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![Immunofluorescence labeling of βII-tubulin in isolated cardiomyocytes with primary and Cy™ 5-conjugated AffiniPure goat secondary anti-mouse IgG (Jackson Immunoresearch). Labeling of mitochondria in parallel rows parallel to long axis of the cell is seen. For further details see Ref. [14].](profile/Vladimir-Chekulayev/publication/51575916/figure/fig3/AS:340417259294738@1458173207801/Immunofluorescence-labeling-of-bII-tubulin-in-isolated-cardiomyocytes-with-primary-and.png)
Immunofluorescence labeling of βII-tubulin in isolated cardiomyocytes with primary and Cy™ 5-conjugated AffiniPure goat secondary anti-mouse IgG (Jackson Immunoresearch). Labeling of mitochondria in parallel rows parallel to long axis of the cell is seen. For further details see Ref. [14].
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The aim of this study was to investigate the possible role of tubulin βII, a cytoskeletal protein, in regulation of mitochondrial oxidative phosphorylation and energy fluxes in heart cells. This isotype of tubulin is closely associated with mitochondria and co-expressed with mitochondrial creatine kinase (MtCK). It can be rapidly removed by mild pr...
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Context 1
... proteolysis the fluorescence intensity decreases significantly and regular arrangement of tubulin disap- pears. Since green fluorescence seen in Fig. 2A and B may be influenced by the autofluorescence of oxidized mitochondrial flavo- proteins [18], localization of tubulin-βII was studied also by using secondary antibodies with red fluorescence (Fig. 3). Again, very regular labeling of mitochondria was seen. Similar to the results presented on Figs. 2B, Fig. 4 shows again that short treatment of permeabilized cardiomyocytes with trypsin completely removes the tubulin-βII, also changing the cell shape due to destruction of tubulin and other cytoskeletal systems, and changes ...
Citations
... Therefore, several factors and signals from mitochondria may influence the cell metabolism (signaling out). The interaction of mitochondria with various cytoskeletal proteins such as beta-tubulin and specific isoforms of plectin can dynamically participate in the regulation of mitochondrial function via the outer membrane protein VDAC (Voltage Dependent Anion Channel) [13,14]. Mitochondria-endoplasmic reticulum (ER) interactions have been observed to be crucial for Ca 2+ homeostasis [15]. ...
Mitochondria have been recognized as the energy (in the form of ATP)-producing cell organelles, required for cell viability, survival and normal cell function [...]
... Other minor β-tubulin isotypes (βII-tubulin and βIVα-tubulin) have also been associated with regulating mitochondrial localization specifically in cardiomyocytes [66][67][68][69], although the mechanisms by which they operate remain unknown. With increasing recognition for the combinatorial manner in which β-tubulin isotypes collectively regulate microtubule functions and microtubule-dependent processes in cells [18,21,70], it is likely that the tubulin code regulating mitochondrial dynamics is a complex spatiotemporal network that depends on the local β-tubulin composition. ...
βIII-tubulin is a neuronal microtubule protein that is aberrantly expressed in epithelial cancers. The microtubule network is implicated in regulating the architecture and dynamics of the mitochondrial network, although the isotype-specific role for β-tubulin proteins that constitute this microtubule network remains unclear. High-resolution electron microscopy revealed that manipulation of βIII-tubulin expression levels impacts the volume and shape of mitochondria. Analysis of the structural domains of the protein identifies that the C-terminal tail of βIII-tubulin, which distinguishes this protein from other β-tubulin isotypes, significantly contributes to the isotype-specific effects of βIII-tubulin on mitochondrial architecture. Mass spectrometry analysis of protein–protein interactions with β-tubulin isotypes identifies that βIII-tubulin specifically interacts with regulators of mitochondrial dynamics that may mediate these functional effects. Advanced quantitative dynamic lattice light sheet imaging of the mitochondrial network reveals that βIII-tubulin promotes a more dynamic and extended reticular mitochondrial network, and regulates mitochondrial volume. A regulatory role for the βIII-tubulin C-terminal tail in mitochondrial network dynamics and architecture has widespread implications for the maintenance of mitochondrial homeostasis in health and disease.
... tubulin and bIII tubulin are reported in cancer (24,38). bII tubulin is shown to regulate Voltage-Dependent Anion Channels (VDACs) in the mitochondrial outer membrane, which act as a critical regulator of the Warburg effect observed in cancer cells (39,40). At the same time, overexpression of bIII-tubulin (TUBB3) is associated with the development of resistance to microtubule-targeting agents, resistance to apoptosis, and development of metastasis (41). ...
