Roles and mechanisms of copper homeostasis and cuproptosis in osteoarticular diseases.

Copper is an essential trace element in the human body that is extensively distributed throughout various tissues. The appropriate level of copper is crucial to maintaining the life activities of the human body, and the excess and deficiency of copper can lead to various diseases. The copper levels in the human body are regulated by copper homeostasis, which maintains appropriate levels of copper in tissues and cells by controlling its absorption, transport, and storage. Cuproptosis is a distinct form of cell death induced by the excessive accumulation of intracellular copper. Copper homeostasis and cuproptosis has recently elicited increased attention in the realm of human health. Cuproptosis has emerged as a promising avenue for cancer therapy. Studies concerning osteoarticular diseases have elucidated the intricate interplay among copper homeostasis, cuproptosis, and the onset of osteoarticular diseases. Copper dysregulation and cuproptosis cause abnormal bone and cartilage metabolism, affecting related cells. This phenomenon assumes a critical role in the pathophysiological processes underpinning various osteoarticular diseases, with implications for inflammatory and immune responses. While early Cu-modulating agents have shown promise in clinical settings, additional research and advancements are warranted to enhance their efficacy. In this review, we summarize the effects and potential mechanisms of copper homeostasis and cuproptosis on bone and cartilage, as well as their regulatory roles in the pathological mechanism of osteoarticular diseases (e.g., osteosarcoma (OS), osteoarthritis (OA), and rheumatoid arthritis (RA)). We also discuss the clinical-application prospects of copper-targeting strategy, which may provide new ideas for the diagnosis and treatment of osteoarticular diseases.

Moderate mechanical stress suppresses chondrocyte ferroptosis in osteoarthritis by regulating NF-κB p65/GPX4 signaling pathway.

Ferroptosis is a recently identified form of programmed cell death that plays an important role in the pathophysiological process of osteoarthritis (OA). Herein, we investigated the protective effect of moderate mechanical stress on chondrocyte ferroptosis and further revealed the internal molecular mechanism. Intra-articular injection of sodium iodoacetate (MIA) was conducted to induce the rat model of OA in vivo, meanwhile, interleukin-1 beta (IL-1ß) was treated to chondrocytes to induce the OA cell model in vitro. The OA phenotype was analyzed by histology and microcomputed tomography, the ferroptosis was analyzed by transmission electron microscope and immunofluorescence. The expression of ferroptosis and cartilage metabolism-related factors was analyzed by immunohistochemical and Western blot. Animal experiments revealed that moderate-intensity treadmill exercise could effectively reduce chondrocyte ferroptosis and cartilage matrix degradation in MIA-induced OA rats. Cell experiments showed that 4-h cyclic tensile strain intervention could activate Nrf2 and inhibit the NF-κB signaling pathway, increase the expression of Col2a1, GPX4, and SLC7A11, decrease the expression of MMP13 and P53, thereby restraining IL-1ß-induced chondrocyte ferroptosis and degeneration. Inhibition of NF-κB signaling pathway relieved the chondrocyte ferroptosis and degeneration. Meanwhile, overexpression of NF-κB by recombinant lentivirus reversed the positive effect of CTS on chondrocytes. Moderate mechanical stress could activate the Nrf2 antioxidant system, inhibit the NF-κB p65 signaling pathway, and inhibit chondrocyte ferroptosis and cartilage matrix degradation by regulating P53, SLC7A11, and GPX4.

Recent advances in polymeric microparticle-based drug delivery systems for knee osteoarthritis treatment.

Due to the poor bioavailability and high joint clearance of drugs, sustained delivery of therapeutic agents has proven difficult in the treatment of osteoarthritis (OA). Intra-articular (IA) drug delivery strategy is an attractive option for enhancing OA patients' prognosis, for which various polymer materials have been used as drug carriers due to their attractive delivery properties, to slow or even reverse the progress of OA by prolonging the duration of therapeutic agent residence in the joint. This article focuses on the recent developments in natural and synthetic polymer-based microsphere drug delivery systems for treating knee osteoarthritis. It evaluates the translational potential of some novel formulations for clinical application.

