High metabolic substrate load induces mitochondrial dysfunction in rat skeletal muscle microvascular endothelial cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Hansen C
PROVIDER: S-EPMC8290479 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:1. Microvascular endothelial cells (MVECs) form a barrier between circulating metabolites, such as adenosine, and the surrounding tissue. We hypothesize that MVECs have a high capacity for the accumulation of nucleosides, such that inhibition of the endothelial nucleoside transporters (NT) would profoundly affect the actions of adenosine in the microvasculature. 2. We assessed the binding of [(3)H]nitrobenzylmercaptopurine riboside (NBMPR), a specific probe for the inhibitor-sensitive subtype of equilibrative NT (es), and the uptake of [(3)H]formycin B (FB), by MVECs isolated from rat skeletal muscle. The cellular expression of equilibrative (ENT1, ENT2, ENT3) and concentrative (CNT1, CNT2, CNT3) NT subtypes was also determined using both qualitative and quantitative polymerase chain reaction techniques. 3. In the absence of Na(+), MVECs accumulated [(3)H]FB with a V(max) of 21+/-1 pmol microl(-1) s(-1). This uptake was mediated equally by es (K(m) 260+/-70 microm) and ei (equilibrative inhibitor-insensitive; K(m) 130+/-20 microm) NTs. 4. A minor component of Na(+)-dependent cif (concentrative inhibitor-insensitive FB transporter)/CNT2-mediated [(3)H]FB uptake (V(i) 0.008+/-0.005 pmol microl(-1) s(-1) at 10 microm) was also observed at room temperature upon inhibition of ENTs with dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine)/NBMPR. 5. MVECs had 122,000 high-affinity (K(d) 0.10 nm) [(3)H]NBMPR binding sites (representing es transporters) per cell. A lower-affinity [(3)H]NBMPR binding component (K(d) 4.8 nm) was also observed that may be related to intracellular es-like proteins. 6. Rat skeletal muscle MVECs express es/ENT1, ei/ENT2, and cif/CNT2 transporters with characteristics typical of rat tissues. This primary cell culture model will enable future studies on factors influencing NT subtype expression, and the consequent effect on adenosine bioactivity, in the microvasculature.
| S-EPMC1575281 | biostudies-literature
Project description:Sepsis is a dysregulated host inflammatory response to infection potentially leading to life-threatening organ dysfunction. The objectives of this study were to determine whether early microvascular dysfunction (MVD) in skeletal muscle can be detected as dynamic changes in microvascular hemoglobin (MVHb) levels using spectroscopy and whether MVD precedes organ histopathology in septic peritonitis. Skeletal muscle of male Sprague-Dawley rats was prepared for intravital microscopy. After intraperitoneal injection of fecal slurry or saline, microscopy and spectroscopy recordings were taken for 6 h. Capillary red blood cell (RBC) dynamics and SO2 were quantified from digitized microscopy frames and MVHb levels were derived from spectroscopy data. Capillary RBC dynamics were significantly decreased by 4 h after peritoneal infection and preceded macrohemodynamic changes. At the same time, low-frequency oscillations in MVHb levels exhibited a significant increase in Power in parts of the muscle and resembled oscillations in RBC dynamics and SO2. After completion of microscopy, tissues were collected. Histopathological alterations were not observed in livers, kidneys, brains, or muscles 6 h after induction of peritonitis. The findings of this study show that, in our rat model of sepsis, MVD occurs before detectable organ histopathology and includes ~ 30-s oscillations in MVHb. Our work highlights MVHb oscillations as one of the indicators of MVD onset and provides a foundation for the use of non-invasive spectroscopy to continuously monitor MVD in septic patients.
