Calcineurin inhibitors (CNIs), including tacrolimus and cyclosporin A, are potent immunosuppressive drugs that exert their effects by inhibiting protein phosphatase 3, also known as calcineurin. CNIs are widely used clinically, particularly for preventing organ rejection following transplantation. However, their use is frequently associated with significant adverse effects. In particular, CNIs commonly affect the kidney and can lead to acute and chronic kidney injury. In addition, they interfere with renal electrolyte reabsorption resulting in electrolyte disturbances (e.g., hyperkalemia, hypomagnesemia) and hypertension. Notably, calcineurin subunits are abundantly expressed throughout the nephron. Consequently, calcineurin inhibition affects electrolyte transport in virtually all nephron segments. In this review, we summarize the current knowledge on the effects of CNIs on renal tubular ion transport processes and highlight the underlying mechanism by which these effects contribute to CNI-induced electrolyte disturbances.
This study investigated the effects of high/low-intensity interval training (HIIT/LIIT) via electrical stimulation (ES) on muscle performance, specifically focusing on fatigue resistance and cytoplasmic free calcium ([Ca2+]i) dynamics in mouse skeletal muscle. Thirty 9-week-old female C57BL6 mice underwent four weeks of involuntary IT-ES in hindlimb plantar flexor muscles in vivo, with stimulation frequencies set at either 20 Hz (LIIT) or 100 Hz (HIIT). Our results showed that IT-ES significantly enhanced fatigue resistance, particularly with HIIT, evidenced by improved muscle torque output and better preservation of tetanic [Ca2+]i levels during repeated contractions. Additionally, IT-ES led to increases in sarcoplasmic reticulum (SR) Ca2+ handling proteins, such as SR Ca2+ ATPase 1 (SERCA1) and the ryanodine receptor 1 (RyR1), as well as mitochondrial respiratory complex proteins, indicating enhanced metabolic adaptations. The results suggest that HIIT-ES is an effective intervention for improving muscle function, intracellular Ca2+ management, and mitochondrial content, providing a foundation for future consideration in rehabilitation and clinical settings.
Glycyrrhiza glabra is one of 30 species of licorice which has been prescribed for centuries for a wide range of ailments and conditions. In particular, licorice root extract, as well as its main isoflavonoid constituent, glabridin, have been used to treat a variety of respiratory diseases, ranging from infection to asthma. As glabridin has been shown to modulate the activity of ion channels in other tissues, we determined the effect of glabridin on K+ and Cl⁻ secretion across primary human bronchial epithelial cells (HBEs), as these may represent therapeutic targets. Glabridin stimulated BKCa-dependent transepithelial potassium secretion across HBEs. In contrast, the glabridin derivative, vutiglabridin failed to stimulate K+ secretion and inhibited the glabridin-dependent K+ secretory current. Whole-cell patch-clamp studies on HEK cells expressing BKCa demonstrate that glabridin activates, whereas vutiglabridin inhibits, BKCa. We further demonstrate glabridin inhibits forskolin-mediated transepithelial Cl⁻ secretion across HBEs, while vutiglabridin has little effect. Using Fisher Rat Thyroid (FRT) cells stably expressing either wild type (FRT-WT) or F508del CFTR (FRT-F508del), we demonstrate glabridin neither potentiates WT CFTR nor corrects F508del CFTR. In contrast, whole-cell patch-clamp studies demonstrate glabridin inhibits KCa3.1 stably expressed in HEK cells. We previously demonstrated a role for KCa3.1 in forskolin-mediated Cl⁻ secretion across HBEs, likely explaining the inhibition of Cl⁻ secretion observed. In summary, we show that glabridin both stimulates BKCa-dependent K+ secretion and inhibits cAMP-mediated Cl⁻ secretion across HBEs. These findings support a role for BKCa in the therapeutic effects of glabridin in airway.
