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. 2017 Jul 15;6(7):e005812.
doi: 10.1161/JAHA.117.005812.

Enhanced Mitochondrial Transient Receptor Potential Channel, Canonical Type 3-Mediated Calcium Handling in the Vasculature From Hypertensive Rats

Bin Wang  1 , Shiqiang Xiong  1 , Shaoyang Lin  1 , Weijie Xia  1 , Qiang Li  1 , Zhigang Zhao  1 , Xing Wei  1 , Zongshi Lu  1 , Xiao Wei  1 , Peng Gao  1 , Daoyan Liu  2 , Zhiming Zhu  2
Affiliations

Enhanced Mitochondrial Transient Receptor Potential Channel, Canonical Type 3-Mediated Calcium Handling in the Vasculature From Hypertensive Rats

Bin Wang et al. J Am Heart Assoc. .

Abstract

Background: Mitochondrial Ca2+ homeostasis is fundamental to the regulation of mitochondrial reactive oxygen species (ROS) generation and adenosine triphosphate production. Recently, transient receptor potential channel, canonical type 3 (TRPC3), has been shown to localize to the mitochondria and to play a role in maintaining mitochondrial calcium homeostasis. Inhibition of TRPC3 attenuates vascular calcium influx in spontaneously hypertensive rats (SHRs). However, it remains elusive whether mitochondrial TRPC3 participates in hypertension by increasing mitochondrial calcium handling and ROS production.

Methods and results: In this study we demonstrated increased TRPC3 expression in purified mitochondria in the vasculature from SHRs, which facilitates enhanced mitochondrial calcium uptake and ROS generation compared with Wistar-Kyoto rats. Furthermore, inhibition of TRPC3 by its specific inhibitor, Pyr3, significantly decreased the vascular mitochondrial ROS production and H2O2 synthesis and increased adenosine triphosphate content. Administration of telmisartan can improve these abnormalities. This beneficial effect was associated with improvement of the mitochondrial respiratory function through recovering the activity of pyruvate dehydrogenase in the vasculature of SHRs. In vivo, chronic administration of telmisartan suppressed TRPC3-mediated excessive mitochondrial ROS generation and vasoconstriction in the vasculature of SHRs. More importantly, TRPC3 knockout mice exhibited significantly ameliorated hypertension through reduction of angiotensin II-induced mitochondrial ROS generation.

Conclusions: Together, we give experimental evidence for a potential mechanism by which enhanced TRPC3 activity at the cytoplasmic and mitochondrial levels contributes to redox signaling and calcium dysregulation in the vasculature from SHRs. Angiotensin II or telmisartan can regulate [Ca2+]mito, ROS production, and mitochondrial energy metabolism through targeting TRPC3.

Keywords: mitochondria; pyruvate dehydrogenase; telmisartan; transient receptor potential channel, canonical type 3; vasoconstriction.