| S-EPMC9012880 | biostudies-literature
Project description:Endothelial dysfunction is an early event in coronary microvascular disease. Integrin-linked kinase (ILK) prevents endothelial nitric oxide synthase (eNOS) uncoupling and, thus, endothelial dysfunction. However, the specific role of endothelial ILK in cardiac function remains to be fully elucidated. We hypothesised that endothelial ILK plays a crucial role in maintaining coronary microvascular function and contractile performance in the heart. We generated an endothelial cell-specific ILK conditional knock-out mouse (ecILK cKO) and investigated cardiovascular function. Coronary endothelial ILK deletion significantly impaired cardiac function: ejection fraction, fractional shortening and cardiac output decreased, whilst left ventricle diastolic internal diameter decreased and E/A and E/E' ratios increased, indicating not only systolic but also diastolic dysfunction. The functional data correlated with extensive extracellular matrix remodelling and perivascular fibrosis, indicative of adverse cardiac remodelling. Mice with endothelial ILK deletion suffered early ischaemic-like events with ST elevation and transient increases in cardiac troponins, which correlated with fibrotic remodelling. In addition, ecILK cKO mice exhibited many features of coronary microvascular disease: reduced cardiac perfusion, impaired coronary flow reserve and arterial remodelling with patent epicardial coronary arteries. Moreover, endothelial ILK deletion induced a moderate increase in blood pressure, but the antihypertensive drug Losartan did not affect microvascular remodelling whilst only partially ameliorated fibrotic remodelling. The plasma miRNA profile reveals endothelial-to-mesenchymal transition (endMT) as an upregulated pathway in endothelial ILK conditional KO mice. Our results show that endothelial cells in the microvasculature in endothelial ILK conditional KO mice underwent endMT. Moreover, endothelial cells isolated from these mice and ILK-silenced human microvascular endothelial cells underwent endMT, indicating that decreased endothelial ILK contributes directly to this endothelial phenotype shift. Our results identify ILK as a crucial regulator of microvascular endothelial homeostasis. Endothelial ILK prevents microvascular dysfunction and cardiac remodelling, contributing to the maintenance of the endothelial cell phenotype.
| S-EPMC10348984 | biostudies-literature
Project description:Recent work has established associations between elevated p21, the accumulation of senescent cells, and skeletal muscle dysfunction in mice and humans. Using a mouse model of p21 overexpression (p21OE), we examined if p21 mechanistically contributes to cellular senescence and pathological features in skeletal muscle. We show that p21 induces several core properties of cellular senescence in skeletal muscle, including an altered transcriptome, DNA damage, mitochondrial dysfunction, and the senescence-associated secretory phenotype (SASP). Furthermore, p21OE mice exhibit manifestations of skeletal muscle pathology, such as atrophy, fibrosis, and impaired physical function when compared to age-matched controls. These findings suggest p21 alone is sufficient to drive a cellular senescence program and reveal a novel source of skeletal muscle loss and dysfunction.
| S-EPMC9800630 | biostudies-literature
Project description:Recently, we showed that administration of the angiotensin-converting enzyme inhibitor enalapril to aged rats attenuated muscle strength decline and mitigated apoptosis in the gastrocnemius muscle. The aim of the present study was to investigate possible mechanisms underlying the muscle-protective effects of enalapril. We also sought to discern the effects of enalapril mediated by nitric oxide (NO) from those independent of this signaling molecule. Eighty-seven male Fischer 344?×?Brown Norway rats were randomly assigned to receive enalapril (n?=?23), the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME; n?=?22), enalapril + L-NAME (n?=?19), or placebo (n?=?23) from 24 to 27 months of age. Experiments were performed on the tibialis anterior muscle. Total NOS activity and the expression of neuronal, endothelial, and inducible NOS isoforms (nNOS, eNOS, and iNOS) were determined to investigate the effects of enalapril on NO signaling. Transcript levels of tumor necrosis factor-alpha (TNF-?) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1?) were assessed to explore actions of enalapril on inflammation and mitochondrial biogenesis, respectively. Protein expression of energy-sensing and insulin signaling mediators, including protein kinase B (Akt-1), phosphorylated Akt-1 (pAkt-1), mammalian target of rapamycin (mTOR), AMP-activated protein kinase subunit alpha (AMPK?), phosphorylated AMPK? (pAMPK?), and the glucose transporter GLUT-4, was also determined. Finally, the generation of hydrogen peroxide (H2O2) was quantified in subsarcolemmal (SSM) and intermyofibrillar (IFM) mitochondria. Enalapril increased total NOS activity, which was prevented by L-NAME co-administration. eNOS protein content was enhanced by enalapril, but not by enalapril + L-NAME. Gene expression of iNOS was down-regulated by enalapril either alone or in combination with L-NAME. In contrast, protein levels of nNOS were unaltered by treatments. The mRNA abundance of TNF-? was reduced by enalapril relative to placebo, with no differences among any other group. PCG-1? gene expression was unaffected by enalapril and lowered by enalapril + L-NAME. No differences in protein expression of Akt-1, pAkt-1, AMPK?, pAMPK?, or GLUT-4 were detected among groups. However, mTOR protein levels were increased by enalapril compared with placebo. Finally, all treatment groups displayed reduced SSM, but not IFM H2O2 production relative to placebo. Our data indicate that enalapril induces a number of metabolic adaptations in aged skeletal muscle. These effects result from the concerted modulation of NO and angiotensin II signaling, rather than from a dichotomous action of enalapril on the two pathways. Muscle protection by enalapril administered late in life appears to be primarily mediated by mitigation of oxidative stress and pro-inflammatory signaling.
