Fiber Cross-sectional Area Research Articles

Effects of some locally isolated Lactobacillus species on general growth, muscular mass and bone development in mice

Probiotics are dietary supplements which contain live microorganisms, mostly different bacterial species that inhabit gut of host and ultimately benefit that host. The current investigation was done to explore the growth-promoting effects of four locally isolated probiotics strains L. paracasei, L. paracaseii, L. delbrueckii and L. rhamnosus. In this regard, 108 colony-forming units (CFU) were administered to the mice of experimental groups for 30 days. Upon administering probiotics, it was observed that the Lactobacilli species had a positive impact on body weight. The group that received L. rhamnosus exhibited the most significant increase, with a 76.53 % improvement over the control group (P < 0.05). Positive effects on somatic indices of different organs including spleen, thymus, kidneys, heart, liver, and lungs were also observed. In addition to all of these, the increases in the number of extensor digitorum longus (EDL) muscle fibers and their cross-sectional area (CSA) up to 40.70 % and 28.94 % respectively in L. rhamnosus group as compared with the control group. In the L. rhamnosus group, there was a 40.70 % increase in the number of EDL muscle fibers and a 28.94 % increase in their cross-sectional area (CSA) of muscle fiber as compared to the control group. On provision of Lacticaseibacillus rhamnosus, 50.81 % higher femur bone density was noted which endorse the overall health-improving abilities of this probiotic. The application of Lacticaseibacillus rhamnosus in animal feed is recommended.

Unilateral hindlimb ischaemia-induced systemic inflammation is associated with non-ischaemic skeletal muscle inflammation.

Skeletal muscle atrophy and dysfunction commonly accompany cardiovascular diseases such as peripheral arterial disease and may be partially attributable to systemic inflammation. We sought to determine whether acute systemic inflammation in a model of hindlimb ischaemia (HLI) could affect skeletal muscle macrophage infiltration, fibre size, or capillarization, independent of the ischaemia. Eight-week-old C57BL/6 male mice underwent either Sham or HLI surgery, and were killed 1, 3, or 7 days post-surgery. Circulating inflammatory cytokine concentrations were measured, as well as immune cell infiltration and morphology of skeletal muscle from both limbs of HLI and Sham mice. In HLI compared with Sham mice at day 1, plasma interleukin-1β levels were 216% higher (0.48±0.10 vs. 0.15±0.01pg/μL, P = 0.005) and decreased by day 3. This was followed by increased macrophage presence in muscle from both ischaemic and non-ischaemic limbs of HLI mice by day 7 (7.3- and 2.3-fold greater than Sham, respectively, P<0.0001). In HLI mice, muscle from the ischaemic limb had 21% lower fibre cross-sectional area than the non-ischaemic limb (724±28 vs. 916±46μm2, P = 0.01), but the non-ischaemic limb of HLI mice was no different from Sham. This shows that HLI induces acute systemic inflammation accompanied by immune infiltration in both ischaemic and remote skeletal muscle; however, this did not induce skeletal muscle atrophy in remote muscle within the 7-day time course of this study. This effect of local skeletal muscle ischaemia on the inflammatory status of remote skeletal muscle may signal a priming of muscle for subsequent atrophy over a longer time course. HIGHLIGHTS: What is the central question of this study? Does hindlimb ischaemia-induced inflammation cause acute immune, inflammatory and morphological alterations in remote non-ischaemic skeletal muscle? What is the main finding and its importance? Hindlimb ischaemia induced systemic inflammation with subsequent neutrophil and macrophage infiltration in both ischaemic and non-ischaemic skeletal muscle; however, morphological changes did not occur in non-ischaemic muscle within 7 days. These immune alterations may have functional implications that take longer than 7 days to manifest, and subsequent or prolonged systemic inflammation and immune infiltration of muscle could lead to morphological changes and functional decline.

A round-robin study on the tensile characterization of single fibres: A multifactorial analysis and recommendations for more reliable results

In this benchmark, the tensile properties of three types of organic fibres – flax, hemp and aramid − were determined using single-fibre tensile tests performed by nine research groups. Flax and hemp were chosen due to their prevalence among European fibre plants. Aramid was selected for its synthetic nature and comparable dimensional properties. Due to the morphological complexity and variability of plant fibres, the scatter in the apparent tangent modulus and strength is more pronounced for flax and hemp compared to aramid. The primary source of scatter in the tensile properties results from human factors and experimental procedures, particularly regarding the fibre selection, the measurement of the fibre cross-sectional area and of the tensile strain. The post-processing procedure also turns out to be a key factor. Finally, recommendations and guidelines for best practices are proposed to reduce the main sources of dispersion associated with the reproducibility of single fibre tensile tests.

