Skeletal Muscle Mitochondrial Function and Fatigability in Older Adults.

BACKGROUND: Fatigability increases while the capacity for mitochondrial energy production tends to decrease significantly with age. Thus, diminished mitochondrial function may contribute to higher levels of fatigability in older adults. METHODS: The relationship between fatigability and skeletal muscle mitochondrial function was examined in 30 participants aged 78.5 ± 5.0 years (47% female, 93% white), with a body mass index of 25.9 ± 2.7 kg/m(2) and usual gait-speed of 1.2 ± 0.2 m/s. Fatigability was defined using rating of perceived exertion (6-20 point Borg scale) after a 5-minute treadmill walk at 0.72 m/s. Phosphocreatine recovery in the quadriceps was measured using (31)P magnetic resonance spectroscopy and images of the quadriceps were captured to calculate quadriceps volume. ATPmax (mM ATP/s) and oxidative capacity of the quadriceps (ATPmax·Quadriceps volume) were calculated. Peak aerobic capacity (VO2peak) was measured using a modified Balke protocol. RESULTS: ATPmax·Quadriceps volume was associated with VO2peak and was 162.61mM ATP·mL/s lower (p = .03) in those with high (rating of perceived exertion ≥10) versus low (rating of perceived exertion ≤9) fatigability. Participants with high fatigability required a significantly higher proportion of VO2peak to walk at 0.72 m/s compared with those with low fatigability (58.7 ± 19.4% vs 44.9 ± 13.2%, p < .05). After adjustment for age and sex, higher ATPmax was associated with lower odds of having high fatigability (odds ratio: 0.34, 95% CI: 0.11-1.01, p = .05). CONCLUSIONS: Lower capacity for oxidative phosphorylation in the quadriceps, perhaps by contributing to lower VO2peak, is associated with higher fatigability in older adults.

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Chronic exercise preserves lean muscle mass in masters athletes.

Aging is commonly associated with a loss of muscle mass and strength, resulting in falls, functional decline, and the subjective feeling of weakness. Exercise modulates the morbidities of muscle aging. Most studies, however, have examined muscle-loss changes in sedentary aging adults. This leaves the question of whether the changes that are commonly associated with muscle aging reflect the true physiology of muscle aging or whether they reflect disuse atrophy. This study evaluated whether high levels of chronic exercise prevents the loss of lean muscle mass and strength experienced in sedentary aging adults. A cross-section of 40 high-level recreational athletes ("masters athletes") who were aged 40 to 81 years and trained 4 to 5 times per week underwent tests of health/activity, body composition, quadriceps peak torque (PT), and magnetic resonance imaging of bilateral quadriceps. Mid-thigh muscle area, quadriceps area (QA), subcutaneous adipose tissue, and intramuscular adipose tissue were quantified in magnetic resonance imaging using medical image processing, analysis, and visualization software. One-way analysis of variance was used to examine age group differences. Relationships were evaluated using Spearman correlations. Mid-thigh muscle area (P = 0.31) and lean mass (P = 0.15) did not increase with age and were significantly related to retention of mid-thigh muscle area (P < 0.0001). This occurred despite an increase in total body fat percentage (P = 0.003) with age. Mid-thigh muscle area (P = 0.12), QA (P = 0.17), and quadriceps PT did not decline with age. Specific strength (strength per QA) did not decline significantly with age (P = 0.06). As muscle area increased, PT increased significantly (P = 0.008). There was not a significant relationship between intramuscular adipose tissue (P = 0.71) or lean mass (P = 0.4) and PT. This study contradicts the common observation that muscle mass and strength decline as a function of aging alone. Instead, these declines may signal the effect of chronic disuse rather than muscle aging. Evaluation of masters athletes removes disuse as a confounding variable in the study of lower-extremity function and loss of lean muscle mass. This maintenance of muscle mass and strength may decrease or eliminate the falls, functional decline, and loss of independence that are commonly seen in aging adults.

