Muscle Cell Membrane Research Articles

Myogenic response of the cerebral vessel: a mathematical model

The myogenic response is most typical for small arterial vessels and consists in constriction of the vessel with an increase in transmural pressure, the pressure difference between the inner and outer surfaces of the vascular wall. There are many models describing this response. In models of last decades, in addition to macrostretchings and macrostrains, their microanalogues are introduced into consideration, that significantly complicates the description. In the presented work a simple model of the wall of the small cerebral artery is described, which includes the main parameters that control the myogenic response and corresponds to physiological processes underlining this response. It is believed that active stresses depend on stretching and the concentration of free calcium ions in the cytoplasm of the smooth muscle cell. In turn, the calcium content depends on the membrane potential, determined by the pressure. An axisymmetric stationary problem is considered; the wall material in the non-activated state is assumed to be pseudoelastic. Calculations showed a qualitative difference in the dependence of active stress on stretching and its distribution in the radial direction before and after the development of the vascular response. The calculated diameter values before and after exposure to substances affecting the polarization of the smooth muscle cell membrane are within the range of experimental values. These results indicate that the introduction of the dependence of calcium concentration on membrane potential expands the area of applicability of simple models of this kind for evaluating the value of vascular diameter.

A Comparative Analysis of Two Agonist Muscle Superset Training Variations on Muscle Damage and Oxidative Stress Markers in Resistance-Trained Men

Background: Evidence suggests that heavy resistance activities can damage muscle cell membranes by increasing free radical production. However, the specific type of resistance activity and the adaptations made by resistance-trained men can influence oxidative stress indicators and muscle damage. Objectives: This study aimed to evaluate the acute impact of two agonist muscle superset resistance training protocols on malondialdehyde (MDA), creatine kinase (CK), lactate dehydrogenase (LDH), total antioxidant capacity (TAC), and catalase (CAT) in resistance-trained men. Methods: A quasi-experimental study with a pretest-posttest design was conducted. Ten resistance-trained men (age: 24.25 ± 4.92 years; height: 175.12 ± 19.6 cm; weight: 74.75 ± 8.44 kg) participated in one of two superset agonist muscle resistance training protocols (post-exhaustion supersets and pre-exhaustion supersets). Subjects performed three sets of ten repetitions at an intensity of 80% of their 1-repetition maximum (1RM). Blood samples were taken immediately before and 24 hours after each session, and serum levels of MDA, CK, LDH, TAC, and CAT were evaluated. Results: Both types of superset resistance training significantly increased MDA, CK, LDH, TAC, and CAT levels, with no significant difference in the effects on MDA, CK, and LDH. However, a significant difference was observed between the two training protocols in terms of TAC and CAT, with post-exhaustion supersets showing a greater increase in these antioxidant indices. Conclusions: A single session of both protocols can induce oxidative stress and muscle damage in resistance-trained men. Additionally, post-exhaustion superset training of agonist muscles may have a greater impact on enhancing antioxidant capacity.

A Voyage on the Role of Nuclear Factor Kappa B (NF-kB) Signaling Pathway in Duchenne Muscular Dystrophy: An Inherited Muscle Disorder.

A recessive X-linked illness called Duchenne muscular dystrophy (DMD) is characterized by increasing muscle weakening and degradation. It primarily affects boys and is one of the most prevalent and severe forms of muscular dystrophy. Mutations in the DMD gene, which codes for the essential protein dystrophin, which aids in maintaining the stability of muscle cell membranes during contraction, are the cause of the illness. Dystrophin deficiency or malfunction damages muscle cells, resulting in persistent inflammation and progressive loss of muscular mass. The pathophysiology and genetic foundation of DMD are thoroughly examined in this review paper, focusing on the function of the NF-κB signaling system in the disease's progression. An important immune response regulator, NF-κB, is aberrantly activated in DMD, which exacerbates the inflammatory milieu in dystrophic muscles. Muscle injury and fibrosis are exacerbated and muscle regeneration is hampered by the pro-inflammatory cytokines and chemokines that are produced when NF-κB is persistently activated in muscle cells. The paper also examines our existing knowledge of treatment approaches meant to inhibit the progression of disease by modifying NF-κB signaling. These include new molecular techniques, gene treatments, and pharmacological inhibitors that are intended to lessen inflammation and improve muscle healing. Furthermore covered in the analysis is the significance of supportive care for DMD patients, including physical therapy and corticosteroid treatment, in symptom management and quality of life enhancement. The article seeks to provide a thorough understanding of the mechanisms causing DMD, possible therapeutic targets, and developing treatment options by combining recent research findings. This will provide clinicians and researchers involved in DMD care and research with invaluable insights.

