Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in Caenorhabditis elegans

Haikel Dridi; Frances Forrester; Alisa Umanskaya; Wenjun Xie; Steven Reiken; Alain Lacampagne; Andrew Marks

doi:10.7554/eLife.75529

. 2022 May 4;11:e75529. doi: 10.7554/eLife.75529

Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in Caenorhabditis elegans

Haikel Dridi ¹, Frances Forrester ¹, Alisa Umanskaya ¹, Wenjun Xie ¹, Steven Reiken ¹, Alain Lacampagne ^2,³, Andrew Marks ^1,^✉

Editors: Mark T Nelson⁴, Richard W Aldrich⁵

PMCID: PMC9113742 PMID: 35506650

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 (Ca²⁺) 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 Ca²⁺ 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 Ca²⁺ stores, reduced body wall muscle Ca²⁺ 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 Ca²⁺ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca²⁺ 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.

Research organism: C. elegans

Introduction

Approximately 50% of humans over the age of 80 develop muscle weakness, which contributes to falls and hip fractures, a leading cause of mortality in the elderly (Marzetti and Leeuwenburgh, 2006; Ganz et al., 2007; Santulli et al., 2013). Strikingly, despite an approximately 2000-fold difference in the lifespans of humans and Caenorhabditis elegans (Herndon et al., 2002; Ljubuncic and Reznick, 2009), both exhibit oxidative overload induced age-dependent reductions in muscle function and motor activity that ultimately contribute to senescence and death. Due to its short lifespan and well-characterized genome, C. elegans has been used as a model to study the genetics of aging and lifespan determination (Guarente and Kenyon, 2000; Kenyon, 2010), including the age-dependent decline in locomotion (Herndon et al., 2002; Hsu et al., 2009). Age-dependent reduction in locomotion in C. elegans has been attributed to degeneration of the nervous system (Liu et al., 2013) and the body wall musculature (Kirkwood, 2013). Here, we investigated the role of the ryanodine receptor (RyR)/intracellular calcium (Ca²⁺) release channel homolog, UNC-68, in age-dependent loss of muscle function in C. elegans.

Mammalian RyR1 is the major intracellular Ca²⁺ release channel in skeletal muscle required for excitation-contraction (E-C) coupling (Zalk et al., 2015). In mammals, peak intracellular Ca²⁺ transients evoked by sarcolemmal depolarization decrease with age (Gonzalez et al., 2003), and this decrease is associated with a reduced SR Ca²⁺ release (Jiménez-Moreno et al., 2008) that directly determines the force production of skeletal muscle. Our group has shown that a mechanism underlying age-dependent loss of muscle function is RyR1 channel oxidation which depletes the channel complex of the stabilizing subunit calstabin1 (calcium channel stabilizing binding protein type 1, or FKBP12), resulting in intracellular Ca²⁺ leak and muscle weakness (Andersson et al., 2011; Umanskaya et al., 2014). RyR1 is a macromolecular complex comprised of homotetramers of four ~565 kDa RyR monomers (; Zalk et al., 2007). Cyclic AMP (cAMP)-dependent protein kinase A (PKA) (Marx et al., 2000), protein phosphatase 1 (Kass et al., 2003), phosphodiesterase PDE4D3 (Lehnart et al., 2005), Ca²⁺-dependent calmodulin kinase II (CaMKII) (Currie et al., 2004; Kushnir et al., 2010), and calstabin1 (Bellinger et al., 2008) are components of the RyR1 macromolecular complex (Santulli and Marks, 2015). Calstabin1 is part of the RyR1 complex in skeletal muscle, and calstabin2 (FKBP12.6) is part of the RyR2 complex in cardiac muscle (Santulli et al., 2017). Calstabins are immunophilins (Marks, 1996) with peptidyl-prolyl isomerase; however, this enzymatic activity does not play a role in regulating RyR channels and rather they stabilize the closed state of RyRs and prevent a Ca²⁺ leak via the channel (Marx et al., 2000; Brillantes et al., 1994).

RyR belongs to a small family of large intracellular Ca²⁺ release channels, the only other member being the inositol 1,4,5-triphosphate receptor (IP₃R) (Harnick et al., 1995; Jayaraman et al., 1995; Jayaraman and Marks, 2000). RyR may have evolved from IP₃R-B, which encoded an IP₃R-like channel that could not bind IP₃ and was replaced by RyR at the Holozoa clade (Alzayady et al., 2015). Invertebrates have one gene for each of RyR and IP₃R, while vertebrates have three (RyR1-3 and IP₃R1-3). RyRs and IP3Rs are intracellular Ca²⁺ release channels on the SR/ER and are tetramers that along with associated proteins comprise the largest known ion channel macromolecular complexes (Marx et al., 2000; DeSouza et al., 2002). Defects in Ca²⁺ signaling linked to stress-induced remodeling that results in leaky RyR channels have been implicated in heart failure (Dridi et al., 2020c; Marks, 2003), cardiac arrhythmias (Dridi et al., 2020c; Lehnart et al., 2006; Lehnart et al., 2004; Vest et al., 2005; Wehrens et al., 2003), diabetes (Santulli et al., 2015), muscle weakness (Kushnir et al., 2020; Dridi et al., 2020b; Matecki et al., 2016; Dridi et al., 2020d), and neurodegenerative disorders (Dridi et al., 2020b; Lacampagne et al., 2017; Liu et al., 2012).

RyR has evolved unique SPRY domains (des Georges et al., 2016) that are absent in IP₃R, one of which (SPRY2) allows RyR1 to directly interact with the L-type calcium channel (Cav1.1) in mammalian skeletal muscle (Cui et al., 2009). This interaction couples excitation of the sarcolemma to muscle contraction to overcome the dependence on extracellular Ca²⁺. RyR1 is remarkably well conserved, suggesting that independence from extracellular Ca²⁺ evolved to support locomotion in higher organisms.

UNC-68 is the RyR gene homolog in the C. elegans genome (Maryon et al., 1996). Worms lacking both exon 1.1 and promoter1 (Marques et al., 2020), and UNC-68 (e540) null mutants exhibit severely defective swimming behavior and locomotive characterized by the ‘unc’, or ‘unco-ordinated’ phenotype (Brenner, 1974). Treatment with ryanodine, a chemical ligand of RyR, induces contractile paralysis in wild-type (WT) C. elegans, whereas UNC-68 (e540) null mutants are unaffected by ryanodine (Maryon et al., 1996; Brenner, 1974; Sakube et al., 1997). Ca²⁺ transients triggered by action potentials in C. elegans body wall muscles require UNC-68.

We previously reported that in aged mice (2 years old equivalent to ~80-year old humans) RyR1 oxidation depletes calstabin1 from the channels and renders them leaky to Ca²⁺, which contributes to the loss of muscle function and impaired muscle strength (Umanskaya et al., 2014). In the present study, we show that UNC-68 is comprised of a macromolecular complex that is remarkably conserved compared to RyR1 and includes the channel-stabilizing subunit, FKB-2. Like calstabin, FKB-2 regulates UNC-68 by directly associating with the channel. Similar to what we previously observed in mice (Andersson et al., 2011), we found age-dependent oxidation of UNC-68 which causes depletion of FKB-2 from the UNC-68 channel complex, and reduces Ca²⁺ transients in aged nematodes. This aging phenotype was accelerated in FKB-2 (ok3007) worms, an FKB-2 deletion mutant that results in leaky UNC-68. Competing FKB-2 from UNC-68 with rapamycin or FK506 (Timerman et al., 1993) resulted in reduced body wall muscle Ca²⁺ transients and defective locomotion. Conversely, pharmacological and genetic oxidation of UNC-68 with the reactive oxygen species (ROS)-generating drug paraquat (Lee et al., 2003) caused FKB-2 dissociation from the channel and reduced contraction-associated Ca²⁺ transients. Reassociating FKB-2 with UNC-68 using the RyR-stabilizing drug S107 improved Ca²⁺ transients and locomotion in aged nematodes. We have recently reported the binding site for S107 and its second generation Rycal, ARM210, using cryogenic electron microscopy (Melville et al., 2022). The compound binds in a cleft in the cytosolic shell and prevents a remodeled RyR channel from sitting in a ‘primed state’ sensitive to activation (Melville et al., 2022; Miotto et al., in-revision Science Advances 2022). A clinical trial using ARM210 to fix the leak in RyR1 channels is currently underway at the NIH (NCT04141670).

Our study provides an underlying mechanism for age-dependent loss of muscle function in C. elegans including progressive oxidation of UNC-68, which depletes the stabilizing binding protein, FKB-2 and, renders the channel leaky within 2 weeks compared to 2 years in mice and 80 years in humans and a potential therapy.

Results

Conserved evolution and architecture of UNC-68

Phylogenic analysis of RyR and FKBP among species reveals remarkable evolutionary conservation (Figure 1A–B). UNC-68, the C. elegans intracellular calcium release channel, shares ~40% homology with the human RyR1 (Figure 1C). C. elegans FKB-2 has ~60% sequence identity with the skeletal muscle isoform calstabin1 (FKBP12) (Figure 1D). Based on these observations, we hypothesized that in C. elegans, UNC-68 comprises a macromolecular complex, similar to that of mammalian RyRs. To test this hypothesis, lysates were prepared from populations of freeze-cracked WT C. elegans, and UNC-68 was immunoprecipitated using mammalian anti-RyR antibody (5029) as previously described (Kushnir et al., 2018). The immunoprecipitates were immunoblotted to detect UNC-68, as well as other components of the RyR macromolecular complex including the catalytic subunit of protein kinase A (PKA_cat), protein phosphatase 1 (PP1), FKB-2, and phosphodiesterase 4 (PDE-4) using mammalian anti-RyR, anti-PKA, anti-PP1, anti-calstabin, and anti-PDE-4 antibodies, respectively (Figure 1E). The previously published C. elegans anti-PDE-4 (Charlie et al., 2006) was used to detect PDE-4 on the channel. Our data show that UNC-68 comprises a macromolecular complex, similar to that found in the mammalian muscle, that includes PKA_cat, PP1, PDE-4, and FKB-2. UNC-68 was depleted of FKB-2 in the FKB-2 (ok3007) null mutant (Figure 1E and G). In the FKB-2 null C. elegans, UNC-68 and the rest of the macromolecular complex could not be immunoprecipitated using an anti-FKBP antibody (Figure 1F and H). Taken together these data indicate remarkable evolutionary conservation of the RyR macromolecular complex.