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Figures

Figure 1
Figure 1
The whole‐cell and mitochondrial TRPC3 expression levels in primary VSMCs from WKY rats and SHRs. A, TRPC3 immunoreactivity was detected by the anti‐TRPC3 antibody (96 kDa) in homogenates of primary VSMCs from SHRs. B and C, The TRPC3 expression levels in cell lysates and purified mitochondria of primarily cultured VSMCs were analyzed by Western blotting, using VDAC protein as a mitochondrial control and GAPDH as an internal reference in (B). D indicates DMSO treatment. Three Western blot bands were quantified and analyzed in the lower panels (C). *P<0.05, **P<0.01. SHR indicates spontaneously hypertensive rats; Telmi, telmisartan; TRPC3, transient receptor potential channel, canonical type 3; VDAC, voltage‐dependent anion‐selective channel; VSMC, vascular smooth muscle cells; WKY, Wistar‐Kyoto.
Figure 2
Figure 2
The effects of TRPC3 on mitochondrial calcium uptake in VSMCs from WKY rats and SHRs. A and B, Mitochondrial Ca2+ concentrations ([Ca2+]mito) in the VSMCs from WKY rats and SHRs treated with 200 μmol/L ATP in either a 0.3 mmol/L extracellular Ca2+ solution (A) or a calcium‐free (B) extracellular solution after preincubation with 10 μmol/L Pyr3 or telmisartan. [Ca2+]mito was monitored based on the fluorescence of R552 nm/R581 nm. N=9 to 12 per group. *P<0.05, **P<0.01, ***P<0.0001. C and D, [Ca2+]mito in the VSMCs from WKY rats and SHRs treated with 10 μmol/L histamine in a 0.3 mmol/L extracellular Ca2+ solution (C) or a calcium‐free (D) extracellular solution after preincubation with 10 μmol/L Pyr3 or telmisartan. N=12 to 15 per group. *P<0.05, **P<0.01, ***P<0.0001. E and F, [Ca2+]mito changes in VSMCs permeabilized with 10 μmol/L digitonin and challenged with the cytosolic loading of 300 μmol/L (E) or 75 μmol/L (F) Ca2+. N=9 to 12 per group. *P<0.05, **P<0.01, ***P<0.0001. G and H, The VSMCs from SHRs were transfected with TRPC3 siRNA and negative control siRNA, followed by the addition of telmisartan, after which the cells were permeabilized with digitonin and [Ca2+]mito was monitored with cytosolic loading of 300 μmol/L (G) or 75 μmol/L (H) Ca2+. N=9 to 12 per group. **P<0.01, ***P<0.0001 vs siNC DMSO in (G); *P<0.05, **P<0.01 vs siNC DMSO as shown in (H). I and J, The VSMCs from WKY rats were transfected with pcDNA‐TRPC3myc and pcDNA3.1, respectively, followed by the addition of telmisartan, after which the cells were permeabilized with digitonin and [Ca2+]mito was monitored with cytosolic loading of either 300 μmol/L (I) or 75 μmol/L (J) Ca2+. N=9 to 12 per group. *P<0.05. Pyr3 indicates specific inhibitor of TRPC3; SHR, spontaneously hypertensive rats; siNC, negative control small interfering RNA; Telmi, telmisartan; TRPC3, transient receptor potential channel, canonical type 3; VSMC, vascular smooth muscle cells; WKY, Wistar‐Kyoto.
Figure 3
Figure 3
Mitochondria participate in calcium handling and vasoconstriction in the vasculature from WKY rats and SHRs. A and B, VSMCs from WKY rats and SHRs were pretreated with 10 μmol/L of the mitochondria‐target reactive oxygen species (ROS) scavenger mitoTEMPO, 10 μmol/L of telmisartan or both for 24 hours, and 1 μmol/L of thapsigargin (TG) was then added to detect the store‐operated calcium entry (SOCE). ***P<0.0001 vs WKY; **P<0.01 vs SHR control; # P<0.0001 vs SHR control. C and D, The effect of 50 μmol/L of mitoTEMPO and 10 μmol/L of the TRPC3 inhibitor Pyr3 incubation on U46619‐ and phenylephrine (PE)‐induced vasoconstriction in mesenteric arteries from SHR, compared with WKY rats. N=6 to 8 per group. *P<0.05, **P<0.01, ***P<0.0001. Pyr3 indicates specific inhibitor of TRPC3; SHR, spontaneously hypertensive rats; Telmi, telmisartan; TRPC3, transient receptor potential channel, canonical type 3; VSMC, vascular smooth muscle cells; WKY, Wistar‐Kyoto.
Figure 4
Figure 4
