Ann-Marie Gustafson

Muscle wasting and the temporal gene expression pattern in a novel rat intensive care unit model.

BackgroundAcute quadriplegic myopathy (AQM) or critical illness myopathy (CIM) is frequently observed in intensive care unit (ICU) patients. To elucidate duration-dependent effects of the ICU intervention on molecular and functional networks that control the muscle wasting and weakness associated with AQM, a gene expression profile was analyzed at time points varying from 6 hours to 14 days in a unique experimental rat model mimicking ICU conditions, i.e., post-synaptically paralyzed, mechanically ventilated and extensively monitored animals.ResultsDuring the observation period, 1583 genes were significantly up- or down-regulated by factors of two or greater. A significant temporal gene expression pattern was constructed at short (6 h-4 days), intermediate (5-8 days) and long (9-14 days) durations. A striking early and maintained up-regulation (6 h-14d) of muscle atrogenes (muscle ring-finger 1/tripartite motif-containing 63 and F-box protein 32/atrogin-1) was observed, followed by an up-regulation of the proteolytic systems at intermediate and long durations (5-14d). Oxidative stress response genes and genes that take part in amino acid catabolism, cell cycle arrest, apoptosis, muscle development, and protein synthesis together with myogenic factors were significantly up-regulated from 5 to 14 days. At 9-14 d, genes involved in immune response and the caspase cascade were up-regulated. At 5-14d, genes related to contractile (myosin heavy chain and myosin binding protein C), regulatory (troponin, tropomyosin), developmental, caveolin-3, extracellular matrix, glycolysis/gluconeogenesis, cytoskeleton/sarcomere regulation and mitochondrial proteins were down-regulated. An activation of genes related to muscle growth and new muscle fiber formation (increase of myogenic factors and JunB and down-regulation of myostatin) and up-regulation of genes that code protein synthesis and translation factors were found from 5 to 14 days.ConclusionsNovel temporal patterns of gene expression have been uncovered, suggesting a unique, coordinated and highly complex mechanism underlying the muscle wasting associated with AQM in ICU patients and providing new target genes and avenues for intervention studies.

Time course analysis of mechanical ventilation‐induced diaphragm contractile muscle dysfunction in the rat

Weaning from mechanical ventilation (MV) of long‐term intensive care unit (ICU) patients is delayed by impaired respiratory muscle function; however, the mechanisms that cause the impairment are not fully understood. A novel experimental rat ICU model was used for time‐resolved analyses (6 h to 2 weeks) of the effects of MV on diaphragm muscle fibre structure and function, and on gene and protein expression. A prompt and progressive decline of diaphragm muscle fibre function, preceding atrophy, occurred with MV, and at the end of 2 weeks residual diaphragm muscle fibre function was <15% of control levels. Cellular and subcellular analyses indicated that oxidative stress‐triggered protein modifications had significantly diminished diaphragm muscle fibre function. The novel finding that activation of proteolytic pathways and regulation of contractile protein synthesis were different in diaphragm and limb muscles has direct implications for the design of muscle‐specific intervention strategies.

Tryptase-catalyzed core histone truncation: a novel epigenetic regulatory mechanism in mast cells.

Background Mast cells are key effector cells in allergic reactions. When activated to degranulate, they release a plethora of bioactive compounds from their secretory granules, including mast cell‐restricted proteases such as tryptase. In a previous study, we showed that tryptase, in addition to its intragranular location, can be found within the nuclei of mast cells where it truncates core histones at their N‐terminal ends. Objective Considering that the N‐terminal portions of the core histones constitute sites for posttranslational modifications of major epigenetic impact, we evaluated whether histone truncation by tryptase could have an impact on epigenetic events in mast cells. Methods Mast cells were cultured from wild‐type and tryptase null mice, followed by an assessment of their profile of epigenetic histone modifications and their phenotypic characteristics. Results We show that tryptase truncates nucleosomal histone 3 and histone 2B (H2B) and that its absence results in accumulation of the epigenetic mark, lysine 5‐acetylated H2B. Intriguingly, the accumulation of lysine 5‐acetylated H2B was cell age–dependent and was associated with a profound upregulation of markers of non–mast cell lineages, loss of proliferative control, chromatin remodeling as well as extensive morphological alterations. Conclusions These findings introduce tryptase‐catalyzed histone clipping as a novel epigenetic regulatory mechanism, which in the mast cell context may be crucial for maintaining cellular identity. Graphical abstract Figure. No Caption available.