Competing endogenous RNA networks were associated with fat accumulation in skeletal muscle of aged male mice

Muscle aging contributed to morbidity and mortality in the elderly adults by leading to severe outcomes such as frailty, falls and fractures. Post-transcriptional regulation especially competing endogenous RNA (ceRNA) mechanism may modulate the process of skeletal muscle aging. RNA-seq was performed in quadriceps of 6-month-old (adult) and 22-month-old (aged) male mice to identify differentially expressed ncRNAs and mRNAs and further construct ceRNA networks. Decreased quadriceps-body weight ratio and muscle fiber cross-sectional area as well as histological characteristics of aging were observed in the aged mice. Besides, there were higher expressions of atrogin-1 and MuRF-1 and lower expression of Myog, Myf4 and Myod1 in the quadriceps of aged mice relative to that of adult mice. The expression of 85 lncRNAs, 52 circRNAs, 10 miRNAs and 277 mRNAs were significantly dysregulated in quadriceps between the two groups, among which two ceRNA networks lncRNA 2700081O15Rik/circRNA_0000820-miR-673–3p-Tmem120b were constructed. Level of triglycerides and expression of PPARγ, C/EBPα, FASN and leptin were elevated and the expression of adiponectin was reduced in quadriceps of aged mice compared with that of adult mice. LncRNA 2700081O15Rik/circRNA_0000820-miR-673–3p-Tmem120b were possibly associated with the adipogenesis and fat accumulation in skeletal muscle of age male mice.

Influence of intramuscular steroid receptor content and fiber capillarization on skeletal muscle hypertrophy.

Multiple intramuscular variables have been proposed to explain the high variability in resistance training induced muscle hypertrophy across humans. This study investigated if muscular androgen receptor (AR), estrogen receptor α (ERα) and β (ERβ) content and fiber capillarization are associated with fiber and whole-muscle hypertrophy after chronic resistance training. Male (n = 11) and female (n = 10) resistance training novices (22.1 ± 2.2 years) trained their knee extensors 3×/week for 10 weeks. Vastus lateralis biopsies were taken at baseline and post the training period to determine changes in fiber type specific cross-sectional area (CSA) and fiber capillarization by immunohistochemistry and, intramuscular AR, ERα and ERβ content by Western blotting. Vastus lateralis volume was quantified by MRI-based 3D segmentation. Vastus lateralis muscle volume significantly increased over the training period (+7.22%; range: -1.82 to +18.8%, p < 0.0001) but no changes occurred in all fiber (+1.64%; range: -21 to +34%, p = 0.869), type I fiber (+1.33%; range: -24 to +41%, p = 0.952) and type II fiber CSA (+2.19%; range: -23 to +29%, p = 0.838). However, wide inter-individual ranges were found. Resistance training increased the protein expression of ERα but not ERβ and AR, and the increase in ERα content was positively related to changes in fiber CSA. Only for the type II fibers, the baseline capillary-to-fiber-perimeter index was positively related to type II fiber hypertrophy but not to whole muscle responsiveness. In conclusion, an upregulation of ERα content and an adequate initial fiber capillarization may be contributing factors implicated in muscle fiber hypertrophy responsiveness after chronic resistance training.

Effects of Sex on Growth Performance, Carcass Traits, Blood Biochemical Parameters, and Meat Quality of XueShan Chickens.

The demand for high-quality chilled chicken has continued to increase in China. Chickens are sexually dimorphic, and to better understand the specific differences in chicken production based on sex, we examined how sex affects growth performance, carcass traits, and meat quality of yellow-feathered chickens. Male and female Xueshan chickens were used as the experimental model. Although males exhibited better growth performance, including body weight (BW), body slope, keel, shank length, and shank girth (p < 0.05), as well as carcass traits, such as dressed weight, leg muscle, and lean meat, females had higher carcass and breast muscle yields (p < 0.05). Males had higher follicle density and yellowness (b*) of the skin and better skin than females (p < 0.05). Among blood biochemical parameters, the serum content of corticosterone (CORT) was higher in males, while those of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), and catalase (CAT) were lower in males than in females (p < 0.05). The pH levels, shear force, and moisture content quality were better in male breast meat, while the intramuscular fat content (IMF) was lower in males than in females (p < 0.05). The redness (a*) and moisture content were higher in male leg meat, while the pH, water-loss rate (WLR), lightness (L*), and IMF were lower (p < 0.05). The muscle fiber diameter and cross-sectional area were also higher in males (p < 0.05). Consumers felt that soup of male chicken was better than female (p < 0.05), while mouthfeel and tenderness acceptance of breast meat were different between the sexes. These results indicate that female chickens can be marketed as a whole carcass, while males are more suitable for processed carcass products. This study provides significant insights into the production and processing methodologies of yellow-feathered chickens.

Altered skeletal muscle function and beneficial effects of exercise training in a rat model of induced pulmonary emphysema.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow obstruction and development of emphysema. Among the comorbidities associated with COPD, skeletal muscle dysfunction is known to affect exercise capacity and the survival rate of patients. Pulmonary rehabilitation (PR), via exercise training, is essential for COPD patients. However, the response to PR is most often moderate. An animal model that recapitulates critical features of chronic human disease and provides access to muscle function should therefore be useful to improve PR benefits. We used a rat model of induced emphysema based on pulmonary instillations of elastase (ELA) and lipopolysaccharides (LPS). We assessed the long-term effects of ELA/LPS and the potential effectiveness of endurance training on the skeletal muscle function. Invivo strength of the animals, and exvivo contractility, endurance, type 1 fiber proportion, fiber cross-sectional area, and capillarization of both soleus and extensor digitorum longus (EDL) were assessed. An impaired overall muscle strength with decreased force, reduced capillarization, and atrophy of type 1 fiber of EDL was observed in ELA/LPS rats. Soleus was not affected. Endurance training was able to reduce fatigability, and increase type 1 fiber proportion and capillarization of soleus, and improve force, endurance, and capillarization of EDL in control and ELA/LPS rats. Our rat model of induced emphysema, which shares some features with the phenotype present in patients with COPD, could represent a suitable model to study skeletal muscle dysfunction and the effects of exercise training on muscle function in patients.

