NLRP3 Contributes to Sarcopenia Associated to Dependency Recapitulating Inflammatory-Associated Muscle Degeneration

Abstract

Sarcopenia, a complex and debilitating condition characterized by progressive deterioration of skeletal muscle, is the primary cause of age-associated disability and significantly impacts healthspan in elderly patients. Despite its prevalence among the aging population, the underlying molecular mechanisms are still under investigation. The NLRP3 inflammasome is crucial in the innate immune response and has a significant impact on diseases related to inflammation and aging. Here, we investigated the expression of the NLRP3 inflammasome pathway and pro-inflammatory cytokines in skeletal muscle and peripheral blood of dependent and independent patients who underwent hip surgery. Patients were categorized into independent and dependent individuals based on their Barthel Index. The expression of NLRP3 inflammasome components was significantly upregulated in sarcopenic muscle from dependent patients, accompanied by higher levels of Caspase-1, IL-1β and IL-6. Among older dependent individuals with sarcopenia, there was a significant increase in the MYH3/MYH2 ratio, indicating a transcriptional shift in expression from mature to developmental myosin isoforms. Creatine kinase levels and senescence markers were also higher in dependent patients, altogether resembling dystrophic diseases and indicating muscle degeneration. In summary, we present evidence for the involvement of the NLRP3/ASC/NEK7/Caspase-1 inflammasome pathway with activation of pro-inflammatory SASP in the outcome of sarcopenia in the elderly.

Keywords: ASC; Caspase-1; MYH3; NLRP3 inflammasome; SASP; dependency; inflammaging; sarcopenia.

Figure 1

Higher expression of the components of NLRP3 pathway in muscle samples from dependent vs. independent patients. RT-PCR analysis of (A) NLRP3, (B) ASC, (C) NEK7, (D) GSDMD relative quantification (RQ) to HPRT1 (n = 30 for each of the two groups). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 2

Higher interleukin expression in plasma samples from dependent patients vs. independent patients. (A) Caspase-Glo^® 1 Inflammasome Assay in muscle lysates evaluated by bioluminescence (n = 25 for each of the two groups). (B–D) Expression of plasma cytokines measured as pg IL/ mg total protein (B) IL-1β (n = 19/19), (C) IL-6 (n = 19/20), (D) IL-10 (n = 20/19), and (E) IL-6/IL-10 ratio (n = 19/19). * p ≤ 0.05.

Figure 3

Increased muscle atrophy in dependent muscle. RT-PCR analysis of (A) MYH2, (B) MYH3, (C) ratio MYH3/MYH2 (n = 30 for each of the two groups); (D) creatine kinase activity in protein lysates from independent- and dependent-skeletal muscle lysates (n = 16 for each of the two groups). * p ≤ 0.05, **** p ≤ 0.0001.

Figure 4

Increase in infiltrated immune cells in sarcopenic muscle from DP. (A) Immunofluorescence staining of skeletal muscle sections from independent- and dependent patients for CD45 and CD68. (B) Quantification of CD45⁺- and CD68⁺-cells in independent and dependent patients (n = 5 for each of the two groups). *** p ≤ 0.001.

Figure 5

NLRP3 induces cellular senescence in dependent muscle. Immunoblots and quantification of (A) p16, (B) RIP3, (C) p-H2AX in muscle lysates from independent- and dependent patients, (D) Western blotting representative images (n = 30 for each of the two groups). * p ≤ 0.05, ** p ≤ 0.01.

Figure 6

Higher damage-associated molecules in dependent muscle. (A) Hemolysis test expressed in hemolysis percentage (%) (n = 30 for each of the two groups); (B,C) ATP levels in (B) plasma (n = 17 for each of the two groups) and (C) erythrocytes (n = 22 for each of the two groups) of independent- and dependent patients. ** p ≤ 0.01.

Figure 7

Overview of NLRP3 inflammasome pathway in skeletal muscle of independent and depenedent elderly patients.