Mitochondria control functional CaV1.2 expression in smooth muscle cells of cerebral arteries

Abstract

Rationale: Physiological functions of mitochondria in contractile arterial myocytes are poorly understood. Mitochondria can uptake calcium (Ca(2+)), but intracellular Ca(2+) signals that regulate mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) and physiological functions of changes in [Ca(2+)](mito) in arterial myocytes are unclear.

Objective: To identify Ca(2+) signals that regulate [Ca(2+)](mito), examine the significance of changes in [Ca(2+)](mito), and test the hypothesis that [Ca(2+)](mito) controls functional ion channel transcription in myocytes of resistance-size cerebral arteries.

Methods and results: Endothelin (ET)-1 activated Ca(2+) waves and elevated global Ca(2+) concentration ([Ca(2+)](i)) via inositol 1,4,5-trisphosphate receptor (IP(3)R) activation. IP(3)R-mediated sarcoplasmic reticulum (SR) Ca(2+) release increased [Ca(2+)](mito) and induced mitochondrial depolarization, which stimulated mitochondrial reactive oxygen species (mitoROS) generation that elevated cytosolic ROS. In contrast, a global [Ca(2+)](i) elevation did not alter [Ca(2+)](mito), mitochondrial potential, or mitoROS generation. ET-1 stimulated nuclear translocation of nuclear factor (NF)-kappaB p50 subunit and ET-1-induced IP(3)R-mediated mitoROS elevated NF-kappaB-dependent transcriptional activity. ET-1 elevated voltage-dependent Ca(2+) (Ca(V)1.2) channel expression, leading to an increase in both pressure (myogenic tone)- and depolarization-induced vasoconstriction. Baseline Ca(V)1.2 expression and the ET-1-induced elevation in Ca(V)1.2 expression were both reduced by IP(3)R inhibition, mitochondrial electron transport chain block, antioxidant treatment, and NF-kappaB subunit knockdown, leading to vasodilation.

Conclusions: IP(3)R-mediated SR Ca(2+) release elevates [Ca(2+)](mito), which induces mitoROS generation. MitoROS activate NF-kappaB, which stimulates Ca(V)1.2 channel transcription. Thus, mitochondria sense IP(3)R-mediated SR Ca(2+) release to control NF-kappaB-dependent Ca(V)1.2 channel expression in arterial myocytes, thereby modulating arterial contractility.

Figure 1

ET-1 regulates local and global Ca²⁺ signals in arterial myocytes. A, Confocal images illustrating average fluo-4 fluorescence in myocytes in the same artery in control and ET-1 (10 nmol/L). White boxes illustrate locations where sparks occurred during 10 seconds of imaging. Colored boxes illustrate locations from where normalized fluorescence (F/F₀) over time traces shown in C were determined. B, Two representative Ca²⁺ sparks that occurred at locations labeled in A for each condition. C, F/F₀ over time traces illustrate ET-1-induced Ca²⁺ wave activation. D, Mean data (n=7 for each condition). P<0.05: * vs control; # vs ET-1.

Figure 2

ET-1-induced IP₃R-mediated SR Ca²⁺ release elevates [Ca²⁺]_mito in arterial myocytes. A, Confocal images illustrate colocalization of punctate 2mt8CG2 fluorescence with MitoTracker Orange in a myocyte. Colocalization quantified using weighted colocalization was 94.8±0.8% (n=10, p<0.001). B, Confocal images of 2mt8CG2 fluorescence in myocytes in the same area of a cerebral artery in control and ET-1. Scale bars=10 µm. C, Mean data for ionomycin (10 µmol/L, n=7), ET-1 (n=8), thapsigargin (100 nmol/L, n=5), ET-1+thapsigargin (n=6), XeC (20 µmol/L, n=5), ET-1+XeC (n=6), Ru360 (10 µmol/L, n=4), ET-1+Ru360 (n=4), CCCP (10 µmol/L, n=4), and 60 mmol/L K⁺ (n=6). ET-1 concentration was 100 nmol/L in all experiments. Thapsigargin was applied 15 minutes prior to ET-1, a time course sufficient to deplete SR Ca²⁺ load . P<0.05: * vs control; # vs ET-1.

Figure 3

ET-1-induced IP₃R-mediated SR Ca²⁺ release depolarizes mitochondria in arterial myocytes. A, ET-1 caused reproducible mitochondrial depolarization which was blocked by XeC (20 µmol/L). B, Concentration-dependent mitochondrial depolarization by ET-1 (n=4–7). C, Mean data for ET-1 (n=37), second ET-1 application (n=12), thapsigargin (100 nmol/L, n=18), ET-1+thapsigargin (n=18), XeC (20 µmol/L, n=12), ET-1+XeC (n=12), 60 mmol/L K⁺ (n=10), and CCCP (10 µmol/L, n= 65). ET-1 concentration was 30 nmol/L in all experiments. P<0.05: * vs control; # vs ET-1.