... But the Kaplan-Meier survival analysis suggested functional cooperation of these two proteins in the aggressiveness of the disease as the high expression of both these proteins or increased expression of even one of these proteins can significantly lead to poor prognosis. It is worthwhile to mention here that, like TUBB4B, its close relatives TUBB2 and TUBB3 have been shown to predict poor survival, the former regulating the cancer energetics through VDAC and the latter by imparting resistance to microtubule targeting agents (24,(38)(39)(40)(41). Our results show that the mechanism by which TUBB4B influences survival is through another means, possibly by regulating the proper localization of Ephrin-B1 in the CSC niche. ...
Recent advancements in cancer research have shown that cancer stem cell (CSC) niche is a crucial factor modulating tumor progression and treatment outcomes. It sustains CSCs by orchestrated regulation of several cytokines, growth factors, and signaling pathways. Although the features defining adult stem cell niches are well-explored, the CSC niche is poorly characterized. Since membrane trafficking proteins have been shown to be essential for the localization of critical proteins supporting CSCs, we investigated the role of TUBB4B, a probable membrane trafficking protein that was found to be overexpressed in the membranes of stem cell enriched cultures, in sustaining CSCs in oral cancer. Here, we show that the knockdown of TUBB4B downregulates the expression of pluripotency markers, depletes ALDH1A1 ⁺ population, decreases in vitro sphere formation, and diminishes the tumor initiation potential in vivo . As TUBB4B is not known to have any role in transcriptional regulation nor cell signaling, we suspected that its membrane trafficking function plays a role in constituting a CSC niche. The pattern of its expression in tissue sections, forming a gradient in and around the CSCs, reinforced the notion. Later, we explored its possible cooperation with a signaling protein, Ephrin-B1, the abrogation of which reduces the self-renewal of oral cancer stem cells. Expression and survival analyses based on the TCGA dataset of head and neck squamous cell carcinoma (HNSCC) samples indicated that the functional cooperation of TUBB4 and EFNB1 results in a poor prognosis. We also show that TUBB4B and Ephrin-B1 cohabit in the CSC niche. Moreover, depletion of TUBB4B downregulates the membrane expression of Ephrin-B1 and reduces the CSC population. Our results imply that the dynamics of TUBB4B is decisive for the surface localization of proteins, like Ephrin-B1, that sustain CSCs by their concerted signaling.
... The study also found that β-tubulin can regulate mitochondrial oxidative phosphorylation and energy flux in the regulation of apoptosis. β-Tubulin is directly related to mitochondrial function and is coexpressed with mitochondrial creatine kinase (MtCK) [38]. Moreover, the normal morphological arrangement of mitochondria is also maintained by the cytoskeleton structure, so β-tubulin directly affects the integrity of the mitochondrial structure and the normal function of the mitochondria [39]. ...
... After treating the cells with microtubule depolymerization agents, it is found that ICa-L was significantly inhibited. β-Tubulin can regulate the expression levels of RyR2 and SERCA2a by regulating ICa-L, participating in the regulation of the "Calcium Clock," thereby maintaining the electrophysiological balance of SANCs, and the normal physiological functions of SANCs [38]. Our study is consistent with the above results. ...
Sick sinus syndrome (SSS) is a disease with bradycardia or arrhythmia. The pathological mechanism of SSS is mainly due to the abnormal conduction function of the sinoatrial node (SAN) caused by interstitial lesions or fibrosis of the SAN or surrounding tissues, SAN pacing dysfunction, and SAN impulse conduction accompanied by SAN fibrosis. Tongyang Huoxue Decoction (TYHX) is widely used in SSS treatment and amelioration of SAN fibrosis. It has a variety of active ingredients to regulate the redox balance and mitochondrial quality control. This study mainly discusses the mechanism of TYHX in ameliorating calcium homeostasis disorder and redox imbalance of sinoatrial node cells (SANCs) and clarifies the protective mechanism of TYHX on the activity of SANCs. The activity of SANCs was determined by CCK-8 and the TUNEL method. The levels of apoptosis, ROS, and calcium release were analyzed by flow cytometry and immunofluorescence. The mRNA and protein levels of calcium channel regulatory molecules and mitochondrial quality control-related molecules were detected by real-time quantitative PCR and Western Blot. The level of calcium release was detected by laser confocal. It was found that after H/R treatment, the viability of SANCs decreased significantly, the levels of apoptosis and ROS increased, and the cells showed calcium overload, redox imbalance, and mitochondrial dysfunction. After treatment with TYHX, the cell survival level was improved, calcium overload and oxidative stress were inhibited, and mitochondrial energy metabolism and mitochondrial function were restored. However, after the SANCs were treated with siRNA (si-β-tubulin), the regulation of TYHX on calcium homeostasis and redox balance was counteracted. These results suggest that β-tubulin interacts with the regulation of mitochondrial function and calcium release. TYHX may regulate mitochondrial quality control, maintain calcium homeostasis and redox balance, and protect SANCs through β-tubulin. The regulation mechanism of TYHX on mitochondrial quality control may also become a new target for SSS treatment.
