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. 2024 May 16:13:RP94616.
doi: 10.7554/eLife.94616.

Remodeling of skeletal muscle myosin metabolic states in hibernating mammals

Christopher T A Lewis  1 , Elise G Melhedegaard  1 , Marija M Ognjanovic  1 , Mathilde S Olsen  1 , Jenni Laitila  1 , Robert A E Seaborne  1   2 , Magnus Gronset  3 , Changxin Zhang  4 , Hiroyuki Iwamoto  5 , Anthony L Hessel  6   7 , Michel N Kuehn  6   7 , Carla Merino  8 , Nuria Amigo  8 , Ole Frobert  9   10 , Sylvain Giroud  11   12 , James F Staples  13 , Anna V Goropashnaya  14 , Vadim B Fedorov  14 , Brian Barnes  14 , Oivind Toien  14 , Kelly Drew  14 , Ryan J Sprenger  15 , Julien Ochala  1
Affiliations

Remodeling of skeletal muscle myosin metabolic states in hibernating mammals

Christopher T A Lewis et al. Elife. .

Abstract

Hibernation is a period of metabolic suppression utilized by many small and large mammal species to survive during winter periods. As the underlying cellular and molecular mechanisms remain incompletely understood, our study aimed to determine whether skeletal muscle myosin and its metabolic efficiency undergo alterations during hibernation to optimize energy utilization. We isolated muscle fibers from small hibernators, Ictidomys tridecemlineatus and Eliomys quercinus and larger hibernators, Ursus arctos and Ursus americanus. We then conducted loaded Mant-ATP chase experiments alongside X-ray diffraction to measure resting myosin dynamics and its ATP demand. In parallel, we performed multiple proteomics analyses. Our results showed a preservation of myosin structure in U. arctos and U. americanus during hibernation, whilst in I. tridecemlineatus and E. quercinus, changes in myosin metabolic states during torpor unexpectedly led to higher levels in energy expenditure of type II, fast-twitch muscle fibers at ambient lab temperatures (20 °C). Upon repeating loaded Mant-ATP chase experiments at 8 °C (near the body temperature of torpid animals), we found that myosin ATP consumption in type II muscle fibers was reduced by 77-107% during torpor compared to active periods. Additionally, we observed Myh2 hyper-phosphorylation during torpor in I. tridecemilineatus, which was predicted to stabilize the myosin molecule. This may act as a potential molecular mechanism mitigating myosin-associated increases in skeletal muscle energy expenditure during periods of torpor in response to cold exposure. Altogether, we demonstrate that resting myosin is altered in hibernating mammals, contributing to significant changes to the ATP consumption of skeletal muscle. Additionally, we observe that it is further altered in response to cold exposure and highlight myosin as a potentially contributor to skeletal muscle non-shivering thermogenesis.

Keywords: biochemistry; cell biology; chemical biology; eliomys quercinus; ictidomys tridecemlineatus; ursus americanus; ursus arctos.

Plain language summary

Many animals use hibernation as a tactic to survive harsh winters. During this dormant, inactive state, animals reduce or limit body processes, such as heart rate and body temperature, to minimise their energy use. To conserve energy during hibernation, animals can use different approaches. For example, garden dormice undergo periodic states of extremely low core temperatures (down to 4–8oC); whereas Eurasian brown bears see milder temperature drops (down to 23–25oC). An important organ that changes during hibernation is skeletal muscle. Skeletal muscle typically uses large amounts of energy, making up around 50% of body mass. To survive, hibernating animals must change how their skeletal muscle uses energy. Traditionally, active myosin – a protein found in muscles that helps muscles to contract – was thought to be responsible for most of the energy use by skeletal muscle. But, more recently, resting myosin has also been found to use energy when muscles are relaxed. Lewis et al. studied myosin and skeletal muscle energy use changes during hibernation and whether they could impact the metabolism of hibernating animals. Lewis et al. assessed myosin changes in muscle samples from squirrels, dormice and bears during hibernation and during activity. Experiments showed changes in resting myosin in squirrels and dormice (whose temperature drops to 4–8oC during hibernation) but not in bears. Further analysis revealed that cooling samples from non-hibernating muscle to 4–8oC increased energy use in resting myosin, thereby generating heat. However, no increase in energy use was found after cooling hibernating muscle samples to 4–8oC. This suggest that resting myosin generates heat at cool temperatures – a mechanism that is switched off in hibernating animals to allow them to cool their body temperature. These findings reveal key insights into how animals conserve energy during hibernation. In addition, the results show that myosin regulates energy use in skeletal muscles, which indicates myosin may be a potential drug target in metabolic diseases, such as obesity.

