Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS)

Abstract

Mitochondrial Ca²⁺ uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO^•) production. However, it is not entirely clear if the organelles support or counteract NO^• biosynthesis by taking up Ca²⁺. The objective of this study was to verify whether or not mitochondrial Ca²⁺ uptake influences Ca²⁺-triggered NO^• generation by endothelial NO^• synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO^• probes, the geNOps, and Ca²⁺ sensors to monitor single cell NO^• and Ca²⁺ dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP₃)-generating agonist. Mitochondrial Ca²⁺ uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca²⁺ uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca²⁺ uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca²⁺-triggered NO^• increase independently of global cytosolic Ca²⁺ signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca²⁺ sequestration and Ca²⁺-induced NO^• signals. The positive correlation between mitochondrial Ca²⁺ elevation and NO^• production was independent of eNOS phosphorylation at serine¹¹⁷⁷. Our findings emphasize that manipulating mitochondrial Ca²⁺ uptake may represent a novel strategy to control eNOS-mediated NO^• production.

Keywords: Calcium; ENOS; Endothelial nitric oxide production; GeNOps; Mitochondria.

Fig. 1. Live-cell imaging of Ca²⁺-triggered NO^• formation in endothelial cells expressing G-geNOp.

(A) Schematic illustration of G-geNOp consisting of the enhanced green fluorescent protein (EGFP) fused to the bacteria-derived NO^• binding domain GAF (grey). Binding of NO^• in the vicinity of EGFP in G-geNOp induces fluorescence quenching. (B) Representative image showing EA.hy926 cells expressing G-geNOp. Notably, the NO^• probe localizes to the nucleus and cytosol. (C) Average curve ( ± SEM) showing NO^• signal over time obtained from single EA.hy926 cells obtained from independent experiments (N =40) upon treatment with 100 μM ATP in the absence of Ca²⁺(1 mM EGTA) and upon Ca²⁺(2 mM) addition. (D) Representative simultaneous recordings of NO^• (solid grey line) and Ca²⁺(dashed black line) signals over time in a single fura-2/am loaded EA.hy926 cell expressing G-geNOp. As indicated the cell was treated with 100 μM ATP in the absence of Ca²⁺(1 mM EGTA) and upon Ca²⁺ addition (2 mM) for 14 min.

Fig. 2. Knock-down of EMRE and MCU reduces Ca²⁺-triggered NO^• production in EA.hy926 cells.

(A) Average curves of [Ca²⁺]_mito (left panel) in EA.hy926 cells treated with either scrambled siRNA (black curve and white bars, N=3) or siRNAs against EMRE and MCU (siEMRE/siMCU, red curve and red bars, N=3) upon stimulation with 100 μM ATP in the absence of extracellular Ca²⁺(1 mM EGTA) followed by addition of Ca²⁺(2 mM). Bar graphs showing average basal Ca²⁺ level (basal, p=0.0581) and Ca²⁺ transients in response to 100 μM ATP-stimulated ER Ca²⁺ release (ATP + EGTA, **p=0.0029) and store-operated Ca²⁺ entry (ATP + Ca²⁺, *p=0.0169). (B) Curves representing average of [Ca²⁺]_cyto in control (Scrambled siRNA, black curve, N=6) versus EMRE/MCU ablated (siEMRE/siMCU, red curve, N=7) EA.hy926 cells (left panel) stimulated with 100 μM ATP in the absence (1 mM EGTA) and addition of Ca²⁺(2 mM) as indicated. Respective statistical analyses of basal levels (basal, p=0.397), maximum cytosolic Ca²⁺ release (ATP + EGTA, p=0.6373) and maximum cytosolic Ca²⁺ entry (ATP + Ca²⁺, p=0.6988). (C) Representative curve of [NO^•]_cyto in response to ATP stimulation. Statistical analyses of average [NO^•]_cyto are shown in bar graphs (right panels). Maximal signals (amplitude) of scrambled siRNA- or siMCU/siEMRE-treated cells are defined as 100%. Cells were first stimulated with 100 μM ATP in the absence of extracellular Ca²⁺ in control cells (Scrambled siRNA, white column, ATP + EGTA, N =17) and cells reduced of EMRE and MCU (siEMRE/siMCU, red column, ATP + EGTA, N =17) and upon Ca²⁺( 2 mM) addition (right middle column pair). **p=0.0068 (ATP+EGTA) and **p=0.0042 (ATP+Ca²⁺) vs Scrambled siRNA. Right columns represent maximal fluorescence changes of G-geNOp^mut in response to 100 μM ATP in EGTA (1 mM) in control cells (white column, N =11) and cells treated with siRNA against EMRE and MCU (red column, N =10). (D) Average curves of Ca²⁺-dependent NO formation upon stimulation with 1 μM (upper left panel) 5 μM (upper middle panel) or 100 μM ATP (upper right panel) in the absence of extracellular Ca²⁺ in EA.hy926 cells treated with either negative control siRNA (Scrambled siRNA, black curves) or siRNA against EMRE and MCU (siEMRE/siMCU, red curves). Statistical analysis of maximum NO^• formation in cells stimulated with either 1 μM ATP (lower left panel) representing Scrambled siRNA (white bar, N=3) and siEMRE/siMCU (red bar, N=3); **p=0.0045, with 5 μM ATP (lower middle panel) representing Scrambled siRNA (white bar, N=3) and siEMRE/siMCU (red bar, N=3); **p=0.0087, or with 100 μM ATP (lower right panel) representing Scrambled siRNA (white bar, N=3) and siEMRE/siMCU (red bar, N=3); **p=0.0012.

