Electric field stimulation in Caenorhabditis elegans as a novel approach to investigate mitochondrial Ca²⁺ homeostasis during in vivo muscle aging

Mitochondrial calcium ([Ca²⁺]mito) homeostasis is a key regulator of cellular physiology, controlling signal transduction, energy metabolism, and cell survival. To examine how these processes change with age in vivo, we used the pharyngeal muscle of Caenorhabditis elegans (C. elegans) as a tractable model for studying [Ca²⁺]mito dynamics. We introduced electric field stimulation as a robust trigger for [Ca²⁺]mito uptake that overcomes limitations of compound stimulation, which relies on pharyngeal pumping and endoplasmic reticulum store filling and is typically confined to single-stimulus protocols. In contrast, electric field stimulation enables physiologically relevant repeated excitation that challenges [Ca²⁺]mito uptake and recovery. Stimulation with 10 V for 10 s reliably evoked reproducible [Ca²⁺]mito transients in the pharyngeal muscle of C. elegans and produced higher responder rates than compound stimulation. Basal [Ca²⁺]mito increased with age, and the first field-evoked transient was significantly larger in aged animals, an effect not detected with pharmacological triggers. Repeated pulses unmasked cumulative [Ca²⁺]mito loading and incomplete recovery in aged pharynx, indicative of [Ca²⁺]mito overload, which was attenuated by inhibition of the mitochondrial Ca²⁺ uniporter (MCU) with mitoxantrone. Regional analyses identified the corpus as a hotspot for electric field-evoked [Ca²⁺]mito uptake and overload, while MCU inhibition reduced repeated responses in both corpus and posterior bulb. Electric field stimulation enables precise, repeated in vivo probing of [Ca²⁺]mito uptake and recovery, revealing overload in the C. elegans pharynx. This approach identified age-enhanced, MCU-dependent, and region-specific [Ca²⁺]mito loading, providing a pathophysiologically relevant readout of impaired Ca²⁺ handling that may contribute to age-related muscle dysfunction.