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Conflict of interest statement

CL, EM, MO, MO, JL, RS, MG, CZ, HI, MK, CM, NA, OF, SG, JS, AG, VF, BB, OT, KD, RS, JO No competing interests declared, AH ALH is an owner of Accelerated Muscle Biotechnologies Consultants LLC, which performed the X-ray data reduction and analysis, but services rendered were not linked to outcome or interpretation

Figures

Figure 1.
Figure 1.. Myosin dynamics and myosin ATP consumption is unchanged in Ursus arctos and Ursus americanus during hibernation.
(A) Representative fluorescence mant-ATP decays from single muscle fibers isolated from Ursus arctos skeletal muscle measured over 300 s. (B–C) Percentage of myosin heads in the P1/DRX (B) or P2/SRX (C) from Ursus arctos single muscle fibers obtained during summer (active) or winter (hibernating) periods. Values were separated based on each individual fiber was MyHC type I or MyHC type II. (D) T1 value in seconds denoting the ATP turnover lifetime of the DRX. (E) T2 value in seconds denoting the ATP turnover lifetime in seconds of the SRX. (F) Calculated myosin ATP consumption values of each single muscle fiber per minute. This was calculated using the equation shown in the Materials and methods section. (G) Representative fluorescence mant-ATP decays from single muscle fibers isolated from Ursus americanus skeletal muscle measured over 300 s. (H–I) Percentage of myosin heads in the P1/DRX (G) or P2/SRX (H) from Ursus americanus single muscle fibers obtained during summer (active) or winter (hibernating) periods. Values were separated based on each individual fiber was MyHC type I or MyHC type II. (J) T1 value in seconds denoting the ATP turnover lifetime of the DRX. (K) T2 value in seconds denoting the ATP turnover lifetime in seconds of the SRX. (L) Calculated myosin ATP consumption values of each single muscle fiber per minute. Grey circles represent the values from each individual muscle fiber which was analyzed. Colored triangles represent the mean value from an individual animal, 8–12 fibers analyzed per animal. Statistical analysis was performed upon mean values. One-way ANOVA was used for statistical testing. n=5 individual animals per group. Figure created using BioRender.com and published using a CC BY-NC-ND license with permission.
Figure 2.
Figure 2.. Myosin ATP turnover lifetime is reduced during hibernation in small hibernators, Eliomys quercinus and Ictidomys tridecemlineatus, resulting in an increase in myosin ATP consumption at ambient temperatures.
(A–B) Percentage of myosin heads in the P1/DRX (A) or P2/SRX (B) from E. quercinus single muscle fibers obtained during active, interbout arousal (IBA) or torpor periods. Values were separated based on each individual fiber was MyHC type I or MyHC type II. (C) T1 value in seconds denoting the ATP turnover lifetime of the DRX in E. quercinus. (D) T2 value in seconds denoting the ATP turnover lifetime in seconds of the SRX in E. quercinus. (E) Calculated myosin ATP consumption values of each single muscle fiber per minute in E. quercinus. This was calculated using the equation shown in the Materials and methods section. (F–G) Percentage of myosin heads in the P1/DRX (F) or P2/SRX (G) from I. tridecemlineatus single muscle fibers obtained during summer active (SA), interbout arousal (IBA) or torpor periods. (H) T1 value in seconds denoting the ATP turnover lifetime of the DRX in I. tridecemlineatus. (I) T2 value in seconds denoting the ATP turnover lifetime in seconds of the SRX in I. tridecemlineatus. (J) Calculated myosin ATP consumption values of each single muscle fiber per minute in I. tridecemlineatus. Grey circles represent the values from each individual muscle fiber which was analyzed. Colored triangles represent the mean value from an individual animal, 8–12 fibers analyzed per animal. Statistical analysis was performed upon mean values. One-way ANOVA was used to calculate statistical significance. *=p < 0.05, **=p < 0.01. n=5 individual animals per group. Figure created using BioRender.com and published using a CC BY-NC-ND license with permission.
Figure 3.
