-
Microgravity simulated by HU resulted in a significantly lower soleus muscle mass (P < 0.001). Soleus muscle-to-body mass ratios decreased significantly (P < 0.001) in the HU and HU + MT rats, which confirmed the efficacy of simulated microgravity by HU. The data are summarized in Table 1.
Table 1. Body mass (g), soleus mass (mg), and soleus: body mass ratio (mg/g) of rats from the control, mitoTEMPO-treated control, hindlimb unweighting, and mitotempo-treated hindlimbunweighting groups (n = 8 in each group)
Group Initial mass (g) Final mass (g) Soleus mass (mg) Soleus: body mass (mg/g) CON 198.60 ± 1.45 345.85 ± 10.47 134.54 ± 3.62 0.39 ± 0.01 HU 196.73 ± 2.07 336.79 ± 10.74 65.28 ± 1.85*** 0.19 ± 0.01*** CON + MT 199.55 ± 2.67 338.57 ± 8.17 138.48 ± 2.82 0.41 ± 0.01 HU + MT 199.87 ± 1.80 341.89 ± 11.26 74.29 ± 2.12*** 0.22 ± 0.01*** Note. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON+MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. ***P < 0.001 vs. control. -
Cytoplasmic, mitochondrial, and SR Ca2+ distribution and content in cerebral VSMCs are shown in Figure 1. After HU, cytoplasmic Ca2+ content significantly increased (P < 0.001) (Figure 1A, 1D) with a significant decrease of Ca2+ in mitochondria (Figure 1B, 1E) and the SR (Figure 1C, 1F) (P < 0.001) compared with CON rat cerebral VSMCs. The chronic treatment with mitoTEMPO restored cytoplasmic (P < 0.001), mitochondrial (P < 0.01), and SR (P < 0.001) Ca2+ distribution and content in HU + MT rat cerebral VSMCs.
Figure 1. The effects of mitoTEMPO on cytoplasmic (A, D), mitochondrial (B, E), and sarcoplasmic reticulum (C, F) Ca2+ distribution in rat cerebral vascular smooth muscle cells. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. **P < 0.01 and ***P < 0.001.
-
We analyzed mitochondrial fusion and fission to investigate the mechanism of cytoplasmic, mitochondrial, and SR Ca2+ redistribution (Figure 2). TEM (Figure 2A) showed more long and narrow mitochondria in the HU rat cerebral VSMCs, while more elliptical mitochondria were observed in the CON, CON + MT, and HU + MT rat cerebral VSMCs (mitochondria are marked by white arrows), indicating that HU enhanced mitochondrial fission and that treatment with mitoTEMPO attenuates mitochondrial fission. The MFN1/2 protein and mRNA levels (Figure 2B, 2F and Figure 2C, 2G) in HU rat cerebral VSMCs decreased significantly compared to those in the CON rats (P < 0.001). The DRP1/FIS1 protein and mRNA levels (Figure 2D, 2H and Figure 2E, 2I) were significantly higher in HU rat cerebral arteries than those in the CON rats (P < 0.01 for protein and P < 0.001 for mRNA). Chronic treatment with mitoTEMPO significantly upregulated the expression of MFN1/2 (P < 0.05 for MFN1 mRNA; P < 0.001 for MFN1/2 protein and MFN2 mRNA) but decreased the expression of FIS1/DRP1 (P < 0.01 for protein and P < 0.001 for mRNA) compared to the HU.
Figure 2. Effects of mitoTEMPO on mitochondrial fission and fusion (A) and the protein and mRNA levels of mitofusion 1 (MFN1) (B, F) and mitofusion 2 (MFN2) (C, G), dynamin-related protein 1 (DRP1) (D, H), and fission protein 1 (FIS1) (E, I) in rat cerebral vascular smooth muscle cells. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. *P < 0.05, **P < 0.01, and ***P < 0.001.
-
To investigate whether cytoplasmic Ca2+ redistribution was IP3R-dependent, we determined the abundance of the IP3R in HU rat cerebral arteries (Figure 3). IP3R protein (P < 0.001) and mRNA (P < 0.001) levels increased significantly (Figure 3A and 3B) in HU rat cerebral arteries compared to the CON. Immunohistochemical staining revealed that the IP3R was more positive in HU rat cerebral VSMCs than that in the CON (Figure 3C). Chronic treatment with mitoTEMPO partially restored the enhanced expression of the IP3R after HU.
Figure 3. The effects of mitoTEMPO on protein (A) and mRNA (B) levels of the inositol 1,4,5-trisphosphate receptor (IP3R) and immunohistochemistry for IP3R (C) in rat cerebral vascular smooth muscle cells. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. ***P < 0.001.