Regulation of the akt signalling in human skeletal muscle atrophy

Abstract : The term "muscle disuse" is often used to refer collectively to reductions in neuromuscular activity as observed with sedentary lifestyles, reduced weight bearing, cancer, chronic obstructive pulmonary disease, chronic heart failure, spinal cord injury, sarcopenia or exposure to microgravity (spaceflight). Muscle disuse atrophy, caused by accelerated proteolysis, is predominantly due to the activation of the ATP-dependent ubiquitin (Ub) proteasome pathway. The current advances in understanding the molecular factors contributing to the Ub-dependent proteolysis process have been made mostly in rodent models of human disease and denervation with few investigations performed directly in humans. Recently, in mice, the genes Atrogin-1 and MuRF1 have been designated as primary candidates in the control of muscle atrophy. Additionally, the decreased activity of the Akt/GSK-3ß and Akt/mTOR pathways has been associated with a reduction in protein synthesis and contributing to skeletal muscle atrophy. Therefore, it is now commonly accepted that skeletal muscle atrophy is the result of a decreased protein synthesis concomitant with an increase in protein degradation (Glass 2003). Atrogin-1 and MuRF1 are genes expressed exclusively in muscle. In mice, their expression has been shown to be directly correlated with the severity of atrophy. KO-mice experiments showed a major protection against atrophy when either of these genes were deleted. Skeletal muscle hypertrophy is an important function in normal postnatal development and in the adaptive response to exercise. It has been shown, in vitro, that the activation of phosphatidylinositol 3-kinase (PI-3K), by insulin growth factor 1 (IGF-1), stimulates myotubes hypertrophy by activating the downstream pathways, Akt/GSK-3ß and Akt/mTOR. It has also been demonstrated in mice, in vivo, that activation of these signalling pathways causes muscle hypertrophy. Moreover, the latter were recently proposed to also reduce muscle atrophy by inhibiting the FKHR mediated transcription of several muscle atrophy genes; Atrogin-1 and MuRF1. Therefore, these targets present new avenues for developing further the understanding of the molecular mechanisms involved in both skeletal muscle atrophy and hypertrophy. The present study proposed to investigate the regulation of the Akt/GSK-3ß and Akt/mTOR signalling pathways, as well as the expression levels of the "atrogenes", Atrogin-1 and MuRF1, in four human models of skeletal muscle atrophy. In the first study, we measured the regulation of the Akt signalling pathway after 8 weeks of both hypertrophy stimulating resistance training and atrophy stimulation de-training. As expected following resistance training, muscle hypertrophy and an increase in the phosphorylation status of the different members of the Akt pathway was observed. This was paralleled by a concomitant decrease in FOXO1 nuclear protein content. Surprisingly, exercise training also induced an increase in the, expression of the atrophy genes and proteins involved in the ATP-dependant ubiquitin-proteasome system. On the opposite, following the de-training period a muscle atrophy, relative to the post-training muscle size, was measured. At the same time, the phosphorylation levels of Akt and GSK-3ß were reduced while the amount of FOXO1 in the nucleus increased. After the atrophy phase, there was also a reduction in Atrogin-1 and MuRF1 contents. In this study, we demonstrate for the first time in healthy human skeletal muscle, that the regulation of Akt and its downstream targets GSK-3ß, mTOR and FOXO1 are associated with both thé skeletal muscle hypertrophy and atrophy processes. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the loss of both upper and lower motor neurons, which leads to severe muscle weakness and atrophy. All measurements were performed in biopsies from 22 ALS patients and 16 healthy controls. ALS patients displayed an increase in Atrogin-1 mRNA and protein content which was associated with a decrease in Akt activity. However there was no difference in the mRNA and phospho-protein content of FOXO1, FOXO3a, p70S6K and GSK-3ß. The transcriptional regulation of human Atrogin-1 may be controlled by an Akt-mediated transcription factor other than FKHR or via an other signalling pathway. Chronic complete spinal cord injury (SCI) is associated with severe muscle atrophy which is linked to co-morbidity factors such as diabetes, obesity, lipid disorders and cardiovascular diseases. Molecular mechanisms associated with chronic complete SCI-related muscle atrophy are not well understood. The aim of the present study was to determine if there was an increase in catabolic signalling targets such as Atrogin-1, MuRF1, FOXO and myostatin, and decreases in anabolic signalling targets such as IGF, Akt, GSK-3ß, mTOR, 4E-BP1 and p-70S6K in chronic complete SCI patients. All measurements were performed in biopsies taken from 8 complete chronic SCI patients and 7 age matched healthy controls. In SCI patients when compared with controls, there was a significant reduction in mRNA levels of Atrogin1, MuRF1 and Myostatin. Protein levels for Atrogin-1, FOX01 and FOX03a were also reduced. IGF-1 and both phosphorylated GSK-3ß and 4E-BP1 were decreased; the latter two in an Akt and mTOR independent manner, respectively. Reductions in Atrogin-1, MuRF1, FOXO and myostatin suggest the existence of an internal mechanism aimed at reducing further loss of muscle proteins during chronic SCI. The downregulation of signalling proteins regulating anabolism such as IGF, GSK3ß and 4E-BP1 would reduce the ability to increase protein synthesis rates in this chronic state of muscle wasting. The molecular mechanisms controlling age-related skeletal muscle loss in humans are poorly understood. The present study aimed to investigate the regulation of several genes and proteins involved in the activation of key signalling pathways promoting muscle hypertrophy such as GH/STAT5/IGF, IGF/Akt/GSK-3ß/4E-BP1 and muscle atrophy such as TNFα/SOCS3 and Akt/FOXO/Atrogin-1 or MuRF1 in muscle biopsies from 13 young and 16 elderly men. In the older, as compared with the young subjects, TNFα and SOCS-3 were increased while growth hormone receptor protein (GHR) and IGF-1 mRNA were both decreased. Akt protein levels were increased however no change in phosphorylated Akt content was observed. GSK-3ß phosphorylation levels were increased while 4E-BP1 was not changed. Nuclear FKHR and FKHRL1 protein levels were decreased, with no changes in their atrophy target genes, Atrogin-1 and MuRF1. Myostatin mRNA and protein levels were significantly elevated. Human sarcopenia may be linked to a reduction in the activity or sensitivity of anabolic signalling proteins such as GHR, IGF and Akt. TNFα, SOCS-3 and myostatin are potential candidates influencing this anabolic perturbation. In conclusion our results support those obtained in rodent or ín vitro models, and demonstrate Akt plays a pivotal role in the control of muscle mass in humans. However, the Akt phosphorylation status was dependant upon the model of muscle atrophy as Akt phosphorylation was reduced in all atrophy models except for SCI. Additionally, the activity pattern of the downstream targets of Akt appears to be different upon the various human models. It seems that under particular conditions such as spinal cord injury or sarcopenia, .the regulation of GSK-3ß, 4eBP1 and p70S6K might be independent of Akt suggesting alternative signalling pathways in the control of these the anabolic response in human skeletal muscle. The regulation of Atrogin-1 and MuRF1 in some of our studies has been shown to be also independent of the well-described Akt/FOXO signalling pathway suggesting that other transcription factors may regulate human Atrogin-1 and MuRF1. These four different models of skeletal muscle atrophy and hypertrophy have brought a better understanding concerning the molecular mechanisms controlling skeletal muscle mass in humans.

