Tirzepatide and Muscle Outcomes in Obesity

Measured primarily as changes in body weight and body composition measured by dual-energy X-ray absorptiometry (DXA).

Change in maximal knee extensor torque normalized to body mass (Nm/kg) will be evaluated using an isokinetic dynamometer.

Muscle volume and muscle fat fraction will be quantified using magnetic resonance imaging (MRI).

Transcriptome-wide RNA-sequencing will be performed on vastus lateralis muscle biopsies from all participants (depending on sample quality and RNA integrity) before and after the intervention to identify differentially expressed genes associated with tirzepatide treatment. Differential gene expression will be reported as log2 fold change (log2FC) between post and pre-intervention, derived from normalized RNA-sequencing read counts. Statistical significance will be summarized using false discovery rate (FDR)-adjusted p-values.

Pathway-level changes will be reported using normalized enrichment scores (NES) derived from gene set enrichment analysis (GSEA), together with false discovery rate (FDR)-adjusted q-values for biological processes including mitochondrial function, lipid metabolism, insulin signaling, inflammation, muscle atrophy, and myogenesis.

Assessed by Oil Red O staining using a standard protocol. IMCL will be quantified as the proportion of Oil Red O-positive area per fibre and averaged across available fibres per section per time point. Microscopy imaging and analysis settings will be kept consistent across time points, and assessors will be blinded to group/time.

Muscle fiber diameter will be measured on the biopsy section using routine light microscopy. Fiber typing (type 1 vs type 2) will be performed by standard immunohistochemistry for myosin heavy chain isoforms. The metric is mean minimal Feret diameter in micrometers, averaged across available fibers of same type.

Change in maximal handgrip strength, normalized to body mass, will be assessed using a handheld dynamometer.

Change in lower-limb functional performance assessed using standardized sit-to-stand testing.

The six-minute walk test (6MWT) will be used to assess functional exercise capacity. The outcome will be the total distance walked (meters) over 6 minutes on a flat corridor, following standardized guidelines.

Potential exploratory: Feasibility-dependent analysis of spatial transcriptomic profiles in skeletal muscle biopsy. The objective is to identify treatment-related changes in spatial gene expression patterns in skeletal muscle tissue sections. A subset of approximately 8 patients (paired pre- and post-treatment muscle biopsy samples) will be selected for downstream analyses based on transcriptomic profiles obtained from the full RNA-seq dataset.

Potential exploratory: Electrophysiological recordings of motor responses (compound muscle action potentials, CMAP) will be assessed to evaluate potential changes in peripheral motor nerve excitability and neuromuscular function.