Nutrition, Metabolism and Cardiovascular Diseases, 2013
Muscle wasting prevails with disuse (bedrest and immobilisation) and is associated with many dise... more Muscle wasting prevails with disuse (bedrest and immobilisation) and is associated with many diseases (cancer, sepsis, diabetes, kidney failure, trauma, etc.). This results first in prolonged hospitalisation with associated high health-care costs and second and ultimately in increased morbidity and mortality. The precise characterisation of the signalling pathways leading to muscle atrophy is therefore particularly relevant in clinical settings. Recent major papers have identified highly complex intricate pathways of signalling molecules, which induce the transcription of the muscle-specific ubiquitin protein ligases MAFbx/Atrogin-1 and MuRF1 that are overexpressed in nearly all muscle wasting diseases. These signalling pathways have been targeted with success in animal models of muscle wasting. In particular, these findings have revealed a finely tuned crosstalk between both anabolic and catabolic processes. Whether or not such strategies may be useful for blocking or at least limiting muscle wasting in weight losing and cachectic patients is becoming nowadays a very exciting clinical challenge.
The age-related loss of skeletal muscle mass and function (sarco-penia) is a consistent hallmark ... more The age-related loss of skeletal muscle mass and function (sarco-penia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonu-clei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl trans-ferase-mediated dUTP nick end-labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostain-ing for dystrophin, paired box protein-7 (Pax7) or laminin-2a, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age-related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre-associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7 + satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age-dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing.
The International Journal of Biochemistry & Cell Biology, 2003
Calpain-3 deficiency leads to muscular dystrophy in humans and mice and to perturbation of the NF... more Calpain-3 deficiency leads to muscular dystrophy in humans and mice and to perturbation of the NFkappaB/IkappaB pathway. As this phenotype is mainly atrophic, this study was performed to determine whether protein turnover and/or proteolytic gene expression was altered in muscles following calpain-3 deficiency. In vitro rates of protein turnover and of substrate ubiquitination, cathepsin B and B+L activities, and mRNA levels for several proteolytic genes were measured in skeletal muscles from 4-5 month-old control and calpain-3 knockout mice. Rates of protein synthesis and breakdown, cathepsin activities, and rates of substrate ubiquitination remained stable in muscles from calpain-3 deficient mice. However, and surprisingly, mRNA levels for cathepsin L, the 14-kDa ubiquitin-conjugating enzyme E2, and the C2 subunit of the 20S proteasome decreased by approximately 47% (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.005) in the gastrocnemius muscle from calpain-3 deficient mice. In contrast, muscle mRNA levels for ubiquitin and subunit S5a of the 26S proteasome were unaffected by calpain-3 deficiency. Taken together these data demonstrate that the expression of some genes that are involved in distinct proteolytic pathways is selectively and coordinately down-regulated without any effect on proteolysis. This suggests new pathophysiological hypotheses, e.g. a lack of maturation of NFkappaB precursor and/or a defect in specific substrate targeting.
We tested the hypothesis that skeletal muscle ubiquitin-proteasome-dependent proteolysis is dysre... more We tested the hypothesis that skeletal muscle ubiquitin-proteasome-dependent proteolysis is dysregulated in ageing in response to feeding. In Experiment 1 we measured rates of proteasome-dependent proteolysis in incubated muscles from 8- and 22-month-old rats, proteasome activities, and rates of ubiquitination, in the postprandial and postabsorptive states. Peptidase activities of the proteasome decreased in the postabsorptive state in 22-month-old rats compared with 8-month-old animals, while the rate of ubiquitination was not altered. Furthermore, the down-regulation of in vitro proteasome-dependent proteolysis that prevailed in the postprandial state in 8-month-old rats was defective in 22-month-old rats. Next, we tested the hypothesis that the ingestion of a 5% leucine-supplemented diet may correct this defect. Leucine supplementation restored the postprandial inhibition of in vitro proteasome-dependent proteolysis in 22-month-old animals, by down-regulating both rates of ubiqui...
