American journal of physiology. Cell physiology最新文献

Turtle hepatocytes are a non-excitable model for metabolic depression during low-temperature and/or anoxic overwintering conditions. Cytoskeletal structure and mitochondrial distribution are continuously modified in cells, and we hypothesized that metabolic depression would inhibit such processes as cell attachment and spreading and promote withdrawal of cell protrusions and peripheral mitochondria. After developing a methodology for culturing painted turtle hepatocytes, maintenance of cell attachment after a media change, and 2D area, were used as indicators of structural rearrangement and spreading/volume. These were measured after incubating cells at varying temperatures and with or without the inclusion of cyanide (chemical proxy for anoxia). Experiments were performed using cells from 22°C- or 5°C-acclimated turtles. Live-cell imaging was used to monitor the effect of cyanide exposure on distribution of mitochondria. We also acclimated cultured cells from 22°C-acclimated turtles to 4°C in vitro and scored withdrawal of protrusions. Only cells isolated from 5°C-acclimated turtles and incubated at 4°C had reduced attachment to fibronectin substrate, but cyanide exposure had no effect. These cells also had a 24% smaller 2D area than those from 22°C-acclimated turtles. There was no change in mitochondrial distribution during cyanide perfusion. Finally, 4°C acclimation in vitro resulted in withdrawal of protrusions over 14 days. Taken together with the results from cells acclimated to low temperature in vivo, this suggests inhibition of structural rearrangement and protrusion stability by low temperature acclimation, but not cyanide exposure. Our cultured primary hepatocyte system will facilitate further study of the role of structural dynamics in reversible metabolic depression.

Cohesin complex is essential for cell division and for regulating cell type-specific gene expression programs. Mutations in genes encoding the cohesin subunits are associated with hematological malignancies, pre-leukemia and clonal hematopoiesis of indeterminate potential. In this study, we examined how cohesin mutation impacts hematopoiesis using adult zebrafish that carry heterozygous germline nonsense mutation in the cohesin subunit, rad21 (rad21+/-) that is orthologous to human RAD21. Single cell RNA sequencing analyses showed that adult zebrafish harboring rad21+/- mutation exhibit significant transcriptional dysregulation within the whole kidney marrow and have altered erythroid and granulocyte output. Erythroid progenitors were expanded in rad21+/- and erythroid differentiation was altered. The expression profile of several erythroid genes, including gata1a, was dysregulated in rad21+/- erythroid cells. Mature granulocyte population declined in rad21+/-, and the transcriptional program of granulocytes was impaired but granulocytic maturation was maintained. Granulocytes from rad21+/- showed upregulation of stress hematopoiesis factor, cebpb. These findings show that normal rad21 is required to maintain steady erythropoiesis and granulopoiesis in the adult zebrafish marrow.

Muscle disuse has rapid and debilitating effects on muscle mass and overall health, making it an important issue from both scientific and clinical perspectives. However, the myocellular adaptations to muscle disuse are not yet fully understood, particularly those related to the myonuclear permanence hypothesis. Therefore, in this study, we assessed fiber size, number of myonuclei, satellite cells, and capillaries in human gastrocnemius muscle after a period of immobilization following an Achilles tendon rupture. Six physically active patients (5M/1F, 43 {plus minus} 15 years) were recruited to participate after sustaining an acute unilateral Achilles tendon rupture. Muscle biopsies were obtained from the lateral part of the gastrocnemius before and after six weeks of immobilization using a plaster cast and orthosis. Muscle fiber characteristics were analyzed in tissue cross-sections and isolated single fibers using immunofluorescence and high-resolution microscopy. Immobilization did not change muscle fiber type composition nor cross-sectional area of type I or type II fibers, but muscle fiber volume tended to decline by 13% (p=0.077). After immobilization, the volume per myonucleus was significantly reduced by 20% (p=0.008). Myonuclei were not lost in response to immobilization but tended to increase in single fibers and type II fibers. No significant changes were observed for satellite cells or capillaries. Myonuclei were not lost in the gastrocnemius muscle after a prolonged period of immobilization, which may provide support to the myonuclear permanence hypothesis in human muscle. Capillaries remained stable throughout the immobilization period, whereas the response was variable for satellite cells, particularly in type II fibers.