Contribution of transient receptor potential vanilloid 1 (TRPV1) channel to cholinergic contraction of rat bladder smooth muscle.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-permeable ion channel that is gated by the pungent constituent of red chili pepper, capsaicin, and by related chemicals from the group of vanilloids, in addition to noxious heat. It is expressed mostly in sensory neurons to act as a detector of painful stimuli produced by pungent chemicals and high temperatures. Although TRPV1 is also found outside the sensory nervous system, its expression and function in the bladder detrusor smooth muscle (DSM) remain controversial. Here, by using Ca2+ imaging and patch clamp on isolated rat DSM cells, in addition to tensiometry on multicellular DSM strips, we show that TRPV1 is expressed functionally in only a fraction of DSM cells, in which it acts as an endoplasmic reticulum Ca2+-release channel responsible for the capsaicin-activated [Ca2+]i rise. Carbachol-stimulated contractions of multicellular DSM strips contain a TRPV1-dependent component, which is negligible in the circular DSM but reaches ≤50% in the longitudinal DSM. Activation of TRPV1 in rat DSM during muscarinic cholinergic stimulation is ensured by phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists. Immunofluorescence detection of TRPV1 protein in bladder sections and isolated DSM cells confirmed both its preferential expression in the longitudinal DSM sublayer and its targeting to the endoplasmic reticulum. We conclude that TRPV1 is an essential contributor to the cholinergic contraction of bladder longitudinal DSM, which might be important for producing spatial and/or temporal anisotropy of bladder wall deformation in different regions during parasympathetic stimulation. KEY POINTS: The transient receptor potential vanilloid 1 (TRPV1) heat/capsaicin receptor/channel is localized in the endoplasmic reticulum membrane of detrusor smooth muscle (DSM) cells of the rat bladder, operating as a calcium-release channel. Isolated DSM cells are separated into two nearly equal groups, within which the cells either show or do not show TRPV1-dependent [Ca2+]i rise. Carbachol-stimulated, muscarinic ACh receptor-mediated contractions of multicellular DSM strips contain a TRPV1-dependent component. This component is negligible in the circular DSM but reaches ≤50% in longitudinal DSM. Activation of TRPV1 in rat DSM during cholinergic stimulation involves phospholipase A2-catalysed derivation of arachidonic acid and its conversion by lipoxygenases to eicosanoids, which act as endogenous TRPV1 agonists.

Caveolin-3 regulates slow oxidative myofiber formation in female mice

Abstract Objectives Caveolin-3 (Cav-3) plays a pivotal role in maintaining skeletal muscle mass and function. Mutations or deletions of Cav-3 can result in the development of various forms of myopathy, which affect the integrity and repair capacity of muscle fiber membranes. However, the potential effect of Cav-3 on myofiber type composition remains unclear. Methods To investigate the effect of Cav-3 on muscle strength and running capacity, we examined the grip force test and the low/high-speed running test. Oxidative and glycolytic myofiber-related genes, proteins, and skeletal muscle fiber composition were measured to determine the role of the Cav-3 in oxidative myofiber formation. Results We report the impact of Cav-3 on enhancing muscle endurance performance in female mice, and the discovery of a new physiological function to increase the proportion of slow-twitch oxidative muscle fiber by analyzing the gastrocnemius and soleus. Skeletal muscle-specific ablation of Cav-3 in female mice increased oxidative myofiber-related gene expression and type I oxidative myofiber composition, with resultant improvements in endurance performance. In male mice, the absence of Cav-3 in skeletal muscle reduced in the expression of glycolytic fiber-related genes and proteins. Conclusions This study identified Cav-3 as a regulator of slow-twitch oxidative muscle fiber formation acting on female mice, which may provide a potential target for improving muscle oxidative function.