Mast cells promote melanoma colonization of lungs.

Mast cells have been implicated in malignant processes, mainly through clinical correlative studies and by experiments performed using animals lacking mast cells due to defective c-kit signaling. However, mast cell-deficient mouse models based on c-kit defects have recently been questioned for their relevance. Here we addressed the effect of mast cells in a tumor setting by using transgenic Mcpt5-Cre+ R-DTA+ mice, in which the deficiency of mast cells is independent of c-kit defects. Melanoma cells (B16.F10) were administered either subcutaneously or intravenously into Mcpt5-Cre+ R-DTA+ mice or Mcpt5-Cre− R-DTA+ littermate controls, followed by the assessment of formed tumors. In the subcutaneous model, mast cells were abundant in the tumor stroma of control mice but were absent in Mcpt5-Cre+ R-DTA+ mice. However, the absence of mast cells did not affect tumor size. In contrast, after intravenous administration of B16.F10 cells, melanoma colonization of the lungs was markedly reduced in Mcpt5-Cre+ R-DTA+ vs. Mcpt5-Cre− R-DTA+ animals. Decreased melanoma colonization of the lungs in Mcpt5-Cre+ R-DTA+ animals was accompanied by increased inflammatory cell recruitment into the bronchoalveolar lavage fluid, suggesting that mast cells suppress inflammation in this setting. Further, qPCR analysis revealed significant alterations in the expression of Twist and E-cadherin in lungs of Mcpt5-Cre+ R-DTA+ vs. control Mcpt5-Cre− R-DTA+ animals, suggesting an impact of mast cells on epithelial-mesenchymal transition. In conclusion, this study reveals that mast cells promote melanoma colonization of the lung.

Increased Bone Mass in Female Mice Lacking Mast Cell Chymase

Here we addressed the potential impact of chymase, a mast-cell restricted protease, on mouse bone phenotype. We show that female mice lacking the chymase Mcpt4 acquired a persistent expansion of diaphyseal bone in comparison with wild type controls, reaching a 15% larger diaphyseal cross sectional area at 12 months of age. Mcpt4-/- mice also showed increased levels of a bone anabolic serum marker and higher periosteal bone formation rate. However, they were not protected from experimental osteoporosis, suggesting that chymase regulates normal bone homeostasis rather than the course of osteoporosis. Further, the absence of Mcpt4 resulted in age-dependent upregulation of numerous genes important for bone formation but no effects on osteoclast activity. In spite of the latter, Mcpt4-/- bones had increased cortical porosity and reduced endocortical mineralization. Mast cells were found periosteally and, notably, bone-proximal mast cells in Mcpt4-/- mice were degranulated to a larger extent than in wild type mice. Hence, chymase regulates degranulation of bone mast cells, which could affect the release of mast cell-derived factors influencing bone remodelling. Together, these findings reveal a functional impact of mast cell chymase on bone. Further studies exploring the possibility of using chymase inhibitors as a strategy to increase bone volume may be warranted.