ACE2, serving as a receptor to SARS-CoV-2, is a key player in RAAS. Consequent ACE2 depletion disrupts the balance between the Ang II/ AT1R and Ang (1-7)/MasR arms of the angiotensin system, promoting intense inflammation. A crosstalk existing between the angiotensin system and ACE2/spike proteins may affect the infection severity, with potential therapeutic implications. Evaluating such potential interactions and their relevance to the severity of COVID-19, which is directly affected by ACE2 abundance or indirectly by RAAS axis dysregulation. The intensity of SARS-CoV-2 infection was assessed using a cell-to-cell fusion assay and AT1R activation was assessed using AT1R-Tango approach. We demonstrate the critical function of ACE2 in mitigating AT1R activation, which is disrupted following SARS-CoV-2 infection. Moreover, we show that the spike protein indirectly intensifies AT1R activation. Beyond its established role in activating MasR, Ang1-7 was found to function as a biased agonist for AT1R, without altering ACE2 levels or affecting SARS-CoV-2 entry. In contrast, AVE0991, a MasR agonist, was observed to increase ACE2 levels and enhance SARS-CoV-2 infection. Angiotensin receptor blockers (ARBs) effectively inhibited AT1R activity and had minimal impact on viral entry. Our data supports the likelihood that AT1R blockers (ARBs) may be effective in managing COVID-19 since they inhibit AT1R activation and its deleterious subsequent effects, with no impact on SARS-CoV-2 entry. By contrast, MasR and its agonist AVE0991, by increasing ACE2 levels, may restore RAAS physiological balance, but facilitate host cell invasion by SARS-CoV-2.
Hypertension and cardiovascular disease (CVD) are associated with elevated plasma levels of ceramides (Cer), a type of membrane sphingolipids (SPLs). Increased cellular Cer levels are known to cause vascular endothelial cell (EC) dysfunction. However, Cer metabolism changes due to EC exposure to high-magnitude “hy-pertensive” cyclic stretch (HCS) and their role in EC dysfunction are poorly defined. Cultured human ECs exposed to HCS (15% elon¬gation at 1 Hz, 24 h) exhibited increased oxidative stress, upreg¬ulation of genes for cytokines and leukocyte adhe¬sion molecules, increased inflammatory response to a low concentration of tumor necrosis factor-α, and increased apoptosis compared to ECs exposed to “normotensive” CS (NCS; 5% elongation at 1 Hz, 24 h). Lipidomics analysis of EC pellets at 4 or 24 h of CS detected no differ¬ence in Cer levels and significantly higher sphingomyelin (SM) levels at 24 h of HCS compared to NCS. Cer immunostaining showed significantly higher Cer levels at both the perinuclear and peripheral subcellular regions in HCS compared to the corresponding regions in NCS. HCS signifi¬cantly increased the concentrations of certain long-chain Cer in the extracellular media compared to NCS. Phar¬macological inhibition of key enzymes in Cer biosynthesis, i.e., de novo synthesis and SM hy¬drolysis pathways, significantly inhibited HCS-induced EC inflammation and apoptosis, sug¬gesting that Cer generated from SM, via the sphingomyelinase family of enzymes, and accumulated at specific sub¬cellular compartments may be respon¬sible for the HCS-induced EC dysfunction. In summary, Cer act as mechanotransducers that connect hypertensive stretch to EC inflamma-tion and apoptosis, and promote CVD.
Infertility affects millions of people globally with recent statistics indicating that one in six adults is experiencing reproductive challenges at some point. For female infertility in particular, the global health burden is continuously intensifying with significant disparities across countries. Female infertility has a complex multifactorial etiology and understanding the underlying mechanisms is crucial in order to reduce the health burden and enhance patient well-being. For a pregnancy, successful embryo implantation is a pivotal step, requiring precise spatiotemporal synchronization between a competent blastocyst and a receptive endometrium during the window of implantation (WOI). This review provides an overview of recent developments around the molecular and clinical aspects of this process, with a specific focus on the extracellular microenvironmental cues and maternal-embryo interaction. Specifically, we detail the extensive extracellular matrix (ECM) remodeling that facilitates embryo adhesion and invasion, while shedding light on microRNAs as signaling and communication mediators that govern cell-cell and maternal-fetal interactions. Finally, we examine current limitations in the clinical assessment of receptivity and discuss future perspectives for non-invasive biomarkers and possibilities for more personalized solutions in assisted reproduction.
