Pitfalls in Monitoring Mitochondrial Temperature Using Charged Thermosensitive Fluorophores

Abstract

Mitochondria are the source of internal heat which influences all cellular processes. Hence, monitoring mitochondrial temperature provides a unique insight into cell physiology. Using a thermosensitive fluorescent probe MitoThermo Yellow (MTY), we have shown recently that mitochondria within human cells are maintained at close to 50 °C when active, increasing their temperature locally by about 10 °C. Initially reported in the HEK293 cell line, we confirmed this finding in the HeLa cell line. Delving deeper, using MTY and MTX (MitoThermo X), a modified version of MTY, we unraveled some caveats related to the nature of these charged fluorophores. While enabling the assessment of mitochondrial temperature in HEK and HeLa cell lines, the reactivity of MTY to membrane potential variations in human primary skin fibroblasts precluded local temperature monitoring in these cells. Chemical modification of MTY into MTX did not result in a temperature probe unresponsive to membrane potential variations that could be universally used in any cell type to determine mitochondrial temperature. Thus, the cell-type dependence of MTY in measuring mitochondrial temperature, which is likely due to the variable binding of this dye to specific internal mitochondrial components, should imply cautiousness while using these nanothermometers for mitochondrial temperature analysis.

Contextualizing the biological relevance of standardized high‐resolution respirometry to assess mitochondrial function in permeabilized human skeletal muscle

“…Next, the role of temperature on respiratory rates was considered to explain the divergence between like in vivo and ex vivo measures. While respiratory chamber temperature has been largely standardized for research at or around physiological temperatures of 37°C, 25,81 respiring mitochondria have been reported to function at temperatures reaching over 50°C or ~10°C higher than the enveloping cell 82,83 . Accordingly, we adjusted all respiratory measures to control for ostensibly lower artificial temperatures during standardized data collection as detailed in the methods.…”

“…Femoral venous temperatures were increased by 10.5°C to account for the thermal gradients between skeletal muscle and venous blood (~0.5°C) 126 as well as between skeletal muscle mitochondria and skeletal muscle (~10°C) 82,83 …”

“…While respiratory chamber temperature has been largely standardized for research at or around physiological temperatures of 37°C, 25,81 respiring mitochondria have been reported to function at temperatures reaching over 50°C or ~10°C higher than the enveloping cell. 82,83 Accordingly, we adjusted all respiratory measures to control for ostensibly lower artificial In vivo (ml kg -1 min -1 ) Ex vivo ( S1 and subgroup data separated by aerobic fitness percentiles are displayed in Figure 3A-C. There is a main effect of aerobic fitness on OCR (Kruskal-Wallis statistic ≥108.7, P < .0001), SOR (Kruskal-Wallis statistic ≥89.7, P < .0001) and APR (Kruskal-Wallis statistic ≥108.7, P < .0001).…”

“…82 These findings have since been verified in HeLa cell lines. 83 Several issues have been raised in opposition of these findings, such as: (i) methodological concerns specific to research utilizing fluorescent probes for determination of cellular temperatures that includes possibly confounding influence(s) of the surrounding environment (ie pH, reactive oxygen species, membrane potential, viscosity and ionic strength); (ii) thermodynamic modelling of the cell describing the so-called "10 −5 gap" theory that renders intracellular temperature gradients as all but impossible and (iii) biological improbabilities of such high in vivo temperatures that would challenge human biological function as we understand it. [96][97][98] Thus, considerations for appropriate temperature corrections range from a minimum that reflects the cellular temperature specific to the tissue being analysed when taking into account the metabolic state also being measures (ie basal vs maximal metabolic states) to a maximum of 10°C above that minimum value.…”

“…Given that blood flow and rates of skeletal muscle oxygen consumption have repeatedly been shown as higher during maximal isolated vs wholebody exercise efforts (eg KE max vs CE max ) and assuming that respiration during maximal isolated exercise efforts remains well-coupled to ATP production, temperature correcting standardized HRR values obtained from permeabilized human skeletal muscle appears to require corrections above skeletal muscle temperatures at maximal exercise efforts. Thus, the current study examined how temperature correcting standardized HRR values to 10°C above respective cellular temperatures influences measures reflecting respiratory control (OCR), metabolic flexibility (SOR) and bioenergetics (APR), which is the maximal temperature correction that currently entertains empirical support, 82,83 albeit contested.…”