Lipidomic signature of acyl chains saturation in aged skeletal muscle

FABP3 expression was dramatically increased in skeletal muscle but not in cardiac muscle with age (Fig. 1a). Since FABP3 has been reported as a lipid “chaperone” which regulates fatty acid metabolism¹⁰, we first investigated a lipidomic signature in “young” (3-month-old) and “aged” (24-month-old) mouse tibialis anterior (TA) muscle using UPLC/QTOF mass spectrometry. Our analysis identified 203 lipid species. Approximately 53% of total identified lipid species were significantly changed, with 45 lipid species increased and 62 decreased (Fig. 1b). We next analyzed the distribution and alteration of the lipid classes. The major lipid species identified in skeletal muscle were the membrane lipids phosphatidylcholine (PC, 75% of total identified lipid), phosphatidylethanolamine (PE, 11%), lysophosphatidylcholine (LPC), sphingomyelin (SM), and phosphatidylinositol (PI) (Supplementary Table 1). Aged muscle showed marked changes in the content of lipid classes (Supplementary Table 1 and Supplementary Fig. 1a). Notably, aged muscle had elevated SM and LPC, showing 2.4-fold and 2.8-fold increases over young muscle, respectively (Fig. 1c and Supplementary Fig. 1a). We next analyzed acyl chain composition of detected PC, PE, SM, and LPC in aged muscle compared to young muscle. In aged muscle, 16:0/20:4 PC significantly decreased by 24% and 16:0/16:0 PC increased 2.1-fold over young muscle. Other PC species changed slightly or insignificantly (Fig. 1d and Supplementary Fig. 1b). When individual contents of acyl chains were summed, polyunsaturated PC acyl chains decreased in aged muscle, but saturated species increased (Fig. 1h and Supplementary Fig. 1c). The PE species containing 40:6, 38:4, and 40:8 acyl chains decreased in aged muscle by 45, 42, and 48%, respectively. However, 40:4, 34:1, and 36:1 PE increased more than 2-fold (Fig. 1e and Supplementary Fig. 1d). Identification of individual acyl chains was not possible for all lipid species, due high isomeric or isobaric lipid levels. When PE species containing the same number of double bonds were combined, PEs with more double bonds decreased in aged muscle, but PEs containing fewer double bonds increased (Supplementary Fig. 1e). These data indicate that PC and PE acyl chains were shifted from polyunsaturated to saturated in aged muscle. Interestingly, all identified SM and LPC species (which mainly contain saturated or monounsaturated acyl chains¹⁸) increased simultaneously in aged muscle (Fig. 1f, g and Supplementary Fig. 1f, g). In parallel, we evaluated PC acyl chain length. Changes in C18 and >C18 fatty acid levels reflect very long chain fatty acid (VLCFA) elongase activity. Chain length is associated with the degree of unsaturation in VLCFAs¹⁹. Physical interactions between the VLCFA-elongase complex and desaturase have been reported in yeast²⁰. Functional crosstalk between the elongase complex and desaturase has been reported in myoblasts²¹. As expected, the acyl chain length and the degree of unsaturation were correlated. Aged muscle increased in C18 acyl chain levels, but >C18 acyl chains decreased (Fig. 1i). Taken together, aged skeletal muscle exhibits a unique lipidomic signature of acyl chain saturation. These results prompted us to investigate whether FABP3 upregulation in aged skeletal muscle might be involved in such lipid remodeling.

