The impact of skeletal muscle disuse on distinct echo intensity bands: A retrospective analysis

Echo intensity (EI) is a novel tool for assessing muscle quality. EI has traditionally been reported as the mean of the pixel histogram, with 0 and 255 arbitrary units (A.U.) representing excellent and poor muscle quality, respectively. Recent work conducted in youth and younger and older adults suggested that analyzing specific EI bands, rather than the mean, may provide unique insights into the effectiveness of exercise and rehabilitation interventions. As our previous work showed deterioration of muscle quality after knee joint immobilization, we sought to investigate whether the increase in EI following disuse was limited to specific EI bands. Thirteen females (age = 21 yrs) underwent two weeks of left knee immobilization and ambulated via crutches. B-mode ultrasonography was utilized to obtain images of the immobilized vastus lateralis. The percentage of the total number of pixels within bands of 0–50, 51–100, 101–150, 151–200, and 201–255 A.U. was examined before and after immobilization. We also sought to determine if further subdividing the histogram into 25 A.U. bands (i.e., 0–25, 26–50, etc.) would be a more sensitive methodological approach. Immobilization resulted in a decrease in the percentage of pixels within the 0–50 A.U. band (-3.11 ± 3.98%), but an increase in the 101–150 A.U. (2.94 ± 2.64%) and 151–200 A.U. (0.93 ± 1.42%) bands. Analyses of variance on the change scores indicated that these differences were large and significant (%EI0-50 vs. %EI101-150: p < .001, d = 1.243); %EI0-50 vs. %EI151-200: p = .043, d = 0.831). The effect size for the %EI51-100 versus %EI101-150 comparison was medium/large (d = 0.762), but not statistically significant (p = .085). Further analysis of the 25 A.U. bands indicated that the percentage of pixels within the 25–50 A.U. band decreased (-2.97 ± 3.64%), whereas the 101–125 (1.62 ± 1.47%) and 126–150 A.U. (1.18 ± 1.07%) bands increased. Comparison of the 50 A.U. and 25 A.U. band methods found that 25 A.U. bands offer little additional insight. Though studies are needed to ascertain the factors that may influence specific bands, changes in EI during muscle disuse are not homogeneous across the pixel histogram. We encourage investigators to think critically about the robustness of data obtained from EI histograms, rather than simply reporting the EImean value, in muscle quality research.

Hindlimb Immobilization Increases IL-1β and Cdkn2a Expression in Skeletal Muscle Fibro-Adipogenic Progenitor Cells: A Link Between Senescence and Muscle Disuse Atrophy

Loss of muscle mass and strength contributes to decreased independence and an increased risk for morbidity and mortality. A better understanding of the cellular and molecular mechanisms underlying muscle atrophy therefore has significant clinical and therapeutic implications. Fibro-adipogenic progenitors (FAPs) are a skeletal muscle resident stem cell population that have recently been shown to play vital roles in muscle regeneration and muscle hypertrophy; however, the role that these cells play in muscle disuse atrophy is not well understood. We investigated the role of FAPs in disuse atrophy in vivo utilizing a 2-week single hindlimb immobilization model. RNA-seq was performed on FAPs isolated from the immobilized and non-immobilized limb. The RNAseq data show that IL-1β is significantly upregulated in FAPs following 2 weeks of immobilization, which we confirmed using droplet-digital PCR (ddPCR). We further validated the RNA-seq and ddPCR data from muscle in situ using RNAscope technology. IL-1β is recognized as a key component of the senescence-associated secretory phenotype, or SASP. We then tested the hypothesis that FAPs from the immobilized limb would show elevated senescence measured by cyclin-dependent kinase inhibitor 2A (Cdkn2a) expression as a senescence marker. The ddPCR and RNAscope data both revealed increased Cdkn2a expression in FAPs with immobilization. These data suggest that the gene expression profile of FAPs is significantly altered with disuse, and that disuse itself may drive senescence in FAPs further contributing to muscle atrophy.

Effects of Various Muscle Disuse States and Countermeasures on Muscle Molecular Signaling

Skeletal muscle is capable of changing its structural parameters, metabolic rate and functional characteristics within a wide range when adapting to various loading regimens and states of the organism. Prolonged muscle inactivation leads to serious negative consequences that affect the quality of life and work capacity of people. This review examines various conditions that lead to decreased levels of muscle loading and activity and describes the key molecular mechanisms of muscle responses to these conditions. It also details the theoretical foundations of various methods preventing adverse muscle changes caused by decreased motor activity and describes these methods. A number of recent studies presented in this review make it possible to determine the molecular basis of the countermeasure methods used in rehabilitation and space medicine for many years, as well as to identify promising new approaches to rehabilitation and to form a holistic understanding of the mechanisms of gravity force control over the muscular system.

