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- Chaillou, Thomas, 1985-, et al.
(author)
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Pitfalls in target mRNA quantification for real-time quantitative RT-PCR in overload-induced skeletal muscle hypertrophy
- 2011
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In: Physiological Genomics. - Bethesda, USA : American Physiological Society. - 1094-8341 .- 1531-2267. ; 43:4, s. 228-235
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Journal article (peer-reviewed)abstract
- Quantifying target mRNA using real-time quantitative reverse transcription-polymerase chain reaction requires an accurate normalization method. Determination of normalization factors (NFs) based on validated reference genes according to their relative stability is currently the best standard method in most usual situations. This method controls for technical errors, but its physiological relevance requires constant NF values for a fixed weight of tissue. In the functional overload model, the increase in the total RNA concentration must be considered in determining the NF values. Here, we pointed out a limitation of the classical geNorm-derived normalization. geNorm software selected reference genes despite that the NF values extensively varied under experiment. Only the NF values calculated from four intentionally selected genes were constant between groups. However, a normalization based on these genes is questionable. Indeed, three out of four genes belong to the same functional class (negative regulator of muscle mass), and their use is physiological nonsense in a hypertrophic model. Thus, we proposed guidelines for optimizing target mRNA normalization and quantification, useful in models of muscle mass modulation. In our study, the normalization method by multiple reference genes was not appropriate to compare target mRNA levels between overloaded and control muscles. A solution should be to use an absolute quantification of target mRNAs per unit weight of tissue, without any internal normalization. Even if the technical variations will stay present as a part of the intergroup variations, leading to less statistical power, we consider this method acceptable because it will not generate misleading results.
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| 4. |
- Cheng, A, et al.
(author)
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Increased fatigue resistance and preserved specific force in intact single muscle fibres from the SOD1G93A mouse model of ALS
- 2017
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In: Acta Physiologica. - : Wiley-Blackwell. - 1748-1708 .- 1748-1716. ; 219:S710, s. 17-17
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Journal article (peer-reviewed)abstract
- Introduction: Amyotrophic lateral sclerosis (ALS) is a motor neurone disease characterized by degeneration and loss of motor neurones, leading to severe muscle weakness and paralysis. Although motor neurone degeneration is already a well-characterized symptom that contributes to muscle weakness in the SOD1G93A mouse model of ALS, the purpose of the current study was to determine whether muscle weakness in ALS can be attributed to impaired intrinsic force generation in skeletal muscles of SOD1G93A mice.Methods: Experiments were performed on whole muscles and mechanically dissected intact single fibres from the flexor digitorum brevis (FDB) muscle of SOD1G93A mice at three age groups of 50, 125 and 150 days of age (P50, P125 and P150). Myoplasmic free [Ca2+] ([Ca2+]i) was measured using the fluorescent indicator, indo-1.Results: Motor neurone loss and decreased force were evident in whole FDB muscles of P125–150 mice. In the intact single muscle fibres however, specific force, tetanic [Ca2+]iand resting [Ca2+]i were similar in single FDB fibres from symptomatic P125–150 SOD1G93A and age-matched wild-type littermates. The most intriguing finding was a markedly greater fatigue resistance in single fibres from P125–150 SOD1G93A vs. wild-type mice, which was not present in asymptomatic young P50 SOD1G93A mice. No shift in fibre-type distribution was observed in whole FDB muscles to explain the increased fatigue resistance of single fibres from P125–150 SOD1G93A mice.Conclusion: These results support the hypothesis that muscle weakness in ALS is not attributed to intrinsicdefects in skeletal muscle fibre force generation.
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- Ferreira, Duarte M. S., et al.
(author)
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LIM and cysteine-rich domains 1 (LMCD1) regulates skeletal muscle hypertrophy, calcium handling, and force
- 2019
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In: Skeletal Muscle. - : BioMed Central. - 2044-5040. ; 9:1
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Journal article (peer-reviewed)abstract
- Background: Skeletal muscle mass and strength are crucial determinants of health. Muscle mass loss is associated with weakness, fatigue, and insulin resistance. In fact, it is predicted that controlling muscle atrophy can reduce morbidity and mortality associated with diseases such as cancer cachexia and sarcopenia.Methods: We analyzed gene expression data from muscle of mice or human patients with diverse muscle pathologies and identified LMCD1 as a gene strongly associated with skeletal muscle function. We transiently expressed or silenced LMCD1 in mouse gastrocnemius muscle or in mouse primary muscle cells and determined muscle/cell size, targeted gene expression, kinase activity with kinase arrays, protein immunoblotting, and protein synthesis levels. To evaluate force, calcium handling, and fatigue, we transduced the flexor digitorum brevis muscle with a LMCD1-expressing adenovirus and measured specific force and sarcoplasmic reticulum Ca2+ release in individual fibers. Finally, to explore the relationship between LMCD1 and calcineurin, we ectopically expressed Lmcd1 in the gastrocnemius muscle and treated those mice with cyclosporine A (calcineurin inhibitor). In addition, we used a luciferase reporter construct containing the myoregulin gene promoter to confirm the role of a LMCD1-calcineurin-myoregulin axis in skeletal muscle mass control and calcium handling.Results: Here, we identify LIM and cysteine-rich domains 1 (LMCD1) as a positive regulator of muscle mass, that increases muscle protein synthesis and fiber size. LMCD1 expression in vivo was sufficient to increase specific force with lower requirement for calcium handling and to reduce muscle fatigue. Conversely, silencing LMCD1 expression impairs calcium handling and force, and induces muscle fatigue without overt atrophy. The actions of LMCD1 were dependent on calcineurin, as its inhibition using cyclosporine A reverted the observed hypertrophic phenotype. Finally, we determined that LMCD1 represses the expression of myoregulin, a known negative regulator of muscle performance. Interestingly, we observed that skeletal muscle LMCD1 expression is reduced in patients with skeletal muscle disease.Conclusions: Our gain- and loss-of-function studies show that LMCD1 controls protein synthesis, muscle fiber size, specific force, Ca2+ handling, and fatigue resistance. This work uncovers a novel role for LMCD1 in the regulation of skeletal muscle mass and function with potential therapeutic implications.