Age-dependent biochemical and functional remodeling of UNC-68

RyR1 channels are oxidized, leaky, and Ca²⁺ transients are reduced in aged mammalian skeletal muscle (Andersson et al., 2011). These changes occur by 2 years of age in mice (Andersson et al., 2011) and by 80 years of age in humans. Similarly, FKB-2 deficient worms exhibited an age-dependent decline in body wall muscle peak Ca²⁺ transients starting at day 7 post-hatching (Figure 2A–B).

Green box denotes peak fluorescence from worm’s muscle during contraction. (B) Ca²⁺ transients in age-synchronized populations of WT and FKB-2 (*ok3007*) nematodes (at day 3 and 7); (C) *UNC-68* was immunoprecipitated from age-synchronized populations of mutant (FKB-2 KO) and WT nematodes (at day 3, 5, 9, and 12) and immunoblotted using anti-RyR, anti-calstabin, and dinitrophenyl (DNP; marker of oxidation) antibodies. (D and E) Quantification of the average band intensity from triplicate experiments: band intensity was defined as the ratio of each complex member’s expression over its corresponding /*UNC-68’s* expression. Data are means ± SEM. * p<0.05 WT vs. FKB-2 KO in panel D, # p<0.05 WT vs. FKB-2 KO in panel E, * p<0.05 WT at day 3 vs. WT at day 5 and day 9. (F) Ca²⁺ leak assay performed with microsomes from WT and FKB-2 KO worms (day 5). Ca²⁺ uptake into the microsomes was initiated by adding 1 mM of ATP. Then, 3 µM of thapsigargin was added to block the sarco/endoplasmic calcium ATPase activity. Increased fluorescence is proportional to the spontaneous Ca²⁺ leakage throughout *UNC-68*. (G) Graph showing number of bends recorded for WT vs. FKB-2 KO worms at six distinct ages (day 3, 5, 7, 9, 12, and 15). (H) The number of curling events was calculated as a percentage of the overall motility (curls/bends). N = ~60 worms per group, except for day 15 (as fewer worms were alive at this timepoint). Day 15 = ~40 worms. (I) Percentage of survival of WT (average survival; 18 days) and FKB-2 KO worms (average survival; 14 days); Gehan-Breslow-Wilcoxon test for survival comparison was performed for statistical significance. Data are means ± SEM from triplicate experiments. One-way ANOVA shows * p<0.05 WT vs. FKB-2 KO, # p<0.05 WT at day 3 vs. WT at day 5, 7, 9, 12, and 15. Figure 2—source data 1.

Figure 2—source data 1. Full incut gels of Figure 2.

elife-75529-fig2-data1.pdf^{(295.8KB, pdf)}

RyR1 oxidation has been linked to SR Ca²⁺ leak and impaired muscle function during extreme exercise and in heart failure and muscular dystrophies (Bellinger et al., 2008; Bellinger et al., 2009; Allen et al., 2008). Furthermore, we have previously reported that oxidation of RyR1 and the subsequent intracellular Ca²⁺ leak are underlying mechanisms of age-related loss of skeletal muscle specific force (force normalized to the cross-sectional area of muscle) (Andersson et al., 2011). WT UNC-68 was oxidized (Figure 2C–D) and depleted of FKB-2 (Figure 2C–E) and in an age-dependent manner. These changes mirror those occurring with extreme exercise in mice and humans (Bellinger et al., 2008) and in a murine model of Duchenne muscular dystrophy (mdx mice) characterized by impaired muscle function (Bellinger et al., 2009). Importantly, by 80 years of age, ~50% of humans develop severe muscle weakness that is a strong predictor of mortality due to falls, gait imbalance, and related factors (Degens, 2007). Similarly, UNC-68 was significantly more oxidized (day 3–9) in FKB-2 (ok3007) worms compared to WT (Figure 2C–D).

To further demonstrate that UNC-68 channels lacking FKB-2 are inherently ‘leaky’, we used an assay that can monitor the rate of Ca²⁺ released from the SR. Age synchronized worms' microsomes (day 5) were mixed with the Ca²⁺ dye Fluo-4 and baseline fluorescence measurements were taken before adding 1 mM of ATP. By activating the sarco/endoplasmic calcium ATPase (SERCA) with ATP, cytosolic Ca²⁺ is pumped into the microsomes, resulting in a decrease in Fluo-4 fluorescence. Once the fluorescence level plateaus, thapsigargin (SERCA antagonist) is added to block Ca²⁺ reuptake into the SR. The rate at which the fluorescence increases directly correlates with the amount of Ca²⁺ passively leaking into the cytoplasm: a higher increase of fluorescence compared to WT control indicates leaky UNC-68 channels. Our data show that UNC-68 from FKB-2 KO worms had a higher rate of SR Ca²⁺ leak following thapsigargin administration compared to the WT channels (Figure 2F). This is corroborated by our previous findings, where disruption of RyR-calstabin binding increases the SR Ca²⁺ leak in mammalian tissues (Umanskaya et al., 2014).

In mammals, calstabin regulation of RyR is tightly coupled to beta-adrenergic signaling (Andersson et al., 2012), and it is known that calstabin KO mice must undergo exercise stress before demonstrating a distinct muscle phenotype (Bellinger et al., 2008). Our method of inducing exercise stress in the worm was to place it in M9 buffer and observe it swimming, a well-described behavioral assay (Lüersen et al., 2014). By using an extended time trial of 2 hr, the worms fatigue and exhibit exercise-induced stress similar to that observed in mammals. Our data show a defect in FKB-2 KO swimming behavior over the course of its lifespan when compared to the WT. FKB-2 KO worms had decreased bending activity earlier in life, beginning at day 5, and an increased proportion of curling, a sign of fatigue (Figure 2G–H). Throughout midlife, the FKB-2 KO worms lag significantly behind their age-matched WT counterparts, suggestive of decreased muscle function. Furthermore, FKB-2 KO worms exhibit reduced lifespan compared to WT (Figure 2I).

Pharmacologically mimicking aging phenotype affects Ca²⁺ transient and impairs exercise capacity

FKB-2 was competed off from the UNC-68 macromolecular complex using rapamycin or FK506 (Figure 3). Both rapamycin and FK-506 bind to calstabin and compete it off from RyR channels, resulting in leaky channels and release of SR Ca²⁺ in the resting state (Kaftan et al., 1996; Tang et al., 2002).

Age-synchronized young C. elegans (5 days) were treated with rapamycin or FK506. Ca²⁺ transients were measured in partially immobilized transgenic nematodes expressing the genetically encoded Ca²⁺ indicator, Pmyo-3::GCaMP2, in the body wall muscle cells (Tallini et al., 2006; Liu et al., 2011; Figure 3A). Pharmacologic depletion of FKB-2 from UNC-68 by rapamycin or FK506 treatment (15 min exposure to each drug) caused reduced body wall muscle Ca²⁺ transients in WT C. elegans (Figure 3B). When FKB-2 was genetically depleted from the UNC-68 complex, as in the FKB-2 (ok3007) nematodes, treatment with rapamycin or FK506 had no effect on the Ca²⁺ transients (Figure 3B).

Continuous Ca²⁺ leak via UNC-68 would be expected to result in depleted SR Ca²⁺ stores; therefore, we utilized a common technique from the mammalian RyR literature to evaluate the SR Ca²⁺ stores. In brief, an activating concentration of caffeine is used to fully open the RyR channel, leading to a rapid release of Ca²⁺ from the SR into the cytoplasm. This increase can be approximated using a previously targeted, fluorescent Ca²⁺ sensitive dye or indicator. Caffeine was applied to day 5 cut worms (Figure 3C), and the amount of fluorescence given off by GCaMP2 was measured. The GCaMP2-WT worms demonstrated a strong Ca²⁺ transient within 10 s after caffeine administration, while GCaMP2-FKB-2 KO worms failed to produce a response, suggesting that their SR Ca²⁺ stores were too low to elicit one. Interestingly, GCaMP2-KO worms were observed as having very high background fluorescence, which may indicate an increase in cytosolic Ca²⁺ from passive UNC-68 leak.

Acute treatment with FK506 or rapamycin, for 15 min, each independently caused depletion of FKB-2 from the channel (Figure 3D, E and F) with no effect on the oxidation of UNC-68. Furthermore, longer treatment (2 and 4 hr) of WT worms with FK506 caused oxidation of UNC-68, demonstrating a relationship between depletion of FKB-2 and oxidation of UNC-68 (Figure 3G,H,I).

Indeed, rapamycin altered swimming behavior of WT but not FKB-2 KO worms in a time-dependent manner (Figure 3J). Taken together with our Ca²⁺ transient data, the observed muscle phenotype appears to be the result of UNC-68 channel leak. These data suggest that rendering UNC-68 channels leaky by removing FKB-2 depletes SR Ca²⁺, resulting in reduced Ca²⁺ transients and weakened muscle contraction.

Oxidation of UNC-68 causes reduced body wall muscle Ca²⁺ transients

To investigate the individual effect of age-dependent UNC-68 oxidation independent of the other confounding variables involved in aging (Herndon et al., 2002), we introduced a pharmacological intervention mimicking the aged state in young adult nematodes. Treating young adult nematodes (at 5 days of age) with the superoxide-generating agent paraquat (Lee et al., 2003) increased oxidation of UNC-68 and depletion of FKB-2 from the channel in a concentration-dependent manner (Figure 4A, B and C). Furthermore, contraction-associated Ca²⁺ transients decreased with paraquat treatment in a concentration-dependent manner (Figure 4D). Indeed, treatment with antioxidant N-Acetyl-L-cysteine improved Ca²⁺ transient in FKB-2 KO worms (Figure 4E). These data indicate that both UNC-68 oxidation and FKB-2 depletion independently contribute to the observed aging body wall muscle deterioration.

