Lipid hydroperoxides promote sarcopenia through carbonyl stress
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eLife assessment
This important paper advances our understanding of the role of lipid peroxidation in loss of muscle mass and force-generating capacity during aging and hind-limb suspension. The evidence is in general solid, drawing from experiments in vivo and cell culture using multiple types of manipulations of the formation of lipid peroxides although some weaknesses were identified. The results should be of interest to those who study the molecular basis for sarcopenia and disuse atrophy of skeletal muscle.
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Abstract
Reactive oxygen species (ROS) accumulation is a cardinal feature of skeletal muscle atrophy. ROS refers to a collection of radical molecules whose cellular signals are vast, and it is unclear which downstream consequences of ROS are responsible for the loss of muscle mass and strength. Here, we show that lipid hydroperoxides (LOOH) are increased with age and disuse, and the accumulation of LOOH by deletion of glutathione peroxidase 4 (GPx4) is sufficient to augment muscle atrophy. LOOH promoted atrophy in a lysosomal-dependent, proteasomal-independent manner. In young and old mice, genetic and pharmacological neutralization of LOOH or their secondary reactive lipid aldehydes robustly prevented muscle atrophy and weakness, indicating that LOOH-derived carbonyl stress mediates age- and disuse-induced muscle dysfunction. Our findings provide novel insights for the role of LOOH in sarcopenia including a therapeutic implication by pharmacological suppression.
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Author Response
Reviewer #1 (Public Review):
In this paper, the authors present evidence from studies of biopsies from human subject and muscles from young and older mice that the enzyme glutathione peroxidase 4 (GPx4) is expressed at reduced levels in older organisms associated with elevated levels of lipid peroxides. A series of studies in mice established that genetic reduction of GPx4 and hindlimb unloading each elevated lipid peroxide levels and reduced muscle contractility in young animals. Overexpression of GPx4 or N- acetylcarnosine blocked atrophy and loss of force generating capacity resulting from hindlimb unloading in young mice. Cell culture experiments in C2C12 myotubes were used to develop evidence linking elevated lipid peroxide levels to atrophy using genetic and pharmacologic approaches. Links between autophagy and …
Author Response
Reviewer #1 (Public Review):
In this paper, the authors present evidence from studies of biopsies from human subject and muscles from young and older mice that the enzyme glutathione peroxidase 4 (GPx4) is expressed at reduced levels in older organisms associated with elevated levels of lipid peroxides. A series of studies in mice established that genetic reduction of GPx4 and hindlimb unloading each elevated lipid peroxide levels and reduced muscle contractility in young animals. Overexpression of GPx4 or N- acetylcarnosine blocked atrophy and loss of force generating capacity resulting from hindlimb unloading in young mice. Cell culture experiments in C2C12 myotubes were used to develop evidence linking elevated lipid peroxide levels to atrophy using genetic and pharmacologic approaches. Links between autophagy and atrophy were suggested.
Experiments on GPx4 expression levels, lipid peroxide levels, muscle mass and muscle force generating capacity were internally consistent and convincing. I thought the experiments supporting the view that autophagy contributed to atrophy were convincing. The hypothesis that altered lipidation of autophagy factors contributed was tested or supported in my view. Evidence for muscle atrophy in response to genetic or pharmacologic manipulations is a bit inconsistent throughout the paper, possibly because the small N of some experiments does not provide sufficient power to detect observed numeric differences in the means. The pattern of muscle fiber atrophy by fiber type is consistent throughout the paper but there is variability in which comparisons reached the threshold for significance, again, possibly because of the small N of the experiments. I agree with the authors that altered activity of enzymes in the contractile apparatus provides one explanation for the observed weakness but respectfully wish to point out there are others such as impaired excitation-contraction coupling which is well known to occur in aging.
We thank Dr. Cardozo for taking time to carefully review our manuscript, and for providing an enthusiastic feedback for the significance of our work. We are grateful for additional suggestions and modified our manuscript accordingly.
Reviewer #2 (Public Review):
This is a well-written paper that reports that the accumulation of LOOH with age and disuse contributes to the loss of skeletal muscle mass and strength. Moreover, the authors report that LOOH neutralization attenuates muscle atrophy and weakness. The mechanism via which LOOH contributes to these phenotypes remains unclear but seems to be mediated by the autophagy- lysosomal axis. In addition, the paper also reports the efficacy of N-acetylcarnosine treatment in ameliorating muscle atrophy in mice.
We thank the reviewer 2 for their positive response to our manuscript. Very much appreciated! Below please find our response to your specific comments.
The authors should consider the following points to improve the manuscript:
- The authors showed that inhibition of the autophagy-lysosome axis by ATG3 deletion or BafA1 was sufficient to reduce LOOH levels induced by GPx4 deletion, erastin, or RSL3. Moreover, they found that 4-HNE co-localizes with LAMP2. However, it remains unclear the precise mechanism via which LOOH contributes to muscle atrophy and how it is amplified by the autophagy-lysosomal axis. The authors could further test the functional interaction of 4-HNE with LAMP2 with additional experiments such as immunoprecipitation.