Signaling pathways regulated by natural active ingredients in the fight against exercise fatigue-a review.

Exercise fatigue is a normal protective mechanism of the body. However, long-term fatigue hinders normal metabolism and exercise capacity. The generation and recovery from exercise fatigue involves alterations in multiple signaling pathways, mainly AMPK, PI3K/Akt, Nrf2/ARE, NF-κB, PINK1/Parkin, and BDNF/TrkB, as well as MAPK signaling pathways that mediate energy supply, reduction of metabolites, oxidative stress homeostasis, muscle fiber type switching, and central protective effects. In recent studies, a rich variety of natural active ingredients have been identified in traditional Chinese medicines and plant extracts with anti-fatigue effects, opening up the field of research in new anti-fatigue drugs. In this review we give an overview of the signaling pathways associated with the activity of natural food active ingredients against exercise fatigue. Such a comprehensive review is necessary to understand the potential of these materials as preventive measures and treatments of exercise fatigue. We expect the findings highlighted and discussed here will help guide the development of new health products and provide a theoretical and scientific basis for future research on exercise fatigue.

Effects of Treadmill Running at Different Light Cycles in Mice with Metabolic Disorders.

Type 2 diabetes mellitus accounts for about 90% of cases of diabetes and is considered one of the most important problems of our time. Despite a significant number of studies on glucose metabolism, the molecular mechanisms of its regulation in health and disease remain insufficiently studied. That is why non-drug treatment of metabolic disorders is of great relevance, including physical activity. Metabolic changes under the influence of physical activity are very complex and are still difficult to understand. This study aims to deepen the understanding of the effect of physical exercise on metabolic changes in mice with diabetes mellitus. We studied the effect of forced treadmill running on body weight and metabolic parameters in mice with metabolic disorders. We developed a high-fat-diet-induced diabetic model of metabolic disorders. We exposed mice to forced treadmill running for 4 weeks. We determined glucose and insulin levels in the blood plasma biochemically and analyzed Glut-4 and citrate synthase in M. gastrocnemius muscle tissue using Western blotting. The research results show that daily treadmill running has different effects on different age groups of mice with metabolic disorders. In young-age animals, forced running has a more pronounced effect on body weight. At week 12, young obese mice had a 17% decrease in body weight. Body weight did not change in old mice. Moreover, at weeks 14 and 16, the decrease in body weight was more significant in the young mice (by 17%) compared to the old mice (by 6%) (p < 0.05). In older animals, it influences the rate of glucose uptake. At 60 min, the blood glucose in the exercised older mice decreased to 14.46 mmol/L, while the glucose concentration in the non-exercised group remained at 17 mmol/L. By 120 min, in mice subjected to exercise, the blood glucose approached the initial value (6.92 mmol/L) and amounted to 8.35 mmol/L. In the non-exercised group, this difference was 45%. The effects of physical activity depend on the time of day. The greater effect is observed when performing shift training or exercise during the time when animals are passive (light phase). In young mice, light phase training had a significant effect on increasing the content of Glut-4 in muscle tissue (84.3 ± 11.3%, p < 0.05 with control group-59.3 ± 7.8%). In aged mice, shift training caused an increase in the level of Glut-4 in muscle tissue (71.3 ± 4.1%, p < 0.05 with control group-56.4 ± 10,9%). In the group of aged mice, a lower CS level was noticed in all groups in comparison with young mice. It should also be noted that we observed that CS increased during exercise in the group of young mice, especially during light phase training. The CS content in the light phase subgroup (135.8 ± 7.0%) was higher than in the dark phase subgroup (113.3 ± 7.7%) (p = 0.0006). The CS decreased in aged chow-fed mice and increased in the high-fat-fed group. The CS content in the chow diet group (58.2 ± 5.0%) was 38% lower than in the HFD group (94.9 ± 8.8%).

Protective effects of curcumin against osteoporosis and its molecular mechanisms: a recent review in preclinical trials.