(**B–C**) Quantification of the band intensity shown in (A): band intensity was defined as the ratio of either DNP (marker of *UNC-68* oxidation) or FKB-2 binding over its corresponding /*UNC-68’s* expression. (D) Contraction-associated Ca²⁺ transients measured in young age-synchronized WT nematodes treated for 15 with FK506 or for 20 min with increasing concentrations of paraquat (day 5). (E) Contraction-associated Ca²⁺ transients measured in FKB-2 KO nematodes treated with the antioxidant N-acetylcysteine (NAC) at 5 mM (day 7). (F) *UNC-68* was immunoprecipitated and immunoblotted using anti-RyR, anti-calstabin, and DNP (marker of oxidation) antibodies in WT, the long lived (CLK-1) and the short lived (MEV-1) nematodes at day 2, 7, and 15. (**G–H**) Quantification of the average band intensity from triplicate experiments: band intensity was defined as the ratio of each complex member’s expression over its corresponding /*UNC-68’s* expression. (I) Graph showing number of bends recorded for WT vs. CLK-1 and MEV-1 worms at three distinct ages (day 2, 7, and 15). (J) The number of curling events was calculated as a percentage of the overall motility (curls/bends). N ≥ 20 per group. Data are means ± SEM from triplicate experiments. One-way ANOVA shows * p<0.05. Two-way ANOVA was used in panel I and J. SK SR; sarcoplasmic reticulum fraction from mouse skeletal muscle used as external control reference and was not quantified in the bar graphs. The time 0 min refers to untreated worms. Figure 4—source data 1.

Figure 4—source data 1. Full incut gels of Figure 4.

elife-75529-fig4-data1.pdf^{(203.8KB, pdf)}

To better clarify the role of oxidative stress in age-dependent UNC-68 remodeling and Ca²⁺ leak, we used two mutant mitochondrial electron transport chain (ETC) worms: the complex I mutant, CLK-1, and the complex II mutant, MEV-1. CLK-1 worms contain a Complex I-associated mutation such that they cannot synthesize their own ubiquinone (UQ), a redox active lipid that accepts and transfers electrons from Complex I or II to Complex III in the ETC. The reduction in Complex I activity of CLK-1 is associated with long-lived worms (Yang et al., 2011; Labuschagne et al., 2013; Kayser et al., 2004). In contrast, MEV-1 worms contain a Complex II (succinate dehydrogenase) cytochrome B560 mutation (Ishii et al., 1998; Senoo-Matsuda et al., 2001; Senoo-Matsuda et al., 2003), preventing electron transfer from succinate to fumarate and causing mitochondrial ROS production, which is associated with decreased lifespan, averaging only 9 days (Senoo-Matsuda et al., 2001). Interestingly, we have seen increased UNC-68 oxidation and FKB-2 depletion in the short-lived mutant (MEV-1) compared to WT and long-lived mutant (CLK-1) worms (Figure 4F, G and H). Indeed, MEV-1 worms exhibited reduced exercise capacity compared to WT and CLK-1 worms (Figure 4I–J).

UNC-68 Ca²⁺ channel is a potential therapeutic target in aging

The small molecule Rycal S107 inhibits SR Ca²⁺ leak by reducing the stress-induced depletion of calstabin from the RyR channel complex (Bellinger et al., 2009; Lehnart et al., 2008). Here, we show that treatment with S107 (10 μM) for 5 hr reassociated FKB-2 with UNC-68 without significant effect on the channel oxidation (Figure 5A, B and C). Furthermore, treatment with S107 improved peak Ca²⁺ in an FKB-2-dependent manner, as demonstrated by the fact that treating the FKB-2 KO worms did not change peak Ca²⁺ (Figure 5D–E). Interestingly, S107 treatment reduced age-dependent impairment of exercise capacity in WT worms at day 15 (Figure 5F). Of note, S107 has no effect on the WT worms' lifespan (Figure 5G). Furthermore, the treatment of the short-lived worms, MEV1, with S107 restored the FKB-2 association with UNC-68, despite the persistence of the channel oxidation (Figure 5H,I,J).

(**B–C**) Quantification of the band intensity shown in (A): band intensity was defined as the ratio of either DNP (marker of *UNC-68* oxidation) or FKB-2 binding over its corresponding /*UNC-68* expression. Data are mean ± SEM. * p<0.05 vs. wild-typd L4 (WTL4), # p<0.05 WT D15 vs. WT D15 + S107. (**D–E**) Contraction-associated Ca²⁺ transients were measured in age-synchronized WT (day 3 and 15) (D) and (E) *FKB-2 KO worms* (day 3 and 7). Contraction-associated Ca²⁺ transients in S107-treated worms were performed at day 15 for WT and day 7 for *FKB-2* worms. (F) Graph showing number of bends recorded for WT vs. WT treated with S107 worms at different ages (day 3, 7, and 15). (G) Percent of survival of WT vs. WT treated with S107 nematodes; Gehan-Breslow-Wilcoxon test for survival comparison was performed for statistical significance. (H) *UNC-68* was immunoprecipitated and immunoblotted with anti-RyR, anti-calstabin, and DNP (marker of oxidation) in short-lived nematodes (MEV-1) with S107 treatment (5 hr). (**I–J**) Quantification of the band intensity shown in (H): band intensity was defined as the ratio of either DNP (marker of *UNC-68* oxidation) or *FKB-2* binding over its corresponding /*UNC-68* expression. N ≥ 20 per group. Data are mean ± SEM from triplicate experiments. One-way ANOVA shows * p < 0.05 vs WT L4 unless otherwise indicated. In panel F, a t-test was used to compare WT and WT + S107 for each day. #p<0.05 MEV-1, vs. MEV-1 +S107 in panel J. Figure 5—source data 1.

Figure 5—source data 1. Full incut gels of Figure 5.

elife-75529-fig5-data1.pdf^{(282.2KB, pdf)}

Discussion

Taken together, our data show that the C. elegans intracellular Ca²⁺ release channel UNC-68 comprises a macromolecular complex which is highly conserved throughout evolution from nematodes to humans. In nematodes, the UNC-68 macromolecular complex is comprised of a similar array of regulatory subunits as the mammalian RyR1 channels: a phosphodiesterase PDE-4, a protein kinase PKA, a protein phosphatase PP1, and the immunophilin, FKB-2. Binding of FKB-2 (the C. elegans homolog of the mammalian RyR stabilizing protein calstabin) to the UNC-68 channel is required to prevent a pathological leak of intracellular Ca²⁺, similar to the manner observed in mammalian muscle (Andersson et al., 2011). C. elegans exhibit reduced Ca²⁺ transients, as well as oxidized UNC-68 channels and depleted FKB-2 by ~2 weeks of age. Genetic FKB-2 deficiency causes an accelerated aging phenotype; Ca²⁺ transients are reduced in younger populations of FKB-2 (ok3007) nematodes and UNC-68 is oxidized at an earlier time point in these mutants relative to WT. Treating aged WT nematodes with the RyR-stabilizing drug, S107, reassociates FKB-2 with UNC-68 and increases the Ca²⁺ transients, indicating that UNC-68 dysfunction is likely an underlying mechanism of age-dependent decrease in Ca²⁺ transients in C. elegans body wall muscle. The mechanism causing age-dependent UNC-68 dysfunction involves the loss of UNC-68/FKB-2 from the UNC-68 channel complex due to oxidation of the channel. Of note, this may create a vicious cycle of intracellular Ca²⁺ leak and oxidative overload in which leaky channels cause mitochondrial Ca²⁺ accumulation and high levels of ROS production which further oxidize UNC-68 and further exacerbate the Ca²⁺ leak over the course of the lifespan ( Dridi et al., 2020a) as has been demonstrated in mice (Andersson et al., 2011).

C. elegans exhibit an aging muscle phenotype similar to age-dependent loss of muscle function in humans (Andersson et al., 2011). This is characterized by impaired locomotion, reduction in muscle cell size associated with loss of cytoplasm and myofibrils, and progressive myofibril disorganization (Herndon et al., 2002). However, specific body wall muscle proteins involved in the C. elegans aging phenotype have not been determined. Here, we show that UNC-68 is oxidized in aged nematodes and depleted of the channel-stabilizing protein, FKB-2. Our group has reported similar remodeling of RyR1 in skeletal muscle from aged mice (Andersson et al., 2011) and in murine models of muscular dystrophies (Bellinger et al., 2009), all of which exhibit intracellular Ca²⁺ leak and reduced muscle specific force production.

Though the oxidative stress theory of aging was first proposed in 1956 (Hagen, 2003; Harman, 1956), there is still substantial controversy surrounding the role of ROS in aging. For example, deletion or overexpression of the ROS detoxification enzyme superoxide dismutase has little effect on lifespan in C. elegans (Gems and Doonan, 2009; Van Raamsdonk and Hekimi, 2012). However, loss of sesn-1, the gene encoding sestrin, an evolutionarily conserved protein required for regenerating hyperoxidized forms of peroxiredoxins and for ROS clearance, causes reduced lifespan (Yang et al., 2013). Furthermore, ROS levels measured in vivo in C. elegans increase with age (Back et al., 2012). Other oxidative/antioxidative genes are involved in ROS production and may play a crucial role in the UNC-68 oxidation (Supplementary file 1).

While the free radical theory of aging has taken a hit due to multiple observations that contradict the notion of a link between reduced oxidative load and longevity, the preponderance of data shows a correlation between oxidative damage and reduced lifespan (Shields et al., 2021). Moreover, there is no doubt that reduced muscle function is detrimental to survival (Wilkinson et al., 2018). The present study shows that a key effector of age-dependent oxidative overload, RyR1 channel leak and the resulting muscle dysfunction, occur approximately 2000 times faster in C. elegans compared to Homo sapiens and 50 times faster than in Mus musculus. Since the target system, RyR1/UNC68, is remarkably conserved and underlies dramatically similar physiological functions (namely SR Ca²⁺ release required for muscle contraction) the cause for the accelerated kinetics of aging must be determined elsewhere and in an unrelentingly constant manner as exemplified by the rigid control of species lifespan. There is however, only one known case of a significant prolongation of average lifespan in a species: Homo sapiens. Indeed, the average lifespan in the U.S. has doubled in the past century (Schanzenbach et al., 2016) largely due to improved sanitation and related public health measures that protect against communicable diseases, the present pandemic notwithstanding. This suggests that both environmental and intrinsic biological constraints can determine average lifespan. Since we are a species that can remodel our environment to a greater extent than others, we have been able to double our average lifespan by improving the environment, although now global warming threatens to reverse this achievement. The unanswered question remains what are the intrinsic biological constraints on a given species' longevity? Although, oxidative stress has been thought to be a major contributor to the skeletal muscle aging phenotype (Andersson et al., 2011), other biological factors, including changes to the nervous, hormonal, circulatory, and respiratory systems likely also play important roles.

