetivo do estudo foi avaliar o efeito da etimulação elétrica (EE) no perfil metabólico e no peso muscular de membro posterior de ratos durante a imobilização e após a retirada da órtese...

This study aimed at assessing the effect of electrical stimulation (ES) in muscles metabolic profile and weight of rat hindlimb during the limb immobilization period and after casts were removed...

Effects of L-alanyl-glutamine upon the blood and kidney biochemical parameters in the rat hind limb model of ischemia/reperfusion

OBJETIVO: Investigar alterações dos parâmetros metabólicos no sangue e rim de ratos submetidos à isquemia/reperfusão do membro pélvico. MÉTODOS: Quarenta e oito ratos machos foram distribuídos aleatoriamente em 2 grupos pré-tratados com administração intragástrica de solução salina 2,0 mL (G-1) ou L-alanil-glutamina 0,75 mgKg-1(G-2), uma vez ao dia (7:00h) durante 7 dias. Uma hora após a última gavagem todos os ratos foram anestesiados com éter dietílico, laparotomizados e submetidos ao pinçamento da artéria de ilíaca esquerda, durante 3 horas. Amostras foram coletadas ao término de isquemia e durante a reperfusão (1-3-6h) para determinação das concentrações in vivo de piruvato, lactato, glicose e corpos cetônicos (rim e sangue) e ATP (rim). RESULTADOS: Lactacemia e cetonemia aumentaram no grupo G-2 quando comparadas às aferidas em ratos não-tratados, durante a reperfusão. As concentrações de piruvato diminuíram e de lactato aumentaram significativamente no rim, durante a reperfusão (1h, 3h) em ratos do G-2 comparados aos respectivos controles. Houve um aumento significante nas concentrações renais de glicose, ATP e corpos cetônicos nos ratos tratados com L-alanil-glutamina durante a reperfusão (3h). CONCLUSÕES: A isquemia do membro pélvico em ratos pré-tratados com L-alanil-glutamina induz aumento da lactacemia e da concentração de lactato renal, indicando atividade glicolítica aumentada na medula renal. A hipercetonemia induzida pela oferta do dipeptídeo sugere cetogênese elevada, sinalizada por possível queda nas concentrações plasmáticas de insulina resultante da maior oxidação de glicose e utilização desse hormônio em tecidos periféricos.

Lack of hypophosphatemic effect on glucose uptake, lactate and pyruvate release by the normal rat perfused hind-limb

In previous experiments, it was observed that in the perfused hind-limb of phosphorus deficient rats there was a faster utilization of glucose when compared with preparations obtained from ad-libitum fed rats but similar to that obtained from pair-weighed rats. A greater release of lactate under the influence of insulin and a marked spontaneous and insulin induced liberation of pyruvate was registered in the perfused hind-limb of the phosphorus deficient rat. Since a hypophosphatemic medium was employed, we though it would be interesting to study the effect of low perfusate Pi in normal rat hind-limb preparations and, additionally, to observe it fast changes in the level of medium orthophosphate from normal to low values and viceversa were able to determine changes in glucose uptake and lactate and pyruvate release. In one group of experiments, the hind-limb of normal rats was perfused successively with orthophosphate deprived medium for thirty minutes in each stage. In another group of experiments, the first stage was carried out with normal. Pi medium, the second with orthophosphate deprived medium, and the third with normal Pi medium; each stage lasted thirty minutes. In all the experiments there was a gradual release of orthophosphate. The glucose uptake was not modified by the fast changes in the medium Pi nor were significant differences in lactate and pyruvate release observed, which showed clear increases in all stages. It is concluded that hypophosphatemia alone is not sufficient to obtain greater increases of lactate and pyruvate release; it is also required that the hind-limb preparations be obtained from ..