Uncovering the time-temperature thresholds of <i>in vitro</i> mitochondrial bioenergetics dysfunction under hyperthermic stress

Mitochondrial respiration is particularly vulnerable to temperature fluctuations, yet time-dependent effects of hyperthermia on mitochondria remain largely unexplored. In this study, we evaluated the thermal sensitivity of mitochondria isolated from mouse liver using high-resolution respirometry (HRR) to assess the combined effects of temperature elevation and exposure time. Our results demonstrated that isolated mitochondria experienced progressive dysfunction of the electron transport chain (ETC) and the phosphorylation system as both temperature and incubation time increased. Notably, even moderate thermal stress led to significant impairments, and damage became irreversible when exceeding 40% at a minimum temperature of 43 °C. A mathematical analysis of the time-temperature relationship further supported these findings, highlighting a threshold beyond which mitochondrial function cannot recover. These results emphasize the importance of accounting for the degree of heat exposure and its duration when evaluating thermal effects on mitochondrial bioenergetics. This study provides valuable insights into mitochondrial resilience's <i>in vitro</i> thermal limits.