-
To investigate whether cytoplasmic Ca2+ redistribution induced by mitochondrial oxidative stress was associated with changes in plasma membrane K+ channels, we analyzed total K+ current, current densities, and open probabilities (Po) of the KV and BKCa channels (Figure 4). Total K+ current decreased (Figure 4A), whereas current densities and open probabilities of KV (Figure 4B and 4C) and BKCa (Figure 4D and 4E) decreased and increased, respectively, compared to control rats, in HU rat cerebral VSMCs, which were restored by chronic treatment with mitoTEMPO.
Figure 4. Effects of mitoTEMPO on total K+ current (A), current activation, and opening probabilities of voltage-gated potassium (KV) channels (B, C), and Ca2+-activated K+ (BKCa) channels (D, E) in rat cerebral vascular smooth muscle cells. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. *P < 0.05, **P < 0.01, and ***P < 0.001.
-
To investigate whether the changes in cytoplasmic Ca2+ were associated with cerebrovascular contraction, we studied the cerebrovascular contraction to vasoconstrictors (Figure 5). Cumulative increases in KCl and 5-HT concentrations induced concentration-dependent vasoconstriction in basilar arteries from the four groups. Three-week HU significantly enhanced the maximal contractile responses to KCl and 5-HT in rat basilar arteries (P < 0.05) compared to the CON, which was attenuated by the chronic mitoTEMPO treatment (P < 0.05).
Figure 5. Effects of mitoTEMPO on vasoconstriction to cumulative KCl or 5-hydroxytryptamine (5-HT) in rat basilar arteries. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. *P < 0.05 for HU vs. CON, ▲P < 0.05 for HU vs. HU + MT.
doi: 10.3967/bes2021.001
Mitochondrial Oxidative Stress Enhances Vasoconstriction by Altering Calcium Homeostasis in Cerebrovascular Smooth Muscle Cells under Simulated Microgravity
-
Abstract:
Objective Exposure to microgravity results in postflight cardiovascular deconditioning in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been reported during this process. To elucidate the mechanism for this condition, we investigated whether mitochondrial oxidative stress regulates calcium homeostasis and vasoconstriction in hindlimb unweighted (HU) rat cerebral arteries. Methods Three-week HU was used to simulate microgravity in rats. The contractile responses to vasoconstrictors, mitochondrial fission/fusion, Ca2+ distribution, inositol 1,4,5-trisphosphate receptor (IP3R) abundance, and the activities of voltage-gated K+ channels (KV) and Ca2+-activated K+ channels (BKCa) were examined in rat cerebral vascular smooth muscle cells (VSMCs). Results An increase of cytoplasmic Ca2+ and a decrease of mitochondrial/sarcoplasmic reticulum (SR) Ca2+ were observed in HU rat cerebral VSMCs. The abundance of fusion proteins (mitofusin 1/2 [MFN1/2]) and fission proteins (dynamin-related protein 1 [DRP1] and fission-mitochondrial 1 [FIS1]) was significantly downregulated and upregulated, respectively in HU rat cerebral VSMCs. The cerebrovascular contractile responses to vasoconstrictors were enhanced in HU rats compared to control rats, and IP3R protein/mRNA levels were significantly upregulated. The current densities and open probabilities of KV and BKCa decreased and increased, respectively. Treatment with the mitochondrial-targeted antioxidant mitoTEMPO attenuated mitochondrial fission by upregulating MFN1/2 and downregulating DRP1/FIS1. It also decreased IP3R expression levels and restored the activities of the KV and BKCa channels. MitoTEMPO restored the Ca2+ distribution in VSMCs and attenuated the enhanced vasoconstriction in HU rat cerebral arteries. Conclusion The present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity. -
Key words:
- Microgravity /
- Mitochondrial oxidative stress /
- Calcium homeostasis /
- Vasoconstriction
注释: -
Figure 1. The effects of mitoTEMPO on cytoplasmic (A, D), mitochondrial (B, E), and sarcoplasmic reticulum (C, F) Ca2+ distribution in rat cerebral vascular smooth muscle cells. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. **P < 0.01 and ***P < 0.001.
Figure 2. Effects of mitoTEMPO on mitochondrial fission and fusion (A) and the protein and mRNA levels of mitofusion 1 (MFN1) (B, F) and mitofusion 2 (MFN2) (C, G), dynamin-related protein 1 (DRP1) (D, H), and fission protein 1 (FIS1) (E, I) in rat cerebral vascular smooth muscle cells. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 3. The effects of mitoTEMPO on protein (A) and mRNA (B) levels of the inositol 1,4,5-trisphosphate receptor (IP3R) and immunohistochemistry for IP3R (C) in rat cerebral vascular smooth muscle cells. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. ***P < 0.001.