Co-treatment with ACE inhibitors/angiotensin II receptor antagonists was obligatory and an aldosterone antagonist optional in trials that established efficacy of β1 adrenergic receptor (β1AR) antagonists in heart failure with reduced ejection fraction (HFrEF). However, with worsening HFrEF, combination treatment is often not tolerated, and patients receive β1AR antagonists only. It cannot be assumed monotherapy is efficacious and might even be harmful because a raised myocardial cytosolic Na + concentration ([Na + c ]) in HFrEF reduces myocardial contractility while β1AR-coupled signaling has been widely reported to activate myocyte Na + -K + pump-mediated Na + c export in previous studies. We ligated the circumflex coronary artery of rabbits or performed sham thoracotomy. Rabbits with left ventricular ejection fraction <25% seven days after coronary ligation were treated with the β1AR antagonist metoprolol or vehicle. Metoprolol reduced heart rate of sham-operated rabbits but not of rabbits that had coronary ligation. Metoprolol had no effect on systolic-, diastolic- or mean blood pressures in either group. The primary index for heart failure, lung: body weight ratios, was ~89% higher after coronary ligation than after sham thoracotomy and metoprolol significantly reduced this to a ~46% difference. The index for Na + -K + pump function, electrogenic pump current (I p ), increased for myocytes from sham-operated rabbits. For myocytes from rabbits that had coronary ligation, metoprolol reversed a 63% decrease of Ip. Results are consistent with enhanced Na + -K + pump-mediated Na + c export in HFrEF contributing to efficacy of β1AR antagonist monotherapy independent of heart rate and afterload but does not support β1AR-coupled cAMP signaling increasing cardiac Na + -K + pump activity.
Analysis of mitochondrial morphology and ultrastructure from transmission electron microscopy images is essential for understanding cellular adaptations in both physiological and pathological conditions. However, these investigations remain time-consuming, poorly standardized, and therefore highly variable. We developed an open-access, standalone MATLAB-based application [Analysis of MITOchondria (AMITO)] that can be run without a license and integrates analyses within a single user-friendly interface. AMITO demonstrated excellent agreement with conventional manual image analysis software, high intra- and interexperimenter reproducibility, and substantial reduction in analysis time. Therefore, AMITO provides a reliable, efficient, and accessible tool to ensure robustness and reproducibility in mitochondrial assessment.NEW & NOTEWORTHY AMITO is an open-access, standalone application for mitochondrial analysis from TEM images that combines automated and manual workflows within a single user-friendly interface. Validated against conventional FIJI analysis, AMITO demonstrated excellent reproducibility while substantially reducing analysis time and experimenter-dependent variability. By integrating morphology and ultrastructure quantification without requiring programming expertise, AMITO provides a reliable and accessible solution to improve robustness, efficiency, and reproducibility in mitochondrial research.