| S-EPMC3705103 | biostudies-literature
Project description:Growth differentiation factor 11 (GDF11), a TGF-beta superfamily member, is highly homologous to myostatin and essential for embryonic patterning and organogenesis. Reports of GDF11 effects on adult tissues are conflicting, with some describing anti-aging and pro-regenerative activities on the heart and skeletal muscle while others opposite or no effects. Herein, we sought to determine the in vivo cardiac and skeletal muscle effects of excess GDF11. Mice were injected with GDF11 secreting cells, an identical model to that used to initially identify the in vivo effects of myostatin. GDF11 exposure in mice induced whole body wasting and profound loss of function in cardiac and skeletal muscle over a 14-day period. Loss of cardiac mass preceded skeletal muscle loss. Cardiac histologic and echocardiographic evaluation demonstrated loss of ventricular muscle wall thickness, decreased cardiomyocyte size, and decreased cardiac function 10 days following initiation of GDF11 exposure. Changes in skeletal muscle after GDF11 exposure were manifest at day 13 and were associated with wasting, decreased fiber size, and reduced strength. Changes in cardiomyocytes and skeletal muscle fibers were associated with activation of SMAD2, the ubiquitin-proteasome pathway and autophagy. Thus, GDF11 over administration in vivo results in cardiac and skeletal muscle loss, dysfunction, and death. Here, serum levels of GDF11 by Western blotting were 1.5-fold increased over controls. Although GDF11 effects in vivo are likely dose, route, and duration dependent, its physiologic changes are similar to myostatin and other Activin receptors ligands. These data support that GDF11, like its other closely related TGF-beta family members, induces loss of cardiac and skeletal muscle mass and function.
| S-EPMC5833306 | biostudies-literature
Project description:Skeletal muscle weakness is a prominent feature in patients with rheumatoid arthritis (RA). In this study, we investigated whether neuromuscular electrical stimulation (NMES) training protects against skeletal muscle dysfunction in rats with adjuvant-induced arthritis (AIA). AIA was produced by intraarticular injection of complete Freund's adjuvant into the knees of Wistar rats. For NMES training, dorsiflexor muscles were stimulated via a surface electrode (0.5 ms pulse, 50 Hz, 2 s on/4 s off). NMES training was performed every other day for three weeks and consisted of three sets produced at three min intervals. In each set, the electrical current was set to achieve 60% of the initial maximum isometric torque and the current was progressively increased to maintain this torque; stimulation was stopped when the 60% torque could no longer be maintained. After the intervention period, extensor digitorum longus (EDL) muscles were excised and used for physiological and biochemical analyses. There was a reduction in specific force production (i.e. force per cross-sectional area) in AIA EDL muscles, which was accompanied by aggregation of the myofibrillar proteins actin and desmin. Moreover, the protein expressions of the pro-oxidative enzymes NADPH oxidase, neuronal nitric oxide synthase, p62, and the ratio of the autophagosome marker LC3bII/LC3bI were increased in AIA EDL muscles. NMES training prevented all these AIA-induced alterations. The present data suggest that NMES training prevents AIA-induced skeletal muscle weakness presumably by counteracting the formation of actin and desmin aggregates. Thus, NMES training can be an effective treatment for muscle dysfunction in patients with RA.
| S-EPMC5479592 | biostudies-literature
Project description:Peripheral leukocytes induce blood-brain barrier (BBB) dysfunction through the release of cytotoxic mediators. These include hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system of activated phagocytes. HOCl targets the endogenous pool of ether phospholipids (plasmalogens) generating chlorinated inflammatory mediators like e.g. 2-chlorohexadecanal and its conversion product 2-chlorohexadecanoic acid (2-ClHA). In the cerebrovasculature these compounds inflict damage to brain microvascular endothelial cells (BMVEC) that form the morphological basis of the BBB. To follow subcellular trafficking of 2-ClHA we synthesized a 'clickable' alkyne derivative (2-ClHyA) that phenocopied the biological activity of the parent compound. Confocal and superresolution structured illumination microscopy revealed accumulation of 2-ClHyA in the endoplasmic reticulum (ER) and mitochondria of human BMVEC (hCMEC/D3 cell line). 2-ClHA and its alkyne analogue interfered with protein palmitoylation, induced ER-stress markers, reduced the ER ATP content, and activated transcription and secretion of interleukin (IL)-6 as well as IL-8. 2-ClHA disrupted the mitochondrial membrane potential and induced procaspase-3 and PARP cleavage. The protein kinase R-like ER kinase (PERK) inhibitor GSK2606414 suppressed 2-ClHA-mediated activating transcription factor 4 synthesis and IL-6/8 secretion, but showed no effect on endothelial barrier dysfunction and cleavage of procaspase-3. Our data indicate that 2-ClHA induces potent lipotoxic responses in brain endothelial cells and could have implications in inflammation-induced BBB dysfunction.