a Immunoblots (top) and quantification (bottom) of the indicated proteins in skeletal muscle and heart isolated from young and aged mice (n = 3 mice per group). b Volcano plot of lipid species altered in aged vs. young muscle (n = 4 mice per group). Lipid species were measured by LC-MS. The x-axis indicates the logarithmic (base 2) fold abundance changes of all identified lipid species and the y-axis indicates negative logarithmic (base 10) t-test p-value. The horizontal dotted line reflects the filtering criterion (p-value = 0.05). c Proportion of major lipid classes in young and aged muscles. d–g Volcano plots of PC (d), PE (e), SM (f), and LPC (g) species altered in aged vs. young muscles. The x-axis indicates the percentage changes (aged-young) in lipid abundance and the y-axis indicates negative logarithmic (base 10) t-test p-values. The horizontal dotted line reflects the filtering criterion (p-value = 0.05). Red and green indicate highly increased and decreased lipid species, respectively. h Proportion of saturated (SFA), monounsaturated (MUFA), or polyunsaturated (PUFA) PC acyl chains in young and aged muscles. i Proportion of <C18, C18, or >C18 PC acyl chains in young and aged muscles. Data are presented as means ± S.E.M. Two-tailed unpaired Student’s t-test was used. Source data are provided as a Source Data file.

FABP3-overexpressing young muscle exhibits a lipid composition similar to aged muscle

To study the impact of FABP3 on intramuscular lipid composition, we transfected plasmid constructs encoding HA-cherry FABP3 or HA-cherry control into young TA muscles using an electroporation gene delivery system (Supplementary Fig. 2a). Using lipidomic analysis, we found that FABP3 overexpression increased 53 lipid species and decreased 32 lipid species (Fig. 2a). Lipid species with significant changes comprised nearly half of the total identified lipid species, indicating that FABP3 plays a major role in muscle lipidome remodeling. We next analyzed lipid class distribution and alteration (Supplementary Table 1). Compared to young muscle controls, FABP3 overexpression increased total SM and LPC contents by 29 and 42%, respectively (Fig. 2b, Supplementary Fig. 2b and Supplementary Table 1). Interestingly, this pattern was similar in aged muscle (Fig. 1c).

a Volcano plot of lipid species altered in FABP3-overexpressing muscle vs. control muscle. Lipid species were measured by LC-MS. The x-axis indicates the logarithmic (base 2) fold abundance changes of all identified lipid species and the y-axis indicates negative logarithmic (base 10) t-test p-value. The horizontal dotted line reflects the filtering criterion (p-value = 0.05). (n = 4 mice per group). b Proportion of major lipid classes in FABP3-overexpressing and control muscles. c–f Volcano plot of PC (c), PE (d), SM (e), and LPC (f) species altered in FABP3-overexpressing vs. control muscles. The x-axis indicates the percentage changes (FABP3 o/e-control) in lipid species and the y-axis indicates negative logarithmic (base 10) t-test p-values. The horizontal dotted line reflects the filtering criterion (p-value = 0.05). Red and green indicate highly increased and decreased lipid species, respectively. g Proportion of SFA, MUFA, or PUFA PC acyl chains in FABP3-overexpressing and control muscles. h Proportion of <C18, C18, or >C18 PC acyl chains in FABP3-overexpressing and control muscles. Data are presented as means ± S.E.M. Two-tailed unpaired Student’s t-test was used. i Correlation analysis of the total identified lipid species in the indicated comparative condition. The x-axis indicates logarithmic (base 2) fold concentration changes of all identified lipid species in aged vs. young muscle, and the y-axis indicates logarithmic (base 2) fold changes in FABP3-overexpressing muscle vs. young muscle. Data in i were analyzed using Spearman’s correlation; correlation coefficient (r) and p-value (p) are in red. Source data are provided as a Source Data file.