It would be interesting to know if the increased UNC-68 oxidation-induced FKB-2 depletion and subsequent reduction in body wall muscle Ca²⁺ transients are a result of globally increased ROS levels or increased ROS levels in UNC-68-surrounding microdomains. For example, we have previously shown that inducing RyR leak in enzymatically dissociated skeletal muscle cells causes increased mitochondrial membrane potential and mitochondrial ROS production (Andersson et al., 2011). Based on these data, we have proposed a model in which RyR1 leak (due to age-dependent oxidation of the channel and subsequent dissociation of calstabin) causes mitochondrial Ca²⁺ overload, resulting in ROS production, thus leading to further oxidation of RyR1 and exacerbation of the SR Ca²⁺ leak. This creates a vicious cycle between RyR1 and mitochondria that contributes to age-dependent loss of muscle function.

We also demonstrate that the putative null mutant, FKB-2 (ok3007), prevents FKB-2 from co-immunoprecipitating with UNC-68. The aging phenotype that we characterize in WT nematodes (biochemically modified UNC-68 and reduced Ca²⁺ transients) is accelerated in FKB-2 (ok3007). There are eight FKBs that are homologous to mammalian calstabin in the C. elegans genome; FKB-1 and FKB-8 both have ~50% sequence identity to calstabin. Further studies could elucidate the possibility that in the absence of FKB-2, another FKB may stabilize UNC-68, in particular the aforementioned FKB-8 (its gene is in close proximity to that of FKB-2 on chromosome 2) and FKB-1 (most similar to FKB-2 in terms of molecular weight). Such a mechanism could provide transitory compensation for the lack of FKB-2, in which other FKB isoform(s) bind to UNC-68 with lower affinity. Because this binding is weak, and the channel is unstable, this compensation ends up failing at day 7 of age and the Ca²⁺ leak is exacerbated. This hypothesis is partially supported by the unaltered Ca²⁺ peaks in FKB2-KO worms at day 5 of age despite a complete depletion of FKB-2 binding protein. Such a compensatory mechanism was not observed with acute rapamycin and FK506 treatment potentially because, first, the Ca²⁺ leak was acute and there was no time for a compensatory response, and, second, these drugs could act on all FKB isoforms.

Another key question is why UNC-68 becomes oxidized within 2 weeks, whereas the same post-translational modification requires 2 years in mice and 80 years in humans (Ljubuncic and Reznick, 2009)? Given the high degree of conservation of RyR and other members of the complex (Figure 1), it is feasible that genetic screens in organisms such as C. elegans and Drosophila will yield additional crucial mediators that are common among species and explain disparities in age-dependent loss of muscle function such as genes-genes interactions, epigenetics or architecture, and gating of key proteins involved in aging such as RyR. Indeed, despite the conserved evolution of UNC-68, the channel contains higher numbers of methionine (3.5%) and serine (7.2%) (Supplementary file 2) compared to the human RyR1 (2.9 and 5.9%, respectively). Methionines are a primary target of oxidative stress that might cause defects in the channel gating and alter Ca²⁺ release. Disparities in RyR1 serine residues among species, which are phosphorylated by protein kinases in response to stress, can cause conformational changes to the channel, exposing more residues to oxidation and could be a potential mechanism contributing to the accelerated UNC-68 oxidation in C. elegans.

Regarding the conservation of EC coupling machinery, the UNC-68 is localized to a specific portion of a vesicular network surrounding the myofilament lattice which suggests that the general architecture of the SR is conserved in metazoans. RyRs in vertebrate striated muscle cluster at internal couplings with Ttubules and, peripheral couplings adjacent to the surface membrane, visible as a ‘feet’ in electron micrographs (Maryon et al., 1998). The 12–14 nm gap between the surface and SR vesicle membranes in C. elegans is identical to analogous gaps in vertebrate triad junctions suggesting that UNC-68 bridges these gaps as seen in vertebrates. These similarities in muscle architecture further support our findings regarding the similar muscle aging phenotype between mammals and nematodes and the validity of C. elegans as a useful model to study age-dependent loss of muscle function.

Finally, UNC-68 null mutants are defective in locomotion, but still propagate coordinated contraction waves by an unknown mechanism (Maryon et al., 1996). The only intracellular Ca²⁺ release channels known in the SR vesicles, other than RyRs, are IP3Rs. The C. elegans genome contains a single IP3R gene, the lfe-1 (or itr-1) (Clandinin et al., 1998). However, it has been reported that antibodies to lfe-1 specifically stain the nerve ring but do not stain the myofilament lattice. Furthermore, lfe-1 mutants exhibit normal motility suggesting the IP3Rs channels are not involved in the regulation of the body wall muscle contraction. Moreover, UNC-68 has been reported to be expressed in neurons (Sakube et al., 1997) which may complicate the interpretation of its function in skeletal muscle. However, it seems that the neuronal expression is minor and does not modulate skeletal muscle function. Indeed, transformation of UNC-68 null mutant animals with the WT UNC-68 gene or the WT UNC-68 coding sequence fused to the myo-3 promoter rescued motility defects and sensitivity to ryanodine-induced paralysis (Maryon et al., 1998). myo-3 is expressed in body wall muscles, as well as in enteric muscles (the enteric muscles do not affect motility). Furthermore, no staining of neurons has been observed with an anti-UNC-68 antibodies, which suggests that the major role of UNC-68 is supporting skeletal muscle contraction (Maryon et al., 1998).

Taken together, our data indicate that the C. elegans homolog of RyR, UNC-68, is comprised of a macromolecular complex and regulated by the immunophilin, FKB-2. We have identified age-dependent reduction in body wall muscle Ca²⁺ transients in nematodes that is coupled to oxidation and remodeling of UNC-68. SR Ca²⁺ stores are depleted in FKB2-KO worms, suggesting passive UNC-68 leak. This observation is supported by the Ca²⁺ leak assay results, which show that FKB-2 regulation is critical in preventing UNC-68 channels from aberrantly ‘leaking’ Ca²⁺ into the cytoplasm. With reduced SR Ca²⁺, UNC-68 fails to release the burst of Ca²⁺ required for normal E-C coupling, leading to impaired muscle function. Loss of muscle function is evident in the FKB-2 KO worms during swimming trials, as middle-aged worms performed worse than their age-matched WT controls. Furthermore, our data strongly suggest a role for FKB-2 and UNC-68 in the age-dependent changes in Ca²⁺ signaling, as treatment with the pharmacological RyR stabilizer S107 increases body wall muscle Ca²⁺ transients. The advantage of targeting leaky RyR channels rather than using antioxidants would be the avoidance of the adverse effects of blocking beneficial oxidative signals.

Materials and methods

Key resources table.

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (worms)	ok3007	Caenorhabditis Genetics Center (University of Minnesota)	WormBase ID: WBVar00094093	Genomic position: I: 2918075.12918967
Strain, strain background (worms)	Pmyo-3:GCaMP2 worms	Kindly provided by Zhao-Wen Wang, University of Connecticut Health Center
Strain, strain background (worms)	mev-1	Caenorhabditis Genetics Center (University of Minnesota)	WormBase ID: WBGene00003225	Genomic position III: 10334277.10335168
Strain, strain background (worms)	clk-1	Caenorhabditis Genetics Center (University of Minnesota)	WormBase ID: WBGene00000536	Genomic position III: 5277894.5279344
Antibody	anti-RyR1 (Rabbit polyclonal)	Marks’ lab, Columbia University, NY, USA	Cat. #: 5,029 Aa 1327–1339	WB (1:1000), (10 μl)
Antibody	anti-PDE4 (Rabbit monoclonal)	Kindly provided by Kenneth Miller, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma		WB (1:1000), (10 μl)
Antibody	anti-PP1 (Rabbit polyclonal)	Santa Cruz	Cat. #: sc6104	WB (1:1000), (10 μl)
Antibody	anti-FKBP12 (Mouse monoclonal)	Santa Cruz	Cat. #: sc6104	WB (1:2500), (10 μl)
Antibody	anti-FKBP12 (Rabbit polyclonal)	Abcam	Cat. #: ab2918	WB (1:2000), (10 μl)
Commercial assay or kit	Oxyblot protein oxydation detection kit	Millipore	Cat. #: S7150	WB (1:1000), (10 μl)
Chemical compound, drug	Rapamacin	Sigma Aldrich	Cat. #: 37,094
Chemical compound, drug	FK506	Sigma Aldrich	Cat. #: Y0001926
Chemical compound, drug	Paraquat	Sigma Aldrich	Cat. #: 36,541
Chemical compound, drug	S107rycal drug	Marks’ lab, Columbia University, NY, USA
Software, algorithm	GraphPad	GraphPad	V8.0

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C. elegans strains and culture conditions

Worms were grown and maintained on standard nematode growth medium (NGM) plates on a layer of OP50 Escherichia coli at 20°C, as described (Brenner, 1974). N2 (Bristol) and fkb-2 (ok3007) were provided by the Caenorhabditis Genetics Center (University of Minnesota). fkb-2 (ok3007) was backcrossed six times. The transgenic strain expressing Pmyo-3: GCaMP2 was kindly provided by Zhao-Wen Wang, University of Connecticut Health Center (Liu et al., 2011). Pmyo-3: GCaMP2 was subsequently crossed into fkb-2 (ok3007) for measurement of contraction-associated Ca²⁺ transients.

Age synchronization

Adult worms at the egg-laying stage were treated with alkaline hypochlorite solution to obtain age-synchronized populations, and eggs were plated on NGM plates, as described (Porta-de-la-Riva et al., 2012). For experiments requiring aged worms, age-synchronized animals at the L4 stage were collected in M9 buffer and plated on NGM plates containing 5-fluoro-2’-deoxyuridine (FUDR, Sigma, 50 μM) to prevent egg-laying (Mitchell et al., 1979).

