Isolated Muscle Cells Research Articles

Actin-driven nanotopography promotes stable integrin adhesion formation in developing tissue

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerization-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MASs). Super-resolution microscopy reveals that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking shows that, during MAS development, integrins become immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerization-driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

Purification and Characterization of a Novel Factor of Crotoxin Inter-CRO (V-1), a New Phospholipase A2 Isoform from Crotalus durissus collilineatus Snake Venom Using an In Vitro Neuromuscular Preparation

The fractionation of Crotalus durissus collilineatus whole venom through an HPLC chromatographic method enabled the purification of a new V-1 neurotoxin. Inter-CRO (V-1) presents similarity in its primary structure to crotoxin B (CB), suggesting another isoform of this toxin. The aim of this study was to compare V-1 to the crotoxin complex (CA/CB) and CB to elucidate aspects related to its functionality. The homogeneity of the purified protein was confirmed with a molecular mass of 1425.45 Da, further verified by mass spectrometry. The sequence of the protein showed high similarity to other viperid snake venom PLA2 proteins. The results of this study report that V-1 is an uncharacterized novel toxin with different biological activities from CB. V-1 maintained catalytic activity but presented neurotoxic activity as observed by the 2.5-fold increase in twitch tension record compared to control values on isolated muscle cells.

The Impact of Varying Caffeine‐Induced Contractile Activity on Metabolic Enzyme Activity

Exercise intensity is widely known to differentially affect skeletal muscle metabolic approaches, with a general understanding that short duration, high intensity exercise is associated with an increased reliance on anaerobic glycolysis, while long duration, low to moderate intensity exercise is associated with a greater reliance on aerobic processes. In cell culture, caffeine is a commonly used method to pharmaceutically stimulate contractile activity in skeletal muscle cells; upon exposure to caffeine, ryanodine receptors are stimulated to release calcium, thereby allowing crossbridge cycling and subsequent contractile activity to occur. This technique for exercising muscle cells in culture offers a controlled, experimental platform to study the effect of contractile activity on isolated muscle cells; presumably, varying doses of caffeine should mimic varying levels of exercise intensity, though the degree to which caffeine dose impacts metabolic shifts has yet to be fully elucidated. Here, we exposed C2C12 cells, an immortalized line of mouse skeletal muscle cells, to varying levels of caffeine supplementation throughout myotube differentiation in an attempt to recapitulate metabolic signatures consistent with varying exercise intensities. Following standard proliferation protocols, mature myotubes were differentiated for 7 days. On differentiation days 4‐7, experimental cohorts of cells were exposed to either a low dosage (10mM) or a high dosage (40 mM) of caffeine‐supplemented differentiation media for 20 minutes daily. All cells were harvested for protein on differentiation day seven. Protein concentration across cohorts was measured using a Bradford protein assay. Metabolic changes were characterized by running functional enzyme assays for lactate dehydrogenase (LDH) and citrate synthase (CS) to compare anaerobic and aerobic changes, respectively. Preliminary data suggest that, relative to controls, cells exposed to 10mM caffeine have a decrease in LDH activity, and a trend toward increased CS activity. Conversely, cells exposed to 40mM caffeine show no change in LDH and a trend toward decreased CS activity. Collectively, these preliminary data suggest that C2C12 cells exposed to low doses of caffeine simulating low intensity exercise may have less reliance on anaerobic metabolism with a potential greater reliance on aerobic metabolism, while those exposed to high doses of caffeine simulating high intensity exercise may have a lesser reliance on aerobic metabolism. While more research needs to be done to characterize the precise impact of varying caffeine dosages on C2C12 cells, these initial findings are consistent with previously established trends in exercise intensity and metabolism.