| S-EPMC5975063 | biostudies-literature
Project description:AimTo uncover sex-related microvascular abnormalities that underlie the early presentation of reduced perfusion in leg skeletal muscle in a type II rat model of diabetic cardiomyopathy.Methods and resultsDiabetes was induced using a non-obese, diet-based, low-dose streptozotocin model in adult female (18 diabetic, 9 control) and male rats (29 diabetic, 11 control). Time-course monitoring over 12 months following diabetes induction was performed using echocardiography, treadmill exercise, photoacoustic imaging, flow-mediated dilation (FMD), histopathology, and immunohistochemistry. Diabetic rats maintained normal weights. Hypertension appeared late in both diabetic males (7 months) and females (10 months), while only diabetic males had elevated cholesterol (7 months). On echocardiography, all diabetic animals maintained normal ejection fraction and exhibited diastolic dysfunction, mild systolic dysfunction, and a slightly enlarged left ventricle. Exercise tolerance declined progressively and early in males (4 months), later in females (8 months); FMD showed lower baseline femoral arterial flow but unchanged reactivity in both sexes (5 months); and photoacoustic imaging showed lower tissue oxygen saturation in the legs of diabetic males (4 months) and diabetic females (10 months). Myocardial perfusion was normal in both sexes. Histopathology at the final timepoint of Month 10 (males) and Month 12 (females) revealed that myocardial microvasculature was normal in both vessel density and structure, thus explaining normal perfusion on imaging. However, leg muscle microvasculature exhibited perivascular smooth muscle thickening around small arterioles in diabetic females and around large arterioles in diabetic males, explaining the depressed readings on photoacoustic and FMD. Histology also confirmed the absence of commonly reported HFpEF markers, including microvessel rarefaction, myocardial fibrosis, and left ventricular hypertrophy.ConclusionExercise intolerance manifesting early in the progression of diabetic cardiomyopathy can be attributed to decreased perfusion to the leg skeletal muscle due to perivascular smooth muscle thickening around small arterioles in females and large arterioles in males. This microvascular abnormality was absent in the myocardium, where perfusion levels remained normal throughout the study. We conclude that although skeletal muscle microvascular dysfunction of the vasculature presents at different levels depending on sex, it consistently presents early in both sexes prior to overt cardiac changes such as rarefaction, fibrosis, or hypertrophy.
| S-EPMC9157579 | biostudies-literature
Project description:BDNF (brain-derived neurotrophic factor) is present in skeletal muscle, controlling muscular metabolism, strength and regeneration processes. However, there is no consensus on BDNF cellular source. Furthermore, while endothelial tissue expresses BDNF in large amount, whether endothelial cells inside muscle expressed BDNF has never been explored. The aim of the present study was to provide a comprehensive analysis of BDNF localization in rat skeletal muscle. Cellular localization of BDNF and activated Tropomyosin-related kinase B (TrkB) receptors was studied by immunohistochemical analysis on soleus (SOL) and gastrocnemius (GAS). BDNF and activated TrkB levels were also measured in muscle homogenates using Western blot analysis and/or Elisa tests. The results revealed BDNF immunostaining in all cell types examined with a prominent staining in endothelial cells and a stronger staining in type II than type I muscular fibers. Endothelial cells but not other cells displayed easily detectable activated TrkB receptor expression. Levels of BDNF and activated TrkB receptors were higher in SOL than GAS. In conclusion, endothelial cells are an important and still unexplored source of BDNF present in skeletal muscle. Endothelial BDNF expression likely explains why oxidative muscle exhibits higher BDNF levels than glycolytic muscle despite higher the BDNF expression by type II fibers.
| S-EPMC8748777 | biostudies-literature