Fig. 3. Knock-down of EMRE/MCU reduces Ca²⁺-evoked NO^• formation in HUVECs.

(A) Cytosolic Ca²⁺ signals in EA.hy926 cells (black curve and white bars) and primary HUVECs (red curve and red bars) in response to 100 μM ATP. [Ca²⁺]_cyto was imaged using fura-2/am loaded cells. Middle bars represent basal fura-2 ratio (F₃₄₀/F₃₈₀) values of EA.hy926 cells (white bars, N =6, p=0.9708) and HUVECs (red bars, N =6). Right bars show maximal fura-2 ratio amplitudes in EA.hy926 (white bars, N =6) and HUVEC cells (red bars, N =6). **p=0.005 vs. EA.hy926 cells. (B) Mitochondrial Ca²⁺ signals in EA.hy926 cells (black curve and white bars) and primary HUVECs (red curve and red bars) in response to 100 μM ATP. [Ca²⁺]_mito was imaged using cells expressing 4mtD3cpV. Middle bars represent basal 4mtD3cpV ratio (F₅₃₅/F₄₈₀) values of EA.hy926 cells (white bars, N =3, p=0.4753) and HUVECs (red bars, N =6). Right bars show maximal 4mtD3cpV ratio amplitudes in EA.hy926 (white bars, N =3) and HUVEC cells (red bars, N =6). (C) Cytosolic NO^• signals in EA.hy926 cells (black curve and white bars) and primary HUVECs (red curve and red bars) in response to 100 μM ATP. [NO^•]_cyto was imaged using cells expressing G-geNOp. Middle bars represent maximal fluorescence changes of G-geNOp (1-F/F₀) expressed in EA.hy926 cells (white bars, N =10) and HUVECs (red bars, N =8); *p=0.0366 vs EA.hy926. Right bars show maximal slopes of G-geNOp fluorescence changes in response to 100 mM ATP in EA.hy926 (white bars, N =10) and HUVEC cells (red bars, N =8). *p=0.015 vs. EA.hy926 cells. (D) Mitochondrial Ca²⁺ signals in HUVECs under control conditions (Scrambled siRNA, black curve, white bars, N =6) and in cells treated with siRNA against EMRE and MCU (siEMRE/siMCU, red curve, red columns, N =6). Middle bars represent basal 4mtD3cpV ratio values (p=0.1502). Cells were stimulated with 100 μM ATP in the absence of extracellular Ca²⁺. Right bars show maximal 4mtD3cpV ratio changes upon cell stimulation.**p=0.0032 vs Scrambled siRNA. (E) Cytosolic NO^• signals ([NO^•]_cyto) in control HUVECs (Scrambled siRNA, black curve and white bars, N =8) and cells treated with siRNA against EMRE and MCU (siEMRE/siMCU, red curve and bars, N =8) in response to 100 μM ATP in EGTA (1 mM). Middle columns represent mean of maximal ATP-evoked G-geNOp responses; *p=0.0193. Right columns show respective maximal slopes of G-geNOp signals; *p=0.0206.

Fig. 4. Cell treatment with oligomycin/antimycin reduces Ca²⁺-triggered NO^• formation.