Abstract 10743: Overexpression of GLUT4 Induces Pulmonary Hypertension

Introduction: Pulmonary arterial hypertension (PAH) induces significantly elevated glucose uptake in the patient lungs and right ventricles (RV), which correlates with the severity of the disease. However, the mechanisms responsible for the increased glucose uptake and its contribution to the PAH pathobiology remain unclear. We discovered that Sugen/hypoxia-induced PAH is associated with a marked overexpression of glucose transporter, GLUT4, starting from the early stage of the disease. Hypothesis: We investigated the role of GLUT4 overexpression in triggering PAH and evaluated the downstream mechanisms responsible for the PAH phenotype. Methods: In this study, we used the mouse model overexpressing human GLUT4 (hGLUT4). RV hemodynamics and function were assessed in hGLUT4 mice treated or not with IMPA1 inhibitor (L690,330, i.p., 22 mg/kg BW, daily for 6 weeks). Isolated pulmonary artery smooth muscle cells (PASMC) were used to perform glucose uptake assays and bioenergetics measurements. Results: hGLUT4 mice showed significantly increased RV systolic pressure (RVSP, 28.98±0.72, p<0.001, N=7-9) and RV hypertrophy (0.319±0.006, p<0.0001, N=7-9) compared to WTs and non-transgenic controls. GLUT4 overexpression significantly increased the glucose uptake in PASMC (~30% increase to control, p<0.01) and induced severe mitochondrial dysfunction in unstimulated and insulin-stimulated cells. Previously, we discovered that an increase in glucose uptake elevates the levels of glucose 6-phosphate (G6P) and activates the G6P metabolizing enzyme, myo-inositol monophosphatase 1 (IMPA1). We confirmed the increased expression of IMPA1, a critical activator of the inositol synthesis and inositol-dependent proliferative pathways in hGLUT4 mice. Notably, treatment with the IMPA1 inhibitor completely reversed PAH by reducing RVSP and RV hypertrophy. Conclusions: We conclude that increased GLUT4 expression is sufficient to induce spontaneous PAH through increased glucose uptake, initiation of mitochondrial dysfunction, and upregulation of IMPA1. This discovery introduces a novel potential target, IMPA1, that connects dysregulated glucose metabolism with activation of the proliferative signaling.

Principal protocols for the processing of cultured meat.

The purpose of this study was to establish a basic principal procedure for the processing of cultured meat. The first stage involved isolating satellite cells from the desired muscle of an animal using enzymatic digestion (i.e., by using proteases, collagenases, and pronases). The second stage involved culturing the isolated muscle satellite cells in a growth medium containing fetal bovine serum and penicillin/streptomycin with growth factors for an optimal period of time. The second stage involved a basic method for the isolated muscle cells to proliferate while sub-culturing to further induce differentiation in gelatin-coated culture dishes with the general culture medium. The third stage involved the induction of differentiation of muscle satellite cells or formation of myotubes using myogenic medium. Lastly, the fourth stage involved the identification of cell differentiation or myotube formation (myogenesis) using fluorescent dyes. Moreover, the principle of these protocols can be applied to perform primary culture of animal cells. This study will assist beginners with the technical aspects of culturing meat (isolation, cultivation, and differentiation of muscle satellite cells as well as identification of myotube formation for myogenesis).

Transcriptional analysis of muscle tissue and isolated satellite cells in spastic cerebral palsy.

To analyze transcriptomes from muscle tissue and cells to improve our understanding of differences in gene expression and molecular function in cerebral palsy (CP) muscle. In this case-control study, eight participants with CP (five males, three females; mean [SD] age 14y 2mo [1y 8mo]) and 11 comparison individuals (eight males, three females; mean [SD] age 14y 0mo [2y 6mo]) were enrolled after informed consent/assent and skeletal muscle was obtained during surgery. RNA was extracted from tissue and from primary satellite cells grown to form myotubes invitro. RNA sequencing data were analyzed using validated informatics pipelines. Analysis identified expression of 6308 genes in the tissue samples and 7459 in the cultured cells. Significant differential expression between CP and control was identified in 87 genes in the tissue and 90 genes in isolated satellite cell-derived myotube cultures. Both tissue and cell analyses identified differential expression of genes associated with muscle development and multiple pathways of interest. What this paper adds Expression differences were found in muscle tissue and in isolated muscle cells. There was low variability in expression among cells isolated from different muscles. Expression differences suggest complex functional alterations in spastic cerebral palsy.