In type 2 diabetes (T2D), kidney fibrosis drives the progression of chronic kidney disease (CKD) and kidney failure. Lower urinary levels of collagen type III degradation (C3M), a biomarker of extracellular matrix (ECM) turnover, have been associated with CKD progression. We investigated the effect of combination therapy with renin-angiotensin system inhibition (RASi) and glucagon-like peptide-1 receptor agonist (GLP-1 RA) on urinary C3M levels in people with T2D. Urinary C3M was measured using the nordicC3M ELISA in 229 participants from the Drug Combinations for Rewriting Trajectories of Renal Pathologies in T2D (DC-ren) project. Longitudinal changes in urinary C3M were analyzed using linear regression stratified by treatment group. Risk of kidney disease progression according to baseline urinary C3M was evaluated using Kaplan-Meier analysis. The kidney endpoint was defined as >10% decline in estimated glomerular filtration rate (eGFR) to <60 mL/min/1.73 m2. Combination therapy (RASi + GLP-1 RA) was associated with higher urinary C3M levels compared with RASi monotherapy (P = 0.036). Urinary C3M was positively associated with future eGFR, with a steeper slope in the combination therapy group than in the RASi monotherapy group (P < 0.001). Low baseline urinary C3M was associated with a higher risk of kidney disease progression (P = 0.022). In people with T2D receiving combination therapy with RASi + GLP-1 RA, urinary C3M levels remained stable, whereas levels decreased with RASi monotherapy, suggesting altered kidney ECM turnover.NEW & NOTEWORTHY Urinary C3M is a dynamic biomarker of ECM turnover with pharmacodynamic relevance in kidney disease. Previous studies in diabetic and nondiabetic kidney disease have shown that therapeutic interventions can increase urinary C3M levels. Here, compared with RASi monotherapy, GLP-1 RA-based combination therapy with RASi was associated with a more favorable longitudinal urinary C3M trajectory and an altered association between urinary C3M and kidney function in type 2 diabetes.
The secretory pathway Ca2+-ATPase, SPCA1 (gene name ATP2C1), is a Golgi-localized calcium pump defective in the autosomal dominant cutaneous disorder known as Hailey-Hailey disease (HHD). Although clinically well characterized by suprabasal acantholysis and intertriginous blistering of the skin, the mechanistic underpinnings of the disease are still unclear. Here we use CRISPR/Cas9-mediated single- and biallelic ATP2C1 knockouts in immortalized human N/TERT keratinocytes to show that SPCA1 is required for dynamic reorganization of actin cytoskeleton in keratinocyte spreading, which is the primary mechanism driving skin reepithelialization. We identify an insulin-activated PI3K-AKT-Rac1 signaling pathway required for lamellipodia formation and keratinocyte spreading, defective in SPCA1 knockout cell lines. Our findings may explain the poor wound healing and impaired keratinocyte migration observed in HHD and may be relevant to the observed effect of insulin on wound healing, including diabetic wounds and burns, reported for nearly a century. Transgenic expression of hSPCA1 or treatment with CDN1163, a small molecule Ca2+-ATPase agonist, restored defective phenotypes in the HHD model, paving the way for future therapeutic approaches to treat this disorder.NEW & NOTEWORTHY Keratinocytes spread and change shape dynamically to maintain skin integrity and facilitate the rapid repair of the skin barrier after injury. Defects in these processes are characteristic of Hailey-Hailey disease (HHD), an ulcerative skin disorder caused by mutations in the Golgi Ca2+-ATPase SPCA1. By developing new keratinocyte HHD models, we uncover a role for SPCA1 in an insulin-activated signaling pathway that drives lamellipodia formation and keratinocyte spreading, linking Ca2+ regulation to actin cytoskeleton reorganization.
Lactate shuttling between glycolytic and oxidative muscle fibers via monocarboxylate transporters MCT1 and MCT4 is fundamental to muscle energy homeostasis, yet the physiological necessity of these two transporters remained obscured by functional redundancy. To investigate the significance of these two transporters in intermuscular lactate shuttle, we generated and characterized skeletal muscle-specific MCT1/4 double-knockout (MCT1/4-mKO) mice alongside with MCT4 single-knockout mice (MCT4-mKO). While MCT4-mKO mice had minimal metabolic disturbances, MCT1/4-mKO mice exhibited markedly reduced lean mass, impaired exercise endurance together with enhancement of insulin-stimulated glucose disposal under normal chow diet condition. The blood lactate level after exercise and the maximum grip strength was mitigated in both MCT4-mKO and MCT1/4-mKO mice. Plasma membrane localization of GLUT4 was robustly increased in the skeletal muscle in MCT1/4-mKO mice upon insulin treatment. These findings highlight that the intramuscular lactate shuttle mediated by MCT1 and MCT4 is critical for maintaining skeletal muscle mass, contractile performance and insulin sensitivity.