The effects of TRPC3 inhibition on mitochondrial function and ROS production in the vasculature of SHRs. A, Mitochondrial oxygen consumption was measured in primary VSMCs from aortas of SHRs and WKY rats using Oxygraph‐2k high‐resolution respirometry. Routine values indicate respiration in the original state; CI+IIL eak, respiration on Complex I plus Complex II substrates to compensate for a proton leak; CIOXPHOS, Complex I‐dependent oxidative phosphorylation; CIIOXPHOS, Complex II‐dependent oxidative phosphorylation; CI+IIOXPHOS, oxidative phosphorylation providing Complex I and Complex II substrates; CI+IIETS, noncoupled respiration with Complex I and Complex II substrates (ie, the maximum capacity of the ETS state); CIIETS, noncoupled Complex II‐dependent respiration; CIVETS, noncoupled Complex IV‐dependent respiration. N=6 to 10 per group. *P<0.05 vs WKY; # P<0.05 vs SHR. The right panel is the representative oxygen graph of the left panel. Dig, digitonin 10 μg per 106 cells; G+M, glutamate 5 mmol/L, malate 2 mmol/L; ADP, 5 mmol/L; SUC, succinate 10 mmol/L; FCCP, 1 to 1.5 μmol/L; ROT, rotenone 0.5 μmol/L; AS+TM, ascorbate sodium 2 mmol/L, TMPD 0.5 mmol/L. B, The effect of telmisartan or Pyr3 on cellular ROS levels in VSMCs as determined by DHE staining. N=6 per group, **P<0.01 vs WKY; ***P<0.0001 vs SHR; # P<0.01 vs SHR. C, The effect of telmisartan or Pyr3 on mitochondrial ROS levels in VSMCs as determined by MitoSOX staining. N=6 per group, ***P<0.0001 vs WKY; # P<0.0001 vs SHR. D, The effect of telmisartan or Pyr3 on cellular H2O2 levels in VSMCs. N=4 per group. **P<0.01 vs WKY; # P<0.01 vs SHR. E, The effect of telmisartan or Pyr3 on cellular ATP levels in VSMCs. N=4 per group. *P<0.05 vs WKY; ***P<0.0001 vs SHR; # P<0.05 vs SHR. F, The expression levels of phospho‐PDHE1α (p‐PDHE1α) and total PDHE1α (t‐PDHE1α) in VSMCs from WKY and SHR, after treatment with either telmisartan or Pyr3, were analyzed by western blotting. T indicates telmisartan; P indicates Pyr3. Three Western blot bands were quantified, and the gray values are analyzed in the right panel. *P<0.05 vs WKY DMSO; **P<0.01 vs SHR DMSO, # P<0.05 vs SHR DMSO. DHE indicates dihydroethidium; MitoSOX, mitochondrial superoxide indicator; Pyr3, specific inhibitor of TRPC3; ROS, reactive oxygen species; SHR, spontaneously hypertensive rats; Telmi, telmisartan; TMPD, (N,N,N',N'‐Tetramethyl‐p‐phenylenediamine dihydrochloride); TRPC3, transient receptor potential channel, canonical type 3; VSMC, vascular smooth muscle cells; WKY, Wistar‐Kyoto.
Figure 5
Figure 5
The effects of chronic oral telmisartan intervention on mitochondrial and vascular function in the vasculature from SHRs. A, The TRPC3 expression levels in isolated mitochondria from the aortic tissues of WKY rats and SHRs, in the presence and absence of telmisartan treatment, as analyzed by Western blotting using voltage‐dependent anion‐selective channel protein as a mitochondrial control. Western blot bands were quantified and analyzed in the below panel. N=4 per group. *P<0.05 vs WKY‐ND (indicates WKY rats with ND treatment); # P<0.05 vs SHR‐ND. B, Representative images of DHE and MitoSOX staining. The relative fluorescence intensity of the aortas from WKY rats and SHRs were quantified in the presence and absence of telmisartan treatment. SHR‐Telmi indicates SHRs after long‐term administration of telmisartan. N=6 per group. ***P<0.0001 vs WKY‐ND; # P<0.0001 vs SHR‐ND. C, The effects of chronic telmisartan intervention on mitochondrial complex I and complex II activities of aorta isolated from WKY rats and SHRs. *P<0.05 vs WKY; # P<0.05 vs SHR. D, The effects of chronic oral telmisartan intervention on U46619‐ and PE‐induced vasoconstriction in MAs from SHRs. N=6 to 8 per group. *P<0.05, **P<0.01, ***P<0.0001. E, The effects of mitoTEMPO and Pyr3 administration on U46619‐ and PE‐induced vasoconstriction in the MAs of SHRs treated with telmisartan. Isolated MAs were preincubated with 50 μmol/L of mitoTEMPO or 10 μmol/L of TRPC3 inhibitor Pyr3 for 30 minutes before experiments. N=6 to 8 per group. *P<0.05, ***P<0.0001. DHE indicates dihydroethidium; MA, mesenteric artery; MitoSOX, mitochondrial superoxide indicator; Pyr3, specific inhibitor of TRPC3; SHR, spontaneously hypertensive rats; Telmi, telmisartan; TRPC3, transient receptor potential channel, canonical type 3; VDAC, voltage‐dependent anion‐selective channel; WKY, Wistar‐Kyoto.
