Functional hypoxia reduces mitochondrial calcium uptake

Abstract

Mitochondrial respiration extends beyond ATP generation, with the organelle participating in many cellular and physiological processes. Parallel changes in components of the mitochondrial electron transfer system with respiration render it an appropriate hub for coordinating cellular adaption to changes in oxygen levels. How changes in respiration under functional hypoxia (i.e., when intracellular O₂ levels limit mitochondrial respiration) are relayed by the electron transfer system to impact mitochondrial adaption and remodeling after hypoxic exposure remains poorly defined. This is largely due to challenges integrating findings under controlled and defined O₂ levels in studies connecting functions of isolated mitochondria to humans during physical exercise. Here we present experiments under conditions of hypoxia in isolated mitochondria, myotubes and exercising humans. Performing steady-state respirometry with isolated mitochondria we found that oxygen limitation of respiration reduced electron flow and oxidative phosphorylation, lowered the mitochondrial membrane potential difference, and decreased mitochondrial calcium influx. Similarly, in myotubes under functional hypoxia mitochondrial calcium uptake decreased in response to sarcoplasmic reticulum calcium release for contraction. In both myotubes and human skeletal muscle this blunted mitochondrial adaptive responses and remodeling upon contractions. Our results suggest that by regulating calcium uptake the mitochondrial electron transfer system is a hub for coordinating cellular adaption under functional hypoxia.

Keywords: Coenzyme Q; Exercise; Membrane potential; Respirometry; Skeletal muscle.

Fig. 1

Functional hypoxia reduces the redox state of electron-transfer-reactive Q and lowers the mitochondrial membrane potential difference. A) Schematic representation of mitochondrial electron transfer system and coupling to oxidative phosphorylation (F₁F_O-ATPase). Electron transferring flavoprotein ETF is the redox carrier between β-oxidation and the respiratory Complex ETF dehydrogenase, CETFDH [29]. B) Schematic representation of the H₂O₂ and catalase-based system for steady-state respirometry used to study functional hypoxia. At steady-state the rate of H₂O₂ injection and conversion of H₂O₂ to O₂ sets the mitochondrial metabolic flux j. C) Measurement of the redox state of the mitochondrial electron-transfer-reactive Q-pool (Q) under steady-states of hypoxia and at maximal O₂ flux in isolated mitochondria from mouse cardiac muscle respiring on NADH-linked substrates, succinate, and fatty-acid in the presence of kinetically saturating ADP. D) Relative Q oxidation under anoxia (j = 0), functional hypoxia (j = 0.1 to 0.5) and at maximal O₂ flux (j = 1) in mitochondria isolated from mouse cardiac muscle (N = 3). j is the ratio of the experimentally set J_O2 to the measured OXPHOS capacity (J_max). E) Mitochondrial membrane potential difference under steady states of hypoxia and at maximal O₂ flux in mitochondria isolated from mouse cardiac muscle respiring on NADH-linked substrates, succinate, and fatty acid in the presence of kinetically saturating ADP. Increase in the TMRM concentration indicates a decrease in the mitochondrial membrane potential difference. F) Relative TMRM signal under anoxia (j = 0), functional hypoxia (j = 0.1 to 0.5) and at maximal O₂ flux (j = 1) in mitochondria isolated from mouse cardiac muscle (N = 5). j is the ratio of the set J_O2 to the measured OXPHOS capacity (J_max at kinetically saturating O₂ concentrations). In panels D and F, the lines represent the means, dots are individual values.

Fig. 2

Functional hypoxia reduced mitochondrial Ca²⁺ flux in mitochondria isolated from mouse cardiac muscle. A) Mitochondrial Ca²⁺ flux under steady-state hypoxia. B) Mitochondrial Ca²⁺ flux at maximal O₂ flux. C) Representative traces of extramitochondrial Ca²⁺ concentration and mitochondrial Ca²⁺ flux at maximal O₂ flux (J_max, green line) and under functional hypoxia (j = 0.3, blue line; superimposed from panels A and B). D) Mitochondrial Ca²⁺ flux under functional hypoxia (j = 0.3) and at maximal O₂ flux (j = 1.0; N = 5) measured during the final 3 min of the 4-min H₂O₂ titration. In panel D the bars represent the means, error bars the standard deviations, dots are individual values.

