Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Microtubular Stability Affects pVHL-Mediated Regulation of HIF-1alpha via the p38/MAPK Pathway in Hypoxic Cardiomyocytes

  • Miao Teng ,

    Contributed equally to this work with: Miao Teng, Xu-pin Jiang, Qiong Zhang

  • Xu-pin Jiang ,

    Contributed equally to this work with: Miao Teng, Xu-pin Jiang, Qiong Zhang

  • Qiong Zhang ,

    Contributed equally to this work with: Miao Teng, Xu-pin Jiang, Qiong Zhang

  • Jia-ping Zhang,
  • Dong-xia Zhang,
  • Guang-ping Liang,
  • Yue-sheng Huang

    yshuang.tmmu@gmail.com

    Affiliation Institute of Burn Research, State Key Laboratory of Trauma, Burns and Combined Injury, Southwest Hospital, The Third Military Medical University, Chongqing, China

Microtubular Stability Affects pVHL-Mediated Regulation of HIF-1alpha via the p38/MAPK Pathway in Hypoxic Cardiomyocytes

  • Miao Teng, 
  • Xu-pin Jiang, 
  • Qiong Zhang, 
  • Jia-ping Zhang, 
  • Dong-xia Zhang, 
  • Guang-ping Liang, 
  • Yue-sheng Huang
PLOS
x

Abstract

Background

Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α.

Methodology/Principal Findings

In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule–depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells.

Conclusions/Significance

This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.

Introduction

Hypoxia is a common pathophysiological process in many human diseases. Despite being a frequent process in the human system, the hypoxic state is implicated in the onset and progression of many life-threatening diseases. Myocardial hypoxia is a common clinical finding in patients with coronary artery disease, hypertensive heart disease and cardiomyopathy [1], [2]. Interestingly, this particular type of tissue damage is also present in patients with severe burns [3], [4]. At the cellular level hypoxia or ischemia elicits cytoskeletal damage, including microtubule network alteration [5], [6]. This alteration in the sub-cellular architecture in turn influences glycolysis in hypoxic cardiomyocytes (CMs), a process mediated by hypoxia-inducible factor (HIF)-1α [7], which is itself regulated by O2 tension and the microtubule network [7][9].

The microtubule network is a major component of the eukaryotic cytoskeleton and has multiple effects on cellular processes. Previous studies have suggested that microtubule-stabilizing agent (paclitaxel) or transfection of physiological microtubule stabilizer (MAP4) stabilizes microtubules [7], [10], [11] and up-regulates HIF-1α in CMs during early hypoxia [7]. In addition, cytoskeletal disruption also results in increased expression of pVHL in renal cell carcinoma cells during hypoxia [12].

pVHL is encoded by a tumor-suppressor gene located on chromosome 3p25 and functions as the E3 ubiquitin ligase for HIF-1α [13], [14]. pVHL-mediated ubiquitination of HIF-1α plays a central role in the cellular responses to changes in oxygen availability [15][17]. Under normoxic conditions, HIF-1α is rapidly degraded by the pVHL-mediated ubiquitin-proteasome pathway [18]. Conversely, under hypoxic conditions, HIF-1α is stabilized through decreasing the affinity of HIF-1α toward pVHL [19], [20].

It is well known that microtubules play a key role in signal transduction [21]. Mitogen-activated protein kinase (MAPK) systems, especially p38/MAPK, are involved in the intracellular signaling events triggered by microtubule-interfering agents [22][24]. Recent data showed that phospho-p38 could be co-immunoprecipitated with MAP4 in normoxic and hypoxic CMs [5]. In addition, p38/MAPK modulates the expression of HIF-1α [25], [26] and pVHL [12] in hypoxic cells. Accordingly, these data promoted us to investigate the functions of p38/MAPK in the regulation of pVHL following microtubule alteration.

Moreover, microtubule stabilization regulates HIF-1α [7][9] and pVHL protein levels [12], and the affinity of HIF-1α toward pVHL decreases during hypoxia [19], [20]. Little is known about the regulation of HIF-1α mediated by pVHL after microtubule alteration during early hypoxia. Here, we report that pVHL is involved in the regulation of HIF-1α in response to both microtubule stabilization and disruption and that the p38/MAPK pathway plays an important role in regulating pVHL during early hypoxia. Our results suggest that, in both hypoxic CMs and H9c2 cells, microtubular stabilization decreases the activity of the p38/MAPK pathway, which in turn suppresses pVHL and leads to the up-regulation of HIF-1α. Thus, our findings provide novel insights into the regulation of HIF-1α by microtubule alternation in hypoxic CMs.