Ascorbic Acid Supplementation Improves Skeletal Muscle Growth in Pacu (Piaractus mesopotamicus) Juveniles: In Vivo and In Vitro Studies.

In fish, fasting leads to loss of muscle mass. This condition triggers oxidative stress, and therefore, antioxidants can be an alternative to muscle recovery. We investigated the effects of antioxidant ascorbic acid (AA) on the morphology, antioxidant enzyme activity, and gene expression in the skeletal muscle of pacu (Piaractus mesopotamicus) following fasting, using in vitro and in vivo strategies. Isolated muscle cells of the pacu were subjected to 72 h of nutrient restriction, followed by 24 h of incubation with nutrients or nutrients and AA (200 µM). Fish were fasted for 15 days, followed by 6 h and 15 and 30 days of refeeding with 100, 200, and 400 mg/kg of AA supplementation. AA addition increased cell diameter and the expression of anabolic and cell proliferation genes in vitro. In vivo, 400 mg/kg of AA increased anabolic and proliferative genes expression at 6 h of refeeding, the fiber diameter and the expression of genes related to cell proliferation at 15 days, and the expression of catabolic and oxidative metabolism genes at 30 days. Catalase activity remained low in the higher supplementation group. In conclusion, AA directly affected the isolated muscle cells, and the higher AA supplementation positively influenced muscle growth after fasting.

Decreased smooth muscle function, peristaltic activity, and gastrointestinal transit in dystrophic (mdx) mice.

Duchenne muscular dystrophy (DMD) is characterized by the lack of dystrophin in skeletal, cardiac, and smooth muscle. Slow colonic transit and constipation are common in DMD patients and animal models of DMD. However, the cause of this hypocontractility and the expression of contractile proteins in smooth muscle are unknown. The aim of the study was to investigate the expression of contractile proteins in the colonic smooth muscle and the function of the colon in control and mdx mice. Muscle contraction was measured in muscle strips and isolated muscle cells. Peristaltic activity was measured in ex vivo preparations by spatiotemporal mapping, and gastrointestinal (GI) transit in vivo was measured by the distribution of fluorescent marker along the intestine and colon. mRNA expression of contractile proteins smoothelin, caldesmon, calponin, and tropomyosin was measured by qRT-PCR. Expression of mRNA for contractile proteins was decreased in colonic smooth muscle of mdx mice compared with control. Contraction in response to acetylcholine and KCl was decreased in colonic muscle strips and in isolated muscle cells of mdx mice. Distension of ex vivo colons with Krebs buffer induced peristalsis in both control and mdx mice; however, significantly fewer full peristaltic waves were recorded in the colons of mdx mice. GI transit was also inhibited in mdx mice. The data indicate that the lack of dystrophin causes decrease in colonic smooth muscle contractility, peristalsis, and GI transit and provides the basis for analysis of mechanisms involved in smooth muscle dysfunction in DMD.

Comprehensive analysis of skeletal muscle- and bone-derived mesenchymal stem/stromal cells in patients with osteoarthritis and femoral neck fracture