Cancer cachexia affects up to 80% of cancer patients and results in reduced quality of life and survival. We previously demonstrated that the transcriptional repressor Forkhead box P1 (FoxP1) is upregulated in skeletal muscle of cachectic mice and people with cancer, and when overexpressed in skeletal muscle is sufficient to induce pathological features characteristic of cachexia. However, the role of myofiber-derived FoxP1 in both normal muscle physiology and cancer-induced muscle wasting remains largely unexplored. To address this gap, we generated a conditional mouse line with myofiber-specific ablation of FoxP1 (FoxP1^SkmKO) and found that in cancer-free mice, deletion of FoxP1 in skeletal myofibers resulted in increased myofiber size in both males and females, with a significant increase in muscle mass in males. In response to murine KPC pancreatic tumor burden, we found that myofiber-derived FoxP1 is required for cancer-induced muscle wasting and diaphragm muscle weakness in male mice. In summary, our findings identify myofiber-specific FoxP1 as a negative regulator of skeletal muscle with sex-specific differences in the context of cancer.

Sodium-glucose cotransporter 2 inhibitors (SGLT2is), initially developed for type 2 diabetes (T2D) treatment, have demonstrated significant cardiovascular and renal benefits in heart failure (HF) and chronic kidney disease (CKD), irrespective of T2D. This review provides an analysis of the multifaceted mechanisms underlying the cardiorenal benefits of SGLT2i in HF and CKD outside of the T2D context. Eight major aspects of the protective effects of SGLT2i beyond glycemic control are explored: 1) the impact on renal hemodynamics and tubuloglomerular feedback; 2) the natriuretic effects via proximal tubule Na⁺/H⁺ exchanger NHE3 inhibition; 3) the modulation of neurohumoral pathways with evidence of attenuated sympathetic activity; 4) the impact on erythropoiesis, not only in the context of local hypoxia but also systemic inflammation and iron regulation; 5) the uricosuria and mitigation of the hyperuricemic environment in cardiorenal syndromes; 6) the multiorgan metabolic reprogramming including the potential induction of a fasting-like state, improvement in glucose and insulin tolerance, and stimulation of lipolysis and ketogenesis; 7) the vascular endothelial growth factor A (VEGF-A) upregulation and angiogenesis, and 8) the direct cardiac effects. The intricate interplay between renal, neurohumoral, metabolic, and cardiac effects underscores the complexity of SGLT2i actions and provides valuable insights into their therapeutic implications for HF and CKD. Furthermore, this review sets the stage for future research to evaluate the individual contributions of these mechanisms in diverse clinical settings.

Type 2 diabetes (T2D) constitutes a major public health problem, and despite prevention efforts, this pandemic disease is one of the deadliest diseases in the world. In 2022, 6.7 million patients with T2D died prematurely from vascular complications. Indeed, diabetes increases the risk of myocardial infarction or stroke eightfold. The identification of the molecular factors involved in the occurrence of cardiovascular complications and their prevention are therefore major axes. Our hypothesis is that factors brought into play during physiological aging appear prematurely with diabetes progression. Our study focused on the aging of the extracellular matrix (ECM), a major element in the maintenance of vascular homeostasis. We characterized the morphological and functional aspects of aorta, with a focus on the collagen and elastic fibers of diabetic mice aged from 6 mo to nondiabetic mice aged 6 mo and 20 mo. The comparison with the two nondiabetic models (young and old) highlighted an exacerbated activity of proteases, which could explain a disturbance in the collagen accumulation and an excessive degradation of elastic fibers. Moreover, the generation of circulating elastin-derived peptides reflects premature aging of the ECM. These extracellular elements contribute to the appearance of vascular rigidity, often the origin of pathologies such as hypertension and atherosclerosis. In conclusion, we show that diabetic mice aged 6 mo present the same characteristics of ECM wear as those observed in mice aged 20 mo. This accelerated aortic wall remodeling could then explain the early onset of cardiovascular diseases and, therefore, the premature death of patients with T2D.NEW & NOTEWORTHY Aortic elastic fibers of young (6-mo old) individuals with diabetes degrade prematurely and exhibit an appearance like that found in aged (20-mo old) nondiabetic mice. Exacerbated elastolysis and elastin-derived peptide production are characteristic elements, contributing to early aortic wall rigidity and hypertension development. Therefore, limiting this early aging could be a judicious therapeutic approach to reduce cardiovascular complications and premature death in patients with diabetes.