In Duchenne muscular dystrophy (DMD), neuronal nitric oxide synthase (nNOS) is mislocalized from the sarcolemmal membrane and exhibits reduced expression and activity, impairing vasomodulation and contributing to increased muscle fatigue. Identification of upstream regulators that restore nNOS localization and function may provide therapeutic strategies to improve muscle performance in dystrophic muscle. In this study, we investigated the role of lipin1 in regulating nNOS expression, sarcolemmal localization, and nitric oxide synthase (NOS) activity in skeletal muscle. Lipin1 deficiency significantly reduced nNOS expression and total NOS enzymatic activity, whereas lipin1 overexpression enhanced these parameters. Skeletal muscle-specific lipin1 knockout mice (lipin1Myf5cKO) exhibited increased muscle fatigue, consistent with impaired nNOS-dependent muscle function. In contrast, transgenic lipin1 restoration in dystrophic muscle (mdx:lipin1Tg/0) restored nNOS expression and sarcolemmal localization and improved fatigue resistance. Our findings suggest that lipin1 promotes nNOS sarcolemmal localization, potentially through stabilization of membrane-associated protein complexes, and enhances nNOS expression through a lipin1/diacylglycerol/protein kinase D/cAMP response element-binding protein signaling axis. Collectively, these findings identify lipin1 as an important regulator of nNOS expression and localization in skeletal muscle and support lipin1 restoration as a potential therapeutic strategy for DMD.NEW & NOTEWORTHY In Duchenne muscular dystrophy (DMD) mislocalization and reduced expression of neuronal nitric oxide synthase (nNOS) impair nitric oxide signaling and contribute to muscle dysfunction. This study demonstrates that lipin1 restoration increases nNOS expression, promotes sarcolemmal nNOS localization, enhances NOS activity, and improves fatigue resistance in dystrophic muscle. These findings identify a previously unrecognized role for lipin1 in regulating nNOS and support lipin1 restoration as a potential therapeutic strategy for DMD.
Oxygen is needed to generate adenosine triphosphate (ATP) via aerobic respiration in mitochondria. Hypoxia - when the tissue's oxygen demand exceeds its supply -characterizes sites of acute inflammation but also many tumors and their microenvironment. But even then, (immune) cells must ensure sufficient ATP synthesis. Under hypoxic conditions, hypoxia-inducible factors (HIFs) are capable of adapting cellular metabolism by induction of hundreds of target genes that amongst others regulate glycolysis, cell survival, and angiogenesis. This may hold true for immune cells under homeostatic conditions when they are forced to fight against pathogens and for tumor cells when the activation of HIFs enables tumor cells to survive in oxygen-deficient tissues. In addition, HIF activation in many tumors promotes the creation of an immunosuppressive tumor microenvironment that prevents immune cells from effectively fighting the tumor. Therefore, promising approaches have been made in the combination of immune therapies with HIF-inhibition. In this review we highlight, that a better understanding of the role of hypoxic adaptation requires considering the HIF stabilization patterns of tumor type, tumor stage, and the immune cells within the tumor microenvironment to optimize therapeutic efficacy.