Identification of key genes affecting ventilator-induced diaphragmatic dysfunction in diabetic mice.

Mechanical ventilation (MV) is often required in critically ill patients. However, prolonged mechanical ventilation can lead to Ventilator-induced diaphragmatic dysfunction (VIDD), resulting in difficulty in extubation after tracheal intubation, prolonged ICU stay, and increased mortality. At present, the incidence of diabetes is high in the world, and the prognosis of diabetic patients with mechanical ventilation is generally poor. Therefore, the role of diabetes in the development of VIDD needs to be discovered. MV modeling was performed on C57 mice and DB mice, and the control group was set up in each group. After 12h of mechanical ventilation, the muscle strength of the diaphragm was measured, and the muscle fiber immunofluorescence staining was used to verify the successful establishment of the MV model. RNA sequencing (RNA-seq) method was used to detect mRNA expression levels of the diaphragms of each group, and then differential expressed gene analysis, Heatmap analysis, WGCNA analysis, Venn analysis, GO and KEGG enrichment analysis were performed. qRT-PCR was used to verify the expression of the selected mRNAs. Our results showed that, compared with C57 control mice, the muscle strength and muscle fiber cross-sectional area of mice after mechanical ventilation decreased, and DB mice showed more obvious in this respect. RNA-seq showed that these differential expressed (DE) mRNAs were mainly related to genes such as extracellular matrix, collagen, elastic fiber and Fbxo32. GO and KEGG enrichment analysis showed that the signaling pathways associated with diabetes were mainly as follows: extracellular matrix (ECM), protein digestion and absorption, PI3K-Akt signaling pathway, calcium signaling pathway, MAPK signaling pathway and AGE-RAGE signaling pathway in diabetic complications, etc. ECM has the closest relationship with VIDD in diabetic mice. The key genes determined by WGCNA and Venn analysis were validated by quantitative real-time polymerase chain reaction (qRT-PCR), which exhibited trends similar to those observed by RNA-seq. VIDD can be aggravated in diabetic environment. This study provides new evidence for mRNA changes after mechanical ventilation in diabetic mice, suggesting that ECM and collagen may play an important role in the pathophysiological mechanism and progression of VIDD in diabetic mice, and provides some clues for the research, diagnosis, and treatment of VIDD in diabetic context.

Comparison of Muscle Regeneration Effects at Different Melittin Concentrations in Rabbit Atrophied Muscle.

This research aimed to explore the healing impacts of Melittin treatment on gastrocnemius muscle wasting caused by immobilization with a cast in rabbits. Twenty-four rabbits were randomly allocated to four groups. The procedures included different injections: 0.2 mL of normal saline to Group 1 (G1-NS); 4 μg/kg of Melittin to Group 2 (G2-4 μg/kg Melittin); 20 μg/kg of Melittin to Group 3 (G3-20 μg/kg Melittin); and 100 μg/kg of Melittin to Group 4 (G4-100 μg/kg Melittin). Ultrasound was used to guide the injections into the rabbits' atrophied calf muscles following two weeks of immobilization via casting. Clinical measurements, including the length of the calf, the compound muscle action potential (CMAP) of the tibial nerve, and the gastrocnemius muscle thickness, were assessed. Additionally, cross-sectional slices of gastrocnemius muscle fibers were examined, and immunohistochemistry and Western blot analyses were performed following two weeks of therapy. The mean regenerative changes, as indicated by clinical parameters, in Group 4 were significantly more pronounced than in the other groups (p < 0.05). Furthermore, the cross-sectional area of the gastrocnemius muscle fibers and immunohistochemical indicators in Group 4 exceeded those in the remaining groups (p < 0.05). Western blot analysis also showed a more significant presence of anti-inflammatory and angiogenic cytokines in Group 4 compared to the others (p < 0.05). Melittin therapy at a higher dosage can more efficiently activate regeneration in atrophied gastrocnemius muscle compared to lower doses of Melittin or normal saline.