Alterations of Neuromuscular Function after the World's Most Challenging Mountain Ultra-Marathon.

We investigated the physiological consequences of the most challenging mountain ultra-marathon (MUM) in the world: a 330-km trail run with 24000 m of positive and negative elevation change. Neuromuscular fatigue (NMF) was assessed before (Pre-), during (Mid-) and after (Post-) the MUM in experienced ultra-marathon runners (n = 15; finish time  = 122.43 hours ±17.21 hours) and in Pre- and Post- in a control group with a similar level of sleep deprivation (n = 8). Blood markers of muscle inflammation and damage were analyzed at Pre- and Post-. Mean ± SD maximal voluntary contraction force declined significantly at Mid- (-13±17% and -10±16%, P<0.05 for knee extensor, KE, and plantar flexor muscles, PF, respectively), and further decreased at Post- (-24±13% and -26±19%, P<0.01) with alteration of the central activation ratio (-24±24% and -28±34% between Pre- and Post-, P<0.05) in runners whereas these parameters did not change in the control group. Peripheral NMF markers such as 100 Hz doublet (KE: -18±18% and PF: -20±15%, P<0.01) and peak twitch (KE: -33±12%, P<0.001 and PF: -19±14%, P<0.01) were also altered in runners but not in controls. Post-MUM blood concentrations of creatine kinase (3719±3045 Ul·(1)), lactate dehydrogenase (1145±511 UI·L(-1)), C-Reactive Protein (13.1±7.5 mg·L(-1)) and myoglobin (449.3±338.2 µg·L(-1)) were higher (P<0.001) than at Pre- in runners but not in controls. Our findings revealed less neuromuscular fatigue, muscle damage and inflammation than in shorter MUMs. In conclusion, paradoxically, such extreme exercise seems to induce a relative muscle preservation process due likely to a protective anticipatory pacing strategy during the first half of MUM and sleep deprivation in the second half.

Double gene deletion reveals the lack of cooperation between PPARalpha and PPARbeta in skeletal muscle.

The peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of most of the pathways linked to lipid metabolism. PPARalpha and PPARbeta isotypes are known to regulate muscle fatty acid oxidation and a reciprocal compensation of their function has been proposed. Herein, we investigated muscle contractile and metabolic phenotypes in PPARalpha-/-, PPARbeta-/-, and double PPARalpha-/- beta-/- mice. Heart and soleus muscle analyses show that the deletion of PPARalpha induces a decrease of the HAD activity (beta-oxidation) while soleus contractile phenotype remains unchanged. A PPARbeta deletion alone has no effect. However, these mild phenotypes are not due to a reciprocal compensation of PPARbeta and PPARalpha functions since double gene deletion PPARalpha-PPARbeta mostly reproduces the null PPARalpha-mediated reduced beta-oxidation, in addition to a shift from fast to slow fibers. In conclusion, PPARbeta is not required for maintaining skeletal muscle metabolic activity and does not compensate the lack of PPARalpha in PPARalpha null mice.

Respiratory muscle training enhances maximal minute ventilation and forced vital capacity in adolescent athletes.

Introduction. Respiratory difficulties in athletes are common, especially in adolescents, even in the absence of exercise-induced bronchoconstriction. Immaturity of the respiratory muscles coupling at high respiratory rates could be a potential mechanism. Whether respiratory muscle training (RMT) can positively influence it is yet unknown. Goal. We investigate the effects of RMT on ventilation and performance parameters in adolescent athletes and hypothesize that RMT will enhance respiratory capacity. Methods. 12 healthy subjects (8 male, 4 female, 17±0.5 years) from a sports/study high school class, competitively involved in various sports (minimum of 10 hours per week) underwent respiratory function testing, maximal minute ventilation (MMV) measurements and a maximal treadmill incremental test with VO2max and ventilatory thresholds (VT1 and VT2) determination. They then underwent one month of RMT (4 times/week) using a eucapnic hyperventilation device, with an incremental training program. The same tests were repeated after RMT. Results. Subjects completed 14.8 sessions of RMT, with an increase in total ventilation per session of 211±29% during training. Borg scale evaluation of the RMT session was unchanged or reduced in all subjects, despite an increase in total respiratory work. No changes (p>0.05) were observed pre/post RMT in VO2max (53.4±7.5 vs 51.6±7.7 ml/kg/min), VT2 (14.4±1.4 vs 14.0±1.1 km/h) or Speed max at end of test (16.1±1.7 vs 15.8±1.7 km/h). MVV increased by 9.2% (176.7±36.9 vs 192.9±32.6 l/min, p<0.001) and FVC by 3.3% (6.70±0.75 vs 4.85±0.76 litres, p<0.05). Subjective evaluation of respiratory sensations during exercise and daily living were also improved. Conclusions. RMT improves MMV and FVC in adolescent athletes, along with important subjective respiratory benefits, although no changes are seen in treadmill maximal performance tests and VO2max measurements. RMT can be easily performed in adolescent without side effects, with a potential for improvement in training capacity and overall well-being.