The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is a fundamental intracellular axis that integrates extracellular stimuli to orchestrate essential cellular processes. The activation of Akt1 represents a key determinant of aggressiveness in estrogen receptor-positive (ER+) breast cancer, a process closely associated with Ca2+-signaling remodeling mediated by Orai1 and Orai3. In ER+ breast cancer cells, Orai3 plays a predominant role in store-operated Ca2+ entry (SOCE), whereas Orai1 indirectly regulates SOCE and contributes to store-independent Ca2+ influx pathways. Here we investigated the regulation of Akt1 activation by Orai3 and Orai1 and characterized the underlying intracellular mechanisms in ER+ breast cancer cells. Our findings reveal distinct, opposing roles for Orai1 and Orai3. Orai3 overexpression in wild-type cells, as well as 2-aminoethoxydiphenyl borate (2-APB)-mediated stimulation of Orai1-knockout (O1KO) MCF-7 cells, enhanced Akt1 phosphorylation, whereas Orai3 knockdown attenuated this effect. This 2-APB-induced activation was impaired by calmodulin-dependent protein kinase kinase (CaMKK) inhibition in a concentration-dependent manner. Conversely, Orai1 deficiency (O1KO) or shRNA-mediated knockdown resulted in enhanced Akt1 phosphorylation, whereas Orai1 overexpression attenuated it. Expression of the pore-dead Orai1E106Q mutant in O1KO cells failed to affect Akt phosphorylation, indicating that Ca2+ influx is essential for this modulation. Mechanistically, Orai1 knockout or knockdown attenuated the expression of adenylyl cyclase 8 (AC8), whereas Orai1 expression enhanced AC8 levels through a Ca2+-influx-dependent mechanism. Finally, silencing AC8 impaired Orai1-mediated inhibition of Akt phosphorylation. Overall, these findings demonstrate an unprecedented functional antagonism between Orai1 and Orai3 in ER+ breast cancer cells, where distinct signaling pathways mediate their opposing effects on Akt1 phosphorylation.NEW & NOTEWORTHY Orai1 and Orai3 exert opposing effects on the PI3K/Akt1 pathway, revealing a complex regulatory mechanism to coordinate cellular processes, such as survival, in ER+ breast cancer cells. Although Orai3 promotes Akt1 activation via CaMKK to drive tumor progression, Orai1 acts as a modulator on this axis through an AC8/cAMP-dependent mechanism. These findings highlight the functional diversity of Orai proteins and identify them as potential therapeutic targets for modulating Akt1-driven aggressiveness in breast cancer.
Previous results indicated that LPS treatment of human macrophages is associated with the induction of glutamate-ammonia-ligase (GLUL)GLUL, the enzyme that synthesizes glutamine, although the effects on cell Gln level were not assessed by those studies. Here, we show that M1 polarization of human THP-1 macrophage-like cells with LPS and IFNγ induces not only GLUL but also the exchange Gln transporter ASCT2 and the bidirectional carrier SNAT5. Consequently, Gln influx markedly increases, with ASCT2 and SNAT5 inhibitors suppressing the effect. Notwithstanding these changes, cell Gln is comparable in human M0 and M1 macrophages. However, under Gln-free conditions, Gln efflux is three-fold faster in M1 macrophages. With the same approach, we have demonstrated that cell glutamate mostly derives from extracellular Gln. Furthermore, Glu efflux is markedly higher in M1 macrophages, consistent with the induction of the Glu-cystine exchanger xCT. Induction of GLUL, SNAT5 and xCT, along with SNAT5-dependent stimulation of Gln transport, are clearly detectable also in primary human macrophages, derived from monocytes of peripheral blood and M1-polarized with LPS/IFNγ. These data indicate that, through a stimulation of Gln and Glu efflux, M1-polarized human macrophages can increase the concentration of both amino acids in the extracellular microenvironment.