1. Introduction
Sick sinus syndrome (SSS), also known as sinoatrial node dysfunction, is a syndrome of arrhythmias caused by the pathological changes of sinoatrial node (SAN) and its adjacent tissues. It is one of the most refractory major cardiovascular diseases in clinic [1]. With population aging, the incidence of SSS is increasing. SAN, the physiological pacemaker of the heart, can trigger and regulate the rhythm of the heart [2, 3]. Cardiac rhythm is produced by the special cardiac myocytes of SAN and transmitted to the whole atrium and ventricle through the cardiac conduction system. Studies have found that calcium homeostasis and oxidative stress can affect SAN function directly [4]. Mitochondrial quality control (MQC) is closely related to calcium homeostasis and redox balance [5, 6], which directly affect the survival level of sinoatrial node cells (SANCs). Mitochondrion, as the central organelle of apoptosis pathway, is also the main site of tricarboxylic acid cycle and oxidative phosphorylation [7]. Its normal structure and function can meet the energy requirements of heart beating and ejection function, maintaining the homeostasis of intracellular environment, and regulating cell growth [8, 9].
Mitochondria can maintain the normal electrophysiological and contractile functions of cardiomyocytes and SANCs by producing adenosine triphosphate (ATP) and directly participate in the regulation of calcium homeostasis in SANCs. As the main site of ROS production and the first target of ROS attack, excessive ROS production can lead to dysfunction of mitochondrial respiratory electron-transport chain and further affect the balance of redox in cells [10]. Therefore, under the stimulation of hypoxia, inflammation, and high glucose, it will lead to excessive production of ROS and induce structural and functional abnormalities of mitochondria, including imbalance of mitochondrial energy metabolism, reduction of mitochondrial biosynthesis, and abnormal opening of mitochondrial membrane permeability transition pore (mPTP) [11, 12].
Moreover, the dysfunction of the balance system of intracellular “calcium release” and “calcium contraction” can also lead to abnormal increase of intracellular calcium concentration or calcium overload, which can cause the disorder of mitochondrial oxidative phosphorylation [13, 14]. Moreover, high concentration of Ca²⁺ in cytoplasm can increase Ca²⁺ uptake by mitochondria, which will lead to calcium phosphate deposition in mitochondria, and then affect ATP synthesis [15]. Calcium overload can further activate calcium-dependent proteases, promote the transformation of xanthine dehydrogenase into xanthine oxidase, and increase the production of ROS [16, 17]. Therefore, the redox imbalance and mitochondrial dysfunction caused by calcium overload will form a vicious circle and further participate in the process of cell damage [18, 19]. Early correction of mitochondrial dysfunction and regulation of calcium homeostasis and redox balance are important targets for protecting SANCs.
Tongyang Huoxue Decoction (TYHX) has been used in clinic for more than 50 years. It is composed of Astragalus membranaceus, ginseng, Rehmannia glutinosa, Angelica sinensis, and licorice. Many studies have reported that ginsenoside Rb1, resveratrol and astragaloside IV in Astragalus membranaceus, ginseng, Rehmannia glutinosa, and Angelica sinensis can protect mitochondria and regulate redox balance and mitochondrial quality control [20, 21]. Our previous experimental study also found that astragaloside IV, an active ingredient of TYHX, can shorten the action potential duration of injured SANCs in rabbits and protect the cytoskeleton [22]. However, the specific mechanism of TYHX in protecting SANCs and regulating the electrophysiological function of SANCs has not been elucidated. Therefore, this study is aimed at exploring the protective pharmacological action of TYHX on SANCs.