Immunoprecipitation and immunoblotting

Nematodes were grown under standard conditions. For protein biochemistry experiments, a procedure to crack nematodes in a solubilizing and denaturing buffer was adapted (Francis and Waterston, 1985). Briefly, worms were washed and collected with M9 buffer, centrifuged for 2 min at 1000 rpm three times to wash. Worms were allowed to settle to the bottom of the collection tube by sitting on ice for ~5 min. Fluid was removed and the worm pellet was snap frozen in liquid nitrogen. Frozen pellets containing whole nematodes were rapidly thawed under warm running water. A volume of nematode solubilization buffer equal to the volume of the worm pellet was added (nematode solubilization buffer: 0.3% ethanolamine, 2 mM EDTA, 1 mM PMSF in DMSO, 5 mM DTT, 1× protease inhibitor), and tubes were microwaved (25 s for 100 μl pellet; time was increased for greater volumes). Lysates were then quickly drawn into a syringe through a 26-gauge needle and forced back through the needle into a new collection tube on ice. Samples were centrifuged at 1000 rpm for 2 min to remove insoluble material, and the supernatant was transferred to a new tube on ice. Lysates were snap frozen and stored in at –80°C.

A anti-mammalian RyR antibody (4 μg 5029 Ab [Jayaraman et al., 1992]) was used to immunoprecipitate UNC-68 from 100 μg of nematode homogenate. Samples were incubated with the antibody in 0.5 ml of a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na3VO4, 1% Triton- X100, and protease inhibitors) for 1 hr at 4°C. The immune complexes were incubated with protein A Sepharose beads (Sigma, St. Louis, MS) at 4°C for 1 hr, after which time the beads were washed three times with buffer. Proteins were size-fractionated by SDS-PAGE (6% for UNC-68, 15% for FKB-2) and transferred onto nitrocellulose membranes for 1 hr at 200 mA (SemiDry transfer blot, Bio-Rad). After incubation with blocking solution (LICOR Biosciences, Lincoln NE) to prevent non-specific antibody binding, immunoblots were developed using antibodies against RyR (5029, 1:5000), PKAcat (Santa Cruz Biotechnology, sc-903, 1:1000), PDE4 (kindly provided to us by Kenneth Miller, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma), PP1 (sc6104, 1:1000), or an anti-calstabin antibody (Santa Cruz 1: 2500). To determine channel oxidation, the carbonyl groups on the protein side chains were derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophenylhydrazine (DNPH) according to manufacturers (Millipore) instructions. The DNP signal on immunoprecipitated UNC-68 was determined by immunoblotting with an anti-DNP antibody (Millipore, 1:1000). All immunoblots were developed and quantified using the Odyssey Infrared Imaging System (LICOR Biosystems, Lincoln, NE) and infrared-labeled secondary antibodies. In addition, immunoblotting and immunoprecipitation of the UNC-68 macromolecular complex were conducted using another anti-calstabin antibody (1:2000, Abcam) and the same methods as described.

Imaging contraction-associated body wall muscle Ca²⁺ transients

Spontaneous changes in body wall muscle Ca²⁺ were measured in nematodes expressing GCaMP2 by fluorescence imaging using a Zeiss Axio Observer inverted microscope with an electron-multiplying CCD camera (Photometrics Evolve 512) and an LED light source (Colibri). Nematodes were partially immobilized by placing them individually into a 5–10 μl drop of M9 buffer, suspended between a glass slide and coverslip. 20-s videos of individual nematodes were recorded.

Analyzing contraction-associated body wall muscle Ca²⁺ transients

Contraction-associated body wall muscle Ca²⁺ transients were analyzed using an interactive data language-based image quantification software that was developed for this purpose in our laboratory. For each 20-s video, signals from the body wall muscles in nematodes expressing GCaMP2 fluorescence were analyzed using an edge-detection algorithm from each frame as ‘line-scan’ images, with the nematode perimeter on the y-axis and time (s) on the x-axis (Xie et al., 2013; Yuan et al., 2014). These images were then quantified based on the average of the peak Ca²⁺ fluorescence signal on the worm muscle wall.

Drug treatment

To pharmacologically deplete FKB-2 from UNC-68, nematodes were treated for 15 min with 15 μM rapamycin or imaging 50 μM FK506, respectively. To re-associate FKB-2 and UNC-68, aged nematodes were treated with 10 μM S107 for 3–5 hr. Oxidative stress was induced in the worms using 20 mM paraquat, a known generator of superoxide (Wu et al., 2017). Nematodes were grown in standard conditions, age-synchronized as described, washed and collected with M9 buffer, then centrifuged for 2 min at 1000 rpm three times. Worms were allowed to settle to the bottom of the collection tube by sitting on ice for ~5 min. Fluid was removed, the worm pellet was gently resuspended in M9 containing the appropriate drug concentration and gently rocked on a shaker at RT for the indicated time periods. Collection tubes were centrifuged for 2 min at 1000 rpm and M9 containing drug was removed and replaced with M9. Biochemistry or Ca²⁺ measurements were then conducted as previously described (Umanskaya et al., 2014).

Measuring SR Ca²⁺ stores using caffeine activation

Age-synchronized GCaMP2: WT and GCaMP2: FKB-2 KO were grown on NGM plates at 20°C they were separated from their progeny and left undisturbed until day 5. Individual worms were placed in a drop of M9 on a coverslip. The liquid was carefully wicked away using KIMTECH wipes until only a sliver of moisture surrounded the worm. The worm was quickly glued down to the coverslip using a tiny drop of DermaWorm applied to the head and tail of the worm before the worm desiccated. 80 μl of M9 buffer was added immediately afterward to polymerize the glue. Once the worm was secure, a clean lateral cut to the immediate tail region was made using a 20 G 1½ needle (adapted from Wang ZW et al., Neuron 2011⁴⁸). An additional 170 μl of M9 buffer was applied for a total of 250 μl. The completed preparation was placed on the platform of a Zeiss confocal microscope; after 1 min at baseline, 25 mM of caffeine was added to an equal volume of M9 solution. The resulting body wall transients were recorded for 1 min.

Calcium leak assay

Microsomes were prepared by centrifuging the C. elegans lysates (5 days synchronized populations) at 45,000× g for 30 min. Pellets were resuspended in lysis buffer containing 300 mM sucrose. Microsomes (5 µg/ml) were diluted into a 20 mM HEPES buffer (pH 7.2) containing 7 mM NaCl, 1.5 mM MgCl2, 120 mM K-gluconate, 5 mM K-phosphate, 8 mM K-phosphocreatine, 1 µM EGTA, and 2 µM CaCl2 mixed with 3 µM Fluo-4 and added to multiple wells of a 96-well plate. Calcium (Ca²⁺) loading of the microsomes was initiated by adding 1 mM ATP. After Ca²⁺ uptake and a new Fluo-4 signal baseline was observed, 3 μM Thapsigargin was added to inhibit the calcium uptake by the calcium pump (SERCA). The ‘leak’ of Ca²⁺ out of the SR is measured by the increase in intensity of the Fluo-4 signal (measured in a Tecan infinite F500 fluorescence plate reader).

Swimming behavior

Standard M9 buffer was mixed with 2% agar and poured into 96-well plates to create a planar surface for analyzing worm swimming behavior. Once the mixture had polymerized, approximately 180 μl of M9 was pipetted on top of the agar bed and age-synchronized worms from one of two groups (WT or FKB-2 KO) were placed individually into each well. To assess differences in exercise fatigue, worms were allowed to swim freely in M9 buffer for 2 hr; swimming bends and curls (Lüersen et al., 2014) were recorded by eye for 1 min. Representative videos were taken of each group, and investigators were blinded over the course of each experiment. All recordings were made in duplicate.

Statistical analysis

All results are presented as mean ± SEM. Statistical analyses were performed using an unpaired two-tailed Student’s t test (for two groups) and one-way ANOVA with Tukey-Kramer test (for three or more groups), unless otherwise indicated. For survival statistical comparison, we used Gehan-Breslow-Wilcoxon test. p-values <0.05 were considered significant. All statistical analyses were performed with Prism 8.0.

Acknowledgements

This work was supported by grants from the NIH to ARM (T32HL120826, R01HL145473, R01DK118240, R01HL142903, R01HL061503, R01HL140934, R01AR070194).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Andrew Marks, Email: arm42@cumc.columbia.edu.

Mark T Nelson, University of Vermont, United States.

Richard W Aldrich, The University of Texas at Austin, United States.

Funding Information

This paper was supported by the following grants:

National Heart, Lung, and Blood Institute R01HL145473 to Andrew Marks.
National Institute of Diabetes and Digestive and Kidney Diseases R01DK118240 to Andrew Marks.
National Heart, Lung, and Blood Institute R01HL142903 to Andrew Marks.
National Heart, Lung, and Blood Institute R01HL061503 to Andrew Marks.
National Heart, Lung, and Blood Institute R01HL140934 to Andrew Marks.
National Institute of Arthritis and Musculoskeletal and Skin Diseases R01AR070194 to Andrew Marks.
National Heart, Lung, and Blood Institute T32HL120826 to Andrew Marks.

Additional information

Competing interests

No competing interests declared.

owns stock in ARMGO, Inc a company developing compounds targeting RyR and has patents on Rycals.US 2014/0378437, and US 7,718,644.

Author contributions

Conceptualization, Methodology, Writing – original draft, Writing – review and editing.

Conceptualization, Data curation, Formal analysis, Methodology.

Formal analysis, Methodology.

Methodology.

Conceptualization, Methodology, Project administration.

Conceptualization, Data curation, Investigation, Methodology.

Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Writing – original draft, Writing – review and editing.

Additional files

Supplementary file 1. Oxidative regulators in C. elegans vs mice vs humans.

Comparison of homologous oxidative and antioxidative genes between C. elegans, mouse and human. Criteria of comparison includes the function, the subcellular location, the enzymatic activity, mutation induces disrupted phenotype and percentage of homology.

elife-75529-supp1.docx^{(51.3KB, docx)}

Supplementary file 2. Amino acid composition of UNC-68 and Human RyR1.

Comparison of amino acid abundance in the C. elegans UNC-68 and the human RyR1 calcium channels. Number and percentage of Serines and methionines for each species are shown in red.

elife-75529-supp2.docx^{(15.8KB, docx)}

Transparent reporting form

elife-75529-transrepform1.pdf^{(684.1KB, pdf)}

Data availability

All data are described/available in the manuscript.