Osteoporosis (OP) is one of the most common metabolic skeletal disorders and is commonly seen in the elderly population and postmenopausal women. It is mainly associated with progressive loss of bone mineral density, persistent deterioration of bone microarchitecture, and increased fracture risk. To date, drug therapy is the primary method used to prevent and treat osteoporosis. However, long-term drug therapy inevitably leads to drug resistance and specific side effects. Therefore, researchers are constantly searching for new monomer compounds from natural plants. As a candidate for the treatment of osteoporosis, curcumin (CUR) is a natural phenolic compound with various pharmacological and biological activities, including antioxidant, anti-apoptotic, and anti-inflammatory. This compound has gained research attention for maintaining bone health in various osteoporosis models. We reviewed preclinical and clinical studies of curcumin in preventing and alleviating osteoporosis. These results suggest that if subjected to rigorous pharmacological and clinical trials, naturally-derived curcumin could be used as a complementary and alternative medicine for the treatment of osteoporosis by targeting osteoporosis-related mechanistic pathways. This review summarizes the mechanisms of action and potential therapeutic applications of curcumin in the prevention and mitigation of osteoporosis and provides reference for further research and development of curcumin.

Effects of cold water immersion after exercise on fatigue recovery and exercise performance--meta analysis.

Cold water immersion (CWI) is very popular as a method reducing post-exercise muscle stiffness, eliminating fatigue, decreasing exercise-induced muscle damage (EIMD), and recovering sports performance. However, there are conflicting opinions as to whether CWI functions positively or negatively. The mechanisms of CWI are still not clear. In this systematic review, we used meta-analysis aims to examine the effect of CWI on fatigue recovery after high-intensity exercise and exercise performance. A total of 20 studies were retrieved and included from PubMed, PEDro and Elsevier databases in this review. Publication years of articles ranged from 2002 to 2022. In selected studies including randomized controlled trials (RCTs) and Crossover design (COD). Analyses of subjective indicators such as delayed-onset muscle soreness (DOMS) and ratings of perceived exertion (RPE), and objective indicators such as countermovement jump (CMJ) and blood plasma markers including creatine kinase(CK), lactate/lactate dehydrogenase(LDH), C-reactive protein(CRP), and IL-6 were performed. Pooled data showed as follows: CWI resulted in a significant decline in subjective characteristics (delayed-onset muscle soreness and perceived exertion at 0 h); CWI reduced countermovement jump(CMJ) significantly at 0 h, creatine kinase(CK) was lowered at 24 h, and lactate at 24 and 48 h. There was no evidence that CWI affects C-reactive protein(CRP) and IL-6 during a 48-h recovery period. Subgroup analysis revealed that different CWI sites and water temperatures have no effect on post-exercise fatigue recovery. Recommended athletes immersed in cold water immediately after exercise, which can effectively reduce muscle soreness and accelerate fatigue recovery.

Associations Between Heart Rate Variability-Derived Indexes and Training Load: Repeated Measures Correlation Approach Contribution.

ABSTRACT: Davletyarova, K, Vacher, P, Nicolas, M, Kapilevich, LV, and Mourot, L. Associations between heart rate variability-derived indexes and training load: repeated measures correlation approach contribution. J Strength Cond Res 36(7): 2005-2010, 2022-This study aimed to evaluate whether similar associations between indexes derived from heart rate variability (HRV) analyses and training load (TL) could be obtained by using the commonly used Pearson correlation technique and the repeated measures correlation (rmcorr). Fourteen well-trained swimmers (18.5 ± 1.6 years) participated. The training period lasted 4 weeks with a gradual increase in TL. Daily external TL (exTL) and internal TL (inTL) were summed to obtain a weekly TL, and HRV analyses were performed every Saturday morning. During the 4-week period, exTL and inTL increased (p < 0.05) together with a decrease (p < 0.05) in heart rate and an increase (p < 0.05) of cardiac parasympathetic indexes. No significant correlation was found using Pearson correlation while significant associations were found using rmcorr; considering exTL, positive (mean R-R interval [MeanRR], root mean square of differences between successive RR interval [RMSSD], low frequency [LF], high frequency [HF], instantaneous beat-to-beat variability [SD1], continuous beat-to-beat variability [SD2], SD1/SD2; r from 0.59 to 0.46, p value from <0.001 to 0.002) and negative (mean heart rate [meanHR]; r = -0.55, p < 0.001) associations were found. Considering inTL, positive (MeanRR, RMSSD, LF, HF, HFnu, SD1, SD2, SD1/SD2; r from 0.56 to 0.34, p-value from <0.001 to 0.025) and negative (meanHR, LFnu, LF/HF; r from -0.49 to -0.34, p value from 0.001 to 0.025) associations were found. The rmcorr statistical method was able to show associations between parasympathetic indexes and TL contrary to Pearson correlation analysis. Because rmcorr is specifically designed to investigate within-individual association for paired measures assessed on 2 or more occasions for multiple individuals, it should constitute a tool for future training monitoring researches based on a repeated-measures protocol.