Small interfering RNAs (siRNAs) are powerful tools to target cellular protein expression, making them promising candidates for therapeutic applications. siRNA-based approaches to target detrimental mechanisms in airway diseases such as asthma and chronic obstructive pulmonary disease are highly appealing. However, such delivery systems must be nontoxic, protect siRNAs from degradation, and enable intracellular uptake that accesses cytosolic RNA machinery. The present study examines the mechanisms by which guanidinium-functionalized poly(oxanorbornene)imide polymer (PONI-Guan) nanoparticles can effectively and safely deliver siRNA in human bronchial epithelial (BEC) and airway smooth muscle cells (ASM). PONI-Guan polymers were engineered to self-assemble with siRNA through electrostatic interactions. Primary BEC and ASM cells were preincubated with methyl-β-cyclodextrin, dynasore, dansylcadaverine chlorpromazine, latrunculin B, or cytochalasin D followed by incubation with nanoparticles at a single concentration but different guanidinium/phosphate ratios (G/P). BEC and ASM treated with methyl-β-cyclodextrin and dynasore demonstrated significant decrease in nanoparticle uptake. In addition, BEC showed decreased uptake with latrunculin B. Minimal BEC toxicity was observed with 20, 30, and 40 G/P ratios; ASM showed some toxicity with 40 G/P. Transepithelial electrical resistance readings were stable with 20 and 30 G/P, whereas 40 G/P showed significant but transient changes, with barrier integrity restored in ∼6 h. Furthermore, 30 G/P did not induce markers of necrosis, apoptosis, or inflammation in BEC or ASM. Transfection of BDNF siRNA in ASM and Arginase 1 and 2 siRNA in BEC showed significant decrease in corresponding mRNA and protein expression. Overall, these data indicate that PONI-Guan polymers can deliver siRNA safely and effectively in bronchial cells, primarily through caveolar or macropinocytosis uptake, offering a promising tool for future siRNA-based therapies.NEW & NOTEWORTHY siRNA delivery to target cellular processes is an appealing area in lung diseases such as asthma. To realize this potential requires efficient, targeted cytosolic delivery without cellular toxicity or siRNA degradation. We demonstrate the efficacy of PONI-Guan nanoparticles in accessing airway epithelial and smooth muscle cells with specific siRNA targeting of mRNAs of interest while maintaining barrier integrity and avoiding cellular toxicity.
Skeletal muscle maintains considerable capacity for regeneration following injury, but successful regeneration is limited in instances of volumetric muscle loss, advanced aging, or muscular dystrophies. Considerable research has been done on muscle stem cell (MuSC) transplantation; however, proliferative exhaustion and donor cell dose requirements have slowed progress. Due to the paramount role of cellular metabolism in regenerative function of stem cells, the clinical potential for MuSC therapy may be improved by minimizing the isolation-induced metabolic perturbations experienced by MuSCs. This study uses a model of simulated cell sorting combined with untargeted, small-molecule metabolomic profiling to outline sorting-induced metabolic perturbations in C2C12 myoblasts. We expand upon this by performing a time course of metabolomic profiling on myoblasts recovering from either fluorescence-activated cell sorting or magnetic-bead-activated cell sorting-based isolation procedures to determine the method and recovery timing for optimal redox and energetic status. Using this metabolism-informed method, we then performed primary MuSC transplantation studies in mice to demonstrate the generalizability from the in vitro system to in vivo MuSC transplantation during regeneration from barium chloride-induced injury. Our findings demonstrate metabolically favorable strategies to isolate MuSC for analysis of the quiescent-to-activated metabolic transition or enhance transplantation efficacy.NEW & NOTEWORTHY Due to the paramount role of cellular metabolism in regenerative function of stem cells, the clinical potential for muscle stem cell (MuSC) therapy may be improved by minimizing isolation-induced metabolic perturbations. This study uses a model of simulated cell sorting combined with untargeted, small-molecule metabolomic profiling to outline sorting-induced metabolic perturbations in myoblasts. Our findings demonstrate metabolically favorable strategies to isolate MuSC for analysis of the quiescent-to-activated metabolic transition or enhance transplantation efficacy.