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eLife. doi: 10.7554/eLife.75529.sa0

Editor's evaluation

Mark T Nelson ¹

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 calcium leak, reduced body wall calcium transients and muscle weakness, changes that are similar to those that occur in aging human skeletal muscle despite the dramatic differences in the lifespan of the two organisms.

eLife. doi: 10.7554/eLife.75529.sa1

Decision letter

Editor: Mark T Nelson¹

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in C. elegans" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Richard Aldrich as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. While researchers in the field may not be misled by the statement in the Introduction implying that muscle contraction is fully independent of extracellular ca²⁺, for the benefit of those who may not have a fully nuanced understanding of EC coupling in skeletal (or cardiac) muscle, the authors should modify the statement to include the concept of triggering-influxes of extracellular ca²⁺.

2. It is recommended that the authors sort out their thinking on the cause and effect relationships among UNC-68 oxidation, FKB-2 dissociation and UNC-68 leakiness, and present a cogent and compelling argument for them. It is not sufficient to present various lines of evidence showing that these things are interrelated and connected via a fuzzy mechanism to reduced ca²⁺ transients and an aging phenotype.

3. 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.

4. Please "dial back" your rhetoric on longevity.

5. Presentation is choppy. The presentation of all results shown in Figure 2 (Age-dependent…, FKB-2 deficiency… UNC-68 channels are leaky… and UNC-68 channel leak impairs…) should be merged into a single section, eliminating any redundancies in the process.

6. The strangely worded last sentence of the first paragraph of the "Pharmacologically mimicking aging phenotype…" section is internally illogical – the conclusion (second phrase) doesn't logically follow from the premise (first phrase). This muddles the message of this paragraph, which is presumably to convey the idea that it is the ability of rapamycin and FK506 to compete off calstabin (FKB-2 in this case), and not their well-known abilities to inhibit mTOR and calcineurin, respectively, that accounts for their observed effects on ca²⁺ transients, therefore justifying their use in mimicking the aging phenotype. The sentence should be revised to more accurately convey the authors' intended meaning.

7. Suggest organizing panels in Figure 3 to mirror organization in Figure 2 since they are similar data, just differing in "stimulus" (age in Figure 2 and pharmacology in Figure 3).

8. Need to clarify what time point the "WT+FK506" label corresponds to in all figure panels where it appears.

9. Nematode age should be indicated for all experiments/figure panels.

10. English quality could be improved (e.g., awkward phrasing, extraneously added or missing articles).

11. "This interaction [between RyR1 and CaV1.1] couples excitation of the sarcolemma to muscle contraction and eliminates dependence on extracellular ca²⁺." Direct coupling of RyR to sarcolemmal ca²⁺ channels via SPRY2 domains does not entirely eliminate the need for extracellular ca²⁺ for EC coupling; it merely reduces it to a very small influx necessary to trigger release from intracellular stores via a ca²⁺-induced ca²⁺-release mechanism.

12. Figure 2B:

– Is the day-15 bar for the FKB-2-KO missing? Or are there no ca²⁺ transients at this time point?

– Are these muscle contraction-induced transients or caffeine-induced transients?

13. FKB-2 deficiency accelerates age-dependent reduction in ca²⁺ transient that drives muscle contraction Not sure why this mini-section is here; recapitulates the first paragraph of the previous paragraph.

14. UNC-68 channels are leaky in the absence of FKB-2

"Once the fluorescence level plateaus…" Fluorescence level clearly has not plateaued in microsome samples from FKB-2-KO worms at the time thapsigargin is added. If the assay system is capable of truly reaching a plateau from which the resulting leak can be more accurately measured, experiments should be repeated; alternatively, a rationale should be provided for "jumping the gun" with thapsigargin addition.

15. UNC-68 channel leak impairs exercise capacity

"FKB-2 KO worms exhibit reduced life span compared to WT (Figure 2I)" – Maximum lifespan was not different; what are the average lifespans for WT and KO worms, and what statistical analysis supports the difference?

16. Pharmacologically mimicking aging phenotype affects ca²⁺ transient and impairs exercise capacity.

"…longer treatment of WT worms with FK506 caused oxidation of UNC-68, demonstrating a relationship between depletion of FKB-2 and oxidation of UNC-68" – This implies that depleting FKB-2 causes oxidation of UNC-68. This is causally backwards relative to the proposed model, where oxidation of UNC-68 (associated with aging) causes dissociation of FKB-2, leading to leaky channels and reduced muscle function and exercise capacity. Although in the Discussion, the authors do obliquely address the idea that dissociating FKB-2 could be expected to increase oxidation through increased UNC-68 leakiness, it comes too late in the presentation to do anything but muddle the message.

17. Figure 3F:

– The only way the labeling for this panel makes sense is if "WT+FK506" and "FKB-2 KO+FK506" labels are switched AND "WT+FK506" corresponds to a measurement at some earlier time point (0 or 1h? Not specified). Labeling in Figure 3D (and Figure 4C) is consistent with the idea that labels in 3F were switched, but the label "WT+FK506" is still ambiguous.

– Also, the "time course" is really a digital response that goes from ~0 at 2 h to max at 4 h. At minimum, data should be collected from a time point between 2 and 4 hours to demonstrate an actual time course.

18. Figure 4D:

– What is"WT+FK506" exactly? Is this the 4-h time point, as would make sense given that the comparison is with FKB-2 KO+FK506 (i.e., both conditions are associated with maximal UNC-68 oxidation), or is this a 0 h (or 1 h) time point, as suggested by the use of "WT+FK506" as the first term in the "WT+FK506", "WT+FK506, 2h" and "WT+FK506, 4h" series?

– Not clear why the WT+FK506 conditions would show a greater reduction in peak ca²⁺ than the FKB-2 KO condition (with or without FK506), since FKB-2 knockout presumably has a greater effect on UNC-68 subunit composition (i.e., FKB-2 completely gone) than competing off FKB-2 with FK506 (partially eliminated).

19. Figure 4A-D:

– Overall effects of FKB-2 KO/KD on ca²⁺ transients are not terribly impressive (Figure 4D) and not all that well correlated with UNC-68 oxidation (Figure 4C) or FKB-2 binding (Figure 4B). According to the authors' model, it is the loss of FKB-2 that ultimately is responsible for the leakiness of UNC-68 and associated phenotypes. Therefore, complete loss of FKB-2 binding should be more closely correlated with diminished ca²⁺ transients than UNC-68 oxidation (which presumably causes graded dissociation of FKB-2). By the same logic, FKB-2 KO should be more closely correlated with diminished ca²⁺ transients than WT+FK506 or paraquat (again, assuming that the paraquat-induced, UNC-68 oxidation-dependent dissociation of FKB-2 is also graded).

– The idea that the loss of FKB-2 is ultimately responsible for the leakiness of UNC-68 and associated phenotypes appeared to be the proposed model up until the presentation of data hinting that oxidation in and of itself was capable of producing this leakiness. In this context, only the paraquat FKB-2 binding/UNC-68 oxidation data were well correlated with peak ca²⁺ data.

20. Nematode age is a key parameter that is rarely specified in the text or figures/legends. Note that "young" is uninformative unless specifically defined.

21. "Another key question is why UNC-68 becomes oxidized within two weeks, whereas the same post-translational modification requires two years in mice and 80 years in humans (5)." This is not a key question. There are many, many contributors to the longevity of animals. It would be hard to find anyone who would argue that worms would live for years if only they had a greater antioxidant capacity.

22. The authors go to great lengths to connect UNC-68 oxidation with FKB-2 dissociation and link this relationship to UNC-68 leakiness and the aging phenotype. But it's never really clear whether it is the dissociation of FKB-2 or the oxidation of UNC-68 that underlies the leakiness. The authors clearly believe that FKB-2 dissociation is an important event in this sequence, otherwise, why use FKB-2-KO and WT+FK506 models throughout? But another formulation of their model (Discussion) is all oxidation all the time: "RyR1 leak (due to age-dependent oxidation of the channel) causes mitochondrial ca²⁺ overload, resulting in ROS production, thus leading to further oxidation of RyR1 and exacerbation of the SR ca²⁺ leak." Adding to this either/or quality is the apparent bidirectionality of the oxidation-FKB-2 dissociation relationship: not only does UNC-68 oxidation lead to FKB-2 dissociation, FKB-2 dissociation also leads to UNC-68 oxidation (presumably through feedback on mitochondrial ca²⁺ overload via increased UNC-68 leakiness). Lost in the presentation is how UNC-68 oxidation causes FKB-2 dissociation and whether (and how) UNC-68 oxidation causes increased UNC-68 leakiness in the absence of FKB-2 dissociation. All in all, the manuscript presents quite the Mobius strip with respect to cause-and-effect relationships.

eLife. 2022 May 4;11:e75529. doi: 10.7554/eLife.75529.sa2

Author response

Essential revisions:

1. While researchers in the field may not be misled by the statement in the Introduction implying that muscle contraction is fully independent of extracellular ca²⁺, for the benefit of those who may not have a fully nuanced understanding of EC coupling in skeletal (or cardiac) muscle, the authors should modify the statement to include the concept of triggering-influxes of extracellular ca²⁺.

We thank the Reviewer for her/his comment. The three mammalian RyRs (RyR1, RyR2, RyR3) exhibit ca²⁺ induced ca²⁺ release (CICR), a process in which ca²⁺ itself activates the channel to release more ca²⁺. However, in physiological conditions extracellular ca²⁺ is not necessary for the activation of the mammalian skeletal muscle RyR1 to be activated. Indeed, ca²⁺ release from mammalian skeletal muscle, RyR1 isoform is essentially triggered by conformational changes to the voltage gated sensor in the DHPR channel, upon depolarization of the T-tubule (Rios and Pizarro 1991, Schneider 1994). This point of view has been supported by studies in which suppression of the extracellular ca²⁺ failed to blunt muscle contraction. Indeed, myofibers exhibit normal twitching even in a Ringer solution with 80 mM EGTA (Luttgau 1968). Ca²⁺ release and transients can be also recorded under voltage clamp at large positive potentials under which the slow inward ca²⁺ current becomes outwards (Brum 1987). Moreover, a series of mutagenesis studies have shown that the amino acid residues 489-503 in the DHPR-β1 subunit directly interact with RyR1 channels and mediate EC coupling (Eltit et al., JBC, 2014). We believe that the Reviewer’s comment refers to the skeletal muscle EC coupling in amphibians (e.g., frog), which exhibit a minor dependence of extracellular ca²⁺ due to the expression of the β-RyR with similar gating properties to mammalian cardiac RyR2 (see Review Takashi 2011). We revised our introduction to clarify that we are describing the mechanism of EC coupling in mammals and in C-elegans (pages 2-3-4-5-6-7-8-10 and 12).