Estimation of glomerular filtration rate in hospitalised patients: are we overestimating renal function?

QUESTIONS UNDER STUDY AND PRINCIPLES: Estimating glomerular filtration rate (GFR) in hospitalised patients with chronic kidney disease (CKD) is important for drug prescription but it remains a difficult task. The purpose of this study was to investigate the reliability of selected algorithms based on serum creatinine, cystatin C and beta-trace protein to estimate GFR and the potential added advantage of measuring muscle mass by bioimpedance. In a prospective unselected group of patients hospitalised in a general internal medicine ward with CKD, GFR was evaluated using inulin clearance as the gold standard and the algorithms of Cockcroft, MDRD, Larsson (cystatin C), White (beta-trace) and MacDonald (creatinine and muscle mass by bioimpedance). 69 patients were included in the study. Median age (interquartile range) was 80 years (73-83); weight 74.7 kg (67.0-85.6), appendicular lean mass 19.1 kg (14.9-22.3), serum creatinine 126 μmol/l (100-149), cystatin C 1.45 mg/l (1.19-1.90), beta-trace protein 1.17 mg/l (0.99-1.53) and GFR measured by inulin 30.9 ml/min (22.0-43.3). The errors in the estimation of GFR and the area under the ROC curves (95% confidence interval) relative to inulin were respectively: Cockcroft 14.3 ml/min (5.55-23.2) and 0.68 (0.55-0.81), MDRD 16.3 ml/min (6.4-27.5) and 0.76 (0.64-0.87), Larsson 12.8 ml/min (4.50-25.3) and 0.82 (0.72-0.92), White 17.6 ml/min (11.5-31.5) and 0.75 (0.63-0.87), MacDonald 32.2 ml/min (13.9-45.4) and 0.65 (0.52-0.78). Currently used algorithms overestimate GFR in hospitalised patients with CKD. As a consequence eGFR targeted prescriptions of renal-cleared drugs, might expose patients to overdosing. The best results were obtained with the Larsson algorithm. The determination of muscle mass by bioimpedance did not provide significant contributions.

Participation of connexin-based channels in the control of vascular function

SUMMARYIntercellular communication is achieved at specialized regions of the plasma membrane by gap junctions. The proteins constituting the gap junctions are called connexins and are encoded by a family of genes highly conserved during evolution. In adult mouse, four connexins (Cxs) are known to be expressed in the vasculature: Cx37, Cx40, Cx43 and Cx45. Several recent studies have provided evidences that vascular connexins expression and blood pressure regulation are closely linked, suggesting a role for connexins in the control of blood pressure. However, the precise function that each vascular connexin plays under physiological and pathophysiological conditions is still not elucidated. In this context, this work was dedicated to evaluate the contribution of each of the four vascular connexins in the control of the vascular function and in the blood pressure regulation.In the present work, we first demonstrated that vascular connexins are differently regulated by hypertension in the mouse aorta. We also observed that endothelial connexins play a regulatory role on eNOS expression levels and function in the aorta, therefore in the control of vascular tone. Then, we demonstrated that Cx40 plays a pivotal role in the kidney by regulating the renal levels of COX-2 and nNOS, two key enzymes of the macula densa known to participate in the control of renin secreting cells. We also found that Cx43 forms the functional gap junction involved in intercellular Ca2+ wave propagation between vascular smooth muscle cells. Finally, we have started to generate transgenic mice expressing specifically Cx40 in the endothelium to investigate the involvement of Cx40 in the vasomotor tone, or in the renin secreting cells to evaluate the role of Cx40 in the control of renin secretion.In conclusion, this work has allowed us to identify new roles for connexins in the vasculature. Our results suggest that vascular connexins could be interesting targets for new therapies caring hypertension and vascular diseases.

Alteration in neuromuscular function after a 5 km running time trial.

The aim of this study was to characterize the effect of a 5 km running time trial on the neuromuscular properties of the plantar flexors. Eleven well-trained triathletes performed a series of neuromuscular tests before and immediately after the run on a 200 m indoor track. Muscle activation (twitch interpolation) and normalized EMG activity were assessed during maximal voluntary contraction (MVC) of plantar flexors. Maximal soleus H-reflexes and M-waves were evoked at rest (i.e. H (MAX) and M (MAX), respectively) and during MVC (i.e. H (SUP) and M (SUP), respectively). MVC significantly declined (-27%; P < 0.001) after the run, due to decrease in muscle activation (-8%; P < 0.05) and M (MAX)-normalized EMG activity (-13%; P < 0.05). Significant reductions in M-wave amplitudes (M (MAX): -13% and M (SUP): -16%; P < 0.05) as well as H (MAX)/M (MAX) (-37%; P < 0.01) and H (SUP)/M (SUP) (-25%; P < 0.05) ratios occurred with fatigue. Following exercise, the single twitch was characterized by lower peak torque (-16%; P < 0.001) as well as shorter contraction (-19%; P < 0.001) and half-relaxation (-24%; P < 0.001) times. In conclusion, the reduction in plantar flexors strength induced by a 5 km running time trial is caused by peripheral adjustments, which are attributable to a failure of the neuromuscular transmission and excitation-contraction coupling. Fatigue also decreased the magnitude of efferent motor outflow from spinal motor neurons to the plantar flexors and part of this suboptimal neural drive is the result of an inhibition of soleus motoneuron pool reflex excitability.