Results

Effects of microtubule network alteration on HIF-1α protein levels in normoxic cardiomyocytes

Cardiomyocytes and H9c2 cells cultured under normoxic conditions were exposed to microtubule-stabilization treatments (paclitaxel, or transfection of MAP4 overexpression plasmids), or microtubule–depolymerization treatments (colchicine, or transfection of MAP4 siRNA). The changes in HIF-1α protein levels were detected in treated and untreated cells by Western blotting. The results showed that the HIF-1α protein levels were low under normoxic conditions, and none of the treatments with microtubule-interfering agents produced any change in the HIF-1α protein levels (Figure S1).

Microtubule network alteration of hypoxic cardiomyocytes resulted in change of pVHL protein levels

When subjected to hypoxic conditions, the microtubule network in CMs and H9c2 cells became disordered. Microtubular shrinkage was observed in the vicinity of the nuclei and the microtubules appeared to curl and buckle along the edges of the cells (Figure 1A). MAP4 is an endogenous stabilizer of microtubules [10]. To change microtubule network, we used paclitaxel and colchicine as well as vectors to increase or decrease MAP4. The protein levels of MAP4 increased in hypoxic CMs and H9c2 cells transfected with MAP4 recombinant adenovirus and decreased in hypoxic cells transfected with MAP4-siRNA adenovirus (Figure 1B and 1C). Overexpression of MAP4 or pretreatment with paclitaxel restabilized the microtubule network of the hypoxic CMs and H9c2 cells. Specifically, the reticular microtubules appeared as bundled and evenly distributed in the cytoplasm and the microtubule density was significantly higher than that in control group. In contrast, knockdown of MAP4 by siRNA or pretreatment with colchicine further disrupted the microtubule network of the hypoxic cells and the microtubule density was low(Figure 1A).

thumbnail
Figure 1. Microtubule network alteration of hypoxic cardiomyocytes resulted in change of pVHL protein levels.

(A) Immunofluorescent confocal micrographs. α-tubulin is green and the nucleus is blue. N, normoxia; H, hypoxia; H+P, hypoxia and paclitaxel; H+MAP4, hypoxia and MAP4 overexpression; H+HK1, hypoxia and a HK plasmid (as a negative control of MAP4 transfection); H+C, hypoxia and colchicine; H+siRNA, hypoxia and transfected for MAP4-siRNA; H+HK2, hypoxia and a HK plasmid (as a negative control of MAP4-siRNA). (B) Western blots showing MAP4 protein expression for the various experimental groups. (C, E) Graphs representing the mean±SD (n = 5) of the relative integrated signal. (D) Western blot analysis of pVHL in microtubule-altered hypoxic cardiomyocytes and H9c2 cells. ΔP<0.05 vs. Hypoxia group.

https://doi.org/10.1371/journal.pone.0035017.g001

We next evaluated the pVHL protein levels in those cells with altered microtubule networks. Under hypoxic conditions, microtubule stabilization decreased the protein levels of pVHL, while depolymerization of the microtubules increased it (Figure 1D and 1E).

Involvement of pVHL in the regulation of HIF-1α following microtubule alteration

It is well documented that microtubule network alteration leads to appreciable changes in HIF-1α levels [7][9]. We sought to determine whether this alteration of HIF-1α protein levels depends on the microtubule alteration-induced pVHL change. CMs and H9c2 cells were transfected with pVHL recombinant adenovirus to overexpress pVHL or pVHL-siRNA adenovirus to knockdown pVHL prior to microtubule interference, after which the cells were subjected to hypoxic conditions as described. In both CMs and H9c2 cells, the protein levels of HIF-1α were down-regulated by pVHL overexpression and up-regulated by knockdown of pVHL. In contrast, cells expressing GFP showed no changes in HIF-1α protein levels compared to the control group (Figure 2). These results suggested that pVHL plays an important role in the regulation of HIF-1α after alteration of the microtubule network in hypoxic CMs and H9c2 cells.

thumbnail
Figure 2. Involvement of pVHL in the regulation of HIF-1α following microtubule alteration.