125 Effects of Saccharmyces yeast postbiotics on nursey pig muscle fiber cross-sectional area and oxidative capacity

Abstract This study aimed to determine effects of dietary supplementation of Saccharomyces yeast postbiotics (celluTEIN, Puretein Bioscience, LLC, Minneapolis, MN) on nursery pig muscle fiber cross-sectional area (CSA), isoform, and succinate dehydrogenase (SDH) staining intensity. Newly weaned pigs [n = 32; 16 barrows and 16 gilts; 21 d of age, body weight (BW) 6.05 ± 0.24 kg] were stratified by BW within sex, placed in individual pens, and randomly assigned to one of two dietary treatments within each two-pig strata. Dietary treatments included pigs supplemented 0 (CON; n = 16) or 175 g/ton (CT; n = 16) of celluTEIN to their standard three-phase nursery ration. Pigs were harvested at d 35 of feeding and the longissimus lumborum muscle was collected for mean SDH staining intensity and immunohistochemistry analyses. Data were analyzed as a completely randomized design with pig as the experimental unit and treatment as the fixed effect. Statistical significance was determined at P ≤ 0.05. There were no treatment effects on type I and type IIB muscle fiber CSA (P &amp;gt; 0.14), but CT pigs had greater type IIA and type IIX muscle fiber CSA compared with CON (P &amp;lt; 0.05). When not analyzed by fiber isoform, pigs fed CT had a greater (P = 0.02) average muscle fiber CSA than CON pigs. There were no treatment effects on muscle fiber type percentage or SDH intensity of all fiber types (P ≥ 0.23); however, when calculated as a muscle fiber CSA ratio, CT type IIA fibers had a smaller ratio (darker staining/µm2) than CON type IIA fibers (P = 0.05). There were no treatment effects for type I, IIX, and IIB ratios (P &amp;gt; 0.21). In conclusion, supplementing Saccharomyces yeast postbiotics during the nursery period increased muscle fiber hypertrophy and type IIA fiber oxidative capacity.

Impact of Reduced Activity on Endurance of Rat Diaphragm Muscle

The diaphragm muscle (DIAm) as the major inspiratory pump is highly active. During breathing, fatigue resistant DIAm motor units are recruited that comprise type I and IIa fibers. Accordingly, these fibers have substantially higher mitochondrial volume density (MVD) and mitochondrial respiratory capacity (SDHmax) compared to more fatigable type IIx/IIb fibers. Cervical spinal cord injury disrupts descending inspiratory drive and markedly reduces eupneic activity of the DIAm. With C2 spinal cord hemisection (C2SH) DIAm activity on the ipsilateral side is reduced. Following a two-week period of C2SH, we found reduced MVD and SDHmax in type I and IIa DIAm fibers. As a result, we hypothesize that endurance of the DIAm is reduced. Female and male Sprague Dawley rats were used. EMG electrodes were placed in the left and right DIAm 3 days prior to C2SH surgery, and baseline recordings of eupneic DIAm activity were obtained in awake animals. The right C2 spinal cord was hemisected, and the extent of injury was confirmed by the absence of eupneic DIAm activity on the ipsilateral side while animals were anesthetized. Subsequently, DIAm EMG activity was recorded at 3, 7 and 14 days post C2SH. After two weeks, rats were anesthetized and the DIAm was excised. DIAm strips were cut and suspended in a tissue chamber containing Rees-Simpson solution maintained at 26oC, with thecostal margin clamped and the central tendon attached to a Cambridge Dual-Mode force/length transducer. DIAm strips were stimulated using platinum plate electrodes with supramaximal current (~250 mA) pulses (1 ms), and maximum specific force was determined at 75 Hz. The force-velocity relationship was then determined by systematically changing load, and maximum velocity (Vmax) was estimated based on curve fitting with the Hill-equation. Based on the force-velocity measurements peak power of the DIAm was calculated. Subsequently, the DIAm was repetitively stimulated and allowed to shorten at 30% maximum velocity (isovelocity approximating peak power output). Endurance of the DIAm was then determined as the duration that peak power output was sustained. Finally, the DIAm was rapidly frozen, and cross-sections were cut for fiber type and cross-sectional area (CSA) measurements. C2SH resulted in a marked decrease in eupneic DIAm EMG activity across the two-week period, which disproportionately affected type I and IIa DIAm fibers. However, there was no impact of C2SH on DIAm fiber type proportions or CSA. Following two-weeks C2SH, maximum specific force, Vmax, and peak power output of the DIAm were comparable between intact and injured sides. However, endurance of the DIAm was reduced on the injured side compared to the intact side. These results support our hypothesis that reduced activity of DIAm, reduces oxidative capacity of type I and IIa fibers and thereby reduces endurance. In clinical conditions such as mechanical ventilation DIAm activity is reduced and if maintained for longer periods of time this may affect DIAm endurance post weaning. Research supported by NIH grant: HL146114 (GCS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal muscle preservation in arctic ground squirrels during hibernation season