2. Materials and Methods
2.1. Drugs and Concentration Selection
All Chinese herbal medicines are provided by the pharmacy department of Guang’anmen Hospital. TYHX is composed of five herbs: Astragalus membranaceus, ginseng, Rehmannia glutinosa, Angelica sinensis, and licorice. Decoct all the herbs for 45 minutes, then filter out the solution, then add 6 times the volume of water to decoct again for 30 minutes, and then filter again. Finally, mix the two portions of filtered solution in a 75°C water bath. Finally, 1 g/ml TYHX stock solution was obtained. Correct the pH value to 7.4~7.6, and after centrifugation in a centrifuge, autoclave the supernatant at 8 pounds of pressure for 15 minutes, and then filter with a filter and microporous membrane before use. The nontoxic concentration of the drug was detected by the MTT method, and it was found that 50 μg/ml was the optimal nontoxic concentration of TYHX.
2.2. Reagents
DMEM was purchased from GIBCO (USA); FBS and trypsin were purchased from GIBCO (USA); collagenase II, Triton X-100, and Na2EDTA were purchased from Sigma Aldrich; preparation of anoxic hypoxia medium (in mM) is as follows: put 1.8 CaCl2, 20 HEPES, 10 KCl, 1.2 MgSO4, 98.5 NaCl, 6 NaHCO3, 0.9 NaH2PO4, and 40 sodium lactate in an anoxic chamber at 37°C for 3 h to induce hypoxia; preparation of reoxygenation medium (in mM) is as follows: 1.8 CaCl2, 5.5 glucose, 20 HEPES, 5 KCl, 1.2 MgSO4, 129.5 NaCl, 20 NaHCO3, and 0.9 NaH2PO4 at 37°C, and finally expose it to an atmosphere with 95% O2 and 5% CO2 for 2 h.
2.3. Isolation of SANCs
A total of 80 rabbits were selected (). The rabbits were sterilized with 75% ethanol before operation, and the suckling rabbits were anesthetized with isoflurane. The heart was completely exposed under the anatomical microscope. The SAN tissue was collected from the venous sinus and anterior vena cava root in the middle of the end ridge (). The tissue was placed in DMEM. The SAN tissue was washed with PBS and cut into pieces (). Discard the supernatant, add 0.08% trypsin (8 ml) to the chopped tissue for digestion, and shake in 37°C water bath for 5 minutes. After precipitation, the supernatant was removed and discarded, and the tissue was further digested with 8 ml 0.025% collagenase II. The supernatant was collected after sedimentation and transferred to a 50 ml centrifuge tube (containing 20 ml DMEM which contains 15% FBS), following the same procedure three times. The supernatant after digestion was collected and filtered by metal sieve (400 meshes). The supernatant after filtrate centrifuged for 7 min at 940 R/min. The supernatant was discarded after centrifugation, leaving cells suspended in the medium with a density of , which were seeded in 5 culture dishes. The culture dishes were incubated in the cell incubator for 90 minutes. After removing fibroblasts by the differential adhesion method, the remaining cells continued to grow after adding 5-bromo-2-deoxyuridine (5-BrdU). After culturing for 24 hours, replace the medium, and then replace it every other day. When replacing the medium, add 5-BrdU (0.1 mmol L⁻¹) [22].
2.4. Grouping and Culturing of Cells
Before the experiment, SANCs were divided into 5 groups: (1) control group: cells were cultured under the above normal conditions; (2) H/R group: cells were cultured in hypoxic medium instead of DMEM (as shown in the section on reagents) and placed in a hypoxic environment for 3 hours. Replace the hypoxic medium with the reoxygenated culture medium (as shown in the section on reagents), and place it in an environment with 95% O2 and 5% CO2 to continue the culturing; (3) TYHX group: SANCs were pretreated with TYHX before H/R modeling; (4) TYHX + siRNA-β-tubulin group: SANCs were treated with β-tubulin siRNA (siRNA-β-tubulin) before modeling and drug treatment; and (5) TYHX + Ad-β-tubulin group: SANCs were treated with an adenoviral vector (Ad-β-tubulin) overexpressing β-tubulin before modeling and drug treatment.
2.5. Apoptosis Detection
The SANCs in each group were washed with PBS. Then, an apoptosis detection kit was used for detection. Flow cytometry and the CXP software (Beckman, Brea, CA, USA) were used for analysis.