The musculoskeletal system, crucial for movement and support, relies on the delicate balance of connective tissue homeostasis. Maintaining this equilibrium is essential for tissue health and function. There has been increasing evidence in the past decade that shows the circadian clock as a master regulator of extracellular matrix (ECM) homeostasis in several connective tissue clocks. Very recently, exercise has emerged as a significant entrainment factor for cartilage and intervertebral disk circadian rhythms. Understanding the implications of exercise on connective tissue peripheral clocks holds promise for enhancing tissue health and disease prevention. Exercise-induced factors such as heat, glucocorticoid release, mechanical loading, and inter-tissue cross talk may play pivotal roles in entraining the circadian rhythm of connective tissues. This mini review underscores the importance of elucidating the mechanisms through which exercise influences circadian rhythms in connective tissues to optimize ECM homeostasis. Leveraging exercise as a modulator of circadian rhythms in connective tissues may offer novel therapeutic approaches to physical training for preventing musculoskeletal disorders and enhancing recovery.

Although studies have identified characteristics of quiescent satellite cells (SCs), their isolation has been hampered by the fact that the isolation procedures result in the activation of these cells into their rapidly proliferating progeny (myoblasts). Thus, the use of myoblasts for therapeutic (regenerative medicine) or industrial applications (cellular agriculture) has been impeded by the limited proliferative and differentiative capacity of these myogenic progenitors. Here we identify a subpopulation of satellite cells isolated from mouse skeletal muscle using flow cytometry that is highly Pax7-positive, exhibit a very slow proliferation rate (7.7 ± 1.2 days/doubling), and are capable of being maintained in culture for at least 3 mo without a change in phenotype. These cells can be activated from quiescence using a p38 inhibitor or by exposure to freeze-thaw cycles. Once activated, these cells proliferate rapidly (22.7 ± 0.2 h/doubling), have reduced Pax7 expression (threefold decrease in Pax7 fluorescence vs. quiescence), and differentiate into myotubes with a high efficiency. Furthermore, these cells withstand freeze-thawing readily without a significant loss of viability (83.1 ± 2.1% live). The results presented here provide researchers with a method to isolate quiescent satellite cells, allowing for more detailed examinations of the factors affecting satellite cell quiescence/activation and providing a cell source that has a unique potential in the regenerative medicine and cellular agriculture fields.NEW & NOTEWORTHY We provide a method to isolate quiescent satellite cells from skeletal muscle. These cells are highly Pax7-positive, exhibit a very slow proliferation rate, and are capable of being maintained in culture for months without a change in phenotype. The use of these cells by muscle researchers will allow for more detailed examinations of the factors affecting satellite cell quiescence/activation and provide a novel cell source for the regenerative medicine and cellular agriculture fields.