We then analyzed phospholipid acyl chain composition. Interestingly, PCs containing polyunsaturated acyl chains such as 16:0/22:6, 18:0/22:6, 18:2/22:6, and 18:1/22:6 decreased in FABP3-overexpressing muscle compared to control young muscle. In contrast, PCs containing 18:1/18:2, 18:0/18:2, and 16:0/16:0 acyl chains increased (Fig. 2c and Supplementary Fig. 2c). When the PC species with the same number of double bonds were combined, PCs containing more double bonds decreased in FABP3-overexpressing muscle, whereas PCs with fewer double bonds increased (Supplementary Fig. 2d). Saturated PC acyl chains increased in FABP3-overexpressing muscle, while polyunsaturated acyl chains decreased (Fig. 2g). Polyunsaturated 40:6, 40:7, and 44:10 PEs significantly decreased, but 36:2, 36:4, 36:3, and 34:2 PEs increased in FABP3-overexpressing muscle (Fig. 2d and Supplementary Fig. 2e). When the PE species containing the same number of double bonds were combined, PEs with more double bonds decreased in FABP3-overexpressing muscle, whereas PEs containing fewer double bonds increased, indicating that PE acyl chains were shifted toward saturation in FABP3-overexpressing muscle (Supplementary Fig. 2f). This pattern of phospholipid saturation in FABP3-overexpressing muscle was similar in aged muscle. SM and LPC species increased in FABP3-overexpressing muscle (Fig. 2e, f and Supplementary Fig. 2g, h). Regarding PC acyl chain length, C18 acyl chains increased in FABP3-overexpressing muscle, while >C18 acyl chains decreased, similar to aged muscle (Fig. 2h). Finally, we analyzed the correlation of the up/down ratio of all identified lipid species between aged and FABP3-overexpressing muscles in comparison with young muscles (Fig. 2i). Surprisingly, a strong linear correlation was found in lipid composition between aged muscle and FABP3-overexpressing young muscle. Based on these lipidomic data, we suggest that FABP3 contributes to lipid alterations during muscle aging.

FABP3 executes PERK–eIF2α-mediated inhibition of protein translation

Given that increased membrane lipid saturation induces ER stress and the unfolded protein response²², we investigated whether ER stress was induced in aged mouse muscle with elevated membrane SFA content. Phosphorylation of an ER stress sensor, PERK (pancreatic ER kinase), increased in aged TA muscle (Fig. 3a). Consistent with evidence that PERK phosphorylates eIF2α, thereby inhibiting protein translation²³, we detected increased eIF2α phosphorylation and ~50% decreased de novo protein synthesis in aged muscle (Fig. 3a). Therefore, we next investigated whether lipid remodeling via FABP3 overexpression could induce ER stress in vivo. Mouse TA muscle overexpressing FABP3 had increased PERK and eIF2α phosphorylation, and ~40% reduced de novo protein synthesis (Fig. 3b). These results suggest that FABP3-induced lipid remodeling might induce an ER stress response and defective protein synthesis in aged skeletal muscle.

a–c Immunoblot analysis (left) and quantification (right) of the PERK and eIF2α phosphorylation and puromycin incorporation in young (6 months) and aged (25 months) TA muscles (n = 3 mice per group) (a), FABP3-overexpressing and control TA muscles (n = 3 mice per group) (b), and FABP-overexpressing myotubes (n = 3 independent experiments) (c). Mice were injected intraperitoneally with puromycin and muscles (a, b) were harvested 30 min post-injection. TA muscles of young mice (b) were transfected with HA-cherry-FABP3 or HA-cherry control plasmid and were harvested 5 days after transfection. Fully differentiated C2C12 myotubes (c) expressing Cre-inducible FABP3 constructs were infected with Cre-carrying adenovirus (Ad-Cre) and cultured for 3 days before puromycin treatment. Palmitate was treated for 12 h as a positive control ER stress inducer. d Membrane fluidity was analyzed by FRAP. Representative confocal images of the BODIPY 500/510 C1, C12 -labeled myotubes before bleaching (pre-bleach) and at 0 and 50 s. after bleaching (post-bleach). White dotted squares, bleached areas. Scale bar, 20 μm. (n = 24 independent experiments). e Time course fluorescence gain in palmitate-treated or FABP3-overexpressing C2C12 myotubes. t_1/2, the half-time for fluorescence recovery; black, control, n = 9; gray, palmitate, n = 7; red, FABP3, n = 8 each myotube. f, g Temperature effect on membrane fluidity and ER stress. FABP3-overexpressing and control C2C12 myotubes were incubated at 32 or 37 °C. Membrane fluidity (f) was measured by FRAP analysis. Note the t_1/2 values in FRAP analyses. Black, 37 °C, n = 6; gray, 32 °C, n = 8; red, FABP3 at 32 °C, n = 6; light red, FABP3 at 37 °C, n = 8 each myotube. g Immunoblot analysis (left) and quantification (right) of the indicated proteins and puromycin incorporation. Data are presented as means ± S.E.M. Two-tailed unpaired Student’s t-test was used. Source data are provided as a Source Data file.