2. It is recommended that the authors sort out their thinking on the cause and effect relationships among UNC-68 oxidation, FKB-2 dissociation and UNC-68 leakiness, and present a cogent and compelling argument for them. It is not sufficient to present various lines of evidence showing that these things are interrelated and connected via a fuzzy mechanism to reduced ca²⁺ transients and an aging phenotype.

We thank the Reviewer for her/his comment which helped us to clarify our message. UNC-68 oxidation causes depletion of FKB-2 from the channel, which results in ca²⁺ leak. This ca²⁺ leak leads to excessive ca²⁺ accumulation in the mitochondria, generates ROS and further oxidizes UNC-68. In disease conditions (e.g. aging), this mechanism results in a vicious cycle in which oxidation promotes FKB-2 depletion which subsequently promotes further oxidation of UNC-68 or in mammalian muscle, RyR1 (Ref Andersson et al., Mol Cell). We revised our manuscript to better clarify this mechanism (Last paragraph, page 10).

3. 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 thank the Reviewer for his comment which helped improve the manuscript. 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 13).

4. Please "dial back" your rhetoric on longevity.

We have largely replaced the word “lifespan” by “age-dependent loss of muscle function” throughout the revised manuscript.

5. Presentation is choppy. The presentation of all results shown in Figure 2 (Age-dependent…, FKB-2 deficiency… UNC-68 channels are leaky… and UNC-68 channel leak impairs…) should be merged into a single section, eliminating any redundancies in the process.

We thank the Reviewer for her/his comment which helped achieve a better organization of the manuscript. In the revised version, we merged the presentation of the results shown in Figure 2 into 1 section and eliminated the redundancies (pages 6 and 7).

6. The strangely worded last sentence of the first paragraph of the "Pharmacologically mimicking aging phenotype…" section is internally illogical – the conclusion (second phrase) doesn't logically follow from the premise (first phrase). This muddles the message of this paragraph, which is presumably to convey the idea that it is the ability of rapamycin and FK506 to compete off calstabin (FKB-2 in this case), and not their well-known abilities to inhibit mTOR and calcineurin, respectively, that accounts for their observed effects on ca²⁺ transients, therefore justifying their use in mimicking the aging phenotype. The sentence should be revised to more accurately convey the authors' intended meaning.

We thank the Reviewer for her/his comment which helped clarifying our message on the use of FK506 and rapamycin to compete off FKB-2. We revised these statement in the new version of the manuscript and added references supporting the significance of these experiments (Last paragraph, page 7).

7. Suggest organizing panels in Figure 3 to mirror organization in Figure 2 since they are similar data, just differing in "stimulus" (age in Figure 2 and pharmacology in Figure 3).

In the revised manuscript, the panels organization in Figure 3 mirrors the panel organization in Figure 2.

8. Need to clarify what time point the "WT+FK506" label corresponds to in all figure panels where it appears.

We apologize for not adding this important information. “WT+FK506” label correspond to a short treatment (15min) of WT worms with FK506 drug. We have revised all our figures and figure legends to show the time point for each experiment.

9. Nematode age should be indicated for all experiments/figure panels.

Again, we thank the Reviewer for her/his comment we apologize for not adding this important information in some experiments. We added the nematode age for all the experiments described in the manuscript.

10. English quality could be improved (e.g., awkward phrasing, extraneously added or missing articles).

We revised our manuscript to improve the English quality and remove potentially confusing words.

11. "This interaction [between RyR1 and CaV1.1] couples excitation of the sarcolemma to muscle contraction and eliminates dependence on extracellular ca²⁺." Direct coupling of RyR to sarcolemmal ca²⁺ channels via SPRY2 domains does not entirely eliminate the need for extracellular ca²⁺ for EC coupling; it merely reduces it to a very small influx necessary to trigger release from intracellular stores via a ca²⁺-induced ca²⁺-release mechanism.

We thank the Reviewer for her/his remark. We agree that the physical interaction between RyR1 and Cav_1.1 does not eliminate the need for extracellular ca²⁺ or the activation of RyR1 by ca²⁺ but it renders it unnecessary to achieve a normal contraction. Please refer to the answer to question 1 for details. In the revised manuscript, we replace “eliminates dependence on extracellular ca²⁺” by “reduces dependence on extracellular ca²⁺” (Page 4)

12. Figure 2B:

– Is the day-15 bar for the FKB-2-KO missing? Or are there no ca²⁺ transients at this time point?

– Are these muscle contraction-induced transients or caffeine-induced transients?

We thank the Reviewer for her/his comment, and we apologize for not being clear enough in the description of Figure 2B. Indeed, we did not measure ca²⁺ in FKB-2-KO worms at day 15 as the mutant already exhibited a significant reduction in ca²⁺ peak at day 7. The ca²⁺ transient at day 15 was only measured in WT since their ca²⁺ peak was not affected at day 7. To be consistent, we removed the WT Day 15 ca²⁺ transient. We also indicated on the top of panel 2B whether these measurements are contraction-induced ca²⁺ transients or caffeine-induced ca²⁺ transients.

13. FKB-2 deficiency accelerates age-dependent reduction in ca²⁺ transient that drives muscle contraction Not sure why this mini-section is here; recapitulates the first paragraph of the previous paragraph.

We have removed this redundancy in the revised version of the manuscript.

14. UNC-68 channels are leaky in the absence of FKB-2

"Once the fluorescence level plateaus…" Fluorescence level clearly has not plateaued in microsome samples from FKB-2-KO worms at the time thapsigargin is added. If the assay system is capable of truly reaching a plateau from which the resulting leak can be more accurately measured, experiments should be repeated; alternatively, a rationale should be provided for "jumping the gun" with thapsigargin addition.

We have repeated this leak assay experiment and adjusted the conditions, mainly by using a higher amount of microsomes, which allowed us to reach a fluorescence plateau after adding thapsigargin. The new results are displayed in the new Figure 2F.

15. UNC-68 channel leak impairs exercise capacity

"FKB-2 KO worms exhibit reduced life span compared to WT (Figure 2I)" – Maximum lifespan was not different; what are the average lifespans for WT and KO worms, and what statistical analysis supports the difference?

The median WT survival was 18 days versus 14 days for FKB-2-KO worms. We performed Gehan-Breslow-Wilcoxon test for survival comparison which gives more weight to death at early time points. Since we do not have censored worms at any time point, we chose this test over the log-rank test to take in consideration the early death of worms. We added these details to the figure 2 legend.

16. Pharmacologically mimicking aging phenotype affects ca²⁺ transient and impairs exercise capacity.

"…longer treatment of WT worms with FK506 caused oxidation of UNC-68, demonstrating a relationship between depletion of FKB-2 and oxidation of UNC-68" – This implies that depleting FKB-2 causes oxidation of UNC-68. This is causally backwards relative to the proposed model, where oxidation of UNC-68 (associated with aging) causes dissociation of FKB-2, leading to leaky channels and reduced muscle function and exercise capacity. Although in the Discussion, the authors do obliquely address the idea that dissociating FKB-2 could be expected to increase oxidation through increased UNC-68 leakiness, it comes too late in the presentation to do anything but muddle the message.

We addressed the concept of “oxidation-ca²⁺ leak” vicious cycle earlier in the discussion (Page 10).

17. Figure 3F:

– The only way the labeling for this panel makes sense is if "WT+FK506" and "FKB-2 KO+FK506" labels are switched AND "WT+FK506" corresponds to a measurement at some earlier time point (0 or 1h? Not specified). Labeling in Figure 3D (and Figure 4C) is consistent with the idea that labels in 3F were switched, but the label "WT+FK506" is still ambiguous.

We thank the Reviewer for her/his comment, and we apologize for the lack of clarity. Indeed, WT+FK506 are WT worms treated with FK506 for 15 min. The worms exhibit complete depletion of FKB-2 but no oxidation. The oxidation only appears when the WT worms are treated with FK506 for 4 hours. The FKB-2 KO worms have basal oxidation independent of FK506 treatment (no difference between FKB-2-KO treated and untreated), (see revised Figure 3, Panel D and G). The basal oxidation in the FKB-2 KO worms could be due to constitutive ca²⁺ leak which mitochondrial ca²⁺ overload and oxidative stress in this strain.

The first line of the gel is not quantified (no bar graphs are shown), it represents an immunoprecipitated RyR1 from mouse skeletal muscle and serves as a positive control for the antibody. The quantification bars reflect only the experiments performed with worms. We revised the figure 3, and 4 legends to better clarify these details.

– Also, the "time course" is really a digital response that goes from ~0 at 2 h to max at 4 h. At minimum, data should be collected from a time point between 2 and 4 hours to demonstrate an actual time course.

We thank the Reviewer for her/his comment, and we apologize for the lack of clarity regarding the time course experiments. Indeed, our time course includes 0, 15 min, 2 h and 4 h. We have added these missing details to the figure and the legend.

18. Figure 4D:

– What is"WT+FK506" exactly? Is this the 4-h time point, as would make sense given that the comparison is with FKB-2 KO+FK506 (i.e., both conditions are associated with maximal UNC-68 oxidation), or is this a 0 h (or 1 h) time point, as suggested by the use of "WT+FK506" as the first term in the "WT+FK506", "WT+FK506, 2h" and "WT+FK506, 4h" series?

“WT+FK506” is WT worms treated with FK506 for 15 min. This group exhibits complete depletion of FKB-2 but no oxidation. Of note, the first line of the gel is not quantified (no bar graphs are shown), it represents an immunoprecipitated RyR1 from mouse skeletal muscle and serves as a positive control for the antibody. This detail has been added to the figure 4 legend.