The combined action of mast cell chymase, tryptase and carboxypeptidase A3 protects against melanoma colonization of the lung

Mast cell secretory granules are densely packed with various bioactive mediators including proteases of chymase, tryptase and CPA3 type. Previous studies have indicated that mast cells can affect the outcome of melanoma but the contribution of the mast cell granule proteases to such effects has not been clear. Here we addressed this issue by assessing mice lacking either the chymase Mcpt4, the tryptase Mcpt6 or carboxypeptidase A3 (Cpa3), as well as mice simultaneously lacking all three proteases, in a model of melanoma dissemination from blood to the lung. Although mice with individual deficiency in the respective proteases did not differ significantly from wildtype mice in the extent of melanoma colonization, mice with multiple protease deficiency (Mcpt4/Mcpt6/Cpa3-deficient) exhibited a higher extent of melanoma colonization in lungs as compared to wildtype animals. This was supported by higher expression of melanoma-specific genes in lungs of Mcpt4/Mcpt6/CPA3-deficient vs. wildtype mice. Cytokine profiling showed that the levels of CXCL16, a chemokine with effects on T cell populations and NKT cells, were significantly lower in lungs of Mcpt4/Mcpt6/Cpa3-deficient animals vs. controls, suggesting that multiple mast cell protease deficiency might affect T cell or NKT cell populations. In line with this, we found that the Mcpt4/Mcpt6/Cpa3-deficiency was associated with a reduction in cells expressing CD1d, a MHC class 1-like molecule that is crucial for presenting antigen to invariant NKT (iNKT) cells. Together, these findings indicate a protective role of mast cell-specific proteases in melanoma dissemination, and suggest that this effect involves a CXCL16/CD1d/NKT cell axis.

The Effect of Lipopolysaccharide-Induced Experimental Bovine Mastitis on Clinical Parameters, Inflammatory Markers, and the Metabolome: A Kinetic Approach

Mastitis is an inflammatory condition of the mammary tissue and represents a major problem for the dairy industry worldwide. The present study was undertaken to study how experimentally induced acute bovine mastitis affects inflammatory parameters and changes in the metabolome. To this end, we induced experimental mastitis in nine cows by intramammary infusion of 100 µg purified Escherichia coli lipopolysaccharide (LPS) followed by kinetic assessments of cytokine responses (by enzyme-linked immunosorbent assay), changes in the metabolome (assessed by nuclear magnetic resonance), clinical parameters (heat, local pain perception, redness, swelling, rectal temperature, clot formation, and color changes in the milk), and milk somatic cell counts, at several time points post LPS infusion. Intramammary LPS infusion induced clinical signs of mastitis, which started from 2 h post infusion and had returned to normal levels within 24–72 h. Milk changes were seen with a delay compared with the clinical signs and persisted for a longer time. In parallel, induction of IL-6 and TNF-α were seen in milk, and there was also a transient elevation of plasma IL-6 whereas plasma TNF-α was not significantly elevated. In addition, a robust increase in CCL2 was seen in the milk of LPS-infused cows, whereas G-CSF, CXCL1, and histamine in milk were unaffected. By using a metabolomics approach, a transient increase of plasma lactose was seen in LPS-induced cows. In plasma, significant reductions in ketone bodies (3-hydroxybutyrate and acetoacetate) and decreased levels of short-chain fatty acids, known to be major products released from the gut microbiota, were observed after LPS infusion; a profound reduction of plasma citrate was also seen. Intramammary LPS infusion also caused major changes in the milk metabolome, although with a delay in comparison with plasma, including a reduction of lactose. We conclude that the LPS-induced acute mastitis rapidly affects the plasma metabolome and cytokine induction with similar kinetics as the development of the clinical signs, whereas the corresponding effects in milk occurred with a delay.

Bones in human CYP26B1 deficiency and rats with hypervitaminosis A phenocopy Vegfa overexpression