Skeletal muscle mitochondria in the elderly: effects of physical fitness and exercise training.

CONTEXT: Sarcopenia is thought to be associated with mitochondrial (Mito) loss. It is unclear whether the decrease in Mito content is consequent to aging per se or to decreased physical activity. OBJECTIVES: The objective of the study was to examine the influence of fitness on Mito content and function and to assess whether exercise could improve Mito function in older adults. DESIGN AND SUBJECTS: Three distinct studies were conducted: 1) a cross-sectional observation comparing Mito content and fitness in a large heterogeneous cohort of older adults; 2) a case-control study comparing chronically endurance-trained older adults and sedentary (S) subjects matched for age and gender; and 3) a 4-month exercise intervention in S. SETTING: The study was conducted at a university-based clinical research center. OUTCOMES: Mito volume density (MitoVd) was assessed by electron microscopy from vastus lateralis biopsies, electron transport chain proteins by Western blotting, mRNAs for transcription factors involved in M biogenesis by quantitative RT-PCR, and in vivo oxidative capacity (ATPmax) by (31)P-magnetice resonance spectroscopy. Peak oxygen uptake was measured by graded exercise test. RESULTS: Peak oxygen uptake was strongly correlated with MitoVd in 80 60- to 80-year-old adults. Comparison of chronically endurance-trained older adults vs S revealed differences in MitoVd, ATPmax, and some electron transport chain protein complexes. Finally, exercise intervention confirmed that S subjects are able to recover MitoVd, ATPmax, and specific transcription factors. CONCLUSIONS: These data suggest the following: 1) aging per se is not the primary culprit leading to Mito dysfunction; 2) an aerobic exercise program, even at an older age, can ameliorate the loss in skeletal muscle Mito content and may prevent aging muscle comorbidities; and 3) the improvement of Mito function is all about content.

A phenotypical study of vascular smooth muscle cells in human arterial and venous stenosis

Abstract Introduction The primary function of the contractile vascular smooth muscle cells (cVSMCs) is the regulation of the vascular contractility which means the adaptation of the vascular tonus in response to the modulation of the blood pressure and blood flow. The cVSMCs are essentially quiescent, and therefore their synthesis rate is very limited. They are characterized by the expression of contractile proteins specific to the muscular tissue including myosin, h-­‐caldesmon and <-­‐smooth muscle actin (〈-­‐SMA). These contractile cells are strongly represented in the media layer of the arterial wall and, in a smaller proportion, of the vein wall. Their typical stretched-­‐out morphology allows recognizing them by a histological analysis. They do not produce any extracellular matrix (ECM), and do not migrate through the different layers of the vessel wall, and are not directly involved in the development of intimal hyperplasia (IH). Neointimal formation occurs after endothelial disruption leading to complex molecular and biological mechanisms. The de-­‐differentiation of cVSMCs into synthetic VSMCs (sVSMCs) is mentioned as a key element. These non mature cells are able to proliferate and produce ECM. The characterization of the vascular smooth muscle cells (VSMCs) from healthy and stenosed vascular tissues will contribue to the understanding of the different biological processes leading to IH and will be useful for the development of new therapies to interfere with the cVSMCs growth and migration. The aim of our research was to quantify the proportion of cVSMCs and sVSMCs into the healthy and pathologic human blood vessel wall and to characterize their phenotype. Methods We selected 23 specimens of arterial and venous segments from 18 patients. All these specimens were stored in the biobank from the thoracic and vascular surgery departement. 4 groups were designed (group 1 :arteries without lesions (n=3) ;group 2 : veins without lesions (n=1); group 3: arteries with stenosis (n=9); group 4: veins with stenosis (n=10)). Histology: 5µm-­‐sections were made from each sample embedded in paraffin wax and further stained with hematoxylin & eosin (HE), Van Gieson's stain (VGEL) and Masson's Trichrome (TMB). Pathologic tissues were defined using the label that was given to the macroscopic samples by the surgeon and also, based on the histological analysis with HE and VGEL evaluating the presence of a thickened intima. The same was done to the control samples evaluating the absence of thickening. Immunohistochemistry : The primary antibodies were used :〈-­‐SMA, vimentin, h-­‐ caldesmon, calponin, smooth muscle-myosin heavy chain (SM-­‐MHC), tropomyosin-­‐4, retinol binding protein-­‐1 (RBP-­‐1), nonmuscle-­‐myosin heavy chain-­‐B (NM-­‐MHC-­‐B), Von Willebrand factor (VWF). A semi-­‐quantitative assessment of the intensity of each sample stained was performed. Western Blot : Segments of arteries and veins were analyzed using the following primary antibodies :〈-­‐SMA, Calponin, SM-­‐MHC, NM-­‐MHC-­‐B. The given results were then normalized with tubulin. Results Our data showed that, when using immunohistochemistry analysis we found that〈-­‐SMA was mostly expressed in control arteries, whereas NM-­‐MHC-­‐B in the pathologic ones. Using SM-­‐MHC, calponin, vimentin and caldesmon we found no significative differences in the expression of these proteins in the control and in the pathologic samples. Western Blot analysis showed an inverse correlation between healthy and pathological samples as <-­‐ SMA was more expressed in the pathological samples, while NM-­‐MHC-­‐B in the control group; SM-­‐MHC and calponin were mostly expressed in the pathologic samples. Conclusion Our study showed no clear differences between stenotic and control arterial and venous segments using semi-­‐quantitative assessement by immunohistochemistry. Western Blot showed a significant increased expression of 〈-­‐SMA, calponin and SM-­‐MHC in the arteries with stenosis, while NM-­‐MHC-­‐B was mostly expressed in the arteries without lesions. Further studies are needed to track the lineage of VSMCs to understand the mechanisms leading toIH.