BackgroundMesenchymal stem/stromal cells (MSCs) can replenish the aged cells of the musculoskeletal system in adult life. Stem cell exhaustion and decrease in their regenerative potential have been suggested to be hallmarks of aging. Here, we investigated whether muscle- and bone-derived MSCs of patients with osteoarthritis and osteoporosis are affected by this exhaustion, compared to healthy donors.MethodsPatients with primary osteoarthritis, femoral neck fractures due to osteoporosis, and healthy donors (controls) were included. MSCs were isolated from the skeletal muscle and subchondral bone from each patient and compared using ex vivo and in vitro analyses, including immunophenotyping, colony-forming unit fibroblast assays, growth kinetics, cell senescence, multilineage potential, and MSC marker gene expression profiling.ResultsFreshly isolated cells from muscle from patients with osteoarthritis showed a lower proportion of CD45/CD19/CD14/CD34-negative cells compared to patients with osteoporosis and healthy donors. Freshly isolated muscle cells from patients with osteoarthritis and osteoporosis also showed higher clonogenicity compared to healthy donors. MSCs from both tissues of osteoarthritis patients showed significantly reduced osteogenesis and MSCs from the bone also reduced adipogenesis. Chondrogenic pellet diameter was reduced in bone-derived MSCs from both patient groups compared to healthy donors. A significant positive correlation was observed between adipogenesis and CD271 expression in muscle-derived MSCs. CD73 was significantly lower in bone-derived MSCs from osteoarthritis patients, compared to osteoporosis patients. Gene expression profiling showed significantly lower expression of MSC marker gene leptin receptor, LEPR, previously identified as the major source of the bone and adipocytes in the adult bone marrow, in bone-derived MSCs from patients with osteoarthritis in comparison with osteoporotic patients and healthy donors.ConclusionsOur results show deficient ex vivo and in vitro properties of both skeletal muscle- and bone-derived MSCs in osteoarthritis and osteoporosis patients, compared to healthy donors. In bone-derived MSCs from patients with osteoarthritis, we also identified a lower expression of the leptin receptor, a marker of MSCs that present a major source of MSCs in the adult bone marrow. This suggests that exhaustion of skeletal muscle- and bone-derived MSCs is a hallmark of osteoarthritis and osteoporosis, which defines the need for further clinical trials of stem cell transplantation in these patients.

Sex‐Differences in Gastric Smooth Muscle Function: Role of G Protein‐Coupled Estrogen Receptor

The regulation of gastrointestinal (GI) motility by estrogen and its receptors has been a topic of great interest owing to sexual dimorphism that exists in motility disorders. Scientific and clinical evidence establishes the fact of GI motility disturbances during estrous cycle but, there is a gap in the knowledge and understanding of estrogen and its receptors role in GI motility physiology during these cycles. Estrogen activates classical nuclear receptors ERα, ERβ and atypical 7‐transmembrane G protein‐coupled estrogen receptor (GPER or GPR30) to initiate both genomic and non‐genomic effects. GPER is involved in the several rapid cellular signaling events. The pleiotropic effects manifested by GPER action include regulation of body weight, glucose homeostasis, anti‐inflammation, anti‐nociception, and vascular muscle relaxation. Ablation of GPER leads to increase in mean arterial pressure in female mice, whereas treatment with GPER‐specific agonist G‐1 lowers arterial blood pressure in hypertensive and ovariectomized female rats. The role of GPER in mediating estrogen effects on GI smooth muscle is unknown.AimTo understand the role of GPER in gastric smooth muscle function in male and female mice.MethodsMuscle strips and muscle cells were isolated from the stomachs of control and GPER knockout mice. Expression of GPER was analyzed by qRT‐PCR and western blot. cAMP levels in response to estrogen and G‐1 was measured by radioimmunoassay. Relaxation was measured in organ bath studies in muscle strips and by scanning micrometry in isolated muscle cells.ResultsExpression of GPER (mRNA and protein) in the gastric smooth muscle was higher in female mice compared to male mice. 17β‐estradiol and G‐1 caused an increase in cAMP levels and inhibited acetylcholine‐induced muscle contraction (i.e., caused muscle relaxation) in gastric muscle strips and isolated muscle cells from both female and male mice, however, the increase in cAMP levels and the amount of relaxation was significantly greater in gastric muscle from female mice compared to male mice. The effect of 17β‐estradiol and G‐1 on muscle relaxation was concentration‐dependent. Relaxation was blocked by the GPER specific antagonist G15 (10 μM) in gastric muscle from control mice and absent in gastric muscle from GPER knockout mice. Relaxation was higher in proestrus and estrus phases compared to diestrus phase in female mice and this is consistent with higher expression of GPER in proestrus and estrus phases compared to diestrus phase.ConclusionActivation of GPER causes relaxation via cAMP/PKA pathway in gastric smooth muscle from female and male mice. Greater relaxation in gastric smooth muscle from female mice is due to higher expression of GPER in female mice compared to male mice. Pilot studies suggests that higher expression of GPER in the stomach of female mice is due to differential regulation of GPER expression by epigenetic mechanisms.Support or Funding InformationSupported by grants from VETAR (SM); IACUC approval AD10001201