2.6. ROS
SANCs were incubated with 2,7-dichlorofluorescein (10 μm) at 37°C for 20 min. Then, the fluorescence was measured by a fluorescence microscope (Olympus, Japan) and a flow cytometry (Beckman, Brea, CA, USA). The ROS in the cells was detected by flow cytometry.
2.7. Determination of Antioxidant Enzyme Activity and MDA Level
The SOD\MDA\GSH-Px\TrxR kit was provided by Nanjing Jiancheng Institute of Biological Engineering. All operations were carried out in accordance with the manufacturer’s instructions. Simply put, cells were collected before and after modeling and drug pretreatment. After ultrasonic treatment, the cell lysate was centrifuged at 3000°C 20 r/min, and then the supernatant was collected to determine the level of SOD\MDA\GSH-Px\TrxR.
2.8. Cellular Energy Metabolism
The oxygen consumption rate (OCR) of the whole cell was analyzed by an XFp extracellular flux analyzer (Seahorse Biosciences) to detect the basic respiration, respiratory reserve, maximum respiration, ATP production level, and proton leakage level of mitochondria. SANCs ( cells/well) were inoculated. Mitochondrial respiration was detected, and mitochondrial energy metabolism was recorded according to the manufacturer’s instructions.
2.9. Laser Confocal and Immunofluorescence
After simulated H/R, cells were rinsed with PBS, then fixed with 4% paraformaldehyde. The treated cells were placed in a 37°C wet box, into which 100 μl of the first antibody was added, and the cells were incubated at 4°C for 16 hours. The cells were rinsed with PBS for four times, each time for 5 minutes. Then, the second antibody was added, and the cells were incubated in the dark for 1 hour. After buffering glycerin was added, the fluorescence image and fluorescence intensity were detected and analyzed by a laser confocal microscope. The average fluorescence intensity was analyzed by the ImageJ software.
2.10. Calcium Release/Calcium Contraction Detection
After SANCs were collected in an EP tube, 0.1% BSA low-calcium solution containing Fluo-4 AM was used to prepare the incubation solution and then cultured at 37°C for 20-30 min. Then, the solution was centrifugated to remove the fluo-4 AM working fluid. A small amount of SANCs were placed in a confocal dish coated with laminin. The concentration of Ca²⁺ was recorded.
2.11. Real-Time PCR
TRIzol was used to extract the total RNA of each group. Under the catalysis of reverse transcriptase, the total RNA in each group was used as template to synthesize cDNA by reverse transcription. The PrimeScript™ RT Kit was used to reverse transcribe RNA into cDNA with a gDNA eraser. Real-time quantitative PCR was used with cDNA as template, and the data was analyzed by .
2.12. Western Blot
Western Blot was used to study the protein expression of SERCA2a/RyR2/CaV1.3/NCX/β-tubulin (β-tubulin, Abcam, ab6046; SERCA2a, Abcam, ab150435; RyR2, Abcam, ab21796; Cav1.3, Abcam, ab84811; and NXC, Abcam, ab177952). The total protein was obtained from the SANCs. Protein samples are separated on 8% and 12% SDS-PAGE gel and transferred to nitrocellulose membranes. Transfer the protein to the PVDF membrane, block it with 5% skimmed milk powder at room temperature for 1 hour, and then incubate it with the primary antibody overnight at 4°C. The cells were incubated with the first antibody overnight. The ImageJ software was used to analyze the data by optical density analysis.
2.13. Statistical Analysis
The data obtained by the SPSS 22.0 statistical software were applied, and normal distribution data were measured by the -form expression. Median or quartile was used for nonnormal distribution data, and -test was used for paired or two groups of continuous measurement data. One-way analysis of variance is used to compare multiple sets of data, and two groups with SNK-q. The nonparametric test was used for nonnormal data, and test was used for pairwise comparison of classification data. The significance standard was .
