The NLRP3 inflammasome contributes to sarcopenia and lower muscle glycolytic potential in old mice

Abstract

The mechanisms underpinning decreased skeletal muscle strength and slowing of movement during aging are ill-defined. "Inflammaging," increased inflammation with advancing age, may contribute to aspects of sarcopenia, but little is known about the participatory immune components. We discovered that aging was associated with increased caspase-1 activity in mouse skeletal muscle. We hypothesized that the caspase-1-containing NLRP3 inflammasome contributes to sarcopenia in mice. Male C57BL/6J wild-type (WT) and NLRP3^-/- mice were aged to 10 (adult) and 24 mo (old). NLRP3^-/- mice were protected from decreased muscle mass (relative to body mass) and decreased size of type IIB and IIA myofibers, which occurred between 10 and 24 mo of age in WT mice. Old NLRP3^-/- mice also had increased relative muscle strength and endurance and were protected from age-related increases in the number of myopathic fibers. We found no evidence of age-related or NLRP3-dependent changes in markers of systemic inflammation. Increased caspase-1 activity was associated with GAPDH proteolysis and reduced GAPDH enzymatic activity in skeletal muscles from old WT mice. Aging did not alter caspase-1 activity, GAPDH proteolysis, or GAPDH activity in skeletal muscles of NLRP3^-/- mice. Our results show that the NLRP3 inflammasome participates in age-related loss of muscle glycolytic potential. Deletion of NLRP3 mitigates both the decline in glycolytic myofiber size and the reduced activity of glycolytic enzymes in muscle during aging. We propose that the etiology of sarcopenia involves direct communication between immune responses and metabolic flux in skeletal muscle.

Keywords: NOD-like receptor family pyrin domain containing 3; aging; caspase; glycolysis; inflammation; muscle.

Fig. 1.

Body and tissue masses for wild-type (WT) and NOD-like receptor family pyrin domain containing 3 (NLPR3^−/−) mice at 10 and 24 mo of age. A–H: body mass and tissues masses of 10- and 24-mo-old WT mice. I–P: body mass and tissue masses of 10- and 24-mo-old NLRP3^−/− mice. All data are means ± SE, and individual data points represent separate mice; n = 10–11 WT mice; n = 9–12 NLRP3^−/− mice. Statistical analyses were performed using Student’s t-test. *P < 0.05.

Fig. 2.

Deletion of NLRP3 attenuates muscle loss, reduces myopathy, and improves muscle function in mice. WT and NLRP3^−/− mice were aged to 10 (adult) and 24 mo (old). A: %change in mass of tibialis anterior (TA), extensor digitorum longus (EDL), and soleus muscles between adult and old WT and NLRP3^−/− mice were calculated relative to body mass and heart mass. B: representative micrographs of hematoxylin and eosin-stained transverse sections from TA muscle in adult and old WT and NLRP3^−/− mice. Scale bar, 250 μm. Inset images of micrographs from 24-mo-old mice shown on the right. C: quantification of changes in mean cross-sectional area (CSA) of TA muscles between 10- and 24-mo-old WT and NLRP3^−/− mice. D: representative immunofluorescence micrographs of TA muscles labeled for type IIB (red), type IIX (unstained), and type IIA (green) myofibers. Scale bar, 200 μm. Inset images of micrographs from 24-mo-old mice shown on the right. E: quantification of the mean CSA changes between 10- and 24-mo-old WT and NLRP3^−/− mice within each fiber type. F: quantification of myopathic fibers in adult and old WT mice. G: quantification of myopathic fibers in adult and old NLRP3^−/−mice. H: quantification of relative muscle strength and endurance using latency to fall during a wire hang test. All data are means ± SE, and individual data points represent separate mice; n = 10–11 WT mice; n = 9–12 NLRP3^−/− mice. Statistical analyses were performed using Student’s t-test. *P < 0.05.

Fig. 3.