Figure 4. Effects of mitoTEMPO on total K+ current (A), current activation, and opening probabilities of voltage-gated potassium (KV) channels (B, C), and Ca2+-activated K+ (BKCa) channels (D, E) in rat cerebral vascular smooth muscle cells. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 5. Effects of mitoTEMPO on vasoconstriction to cumulative KCl or 5-hydroxytryptamine (5-HT) in rat basilar arteries. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON + MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. *P < 0.05 for HU vs. CON, ▲P < 0.05 for HU vs. HU + MT.
Table 1. Body mass (g), soleus mass (mg), and soleus: body mass ratio (mg/g) of rats from the control, mitoTEMPO-treated control, hindlimb unweighting, and mitotempo-treated hindlimbunweighting groups (n = 8 in each group)
Group Initial mass (g) Final mass (g) Soleus mass (mg) Soleus: body mass (mg/g) CON 198.60 ± 1.45 345.85 ± 10.47 134.54 ± 3.62 0.39 ± 0.01 HU 196.73 ± 2.07 336.79 ± 10.74 65.28 ± 1.85*** 0.19 ± 0.01*** CON + MT 199.55 ± 2.67 338.57 ± 8.17 138.48 ± 2.82 0.41 ± 0.01 HU + MT 199.87 ± 1.80 341.89 ± 11.26 74.29 ± 2.12*** 0.22 ± 0.01*** Note. CON, control; HU, hindlimb unweighting; MT, mitoTEMPO; CON+MT, mitoTEMPO-treated control; HU + MT, mitoTEMPO-treated HU. Values are mean ± standard error. ***P < 0.001 vs. control. -
[1] Convertino VA. Mechanisms of microgravity induced orthostatic intolerance: implications for effective countermeasures. J Gravit Physiol, 2002; 9, 1−13. [2] Hargens AR, Watenpaugh DE. Cardiovascular adaptation to spaceflight. Med Sci Sports Exerc, 1996; 28, 977−82. doi: 10.1097/00005768-199608000-00007 [3] Zhang LF. Vascular adaptation to microgravity: what have we learned? J Appl Physiol, 2001; 91, 2415−30. doi: 10.1152/jappl.2001.91.6.2415 [4] Zhang LF. Region-specific vascular remodeling and its prevention by artificial gravity in weightless environment. Eur J Appl Physiol, 2013; 113, 2873−95. doi: 10.1007/s00421-013-2597-8 [5] Maier JAM, Cialdai F, Monici M, et al. The impact of microgravity and hypergravity on endothelial cells. BioMed Res Int, 2015; 2015, 434803. [6] Zhang R, Bai YG, Lin LJ, et al. Blockade of AT1 receptor partially restores vasoreactivity, NOS expression, and superoxide levels in cerebral and carotid arteries of hindlimb unweighting rats. J Appl Physiol, 2009; 106, 251−8. doi: 10.1152/japplphysiol.01278.2007 [7] Zhang R, Ran HH, Ma J, et al. NAD(P)H oxidase inhibiting with apocynin improved vascular reactivity in tail-suspended hindlimb unweighting rat. J Physiol Biochem, 2012; 68, 99−105. doi: 10.1007/s13105-011-0123-1 [8] Shi F, Wang YC, Zhao TZ, et al. Effects of simulated microgravity on human umbilical vein endothelial cell angiogenesis and role of the PI3K-Akt-eNOS signal pathway. PLoS One, 2012; 7, e40365. doi: 10.1371/journal.pone.0040365 [9] Versari S, Villa A, Bradamante S, et al. Alterations of the actin cytoskeleton and increased nitric oxide synthesis are common features in human primary endothelial cell response to changes in gravity. Biochim Biophys Acta, 2007; 1773, 1645−52. doi: 10.1016/j.bbamcr.2007.05.014 [10] Xie MJ, Ma YG, Gao F, et al. Activation of BKCa channel is associated with increased apoptosis of cerebrovascular smooth muscle cells in simulated microgravity rats. Am J Physiol Cell Physiol, 2010; 298, C1489−500. doi: 10.1152/ajpcell.00474.2009 [11] Xue JH, Zhang LF, Ma J, et al. Differential regulation of L-type Ca2+ channels in cerebral and mesenteric arteries after simulated microgravity in rats and its intervention by standing. Am J Physiol Heart Circ Physiol, 2007; 293, H691−701. doi: 10.1152/ajpheart.01229.2006 [12] Islam MS. Calcium signaling: from basic to bedside. In: Islam M. Calcium Signaling. Springer. 