Fig. 3

Mitochondrial Ca²⁺ uptake was lower under functional hypoxia in mouse myotubes. A) Culture conditions. B) Partial pressure of O₂ in culture media upon exposure to 1 % O₂ in the gas phase (N = 3). C) HIF1α protein content in myotubes 120 min after exposure to 1 % O₂ in the gas phase (N = 4). Representative blot of HIF1α with ponceau (loading) stain in myotubes 120 min after exposure to 1 % O₂ in the gas phase (N = 4). E) Hypoxia and glycolysis hallmark gene sets enriched in myotubes 120 min after exposure to 1 % O₂ compared with 5 % O₂ in the gas phase (N = 6). False discovery rate FDR. Normalized enrichment score NES. F) TMRM mean intensity under 5 % (t = 0 min) and after 120 min of exposure to 1 % O₂ in the gas phase (t = 120 min; N = 4) in C2C12 myotubes. G) Representative images of C2C12 myotubes stained with TMRM exposed to 5 % or 120 min after exposure to 1 % O₂ in the gas phase. Scale bar is 50 μm. An increase in the TMRM intensity indicates a decrease in the mitochondrial membrane potential difference. H) Quantification of Fluo-4AM mean intensity in response to caffeine stimulation in myotubes 120 min after exposure to 5 % or 1 % O₂ in the gas phase (N = 5). I) Representative images of C2C12 myotubes stained with Fluo-4AM, exposed to 5 % or 1 % O₂ and stimulated with caffeine. Scale bar is 50 μm. J) Response of Rhod-2AM fluorescence to caffeine stimulation in C2C12 myotubes 120 min after exposure to 5 % or 1 % O₂ in the gas phase (N = 4). K) Representative images of C2C12 myotubes stained with Mitotracker Green and Rhod-2AM, exposed to 5 % or 1 % O₂ and stimulated with caffeine. Scale bar is 20 μm. In panels B, C, F, H and J, the bars represent the means, error bars the standard deviations, dots are individual values.

Fig. 4

Functional hypoxia altered mitochondrial calcium uptake and mitochondrial responses to contractions in mouse myotubes. A) Culture conditions and electrical stimulation protocol. B) Hallmark gene sets enriched in myotubes immediately after stimulation in 5 % O₂ compared with non-stimulated myotubes (N = 6). C) Response of Rhod-2AM fluorescence to electrical stimulation in C2C12 myotubes 120 min after exposure to 5 % or 1 % O₂ in the gas phase (N = 4). D) Representative images of C2C12 myotubes stained with Mitotracker Green and Rhod-2AM, exposed to 5 % or 1 % O₂ and electrically stimulated using the protocol shown in panel A. Images are taken immediately before- and after the electrical stimulation protocol. Scale bar is 50 μm. E-I) ATP5A (E), CIV-MTCO1 (F), CIII-UQCRC2 (G), CII-SDHB (H) and CI-NDUFB8 (I) protein content in C2C12 myotubes 48 h after stimulation (N = 7). J) Representative blot of ATP5A, CIII-UQCRC2, CIV-MTCO1, CII-SDHB and CI-NDUFB8 with loading total protein stain from C2C12 myotubes 48 h after stimulation. K) Hallmark gene sets enriched in myotubes immediately after stimulation in 1 % O₂ compared with myotubes stimulated in 5 % O₂ (N = 6). In panels B and K the bar represents the false discovery rate (FDR) for the specified group comparison. In panels C and E-I, the bars represent the means, error bars the standard deviations, dots are individual values.

Fig. 5

Functional hypoxia altered mitochondrial responses to contractions in human skeletal muscle. A) Human exercise study protocol. B) Total work performed (expressed relative to body mass) during the SIT session (N = 8). C) Representative blot of ATP5A, CIII-UQCRC2, CIV-MTCO1, CII-SDHB, CI-NDUFB8 and Gapdh (loading control) from human vastus lateralis biopsies. D-H) Relative changes in ATP5A (D), CIV-MTCO1 (E), CIII-UQCRC2 (F), CII-SDHB (G) and CI-NDUFB8 (H) protein content from Pre to +24 h in human vastus lateralis biopsies (N = 8). In panels B and D-H, the bars represent the means, error bars the standard deviations, dots are individual values.