pH-Resolved ATP Synthesis in Skeletal Muscle: Concept, Implementation, and Assessment Using Dynamic 31P Magnetic Resonance Spectroscopy at 7T

Background/Objectives: Dynamic changes in inorganic phosphate (Pi), phosphocreatine (PCr), and pH during post-exercise recovery reflect underlying muscle energetics and mitochondrial ATP synthesis, but the conventional single-pool model assuming uniform metabolic response fails to address myofiber composition and pH-dependent metabolic heterogeneity in skeletal muscle. This study aimed to characterize the interplay between pH, Pi, and PCr, and to develop an analytical method for assessing pH-resolved ATP synthesis using 31P MRS. Methods: Five healthy subjects underwent dynamic 31P MRS scans during plantar flexion exercise. ATP synthesis was evaluated from post-exercise PCr and Pi recovery time courses using the single-pool model, and from Pi recovery time courses using a multi-pool model in which the Pi signal lineshape was segmented into four pH-specific pools: alkaline (pH 7.3 ± 0.2), neutral (pH 7.0 ± 0.1), mildly acidic (pH 6.8 ± 0.1), and moderately acidic (pH 6.6 ± 0.1). Results: The single-pool model showed that during exercise, Pi increased proportionally to PCr depletion, and both Pi and PCr recovered monoexponentially immediately after exercise with 𝜏pH ≫ 𝜏PCr ). The four-pool model demonstrated a pro-nounced pH dependence of Pi recovery, with slower recovery at lower pH (𝜏Pi: 19 ± 6 s at pH 7.3, 25 ± 7 s at pH 7.0, 32 ± 11 s at pH 6.8, and 46 ± 17 s at pH 6.6). Pi recovery is slowed with aging under acidic conditions, with little or no effect observed at neutral or alkaline pH. These results provide new insights into skeletal muscle metabolic heterogeneity, re-flecting how different myofiber microenvironments modulate ATP synthesis. Conclusions: By overcoming the constraints of the single-pool model, the proposed multi-pool framework uncovers pH-dependent ATP synthesis and provides direct evidence of pro-nounced metabolic heterogeneity in skeletal muscle during exercise and recovery.