Figure 4

ET-1-induced IP₃R-mediated SR Ca²⁺ release elevates mitoROS generation, leading to an increase in cytosolic ROS in arterial myocytes. A, Confocal images illustrating mt-cpYFP and MitoTracker Orange fluorescence in the same myocyte. Colocalization quantified using weighted colocalization was 94.7±1.2% (n=10, p<0.001). Scale bar=10 µm. B, Mean mt-cpYFP fluorescence changes in myocytes of intact arteries. ET-1, XeC (20 µmol/L), ET-1+XeC, Ru360 (10 µmol/L), ET-1+Ru360, rotenone (10 µmol/L), ET-1+rotenone, CCCP (10 µmol/L), ET-1+CCCP, CCCP (1 nmol/L), and 60 mmol/L K⁺. C, Mean HyPer-CYTO fluorescence. H₂O₂ (100 µmol/L), ET-1 (endothelium-intact), ET-1 (endothelium-denuded), rotenone (10 µmol/L), ET-1+rotenone, MnTMPyP (10 µmol/L), and ET-1+MnTMPyP. ET-1 concentration was 100 nmol/L in all experiments. n=5 for each condition. P<0.05: * vs control; # vs ET-1.

Figure 5

ET-1 stimulates p50 nuclear translocation and NF-κB-dependent transcription through SR Ca²⁺ release and mitoROS elevation in arterial myocytes. A, Immunofluorescence images of myocytes in arteries illustrating YOYO-1 (nuclear stain, green), p50 (red), overlay, and DIC (lumenally-inserted rectangular glass cannula can be seen). B, Enlarged images indicated by boxes in A illustrate ET-1-induced elevation in p50 and YOYO-1 pixel colocalization (purple). Scale bars=20 µm. C, Mean data (n=10 for each). D, Average NF-κB-p-Luc luciferase activity with TNF-α (100 ng/ml, n=4), ET-1 (n= 5), thapsigargin (100 nmol/L, n=5), ET-1+thapsigargin (n=5), XeC (20 µmol/L, n=5), ET-1+XeC (n=5), rotenone (1 µmol/L, n=5), ET-1+rotenone (n=5), MnTMPyP (10 µmol/L, n=4), ET-1+MnTMPyP (n=4), H₂O₂ (100 µmol/L, n=4), and H₂O₂+rotenone (n=4). ET-1 concentration was 100 nmol/L in all experiments. P<0.05: * vs control; # vs ET-1; § vs rotenone or MnTMPyP.

Figure 6

ET-1-induced IP₃R-mediated SR Ca²⁺ release and mitoROS elevation stimulate Ca_V1.2 expression in cerebral arteries. A, ET-1 elevated mean Ca_V1.2 mRNA (n=7). B, Western blot indicating that XeC (20 µmol/L) blocked ET-1-induced elevation in Ca_V1.2 expression. C, Mean data for ET-1 (n=12), XeC (20 µmol/L, n=7), ET-1+XeC (n=7), rotenone (1µmol/L, n=6), ET-1+rotenone (n=6), MnTMPyP (10 µmol/L, n=5), ET-1+MnTMPyP (n=5), H₂O₂ (100 µmol/L, n=5), and H₂O₂+rotenone (n=5). ET-1 concentration was 10 nmol/L in all experiments. P<0.05: * vs untreated; # vs ET-1; § vs rotenone or MnTMPyP.

Figure 7

NF-κB controls basal and ET-1-induced elevation in functional Ca_V1.2 expression in cerebral arteries. A, Western blot illustrating effects of p105siRNAs (10 µg/ml each) on basal and ET-1-induced Ca_V1.2 expression. B, Mean data. n=5 for each. C, Representative traces illustrating diameter in arteries pressurized to 60 mm Hg. Nimodipine (1 µmol/L) fully dilated p105scrm- and p105siRNAs-treated arteries. D, Mean myogenic tone at 60 mm Hg, in 60 mmol/L K⁺ at 60 mm Hg, and nimodipine at 60 mm Hg. n=5–6 for each. ET-1 concentration was 10 nmol/L in all experiments. P<0.05: * vs p105scrm; # vs p105siRNAs.

Figure 8

Proposed signaling pathways that control basal and ET-1-induced Ca_V1.2 expression in cerebral artery myocytes. Δψ_m indicates mitochondrial potential.