Figure 3.. X-ray diffraction experiments of skeletal muscle from Ictidomys tridecemlineaus demonstrate changes in M6 myosin meridional spacing during torpor.
(A) Representative X-ray diffraction recordings from permeabilized skeletal muscle bundles from Ictidomys tridecemlineatus from summer active (SA), interbout arousal (IBA) and torpor. The M3 and M6 meridional reflections and the 1,0 and 1,1 equatorial reflections are indicated. (B) Ratio of the 1,1–1,0 equatorial reflections from active, IBA and torpor skeletal muscle. (C) M3 meridional spacing, measured in nm. (D) Normalized intensity (A.U.) of the M3 meridional reflection. (E) M6 meridional spacing, measured in nm. Colored circles represent the mean value obtained from each skeletal muscle bundle which was recorded. Data is displayed as mean ± SEM. One-way ANOVA was used to calculate statistical significance. *=p < 0.05, **=p < 0.01, ***=p < 0.001. n=5 individual animals per group.
Figure 4.
Figure 4.. Myosin dynamics of Ictidomys tridecemlineatus are protected from temperature induced change during torpor, preventing an increase in myosin ATP consumption.
(A) T1 value in seconds denoting the ATP turnover lifetime of the DRX in I. tridecemlineatus at 8 °C. (B) T2 value in seconds denoting the ATP turnover lifetime in seconds of the SRX in I. tridecemlineatus at 8 °C. (C) Ratio of the T1 expressed as the mean value for each matched animal at 20 °C/8 °C, separated for fiber type. (D) Ratio of the T2 expressed as the mean value for each matched animal at 20 °C/8 °C, separated for fiber type. (E) Ratio of calculated myosin ATP consumption expressed as 20 °C/8 °C, separated for fiber type. Black triangles represent the mean ratio value for each animal. One-way ANOVA was used to calculate statistical significance. *=p < 0.05, **=p < 0.01, ***=p < 0.001. n=5 individual animals per group. Figure created using BioRender.com and published using a CC BY-NC-ND license with permission.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. Myosin ATP turnover lifetime is altered following exposure to cold temperature in MyHC-II muscle fibers from I. tridecemlineatus during active and IBA periods but not torpor.
(A–B) Percentage of myosin heads in the P1/DRX (A) or P2/SRX (B) from I. tridecemlineatus single muscle fibers obtained during summer active (SA), interbout arousal (IBA) or torpor periods at 8 °C. Grey circles represent the values from each individual muscle fiber which was analyzed. Colored triangles represent the mean value from an individual animal, 8–12 fibers analyzed per animal. Statistical analysis was performed upon mean values. One-way ANOVA was used to calculate statistical significance. n=5 individual animals per group. Figure created using BioRender.com and published using a CC BY-NC-ND license with permission.
Figure 5.
Figure 5.. MYH2 protein in Ictidomys tridecemlineatus is hyper-phosphorylated during torpor, which is predicted to increase protein stability.
(A) Peptide mapping of differentiated phosphorylation sites upon MYH2 protein during SA, IBA and torpor periods. Heat map demonstrates all sites observed to be differentiated following the calculation of z-scores for each site. Z-scores >0 equal hyper-phosphorylation and z-scores<0 equal hypo-phosphorylation for each residue. Violin plot demonstrates significantly differentiated residues using z-scores. Two-way ANOVA with Šídák’s multiple comparisons test was used to calculate statistical significance. *=p < 0.05, **=p < 0.01, ***=p < 0.001, ****=p < 0.0001. n=5 individual animals per group. (B) Chimera of MYH2 protein created using ChimeraX software. Important regions of the protein are annotated including coiled-coil region, ATP binding domain, actin binding domain and N-terminal SH3-like domain. Also, significantly hyper-phosphorylated residues are highlighted in red. (C) Schematic of MYH2 protein with regions and hyper-phosphorylated resides annotated in red. Figure made in BioRender. (D) EvoEF calculations of protein stability in both wild type and phosphor-mimetic mutants. Aspartic acid was used to mimic phospho-threonine/phospho-serine due to their chemical similarity. ΔGStability indicates the stability score for the protein in its corresponding configuration. ΔΔGStability represents the change in stability in mutant proteins versus the wild type protein. ΔΔGStability of >0 represents an increase in the stability of a mutant versus wild type. Panel C created using BioRender.com and published using a CC BY-NC-ND license with permission.