(A, C) CMs and H9c2 cells were transfected with pVHL recombinant adenovirus or pVHL-siRNA adenovirus before treatment with microtubule-interfering agents (paclitaxel and colchicine) prior to hypoxia. Western blots are shown for HIF-1α in the microtubule-altered hypoxic CMs and H9c2 cells. H, hypoxia; H+P, hypoxia and paclitaxel; H+P+pVHL, hypoxia+paclitaxel+pVHL overexpression; H+C, hypoxia+colchicine; H+C+pVHL, hypoxia+colchicine+pVHL overexpression; H+P+siRNA, hypoxia+paclitaxel+transfected for pVHL-siRNA; H+C+siRNA; hypoxia+colchicine+transfected for pVHL-siRNA; H+GFP, hypoxia and GFP transfection (as a negative control of pVHL transfection or pVHL-siRNA). (B, D) Graphs represent the mean±SD (n = 5) of the relative integrated signal. ΔP<0.05 vs. H group (hypoxia); *P<0.05 vs.H+T group; **P<0.05 vs.H+C group.

https://doi.org/10.1371/journal.pone.0035017.g002

Microtubule network depolymerization of hypoxic cardiomyocytes activated p38/MAPK signaling pathway

To elucidate the signaling events of microtubule alteration-induced pVHL change, we investigated the status of the p38/MAPK signaling pathway in hypoxic CMs and H9c2 cells. Phospho-p38 in hypoxic CMs and H9c2 cells was found by immunoblotting to be elevated significantly in response to knockdown of MAP4 or pretreatment with colchicine. While in microtubule-stabilized hypoxic cells, pretreatment with MAP4 recombinant adenovirus or paclitaxel decreased phospho-p38, as compared to that in control group. (Figure 3).

thumbnail
Figure 3. Microtubule network depolymerization of hypoxic cardiomyocytes activated p38/MAPK signaling pathway.

Representative Western blots (A) and data summary of phospho-p38 (p-p38) (B) and p38 (C) in the microtubule-altered CMs and H9c2 cells under hypoxic conditions. H, hypoxia; H+P, hypoxia+paclitaxel; H+MAP4, hypoxia+MAP4 overexpression; H+HK1, hypoxia+a HK plasmid (as a negative control of MAP4 transfection); H+C, hypoxia+colchicine; H+siRNA, hypoxia+transfection of MAP4-siRNA; H+HK2, hypoxia+a HK plasmid (as a negative control of MAP4-siRNA). Graph represents the mean±SD (n = 5) of the relative gray values. ΔP<0.05 vs. H group.

https://doi.org/10.1371/journal.pone.0035017.g003

Microtubule alteration-induced change of pVHL relied on p38/MAPK signaling

To test whether the changes of pVHL induced by microtubule alteration depend on the activity of p38/MAPK, we treated CMs and H9c2 cells with the p38/MAPK inhibitor SB203580. SB203580 downregulated pVHL in the hypoxic cells pretreated with either paclitaxel or colchicine (Figure 4). We then further overexpressed p38 kinase activator MKK6 (Glu) in these cells. MKK6 is known to phosphorylate p38/MAPK on Thr-180 and Tyr-182, which leads to the activation of p38/MAPK [27]. The cells were transfected with MKK6 (Glu) recombinant adenoviruses before pretreating with microtubule interfering agents (paclitaxel or colchicine). Three hours after hypoxia, pVHL was found to have increased significantly and no changes were observed in the cells expressing GFP (Figure 5). These results indicated that p38/MAPK signaling plays an important role in the change of pVHL protein levels in hypoxic CMs after microtubule alteration.

thumbnail
Figure 4. Effect of the p38/MAPK inhibitor, SB203580, on pVHL in the microtubule-altered hypoxic cardiomyocytes.

CMs and H9c2 cells pretreated with microtubule-interfering agents (paclitaxel and colchicine) were incubated with or without SB203580 (SB) prior to hypoxia. (A) Western blots are shown for pVHL. (B) Graph represents the mean±SD (n = 5) of the relative integrated signal. ΔP<0.05 vs.H group (hypoxia);*P<0.05 vs.H+T group;**P<0.05 vs.H+C group.

https://doi.org/10.1371/journal.pone.0035017.g004

thumbnail
Figure 5. Effect of MKK (Glu) overexpression on pVHL in the microtubule-altered hypoxic CMs and H9c2 cells.