Cross-sectional area of specific fiber types in TA muscle from adult and old WT and NLRP3^−/− mice. Frequency histograms of the distribution of fiber cross-sectional areas (μm²) in 10- and 24-mo-old WT and NLRP3^−/− mice for type IIB (A), type IIX (B), and type IIA fibers (C). All data are means ± SE; n = 10–11 WT mice; n = 9–12 NLRP3^−/− mice. Statistical analyses were performed using 2-way ANOVA. *P < 0.05.

Fig. 4.

Aging increases caspase-1 activity and decreases GAPDH activity in mouse skeletal muscle via NLRP3. A: caspase-1 enzymatic activity in TA muscles from 10- and 24-mo-old WT mice. B: caspase-1 enzymatic activity in TA muscles from 10- and 24-mo-old NLRP3^−/− mice. C: GAPDH enzymatic activity in TA muscles from 10- and 24-mo-old WT mice. D: GAPDH enzymatic activity in TA muscles from 10- and 24-mo-old NLRP3^−/− mice. All data are means ± SE, and individual data points represent separate mice; n = 10–11 WT mice; n = 8–10 NLRP3^−/− mice for caspase-1; n = 9–12 NLRP3^−/− mice for GAPDH activity. Statistical analyses were performed using Student’s t-test. *P < 0.05.

Fig. 5.

Aging increases NLRP3-dependent proteolysis of GAPDH in mouse TA muscle. A: representative immunoblot for GAPDH using TA muscle lysates prepared from 10- and 24-mo-old WT and NLRP3^−/− mice and from TA muscles that were an internal loading control (CON). B and C: quantification of immunoreactive bands at ∼37 kDa predicted to be full-length GAPDH in TA muscle lysates prepared from 10- and 24-mo-old WT and NLRP3^−/− mice. D: representative immunoblot for GAPDH after longer exposure times (i.e., higher exposure) using TA muscle lysates prepared from 10- and 24-mo-old WT and NLRP3^−/− mice and from TA muscles that were an internal loading CON. E and F: quantification of immunoreactive bands at ∼25 kDa predicted to be a fragment of GAPDH in TA muscle lysates prepared from 10- and 24-mo-old WT and NLRP3^−/− mice. G: Ponceau stain of the representative immunoblot showing equal loading of protein between samples; n = 10–11 WT mice; n = 9–10 NLRP3^−/− mice. All data are means ± SE, and individual data points represent separate mice. Statistical analyses were performed using Student’s t-test. *P < 0.05. KO, knockout.

Fig. 6.

Aging does not alter muscle mitochondrial capacity or markers of inflammation in WT or NLRP3^−/− mice. A: representative immunoblot for mitochondrial complexes I–V using TA muscle lysates prepared from 10- and 24-mo-old WT and NLRP3^−/− mice. B: quantification of mitochondrial complexes I–V in TA muscles from 10- and 24-mo-old WT and NLRP3^−/− mice. C: Ponceau stain of the representative immunoblot showing equal loading of protein between samples. D: TaqMan-based quantitative PCR detection of transcript levels of selected inflammatory targets in TA muscles of 10- and 24-mo-old WT and NLRP3^−/− mice. All data are means ± SE; n = 8–9 WT mice, n = 8–9 NLRP3^−/− mice for mitochondrial assessments, n = 8–9 WT mice, and n = 6–11 NLRP3^−/− mice for detection of transcripts based on certain transcripts reporting lower than the limit of detection by quantitative PCR in a subset of mice. Statistical analyses were performed using Student’s t-test to compare 10- and 24-mo-old mice within each genotype for each measure.

Fig. 7.

Systemic inflammation in adult and old WT and NLRP3^−/− mice. A panel of 23 cytokines/chemokines was analyzed in the serum of WT (A) and NLRP3^−/− (B) 10- and 24-mo-old mice. All data are means ± SE; n = 6–9 WT mice and 5–9 NLRP3^−/− mice for reported values upon detection of analytes based on certain analytes being lower than the limit of detection by ELISA in a subset of mice. Statistical analyses were performed using Student’s t-test within each analyte.