To further confirm the role of FABP3 on ER stress signaling and protein synthesis, we established a stable C2C12 cell line that overexpressed FABP3 under the control of Cre recombinase. Acutely overexpressing FABP3 via Cre recombinase-expressing adenovirus increased PERK phosphorylation in differentiated C2C12 myotubes. Further, FABP3 overexpression increased eIF2α phosphorylation and inhibited de novo protein synthesis (Fig. 3c and Supplementary Fig. 3a), which was more severe than in myotubes treated with the well-known ER stress inducer, palmitate. PERK-eIF2α regulates cell death via ATF4 and CHOP activation upon ER stress²⁴, and inhibits protein synthesis via the 43S preinitiation complex²⁵. We excluded a possibility that FABP3 induced cell death via PERK–eIF2α, because FABP3 expression did not induce Atf4 and Chop mRNA expression (Supplementary Fig. 3b). Since we found that PERK inhibition almost totally rescued FABP3-induced inhibition of protein synthesis (Supplementary Fig. 3c), we postulated that FABP3-driven eIF2α phosphorylation inhibits protein synthesis by directly inhibiting the 43S pre-initiation complex.

Next, we investigated whether alternative ER membrane-associated sensors including inositol requiring transmembrane kinase/endonucleases-1α (IRE-1α) and activating transcription factor 6 (ATF6) could be involved in FABP3-driven ER stress. We found that neither did FABP3 induce phosphorylation of IRE-1α or its downstream effectors, JNK, p65, and SEK, nor did it induce ATF6 cleavage (Supplementary Fig. 3d, f). Moreover, no significant change was observed in the mRNA expression level of unfolded protein response genes, Xbp1-s, Grp78/Bip Erdj4, and Edem, which are downstream of IRE-1α and ATF6 (Supplementary Fig. 3e). While palmitate induces ER stress via simultaneous activation of the PERK, IRE1α, and ATF6 axis in myotubes²⁶, our data suggest that FABP3 induces ER stress primarily via PERK–eIF2α-43S pre-initiation complex mediated inhibition of protein translation. Meanwhile, either was FABP3-dependent inhibition of de novo protein synthesis associated with AKT–GSK-3β, nor with the mTOR signaling pathway (Supplementary Fig. 3g, h).

FABP3 overexpression decreased membrane fluidity in myotubes

Since lipid composition affects membrane fluidity²⁷, we investigated whether FABP3-induced lipid remodeling could affect muscle cell membrane fluidity. To this end, we measured the membrane lipid diffusion rate by FRAP analysis of 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY 500/510 C1, C12) in myotubes (Fig. 3d). Consistent with a previous report²⁸, palmitate-treated, BODIPY 500/510 C1, C12-labeled myotubes decreased in fluorescence gain by ~36% at 50 s after bleaching (Fig. 3e). Surprisingly, FABP3-overexpressing myotube membranes had diffusion rates similar to palmitate-treated myotubes. Half-maximal fluorescence gain time in control, palmitate-treated, and FABP3-overexpressing myotubes were 41.33, 49.58, and 52.87 s, respectively (Fig. 3e). These results indicate that FABP3 decreases membrane fluidity. We then sought to determine whether the FABP3-altered membrane fluidity influences ER stress. High temperatures cause membrane fluidization, while low temperatures decrease membrane fluidity²⁹. Low temperature (32 °C) markedly aggravated membrane fluidity in FABP3-overexpressing myotubes (Fig. 3f), along with elevating PERK and eIF2α phosphorylation, indicating a more severe ER stress response, and enhanced inhibition of protein synthesis (Fig. 3g). These results suggest that FABP3 induces ER stress by modulating membrane fluidity in aged muscle.