Phase Angle as Surrogate Marker of Muscle Weakness in Kidney Transplant Candidates Referred to Prehabilitation.

Phase angle (PhA), a marker of nutritional status obtained by bioelectrical impedance analysis (BIA), is associated with the integrity of cell membranes. Damage to muscle fiber membranes can impact muscle strength, which is related to adverse outcomes in adults with advanced chronic kidney disease (CKD). The main objective of this study was to determine the usefulness of the PhA in identifying muscle weakness in candidates for kidney transplants (KTs). Secondly, it aimed to examine the associations of PhA with other parameters of body composition, exercise performance, and muscle structure. Sensitivity, specificity, and area under the receiver operating characteristics curve were used to evaluate the PhA (index test) as a biomarker of muscle weakness. Muscle strength was estimated with maximal voluntary isometric contraction of the quadriceps (MVCI-Q) of the dominant side. Muscle weakness was defined as MVIC-Q < 40% of body weight. A total of 119 patients were evaluated (mean age 63.7 years, 75.6% men). A phase angle cut-off of 5.1° was identified to classify men with a higher likelihood of having low muscle strength in upper limbs (MVIC-Q 40% of their body weight). Male KT candidates with PhA < 5.1° had poorer exercise capacity, lower muscle strength, less muscle mass, and smaller muscle size. A PhA < 5.1° was significantly associated with an eight-fold higher muscle weakness risk (OR = 8.2, 95%CI 2.3-29.2) in a binary regression model adjusted by age, frailty, and hydration status. Remarkably, PhA is an easily obtainable objective parameter in CKD patients, requiring no volitional effort from the individual. The associations of PhA with aerobic capacity, physical activity, muscle mass, and muscle size underscore its clinical relevance and potential utility in the comprehensive evaluation of these patients.

A conserved RNA switch for acetylcholine receptor clustering at neuromuscular junctions in chordates.

In mammals, neuromuscular synapses rely on clustering of acetylcholine receptors (AChRs) in the muscle plasma membrane, ensuring optimal stimulation by motor neuron-released acetylcholine neurotransmitter. This clustering depends on a complex pathway based on alternative splicing of Agrin mRNAs by the RNA-binding proteins Nova1/2. Neuron-specific expression of Nova1/2 ensures the inclusion of small "Z" exons in Agrin, resulting in a neural-specific form of this extracellular proteoglycan carrying a short peptide motif that is required for binding to Lrp4 receptors on the muscle side, which in turn stimulate AChR clustering. Here we show that this intricate pathway is remarkably conserved in Ciona robusta, a non-vertebrate chordate in the tunicate subphylum. We use in vivo tissue-specific CRISPR/Cas9-mediated mutagenesis and heterologous "mini-gene" alternative splicing assays in cultured mammalian cells to show that Ciona Nova is also necessary and sufficient for Agrin Z exon inclusion and downstream AChR clustering. We present evidence that, although the overall pathway is well conserved, there are some surprising differences in Nova structure-function between Ciona and mammals. We further show that, in Ciona motor neurons, the transcription factor Ebf is a key activator of Nova expression, thus ultimately linking this RNA switch to a conserved, motor neuron-specific transcriptional regulatory network.

Dietary protein re-alimentation following restriction improves protein deposition via changing amino acid metabolism and transcriptional profiling of muscle tissue in growing beef bulls