Skeletal muscle proteolysis is inhibited by oral feeding in the young and mature but not in the e... more Skeletal muscle proteolysis is inhibited by oral feeding in the young and mature but not in the elderly. However, the proteolytic pathway(s) responsible for the decreased muscle proteolysis in the postprandial (PP) state is (are) unknown in the young. Moreover, muscle proteolysis is inhibited by both insulin (INS) and amino acids (AA) in vitro, but their respective roles on specific proteolytic pathways in vivo remain to be elucidated. The aim of this study was to investigate the respective role of INS and AA on the inhibition of proteolytic pathways in the PP state in skeletal muscles from young, mature and old rats. Rats were fed over 1 h either a 25% (AA+) or a 0% (AA-) amino acid/protein meal. In each nutritional condition, PP insulin secretion was maintained (AA+/INS+ and AA-/INS+) or blocked (AA+/INS- and AA-/INS-) with diazoxide injections. We report that the PP inhibition of proteolysis in young rats was mediated by the increased INS secretion and resulted from a down-regulation of both lysosomal and Ca(2+)-dependent proteolysis. Moreover, our data showed that proteasome activities are inhibited by either INS or AA in mature rats, whereas they become selectively insensitive to AA in old rats. In conclusion, the present work provides direct evidence that the lack of PP regulation of proteasome-dependent proteolysis in old rats resulted from a selective resistance to AA.
The International Journal of Biochemistry & Cell Biology, 2005
Lysosomal proteases are abundantly expressed in fetal muscles, but poorly represented in the adul... more Lysosomal proteases are abundantly expressed in fetal muscles, but poorly represented in the adult skeletal muscles. The lysosomal proteolytic system is nonetheless stimulated in adult muscles in a variety of pathological conditions. Furthermore, recent investigations describe autophagosomes in muscle fibers in vitro and in vivo, and report myopathies with excessive autophagy. This review presents our current knowledge about the lysosomal proteolytic system and summarizes the evidences pertaining to the role of lysosomes and autophagosomes in muscle physiology and pathology.
The international journal of biochemistry & cell biology, 2005
We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and s... more We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery ...
The ubiquitin-proteasome system (UPS) is believed to degrade the major contractile skeletal muscl... more The ubiquitin-proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin-protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.
The immobilization-induced tibialis anterior (TA) muscle atrophy worsens after cast removal and i... more The immobilization-induced tibialis anterior (TA) muscle atrophy worsens after cast removal and is associated with altered extracellular matrix (ECM) composition. The secreted protein acidic and rich in cysteine (Sparc) is an ECM component involved in Akt activation and in β-catenin stabilization, which controls protein turnover and induces muscle regulatory factors (MRFs), respectively. We hypothesized that ECM alterations may influence these intracellular signalling pathways controlling TA muscle mass. Six-month-old Wistar rats were subjected to hindlimb cast immobilization for 8 days (I8) or not (I0) and allowed to recover for 1 to 10 days (R1-10). The TA atrophy during remobilization correlated with reduced fibre cross-sectional area and thickening of endomysium. mRNA levels for Sparc increased during remobilization until R10 and for integrin-α7 and -β1 at I8 and R1. Integrin-linked kinase protein levels increased during immobilization and remobilization until R10. This was inve...
Nutrition, Metabolism and Cardiovascular Diseases, 2013
Muscle wasting prevails with disuse (bedrest and immobilisation) and is associated with many dise... more Muscle wasting prevails with disuse (bedrest and immobilisation) and is associated with many diseases (cancer, sepsis, diabetes, kidney failure, trauma, etc.). This results first in prolonged hospitalisation with associated high health-care costs and second and ultimately in increased morbidity and mortality. The precise characterisation of the signalling pathways leading to muscle atrophy is therefore particularly relevant in clinical settings. Recent major papers have identified highly complex intricate pathways of signalling molecules, which induce the transcription of the muscle-specific ubiquitin protein ligases MAFbx/Atrogin-1 and MuRF1 that are overexpressed in nearly all muscle wasting diseases. These signalling pathways have been targeted with success in animal models of muscle wasting. In particular, these findings have revealed a finely tuned crosstalk between both anabolic and catabolic processes. Whether or not such strategies may be useful for blocking or at least limiting muscle wasting in weight losing and cachectic patients is becoming nowadays a very exciting clinical challenge.