3. Results
3.1. TYHX Can Effectively Improve the Activity of SANCs and Inhibit Apoptosis
We first verified the effect of TYHX on activity of SANCs, and it was found that TYHX can further improve cell viability through CCK-8 detection (Figure 1(a)), while effectively inhibiting cell apoptosis (Figures 1(e) and 1(f)). In order to further verify the role of skeleton protein (β-tubulin) in SANC damage induced by hypoxia/reoxygenation (H/R), we detected the levels of β-tubulin by PCR/Western Blot and immunofluorescence. It was found that H/R can reduce the mRNA and protein expression levels of SANCs, and TYHX can reverse this phenomenon (Figures 1(b) and 1(c)), so it was inferred that the mechanism of TYHX to improve the viability of SANCs may be related to β-tubulin. To further verify this hypothesis, SANCs were treated with an adenoviral vector overexpressing β-tubulin (Ad-β-tubulin) and β-tubulin siRNA (siRNA-β-tubulin). Then, through CCK-8 and cell apoptosis detection, it was found that SANCs treated with siRNA-β-tubulin eliminated the protective effect of TYHX on SANCs (Figures 1(d)–1(f)). However, the SANCs treated with Ad-β-tubulin under the intervention of TYHX further increased the protective effect of TYHX on SANCs (Figures 1(d)–1(f)). The experimental results suggest that TYHX can protect against H/R-induced SANC damage. The protective effect of TYHX on SANCs may be accomplished through the regulation of β-tubulin, and the specific mechanism needs further verification.
(a)
... Reliance on aerobic glycolysis alone is seen in cancerous cells. Different isoforms of human tubulin, for example, tubulin βII and βIII, have been shown to associate with the OMM and block the pore of VDAC [79,80]. Blockage of VDAC by tubulin βII is important in cardiomyocytes where ATP/ADP is compartmentalized near complexes. ...
The voltage-dependent anion channel (VDAC) is a β-barrel membrane protein located in the outer mitochondrial membrane (OMM). VDAC has two conductance states: an open anion selective state, and a closed and slightly cation-selective state. VDAC conductance states play major roles in regulating permeability of ATP/ADP, regulation of calcium homeostasis, calcium flux within ER-mitochondria contact sites, and apoptotic signaling events. Three reported structures of VDAC provide information on the VDAC open state via X-ray crystallography and nuclear magnetic resonance (NMR). Together, these structures provide insight on how VDAC aids metabolite transport. The interaction partners of VDAC, together with the permeability of the pore, affect the molecular pathology of diseases including Parkinson’s disease (PD), Friedreich’s ataxia (FA), lupus, and cancer. To fully address the molecular role of VDAC in disease pathology, major questions must be answered on the structural conformers of VDAC. For example, further information is needed on the structure of the closed state, how binding partners or membrane potential could lead to the open/closed states, the function and mobility of the N-terminal α-helical domain of VDAC, and the physiological role of VDAC oligomers. This review covers our current understanding of the various states of VDAC, VDAC interaction partners, and the roles they play in mitochondrial regulation pertaining to human diseases.
... Mitochondrial injuries are implicated in intracellular signaling and mitochondrial respiratory function plays a central role in cellular energy metabolism and redox regulation, particularly in the heart as a continuously active tissue which depends on aerobic energy supply. Studies of the delicate bioenergetic mechanisms in the heart have demonstrated a key role for the mitochondrial creatine kinase (mitCK) for metabolic channeling and intracellular micro-compartmentalization, and resulted in the discovery of mitochondrial functional complexes with other cellular organelles, such as myofibrils and the sarcoplasmic reticulum, forming intracellular energetic units [83,187,188]. Importantly, mitCK and, in particular, its functional links with energy transferring systems [189][190][191], can be very sensitive to cardiac ischemia (due to increases in cellular inorganic phosphate level) and various cardiomyopathies [12,[192][193][194][195]. Moreover, the mitCK system can be damaged by oxidative stress, due to possible oxidation of the enzyme-essential -SH residues by ROS [196]. ...
... The exact mechanisms of how mitochondria precisely respond to the heart energy demand remained unknown for a long time and require further investigations. A growing body of evidence shows that the cells contain intracellular metabolic micro-compartments provided by multidirectional mitochondrial interactions with other subcellular organelles and macromolecules, in particular, specific cytoskeletal proteins [26][27][28][29][30][31][32][33][34]. In this book chapter, we summarize and discuss previous studies that provide strong evidence for the role of cytoskeletal proteins, in particular, tubulin beta-II and plectin 1b, in the regulation of mitochondrial bioenergetics and energy fluxes via the energy-transferring supercomplex VDAC-mitochondrial creatine kinase (MitCK)-ATP-ADP translocase (ANT) under physiological and pathological conditions. ...