Reduced skeletal loading leads to muscle atrophy in humans and most mammals. By contrast, hibernating mammals demonstrate limited loss of skeletal muscle mass and strength by the end of winter after being physically inactive for several months. The present study objective was to establish potential underlying processes for the lack of muscle loss during the hibernation season of arctic ground squirrels (AGS). Our hypothesis was that early in the the hibernation season, protective mechanisms of AGS are not yet mobilized and muscles show signs of atrophy, while compensatory processes for preventing muscle loss appear at a later time point. Quadriceps muscles of juvenile male AGS were collected shortly before hibernation, and on 2, 6, 10-12 and 16-22 weeks during hibernation. Pre-hibernating animals were used as controls. Fiber cross-sectional area (CSA) and fiber type composition were determined with immunohistochemistry. Amount of total RNA, ribosomal RNA content and expression levels of genes involved in 2 pathways of protein degradation, ubiquitination and autophagy, were analyzed by real-time PCR, to evaluate molecular mechanisms of muscle maintenance. One-Way ANOVA was used to statistically analyze the group differences. We found that CSA was not different between the groups (P &gt; 0.05). CSA frequency distribution curves did not reveal any shift to a smaller or larger size range. No difference was detected in myofiber composition between the hibernation groups compared to the control. Total RNA and ribosomal RNA content were not significantly different between the groups during hibernation. Muscle atrophy marker FBXO32 (Atrogin-1, MAFBX) and autophagy related genes MAP1LC3A and BECN1 did not show different level of expression between the groups. Only another muscle atrophy marker TRIM63 (MURF-1, Iris Ring Finger Protein) was significantly over-expressed at the time point of 2 weeks of hibernation. These results indicate that throughout the hibernation season, AGS preserve muscle fiber CSA showing limited signs of muscle atrophy only during the first weeks of hibernation, and yet-to-be determined processes exist to suppress protein degradation in AGS muscles during hibernation. The work was supported by Center of Biomedical Research Excellence under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High-fat diet induces sarcopenic obesity in natural aging rats through the gut–trimethylamine N-oxide–muscle axis

IntroductionThe lack of suitable animal models for sarcopenic obesity (SO) limits in-depth research into the disease. Emerging studies have demonstrated that gut dysbiosis is involved in the development of SO. As the importance of microbial metabolites is starting to unveil, it is necessary to comprehend the specific metabolites associated with gut microbiota and SO. ObjectivesWe aimed to investigate whether high-fat diet (HFD) causes SO in natural aging animal models and specific microbial metabolites that are involved in linking HFD and SO. MethodsYoung rats received HFD or control diet for 80 weeks, and obesity-related metabolic disorders and sarcopenia were measured. 16S rRNA sequencing and non-targeted and targeted metabolomics methods were used to detect fecal gut microbiota and serum metabolites. Gut barrier function was evaluated by intestinal barrier integrity and intestinal permeability. Trimethylamine N-oxide (TMAO) treatment was further conducted for verification. ResultsHFD resulted in body weight gain, dyslipidemia, impaired glucose tolerance, insulin resistance, and systemic inflammation in natural aging rats. HFD also caused decreases in muscle mass, strength, function, and fiber cross-sectional area and increase in muscle fatty infiltration in natural aging rats. 16S rRNA sequencing and nontargeted and targeted metabolomics analysis indicated that HFD contributed to gut dysbiosis, mainly characterized by increases in deleterious bacteria and TMAO. HFD destroyed intestinal barrier integrity and increased intestinal permeability, as evaluated by reducing levels of colonic mucin-2, tight junction proteins, goblet cells and elevating serum level of fluorescein isothiocyanate-dextran 4. Correlation analysis showed a positive association between TMAO and SO. In addition, TMAO treatment aggravated the development of SO in HFD-fed aged rats through regulating the ROS-AKT/mTOR signaling pathway. ConclusionHFD leads to SO in natural aging rats, partially through the gut–microbiota–TMAO–muscle axis.

Parathyroid hormone promotes induction of beige adipocytes and reversibly ameliorates muscle quality and atrophy following chronic rotator cuff tear in a rat model

Fatty infiltration (FI) and muscle atrophy (MA) in the rotator cuff muscles following rotator cuff tears (RCT) persist postrepair, increasing the risk of re-tears. Brown adipocyte-like "beige adipocytes" are expected to have a therapeutic effect on intramuscular FI and MA due to their lipolytic activity and the muscle regenerative effects of their secreted factors. However, whether parathyroid hormone (PTH) ameliorates the already advanced FI and MA remains unknown. Therefore, this study aimed to clarify whether PTH promotes the expression of beige adipocytes and ameliorates advanced FI and MA following chronic RCT in rats. Supraspinatus muscles were harvested from rats with chronic RCT after 4 or 8weeks of PTH treatment and compared to those in the control group or to those at the start of treatment. FI was assessed by Oil Red O staining, and the staining area was evaluated as a percentage of the muscle cross-sectional area. MA was evaluated by measuring muscle wet weight and cross-sectional area of muscle fiber. Beige adipocyte expression was evaluated by immunostaining for uncoupling protein 1 (UCP1). Fibro-adipogenic progenitors (FAPs) were separated from muscle-injured mice. We assessed whether PTH could diminish fat droplet accumulation by promoting the differentiation of FAPs into beige adipocytes. After 4 weeks, PTH reduced the area fraction of FI in the rat supraspinatus muscle following chronic RCT compared with that at the beginning of treatment (P=.028). In addition, PTH increased wet muscle mass (P<.001), and muscle fiber cross-sectional area (P=.018) compared with measurements at the start of treatment. PTH administration promoted the expression of UCP1, a beige adipocyte marker, in the supraspinatus muscle (P=.019). PTH increased gene expression of beige adipocyte-related markers and suppressed fat droplet accumulation even after adipogenic differentiation of FAPs (P=.004) but did not reduce fat droplets that had already accumulated in invitro experiments. PTH facilitated beige adipocyte expression and reversibly ameliorated muscle quality and atrophy following chronic RCT by hindering fat droplet accumulation and facilitating muscle regeneration. Therefore, PTH may be a medical treatment for FI and MA following RCT, leading to expanded rotator cuff repair indications.