This study aimed to develop a compensatory growth model using growing beef cattle by changing dietary protein and to investigate the underlying mechanisms of compensatory protein deposition in muscle tissue. Twelve Charolais bulls were randomly assigned to one of two groups with two periods: 1) a control group (CON) fed a 13% crude protein (CP) diet for 6 weeks; 2) a treatment group (REC) fed a 7% CP diet for 4 weeks (restriction period) and fed a 13% CP diet in the following 2 weeks (re-alimentation period). Growth performance, digestibility, nitrogen balance, targeted metabolomics of amino acids (AA) in plasma, and transcriptional profiling in muscle tissue were analyzed. Protein restriction decreased average daily gain (ADG; P < 0.05), while protein re-alimentation increased ADG relative to the CON (P < 0.05). Compared to the CON, REC reduced retained N (P < 0.05), and protein re-alimentation increased retained N and N utilization efficiency (P < 0.05), due to reduced urinary urea, hippuric acid, and creatinine excretions (P < 0.05). Ruminal NH3-N in the REC was lower than that in the CON in the protein re-alimentation period (P < 0.05). However, there was no difference in microbial protein and plasma urea nitrogen concentrations. Dietary protein restriction decreased plasma valine and aspartic acid concentrations relative to the CON (P < 0.05), and increased proline and 3-methyl-L-histidine concentrations (P < 0.05). After dietary protein re-alimentation, REC increased plasma citrulline concentrations (P < 0.05). The transcriptional profiling revealed that REC upregulated the AA transporter SLC3A1, and protein re-alimentation downregulated SLC7A8 in the muscle cell membrane. Within the muscle cell, upregulated cytosolic arginine sensor for mTORC1 subunit 2 (CASTOR2) inhibited protein synthesis by inhibiting the mammalian target of rapamycin complex 1 phosphorylation in the protein restriction period, while DNA-damage-inducible transcript 4 (DDIT4) activated the mTOR signaling pathway and promoted protein synthesis in the protein re-alimentation period. In summary, the targeted metabolomics and transcriptomics analyses demonstrated that protein re-alimentation following restriction can promote protein synthesis and reduce muscle breakdown by regulating AA metabolism and functional transcripts related to AA transporters and the mTOR signaling pathway.

Caveolae Disassembly using Methyl-β-cyclodextrin Causes the Abolition of Coupling of the Caveolae and the Sarcoplasmic Reticulum in the Rat Femoral Artery

Background: Caveolae are essential in regulating signal transduction mechanisms of ion channels in vascular tissue, including BKCa channels (maxi-K). The current study investigated the localization of maxi-K channels within caveolae. Methods: Sixteen rats were divided into two groups: A control group and a treated group, where arteries in the treated group were incubated with methyl-β-cyclodextrin (MβCD) to disassemble caveolae from artery tissue. Immunohistochemistry (IHC), immunocytochemistry (ICC), transmission electron microscopy (TEM) and Western blot techniques were used in this study. Result: IHC of intact arteries showed colocalization of maxi-K channels with caveolin-1 in smooth muscle and endothelial cells and colocalized of maxi-K channels with caveolin-3 in smooth muscle cells only. These findings were also corroborated with ICC in a single smooth muscle cell. TEM revealed caveolae covering most plasma membranes of smooth muscle and endothelial cells and showed that caveolae sit close to the sarcoplasmic reticulum only in smooth muscle cells. TEM showed incubating arteries with MβCD led to the disassembly of caveolae from artery tissue. This study concluded that maxi-K channels localize to caveolae and that caveolae abolishment by MβCD led to the abolition of the coupling of caveolae and the sarcoplasmic reticulum.

Ca2+-Dependent Cl- Channels in Vascular Tone Regulation during Aging.

Identifying alterations caused by aging could be an important tool for improving the diagnosis of cardiovascular diseases. Changes in vascular tone regulation involve various mechanisms, like NO synthase activity, activity of the sympathetic nervous system, production of prostaglandin, endothelium-dependent relaxing, and contracting factors, etc. Surprisingly, Ca2+-dependent Cl- channels (CaCCs) are involved in all alterations of the vascular tone regulation mentioned above. Furthermore, we discuss these mechanisms in the context of ontogenetic development and aging. The molecular and electrophysiological mechanisms of CaCCs activation on the cell membrane of the vascular smooth muscle cells (VSMC) and endothelium are explained, as well as the age-dependent changes that imply the activation or inhibition of CaCCs. In conclusion, due to the diverse intracellular concentration of chloride in VSMC and endothelial cells, the activation of CaCCs depends, in part, on intracellular Ca2+ concentration, and, in part, on voltage, leading to fine adjustments of vascular tone. The activation of CaCCs declines during ontogenetic development and aging. This decline in the activation of CaCCs involves a decrease in protein level, the impairment of Ca2+ influx, and probably other alterations in vascular tone regulation.