Thank you for these comments. We agree with the reviewer that our observations on autophagy-lysosomal axis is yet backed by a tangible mechanism. To clarify, we only show 4HNE and LAMP2 colocalization to show that they are proximate to each other. We do not necessarily claim that LAMP2 is the protein that becomes 4-HNE-ylated. We are currently developing a proteomic platform to detect 4-HNE conjugations on peptides, and this should hopefully shed light to the nature of interaction between LOOH and the autophagy-lysosomal axis. We now include additional discussion on autophagy-lysosomal axis with LOOH in lines 280-291.
- A weak point of the paper is not having performed the experiments on 24-month-old-mice. At 20 months of age, the mice do not display any muscle wasting and myofiber atrophy compared to young mice that have completed postnatal muscle growth (=6-month-old-mice). It would be interesting to see the levels of 4-HNE in 24- or 30-month-old mice, and if N-acetylcarnosine treatment in older mice is able to rescue muscle atrophy induced by aging.
This is a nuanced but a very important point. We initially set out to study mice in the 24 months old mice, but these mice did not tolerate the hindlimb unloading procedure well and ended up using the 20 months old mice instead. While mice at this age tolerated our HU procedure well, they did not manifest significant reduction in muscle mass compared to young. We included additional discussions in lines 298-300 and 310-314. To address this point, we are currently performing a 6-month N-acetylcarnosine intervention in 24 months old mice, and examine the effect that this compound has on the effect of aging (without HU) in multiple organ systems. We have thus completed 2 cohorts for this preclinical trial. Results on the effects of long-term N- acetylcarnosine treatment on muscle will be included in the separate manuscript.
Previous studies have shown that inhibition of autophagy accelerates (rather than protect) from sarcopenia, and that autophagy is required to maintain muscle mass (Masiero 2009, PMID: 19945408; Castets 2013, PMID: 23602450; Carnio 2014, PMID: 25176656). On this basis, the authors should test whether their findings are valid only in the context of disuse atrophy or also in the context of sarcopenia (=24-30-month-old mice).
We agree with the reviewer that the role of autophagy and muscle mass is likely complex. In the current study, we only showed that a SHORT-TERM inhibition of autophagy by ATG3 deletion prevents muscle atrophy induced by a SHORT-TERM disuse intervention. Inhibition of autophagic machinery long-term will likely be detrimental, and as shown in references provided by the reviewer, accelerates sarcopenia. We now include these discussions in lines 280-287. We respectfully request that the experiments in 24-30 month old ATG3-MKO mice be beyond the scope of the study. As discussed above, there is much more to study regarding the nature of interaction between the autophagy-lysosomal axis and LOOH.
- In Fig.2 the authors report that GPx4 KD, erastin, and RSL3 reduce the diameter of myotubes. For how long and when was the treatment done? Looking at the images, it seems that there are some myoblasts in the cultures treated with GPx4 KD, erastin, and RSL3. Is it possible that these compounds reduce myotube size by inhibiting myoblast fusion rather than by inducing myotube atrophy?
Thank you for point this out. We now provide further details in the method section (lines 439- 443). For KD experiments, we treat myoblasts with virus simultaneous to differentiation, due to lower infection efficiency in myotubes. This is certainly a caveat. However, erastin and RSL3 experiments were done on fully differentiated myotubes. It is common to have non- differentiated myoblasts under differentiated myotubes.
- MDA quantification was done in the gastrocnemius although all the experiments in this paper were performed in the soleus and EDL. It would be good if the authors could explain the reason for this.
MDA and 4-HNE WB were done on gastroc for all mouse models because some soleus and EDL muscles are below 7 mg and provided insufficient materials to perform MDA or 4-HNE. Soleus and EDL were used for contractile experiments (gastr0c cannot be used for this experiment) and for histological analyses.
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eLife assessment
This important paper advances our understanding of the role of lipid peroxidation in loss of muscle mass and force-generating capacity during aging and hind-limb suspension. The evidence is in general solid, drawing from experiments in vivo and cell culture using multiple types of manipulations of the formation of lipid peroxides although some weaknesses were identified. The results should be of interest to those who study the molecular basis for sarcopenia and disuse atrophy of skeletal muscle.