Postnatal diaphragm muscle and phrenic motor neuron development is impaired in Spastic mice

In rats, there is a strong association between the emergence of mature diaphragm muscle (DIAm) myosin heavy chain isoform expression and the expansion of phrenic motor neuron (PhMN) size (from ~21-28 days postnatal (P)), alongside the manifestation of mature morphological and functional properties. In the Spastic ( Spa) mutant mouse, mutated glycine receptors lead to a spasticity phenotype emerging between P14-28, concomitant with DIAm and PhMN development. In adult Spa mice, DIAm weakness, fiber atrophy and loss of larger PhMNs is evident. We aim to characterize whether these DIAm and PhMN deficits occur contemporaneously to spasticity. Using wildtype (WT) and Spa mice at P14, P21, P28, and P90 (maturity) we evaluated DIAm and PhMN physiologic properties. Ex vivo DIAm strips were used for functional assessments of maximum specific force (Po) and fatigue. DIAm strips were also fresh-frozen, and 8 μm cross-sections were cut and stained with H&amp;E to evaluate cross-sectional area (CSA) of individual DIAm fibers. PhMNs were labelled via rhodamine phrenic nerve dip, processed for longitudinal cryosectioning (70 μm) and prepared for confocal imaging and PhMN assessment. In WT mice, DIAm specific force increased at each age (P14, P21, P28 and P90), whilst in Spa mice, DIAm specific force was reduced from P21 compared to WT. Similarly, DIAm fiber CSA was reduced in Spa compared to WT mice at P21, P28 and P90. The number of PhMNs was similar between genotypes at P21, with a reduction in the number of PhMNs in Spa mice compared to WT evident at P28 continuing to P90. In Spa mice, PhMN size was smaller compared to WT at P21, unchanged at P28 and reduced at P90. The changes in Spa DIAm and PhMNs during the first postnatal month are consistent with the emergence of spasticity from P14 to P28. As most changes occur at ~P21, we are optimistic that interventions prior to weaning may promote the normal growth of DIAm and survival of PhMNs and may stave off later life DIAm behavioral deficits. HL166204 and HL146114. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Peripheral artery disease patients with chronic kidney disease have lower muscle strength and mitochondrial function compared to those with normal kidney function

Introduction: Chronic kidney disease is a significant risk factor for both the development of peripheral artery disease (PAD) and major adverse limb events. However, little is known about how the coalescence of these two conditions impacts limb muscle pathophysiology. The objective of the current study was to determine the impact of CKD on skeletal muscle strength and mitochondrial health in patients with and without PAD. Methods: A cross-sectional prospective study design was employed to assess plantar flexor muscle strength, muscle fiber cross-sectional area (CSA), and mitochondrial health in four groups of patients (61 males and 29 females): PAD only (Toe brachial index, TBI&lt;0.5 and eGFR&gt;60 mL/min), CKD only (TBI&gt;0.75 and eGFR:15-45 mL/min), PAD with CKD (TBI&lt;0.5 and eGFR:15-45 mL/min), and adult controls without PAD and CKD (TBI&gt;0.75 and eGFR&gt;60 mL/min). Results: Plantar flexor muscle strength was lowest in PAD patients with CKD compared to adult controls ( P&lt;0.0001), PAD only ( P=0.001), and CKD only ( P=0.003). No differences in the mean muscle fiber CSA were detected. The percentage of COX+ fibers was significantly lower in PAD patients with CKD compared to adult controls ( P=0.03). High resolution respirometry analysis of mitochondrial oxidative phosphorylation (OXPHOS) function demonstrated that CKD has a significant negative impact on muscle bioenergetics. PAD patients with CKD had lower OXPHOS function than adult controls ( P=0.0016) and PAD patients without CKD ( P=0.0015). Notably, CKD patients without PAD also had lower OXPHOS function than adult controls ( P=0.0054) and PAD patients without CKD ( P=0.005). Conclusion: The current results demonstrate a profound negative impact of CKD on the associated myopathy of PAD patients. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (T.E.R.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Expression of intramuscular extracellular matrix proteins in vastus lateralis muscle fibres between atrophic and non-atrophic COPD.