Investigating the novel role of sarcospan in modulating immune function in mice

Objective: The tetraspanin-like protein sarcospan (SSPN) has been shown to have an important in stabilizing muscle cell membranes and promoting cell adhesion. SSPN has documented expression in immune cells with the highest level of expression in B cells. Examination of SSPN-deficient (SSPN−/−) mice revealed alterations in important immune pathways, which remain uncharacterized. This led us to hypothesize that SSPN may play a role in modulating immune cell development and/or function. Methods: Flow cytometry was used to perform immunophenotyping of spleens obtained from SSPN−/− and WT mice. Cytokine analysis was performed using Luminex to assess cytokines that serve as important signals for immune cell maturation. Hematopoietic stem cells were isolated from red bone marrow and stimulated with phorbol myristate acetate (PMA), a mitogen and polyclonal activator of B cells, to assess whether SSPN deficiency affected their proliferative capacity. To determine whether SSPN plays a role in immunoglobulin isotype switching — antibody isotyping was performed. The presence of immune (antibody-antigen) complexes was investigated in kidney sections obtained from WT and SSPN−/− mice and visualized using biotinylated IgG and IgM antibodies. Autoantigen screening was also performed to assess whether SSPN−/− mice generated abnormal levels of autoantibodies that could be responsible for circulating antibody-antigen complexes. Results: The presence of SSPN on major immune cell types was confirmed using flow cytometry. In addition, flow cytometry analysis showed insignificant baseline differences in the percentage of B cells, T cells, plasmacytoid dendritic cells in SSPN−/− compared to WT spleens. One difference was noted in SSPN−/− females with increased counts of dendritic cells. Spleens in female SSPN−/− mice were found to be slightly larger compared to WT. Levels of B-cell activating factor (BAFF) and B-cell maturation antigen (BCMA) were elevated in SSPN−/− mice, therefore it is expected that SSPN−/− B cells received appropriate signals. Overall B cell proliferation was assessed by CD19+ and appeared unaffected by the absence of SSPN. Subtle increases in IgG1 and IgG2b were detected in SSPN−/− mice, however immunoglobulin isotypes were within normal ranges. SSPN−/− mice were also assessed for signs of dysregulated immune reactivity, evidenced by the presence of immune (antibody-antigen) complexes within filtration units of the kidney. Overall, male SSPN−/− mice were found to have a larger subset of increased IgG autoantibodies compared to male WT mice while female SSPN−/− mice exhibited a larger subset of increased IgM autoantibodies compared to female WT mice. Conclusions: This study examines potentially important functions of SSPN in the immune system. Overall, deletion of SSPN has not been found to affect maturation of major immune cell classes. However, SSPN does appear to influence formation of antigen-antibody complexes in mice, which may contribute to kidney dysfunction. These studies are ongoing and directed toward understanding the role SSPN plays in immune function. FSU Funding, Florida Department of Health, James and Esther King Biomedical Research Program #21K12. 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.

Focal Traumatic Brain Injury Impairs the Integrity of the Basement Membrane of Hindlimb Muscle Fibers Revealed by Extracellular Matrix Immunoreactivity.

Traumatic brain injury (TBI) stands as a prominent global cause of disability, with motor deficits being a common consequence. Despite its widespread impact, the precise pathological mechanisms underlying motor deficits after TBI remain elusive. In this study, hindlimb postural asymmetry (HL-PA) development in rats subjected to focal TBI was investigated to explore the potential roles of collagen IV and laminin within the extracellular matrix (ECM) of selected hindlimb muscles in the emergence of motor deficits following TBI. A focal TBI was induced by ablating the left sensorimotor cortex in rats and motor deficits were assessed by measuring HL-PA. The expression of laminin and collagen IV in eight selected muscles on each side of the hindlimbs from both TBI- and sham-operated rats were studied using immunohistochemistry and semi-quantitatively analyzed. The results indicated that the TBI rats exhibited HL-PA, characterized by flexion of the contralateral (right) hindlimb. In the sham-operated rats, the immunoreactive components of laminin and collagen IV were evenly and smoothly distributed along the border of the muscle fibers in all the investigated muscles. In contrast, in the TBI rats, the pattern was broken into aggregated, granule-like, immunoreactive components. Such a labeling pattern was detected in all the investigated muscles both from the contra- and ipsilateral sides of the TBI rats. However, in TBI rats, most of the muscles from the contralateral hindlimb showed a significantly increased expression of these two proteins in comparison with those from the ipsilateral hindlimb. In comparison to sham-operated rats, there was a significant increase in laminin and collagen IV expression in various contralateral hindlimb muscles in the TBI rats. These findings suggest potential implications of laminin and collagen IV in the development of motor deficits following a focal TBI.