Mitochondrial Bioenergetics and Turnover during Chronic Muscle Disuse

Periods of muscle disuse promote marked mitochondrial alterations that contribute to the impaired metabolic health and degree of atrophy in the muscle. Thus, understanding the molecular underpinnings of muscle mitochondrial decline with prolonged inactivity is of considerable interest. There are translational applications to patients subjected to limb immobilization following injury, illness-induced bed rest, neuropathies, and even microgravity. Studies in these patients, as well as on various pre-clinical rodent models have elucidated the pathways involved in mitochondrial quality control, such as mitochondrial biogenesis, mitophagy, fission and fusion, and the corresponding mitochondrial derangements that underlie the muscle atrophy that ensues from inactivity. Defective organelles display altered respiratory function concurrent with increased accumulation of reactive oxygen species, which exacerbate myofiber atrophy via degradative pathways. The preservation of muscle quality and function is critical for maintaining mobility throughout the lifespan, and for the prevention of inactivity-related diseases. Exercise training is effective in preserving muscle mass by promoting favourable mitochondrial adaptations that offset the mitochondrial dysfunction, which contributes to the declines in muscle and whole-body metabolic health. This highlights the need for further investigation of the mechanisms in which mitochondria contribute to disuse-induced atrophy, as well as the specific molecular targets that can be exploited therapeutically.

Chronic Ouabain Prevents Na,K-ATPase Dysfunction and Targets AMPK and IL-6 in Disused Rat Soleus Muscle

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.

Early Lipid Raft-Related Changes: Interplay between Unilateral Denervation and Hindlimb Suspension

Muscle disuse and denervation leads to muscle atrophy, but underlying mechanisms can be different. Previously, we have found ceramide (Cer) accumulation and lipid raft disruption after acute hindlimb suspension (HS), a model of muscle disuse. Herein, using biochemical and fluorescent approaches the influence of unilateral denervation itself and in combination with short-term HS on membrane-related parameters of rat soleus muscle was studied. Denervation increased immunoexpression of sphingomyelinase and Cer in plasmalemmal regions, but decreased Cer content in the raft fraction and enhanced lipid raft integrity. Preliminary denervation suppressed (1) HS-induced Cer accumulation in plasmalemmal regions, shown for both nonraft and raft-fractions; (2) HS-mediated decrease in lipid raft integrity. Similar to denervation, inhibition of the sciatic nerve afferents with capsaicin itself increased Cer plasmalemmal immunoexpression, but attenuated the membrane-related effects of HS. Finally, both denervation and capsaicin treatment increased immunoexpression of proapoptotic protein Bax and inhibited HS-driven increase in antiapoptotic protein Bcl-2. Thus, denervation can increase lipid raft formation and attenuate HS-induced alterations probably due to decrease of Cer levels in the raft fraction. The effects of denervation could be at least partially caused by the loss of afferentation. The study points to the importance of motor and afferent inputs in control of Cer distribution and thereby stability of lipid rafts in the junctional and extrajunctional membranes of the muscle.

Pre-Clinical Rodent Models of Physical Inactivity-Induced Muscle Insulin Resistance: Challenges and Solutions

The burgeoning rise of health complications emanating from metabolic disease presents an alarming issue with mounting costs for health care and a reduced quality of life. There exists a pressing need for more complete understanding of mechanisms behind the development and progression of metabolic dysfunction. Since lifestyle modifications such as poor diet and lack of physical activity are primary catalysts of metabolic dysfunction, rodent models have been formed to explore mechanisms behind these issues. Particularly, the use of high-fat diet has been pervasive and has been an instrumental model to gain insight into mechanisms underlying diet-induced insulin resistance (IR). However, physical inactivity (and to some extent muscle disuse) is an often overlooked and much less frequently studied lifestyle modification, which some have contended is the primary contributor in the initial development of muscle IR. In this mini-review we highlight some of the key differences between diet- and physical inactivity-induced development of muscle IR and propose reasons for the sparse volume of academic research into physical inactivity-induced IR including infrequent use of clearly translatable rodent physical inactivity models.