The age-related loss of skeletal muscle mass and function (sarco-penia) is a consistent hallmark ... more The age-related loss of skeletal muscle mass and function (sarco-penia) is a consistent hallmark of ageing. Apoptosis plays an important role in muscle atrophy, and the intent of this study was to specify whether apoptosis is restricted to myofibre nuclei (myonu-clei) or occurs in satellite cells or stromal cells of extracellular matrix (ECM). Sarcopenia in mouse gastrocnemius muscle was characterized by myofibre atrophy, oxidative type grouping, delocalization of myonuclei and ECM fibrosis. Terminal deoxynucleotidyl trans-ferase-mediated dUTP nick end-labelling (TUNEL) indicated a sharp rise in apoptosis during ageing. TUNEL coupled with immunostain-ing for dystrophin, paired box protein-7 (Pax7) or laminin-2a, respectively, was used to identify apoptosis in myonuclei, satellite cells and stromal cells. In adult muscle, apoptosis was not detected in myofibres, but was restricted to stromal cells. Moreover, the age-related rise in apoptotic nuclei was essentially due to stromal cells. Myofibre-associated apoptosis nevertheless occurred in old muscle, but represented < 20% of the total muscle apoptosis. Specifically, apoptosis in old muscle affected a small proportion (0.8%) of the myonuclei, but a large part (46%) of the Pax7 + satellite cells. TUNEL coupled with CD31 immunostaining further attributed stromal apoptosis to capillary endothelial cells. Age-dependent rise in apoptotic capillary endothelial cells was concomitant with altered levels of key angiogenic regulators, perlecan and a perlecan domain V (endorepellin) proteolytic product. Collectively, our results indicate that sarcopenia is associated with apoptosis of satellite cells and impairment of capillary functions, which is likely to contribute to the decline in muscle mass and functionality during ageing.
The International Journal of Biochemistry & Cell Biology, 2003
Calpain-3 deficiency leads to muscular dystrophy in humans and mice and to perturbation of the NF... more Calpain-3 deficiency leads to muscular dystrophy in humans and mice and to perturbation of the NFkappaB/IkappaB pathway. As this phenotype is mainly atrophic, this study was performed to determine whether protein turnover and/or proteolytic gene expression was altered in muscles following calpain-3 deficiency. In vitro rates of protein turnover and of substrate ubiquitination, cathepsin B and B+L activities, and mRNA levels for several proteolytic genes were measured in skeletal muscles from 4-5 month-old control and calpain-3 knockout mice. Rates of protein synthesis and breakdown, cathepsin activities, and rates of substrate ubiquitination remained stable in muscles from calpain-3 deficient mice. However, and surprisingly, mRNA levels for cathepsin L, the 14-kDa ubiquitin-conjugating enzyme E2, and the C2 subunit of the 20S proteasome decreased by approximately 47% (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.005) in the gastrocnemius muscle from calpain-3 deficient mice. In contrast, muscle mRNA levels for ubiquitin and subunit S5a of the 26S proteasome were unaffected by calpain-3 deficiency. Taken together these data demonstrate that the expression of some genes that are involved in distinct proteolytic pathways is selectively and coordinately down-regulated without any effect on proteolysis. This suggests new pathophysiological hypotheses, e.g. a lack of maturation of NFkappaB precursor and/or a defect in specific substrate targeting.
We tested the hypothesis that skeletal muscle ubiquitin-proteasome-dependent proteolysis is dysre... more We tested the hypothesis that skeletal muscle ubiquitin-proteasome-dependent proteolysis is dysregulated in ageing in response to feeding. In Experiment 1 we measured rates of proteasome-dependent proteolysis in incubated muscles from 8- and 22-month-old rats, proteasome activities, and rates of ubiquitination, in the postprandial and postabsorptive states. Peptidase activities of the proteasome decreased in the postabsorptive state in 22-month-old rats compared with 8-month-old animals, while the rate of ubiquitination was not altered. Furthermore, the down-regulation of in vitro proteasome-dependent proteolysis that prevailed in the postprandial state in 8-month-old rats was defective in 22-month-old rats. Next, we tested the hypothesis that the ingestion of a 5% leucine-supplemented diet may correct this defect. Leucine supplementation restored the postprandial inhibition of in vitro proteasome-dependent proteolysis in 22-month-old animals, by down-regulating both rates of ubiqui...