Electrophysiological Recording from a "Model" Cell.

The muscle cell or neuron membrane is functionally equivalent to a resistor-capacitor (RC) circuit with the membrane resistance and capacitor in parallel. Once inserted inside the membrane, an electrode introduces a serial resistance and small capacitance to the RC circuit. Through a narrow opening at its tip (∼0.1-μm), current can pass through the electrode, into the cell, and back to the outside (ground) across the membrane to complete the circuit. This arrangement enables a voltage difference between the outside and inside of the cell membrane to be recorded. To determine cell membrane properties, a current can be injected into the cell through the electrode. One complication with this approach, however, is that the voltage difference measured with the electrode includes the voltage drop across the cell membrane and that across the electrode. Furthermore, a small amount of current is drawn by the electrode capacitor, thereby slowing the current flow across the membrane. Fortunately, most amplifiers are equipped with bridge balance and capacitance compensation functions so that the effects of the electrode on cell membrane properties can be canceled out or minimized. This protocol describes the basics of setting up and conducting electrophysiological experiments using a model cell. For the novice, a model cell provides a way to learn the operation of electrophysiology equipment and software without the anxiety of damaging living cells. This protocol also illustrates passive membrane properties such as the input resistance, capacitance, and time constant.

Kinetic regularities of thiacalix[4]arene C-1087 inhibitory effect on the activity of Mg(2+)-dependent Ca(2+)-transporting ATP hydrolase in the plasma membrane of smooth muscle cells

The experiments with the suspension of plasma membranes of myometrium cells, treated with 0.1% digitonin solution, were used to study kinetic regularities of the inhibitory effect of tetra-N-phenylsulfonyl trifluoroacetamidine-thiacalixarene (С-1087) on the activity of Са2+,Mg2+-ATPase. The studies demonstrated the impact of C-1087 on the cumulative effect and the maximal velocity of ATP hydrolysis. No effect of С-1087 on the affinity between Са2+,Mg2+-ATPase, and АТР, affinity and cumulative effect of Ca ions and activation coefficient for Mg ions was revealed. A considerable decrease in the maximal velocity of ATP hydrolysis evidenced a complete non-competitive mechanism of inhibiting Са2+,Mg2+-АТРase activity with thiacalix[4]arene С-1087. Computer simulation demonstrated that thiacalix[4]arene С-1087 inhibiting effect on Са2+,Mg2+-ATPase may be conditioned by the cumulative effect of four spatially oriented N-sulfonylamidine groups on the upper rim of its macrocyclic platform.

The impact of bed rest on human skeletal muscle metabolism

Insulin sensitivity and metabolic flexibility decrease in response to bed rest, but the temporal and causal adaptations in human skeletal muscle metabolism are not fully defined. Here, we use an integrative approach to assess human skeletal muscle metabolism during bed rest and provide a multi-system analysis of how skeletal muscle and the circulatory system adapt to short- and long-term bed rest (German Clinical Trials: DRKS00015677). We uncover that intracellular glycogen accumulation after short-term bed rest accompanies a rapid reduction in systemic insulin sensitivity and less GLUT4 localization at the muscle cell membrane, preventing further intracellular glycogen deposition after long-term bed rest. We provide evidence of a temporal link between the accumulation of intracellular triglycerides, lipotoxic ceramides, and sphingomyelins and an altered skeletal muscle mitochondrial structure and function after long-term bed rest. An intracellular nutrient overload therefore represents a crucial determinant for rapid skeletal muscle insulin insensitivity and mitochondrial alterations after prolonged bed rest.