Extracellular matrix (ECM) proteins are the major constituents of the muscle cell micro-environment, imparting instructive signalling, steering cell behaviour and controlling muscle regeneration. ECM remodelling is among the most affected signalling pathways in COPD and aged muscle. As a fraction of COPD patients present muscle atrophy, we questioned whether ECM composition would be altered in patients with peripheral muscle wasting (atrophic COPD) compared to those without muscle wasting (non-atrophic COPD). A set of ECM molecules with known impact on myogenesis were quantified in vastus lateralis muscle biopsies from 29 COPD patients (forced expiratory volume in 1 s 55±12% predicted) using ELISA and real-time PCR. COPD patients were grouped to atrophic or non-atrophic based on fat-free mass index (<17 or ≥17 kg·m-2). Atrophic COPD patients presented a lower average vastus lateralis muscle fibre cross-sectional area (3872±258 μm2) compared to non-atrophic COPD (4509±198 μm2). Gene expression of ECM molecules was found significantly lower in atrophic COPD compared to non-atrophic COPD for collagen type I alpha 1 chain (COL1A1), fibronectin (FN1), tenascin C (TNC) and biglycan (BGN). In terms of protein levels, there were no significant differences between the two COPD cohorts for any of the ECM molecules tested. Although atrophic COPD presented decreased contractile muscle tissue, the differences in ECM mRNA expression between atrophic and non-atrophic COPD were not translated at the protein level, potentially indicating an accumulation of long-lived ECM proteins and dysregulated proteostasis, as is typically observed during deconditioning and ageing.

Muscle regeneration is not impaired by ablation of UCP-1-expressing cells

INTRODUCTION: Recently, cells expressing uncoupling protein 1 (UCP-1) have been proposed to impact muscle physiology. First, transplant of brown adipose tissue (which has high expression of UCP-1) enhanced regeneration in the injured mouse rotator cuff. Second, isolated UCP-1+ muscle fibroadipogenic progenitors (FAPs) treated to enhance UCP-1 expression improved injured mouse rotator cuff muscle pathology. However, it remains unknown whether endogenous UCP-1-expressing cells contribute to muscle regeneration. Therefore, the objective of this study was to assess whether mice that lack UCP-1-expressing cells demonstrate impaired muscle regeneration following toxin administration. METHODS: Experiments were performed using tibialis anterior (TA) and 5th toe extensor digitorum longus (EDL) muscle of wild type C57BL/6J (WT; n=22), and uncoupling protein 1 diphtheria Toxin A (UCP1-DTA; n=18) mice, which lack all UCP-1 expressing cells. All procedures were performed in accordance with the National Institute of Health’s Guide for the Use and Care of Laboratory Animals and were approved by Washington University School of Medicine IACUC. Muscle injury was induced using intramuscular injection of 50%v/v solution of glycerol. Regeneration was assessed at “early” (7 days post-injury (dpi)), “intermediate” (14 dpi) and “late” (21 dpi) stages. Outcome measures were cell population dynamics by flow cytometry, ex-vivo physiology testing and histological scoring as appropriate for each timepoint. RESULTS: Our preliminary data in early regeneration show that UCP1-DTA mice have significantly greater percentage of FAPs compared to WT mice (29.84 ± 1.24% higher cell population, p&lt;0.0134), however no differences in muscle satellite (stem) cells between genotypes. Physiological and histological outcomes at intermediate and late-stage regeneration find a baseline deficit in muscle mass (23.69 ± 2.3% smaller, p&lt;0.0001), type 2B fiber cross-sectional area (32.56 ± 4.8% smaller, p&lt;0.0001) and normalized muscle active force generation (16.2 ± 3.3% lower force, p&lt;0.0154) in UCP1-DTA compared with WT. However, mass and contractile forces recover to a similar extent between the genotypes following glycerol injection suggesting the phenotypic drivers of the reduced mass and contractile function do not affect muscle regeneration. CONCLUSIONS: Taken together, the data from this study point to a baseline growth deficit in mice lacking UCP-1-expressing cells. However, in two separate timepoints of regeneration (14 and 21dpi), we see that UCP1-DTA mice have similar recovery of mass and normalized contractile force to baseline values suggesting regeneration can proceed normally without UCP-1 expressing cells. However, further data collection will be needed at early-stage regeneration to help discern whether mice lacking UCP-1-expressing cells have initial changes in cell population dynamics given the increased FAP population at 7dpi. ACKNOWLEDGMENTS: This work was supported by R01AR075773 and R21AR071582 to GAM. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

HDAC4 Genomic Methylation and Subsequent DNA Binding Following Joint Injury Confers a Sustained Epigenetic Memory of Disrupted Muscle Plasticity

