Author(s): FERRUSOLA-PASTRANA, A., PRATS-PUIG, A., HOMS, J., VASILEVA, F., JÒDAR, A., FONT-LLADÓ, R., Institution: EUSES - UNIVERSITAT DE GIRONA (UDG), Country: SPAIN, Abstract-ID: 2473

INTRODUCTION:
Physical exercise can induce coordinated molecular adaptations across multiple tissues, including skeletal muscle. Preliminary salivary proteomic analyses that we performed in 39 school-aged children showed that several skeletal muscle-associated proteins (i.e., SPEG, PFKM, AK1, SEMA7A, MYH13) could be identified within enriched muscle-related networks following integrative neuromuscular training (INT). The present study aims to validate selected protein candidates using targeted immunoassays in an independent paediatric cohort and explore associations with anthropometric, functional and cardiovascular parameters.
METHODS:
Saliva samples were collected from school children (age 7.4 ± 0.4) participating in a 7-month structured INT programme (n = 10) or a control group following standard physical education classes (CON; n = 10). Based on preliminary findings, biological relevance, and assay feasibility, three proteins were selected for validation: 6-phosphofructokinase muscle type (PFKM), adenylate kinase 1 (AK1), and striated muscle preferentially expressed protein kinase (SPEG). Salivary protein concentrations were measured using enzyme-linked immunosorbent assays (ELISA). Data were analysed using linear mixed-effects models with time (pre, post), group (INT, CON), and their interaction as fixed effects, and subject as a random intercept. Correlation analyses were performed to explore associations between changes in protein concentrations and the studied parameters. Statistical analyses were performed using R Statistical Software (v4.4.1; R Core Team 2024).
RESULTS:
PFKM was consistently detectable, whereas AK1 and SPEG were only measurable in a subset of samples, precluding statistical analysis. PFKM significantly increased within the INT group (p < .001), showing higher concentrations compared with CON (p < .004); however, the time x group interaction was not statistically significant (p = .150). No significant associations were observed between changes in PFKM and BMI, muscle mass, motor competence, grip strength, resting heart rate, or blood pressure, in either the INT or CON group.
CONCLUSION:
This study extends previous findings by examining the feasibility of targeted validation of skeletal muscle-associated proteins in saliva samples of children. PFKM was consistently detectable by ELISA and showed a within-group responsiveness following neuromuscular training. However, the absence of a significant interaction and lack of associations with functional or health parameters highlights the complexity of validating skeletal muscle-related proteins in saliva. These findings emphasise the need for larger samples and methodological refinement when exploring salivary proteins as potential markers of exercise-related molecular adaptations in paediatric populations. Future studies incorporating more proximal indicators of exercise intensity, metabolic demand and training load may help capture the specific metabolic processes reflected by PFKM and clarify potential associations.