ИЗМЕНЕНИЯ САРКОЛЕММАЛЬНОГО КАВЕОЛИНА-3 КАК РЕЗУЛЬТАТ ЦЕРАМИД-АССОЦИИРОВАННЫХ ЭФФЕКТОВ КИСЛОЙ СФИНГОМИЕЛИНАЗЫ ПРИ 14-ДНЕВНОЙ ФУНКЦИОНАЛЬНОЙ РАЗГРУЗКЕ КАМБАЛОВИДНОЙ МЫШЦЫ КРЫС

It is well known that functional unloading leads to a number of complex changes in the lipid-protein structural components in the sarcolemmal compartment of postural muscles' fibers. Among these changes are an increase of the sarcolemmal ceramide content, loss in cholesterol density in the lipid rafts fraction, a decrease of membrane sphingomyelin as a result of acid sphingomyelinase (ASM) activation and enhanced hydrolysis of sphingomyelin. These changes may underlie damages and primary elimination of damaged parts of muscle fibers' plasma membrane. Secondary reparation of sarcolemma depends on reorganization of the subsarcolemmal cytoskeleton. It appears to be exocytosis of caveolin-enriched vesicles in response to the growth of ASM dependent hydrolysis in the plasma membrane of muscle fibers. In our study, immunofluorescence microscopy and Western blot analysis were used to investigate ceramide and caveolin-3 changes in the sarcolemmal compartment of muscle fibers and total m. soleus from rats subjected to 14-d hindlimb unloading or unloading with administration of ASM inhibitor amitriptyline. In addition, muscle preparations with added exogenous sphingomyelinase were subjected to ex vivo analysis of ceramide and caveolin-3 immunofluorescence. The 14-d unloading led to the growth of ceramide and caveolin-3 immunofluorescence on muscle sections. Ex vivo analysis also demonstrated a significant increase of ceramide and caveolin-3 content in muscle fibers exposed to exogenous sphingomyelinase. The increase of caveolin-3 sarcolemmal density was confirmed by Western blot assay which indicated the growth of its amount in isolated sarcolemmal fraction. These changes were not observed in total homogenates of the unloaded m. soleus. Thus, functional unloading of the postural muscle leads to the accumulation of ceramide in the plasma membrane of muscle fibers and an increase in the level of membrane caveolin-3.

Biochemical Toxicological Study of Insulin Overdose in Rats: A Forensic Perspective.

Due to nonspecific pathological changes and the rapid degradation of insulin in postmortem blood samples, the identification of the cause of death during insulin overdose has always been a difficulty in forensic medicine. At present, there is a lack of studies on the toxicological changes and related mechanisms of an insulin overdose, and the specific molecular markers of insulin overdose are still unclear. In this study, an animal model of insulin overdose was established, and 24 SD rats were randomly divided into a control group, insulin overdose group, and a recovery group (n = 8). We detected the biochemical changes and analyzed the toxicological mechanism of an insulin overdose. The results showed that after insulin overdose, the rats developed irregular convulsions, Eclampsia, Opisthotonos, and other symptoms. The levels of glucose, glycogen, and C-peptide in the body decreased significantly, while the levels of lactate, insulin, and glucagon increased significantly. The decrease in plasma K+ was accompanied by the increase in skeletal muscle K+. The PI3K-AKT signaling pathway was significantly activated in skeletal muscle, and the translocation of GLUT4/Na+-K+-ATPase to sarcolemma was significantly increased. Rare glycogenic hepatopathy occurred in the recovery group after insulin overdose. Our study showed that insulin overdose also plays a role in skeletal muscle cells, mainly through the PI3K-Akt signaling pathway. Therefore, the detection of signaling pathway proteins of the skeletal muscle cell membrane GLUT4 and Na+-K+-ATPase has a certain auxiliary diagnostic value for forensic insulin overdose identification. Glycogen detection in the liver and skeletal muscle is important for the diagnosis of insulin overdose, but it still needs to be differentiated from other causes of death. Skeletal muscle has great potential for insulin detection, and the ratio of insulin to the C-peptide (I:C) can determine whether an exogenous insulin overdose is present.

Expression and clinical implications of PARs in the stenotic tissue of ureteropelvic junction obstruction.