CREB depletion in smooth muscle cells promotes medial thickening, adventitial fibrosis and elicits pulmonary hypertension.

Levels of the cAMP-responsive transcription factor, CREB, are reduced in medial smooth muscle cells in remodeled pulmonary arteries from hypertensive calves and rats with chronic hypoxia-induced pulmonary hypertension. Here, we show that chronic hypoxia fails to promote CREB depletion in pulmonary artery smooth muscle cells or elicit significant remodeling of the pulmonary arteries in mice, suggesting that sustained CREB expression prevents hypoxia-induced pulmonary artery remodeling. This hypothesis was tested by generating mice, in which CREB was ablated in smooth muscle cells. Loss of CREB in smooth muscle cells stimulated pulmonary artery thickening, right ventricular hypertrophy, profound adventitial collagen deposition, recruitment of myeloid cells to the adventitia, and elevated right ventricular systolic pressure without exposure to chronic hypoxia. Isolated murine CREB-null smooth muscle cells exhibited serum-independent proliferation and hypertrophy in vitro and medium conditioned by CREB-null smooth muscle cells stimulated proliferation and expression of extracellular matrix proteins by adventitial fibroblasts. We conclude that CREB governs the pathologic switch from homeostatic, quiescent smooth muscle cells to proliferative, synthetic cells that drive arterial remodeling contributing to the development or pulmonary hypertension.

Branched Short-Chain Fatty Acid Isovaleric Acid Causes Colonic Smooth Muscle Relaxation via cAMP/PKA Pathway.

Isovaleric acid (IVA) is a 5-carbon branched-chain fatty acid present in fermented foods and produced in the colon by bacterial fermentation of leucine. We previously reported that the shorter, straight-chain fatty acids acetate, propionate and butyrate differentially affect colonic motility; however, the effect of branched-chain fatty acids on gut smooth muscle and motility is unknown. To determine the effect of IVA on contractility of colonic smooth muscle. Murine colonic segments were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with acetylcholine (ACh), and the effects of IVA on ACh-induced contraction were measured in the absence and presence of tetrodotoxin (TTx) or inhibitors of nitric oxide synthase [L-N-nitroarginine (L-NNA)] or adenylate cyclase (SQ22536). The effect of IVA on ACh-induced contraction was also measured in isolated muscle cells in the presence or absence of SQ22536 or protein kinase A (PKA) inhibitor (H-89). Direct activation of PKA was measured in isolated muscle cells. In colonic segments, ACh-induced contraction was inhibited by IVA in a concentration-dependent fashion; the IVA response was not affected by TTx or L-NNA but inhibited by SQ22536. Similarly, in isolated colonic muscle cells, ACh-induced contraction was inhibited by IVA in a concentration-dependent fashion and the effect blocked by SQ22536 and H-89. IVA also increased PKA activity in isolated smooth muscle cells. The branched-chain fatty acid IVA acts directly on colonic smooth muscle and causes muscle relaxation via the PKA pathway.