Artificial muscle to wash blood out of fibrillating atrium: an alternative to lifelong anticoagulation.

The Atripump is a motorless, volume displacement pump based on artificial muscle technology that could reproduce the pump function of normal atrium. It could help prevent blood clots due to blood stagnation and eventually avoid anticoagulation therapy in atrial fibrillation (AF). An animal study has been designed to assess mechanical effects of this pump on fibrillating atrium. The Atripump is a dome shaped silicone coated nitinol actuator. A pacemaker like control unit drives the actuator. In five adult sheep, the right atrium (RA) was exposed and dome sutured onto the epicardium. Atrial fibrillation was induced using rapid epicardial pacing (600 beats/min). Ejection fraction of the RA was obtained with intracardiac ultrasound in baseline, AF and Atripump assisted AF conditions. The dome's contraction rate was 60/min with power supply of 12V, 400 mA for 200 ms and ran for 2 hours in total. Mean temperature on the RA was 39+/-1.5 degrees C. Right atrium ejection fraction was 31% in baseline conditions, 5% and 20% in AF and assisted AF, respectively. In two animals a thrombus appeared in the right appendix and washed out once the pump was turned on. The Atripump washes blood out the RA acting as an anticoagulant device. Possible clinical implications in patients with chronic AF are prevention of embolism of cardiac origin and avoidance of hemorrhagic complication due to chronic anticoagulation.

Closure of large intrathoracic airway defects using extrathoracic muscle flaps.

BACKGROUND: Prospective assessment of pedicled extrathoracic muscle flaps for the closure of large intrathoracic airway defects after noncircumferential resection in situations where an end-to-end reconstruction seemed risky (defects of > 4-cm length, desmoplastic reactions after previous infection or radiochemotherapy). METHODS: From 1996 to 2001, 13 intrathoracic muscle transpositions (6 latissimus dorsi and 7 serratus anterior muscle flaps) were performed to close defects of the intrathoracic airways after noncircumferential resection for tumor (n = 5), large tracheoesophageal fistula (n = 2), delayed tracheal injury (n = 1) and bronchopleural fistula (n = 5). In 2 patients, the extent of the tracheal defect required reinforcement of the reconstruction by use of a rib segment embedded into the muscle flap followed by temporary tracheal stenting. Patient follow-up was by clinical examination bronchoscopy and biopsy, pulmonary function tests, and dynamic virtual bronchoscopy by computed tomographic (CT) scan during inspiration and expiration. RESULTS: The airway defects ranged from 2 x 1 cm to 8 x 4 cm and involved up to 50% of the airway circumference. They were all successfully closed using muscle flaps with no mortality and all patients were extubated within 24 hours. Bronchoscopy revealed epithelialization of the reconstructions without dehiscence, stenosis, or recurrence of fistulas. The flow-volume loop was preserved in all patients and dynamic virtual bronchoscopy revealed no significant difference in the endoluminal cross surface areas of the airway between inspiration and expiration above (45 +/- 21 mm(2)), at the site (76 +/- 23 mm(2)) and below the reconstruction (65 +/- 40 mm(2)). CONCLUSIONS: Intrathoracic airway defects of up to 50% of the circumference may be repaired using extrathoracic muscle flaps when an end-to-end reconstruction is not feasible.

Function and regulation of the scaffold protein IB1/JIP-1 in the uro-genital tract.