RhoA and its effectors, the transcriptional coactivators myocardin-related transcription factor (MRTF) and serum response factor (SRF), control epithelial phenotype and are indispensable for profibrotic epithelial reprogramming during fibrogenesis. Context-dependent control of RhoA and fibrosis-associated changes in its regulators, however, remain incompletely characterized. We previously identified the guanine nucleotide exchange factor GEF-H1 as a central mediator of RhoA activation in renal tubular cells exposed to inflammatory or fibrotic stimuli. Here we found that GEF-H1 expression and phosphorylation were strongly elevated in two animal models of fibrosis. In the Unilateral Ureteral Obstruction mouse kidney fibrosis model, GEF-H1 was upregulated predominantly in the tubular compartment. GEF-H1 was also elevated and phosphorylated in a rat pulmonary artery banding (PAB) model of right ventricular fibrosis. Prolonged stimulation of LLC-PK₁ tubular cells with tumor necrosis factor (TNF)-α or transforming growth factor (TGF)-β1 increased GEF-H1 expression and activated a luciferase-coupled GEF-H1 promoter. Knockdown and overexpression studies revealed that these effects were mediated by RhoA, cytoskeleton remodeling, and MRTF, indicative of a positive feedback cycle. Indeed, silencing endogenous GEF-H1 attenuated activation of the GEF-H1 promoter. Of importance, inhibition of MRTF using CCG-1423 prevented GEF-H1 upregulation in both animal models. MRTF-dependent increase in GEF-H1 was prevented by inhibition of the transcription factor Sp1, and mutating putative Sp1 binding sites in the GEF-H1 promoter eliminated its MRTF-dependent activation. As the GEF-H1/RhoA axis is key for fibrogenesis, this novel MRTF/Sp1-dependent regulation of GEF-H1 abundance represents a potential target for reducing renal and cardiac fibrosis.NEW & NOTEWORTHY We show that expression of the RhoA regulator GEF-H1 is upregulated in tubular cells exposed to fibrogenic cytokines and in animal models of kidney and heart fibrosis. We identify a pathway wherein GEF-H1/RhoA-dependent MRTF activation through its noncanonical partner Sp1 upregulates GEF-H1. Our data reveal the existence of a positive feedback cycle that enhances Rho signaling through control of both GEF-H1 activation and expression. This feedback loop may play an important role in organ fibrosis.

Intervertebral disk degeneration (IDD) is a significant cause of low back pain, characterized by excessive senescence and apoptosis of nucleus pulposus cells (NPCs). However, the precise mechanisms behind this senescence and apoptosis remain unclear. This study aimed to investigate the role of T-box transcription factor T (Tbxt) in IDD both in vitro and in vivo, using a hydrogen peroxide (H₂O₂)-induced NPCs senescence and apoptosis model, as well as a rat acupuncture IDD model. First, the expression of p16 and cleaved-caspase 3 significantly increased in degenerated human NPCs, accompanied by a decrease in Tbxt expression. Knockdown of Tbxt exacerbated senescence and apoptosis in the H₂O₂-induced NPCs degeneration model. Conversely, upregulation of Tbxt alleviated these effects induced by H₂O₂. Mechanistically, bioinformatic analysis revealed that the direct downstream target genes of Tbxt were highly enriched in autophagy-related pathways, and overexpression of Tbxt significantly activated autophagy in NPCs. Moreover, the administration of the autophagy inhibitor, 3-methyladenine, impeded the impact of Tbxt on the processes of senescence and apoptosis in NPCs. Further investigation revealed that Tbxt enhances autophagy by facilitating the transcription of ATG7 through its interaction with a specific motif within the promoter region. In conclusion, this study suggests that Tbxt mitigates H₂O₂-induced senescence and apoptosis of NPCs by activating ATG7-mediated autophagy.NEW & NOTEWORTHY This study investigates the role of Tbxt in IDD. The results demonstrate that knockdown of Tbxt exacerbates H₂O₂-induced senescence and apoptosis in NPCs and IDD, whereas upregulation of Tbxt significantly protects against IDD both in vivo and in vitro. Mechanistically, in the nucleus, Tbxt enhances the transcription of ATG7, leading to increased expression of ATG7 protein levels. This, in turn, promotes elevated autophagy levels, ultimately alleviating IDD.