FABP3 overexpression deteriorates muscle mass and force

We next investigated the physiological significance of FABP3 levels in skeletal muscle. Muscle recovery after immobilization in aged mice is incomplete or delayed compared to young mice³⁰. We, therefore, assessed muscle atrophy after 5 days of immobilization and muscle recovery after 5 days of remobilization in FABP3-overexpressing TA muscle (Fig. 4a). Although the atrophic morphology between FABP3-overexpressing TA muscle and control TA muscle was similar, control young muscle regained muscle mass during the remobilization, while FABP3-overexpressing muscle did not (Fig. 4b). Moreover, immunohistochemical analysis revealed that FABP3-overexpressing muscle displayed significantly smaller muscle fibers than control muscle after remobilization, similar to aged muscle (Fig. 4c, d); meanwhile, western blot analysis revealed that FABP3 maintained high levels of PERK and eIF2α phosphorylation and inhibited protein synthesis during remobilization (Fig. 4e). However, transient FABP3 overexpression per se did not induce a significant difference in muscle mass before immobilization (Supplementary Fig. 4). FABP3 overexpression also did not induce expression of atrophy-related ubiquitin ligases, such as Atrogin-1 and MuRF1, nor impair autophagy by Atg5, 7, 12, 16L, and SQSTM1 expression (Supplementary Fig. 3i–k). Next, to evaluate the effect of FABP3 on skeletal muscle function in intact muscles independent of other parameters, we compared ex vivo contractile properties, force, and fatigability, of FABP3-overexpressing and control young TA muscles. The maximum twitch forces were not different between FABP3-overexpressing and control TA muscles (Fig. 4f). However, at increasing stimulation frequencies in the tetanic force test (10–200 Hz, 100 V), the tetanic force was lower in the FABP3-overexpressing muscle than in the control muscle (Fig. 4g, h), similar to the aged muscle³¹. When subjected to fatigue-inducing repetitive stimulations at 1 Hz, 100 V for 10 min, FABP3-overexpressing muscles were more fatigue-sensitive than control muscles (Fig. 4i). These results together suggest that FABP3 upregulation during aging reduces both muscle mass and force.

a Scheme of the experimental procedures. TA muscles of young mice were transfected with HA-cherry-FABP3 or HA-cherry control plasmid, immobilized with a surgical staple 5 days after transfection, and allowed to recover (remobilization, n = 5) after 5-day immobilization (n = 5). b TA muscle mass was measured 5 days after immobilization and 5 days after remobilization. Changes in muscle mass were expressed as percentage of control. c Representative images of the transfected myofibers (red). (n = 5 per group). Scale bars, 100 μm. d Frequency histograms of the cross-sectional area (left) of HA-cherry-FABP3- or HA-cherry control plasmid-transfected muscle fibers 5 days after remobilization. Box plot representing the mean cross-sectional areas (right) of transfected muscle fibers. Box represent the 25th–75th percentiles of the data; whiskers show the min and max range of the data; horizontal lines indicate the median value. e Immunoblot analysis of PERK and eIF2α phosphorylation, and puromycin incorporation in FABP3-overexpressing and control TA muscles after remobilization. Five days after remobilization, mice were injected intraperitoneally with puromycin. TA muscles were harvested 30 min post-injection (n = 3 mice per group). f–i Muscle forces were measured in intact HA-cherry FABP3 or HA-cherry control plasmid-transfected TA muscles mounted on a force transducer. The maximum twitch force (f) at supramaximal voltage, 100 V for 1 ms. Frequency dependence (at 30–200 Hz, 100 V, 500 ms) of average tetanic force (g). Tetanic force traces (h) at 200 Hz for 500 ms (n = 7 mice per group). Fatigue index (i) was measured at 1 Hz and 100 V for 10 min. Generated force was recorded and expressed as a percentage of the initial force (left). Insert represents area under curve (AUC) (right) (n = 7 mice per group). Data are presented as means ± S.E.M. Two way ANOVA withBonferroni’s post hoc test was used (left in i) and two-tailed unpaired Student’s t-test was used (b, d, right in i). Source data are provided as a Source Data file.

