Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in Caenorhabditis elegans
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Evaluation Summary:
This manuscript will appeal to all with an interest in comparative physiology and the molecular biology of age-associated changes in muscle function. The authors draw parallels between aging skeletal muscle in humans and C. elegans, with evidence in support of age-dependent oxidation of the C. elegans ryanodine receptor ortholog, UNC-68, causing loss of the calstabin ortholog, FKB-2. This in turn results in UNC-68 "leakiness", reduced body wall Ca2+ transients and muscle weakness-changes in ryanodine receptor complex structure and function, changes that are similar to those that occur in aging human skeletal muscle despite the dramatic differences in the lifespan of the two organisms. The experimental approaches are generally sound, although the intriguing dataset that is open to multiple interpretations.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Age-dependent loss of body wall muscle function and impaired locomotion occur within 2 weeks in Caenorhabditis elegans (C. elegans) ; however, the underlying mechanism has not been fully elucidated. In humans, age-dependent loss of muscle function occurs at about 80 years of age and has been linked to dysfunction of ryanodine receptor (RyR)/intracellular calcium (Ca 2+ ) release channels on the sarcoplasmic reticulum (SR). Mammalian skeletal muscle RyR1 channels undergo age-related remodeling due to oxidative overload, leading to loss of the stabilizing subunit calstabin1 (FKBP12) from the channel macromolecular complex. This destabilizes the closed state of the channel resulting in intracellular Ca 2+ leak, reduced muscle function, and impaired exercise capacity. We now show that the C. elegans RyR homolog, UNC-68 , exhibits a remarkable degree of evolutionary conservation with mammalian RyR channels and similar age-dependent dysfunction. Like RyR1 in mammals, UNC- 68 encodes a protein that comprises a macromolecular complex which includes the calstabin1 homolog FKB-2 and is immunoreactive with antibodies raised against the RyR1 complex. Furthermore, as in aged mammals, UNC-68 is oxidized and depleted of FKB-2 in an age-dependent manner, resulting in ‘leaky’ channels, depleted SR Ca 2+ stores, reduced body wall muscle Ca 2+ transients, and age-dependent muscle weakness. FKB-2 ( ok3007)- deficient worms exhibit reduced exercise capacity. Pharmacologically induced oxidization of UNC-68 and depletion of FKB-2 from the channel independently caused reduced body wall muscle Ca 2+ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca 2+ transients and function. Taken together, these data suggest that UNC-68 oxidation plays a role in age-dependent loss of muscle function. Remarkably, this age-dependent loss of muscle function induced by oxidative overload, which takes ~2 years in mice and ~80 years in humans, occurs in less than 2–3 weeks in C. elegans , suggesting that reduced antioxidant capacity may contribute to the differences in lifespan among species.
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Author Response
Joint Public Review
The authors sought to demonstrate that for studies of aging, the 20-day life span of the nematode C. elegans gives it an advantage as a model, over mice (2 years) or humans (80 years). They were studying muscle aging and they showed that UNC-68, a single protein which is a homolog of three mammalian calcium release channel proteins RyR1, 2 and 3 is complexed with homolog proteins as part of a large multi-protein complex. The methods used were largely biochemical, using antibodies to identify the proteins they were interested in and a compound DNP to determine the degree of oxidation of UNC-68. There could be stronger support for the conclusion that antioxidant capacity contributes to life span.
We thank the Reviewer for her/his remarks. The present study focuses on UNC-68 as a target for oxidative …
Author Response
Joint Public Review
The authors sought to demonstrate that for studies of aging, the 20-day life span of the nematode C. elegans gives it an advantage as a model, over mice (2 years) or humans (80 years). They were studying muscle aging and they showed that UNC-68, a single protein which is a homolog of three mammalian calcium release channel proteins RyR1, 2 and 3 is complexed with homolog proteins as part of a large multi-protein complex. The methods used were largely biochemical, using antibodies to identify the proteins they were interested in and a compound DNP to determine the degree of oxidation of UNC-68. There could be stronger support for the conclusion that antioxidant capacity contributes to life span.
We thank the Reviewer for her/his remarks. The present study focuses on UNC-68 as a target for oxidative stress generated by multiple sources (mitochondria, oxidative enzymes…). These channels are promising therapeutic target downstream of oxidative stress and independent of its source of production. One advantage of targeting leaky calcium release channels rather than using anti-oxidants is the avoidance of the adverse effects of blocking beneficial oxidative signals. (We have added these points to the discussion on pages 15).
They do show that a compound which was invented by one of the authors appeared to stabilize the association of FKB2 (the C. elegans homolog of the mammalian FKBP12 and 12.6 expressed in muscle and heart respectively) with UNC-68 but they did not show that if the association is strengthened, it reduces oxidation of UNC-68. Overall the data shown is consistent with what the same authors have shown in mammals.
The compound binds to RyR (UNC-68 in C-elegans) and enables FKB-2 to rebind to the channel but without reversing the oxidation of the channel. We have recently reported the binding site for the Rycal ARM210 and S107 in RyR1 (Melville et al, Structure 2022) using cryogenic electron microscopy and for ARM210 in RyR2 (Miotto et al in-revision Science Advances 2022). The compound has no antioxidative properties and does not affect the posttranslational modifications of the channel. (We have added these points to the introduction page 5).
Some weaknesses: It is unclear if there is sufficient evidence that antioxidant capacity contributes to life span, and because UNC-68 is not solely expressed in muscles they cannot be sure that the effect that they see is related to muscle function as opposed to nerve function.