– Not clear why the WT+FK506 conditions would show a greater reduction in peak ca²⁺ than the FKB-2 KO condition (with or without FK506), since FKB-2 knockout presumably has a greater effect on UNC-68 subunit composition (i.e., FKB-2 completely gone) than competing off FKB-2 with FK506 (partially eliminated).

We thank the Reviewer for her/his very interesting comment. We agree with the Reviewer that the FKB-2-KO worms were expected to have a greater reduction in peak ca²⁺ compared to WT+FK506 at any investigated age. However, this was not the case at day 5. One possibility is that the FKB2-KO worms develop a compensatory mechanism over time due to the chronic ca²⁺ leak to protect the nematode from severe pathogenic ca²⁺ overload at an early age. This mechanism ends up failing over time and the ca²⁺ leak is exacerbated around day 7 of age. However, the adaptation to chronic ca²⁺ leak is an important question that needs further investigation in future and is beyond the scope in the current study. Please see discussion, first paragraph of page 13 for proposed potential mechanisms.

19. Figure 4A-D:

– Overall effects of FKB-2 KO/KD on ca²⁺ transients are not terribly impressive (Figure 4D) and not all that well correlated with UNC-68 oxidation (Figure 4C) or FKB-2 binding (Figure 4B). According to the authors' model, it is the loss of FKB-2 that ultimately is responsible for the leakiness of UNC-68 and associated phenotypes. Therefore, complete loss of FKB-2 binding should be more closely correlated with diminished ca²⁺ transients than UNC-68 oxidation (which presumably causes graded dissociation of FKB-2). By the same logic, FKB-2 KO should be more closely correlated with diminished ca²⁺ transients than WT+FK506 or paraquat (again, assuming that the paraquat-induced, UNC-68 oxidation-dependent dissociation of FKB-2 is also graded).

We thank the Reviewer for her/his comment which help clarifying the results displayed in Figure 4A-D. These experiments were done at 5 days of age. Despite a significant UNC-68 oxidation and absence of FKB-2, the FKB-2-KO worms do not exhibit significant reduction in ca²⁺ transients until day 7. Again, as stated in response to question 18, this could be due to a compensatory mechanism by which these worms overcome the chronic ca²⁺ leak early in life but ends up failing at 7 days. In contrast, the WT worms do not develop a compensatory mechanism upon acute oxidation (paraquat treatment) or FKB-2 depletion (FK506 treatment) and therefore rapidly exhibit a leaky ca²⁺ phenotype (reduced ca²⁺ transient). We discussed these details and added the age of worms for each experiment in the revised version of the manuscript as requested by the reviewer (pages 13).

– The idea that the loss of FKB-2 is ultimately responsible for the leakiness of UNC-68 and associated phenotypes appeared to be the proposed model up until the presentation of data hinting that oxidation in and of itself was capable of producing this leakiness. In this context, only the paraquat FKB-2 binding/UNC-68 oxidation data were well correlated with peak ca²⁺ data.

The FKB-2 KO worms do not exhibit a reduction in the ca²⁺ transient at 5 days of age. See answer to question 18 and 19. In WT worms, paraquat treatment causes acute UNC-68 oxidation and subsequently depletes FKB-2 of channel resulting in an acute ca²⁺. Under these conditions, the WT worms have no time to adapt their ca²⁺ machinery and as such exhibit a more severe ca²⁺ leak phenotype at this age (day 5). Again, the pharmacological depletion of FKB-2 is acute, and its consequences are rapidly exacerbated. We discussed these aspects in the revised manuscript (page 13).

20. Nematode age is a key parameter that is rarely specified in the text or figures/legends. Note that "young" is uninformative unless specifically defined.

We added the specific age for each experiment in the revised text and in the figures/legends.

21. "Another key question is why UNC-68 becomes oxidized within two weeks, whereas the same post-translational modification requires two years in mice and 80 years in humans (5)." This is not a key question. There are many, many contributors to the longevity of animals. It would be hard to find anyone who would argue that worms would live for years if only they had a greater antioxidant capacity.

We think this is an important question because the enzymes involved in UNC-68 oxidation are very similar to those causing age-dependent oxidation of RyR1 in mammals which takes orders of magnitude longer. So, if one could answer this question, it would shed some light on the unanswered question of what determines species-specific differences in lifespan. The point is not that the oxidation is the most important issue, but rather the etiology of the tremendous difference in kinetics of the age-dependent signals between species. Despite all of the research on aging this question has yet to be answered.

22. The authors go to great lengths to connect UNC-68 oxidation with FKB-2 dissociation and link this relationship to UNC-68 leakiness and the aging phenotype. But it's never really clear whether it is the dissociation of FKB-2 or the oxidation of UNC-68 that underlies the leakiness. The authors clearly believe that FKB-2 dissociation is an important event in this sequence, otherwise, why use FKB-2-KO and WT+FK506 models throughout? But another formulation of their model (Discussion) is all oxidation all the time: "RyR1 leak (due to age-dependent oxidation of the channel) causes mitochondrial ca²⁺ overload, resulting in ROS production, thus leading to further oxidation of RyR1 and exacerbation of the SR ca²⁺ leak." Adding to this either/or quality is the apparent bidirectionality of the oxidation-FKB-2 dissociation relationship: not only does UNC-68 oxidation lead to FKB-2 dissociation, FKB-2 dissociation also leads to UNC-68 oxidation (presumably through feedback on mitochondrial ca²⁺ overload via increased UNC-68 leakiness). Lost in the presentation is how UNC-68 oxidation causes FKB-2 dissociation and whether (and how) UNC-68 oxidation causes increased UNC-68 leakiness in the absence of FKB-2 dissociation. All in all, the manuscript presents quite the Mobius strip with respect to cause-and-effect relationships.

Oxidation of UNC-68 causes a loss of FKB-2 from the channel. The precise mechanism by which this occurs is unknown and the subject of ongoing structural studies in our laboratory. The result is leaky UNC-68 channels. The same leak can be observed by removing FKB-2 without oxidation (e.g with FK506 or in the FKB-2 KO worms). Conversely, preventing FKB-2 removal with a Rycal drug (S107) can prevent leak even when the channels are oxidized. Thus, we conclude that it is the loss of FKB-2 from the channel that destabilizes the closed state and causes leak, not the oxidation by itself. The oxidation becomes important in the context of lifespan because it is the age-dependent increase in oxidative load in the worms that ultimately leads to UNC-68 oxidation in vivo.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure 1—source data 1. Full incut gels of Figure 1.

elife-75529-fig1-data1.pdf^{(258KB, pdf)}

Figure 2—source data 1. Full incut gels of Figure 2.

elife-75529-fig2-data1.pdf^{(295.8KB, pdf)}

Figure 3—source data 1. Full incut gels of Figure 3.

elife-75529-fig3-data1.pdf^{(417.3KB, pdf)}

Figure 4—source data 1. Full incut gels of Figure 4.

elife-75529-fig4-data1.pdf^{(203.8KB, pdf)}

Figure 5—source data 1. Full incut gels of Figure 5.

elife-75529-fig5-data1.pdf^{(282.2KB, pdf)}

Supplementary file 1. Oxidative regulators in C. elegans vs mice vs humans.

elife-75529-supp1.docx^{(51.3KB, docx)}

Supplementary file 2. Amino acid composition of UNC-68 and Human RyR1.

Comparison of amino acid abundance in the C. elegans UNC-68 and the human RyR1 calcium channels. Number and percentage of Serines and methionines for each species are shown in red.

elife-75529-supp2.docx^{(15.8KB, docx)}

Transparent reporting form

elife-75529-transrepform1.pdf^{(684.1KB, pdf)}

Data Availability Statement

All data are described/available in the manuscript.

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PERMALINK

Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in Caenorhabditis elegans

Haikel Dridi

Frances Forrester

Alisa Umanskaya

Wenjun Xie

Steven Reiken

Alain Lacampagne

Andrew Marks

Roles

Abstract

Introduction

Results

Conserved evolution and architecture of UNC-68

Figure 1. UNC-68 comprises a macromolecular complex comparable to its mammalian homolog ryanodine receptor (RyR); RyR (A) and FKBP (B) evolution among species was inferred by the maximum likelihood method based on the JTT matrix-based model.

Age-dependent biochemical and functional remodeling of UNC-68

Figure 2. Remodeling of UNC-68 and age-dependent reduction in intracellular calcium (Ca2+) transients is accelerated in FKB-2 (ok3007) (A) Representative trace of Ca2+ transients from GCaMP2 wild type (WT) and FKB-2 KO (at day 7).

Pharmacologically mimicking aging phenotype affects Ca2+ transient and impairs exercise capacity

Figure 3. Depleting FKB-2 from UNC-68 causes UNC-68 oxidation (A) Representative image of caffeine activated calcium transient in GCaMP2 wild type (WT) at day 5; arrow denotes peak fluorescence in body wall muscle.

Oxidation of UNC-68 causes reduced body wall muscle Ca2+ transients

UNC-68 Ca2+ channel is a potential therapeutic target in aging

Discussion

Materials and methods

Key resources table.

C. elegans strains and culture conditions

Age synchronization

Immunoprecipitation and immunoblotting

Imaging contraction-associated body wall muscle Ca2+ transients

Analyzing contraction-associated body wall muscle Ca2+ transients

Drug treatment

Measuring SR Ca2+ stores using caffeine activation

Calcium leak assay

Swimming behavior

Statistical analysis

Acknowledgements

Funding Statement

Contributor Information

Funding Information

Additional information

Competing interests

Author contributions

Additional files

Data availability

References

Editor's evaluation

Mark T Nelson

Roles

Decision letter

Roles

Author response

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Figure 2. Remodeling of UNC-68 and age-dependent reduction in intracellular calcium (Ca²⁺) transients is accelerated in FKB-2 (ok3007) (A) Representative trace of Ca²⁺ transients from GCaMP2 wild type (WT) and FKB-2 KO (at day 7).

Pharmacologically mimicking aging phenotype affects Ca²⁺ transient and impairs exercise capacity

Oxidation of UNC-68 causes reduced body wall muscle Ca²⁺ transients

UNC-68 Ca²⁺ channel is a potential therapeutic target in aging

Imaging contraction-associated body wall muscle Ca²⁺ transients

Analyzing contraction-associated body wall muscle Ca²⁺ transients

Measuring SR Ca²⁺ stores using caffeine activation