FABP3 inhibition in aged muscle resulted in young-like lipid composition

To confirm the relevance of FABP3 in age-associated lipid remodeling, FABP3 was knocked down in aged TA muscle using shRNA against FABP3 (Supplementary Fig. 5a). Lipidomic analysis of FABP3 knockdown muscle revealed 64% of total lipid species identified in aged muscle were altered, with 21 lipid species increased and 79 decreased (Fig. 5a). FABP3 knockdown muscle had prominent changes in the lipid classes (Supplementary Table 1). SM and LPC levels decreased by 54 and 53%, respectively (Fig. 5b and Supplementary Fig. 5b, Supplementary Table 1). These results show an inverse correlation with those observed in both FABP3-overexpressing muscle and aged muscle (Figs. 1c, 2b). We next analyzed phospholipid acyl chain composition. Notably, PCs containing polyunsaturated acyl chain such as 16:0/22:6, 18:0/22:6, and 18:2/22:6 increased in FABP3-knockdown muscles (Fig. 5c and Supplementary Fig. 5c), while PCs containing 16:0/16:0, 18:1/18:2, and 18:0/18:2 acyl chains decreased. When PC species containing the same number of double bonds were combined, PCs with more double bonds increased in FABP3-knockdown muscle, whereas PCs containing fewer double bonds decreased (Supplementary Fig. 5d). Polyunsaturated PC acyl chains increased in FABP3-knockdown muscle, while saturated acyl chains decreased (Fig. 5g). Consistently, PEs with more acyl chain double bonds increased, but PEs with fewer double bonds decreased in FABP3-knockdown muscles (Supplementary Fig. 5f). For instance, 40:6, 38:6, and 40:8 PEs increased by 60, 79, and 59%, respectively, while 36:2, 36:3, 34:2, and 34:1 PEs decreased by 35, 50, 32, and 61%, respectively (Fig. 5d and Supplementary Fig. 5e). This phospholipid desaturation pattern in FABP3-knockdown muscle contrasted with FABP3-overexpressing muscle and aged muscle. SM and LPC species decreased simultaneously in FABP3-knockdown muscle (Fig. 5e, f and Supplementary Fig. 5g, h). Additionally, FABP3 knockdown reduced PC C18 acyl chain content but increased >C18 acyl chain content (Fig. 5h). These results were also opposite to those in both FABP3-overexpressing muscle and aged muscle (Figs. 1, 2). The up/downregulation of all identified lipid species displayed a strong correlation between FABP3-knockdown muscles and young muscles in comparison with aged muscles (Fig. 5i). These results together indicate that FABP3 inhibition could recapitulate a young muscle-like lipid composition in aged muscles. Further, principal component analysis (PCA) of total identified lipid species revealed unique lipidomic signatures in young versus aged muscles and in FABP3-overexpressing or FABP3-knockdown muscles. FABP3 overexpression in the young muscle led to clustering toward aged muscles, while FABP knockdown in aged muscles led to clustering toward young muscles (Supplementary Fig. 5i), suggesting that FABP3 drives age-dependent lipid remodeling.