... On the other hand, the full activation of mitochondrial respiration requires at least 250-300 µM of ADP in isolated mitochondrial preparations. The detailed mechanisms of precise matches and synchronizations of mitochondrial respiratory function and heart contractility (excellently tuned cellular energy production and demand) still remain unclear and are under active investigation by several groups [27,28,[30][31][32][33][34]. Apparently, mitochondria-cytoskeleton interactions play a certain role in these crosstalk mechanisms. ...
... All these observations pointed to the involvement of cytoskeletal proteins as primary candidates in the control of mitochondrial respiratory function. Imaging analysis (fluorescence and immunofluorescence confocal microscopy) of cardiac cells and muscle fibers by using specific mitochondrial markers and various antibodies revealed full colocalization of mitochondria with cytoskeletal protein tubulin beta-II, suggesting its structural and functional interactions with mitochondrial VDAC [22,32,46,47]. Notably, in HL-1 cardiac cells that are devoid of tubulin beta-II, mitochondrial respiratory behavior and sensitivity to ADP (appKm) were similar to that of isolated mitochondria [47]. ...
... The second objective was to explore whether this potential relationship was related to altered tubulin-VDAC binding stemming from disorganized microtubules. Specific attention was given to α-tubulin considering it binds various isotypes of β-tubulin as an α/β heterodimer with the CTT tail of both components having affinity for VDAC [10, [14][15][16]. VDAC2 was selected given 1) its deficiency results in embryonic death thereby demonstrating its importance [17], 2) it has been proposed that VDAC2 may uniquely regulate the more efficient creatine-dependent mitochondrial phosphate shuttling mechanism [18][19][20][21], and 3) we have previously shown that tubulin-VDAC2 interactions are changed when microtubule organization is altered by paclitaxel [13]. The results demonstrate a relationship between microtubule disorganization and impaired ADP attenuation of H 2 O 2 emission. ...
... Furthermore, the affinity of tubulin binding can be modulated through post translational modifications [11] such as phosphorylation [43] or altered mitochondrial membrane lipid composition [44] which highlights the complexity of the potential regulation of this pathway. Lastly, it has been proposed that free tubulin regulates VDAC permeability based on experiments that observed the effect of adding or removing exogenous free tubulin to various preparations [10,16]. Future investigations could develop novel approaches that capture the degree to which free tubulin binds VDAC without altering their interactions during specimen processing. ...
In Duchenne muscular dystrophy, a lack of dystrophin leads to extensive muscle weakness and atrophy that is linked to cellular metabolic dysfunction and oxidative stress. This dystro-phinopathy results in a loss of tethering between microtubules and the sarcolemma. Micro-tubules are also believed to regulate mitochondrial bioenergetics potentially by binding the outer mitochondrial membrane voltage dependent anion channel (VDAC) and influencing permeability to ADP/ATP cycling. The objective of this investigation was to determine if a lack of dystrophin causes microtubule disorganization concurrent with mitochondrial dys-function in skeletal muscle, and whether this relationship is linked to altered binding of tubu-lin to VDAC. In extensor digitorum longus (EDL) muscle from 4-week old D2.mdx mice, microtubule disorganization was observed when probing for α-tubulin. This cytoskeletal disorder was associated with a reduced ability of ADP to stimulate respiration and attenuate H 2 O 2 emission relative to wildtype controls. However, this was not associated with altered α-tubulin-VDAC2 interactions. These findings reveal that microtubule disorganization in dys-trophin-deficient EDL is associated with impaired ADP control of mitochondrial bioenerget-ics, and suggests that mechanisms alternative to α-tubulin's regulation of VDAC2 should be examined to understand how cytoskeletal disruption in the absence of dystrophin may cause metabolic dysfunctions in skeletal muscle.
... Association of tubulin with mitochondria is required for maintaining mitochondrial function 18 . Interestingly, removal of β-tubulin from mitochondrial outer membrane by short proteolysis results in altered mitochondrial sensitivity for ADP and increased permeability of VDAC in cardiac cells 19 . Therefore, the increased tubulin in mitochondrial fraction observed in our present study might be related to its mitochondrial anchoring that the cells required for maintaining mitochondrial activity under the high glucose condition. ...