Identification of expression and function of the glucagon-like peptide-1 receptor in colonic smooth muscle

The insulinotropic effects of the incretin hormone, glucagon-like peptide-1 (GLP-1) are mediated via GLP-1 receptors (GLP-1R) present on pancreatic β cells. GLP-1 causes a decrease in the motility of stomach and intestine which involves both central and peripheral nervous systems. The expression and function of GLP-1R in gastrointestinal smooth muscle, however, are not clear. Muscle strips and isolated muscle cells were prepared from mouse colon and the effect of GLP-1(7–36) amide on acetylcholine (ACh)-induced contraction was measured. Muscle cells in culture were used to identify the expression of GLP-1R and the signaling pathways activated by GLP-1(7–36) amide. GLP-1R was expressed in the mucosal and non-mucosal tissue preparations derived from colon, and in smooth muscle cell cultures devoid of other cells such as enteric neurons. In colonic muscle strips, the addition of GLP-1(7–36) amide caused dose-dependent inhibition of acetylcholine-induced contractions. The effect of GLP-1(7–36) amide was partly inhibited by the neuronal blocker tetrodotoxin and nitric oxide (NO) synthase inhibitor l-NNA suggesting both NO-dependent neural and NO-independent direct effects on smooth muscle. In isolated colonic smooth muscle cells, GLP-1(7–36) amide caused an increase in Gαs activity, cAMP levels, and PKA activity, and inhibited ACh-induced contraction. The effect of GLP-1(7–36) amide on Gαs activity and cAMP levels was blocked by NF449, an inhibitor of Gαs, and the effect of GLP-1(7–36) amide on contraction was blocked by NF449 and myristoylated PKI, an inhibitor of PKA. We conclude that colonic smooth muscle cells express GLP-1R, and GLP-1(7–36) amide inhibits acetylcholine-induced contraction via GLP-1R coupled to the Gαs/cAMP/PKA pathway.

Inhibition of RhoA/Rho kinase pathway and smooth muscle contraction by hydrogen sulfide.

Hydrogen sulfide (H2S) plays an important role in smooth muscle relaxation. Here, we investigated the expression of enzymes in H2S synthesis and the mechanism regulating colonic smooth muscle function by H2S. Expression of cystathionine‐γ‐lyase (CSE), but not cystathionine‐β‐synthase (CBS), was identified in the colonic smooth muscle of rabbit, mouse, and human. Carbachol (CCh)‐induced contraction in rabbit muscle strips and isolated muscle cells was inhibited by l‐cysteine (substrate of CSE) and NaHS (an exogenous H2S donor) in a concentration‐dependent fashion. H2S induced S‐sulfhydration of RhoA that was associated with inhibition of RhoA activity. CCh‐induced Rho kinase activity also was inhibited by l‐cysteine and NaHS in a concentration‐dependent fashion. Inhibition of CCh‐induced contraction by l‐cysteine was blocked by the CSE inhibitor, dl‐propargylglycine (DL‐PPG) in dispersed muscle cells. Inhibition of CCh‐induced Rho kinase activity by l‐cysteine was blocked by CSE siRNA in cultured cells and DL‐PPG in dispersed muscle cells. Stimulation of Rho kinase activity and muscle contraction in response to CCh was also inhibited by l‐cysteine or NaHS in colonic muscle cells from mouse and human. Collectively, our studies identified the expression of CSE in colonic smooth muscle and determined that sulfhydration of RhoA by H2S leads to inhibition of RhoA and Rho kinase activities and muscle contraction. The mechanism identified may provide novel therapeutic approaches to mitigate gastrointestinal motility disorders.

Application based on the Canny edge detection algorithm for recording contractions of isolated cardiac myocytes

Background and objectiveThe isolated cardiomyocyte preparation is amenable to several experimental approaches not suitable to the myocardial tissue, which has allowed the gain of important information on the pathophysiology of the cardiac muscle. Thus, the development of techniques for functional studies in this preparation is important. The goal of the present study was to develop a computer program to extract contraction traces generated by cyclic cell shortening from cardiomyocyte video image files. MethodsThe Canny algorithm, widely used for computer vision, was implemented for cell edge recognition and continuous tracking, so that changes in cardiomyocyte length could be monitored. The program was applied to demonstrate the effect of classical inotropic maneuvers on contraction parameters, as well as to assess the development of spontaneous activity in response to defibrillator-like electrical shocks in rat isolated cardiomyocytes. ResultsThe method resulted in successful monitoring of variations in cell length during both electrically-triggered and post-shock spontaneous contractions, of which the rate was significantly related to shock strength. ConclusionsThe proposed approach might be useful for analysis of contractile activity of isolated muscle cells, and allows detection of even the typically low-amplitude noisy spontaneous contractile events. Additionally, the experimental data suggest that the rate of spontaneous contraction could be used as an index of shock-induced electrical membrane damage.