2020, 1-6. [13] Liu ZW, Khalil RA. Evolving mechanisms of vascular smooth muscle contraction highlight key targets in vascular disease. Biochem Pharmacol, 2018; 153, 91−122. doi: 10.1016/j.bcp.2018.02.012 [14] Cioffi DL. Redox regulation of endothelial canonical transient receptor potential channels. Antioxid Redox Signal, 2011; 15, 1567−82. doi: 10.1089/ars.2010.3740 [15] Görlach A, Bertram K, Hudecova S, et al. Calcium and ROS: a mutual interplay. Redox Biol, 2015; 6, 260−71. doi: 10.1016/j.redox.2015.08.010 [16] Zhang X, Yan SM, Zheng HL, et al. A mechanism underlying hypertensive occurrence in the metabolic syndrome: cooperative effect of oxidative stress and calcium accumulation in vascular smooth muscle cells. Horm Metab Res, 2014; 46, 126−32. [17] Hall AR, Burke N, Dongworth RK, et al. Mitochondrial fusion and fission proteins: novel therapeutic targets for combating cardiovascular disease. Br J Pharmacol, 2014; 171, 1890−906. doi: 10.1111/bph.12516 [18] Wu SN, Lu QL, Wang QL, et al. Binding of FUN14 domain containing 1 with inositol 1, 4, 5-trisphosphate receptor in mitochondria-associated endoplasmic reticulum membranes maintains mitochondrial dynamics and function in hearts in Vivo. Circulation, 2017; 136, 2248−66. doi: 10.1161/CIRCULATIONAHA.117.030235 [19] Youle RJ, Van Der Bliek AM. Mitochondrial fission, fusion, and stress. Science, 2012; 337, 1062−5. doi: 10.1126/science.1219855 [20] Yu R, Jin SB, Lendahl U, et al. Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery. EMBO J, 2019; 38, e99748. [21] Zhang R, Ran HH, Cai LL, et al. Simulated microgravity-induced mitochondrial dysfunction in rat cerebral arteries. FASEB J, 2014; 28, 2715−24. doi: 10.1096/fj.13-245654 [22] Zhang R, Ran HH, Peng L, et al. Mitochondrial regulation of NADPH oxidase in hindlimb unweighting rat cerebral arteries. PLoS One, 2014; 9, e95916. doi: 10.1371/journal.pone.0095916 [23] Peng L, Ran HH, Zhang Y, et al. NADPH oxidase accounts for changes in cerebrovascular redox status in hindlimb unweighting rats. Biomed Environ Sci, 2015; 28, 799−807. doi: 10.1016/S0895-3988(15)30110-0 [24] Ren XL, Zhang R, Zhang YY, et al. Nitric oxide synthase activity in the abdominal aorta of rats is decreased after 4 weeks of simulated microgravity. Clin Exp Pharmacol Physiol, 2011; 38, 683−7. doi: 10.1111/j.1440-1681.2011.05565.x [25] Zhang R, Jia GL, Bao JX, et al. Increased vascular cell adhesion molecule-1 was associated with impaired endothelium-dependent relaxation of cerebral and carotid arteries in simulated microgravity rats. J Physiol Sci, 2008; 58, 67−73. doi: 10.2170/physiolsci.RP010707 [26] Zhang R, Jiang M, Zhang JB, et al. Regulation of the cerebrovascular smooth muscle cell phenotype by mitochondrial oxidative injury and endoplasmic reticulum stress in simulated microgravity rats via the PERK-eIF2α-ATF4-CHOP pathway. Biochim Biophys Acta Mol Basis Dis, 2020; 1866, 165799. doi: 10.1016/j.bbadis.2020.165799 [27] Xue JH, Chen LH, Zhao HZ, et al. Differential regulation and recovery of intracellular Ca2+ in cerebral and small mesenteric arterial smooth muscle cells of simulated microgravity rat. PLoS One, 2011; 6, e19775. doi: 10.1371/journal.pone.0019775 [28] Shah VN, Chagot B, Chazin WJ. Calcium-dependent regulation of ion channels. Calcium Bind Proteins, 2006; 1, 203−12. [29] Cheng J, Wen J, Wang N, et al. Ion channels and vascular diseases. Arterioscler Thromb Vasc Biol, 2019; 39, e146−56. [30] Csordás G, Renken C, Várnai P, et al. Structural and functional features and significance of the physical linkage between ER and mitochondria. J Cell Biol, 2006; 174, 915−21. doi: 10.1083/jcb.200604016 [31] Rowland AA, Voeltz GK. Endoplasmic reticulum-mitochondria contacts: function of the junction. Nat Rev Mol Cell Biol, 2012; 13, 607−25. doi: 10.1038/nrm3440 [32] Sterea AM, El Hiani Y. The role of mitochondrial calcium signaling in the pathophysiology of cancer cells. In: Islam M. Calcium Signaling. Springer. 