Mitochondrial dysfunction has been thought to play roles in the pathogenesis of diabetic nephropathy (DN). However, precise mechanisms underlying mitochondrial dysfunction in DN remained unclear. Herein, mitochondria were isolated from renal tubular cells after exposure to normal glucose (5.5 mM glucose), high glucose (25 mM glucose), or osmotic control (5.5 mM glucose + 19.5 mM mannitol) for 96 h. Comparative proteomic analysis revealed six differentially expressed proteins among groups that were subsequently identified by tandem mass spectrometry (nanoLC-ESI-ETD MS/MS) and confirmed by Western blotting. Several various types of post-translational modifications (PTMs) were identified in all of these identified proteins. Interestingly, phosphorylation and oxidation were most abundant in mitochondrial proteins whose levels were exclusively increased in high glucose condition. The high glucose-induced increases in phosphorylation and oxidation of mitochondrial proteins were successfully confirmed by various assays including MS/MS analyses. Moreover, high glucose also increased levels of phosphorylated ezrin, intracellular ATP and ROS, all of which could be abolished by a p38 MAPK inhibitor (SB239063), implicating a role of p38 MAPK-mediated phosphorylation in high glucose-induced mitochondrial dysfunction. These data indicate that phosphorylation and oxidation of mitochondrial proteins are, at least in part, involved in mitochondrial dysfunction in renal tubular cells during DN.
... The exact mechanisms of how mitochondria precisely respond to the heart energy demand remained unknown for a long time and require further investigations. A growing body of evidence shows that the cells contain intracellular metabolic micro-compartments provided by multidirectional mitochondrial interactions with other subcellular organelles and macromolecules, in particular, specific cytoskeletal proteins [26][27][28][29][30][31][32][33][34]. In this review, we summarize and discuss previous studies that provide strong evidence for the role of cytoskeletal proteins, in particular, tubulin beta-II and plectin 1b, in the regulation of mitochondrial bioenergetics and energy fluxes via the energy-transferring supercomplex VDAC-mitochondrial creatine kinase (MitCK)-ATP-ADP translocase (ANT) under physiological and pathological conditions. ...
... On the other hand, the full activation of mitochondrial respiration requires at least 250-300 µM of ADP in isolated mitochondrial preparations. The detailed mechanisms of precise matches and synchronizations of mitochondrial respiratory function and heart contractility (excellently tuned cellular energy production and demand) still remain unclear and are under active investigation by several groups [27,28,[30][31][32][33][34]. Apparently, mitochondria-cytoskeleton interactions play a certain role in these crosstalk mechanisms. ...
... All these observations pointed to the involvement of cytoskeletal proteins as primary candidates in the control of mitochondrial respiratory function. Imaging analysis (fluorescence and immunofluorescence confocal microscopy) of cardiac cells and muscle fibers by using specific mitochondrial markers and various antibodies revealed full colocalization of mitochondria with cytoskeletal protein tubulin beta-II, suggesting its structural and functional interactions with mitochondrial VDAC [22,32,46,47]. Notably, in HL-1 cardiac cells that are devoid of tubulin beta-II, mitochondrial respiratory behavior and sensitivity to ADP (appKm) were similar to that of isolated mitochondria [47]. ...
Elucidation of the mitochondrial regulatory mechanisms for the understanding of muscle bioenergetics and the role of mitochondria is a fundamental problem in cellular physiology and pathophysiology. The cytoskeleton (microtubules, intermediate filaments, microfilaments) plays a central role in the maintenance of mitochondrial shape, location, and motility. In addition, numerous interactions between cytoskeletal proteins and mitochondria can actively participate in the regulation of mitochondrial respiration and oxidative phosphorylation. In cardiac and skeletal muscles, mitochondrial positions are tightly fixed, providing their regular arrangement and numerous interactions with other cellular structures such as sarcoplasmic reticulum and cytoskeleton. This can involve association of cytoskeletal proteins with voltage-dependent anion channel (VDAC), thereby, governing the permeability of the outer mitochondrial membrane (OMM) to metabolites, and regulating cell energy metabolism. Cardiomyocytes and myocardial fibers demonstrate regular arrangement of tubulin beta-II isoform entirely co-localized with mitochondria, in contrast to other isoforms of tubulin. This observation suggests the participation of tubulin beta-II in the regulation of OMM permeability through interaction with VDAC. The OMM permeability is also regulated by the specific isoform of cytolinker protein plectin. This review summarizes and discusses previous studies on the role of cytoskeletal proteins in the regulation of energy metabolism and mitochondrial function, adenosine triphosphate (ATP) production, and energy transfer.