a Volcano plot of lipid species altered in FABP3-knockdown aged vs. control muscle. Lipid species were measured by LC-MS. The x-axis indicates logarithmic (base 2) fold abundance changes of all identified lipid species and the y-axis indicates negative logarithmic (base 10) t-test p-value. The horizontal dotted line reflects the filtering criterion (p-value = 0.05). (n = 4 mice per group) b Proportion of major lipid classes in FABP3-knockdown and control aged muscles. c–f Volcano plot of PC (c), PE (d), SM (e), LPC (f) species altered in FABP3-knockdown vs. control aged muscles. The x-axis indicates the percentage changes (FABP3KD-control-aged muscle) of lipid species and the y-axis indicates negative logarithmic (base 10) t-test p-values. The horizontal dotted line reflects the filtering criterion (p-value = 0.05). Red and green indicate highly increased and decreased lipid species, respectively. g Proportion of SFA, MUFA, or PUFA PC acyl chains in FABP3-knockdown and control-aged muscles. h Proportion of <C18, C18, or >C18 PC acyl chains in FABP3-knockdown and control-aged muscles. The data are presented as means ± S.E.M. Two-tailed unpaired Student’s t-test was used. i Correlation analysis of the total identified lipid species in the indicated comparative condition. The x-axis indicates logarithmic (base 2) fold concentration changes of all identified lipid species in FABP3-knockdown aged vs. aged muscle and the y-axis indicates logarithmic (base 2) fold changes in aged vs. young muscle. Data in i were analyzed using Spearman’s correlation; correlation coefficient (r) and p-value (p) are in red. Source data are provided as a Source Data file.

FABP3 inhibition ameliorated membrane fluidity and alleviated ER stress

To investigate whether inhibition of FABP3 could protect against ER stress, we examined FABP3 knockdown in aged mouse muscle with ER stress. FABP3 knockdown decreased PERK and eIF2α phosphorylation and improved protein synthesis (Fig. 6a). Consistently in C2C12 myotubes, FABP3 knockdown reduced palmitate-induced PERK–eIF2α phosphorylation and improved protein synthesis (Fig. 6b). To investigate whether FABP3 inhibition could re-establish membrane fluidity that was lost after palmitate treatment, we performed FRAP analysis in FABP3-knockdown C2C12 myotubes. The FRAP signal was significantly increased in FABP3 knockdown myotubes in the presence of palmitate compared to control cells (light blue vs. deep blue symbol in Fig. 6c). Taken together with FABP3 overexpression data showing decreased FRAP signal (Fig. 3), we propose that increased FABP3 in aged muscle may reduce membrane fluidity and the ER stress response.

To test whether lipid remodeling is an underlying mechanism for FABP3-induced membrane rigidity and ER stress, we evaluated the effect of polyunsaturated fatty acids (PUFAs) in FABP3-overexpressing C2C12 myotubes. Addition of docosahexaenoic acid (22:6, DHA) into the culture media markedly enhanced the FRAP signal that was lost both in FABP3-overexpressing (light red vs. deep red symbol) and palmitate-treated myotubes (light blue vs. deep blue symbol) (Fig. 7a). These results suggest that PUFAs restore membrane fluidity in such myotubes. DHA supplementation also significantly suppressed FABP3-induced PERK and eIF2α phosphorylation, while improving protein synthesis (Fig. 7b), resulting in improved myotube recovery that was defective in FABP3-overexpressing myotubes (Fig. 7c). This result is consistent with previous reports showing DHA inhibits palmitate-induced ER stress in C2C12 myotubes²⁶. We suggest that FABP3-induced lipid remodeling may be an underlying mechanism in age-related membrane rigidity and ER stress, which is partially rescued by increased PUFA content.

Effects of DHA on fluidity (a), ER stress signal (b), and morphology (c). FABP3 expression was induced with Cre recombinase-carrying adenovirus (Ad-Cre) infected into fully differentiated C2C12 myotubes harboring Cre-inducible FABP3 constructs. FABP3-overexpressing myotubes or palmitate (500 μM)-treated myotubes were treated with DHA (100 μM) or vehicle. a Membrane fluidity was measured by FRAP analysis. Note the t_1/2 values in FRAP analyses. Black, control, n = 6; gray, DHA, n = 6; deep blue, palmitate, n = 5; light blue, palmitate+DHA, n = 9; red, FABP3, n = 5; light red, FABP3+DHA, n = 9 each myotube. b Immunoblot analysis (left) and quantification (right) of the indicated proteins and puromycin incorporation. Myotubes were incubated with puromycin (1 μM) for 30 min (n = 3 independent experiments). c the effect of DHA supplementation on FABP3-induced loss of atrophy recovery. FABP3-overexpressing or palmitate-treated myotubes were treated with dexamethasone (10 μM). Representative images (left) of MyHC (green) and DAPI (blue) staining in myotubes following DHA supplementation (100 μM) for 24 h (n = 3 independent experiments). Scale bar, 100 μm. Quantification (right) of myotubes diameter treated with dexamethasone. Data are presented as means ± S.E.M. Two-tailed unpaired Student’s t-test was used. Source data are provided as a Source Data file.