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. MYH7 protein phosphorylation and acetylation in U. arctos is relatively unchanged during winter periods.
(A) Peptide mapping of differentiated phosphorylation sites upon MYH7 protein during summer and winter periods. Heat map demonstrates all sites observed to be differentiated following the calculation of z-scores for each site. Z-scores >0 equal hyper-phosphorylation and z-scores <0 equal hypo-phosphorylation for each residue. (B) Violin plot demonstrates significantly differentiated residue using z-scores. (C) Peptide mapping of differentiated acetylation sites upon MYH7 protein during summer and winter periods. Heat map demonstrates all sites observed to be differentiated following the calculation of z-scores for each site. Z-scores >0 equal hyper-acetylation and z-scores <0 equal hypo-acetylation for each residue. Two-way ANOVA with Šídák’s multiple comparisons test was used to calculate statistical significance. *=p < 0.05. n=5 individual animals per group.
Figure 5—figure supplement 2.
Figure 5—figure supplement 2.. MYH2 protein phosphorylation and acetylation in U.
arctos is altered during winter periods. (A) Peptide mapping of differentiated phosphorylation sites upon MYH2 protein during summer and winter periods. Heat map demonstrates all sites observed to be differentiated following the calculation of z-scores for each site. Z-scores >0 equal hyper-phosphorylation and z-scores <0 equal hypo-phosphorylation for each residue. (B) Violin plot demonstrates significantly differentiated residues using z-scores. (C) Peptide mapping of differentiated acetylation sites upon MYH7 protein during summer and winter periods. Heat map demonstrates all sites observed to be differentiated following the calculation of z-scores for each site. Z-scores >0 equal hyper-acetylation and z-scores <0 equal hypo-acetylation for each residue. Two-way ANOVA with Šídák’s multiple comparisons test was used to calculate statistical significance. *=p < 0.05, **=p < 0.01. n=5 individual animals per group.
Figure 6.
Figure 6.. Global proteome analysis demonstrates changes to metabolic and sarcomeric changes in skeletal muscle fibers from Ictidomys tridecemlineatus during IBA and torpor.
(A) Principal component analysis for all animals analyzed during SA, IBA and torpor periods. (B) Volcano plot displaying proteins which are differentially expressed during torpor vs active periods. FDR < 0.01. Red circles are upregulated proteins and blue circles are downregulated proteins. Highly differentiated proteins of interest are annotated with their respective protein name. (C) Volcano plot displaying proteins which are differentially expressed during IBA vs SA periods. Red circles are upregulated proteins and blue circles are downregulated proteins. Highly differentiated proteins of interest are annotated with their respective protein name. (D) Ontological associations between proteins upregulated during torpor vs SA periods. The top five association clusters are annotated on the network. A full list of clusters and the proteins lists included in clusters are available in Figure 6—figure supplement 1 and Supplementary file 2. (E) Ontological associations between proteins downregulated during torpor vs SA periods. The top five association clusters are annotated on the network. A full list of clusters and the proteins lists included in clusters are available in Figure 6—figure supplement 1 and Supplementary file 2. (F) Ontological associations between proteins upregulated during IBA vs SA periods. The top five association clusters are annotated on the network. A full list of clusters and the proteins lists included in clusters are available in Figure 6—figure supplement 2 and Supplementary file 3. (G) Ontological associations between proteins downregulated during IBA vs SA periods. The top five association clusters are annotated on the network. A full list of clusters and the proteins lists included in clusters are available in Figure 6—figure supplement 2 and Supplementary file 3. Gene ontology networks were established using Metascape and visualized using Cytoscape. Detailed information upon the statistical testing used is available in the methods section. FDR < 0.01 significantly differentially expressed proteins were used to establish networks. Purple lines indicate a direct interaction. Circle size is determined by enrichment and color is determined by p value. n=5 individual animals per group.