As the reviewer notes, UNC-68 is not solely expressed in muscles but also in neurons, in eggs, in male tail and enteric muscles. The UNC-68 expression in tissues other than muscles seem to be minor and the channel seems to NOT be impacting the muscle function; support of this point of view emerged from a set of experiments showing that WT-UNC-68 coding sequence fused to the muscle-restricted myo-3 promoter rescued motility defects and sensitivity to ryanodine paralysis. The same experiment has been performed to successfully rescue the locomotion defects in UNC-68 null mutant worms (see Ed B. Maryon et al, Journal of Cell Science 1998). We have added these points to the discussion on pages 14.
They do show that a compound which was invented and marketed by one of the authors appeared to stabilize the association of FKB2 (the C. elegans homolog of the mammalian FKBP12 and 12.6 expressed in muscle and heart respectively) with UNC-68.
The Rycal compound S107 was invented by ARMGO Pharma, a company in which I own stock, but S107 is NOT marketed by me. While ARMGO and Columbia hold patents on S107, it is being sold illegally for research purposes by several companies none of which are connected to me. I make no income from the sale of S107.
The authors should discuss differences in EC coupling in C. elegans relative to that of mammals and comment on the validity of C. elegans as a model for aging human muscle.
We have discussed the differences in EC coupling in C. elegans vs. mammals and commented on the validity of C. elegans as a model for aging human muscle (see revised discussion-page 14).
The authors do provide evidence for a remarkable degree of evolutionary conservation of excitation-contraction and in particular with respect to the calcium release channel. They provide a model system that might be important for the field including with respect to aging.
We thank the Reviewer for her/his positive comment on our study.
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Evaluation Summary:
This manuscript will appeal to all with an interest in comparative physiology and the molecular biology of age-associated changes in muscle function. The authors draw parallels between aging skeletal muscle in humans and C. elegans, with evidence in support of age-dependent oxidation of the C. elegans ryanodine receptor ortholog, UNC-68, causing loss of the calstabin ortholog, FKB-2. This in turn results in UNC-68 "leakiness", reduced body wall Ca2+ transients and muscle weakness-changes in ryanodine receptor complex structure and function, changes that are similar to those that occur in aging human skeletal muscle despite the dramatic differences in the lifespan of the two organisms. The experimental approaches are generally sound, although the intriguing dataset that is open to multiple interpretations.
(This …
Evaluation Summary:
This manuscript will appeal to all with an interest in comparative physiology and the molecular biology of age-associated changes in muscle function. The authors draw parallels between aging skeletal muscle in humans and C. elegans, with evidence in support of age-dependent oxidation of the C. elegans ryanodine receptor ortholog, UNC-68, causing loss of the calstabin ortholog, FKB-2. This in turn results in UNC-68 "leakiness", reduced body wall Ca2+ transients and muscle weakness-changes in ryanodine receptor complex structure and function, changes that are similar to those that occur in aging human skeletal muscle despite the dramatic differences in the lifespan of the two organisms. The experimental approaches are generally sound, although the intriguing dataset that is open to multiple interpretations.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
-
Joint Public Review:
The authors sought to demonstrate that for studies of aging, the 20-day life span of the nematode C. elegans gives it an advantage as a model, over mice (2 years) or humans (80 years). They were studying muscle aging and they showed that UNC-68, a single protein which is a homolog of three mammalian calcium release channel proteins RyR1, 2 and 3 is complexed with homolog proteins as part of a large multi-protein complex. The methods used were largely biochemical, using antibodies to identify the proteins they were interested in and a compound DNP to determine the degree of oxidation of UNC-68. There could be stronger support for the conclusion that antioxidant capacity contributes to life span.
They do show that a compound which was invented by one of the authors appeared to stabilize the association of FKB2 (the C. …
Joint Public Review:
The authors sought to demonstrate that for studies of aging, the 20-day life span of the nematode C. elegans gives it an advantage as a model, over mice (2 years) or humans (80 years). They were studying muscle aging and they showed that UNC-68, a single protein which is a homolog of three mammalian calcium release channel proteins RyR1, 2 and 3 is complexed with homolog proteins as part of a large multi-protein complex. The methods used were largely biochemical, using antibodies to identify the proteins they were interested in and a compound DNP to determine the degree of oxidation of UNC-68. There could be stronger support for the conclusion that antioxidant capacity contributes to life span.
They do show that a compound which was invented by one of the authors appeared to stabilize the association of FKB2 (the C. elegans homolog of the mammalian FKBP12 and 12.6 expressed in muscle and heart respectively) with UNC-68 but they did not show that if the association is strengthened, it reduces oxidation of UNC-68. Overall the data shown is consistent with what the same authors have shown in mammals.
Some weaknesses: It is unclear if there is sufficient evidence that antioxidant capacity contributes to life span, and because UNC-68 is not solely expressed in muscles they cannot be sure that the effect that they see is related to muscle function as opposed to nerve function.
They do show that a compound which was invented and marketed by one of the authors appeared to stabilize the association of FKB2 (the C. elegans homolog of the mammalian FKBP12 and 12.6 expressed in muscle and heart respectively) with UNC-68.
The authors should discuss differences in EC coupling in C. elegans relative to that of mammals and comment on the validity of C. elegans as a model for aging human muscle.
The authors do provide evidence for a remarkable degree of evolutionary conservation of excitation-contraction and in particular with respect to the calcium release channel. They provide a model system that might be important for the field including with respect to aging.
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