Microtubules are integral components of the highly regulated and dynamic cytoskeleton, which is vital for cell function and the maintenance of homeostasis. Microtubule disruption is associated with multiple disease states including colorectal cancer and neurodegenerative disorders. Although much is known about the mechanisms by which microtubules are regulated under physiologic conditions, the effect of pathological stimuli and how this contributes to disease progression is less clear. Hypoxia is a prominent microenvironmental feature of a range of pathological states including inflammation, ischemia, neurodegenerative disease, and cancer. However, our knowledge on the effect of hypoxia on microtubules and whether this impacts disease progression remains limited. Understanding the impact of hypoxia on microtubules is therefore of fundamental importance to understanding disease progression mediated by cytoskeletal changes and may identify new therapeutic targets. In this study, we found that hypoxia decreases intestinal epithelial cell migration. This is associated with a rapid and reversible change in microtubule structure. This cytoskeletal rearrangement occurs independently of changes in α-tubulin protein expression or free-to-polymerized α-tubulin ratio and is also independent of the hypoxia-inducible factor 1α (HIF-1α) pathway. Mechanistically, we found that it is hypoxia-induced changes in glycolytic metabolism that mediate the structural rearrangement of α-tubulin. We hypothesize that these data identify a potential opportunity to utilize drugs targeting glycolytic metabolism to sensitize drug-resistant colorectal epithelial cancer cells to microtubule-based chemotherapies.NEW & NOTEWORTHY We found that in response to hypoxia, epithelial cells reorganize their microtubular structure into intense, punctate clusters with no change in polymerization. Furthermore, while this structural rearrangement is rapidly inducible, reversible, and oxygen-dependent, it is mediated in a HIF-1α-independent manner. We noted that PGAM1, a key glycolytic enzyme, facilitates this structural reorganization of the microtubules via association with α-tubulin and its metabolic activity, in response to hypoxia or other forms of glycolytic stress.
Prolonged exposure to high oxygen levels (hyperoxia) is unavoidable in managing severe acute respiratory distress syndrome (ARDS), but can itself worsen lung injury and increase mortality. Rats conditioned to be hyperoxia-tolerant (H-T) or hyperoxia-susceptible (H-S) provide a system for assessing the contribution of mitochondrial bioenergetics to the differential susceptibility to hyperoxia-induced ARDS and for identifying potential therapeutic targets. Due to the system's complexity, interpreting lung mitochondrial bioenergetics data from these rat models requires a computational model to define which processes are altered and how changes influence overall lung tissue bioenergetics. We developed a thermodynamically constrained computational model of lung mitochondrial bioenergetics that extends prior models by incorporating regulation by ions (Ca2+, H+, etc.) and metabolites. The model was parameterized using experimental respirometry data from isolated lung mitochondria of conditioned (H-T and H-S) and control rats with different substrates and ADP concentrations. Model parameterization showed distinct bioenergetic changes. H-S mitochondria had reduced activity in adenine nucleotide translocase (ANT), cytochrome c oxidase (CIV), complex I (CI), and glutamate-oxaloacetate transaminase (GOT). Conversely, H-T mitochondria showed increased activity of ANT and CIV. This supports greater metabolic flexibility in H-T mitochondria compared with H-S. Simulations of ARDS-related changes predicted divergent outcomes. H-S mitochondria underwent rapid failure, with redox collapse, loss of membrane potential, and ATP depletion. H-T mitochondria maintained bioenergetic homeostasis by enhancing electron supply via CI and complex II, with higher CIV activity. This comprehensive computational model provides a framework for identifying critical mitochondrial processes and therapeutic strategies to mitigate mitochondrial dysfunction in ARDS.NEW & NOTEWORTHY We developed a comprehensive computational model of lung mitochondrial bioenergetics that integrates key regulatory mechanisms. The model, parameterized using respirometry data from lung mitochondria of hyperoxia-tolerant and -susceptible rats, identified adenine nucleotide translocase, cytochrome c oxidase, and proton leak as critical determinants of lung mitochondrial bioenergetic homeostasis. Model simulations predict that deficits in these processes drive rapid bioenergetic failure in the lungs of hyperoxia-susceptible rats.