Treadmill Training Effect on the Myokines Content in Skeletal Muscles of Mice With a Metabolic Disorder Model.

The effect of treadmill training loads on the content of cytokines in mice skeletal muscles with metabolic disorders induced by a 16 week high fat diet (HFD) was studied. The study included accounting the age and biorhythmological aspects. In the experiment, mice were used at the age of 4 and 32 weeks, by the end of the experiment-respectively 20 and 48 weeks. HFD feeding lasted 16 weeks. Treadmill training were carried out for last 4 weeks six times a week, the duration 60 min and the speed from 15 to 18 m/min. Three modes of loading were applied. The first subgroup was subjected to stress in the morning hours (light phase); the second subgroup was subjected to stress in the evening hours (dark phase); the third subgroup was subjected to loads in the shift mode (the first- and third-weeks treadmill training was used in the morning hours, the second and fourth treadmill training was used in the evening hours). In 20-week-old animals, the exercise effect does not depend on the training regime, however, in 48-week-old animals, the decrease in body weight in mice with the shift training regime was more profound. HFD affected muscle myokine levels. The content of all myokines, except for LIF, decreased, while the concentration of CLCX1 decreased only in young animals in response to HFD. The treadmill training caused multidirectional changes in the concentration of myokines in muscle tissue. The IL-6 content changed most profoundly. These changes were observed in all groups of animals. The changes depended to the greatest extent on the training time scheme. The effect of physical activity on the content of IL-15 in the skeletal muscle tissue was observed mostly in 48-week-old mice. In 20-week-old animals, physical activity led to an increase in the concentration of LIF in muscle tissue when applied under the training during the dark phase or shift training scheme. In the HFD group, this effect was significantly more pronounced. The content of CXCL1 did not change with the use of treadmill training in almost all groups of animals. Physical activity, introduced considering circadian rhythms, is a promising way of influencing metabolic processes both at the cellular and systemic levels, which is important for the search for new ways of correcting metabolic disorders.

Effect of Dynamic and Static Load on the Concentration of Myokines in the Blood Plasma and Content of Sodium and Potassium in Mouse Skeletal Muscles.

Modulation of cytokine production by physical activity is of considerable interest, since it might be a promising strategy for correcting metabolic processes at both cellular and systemic levels. The content of IL-6, IL-8, and IL-15 in the plasma and the concentration of monovalent cations in the skeletal muscles of trained and untrained mice were studied at different periods after static and dynamic exercises. Dynamic loads caused an increase in the IL-6 content and decrease in the IL-15 content in the plasma of untrained mice, but produced no effect on the concentration of IL-8. In trained mice, the effect of a single load on the concentration of IL-6 and IL-15 in the plasma was enhanced, while the concentration of IL-8 decreased. Static loads produced a similar, but more pronounced effect on the plasma concentration of IL-6 and IL-15 compared the dynamic exercises; however, the concentration of IL-8 in response to the static exercise increased significantly. Prior training reinforced the described response for all the myokines studied. Dynamic load (swimming) increased the intracellular content of sodium but decreased the content of potassium in the mouse musculus soleus. Similar response was observed after the static load (grid hanging) in the musculus biceps; but no correlation of this response with the prior training was found. Possible mechanisms involved in the regulation of cytokine secretion after exercise are discussed, including triggering of gene transcription in response to changes in the [Na+]i/[K+]I ratio.