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Reviewer #1 (Public Review):
In this paper, the authors present evidence from studies of biopsies from human subject and muscles from young and older mice that the enzyme glutathione peroxidase 4 (GPx4) is expressed at reduced levels in older organisms associated with elevated levels of lipid peroxides. A series of studies in mice established that genetic reduction of GPx4 and hindlimb unloading each elevated lipid peroxide levels and reduced muscle contractility in young animals. Overexpression of GPx4 or N-acetylcarnosine blocked atrophy and loss of force generating capacity resulting from hindlimb unloading in young mice. Cell culture experiments in C2C12 myotubes were used to develop evidence linking elevated lipid peroxide levels to atrophy using genetic and pharmacologic approaches. Links between autophagy and atrophy were …
Reviewer #1 (Public Review):
In this paper, the authors present evidence from studies of biopsies from human subject and muscles from young and older mice that the enzyme glutathione peroxidase 4 (GPx4) is expressed at reduced levels in older organisms associated with elevated levels of lipid peroxides. A series of studies in mice established that genetic reduction of GPx4 and hindlimb unloading each elevated lipid peroxide levels and reduced muscle contractility in young animals. Overexpression of GPx4 or N-acetylcarnosine blocked atrophy and loss of force generating capacity resulting from hindlimb unloading in young mice. Cell culture experiments in C2C12 myotubes were used to develop evidence linking elevated lipid peroxide levels to atrophy using genetic and pharmacologic approaches. Links between autophagy and atrophy were suggested.
Experiments on GPx4 expression levels, lipid peroxide levels, muscle mass and muscle force generating capacity were internally consistent and convincing. I thought the experiments supporting the view that autophagy contributed to atrophy were convincing. The hypothesis that altered lipidation of autophagy factors contributed was tested or supported in my view. Evidence for muscle atrophy in response to genetic or pharmacologic manipulations is a bit inconsistent throughout the paper, possibly because the small N of some experiments does not provide sufficient power to detect observed numeric differences in the means. The pattern of muscle fiber atrophy by fiber type is consistent throughout the paper but there is variability in which comparisons reached the threshold for significance, again, possibly because of the small N of the experiments. I agree with the authors that altered activity of enzymes in the contractile apparatus provides one explanation for the observed weakness but respectfully wish to point out there are others such as impaired excitation-contraction coupling which is well known to occur in aging.
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Reviewer #2 (Public Review):
This is a well-written paper that reports that the accumulation of LOOH with age and disuse contributes to the loss of skeletal muscle mass and strength. Moreover, the authors report that LOOH neutralization attenuates muscle atrophy and weakness. The mechanism via which LOOH contributes to these phenotypes remains unclear but seems to be mediated by the autophagy-lysosomal axis. In addition, the paper also reports the efficacy of N-acetylcarnosine treatment in ameliorating muscle atrophy in mice.
The authors should consider the following points to improve the manuscript:
- The authors showed that inhibition of the autophagy-lysosome axis by ATG3 deletion or BafA1 was sufficient to reduce LOOH levels induced by GPx4 deletion, erastin, or RSL3. Moreover, they found that 4-HNE co-localizes with LAMP2. However, …
Reviewer #2 (Public Review):
This is a well-written paper that reports that the accumulation of LOOH with age and disuse contributes to the loss of skeletal muscle mass and strength. Moreover, the authors report that LOOH neutralization attenuates muscle atrophy and weakness. The mechanism via which LOOH contributes to these phenotypes remains unclear but seems to be mediated by the autophagy-lysosomal axis. In addition, the paper also reports the efficacy of N-acetylcarnosine treatment in ameliorating muscle atrophy in mice.
The authors should consider the following points to improve the manuscript:
- The authors showed that inhibition of the autophagy-lysosome axis by ATG3 deletion or BafA1 was sufficient to reduce LOOH levels induced by GPx4 deletion, erastin, or RSL3. Moreover, they found that 4-HNE co-localizes with LAMP2. However, it remains unclear the precise mechanism via which LOOH contributes to muscle atrophy and how it is amplified by the autophagy-lysosomal axis. The authors could further test the functional interaction of 4-HNE with LAMP2 with additional experiments such as immunoprecipitation.
- A weak point of the paper is not having performed the experiments on 24-month-old-mice. At 20 months of age, the mice do not display any muscle wasting and myofiber atrophy compared to young mice that have completed postnatal muscle growth (=6-month-old-mice). It would be interesting to see the levels of 4-HNE in 24- or 30-month-old mice, and if N-acetylcarnosine treatment in older mice is able to rescue muscle atrophy induced by aging.
Previous studies have shown that inhibition of autophagy accelerates (rather than protect) from sarcopenia, and that autophagy is required to maintain muscle mass (Masiero 2009, PMID: 19945408; Castets 2013, PMID: 23602450; Carnio 2014, PMID: 25176656). On this basis, the authors should test whether their findings are valid only in the context of disuse atrophy or also in the context of sarcopenia (=24-30-month-old mice).
- In Fig.2 the authors report that GPx4 KD, erastin, and RSL3 reduce the diameter of myotubes. For how long and when was the treatment done? Looking at the images, it seems that there are some myoblasts in the cultures treated with GPx4 KD, erastin, and RSL3. Is it possible that these compounds reduce myotube size by inhibiting myoblast fusion rather than by inducing myotube atrophy?
- MDA quantification was done in the gastrocnemius although all the experiments in this paper were performed in the soleus and EDL. It would be good if the authors could explain the reason for this.
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