Targeting Heat Shock Proteins Mitigates Ventilator Induced Diaphragm Muscle Dysfunction in an Age-Dependent Manner.

Intensive care unit (ICU) patients are often overtly subjected to mechanical ventilation and immobilization, which leads to impaired limb and respiratory muscle function. The latter, termed ventilator-induced diaphragm dysfunction (VIDD) has recently been related to compromised heat shock protein (Hsp) activation. The administration of a pharmacological drug BGP-15 acting as a Hsp chaperone co-inducer has been found to partially alleviate VIDD in young rats. Considering that the mean age in the ICU is increasing, we aimed to explore whether the beneficial functional effects are also present in old rats. For that, we exposed young (7–8 months) and old (28–32 months) rats to 5-day controlled mechanical ventilation and immobilization with or without systemic BGP-15 administration. We then dissected diaphragm muscles, membrane–permeabilized bundles and evaluated the contractile function at single fiber level. Results confirmed that administration of BGP-15 restored the force-generating capacity of isolated muscle cells from young rats in conjunction with an increased expression of Hsp72. On the other hand, our results highlighted that old rats did not positively respond to the BGP-15 treatment. Therefore, it is of crucial importance to comprehend in more depth the effect of VIDD on diaphragm function and ascertain any further age-related differences.

Lipopolysaccharide Disrupts Mitochondrial Physiology in Skeletal Muscle via Disparate Effects on Sphingolipid Metabolism.

ABSTRACTLipopolysaccharides (LPS) are prevalent pathogenic molecules that are found within tissues and blood. Elevated circulating LPS is a feature of obesity and sepsis, both of which are associated with mitochondrial abnormalities that are key pathological features of LPS excess. However, the mechanism of LPS-induced mitochondrial alterations remains poorly understood. Herein we demonstrate the necessity of sphingolipid accrual in mediating altered mitochondrial physiology in skeletal muscle following LPS exposure. In particular, we found LPS elicited disparate effects on the sphingolipids dihydroceramides (DhCer) and ceramides (Cer) in both cultured myotubes and in muscle of LPS-injected mice. Although LPS-treated myotubes had reduced DhCer and increased Cer as well as increased mitochondrial respiration, muscle from LPS-injected mice manifested a reverse trend, namely elevated DhCer, but reduced Cer as well as reduced mitochondrial respiration. In addition, we found that LPS treatment caused mitochondrial fission, likely via dynamin-related protein 1, and increased oxidative stress. However, inhibition of de novo sphingolipid biosynthesis via myriocin protected normal mitochondrial function in spite of LPS, but inhibition of DhCer desaturase 1, which increases DhCer, but not Cer, exacerbated mitochondrial respiration with LPS. In an attempt to reconcile the incongruent effects of LPS in isolated muscle cells and whole muscle tissue, we incubated myotubes with conditioned medium from treated macrophages. In contrast to direct myotube LPS treatment, conditioned medium from LPS-treated macrophages reduced myotube respiration, but this was again mitigated with sphingolipid inhibition. Thus, macrophage sphingolipid production appears to be necessary for LPS-induced mitochondrial alterations in skeletal muscle tissue.