Ouabain Suppresses IL-6/STAT3 Signaling and Promotes Cytokine Secretion in Cultured Skeletal Muscle Cells.

The cardiotonic steroids (CTS), such as ouabain and marinobufagenin, are thought to be adrenocortical hormones secreted during exercise and the stress response. The catalytic α-subunit of Na,K-ATPase (NKA) is a CTS receptor, whose largest pool is located in skeletal muscles, indicating that muscles are a major target for CTS. Skeletal muscles contribute to adaptations to exercise by secreting interleukin-6 (IL-6) and plethora of other cytokines, which exert paracrine and endocrine effects in muscles and non-muscle tissues. Here, we determined whether ouabain, a prototypical CTS, modulates IL-6 signaling and secretion in the cultured human skeletal muscle cells. Ouabain (2.5-50 nM) suppressed the abundance of STAT3, a key transcription factor downstream of the IL-6 receptor, as well as its basal and IL-6-stimulated phosphorylation. Conversely, ouabain (50 nM) increased the phosphorylation of ERK1/2, Akt, p70S6K, and S6 ribosomal protein, indicating activation of the ERK1/2 and the Akt-mTOR pathways. Proteasome inhibitor MG-132 blocked the ouabain-induced suppression of the total STAT3, but did not prevent the dephosphorylation of STAT3. Ouabain (50 nM) suppressed hypoxia-inducible factor-1α (HIF-1α), a modulator of STAT3 signaling, but gene silencing of HIF-1α and/or its partner protein HIF-1ß did not mimic effects of ouabain on the phosphorylation of STAT3. Ouabain (50 nM) failed to suppress the phosphorylation of STAT3 and HIF-1α in rat L6 skeletal muscle cells, which express the ouabain-resistant α1-subunit of NKA. We also found that ouabain (100 nM) promoted the secretion of IL-6, IL-8, GM-CSF, and TNF-α from the skeletal muscle cells of healthy subjects, and the secretion of GM-CSF from cells of subjects with the type 2 diabetes. Marinobufagenin (10 nM), another important CTS, did not alter the secretion of these cytokines. In conclusion, our study shows that ouabain suppresses the IL-6 signaling via STAT3, but promotes the secretion of IL-6 and other cytokines, which might represent a negative feedback in the IL-6/STAT3 pathway. Collectively, our results implicate a role for CTS and NKA in regulation of the IL-6 signaling and secretion in skeletal muscle.

Elevation of Intracellular Na+ Contributes to Expression of Early Response Genes Triggered by Endothelial Cell Shrinkage.

BACKGROUND/AIMS: Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC. METHODS: Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR. RESULTS: Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition. CONCLUSION: Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.

Transcriptomic changes triggered by ouabain in rat cerebellum granule cells: Role of α3- and α1-Na+,K+-ATPase-mediated signaling.

It was shown previously that inhibition of the ubiquitous α1 isoform of Na+,K+-ATPase by ouabain sharply affects gene expression profile via elevation of intracellular [Na+]i/[K+]i ratio. Unlike other cells, neurons are abundant in the α3 isoform of Na+,K+-ATPase, whose affinity in rodents to ouabain is 104-fold higher compared to the α1 isoform. With these sharp differences in mind, we compared transcriptomic changes in rat cerebellum granule cells triggered by inhibition of α1- and α3-Na+,K+-ATPase isoforms. Inhibition of α1- and α3-Na+,K+-ATPase isoforms by 1 mM ouabain resulted in dissipation of transmembrane Na+ and K+ gradients and differential expression of 994 transcripts, whereas selective inhibition of α3-Na+,K+-ATPase isoform by 100 nM ouabain affected expression of 144 transcripts without any impact on the [Na+]i/[K+]i ratio. The list of genes whose expression was affected by 1 mM ouabain by more than 2-fold was abundant in intermediates of intracellular signaling and transcription regulators, including augmented content of Npas4, Fos, Junb, Atf3, and Klf4 mRNAs, whose upregulated expression was demonstrated in neurons subjected to electrical and glutamatergic stimulation. The role [Na+]i/[K+]i-mediated signaling in transcriptomic changes involved in memory formation and storage should be examined further.