Angulated femurs are present prenatally both in CYP26B1 deficient humans with a reduced capacity to degrade retinoic acid (RA, the active metabolite of vitamin A), and mice overexpressing vascular endothelial growth factor a (Vegfa). Since excessive ingestion of vitamin A is known to induce spontaneous fractures and as the Vegfa-induced femur angulation in mice appears to be caused by intrauterine fractures, we analyzed bones from a CYP26B1 deficient human and rats with hypervitaminosis A to further explore Vegfa as a mechanistic link for the effect of vitamin A on bone. We show that bone from a human with CYP26B1 mutations displayed periosteal osteoclasts in piles within deep resorption pits, a pathognomonic sign of hypervitaminosis A. Analysis of the human angulated fetal femur revealed excessive bone formation in the marrow cavity and abundant blood vessels. Normal human endothelial cells showed disturbed cell-cell junctions and increased CYP26B1 and VEGFA expression upon RA exposure. Studies in rats showed increased plasma and tissue Vegfa concentrations and signs of bone marrow microhemorrhage on the first day of excess dietary vitamin A intake. Subsequently hypervitaminosis A rats displayed excess bone formation, fibrosis and an increased number of megakaryocytes in the bone marrow, which are known characteristics of Vegfa overexpression. This study supports the notion that the skeletal phenotype in CYP26B1 deficient human bone is caused by excess RA. Our findings suggest that an initial part of the vitamin A mechanism causing bone alterations is mediated by excess Vegfa and disturbed bone marrow microvessel integrity.

Time Resolved Analyses of Gene Expression in a Rodent Icu Model

Intensive care unit (ICU) patients commonly develop severe skeletal muscle wasting that aggravate the recovery from the primary disease and weaning from respirator. The modern treatment in anesthesiology and intensive care can progress in Acute Quadriplegic Myopathy (AQM). This study aims at improving our understanding of the mechanisms underlying the muscle wasting and weakness in ICU patients with AQM. Specific interest is focused on duration-dependent effects on intracellular signaling and myofibrillar gene and protein expression. For that reason, a unique experimental rat model mimicking ICU such as mechanical ventilation, muscle unloading, neuromuscular blocking agents (NMBA) administration and monitoring at different time points from 6h to 14 days, was used. Gene expression profile was analyzed in gastrocnemius muscle, showing an increased expression in the muscle-specific ubiquitin ligases, atrogin-1 and MuRF-1 after 6 h, as well as other genes involved in translational repression, authophagic/lysosomal genes (LC3b, cathepsins), oxidative stress response and up-regulation of pro-apoptosis signaling, except caspase-3 that increased after 9 days of intervention. Metalloproteins, GST, cystatins and cell cycle repressors were up-regulated in the early stages in response to oxidative stress and cellular damage among other genes, while LIM and sarcomeric proteins, collagen, extracellular matrix transcripts, carbohydrate metabolism, mitochondrial genes and the muscle-specific calpain-3 were down-regulated mainly after 5 days.These results suggest a very complex, unique and highly temporally coordinated activation of protein synthesis, degradation, protective mechanisms and intracellular signaling activation at different time points during ICU conditions.

G.P.2.01 Acute Quadriplegic Myopathy: Underlying mechanisms, improved diagnostic methods and specific intervention strategies

Exertional heat stroke (EHS) is usually triggered by strenuous exercise performed under hot and humid environmental conditions. It is associated with a classical symptomatology, i.e. increased body temperature and neurologic impairment that could lead to coma, with the addition of rhabdomyolysis-induced myoglobinuria and acute renal failure. Malignant hyperthermia (MH) is an autosomal condition triggered by exposition to halogenated volatile anaesthetics and usually associated to mutations of a calcium channel, the ryanodine receptor (RYR1).While the pathogenesis of EHS differs from a MH crisis, both conditions share some similarities in symptoms, such as the abnormal increase in core temperature. Furthermore, the connection between both syndromes has been substantiated by the presence of both positive IVCT and the presence of RYR1 mutations in patients with a history of exercise induced heat stroke or rhabdomyolysis. The purpose of the present study was to further characterize the genetic and phenotypic connection, if any, between MH and EHS. A large panel of military patients who had developed a clear and well documented EHS was investigated retrospectively. At least 6 months after the EHS episode, in vitro halothane/caffeine contracture tests (IVCT) were performed in order to define their MH status. In addition, 31P magnetic resonance spectroscopy data were recorded during a standardized rest-exercise-recovery protocol in order to assess muscle energetics. Mutations in the RYR1 gene were investigated from the entire cDNA obtained from muscle samples belonging to 20 patients with an MHS-IVCT status. The corresponding data will be presented and discussed.