Figure 6
Figure 6
The effects of TRPC3 on angiotensin II (Ang II)‐induced vascular mitochondrial dysfunctions. A, The effects of Pyr3 and mitoTEMPO administration on vascular constrictions of MAs in Ang II‐infused hypertensive mice. N=6 to 8 per group. *P<0.05, **P<0.01, ***P<0.0001. B and C, [Ca2+]mito changes in permeabilized primary VSMCs from WT and Trpc3 −/− mice, pretreated with 200 nmol/L Ang II and challenged with the cytosolic loading of 300 μmol/L (B) or 75 μmol/L (C) Ca2+. N=9 per group. ***P<0.0001. D, Representative images of DHE‐ and MitoSOX‐stained aortic sections from Ang II‐infused WT and Trpc3 −/− mice. N=7 per group. ***P<0.0001. E, Immunoblotting of the expression levels of TRPC3, p‐PDHE1α and t‐PDHE1α in aortic tissues isolated from WT and Trpc3 −/− mice, using GAPDH as a loading control. Four Western blot bands in each group were quantified and analyzed in the right panels. *P<0.05, ***P<0.0001. DHE indicates dihydroethidium; MA, mesenteric artery; MitoSOX, mitochondrial superoxide indicator; Pyr3, specific inhibitor of TRPC3; TRPC3, transient receptor potential channel, canonical type 3; VSMC, vascular smooth muscle cells; WT, wild type.
Figure 7
Figure 7
The effects of TRPC3 on angiotensin II (Ang II)–induced hypertension and vascular reactivity. A and B, The effects of TRPC3 on the tail‐cuff SBP (A) and the 24‐hour ambulatory blood pressure levels (B) in Ang II–infused hypertensive mice. N=5 per group. A, ***P<0.0001, # P<0.0001, vs corresponding tail‐cuff SBP levels at baseline via repeated measures ANOVA within each group; **P<0.01 vs WT‐Ang II (indicates WT mice with Ang II infusion) at the same time point. B, **P<0.01, ***P<0.0001. C, The effects of TRPC3 on vascular constrictions in Ang II‐infused hypertensive mice. N=7 to 8 per group. *P<0.05, **P<0.01, ***P<0.0001. D and E, The expression levels of phosphorylated and total MYPT1 and MLC2 in aortic tissues from these groups were detected using Western blot (D). E, Quantification of Western blot bands presented as the amount of phosphorylated/total levels. N=4 per group. *P<0.05, **P<0.01, ***P<0.0001. F, Representative images of the immunofluorescence staining of TRPC3, p‐MLC2 and DAPI using aortic sections from Ang II‐infused mice. Quantification of the intensities in each graph was presented in the right 2 panels. N=6 per group. **P<0.01, ***P<0.0001. DAPI, 4′,6‐diamidino‐2‐phenylindole; DBP, diastolic blood pressure; MLC2, myosin light chain 2; MYPT1, myosin phosphatase targeting protein; SBP, systolic blood pressure; transient receptor potential channel, canonical type 3; WT, wild type.
Figure 8
Figure 8
A schematic illustration depicts the mechanism by which increased mitochondrial TRPC3 contributes to hypertension. SHR or Ang II–infusion hypertension have increased mitochondrial TRPC3 expression levels, which induced an enhanced mitochondrial Ca2+ uptake, impaired mitochondrial respiratory functions, and increased ROS production. These abnormalities lead to enhanced ROS production, increased [Ca2+]c, and activation of the RhoA/Rho kinase pathway in hypertension. Inhibition of TRPC3 by telmisartan or Pyr3 ameliorated mitochondrial Ca2+ overload, restored PDH activities, and improved mitochondrial respiratory functions. Targeting TRPC3 may become a new strategy to antagonizing hypertension. Ang indicates angiotensin; PDH, pyruvate dehydrogenase; Pyr3, specific inhibitor of TRPC3; ROS, reactive oxygen species; SHR, spontaneously hypertensive rats; TRPC3, transient receptor potential channel, canonical type 3; VSMC, vascular smooth muscle cells.
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