To explore the expression and clinical implications of protease activated receptors (PARs) in the pathogenesis of children with ureteropelvic junction obstruction (UPJO). Immunohistochemistry was employed to investigate the distribution of PARs in both normal human ureteropelvic junction (UPJ) and cases of UPJO. Furthermore, PAR gene expression levels were assessed using real-time PCR (RT-PCR), and the patients in the UPJO group were stratified according to the Onen grading system. Subsequently, the clinical implications of PARs in UPJO were explored through RT-PCR analysis. Immunofluorescence showed robust PAR2 expression in the control group compared with the UPJO group. The results of RT-PCR analysis revealed a significant decrease in the relative mRNA expression of PAR2 in the UPJO group compared to the control group. Notably, the relative RNA expression of PAR1 was significantly lower in the Onen-4 group compared to the control group. Furthermore, the relative mRNA expression of PAR2 exhibited a statistically significant difference among the Onen-3 group, Onen-4 group, and control group. PARs are widely distributed throughout the SIP syncytium of the UPJ and play a role in maintaining smooth muscle cells (SMCs) membrane potential by interacting with interstitial cells of Cajal (ICCs), as well as platelet-derived growth factor receptor alpha-positive cells (PDGFR α+ cells). The decreased expression of PAR1 suggests a higher preoperative Onen grade in UPJO patients. Furthermore, the downregulation of PAR2 effects at the UPJ may be involved in the loss of inhibitory neuromuscular transmission, disrupting the rhythmic peristalsis of the UPJ.

Pathogenesis of ARIA

AbstractBackgroundAD is characterised pathologically by the deposition of amyloid‐beta peptides in the extracellular spaces of the brain as plaques and cerebral amyloid angiopathy (CAA). Observations on human post‐mortem brains and experimental studies in mice demonstrate that amyloid‐beta is deposited in the basement membranes of capillaries and basement membranes of arterial smooth muscle cells, within the Intramural Periarterial Drainage (IPAD) pathways.MethodPathological studies performed on human post‐mortem brains from the first ELAN trial demonstrated that after active immunization against amyloid‐beta, despite the disappearance of plaques, CAA worsened and both Abeta 40 as well as Abeta42 were present in the vascular wall. This suggests that Abeta42 solubilised from plaques has become entrapped in the IPAD pathways.ResultExperimental immunization in rodents against a cerebral protein demonstrate that within 24h, immune complexes are formed in the basement membranes of capillaries, impairing IPAD. By 7 days after immunization, inflammatory cuffs of perivascular cells are present, but, in a normal adult mouse, IPAD is restored.ConclusionThis suggests that clearance of fluid and solutes can be restored even in the context of immune cell recruitment if the vascular wall is healthy.

Regulated dynamic subcellular GLUT4 localization revealed by proximal proteome mapping in human muscle cells.

Regulation of glucose transport, which is central for control of whole-body metabolism, is determined by the amount of GLUT4 glucose transporter (also known as SLC2A4) in the plasma membrane (PM) of fat and muscle cells. Physiologic signals [such as activated insulin receptor or AMP-activated protein kinase (AMPK)] increase PM GLUT4. Here, we show that the distribution of GLUT4 between the PM and interior of human muscle cells is dynamically maintained, and that AMPK promotes PM redistribution of GLUT4 by regulating exocytosis and endocytosis. Stimulation of exocytosis by AMPK is mediated by Rab10 and the Rab GTPase-activating protein TBC1D4. APEX2 proximity mapping reveals that GLUT4 traverses both PM-proximal and PM-distal compartments in unstimulated muscle cells, further supporting retention of GLUT4 by a constitutive retrieval mechanism. AMPK-stimulated translocation involves GLUT4 redistribution among the same compartments traversed in unstimulated cells, with a significant recruitment of GLUT4 from the Golgi and trans-Golgi network compartments. Our comprehensive proximal protein mapping provides an integrated, high-density, whole-cell accounting of the localization of GLUT4 at a resolution of ∼20 nm that serves as a structural framework for understanding the molecular mechanisms regulating GLUT4 trafficking downstream of different signaling inputs in a physiologically relevant cell type.