RESUME Ce mémoire de thèse traite de l'étude de la « scaffold »protéine ou protéine «échafaud», « Islet-Brain1/ JNK Interacting Protein 1 » (IB1/JIP-1) dans la vessie et la prostate, deux organes importants de l'appareil uro-genital. Cette protéine, mise en évidence dans notre laboratoire à la fin des année 90, a été reconnue pour réguler la voie de signalisation des « Mitogen-Activated Protein Kinases » (MAPKs), et en particulier de la MAPK appelée c-Jun N-terminal Kinase (JNK). Le réseau de voie de signalisation permet aux cellules de percevoir les changements dans le milieu extracellulaire et de permettre une réponse appropriée à ces différents stimuli. La connaissance des voies de signalisation a permis de mettre en évidence leur rôle crucial tant dans l'homéostase des tissus sains que dans des processus pathologiques comme l'oncogenèse. Parmi une vingtaine de voie de signalisation, la voie de signalisation des «MAPKinases » est une des plus importantes et a été montrée pour participer à diverses fonctions cellulaires telles que la différentiation, la motilité, la division et la mort cellulaire. La voie de signalisation des « MAPKinases » est typiquement constituée d'un module de trois kinases qui s'activent séquentiellement par phosphorylation. On note la présence d'une MAPK, d'un activateur de MAPK et d'un activateur de l'activateur de MAPK. Une fois la MAPK activée, elle permettra la régulation de différentes cibles dont certain facteur de transcription. Chez les mammifères, il existe 3 grands groupes de MAPKs : the extracellular signal-regulated kinase 1 and 2 (ERK 1/2) cascade, qui régule préférentiellement la croissance et la différentiation cellulaire, ainsi que les cascades JNK et p38 qui régulent préférentiellement la réponse à différents stress cellulaires telle que l'inflammation ou l'apoptose. JNK est activé par différents stress cellulaire telle que les cytokines inflammatoires. JNK est également requis au cours du développement embryonnaire et contribue à la mort (apoptose) ou à la prolifération cellulaire. Plusieurs études ont mis en évidence le rôle de JNK durant le processus tumoral, sans que son rôle soit clairement identifié. JNK pourrait avoir des fonctions différentes durant l'initiation puis de la progression tumorale. Chez les mammifères, les voies de signalisation intracellulaires forment un réseau complexe et elles interagissent entre elles, ce qui permet aux cellules une réponse adéquate aux multitudes de stimuli existants dans les organismes pluricellulaires. Parmi plusieurs mécanismes de régulation, les protéines dites « scaffold » ou «échafaud » jouent un rôle crucial dans l'homéostase de la voie de signalisation des «MAPKinase ». L'introduction revoit brièvement ces différents aspects, de la voie de signalisation des «MAPKinase et des connaissance sur IB1/JIP-1. Les premières études effectuées sur IB1/JIP-1 ont montré une expression relativement spécifique de cette protéine dans certains types de neurones ainsi que dans la cellule beta-sécrétrice d'insuline. IB1/JIP-1 régule la voie de signalisation JNK par interaction avec les différents composants du module, modifiant ainsi le spectre de substrats activés par JNK. La fonction précise de IB1/JIP-1 n'était pas encore élucidée, mais plusieurs travaux mettaient en lumière un rôle dans la régulation, et la sous-location cellulaire des composants de la voie de signalisation JNK, ainsi que dans la survie cellulaire à certain stress. Cette expression relativement spécifique est intrigante car elle suggère que sa présence serait nécessaire à une régulation spécifique de la MAPKinase JNK ou à certaines autres fonctions cellulaires également spécifiques de certains tissus. Le premier but de ce travail a consisté à mettre en évidence l'expression de IB1/JIP-1 dans l'appareil uro-génital et plus particulièrement dans la vessie et la prostate. Nos résultats ont montré que IB1/JIP-1 est spécifiquement exprimé au niveau de l'urothélium vésical, mais pas dans le muscle lisse. Il en est de même au niveau de la prostate où IB1/JIP-1 est exprimé spécifiquement au niveau de l'épithélium sécrétoire et absent au niveau du stroma fibro-musculaire. La vessie et la prostate sont des organes ou l'activité JNK pourrait être crucial tant dans l' homeostase tissulaire que dans le développement de pathologies bénignes ou malignes. La vessie et la prostate sont le siège fréquent de tumeur. La base pour le développement du cancer est complexe et implique plusieurs anomalies génétiques. Ce processus complexe lié au développement tumoral est encore loin d`être complètement élucidé, raison pour laquelle il est crucial de poursuivre l'étude des différents gènes pouvant être impliqué dans ces processus ou pouvant être utilisé comme outil thérapeutique. Dans l'urothelium de la vessie, la fonction de la MAPK JNK n'a été que très peu étudiée. Il existe quelques études, in vitro, suggérant une implication possible de cette voie de signalisation dans des processus telle que le développement ou la progression tumorale. Le chapitre 1 décrit une étude in vivo dans la vessie un modèle de stress mécanique, connu pour activer les MAPKinase. La dilatation vésicale, due à une obstruction urétrale, a mis en évidence une diminution de l'expression de IB1/JIP-1 ainsi qu'une activation de la MAPKinase JNK. Dans ce modèle, la régulation de IB1/JIP-1, par l'intermédiaire d'un vecteur viral, a permis de démontrer que IB1/JIP-1 régulait l'activité de JNK dans ce tissu. Pour poursuivre l'étude de cette fonction d' IB1/JIP-1 dans l'urothélium, nous avons investigué l'activité JNK dans des souris génétiquement modifiées et porteuse d'une délétion de 1 des 2 allèles du gène codant pour IB1/JIP-1, avec un contenu en IB1/JIP-1 diminué de moitié. L'activation de JNK est également augmentée dans l'urothelium au repos de ces souris, ce qui confirme la