Joint injury is associated with protracted atrophy and weakness that poorly recovers; our objective was to define molecular effectors of lost muscle size, strength, and plasticity following joint injury. We hypothesized that epigenetic imprinting of joint injury in muscle impairs muscle plasticity and contributes to poor functional recovery following surgical repair. We recruited a total of sixteen ACL-injured participants from whom we obtained vastus lateralis muscle biopsies Pre-, 7-days post-, and 4-months post- reconstruction surgery (Sx) and strength measures up to 6-months post-Sx. We performed immunohistochemistry, RNA-seq, reduced representation bisulfite sequencing (RRBS), and HDAC4 chromatin immunoprecipitation sequencing (HDAC4 ChIP-seq) to determine the impact of epigenetics and gene expression on histological profiles, muscle size and muscle strength following injury. Binding and Expression Target Analysis (BETA) of DNA methylation revealed a conserved 985bp regulatory genomic region that was differentially methylated upstream of the HDAC4 promoter. We observed 108 (Pre-Sx), 111 (7d post-Sx), and 110 (4-months post-Sx) differentially methylated promoter sites of which 95 sites were common across all timepoints in the injured limb. Additionally, we observed 1293 differentially methylated intragenic sites within the HDAC4 locus that were also common across all timepoints following injury. Altered HDAC4 methylation was associated with a 2.2 and 4.3 fold change in HDAC4 transcript abundance Pre-Sx and 7d post-Sx, respectively (FDR &lt; 0.05). Notably, HDAC4 binding to critical contractile genes coincided with their downregulation 7d post-Sx (Log2 fold change: ACTA1: -2.02; CKM: -0.97; ATP2A1: -2.34; HOMER2: -1.09; all FDR&lt;0.05). Altered transcript abundance of these genes coincided with lower mean muscle fiber cross-sectional area in the injured limb compared to Healthy (Healthy: 4845 ± 213.4μm2; pre-Sx: 4313 ± 190.6μm2, p&lt;0.05; 7d post-Sx: 3685 ± 228.1μm2, p&lt;0.05; 4-months post-Sx: 3422 ± 142.8μm2, p&lt;0.05) and lower peak torque compared to Healthy (Healthy: 174.8 ± 10.72nm; pre-Sx: 135.2 ± 10.19nm, p&lt;0.05; 4-months post-Sx: 82.64 ± 6.80nm, p&lt;0.05; 6-months post-Sx: 105.9 ± 8.26nm, p&lt;0.05) that was sustained throughout follow-up. Our results suggest that joint injury confers HDAC4 promoter-proximal and intragenic epigenetic imprinting by DNA methylation that increases HDAC4 transcript abundance and HDAC4-DNA binding to represses expression of genes regulating muscle contractile function. This HDAC4 gene regulatory program likely contributes to prolonged deficits in muscle size and strength following joint injury. This work was supported by NIH-NIAMS R01 AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic Hindlimb Ischemia Induces Alterations of Muscle Transcriptomes Based on Muscle Type

Introduction: Peripheral artery disease (PAD) is a progressive atherothrombotic disorder of the arteries supplying the lower limb and is the most prevalent cardiovascular disease, impacting over 230 million people worldwide. PAD is associated with lower extremity myopathy characterized by myofiber degeneration and fibrosis. Increased proportions of type I muscle fibers are associated with improved patient outcomes, but most studies, human and animal, only analyze the gastrocnemius (gastroc), which contains mixed amounts of type I and type II fibers. To elucidate the impacts of ischemia on muscles comprising mainly type I fibers, we evaluated the differential effect of hindlimb ischemia (HLI) on the soleus and gastroc, hypothesizing that chronic HLI would more severely impact the soleus. Methods: Chronic HLI was induced in Sprague-Dawley male rats with an endovascular iliofemoral artery occlusion approach. Time-dependent laser Doppler perfusion imaging assessed hindlimb perfusion before and after HLI up to 42 days. Myopathy was evaluated 28 days post-HLI by staining muscle cross-sections with Hematoxylin and Eosin (H&amp;E) and Masson trichrome. Quantification of muscle fibrosis, average fiber cross-sectional area, and presence of central nuclei were used to determine muscle pathology. RNAsequencing was used to determine the differences in transcriptomes of the gastroc and soleus in both sham and HLI conditions. Results: Histological analysis revealed that there were significantly increased levels of fibrosis after HLI, but only in the soleus (18.24±7.23 vs. 7.71±2.54 %, n = 8-16, p-value &lt;0.0001). The average fiber cross-sectional area was significantly reduced in the HLI soleus compared to the sham (1556.00±459.60 vs. 3072.00±831.50 μm, n = 8-16, p-value &lt;0.0001), but not between HLI and sham gastroc. Conversely, HLI increased the proportions of centralized nuclei in both muscles (soleus — 22.72±12.46 vs. 1.12±1.49 %, n = 8-16, p-value = 0.0002, gastroc — 12.75±10.17 vs. 0.00±0.00 %, n = 8-16, p-value = 0.0002), indicating prior muscle damage. The HLI soleus exhibited worse outcomes in all parameters compared to the HLI gastroc. These data were substantiated by RNAseq, which showed that the HLI soleus transcriptome contained more differentially expressed genes (DEGs, ~1000 altered genes) than its sham counterpart compared to HLI gastroc, which possessed &lt;100 DEGs (n = 5-6). Moreover, the HLI soleus transcriptome was highly fibrotic, pro-angiogenic, and promoted myoblast fusion, a combination that could explain the histological myopathy findings. Conclusions: Chronic HLI induces alterations of muscle ultrastructure and transcription. These effects occur predominantly in the soleus rather than the gastroc. The results of the present study implicate that the pathophysiology is different for distinct muscles and may result from varying fiber composition. This work can help identify the mechanisms responsible for the myopathy of PAD and may lead to the design of targeted interventions for the care of PAD patients. R01HL169205-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.