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- Liu, Zhengye, et al.
(author)
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Mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force
- 2021
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In: The FASEB Journal. - : John Wiley & Sons. - 0892-6638 .- 1530-6860. ; 35:12
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Journal article (peer-reviewed)abstract
- The hypoxia-inducible nuclear-encoded mitochondrial protein NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2) has been demonstrated to decrease oxidative phosphorylation and production of reactive oxygen species in neonatal cardiomyocytes, brain tissue and hypoxic domains of cancer cells. Prolonged local hypoxia can negatively affect skeletal muscle size and tissue oxidative capacity. Although skeletal muscle is a mitochondrial rich, oxygen sensitive tissue, the role of NDUFA4L2 in skeletal muscle has not previously been investigated. Here we ectopically expressed NDUFA4L2 in mouse skeletal muscles using adenovirus-mediated expression and in vivo electroporation. Moreover, femoral artery ligation (FAL) was used as a model of peripheral vascular disease to induce hind limb ischemia and muscle damage. Ectopic NDUFA4L2 expression resulted in reduced mitochondrial respiration and reactive oxygen species followed by lowered AMP, ADP, ATP, and NAD(+) levels without affecting the overall protein content of the mitochondrial electron transport chain. Furthermore, ec-topically expressed NDUFA4L2 caused a similar to 20% reduction in muscle mass that resulted in weaker muscles. The loss of muscle mass was associated with increased gene expression of atrogenes MurF1 and Mul1, and apoptotic genes caspase 3 and Bax. Finally, we showed that NDUFA4L2 was induced by FAL and that the Ndufa4l2 mRNA expression correlated with the reduced capacity of the muscle to generate force after the ischemic insult. These results show, for the first time, that mitochondrial NDUFA4L2 is a novel regulator of skeletal muscle mass and force. Specifically, induced NDUFA4L2 reduces mitochondrial activity leading to lower levels of important intramuscular metabolites, including adenine nucleotides and NAD(+), which are hallmarks of mitochondrial dysfunction and hence shows that dysfunctional mitochondrial activity may drive muscle wasting.
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| 8. |
- Pugnière, P, et al.
(author)
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Pitfalls of reverse transcription quantitative polymerase chain reaction standardization : Volume-related inhibitors of reverse transcription
- 2011
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In: Analytical Biochemistry. - : Academic Press. - 0003-2697 .- 1096-0309. ; 415:2, s. 151-157
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Journal article (peer-reviewed)abstract
- A large part of the reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) data depends on technical variations. Such variations are mainly attributable to the reverse transcription step. Standardization is a key factor in decreasing the intersample variability. However, an ideal standardization is not always possible, and compromises must be found. Due to technical requirements, the current consensus is that a constant amount of total RNA should be used for the RT step (CA-RT). Because RNA isolation yields are variable, such a practice requires the use of variable volumes of nucleic acid extracts in RT reaction. We demonstrate that some RNA extracts contain both exogenous and endogenous inhibitors. These inhibitors induce a decrease in RT efficiency that significantly impairs the reliability of RT-qPCR data. Conversely, these inhibitors have a slight effect on the qPCR step. To overcome such drawbacks, we proposed to carry out the RT reaction with a constant volume of RNA extract by preserving a constant RNA amount through the supplementation of yeast transfer RNA (CV-RT). We show that CV-RT, compared with the usual CA-RT, allows us to decrease the RT-qPCR variability induced by intersample differences. Such a decrease is a prerequisite for the reliability of messenger RNA quantification.
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| 9. |
- Pugnière, P., et al.
(author)
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Volume-related inhibitors standardization for reverse transcription quantitative polymerase chain reaction experiments
- 2012
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Conference paper (peer-reviewed)abstract
- A large part of the reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) data depends on technical variations. Such variations are mainly attributable to the reverse transcription step. Standardization is a key factor in decreasing the intersample variability. However, an ideal standardization is not always possible, and compromises must be found. Due to technical requirements, the current consensus is that a constant amount of total RNA should be used for the RT step (CA-RT). Because RNA isolation yields are variable, such a practice requires the use of variable volumes of nucleic acid extracts in RT reaction. We demonstrate that some RNA extracts contain both exogenous and endogenous inhibitors. These inhibitors induce a decreased RT efficiency that significantly impairs the reliability of RT-qPCR data. Conversely, these inhibitors have slight effects on the qPCR step. To overcome such drawbacks, we proposed to carry out the RT reaction with a constant volume of RNA extract by preserving a constant RNA amount through the supplementation of yeast transfer RNA (CV-RT). We show that CV-RT, compared with the usual CA-RT, allows us to decrease the RT-qPCR variability induced by intersample differences. Such a decrease is a prerequisite for the reliability of messenger RNA quantification.
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