(A) Average [Ca²⁺]_mito signals ± SEM of EA.hy926 cells expressing 4mtD3cpv under control conditions (black curve N =8) and in the presence of 1 μM oligomycin and 5 μM antimycin (red curve, N =12) in response to 100 μM ATP first in the absence of extracellular Ca²⁺(1 mM EGTA) and upon addition of Ca²⁺(2 mM).**p=0.0096 (in EGTA) and *p=0.0159 (in Ca²⁺) vs. Control. (B) TMRM signals of EA.hy926 cells over time to estimate changes in Ψ_mito (mitochondrial membrane potential) under control conditions (black curve N =6) and upon addition of 1 μM oligomycin and 5 μM antimycin (red curve, N =6). *p=0.0286. (C) NO^• signals of EA.hy926 cells over time expressing G-geNOp in the absence (control, black curve, white bars, N =11) or presence of 1 μM oligomycin and 5 μM antimycin (red curve and bars, N =9). Maximal signals under control conditions in response to 100 μM ATP were defined as 100% (middle right bars). **p=0.0074 (in EGTA) and *p=0.0200 (in Ca²⁺) vs. Control. Right bars represent maximal fluorescence changes of the NO^•-insensitive G-geNOp^mut in control cells (white bars N =6) and in the presence of the mitochondria toxins (red bars, N =6). p=0.8016 (in EGTA) and p=0.1508 (in Ca²⁺). (D) Fura-2 ratio signals of EA.hy926 cells over time under control conditions (black curve, N =6) and in the presence of 1 μM oligomycin and 5 μM antimycin (red curve, N =6). *p=0.0286 (in EGTA) and *p=0.0283 (in Ca²⁺) vs. Control.

Fig. 5. Silencing of MICU1 increases Ca²⁺-triggered NO^• production in endothelial cells.

(A) Average curves of [Ca²⁺]_mito (left panel) in EA.hy926 cells treated with either scrambled siRNA (black curve and white bars, N=3) or siRNAs against MICU1 (siMICU1, red curve and red bars, N=3) upon stimulation with 100 μM ATP in the absence of extracellular Ca²⁺(1 mM EGTA) followed by addition of Ca²⁺(2 mM). Bar graphs showing average basal Ca²⁺ level (basal, *p=0.0356) and Ca²⁺ transients in response to 100 μM ATP in 1 mM EGTA (ATP + EGTA, **p=0.0098) and upon Ca²⁺ addition (ATP + Ca²⁺, *p=0.0151). (B) Curves representing average of [Ca²⁺]_cyto in control (Scrambled siRNA, black curve, N=6) versus MICU1 ablated (siMICU1, red curve, N=6) EA.hy926 cells (left panel) stimulated with 100 μM ATP in the absence (1 mM EGTA) and addition of Ca²⁺(2 mM) as indicated. Respective statistical analyses of basal levels (basal, p=0.9621), maximum cytosolic Ca²⁺ release (ATP + EGTA, p=0.9911) and maximum cytosolic Ca²⁺ entry (ATP + Ca²⁺, p=0.4977). (C) Representative curve of [NO^•]_cyto in response to ATP stimulation. Statistical analyses of average [NO^•]_cyto are shown in bar graphs (right panels). Maximal signals (amplitude) of scrambled siRNA- or siMICU1-treated cells are defined as 100%. Cells were first stimulated with 100 μM ATP in the absence of extracellular Ca²⁺ in control cells (Scrambled siRNA, white column, ATP + EGTA, N =17) and cells reduced of MICU1 (siMICU1, red column, ATP + EGTA, N =16) and upon Ca²⁺(2 mM) addition (right middle column pair). **p=0.0083 (ATP + EGTA) and **p=0.0079 (ATP+Ca²⁺) vs Scrambled siRNA. Right columns represent maximal fluorescence changes of G-geNOp^mut in response to 100 μM ATP in EGTA (1 mM) in control cells (white column, N =11) and cells treated with siRNA against MICU1 (siMICU1, red column, N =8); p=0.4424 vs Scrambled siRNA. (D) Mitochondrial Ca²⁺ signals in HUVECs under control conditions (Scrambled siRNA, black curve, white bars, N =6) and in cells treated with siRNA against MICU1 (siMICU1, red curve, red columns, N =6). Middle bars represent basal 4mtD3cpV ratio values (p=0.1502). Cells were stimulated with 100 μM ATP in the absence of extracellular Ca²⁺. Right bars show maximal 4mtD3cpV ratio changes upon cell stimulation.*p=0.0211 vs Scrambled siRNA. (E) Cytosolic NO^• signals ([NO^•]_cyto) in control HUVECs (Scrambled siRNA, black curve and white bars, N =8) and cells treated with siRNA against MICU1 (siMICU1, red curve and bars, N =10) in response to 100 μM ATP in EGTA (1 mM). Middle columns represent mean of maximal ATP-evoked G-geNOp responses; p=0.2532. Right columns show respective maximal slopes of G-geNOp signals; p=0.4567.