Skeletal muscle proteolysis is inhibited by oral feeding in the young and mature but not in the e... more Skeletal muscle proteolysis is inhibited by oral feeding in the young and mature but not in the elderly. However, the proteolytic pathway(s) responsible for the decreased muscle proteolysis in the postprandial (PP) state is (are) unknown in the young. Moreover, muscle proteolysis is inhibited by both insulin (INS) and amino acids (AA) in vitro, but their respective roles on specific proteolytic pathways in vivo remain to be elucidated. The aim of this study was to investigate the respective role of INS and AA on the inhibition of proteolytic pathways in the PP state in skeletal muscles from young, mature and old rats. Rats were fed over 1 h either a 25% (AA+) or a 0% (AA-) amino acid/protein meal. In each nutritional condition, PP insulin secretion was maintained (AA+/INS+ and AA-/INS+) or blocked (AA+/INS- and AA-/INS-) with diazoxide injections. We report that the PP inhibition of proteolysis in young rats was mediated by the increased INS secretion and resulted from a down-regulation of both lysosomal and Ca(2+)-dependent proteolysis. Moreover, our data showed that proteasome activities are inhibited by either INS or AA in mature rats, whereas they become selectively insensitive to AA in old rats. In conclusion, the present work provides direct evidence that the lack of PP regulation of proteasome-dependent proteolysis in old rats resulted from a selective resistance to AA.
The International Journal of Biochemistry & Cell Biology, 2005
Lysosomal proteases are abundantly expressed in fetal muscles, but poorly represented in the adul... more Lysosomal proteases are abundantly expressed in fetal muscles, but poorly represented in the adult skeletal muscles. The lysosomal proteolytic system is nonetheless stimulated in adult muscles in a variety of pathological conditions. Furthermore, recent investigations describe autophagosomes in muscle fibers in vitro and in vivo, and report myopathies with excessive autophagy. This review presents our current knowledge about the lysosomal proteolytic system and summarizes the evidences pertaining to the role of lysosomes and autophagosomes in muscle physiology and pathology.
The international journal of biochemistry & cell biology, 2005
We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and s... more We studied the role of the ubiquitin-proteasome system in rat skeletal muscle during sepsis and subsequent recovery. Sepsis was induced with intraperitoneal zymosan injections. This model allows one to study a sustained and reversible catabolic phase and mimics the events that prevail in septic and subsequently recovering patients. In addition, the role of the ubiquitin-proteasome system during muscle recovery is poorly documented. There was a trend for increased ubiquitin-conjugate formation in the muscle wasting phase, which was abolished during the recovery phase. The trypsin- and chymotrypsin-like peptidase activities of the 20S proteasome peaked at day 6 following zymosan injection (i.e. when both muscle mass and muscle fiber cross-sectional area were reduced the most), but remained elevated when muscle mass and muscle fiber cross-sectional area were recovering (11 days). This clearly suggests a role for the ubiquitin-proteasome pathway in the muscle remodeling and/or recovery ...
The ubiquitin-proteasome system (UPS) is believed to degrade the major contractile skeletal muscl... more The ubiquitin-proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin-protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.
The immobilization-induced tibialis anterior (TA) muscle atrophy worsens after cast removal and i... more The immobilization-induced tibialis anterior (TA) muscle atrophy worsens after cast removal and is associated with altered extracellular matrix (ECM) composition. The secreted protein acidic and rich in cysteine (Sparc) is an ECM component involved in Akt activation and in β-catenin stabilization, which controls protein turnover and induces muscle regulatory factors (MRFs), respectively. We hypothesized that ECM alterations may influence these intracellular signalling pathways controlling TA muscle mass. Six-month-old Wistar rats were subjected to hindlimb cast immobilization for 8 days (I8) or not (I0) and allowed to recover for 1 to 10 days (R1-10). The TA atrophy during remobilization correlated with reduced fibre cross-sectional area and thickening of endomysium. mRNA levels for Sparc increased during remobilization until R10 and for integrin-α7 and -β1 at I8 and R1. Integrin-linked kinase protein levels increased during immobilization and remobilization until R10. This was inve...
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