Ubiquitous and cell type-specific transcriptomic changes triggered by dissipation of monovalent cation gradients in rodent cells: Physiological and pathophysiological implications.

Elevation of [Na+]i/[K+]i-ratio is considered as one of the major signals triggering transcriptomic changes in various cells types. In this study, we identified ubiquitous and cell type-specific [Formula: see text] -sensitive genes by comparative analysis of transcriptomic changes in ouabain-treated rat aorta smooth muscle cells and rat aorta endothelial cells (RASMC and RAEC, respectively), rat cerebellar granule cells (RCGC), and mouse C2C12 myoblasts. Exposure of the cells to ouabain increased intracellular Na+ content by ~14, 8, 7, and 6-fold and resulted in appearance of 7577, 2698, 2120, and 1146 differentially expressed transcripts in RAEC, RASMC, C2C12, and RCGC, respectively. Eighty-three genes were found as the intersection of the four sets of identified transcripts corresponding to each cell type and are classified as ubiquitous. Among the 10 top upregulated ubiquitous transcripts are the following: Dusp6, Plk3, Trib1, Ccl7, Mafk, Atf3, Ptgs2, Cxcl1, Spry4, and Coq10b. Unique transcripts whose expression is cell-specific include 4897, 1523, 789, and 494 transcripts for RAEC, RASMC, C2C12, and RCGC, respectively. The role of gene expression and signal pathways induced by dissipation of transmembrane gradient of monovalent cations in the development of various diseases is discussed with special attention to cardiovascular and pulmonary illnesses.

Transcriptomic changes in C2C12 myotubes triggered by electrical stimulation: Role of Ca2+i-mediated and Ca2+i-independent signaling and elevated [Na+]i/[K+]i ratio.

Elevation of Ca2+i and AMP-activated protein kinase (AMPK) are considered as major signals triggering transcriptomic changes in exercising skeletal muscle. Electrical pulse stimulation (EPS) of cultured myotubes is widely employed as an in vitro model of muscle contraction. This study examines the impact of Ca2+i-mediated and Ca2+i-independent signaling in transcriptomic changes in EPS-treated C2C12 myotubes. Electrical pulse stimulation (40 V, 1 Hz, 10 ms, 2 h) resulted in [Ca2+]i oscillations, gain of Na+i, loss of K+i, and differential expression of 3215 transcripts. Additions of 10 µM nicardipine abolished [Ca2+]i oscillations but did not affect elevation of the [Na+]i/[K+]i ratio seen in EPS-treated myotubes. Differential expression of 1018 transcripts was preserved in the presence of nicardipine, indicating a Ca2+i-independent mechanism of excitation-transcription coupling. Among nicardipine-resistant transcripts, we noted 113 transcripts whose expression was also affected by partial Na+,K+-ATPase inhibition with 30 µM ouabain providing the same elevation of the [Na+]i/[K+]i ratio as in EPS-treated cells. Electrical pulse stimulation increased phosphorylation of CREB, ATF-1, Akt, ERK, and p38 MAPK without any impact on phosphorylation of acetyl-CoA carboxylase and Unc-51 like autophagy activating kinase-1, i.e. downstream markers of AMPK activation. Unlike CREB, ATF-1, and MAPKs, an increment in Akt phosphorylation was abolished by nicardipine. Thus, our results show that Ca2+i-independent signaling plays a key role in altered expression of 30% of studied genes in EPS-treated myotubes. This signaling pathway is at least partially triggered by dissipation of transmembrane gradients of monovalent cations.

Electrical pulse stimulation decreases electrochemical Na+ and K+ gradients in C2C12 myotubes.

Electrical pulse stimulation (EPS)-treated cultured myotubes are widely employed as an in vitro model of muscle contraction. Here we examined time-dependent EPS action and dose-dependent ouabain action on [Na+]i and [K+]i in C2C12 myotubes. After 2 h of EPS (40 V, 1 Hz, 10 ms) [Na+]i increased by ∼150% whereas [K+]i declined by ∼20%. 3 µM ouabain had a negligible impact on [Na+]i and [K+]i in control cells but increased the [Na+]i/[K+]i ratio in EPS-treated myotubes by 85%. Thus, our results show for the first time that EPS results in dissipation of Na+ and K+ gradients in cultured myotubes and suggest that the augmented production of endogenous cardiotonic steroids may contribute to elevation of the [Na+]i/[K+]i ratio in exercising muscle.

Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis.

Recent studies demonstrated that in addition to Na+,K+-ATPase inhibition cardiotonic steroids (CTSs) affect diverse intracellular signaling pathways. This study examines the relative impact of [Na+]i/[K+]i-mediated and -independent signaling in transcriptomic changes triggered by the endogenous CTSs ouabain and marinobufagenin (MBG) in human umbilical vein endothelial cells (HUVEC). We noted that prolongation of incubation increased the apparent affinity for ouabain estimated by the loss of [K+]i and gain of [Na+]i. Six hour exposure of HUVEC to 100 and 3,000 nM ouabain resulted in elevation of the [Na+]i/[K+]i ratio by ~15 and 80-fold and differential expression of 258 and 2185 transcripts, respectively. Neither [Na+]i/[K+]i ratio nor transcriptome were affected by 6-h incubation with 30 nM ouabain. The 96-h incubation with 3 nM ouabain or 30 nM MBG elevated the [Na+]i/[K+]i ratio by ~14 and 3-fold and led to differential expression of 880 and 484 transcripts, respectively. These parameters were not changed after 96-h incubation with 1 nM ouabain or 10 nM MBG. Thus, our results demonstrate that elevation of the [Na+]i/[K+]i ratio is an obligatory step for transcriptomic changes evoked by CTS in HUVEC. The molecular origin of upstream [Na+]i/[K+]i sensors involved in transcription regulation should be identified in forthcoming studies.

Dynamic and Static Exercises Differentially Affect Plasma Cytokine Content in Elite Endurance- and Strength-Trained Athletes and Untrained Volunteers.

Extensive exercise increases the plasma content of IL-6, IL-8, IL-15, leukemia inhibitory factor (LIF), and several other cytokines via their augmented transcription in skeletal muscle cells. However, the relative impact of aerobic and resistant training interventions on cytokine production remains poorly defined. In this study, we compared effects of dynamic and static load on cytokine plasma content in elite strength- and endurance-trained athletes vs. healthy untrained volunteers. The plasma cytokine content was measured before, immediately after, and 30 min post-exercise using enzyme-linked immunosorbent assay. Pedaling on a bicycle ergometer increased IL-6 and IL-8 content in the plasma of trained athletes by about 4- and 2-fold, respectively. In contrast to dynamic load, weightlifting had negligible impact on these parameters in strength exercise-trained athletes. Unlike IL-6 and IL-8, dynamic exercise had no impact on IL-15 and LIF, whereas static load increases the content of these cytokines by ~50%. Two-fold increment of IL-8 content seen in athletes subjected to dynamic exercise was absent in untrained individuals, whereas the ~50% increase in IL-15 triggered by static load in the plasma of weightlifting athletes was not registered in the control group. Thus, our results show the distinct impact of static and dynamic exercises on cytokine content in the plasma of trained athletes. They also demonstrate that both types of exercises differentially affect cytokine content in plasma of athletes and untrained persons.

Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel.

NKCC1 and NKCC2: The pathogenetic role of cation-chloride cotransporters in hypertension.

This review summarizes the data on the functional significance of ubiquitous (NKCC1) and renal-specific (NKCC2) isoforms of electroneutral sodium, potassium and chloride cotransporters. These carriers contribute to the pathogenesis of hypertension via regulation of intracellular chloride concentration in vascular smooth muscle and neuronal cells and via sensing chloride concentration in the renal tubular fluid, respectively. Both NKCC1 and NKCC2 are inhibited by furosemide and other high-ceiling diuretics widely used for attenuation of extracellular fluid volume. However, the chronic usage of these compounds for the treatment of hypertension and other volume-expanded disorders may have diverse side-effects due to suppression of myogenic response in microcirculatory beds.