The PGC-1 coactivators promote an anti-inflammatory environment in skeletal muscle in vivo

The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is abundantly expressed in trained muscles and regulates muscle adaptation to endurance exercise. Inversely, mice lacking a functional PGC-1α allele in muscle exhibit reduced muscle functionality and increased inflammation. In isolated muscle cells, PGC-1α and the related PGC-1β counteract the induction of inflammation by reducing the activity of the nuclear factor κB (NFκB). We now tested the effects of these metabolic regulators on inflammatory reactions in muscle tissue of control and muscle-specific PGC-1α/-1β transgenic mice in vivo in the basal state as well as after an acute inflammatory insult. Surprisingly, we observed a PGC-1-dependent alteration of the cytokine profile characterized by an increase in anti-inflammatory factors and a strong suppression of the pro-inflammatory interleukin 12 (IL-12). In conclusion, the anti-inflammatory environment in muscle that is promoted by the PGC-1s might contribute to the beneficial effects of these coactivators on muscle function and provides a molecular link underlying the tight mutual regulation of metabolism and inflammation.

Role of various kinases in muscarinic M3 receptor-mediated contraction of longitudinal muscle of rat colon.

The longitudinal muscle layer in gut is the functional opponent to the circular muscle layer during peristalsis. Differences in innervation of the layers allow for the contraction of one layer concurrently with the relaxation of the other, enabling the passage of gut contents in a controlled fashion. Differences in development have given the cells of the two layers differences in receptor populations, membrane lipid handling, and calcium handling profiles/behaviors. The contractile activity of the longitudinal muscle is largely mediated by cholinergic neural input from myenteric plexus. Activation of muscarinic receptors leads to rapid activation of several kinases including MLC kinase, ERK1/2, CaMKII and Rho kinase. Phosphorylation of myosin light chain (MLC20) by MLC kinase (MLCK) is a prerequisite for contraction in both circular and longitudinal muscle cells. In rat colonic longitudinal muscle strips, we measured muscarinic receptor-mediated contraction following incubation with kinase inhibitors. Basal tension was differentially regulated by Rho kinase, ERK1/2, CaMKII and CaMKK. Selective inhibitors of Rho kinase, ERK1/2, CaMKK/AMPK, and CaMKII each reduced carbachol-induced contraction in the innervated muscle strips. These inhibitors had no direct effect on MLCK activity. Thus unlike previously reported for isolated muscle cells where CaMKII and ERK1/2 are not involved in contraction, we conclude that the regulation of carbachol-induced contraction in innervated longitudinal muscle strips involves the interplay of Rho kinase, ERK1/2, CaMKK/AMPK, and CAMKII.

Brain-derived neurotrophic factor enhances cholinergic contraction of longitudinal muscle of rabbit intestine via activation of phospholipase C

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of proteins best known for its role in neuronal survival, differentiation, migration, and synaptic plasticity in central and peripheral neurons. BDNF is also widely expressed in nonneuronal tissues including the gastrointestinal tract. The role of BDNF in intestinal smooth muscle contractility is not well defined. The aim of this study was to identify the role of BDNF in carbachol (CCh)- and substance P (SP)-induced contraction of intestinal longitudinal smooth muscle. BDNF, selective tropomyosin-related kinase B (TrkB) receptor agonists, and pharmacological inhibitors of signaling pathways were examined for their effects on contraction of rabbit intestinal longitudinal muscle strips induced by CCh and SP. BDNF activation of intracellular signaling pathways was examined by Western blot in homogenates of muscle strips and isolated muscle cells. One-hour preincubation with BDNF enhanced intestinal muscle contraction induced by CCh but not by SP. The selective synthetic TrkB agonists LM 22A4 and 7,8-dihydroxyflavone produced similar effects to BDNF. The Trk antagonist K-252a, a TrkB antibody but not p75NTR antibody, blocked the effect of BDNF. The enhancement of CCh-induced contraction by BDNF was blocked by the phospholipase C (PLC) antagonist U73122, but not by ERK1/2 or Akt antagonists. Direct measurement in muscle strips and isolated muscle cells showed that BDNF caused phosphorylation of TrkB receptors and PLC-γ, but not ERK1/2 or Akt. We conclude that exogenous BDNF augments the CCh-induced contraction of longitudinal muscle from rabbit intestine by activating TrkB receptors and subsequent PLC activation.