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. 2023 Jul 25;42(7):112735.
doi: 10.1016/j.celrep.2023.112735. Epub 2023 Jul 7.

Increased mitochondrial free Ca2+ during ischemia is suppressed, but not eliminated by, germline deletion of the mitochondrial Ca2+ uniporter

Courtney E Petersen  1 , Junhui Sun  1 , Kavisha Silva  1 , Anna Kosmach  1 , Robert S Balaban  2 , Elizabeth Murphy  3
Affiliations

Increased mitochondrial free Ca2+ during ischemia is suppressed, but not eliminated by, germline deletion of the mitochondrial Ca2+ uniporter

Courtney E Petersen et al. Cell Rep. .

Abstract

Mitochondrial Ca2+ overload is proposed to regulate cell death via opening of the mitochondrial permeability transition pore. It is hypothesized that inhibition of the mitochondrial Ca2+ uniporter (MCU) will prevent Ca2+ accumulation during ischemia/reperfusion and thereby reduce cell death. To address this, we evaluate mitochondrial Ca2+ in ex-vivo-perfused hearts from germline MCU-knockout (KO) and wild-type (WT) mice using transmural spectroscopy. Matrix Ca2+ levels are measured with a genetically encoded, red fluorescent Ca2+ indicator (R-GECO1) using an adeno-associated viral vector (AAV9) for delivery. Due to the pH sensitivity of R-GECO1 and the known fall in pH during ischemia, hearts are glycogen depleted to decrease the ischemic fall in pH. At 20 min of ischemia, there is significantly less mitochondrial Ca2+ in MCU-KO hearts compared with MCU-WT controls. However, an increase in mitochondrial Ca2+ is present in MCU-KO hearts, suggesting that mitochondrial Ca2+ overload during ischemia is not solely dependent on MCU.

Keywords: CP: Developmental biology; MCU; calcium; cardioprotection; ischemia-reperfusion; mitochondria.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Intrathoracic injections of AAV9-mito-R-GECO1 localize to cardiac mitochondria
Wild-type control and TOMM20-mNeonGreen-expressing P3–P5 neonates were intrathoracically injected with 50 μL 6.9 × 1013 GC/mL AAV9-mito-R-GECO1. Approximately 4–6 weeks postinjection, hearts were excised, Langendorff perfused, and imaged. (A) Representative images obtained on a dissecting stereoscope of non-injected control and AAV9-mito-R-GECO1-injected heart in wide view (top panels) and fluorescence (bottom panels) after 532 nm excitation for 30 ms exposure. Scale bar, 1 mM. (B) Representative image obtained on a Leica SP8 microscope approximately 6 weeks post-AAV9-mito-R-GECO1 injection. AAV9-mito-R-GECO1 (red) was found to be contained with TOMM20-mNeonGreen (green) delineated mitochondria. Scale bar, 5 μm.
Figure 2.
Figure 2.. R-GECO1 fluorescence decreases in MCU-KO and -WT hearts during ischemia
(A) Representative line graph of fluorescence emissions with excitation at 532 nm in a control perfused mouse heart that was not injected with AAV9-R-GECO1 (background/black) compared with an AAV9-R-GECO1-injected heart (blue). (B) Protocol for imaging mitochondrial Ca2+ using R-GECO1 during I/R in ex-vivo-perfused hearts. (C) Line graph of R-GECO1 fluorescence throughout ex vivo I/R experiment shown as fold change from baseline. Baseline fluorescence was measured as the average R-GECO1 fluorescence over 30 min of perfusion (MCU-WT n = 6; MCU-KO n = 4 [biological replicates]). Results indicate a non-significant difference in R-GECO1 fluorescence between MCU-KO and -WT hearts as shown by two-way ANOVA with mixed-effect analysis and Sidak’s multiple comparisons test. Error bars indicate mean ± SEM.
Figure 3.
Figure 3.. R-GECO1 is highly pH sensitive
(A) Fluorescent emission spectral results for purified R-GECO1 with 532 nm excitation show R-GECO1 fluorescence intensity decreases in response to acidifying pH changes. (B) Protocol for imaging pHrodo Red during I/R in ex-vivo-perfused MCU-KO and -WT hearts. (C) Glycogen depletion protocol for imaging pHrodo Red during I/R in ex-vivo-perfused MCU-KO and -WT hearts. (D) Line graph of pHrodo Red fluorescence intensity throughout ex vivo I/R experiment shown as fold change from baseline (n = 3 biological replicates per group). No significant difference was observed between MCU-KO or -WT hearts with or without glycogen depletion (GD) by two-way ANOVA analysis with repeated measures and Sidak’s multiple comparison. Error bars indicate mean ± SEM.
Figure 4.
Figure 4.. MCU-KO suppressed mitochondrial Ca2+ increases during I/R
(A) Protocol for imaging mitochondrial Ca2+ using R-GECO1 during I/R following GD in ex-vivo-perfused MCU-KO and -WT hearts. (B) Representative line graph of fluorescence emissions with excitation at 532 nm in a control perfused mouse heart that was not injected with AAV9-R-GECO1 (background/black) compared with an AAV9-R-GECO1-injected heart prior to ischemic onset (blue) and during ischemia (red). Dotted black box denotes fluorescence emissions at 650–680 nm, which were used in analysis. (C) Line graph of R-GECO1 fluorescence intensity during perfusion with Krebs (KH) buffer and the switch to acetate-KH buffer. There was no significant difference in R-GECO1 fluorescence in either buffer as shown by two-way ANOVA analysis and Sidak’s multiple comparisons test (MCU-WT n = 3 biological replicates; MCU-KO n = 4 biological replicates). (D) Line graph of R-GECO1 fluorescence intensity throughout ex vivo I/R following GD shown as fold change from baseline. Baseline fluorescence was measured as the average R-GECO1 fluorescence over 30 min of perfusion (MCU-WT n = 14; MCU-KO n = 16 [biological replicates]). (E) Column chart showing R-GECO1 fluorescence intensity in MCU-KO and -WT ex-vivo–perfused hearts at 20 min of ischemia. Each point in the column charts represents measurements made from a single heart, and results indicate a significant difference between MCU-KO and -WT hearts by two-way ANOVA analysis with repeated measures and Sidak’s multiple comparison (p = 0.0319 ± SEM). (F) Column chart showing R-GECO1 fluorescence intensity in MCU-KO ex-vivo-perfused hearts before ischemia and after 20 min of ischemia. Each point represents measurements made from a single heart, and results indicate a significant difference between R-GECO1 fluorescence in MCU-KO hearts before and at the end of ischemia by paired and unpaired t tests (p < 0.0001 ± SEM).
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