FABP3 knockdown ameliorated age-associated impairment of muscle recovery

We investigated whether FABP3 knockdown ameliorates impaired TA muscle recovery in aged mice (Fig. 8a). FABP3-knockdown tended to slow the muscle loss during immobilization, and significantly increased muscle mass during remobilization. (Fig. 8b). Immunohistochemical analysis revealed significantly larger muscle fibers in FABP3-knockdown aged muscle than in control aged muscle after remobilization (Fig. 8c, d). FABP3-knockdown maintained low levels of PERK and eIF2α phosphorylation and improved protein synthesis during remobilization (Fig. 8e). Next, to evaluate the effect of FABP3 inhibition on aged skeletal muscle function, we compared the ex vivo muscle contractility of FABP3-knockdown and aged muscle. While the maximum twitch force remained unchanged (Fig. 8f), the tetanic force at high frequencies (150–200 Hz) was slightly higher in FABP3-knockdown muscle than in control aged muscle (Fig. 8g, h). While aged muscles displayed a gradual reduction in tetanic forces over 120 Hz, FABP3-knockdown muscle retained tetanic force up to 200 Hz (Fig. 8g). When subjected to fatigue-inducing repetitive stimulations, FABP3-knockdown muscle was more fatigue-resistant than aged muscles (Fig. 8i). Together, these results indicate that FABP knockdown ameliorates age-related decline in muscle mass and strength, thus serving as a potentially valuable therapeutic target.

a Scheme of the experimental procedures. TA muscles of aged mice were infected with Ad-shFABP3 or Ad-shControl virus, immobilized with a surgical staple 5 days after infection, and allowed to recover (remobilization) after 5-day immobilization. b TA muscle mass was measured at 5 days after immobilization (n = 5) and 5 days after remobilization (n = 7). Changes in muscle weight for immobilization and remobilization were expressed as the percentage of the contralateral non-immobilized muscle weight. c Representative images of laminin (red) and DAPI (blue) staining of myofibers infected with Ad-shFABP3 or Ad-shControl virus Scale bar, 200 μm. d Frequency histograms of the cross-sectional area (left) of Ad-shFABP3 or Ad-shControl virus-infected fibers 5 days after remobilization. Box plot representing the mean fiber cross-sectional areas (right). Box represent the 25th–75th percentiles of the data; whiskers show the min and max range of the data; horizontal lines indicate the median value. e Immunoblot analysis of PERK and eIF2α phosphorylation, and puromycin incorporation in Ad-shFABP3 or Ad-shControl virus-infected TA muscles after remobilization. TA muscles were harvested 30 min post-interperitoneal puromycin injection. (n = 3 mice per group). f–i Ad-shFABP3 or Ad-shControl virus-infected TA muscles were mounted on a force transducer. The maximum twitch force (f) at supra-maximal voltage, 100 V for 1 ms. Frequency dependence of average tetanic force curves (g) at 30–200 Hz, 100 V, 500 ms for each frequency. Tetanic force traces (h) upon stimulation at 200 Hz for 500 ms. Data are presented as means. (n = 7 mice per group) Fatigue index (i) was measured at 1 Hz and 100 V by repeated stimuli for 10 min. Generated force was analyzed as a percentage of the initial contractile force (n = 7 mice per group) (left). Insert represents area under curve (AUC) (right). Data are presented as means ± S.E.M. Two way ANOVA with Bonferroni’s post hoc test was used (left in i) and two-tailed unpaired Student’s t-test was used (b, d, right in i). j Proposed model for age-dependent lipid remodeling by FABP3. Source data are provided as a Source Data file.