Fig. 6. The expression level and phosphorylation status of eNOS remain unaffected by transient down-regulation of MCU, EMRE and MICU1.

(A) Representative Western blot of total homogenates of transfected EA.hy926 cells (30 μg of protein) showing phosphorylated eNOS (peNOS; 140 kDa), total eNOS (140 kDa) and β-actin (43 kDa) under basal conditions (unstimulated) or stimulated with 100 μM ATP for 3 or 5 min. Purified human eNOS (30 and 50 ng) was used as standard. Bands of control cells (Scrambled siRNA) and cells treated with siRNA against both EMRE and MCU (siEMRE/siMCU) or against MICU1 (siMICU1) are shown. (B) Bars show the ratio of phosphorylated to total eNOS protein in transfected EA.hy926 cells (Scrambled siRNA, siEMRE/siMCU and siMICU1) under basal conditions (unstimulated), after 3 (left panel) and 5 (right panel) minutes of incubation with 100 μM ATP. N =3 for all conditions.

Fig. 7. Mitochondrial Ca²⁺ uptake controls NO^• formation in HEK293 cells expressing eNOS-RFP.

(A) Representative image showing HEK293 cells expressing eNOS-RFP. (B) Representative curves of cytosolic NO^• signals ([NO^•]_cyto) over time in response to 100 μM ATP and 1 mM SNP in control HEK293 cells (black curve and bars, N =5) and HEK293 cells co-expressing eNOS-RFP (red curve and bars, N =3). Left Bars show maximal changes of G-geNOp fluorescence in response to ATP; ***p=0.0005 vs HEK293. Right bars show maximal G-geNOps signals in response to SNP, p=0.9386. (C) Mitochondrial Ca²⁺ signals in HEK293 cells under control conditions (Scrambled siRNA, grey curve and white columns, N =8), in cells treated with siRNA against EMRE and MCU (siEMRE/siMCU, red curve and red columns, N =8) or against MICU1 (siMICU1, black curve and columns, N =7). Cells were stimulated with 100 μM ATP in EGTA (1 mM). Left columns represent basal 4mtD3cpV ratio values; p=0.4088 (siEMRE/siMCU) vs Scrambled siRNA; ***p=0.0004 (siMICU1) vs Scrambled siRNA. Right columns show mean values of maximal 4mtD3cpV ratio signals; *p=0.0145 (siEMRE/siMCU) vs Scrambled siRNA; *p=0.0152 (siMICU1) vs Scrambled siRNA. (D) Representative cytosolic Ca²⁺ signals of fura-2/am loaded HEK293 cells treated with scrambled siRNA (Scrambled siRNA, grey curve and white columns, N =3) siRNA against EMRE and MCU (siEMRE/siMCU, red curve and red columns, N =3) or MICU1 (siMICU1, black curve and columns, N =4). Cells were stimulated with 100 μM ATP in EGTA (1 mM). Columns represent mean values of maximal fura-2 ratio signals; p=0.6842 (siEMRE/siMCU) vs Scrambled siRNA; p=0.4253 (siMICU1) vs Scrambled siRNA. (E) Representative cytosolic NO^• signals eNOS-RFP expressing HEK293 cells treated with scrambled siRNA (Scrambled siRNA, grey curve and white columns, N =9) siRNA against EMRE and MCU (siEMRE/siMCU, red curve and red columns, N =11) or MICU1 (siMICU1, black curve and columns, N =8). Cells were stimulated with 100 μM ATP in EGTA (1 mM). Columns represent mean values of maximal G-geNOp signals; ***p=0.0005 (siEMRE/siMCU) vs Scrambled siRNA; p=0.0829 (siMICU1) vs Scrambled siRNA.