fonction régulatrice de JNK par IB1/JIP-1. Ces résultats ont permis de mettre en évidence un rôle critique de celle-ci dans l'homéostase de I`urothelium et suggère une nouvelle cible pour réguler la voie de signalisation dans ce tissu. En outre, la modulation des niveaux d'expression d'IB1/JIP-1 dans la vessie, in vivo, par l'intermédiaire de vecteurs viraux s'est révélée réalisable et indique un moyen élégant pour développer une thérapie génique dans cet organe. Un autre élément de ce travail de thèse, révélée au chapitre 2, a été d'étudier la régulation dans la vessie de rat de la communication intercellulaire de type « GAP ». Les cellules adjacentes partagent des ions, messagers secondaires et des petits métabolites par l'intermédiaire de canaux intercellulaire qui forment les jonctions de type « GAP ». Ce type de communications intercellulaire permet une activité cellulaire coordonnée, une caractéristique importante pour l'homéostase des organismes multicellulaire. Ce type de communication intercellulaire est formé de 2 demi-canaux appelés connexons. Chaque connexon est formé de six protéines appelées connexins (Cx). Il existe environ vingt connexines différentes nommées par leur poids moléculaire respectif. Les jonctions de type canaux "GAP" permettent aux cellules de communiquer avec les cellules voisines au quelles elles sont mécaniquement ou électriquement couplées. La vessie peut être particulièrement dépendante de la communication intercellulaire par les canaux « Gap » qui permettrait de coordonner la réponse de la musculature ainsi que de l'urothélium à l'augmentation de la pression transmurale du à l'accumulation d'urine, situation fréquemment observée dans le cadre de l'hyperplasie bénigne de la prostate. Dans la vessie de rat, la connexine26 est exprimée uniquement dans l'urothelium. La Cx26, a été montrée pour être un possible « tumor suppressor gene » dans le cancer de vessie. Une augmentation de la Cx26 ainsi que du couplage des cellules urothéliales a été démontré dans notre modèle de stress mécanique sur la vessie de rat et est dépendante de 2 éléments de réponses connues pour interagir avec AP-1. La régulation de IB1/JIP-1 a permis de montrer que celle-ci régulait l'activité JNK, ainsi que l'activité du facteur de transcription AP-1, composé de c-Jun lui-même cible de JNK. Cette réduction de l'activité de AP-1 est associée à une diminution de l'expression du transcipt de la Cx26. En résumé, la Cx26 pourrait être régulée par le complexe AP-1 lui-même dépendant du contenu en IB1/JIP-1. Dans le chapitre 3, l'étude de IB1/J1P-1 s'est portée sur la prostate. Cet organe, siège fréquent de pathologie telle que le cancer ou l'hyperplasie bénigne de la prostate, exprime IB1/JIP-1 au niveau de son épithélium sécrétoire. Cette expression est maintenue dans une lignée cellulaire humaine largement étudiée est reconnue comme un modèle adéquat de cellules tumorales de type androgène-sensible. IB1/JIP-1 a été investigué dans un modèle in vitro d'apoptose en réponse à un agent appelé N-(4-hydroxyphenyl)retinamide (4-HPR) qui induit une activation de la MAPK JNK ainsi que également un diminution du contenu en IB1/JIP-1. La surexpression de IB1/JIP-1 en utilisant à nouveau des virus comme vecteur a démontré que IB1/JIP-1 était capable de réguler l'activité de JNK ainsi que les taux d'apoptose. Dans le cancer de la prostate, certains travaux ont montré que la différentiation neuroendocrine des cellules tumorales est associée à la progression tumorale et à la perte de sensibilité aux androgènes. Ce travail a permis de dévoiler l'augmentation d'expression de IB1/JIP-1 dans un modèle de neurodifferentiation des cellules d'une lignée prostatique humaine (LNCaP). Les mécanismes qui permettent une expression spécifique de IB1/JIP-1 ont été partiellement investiguée dans notre laboratoire. Son promoteur humain contient un « Neuron Restricive Silencer Element » (NRSE) connu pour se lier a répresseur transcriptionel appelé « RE-1 Silencer Transcription Factor » ou « Neuron Restrictive Silencer Factor » (REST/NRSF). NRSF/REST est capable de réprimer l'expression de gènes neuronaux en dehors du système neuronal. Il prend part à la différentiation terminale des gènes neuronaux. Dans le chapitre 3, on observe que l'activité de REST/NRSF est diminuée dans les cellules LNCaP qui se transdifferencient de manière neuroendocrine, et que REST/NRSF est capable de moduler l'expression de ces gènes cibles dans ce type cellulaire. Ces travaux laissent suggérer que NRSF/REST participe à l'acquisition du phénotype neuroendocrinien et pourrait être une cible pour réguler ce phénomène. En conclusion, ce travail de thèse présente l'expression de IB1/JIP-1 dans 2 organes de l'appareil uro-génital ; la vessie et la prostate. La fonction de IB1/JIP-1 a été étudiée in vivo dans la vessie de rat, ce qui a mis en évidence sa fonction régulatrice de l'activité de la MAPKinase JNK, et de l'activité du facteur de transcription AP-1 ; ainsi que sa possible implication régulatrice de gène cible tel que la Connexin 26 (Cx26). AP-1 et la Cx26 pourraient jouer un rôle dans le processus oncologique, tant dans le control de l'invasion cellulaire ou le control de la croissance cellulaire. Dans la prostate, IB1/JIP-1 régule également l'activité JNK; crucial dans la transmission de certains stimulis pro-apoptotiques. Dans un modèle de transdifférenciation neuroendocrinienne, phénotype possiblement lié au caractère agressif du cancer de la prostate, l'expression de IB1/JIP-1 est augmenté, suggérant soit un rôle possible dans le développement du phénotype neuronal ou une implication dans une fonction anti-apoptotique. Ce travail a donc permis d'élargir nos connaissances sur la régulation et le control de la voie de signalisation des MAPKinases par IB1/JIP-1, qui pourrait avoir encore d'autres fonctions dans ces tissus.