MAP4 mechanism that stabilizes mitochondrial permeability transition in hypoxia: microtubule enhancement and DYNLT1 interaction with VDAC1

Abstract

Mitochondrial membrane permeability has received considerable attention recently because of its key role in apoptosis and necrosis induced by physiological events such as hypoxia. The manner in which mitochondria interact with other molecules to regulate mitochondrial permeability and cell destiny remains elusive. Previously we verified that hypoxia-induced phosphorylation of microtubule-associated protein 4 (MAP4) could lead to microtubules (MTs) disruption. In this study, we established the hypoxic (1% O(2)) cell models of rat cardiomyocytes, H9c2 and HeLa cells to further test MAP4 function. We demonstrated that increase in the pool of MAP4 could promote the stabilization of MT networks by increasing the synthesis and polymerization of tubulin in hypoxia. Results showed MAP4 overexpression could enhance cell viability and ATP content under hypoxic conditions. Subsequently we employed a yeast two-hybrid system to tag a protein interacting with mitochondria, dynein light chain Tctex-type 1 (DYNLT1), by hVDAC1 bait. We confirmed that DYNLT1 had protein-protein interactions with voltage-dependent anion channel 1 (VDAC1) using co-immunoprecipitation; and immunofluorescence technique showed that DYNLT1 was closely associated with MTs and VDAC1. Furthermore, DYNLT1 interactions with MAP4 were explored using a knockdown technique. We thus propose two possible mechanisms triggered by MAP4: (1) stabilization of MT networks, (2) DYNLT1 modulation, which is connected with VDAC1, and inhibition of hypoxia-induced mitochondrial permeabilization.

Figure 1. MAP4 overexpression increases the amount of α-tubulin and MT networks.

A and B, Western blots showed that the expression of MAP4 and gross quantity of α-tubulin in HeLa cells and CMs after Ad-MAP4 was elevated after transfection. Graphs represent the mean±SEM of the relative optical density signals for three separate experiments (n = 3). N - non-transfected cells. # P<0.01 vs. N and Ad-GFP, Student's t test analysis. Cytosol GAPDH was chosen as the internal control. C and D, Immunofluorescent confocal micrographs of HeLa cells and CMs. Micrographs show that cells contain a larger amount of MAP4 (FITC-green) and more luxuriant MT network structure (TRITC-red) after Ad-MAP4 transfection compared with Con (Non-transfected cells). Scale bar, 10 µm.

Figure 2. MAP4 overexpression alleviates MT disruption during the early stages of hypoxia.

Immunofluorescent confocal micrographs of +MAP4 and Con (Non-transfected) HeLa cells (A) and CMs (B) under normoxia and hypoxia. Micrographs show the changes in MTs as hypoxia duration is increased. Graphs to the right represent the corresponding % Integral optical density of MTs (green) (IOD; values were normalized as percentage after comparison with normal, which were set to 100%; n = 3). Results show that the structure of MTs was significantly disrupted after 30 min of hypoxia in Con groups, while in +MAP4 groups this disruption was less apparent until 60 min. +MAP4 cells seemed to contain more MTs than Con cells undergoing the same level of hypoxia, especially at the earlier time points (≤60 min in CMs; ≤180 min in HeLa cells). All values are mean±SEM. * P<0.05, # P<0.01, vs. Con at each time point, Student's t test analysis; † P<0.05, € P<0.01, vs. Norm (Normoxia) within each group, One-way ANOVA followed by Tukey's post-hoc tests. Scale bar, 10 µm.

Figure 3. Interaction and co-localization of VDAC1, DYNLT1 and MTs.

A, Example of Yeast two-hybrid technique. hVDAC1 was used as bait to tag new target molecules (arrows indicate the positive results detailed in Table 1). B, A co-IP approach followed by immunoblot analysis was used to test the protein-protein interaction between DYNLT1 and VDAC1. In whole-cell lysate of CMs and HeLa cells, abundant VDAC1 was detected in the antigen-antibody complex when applying anti-DYNLT1 (Ab-go / Ab-ra) compared with using IC-go / IC-ra. Anti-VDAC1 antibody co-immunoprecipitated with DYNLT1. IP- immunoprecipitate, IB- immunoblotting, Ab- antibody, IC- isotype control, go- goat, ra- rabbit. C, Immunofluorescent confocal micrographs showing the co-localization between DYNLT1 (FITC-green) and VDAC1 (TRITC-red) in CMs. D, Confocal micrographs showing the co-localization between DYNLT1 (FITC-green) and MTs (TRITC-red). Scale bar 10 µm. Areas in the boxed regions are shown at higher magnification, scale bar 1 µm.