Figure 6—figure supplement 1.
Figure 6—figure supplement 1.. All ontological clusters altered in I. tridecemlineatus in torpor vs SA periods.
(A) All upregulated pathways identified in Metascape in torpor vs SA periods. Pathways are listed in order of significance by -log10(p). (B) All downregulated pathways identified in Metascape in torpor vs SA periods. Pathways are listed in order of significance by -log10(p).
Figure 6—figure supplement 2.
Figure 6—figure supplement 2.. All ontological clusters altered in I. tridecemlineatus in IBA vs SA periods.
A. All upregulated pathways identified in Metascape in IBA vs SA periods. Pathways are listed in order of significance by -log10(p). (B) All downregulated pathways identified in Metascape in IBA vs SA periods. Pathways are listed in order of significance by -log10(p).
Figure 6—figure supplement 3.
Figure 6—figure supplement 3.. Metabolite and lipid quantification of skeletal muscle from I.tridecemlineatus reveals a decrease in lipid levels during torpor.
(A) Quantification of metabolites from the skeletal muscle tissue of I. tridecemlineatus during SA, IBA, and torpor periods. Presented in µmol of metabolite per gram of tissue. (B) Quantification of lipids from the skeletal muscle tissue of I. tridecemlineatus during SA, IBA, and torpor periods. Presented in A.U. of lipid per mg of tissue. Two-way ANOVA with Turkey’s multiple comparisons test was used to calculate statistical significance. *=p < 0.05, **=p < 0.01, ***=p < 0.001, ****=p < 0.0001. n=5 individual animals per group.
Figure 6—figure supplement 4.
Figure 6—figure supplement 4.. Global proteome analysis of U.arctos skeletal muscle fibers reveal metabolic changes but not sarcomeric changes.
(A) Principal component analysis for all animals analyzed during summer and winter periods. (B) Volcano plot displaying proteins which are differentially expressed during winter vs summer periods. FDR < 0.01. Red circles are upregulated proteins and blue circles are downregulated proteins. Highly differentiated proteins of interest are annotated with their respective protein name. (C) Ontological associations between proteins upregulated during winter vs summer periods. The top five association clusters are annotated on the network. A full list of clusters and the proteins lists included in clusters are available in Figure 6—figure supplement 5 of this figure and Supplementary file 4. (D) Ontological associations between proteins downregulated during winter vs summer periods. The top five association clusters are annotated on the network. A full list of clusters and the proteins lists included in clusters are available in Figure 6—figure supplement 5 of this figure and Supplementary file 4. Gene ontology networks were established using Metascape and visualized using Cytoscape. Detailed information of the statistical testing used is available in the methods section. FDR < 0.01 significantly differentially expressed proteins were used to establish networks. Purple lines indicate a direct interaction. Circle size is determined by enrichment and color is determined by p value. n=5 individual animals per group.
Figure 6—figure supplement 5.
Figure 6—figure supplement 5.. All ontological clusters altered in U.arctos in summer vs winter periods.
(A) All upregulated pathways identified in Metascape in winter vs summer periods. Pathways are listed in order of significance by -log10(p). (B) All downregulated pathways identified in Metascape in winter vs summer periods. Pathways are listed in order of significance by -log10(p).
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