2020, 747-70. [33] De Brito OM, Scorrano L. Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature, 2008; 456, 605−10. doi: 10.1038/nature07534 [34] Chernorudskiy AL, Zito E. Regulation of calcium homeostasis by ER redox: a close-up of the er/mitochondria connection. J Mol Biol, 2017; 429, 620−32. doi: 10.1016/j.jmb.2017.01.017 [35] Eid AH, El-Yazbi AF, Zouein F, et al. Inositol 1, 4, 5-trisphosphate receptors in hypertension. Front Physiol, 2018; 9, 1018. doi: 10.3389/fphys.2018.01018 [36] Finkel T, Menazza S, Holmstrom KM, et al. The ins and outs of mitochondrial calcium. Circ Res, 2015; 116, 1810−9. doi: 10.1161/CIRCRESAHA.116.305484 [37] Vianello A, Casolo V, Petrussa E, et al. The mitochondrial permeability transition pore (PTP)-an example of multiple molecular exaptation? Biochim Biophys Acta Bioenerg, 2012; 1817, 2072−86. doi: 10.1016/j.bbabio.2012.06.620 [38] Feno S, Butera G, Reane DV, et al. Crosstalk between calcium and ROS in pathophysiological conditions. Oxid Med Cell Longev, 2019; 2019, 9324018. [39] Kozlov AV, Lancaster Jr JR, Meszaros AT, et al. Mitochondria-meditated pathways of organ failure upon inflammation. Redox Biol, 2017; 13, 170−81. doi: 10.1016/j.redox.2017.05.017 [40] Avila G, De La Rosa JA, Monsalvo-Villegas A, et al. Ca2+ channels mediate bidirectional signaling between sarcolemma and sarcoplasmic reticulum in muscle cells. Cells, 2019; 9, 55. doi: 10.3390/cells9010055 [41] Bartok A, Weaver D, Golenár T, et al. IP3 receptor isoforms differently regulate ER-mitochondrial contacts and local calcium transfer. Nature Communications, 2019; 10, 3726. doi: 10.1038/s41467-019-11646-3 [42] Balaban RS, Nemoto S, Finkel T. Mitochondria, oxidants, and aging. Cell, 2005; 120, 483−95. doi: 10.1016/j.cell.2005.02.001 [43] Archer SL. Mitochondrial dynamics-mitochondrial fission and fusion in human diseases. N Engl J Med, 2013; 369, 2236−51. doi: 10.1056/NEJMra1215233 [44] Cipolat S, De Brito OM, Dal Zilio B, et al. OPA1 requires mitofusin 1 to promote mitochondrial fusion. Proc Natl Acad Sci USA, 2004; 101, 15927−32. doi: 10.1073/pnas.0407043101 [45] Guerrero-Hernandez A, Sanchez-Vazquez VH, Martinez-Martinez E, et al. Sarco-endoplasmic reticulum calcium release model based on changes in the luminal calcium content. In: Islam M. Calcium Signaling. Springer. 2020, 337-70. [46] Alirol E, James D, Huber D, et al. The mitochondrial fission protein hFis1 requires the endoplasmic reticulum gateway to induce apoptosis. Mol Biol Cell, 2006; 17, 4593−605. doi: 10.1091/mbc.e06-05-0377 [47] Vallese F, Barazzuol L, Maso L, et al. ER-mitochondria calcium transfer, organelle contacts and neurodegenerative diseases. In: Islam M. Calcium Signaling. Springer. 2020, 719-46. [48] Zhang R, Ran HH, Gao YL, et al. Differential vascular cell adhesion molecule-1 expression and superoxide production in simulated microgravity rat vasculature. EXCLI J, 2010; 9, 195−204. [49] Jiang M, Wang HM, Liu ZF, et al. Endoplasmic reticulum stress-dependent activation of iNOS/NO-NF-κB signaling and NLRP3 inflammasome contributes to endothelial inflammation and apoptosis associated with microgravity. FASEB J, 2020; 34, 10835−49. doi: 10.1096/fj.202000734R