Figure 4. MAP4 overexpression leads to the elevated expression of DYNLT1.

A, Immunoblot of DLNLT1 after MAP4 transfection. HeLa cells with MAP4 overexpression (Ad-MAP4) showed an elevated expression of DYNLT1 compared with non-transfected cells (N) and Ad-GFP transfected cells (Ad-GFP). # P<0.01 vs. N and Ad-GFP. B, Immunoblot of DYNLT1 after transient transfection of the plasmid. DYNLT1 was overexpressed in pFLAG-DYNLT1 cells. * P<0.05 vs. pcDNA3.1-GFP. C, Immunoblot of MAP4 and α-tubulin following up-regulation of DYNLT1 (pFLAG-DYNLT1). There seemed no influence on MAP4 and α-tubulin levels. Graphs represent the mean±SEM (n = 3) of the relative optical density signals.

Figure 5. MAP4 overexpression contributes to cellular viability (measured by MTT) and energy metabolism maintenance (measured by ATP) during hypoxia.

A, MTT reduction in +MAP4 groups (CMs and HeLa cells) was less when compared with Con (non-transfected) cells. B, ATP reduction in +MAP4 groups was also less when compared with Con cells. Values were compared to normal values (Norm; first bar), which were set to 100% and the other values normalized accordingly. Graph represents the mean±SEM (n = 6, Separate six experiments) of the relative luminescence signals. * P<0.05, # P<0.01 vs. Con.

Figure 6. Verification of DYNLT1 knockdown in HeLa and H9c2 cell lines.

H9c2 is a subclone of the original clonal cell line derived from embryonic BD1X rat heart tissue; we chose H9c2 and HeLa cell lines to establish stable cell lines with low expression of DYNLT1 (−DYNLT1) by RNAi technique. A, Immunoblot and quantified relative optical density analysis showed a stable low expression of DYNLT1 in cell clones using a RNAi approach (DYNLT1-shRNA) compared with non-transfected cells (N) and control plasmid DNA transfected cells (Con-shRNA). Graph represents the mean±SEM (n = 3). # P<0.01 vs. N and Con-shRNA.

Figure 7. DYNLT1 knockdown aggravates hypoxia-induced damage of mitochondria: mPT induction, MMP disruption and reduction of cellular viability.

A, −DYNLT1 (RNAi by DYNLT1-shRNA) groups showed a sharp reduction in calcein fluorescence after 60 min of hypoxia (cf. H30, H60 and H180); while +DYNLT1 (pFLAG-DYNLT1 transfected) cells showed no significant difference from WT (non-transfected) cells. B, −DYNLT1 cells showed a significant hypoxia-induced MMP damage, whereas +DYNLT1 cells appeared showed a slower decline in MMP that was similar to WT cells. C, −DYNLT1 cells showed a dramatic decrease in cell viability compared with WT and +DYNLT1 cells; WT and +DYNLT1 cells were not significantly different. Values were compared to Norm (first bar), which was set at 100% and the other values were normalized accordingly. Graphs represent the mean±SEM (n = 6). * P<0.05, # P<0.01 vs. WT group or +DYNLT1 group.

Figure 8. Model of MAP4, MTs and DYNLT1 interactions that might prevent hypoxia-induced cell damage.

The proposed model was built to describe a different cell destiny with the absence or presence of a hypothetical modulation during hypoxia. MAP4 overexpression may be a trigger in stabilizing mitochondrial function by enhancing the structure of MTs and promoting DYNLT1 expression. We demonstrated that DYNLTI interacts with VDAC1, which is considered responsible for mPT and consequent cell death. MT enhancement might be another potential mediator by binding tubulin to VDAC1 in addition to its supporting role with mitochondria.