Cells were transfected with MKK recombinant adenovirus (MKK) before treatment with microtubule-interfering agents (paclitaxel and colchicine) prior to hypoxia. (A) Western blots are shown for pVHL. (B) Graph represents the mean±SD (n = 5) of the relative integrated signal. ΔP<0.05 vs.H group (hypoxia); *P<0.05 vs.H+T group; **P<0.05 vs.H+C group.

https://doi.org/10.1371/journal.pone.0035017.g005

Phospho-p38 physically interacts with α-tubulin

The microtubule interfering agents (paclitaxel or colchicine) are well-documented for their ability to change the microtubule network through interaction with α/β-tubulin heterodimers [28]. The microtubule network alteration induced by paclitaxel or colchicine may affect the p38/MAPK signaling pathway by forming a complex with α-tubulin. We tested this hypothesis by immunoprecipitation. In both CMs and H9c2 cells pretreated with paclitaxel or colchicine, α-tubulin was co-immunoprecipitated by phospho-p38. Conversely, phospho-p38 could be co-immunoprecipitated by α-tubulin under hypoxic conditions (Figure 6).

thumbnail
Figure 6. The physical interaction between phospho-p38 and α-tubulin detected by immunoprecipitation.

Hypoxic CMs and H9c2 cells were pretreated with paclitaxel or colchicine. (A) Hypoxic CMs; (B) Hypoxic H9c2 cells. Cell extracts were immunoprecipitated by anti-phospho-p38 and anti-α-tubulin antibodies and then immunoblotted with anti-α-tubulin or anti-phospho-p38 antibody. IP, immunoprecipitation; IB, immunoblotting; IS, isotype.

https://doi.org/10.1371/journal.pone.0035017.g006

Discussion

The microtubule network is a major component of the cytoskeleton and is crucial for the maintenance of normal cellular physiology. We previously found that microtubule interference affects glycolysis of CMs during early hypoxia through regulation of HIF-1α, and HIF-1α content in response to hypoxic microtubular structural changes occured only at the post-transcriptional level [7]. However, little is known about how such a microtubule network alteration results in the change of HIF-1α expression in CMs during the early stages of hypoxia. In this study, we sought to address this question using primary rat CMs as well as H9c2 cells, a permanent cell line derived from rat cardiac tissue that offers a unique in vitro model to study the metabolic activity of the heart [29], [30]. In initial experiments, we found that the protein levels of HIF-1α were low and were not changed by microtubule interference in normoxic CMs and H9c2 cells (Figure S1). Additionally, our previous results indicated that the levels of HIF-1α had no differences in normoxic CMs treated with or without microtubule interfering agents [7]. Therefore, we focused on the intracellular responses under hypoxia environment in this study.

Microtubule-associated proteins, as microtubule regulators, have the ability to polymerize and stabilize microtubules [31]. In CMs and H9c2 cells, MAP4 represents the major MAP subtype and changes in MAP4 expression interfere with the stability of microtubules [7], [10]. Likewise, we also found that overexpression of MAP4 stabilizes microtubules, and vice versa (Figure 1). To better observe the changes of the downstream signal molecules following microtubule network alteration, we used the microtubule-stabilizing agent paclitaxel and the microtubule-depolymerizing agent colchicine.

HIF-1α is a transcription factor that modulates the cellular responses to hypoxia [32]. Among its transcriptional targets are genes that stimulate erythropoiesis, angiogenesis and intracellular metabolism [33]. It has been demonstrated that microtubule network alteration leads to change in the protein levels of HIF-1α [7][9], [34]. Our previous study found that microtubular structural changes regulate the protiein levels of HIF-1α but not mRNA levels of HIF-1α in the CMs during early stages of hypoxia. Stabilizing microtubular structures of hypoxic CMs increases HIF-1α expression, while microtubular structure breakdown decreased it [7].

pVHL is a component of an E3 ubiquitin protein-ligase complex that targets HIF-1α for degradation by the ubiquitin-proteasome pathway [15][17], [35], [36]. pVHL is known to associate with at least four other partners: elongin B, elongin C, Cullin-2, and Rbx1 [37]. The pVHL/HIF-1α interaction within the cytoplasmic compartment requires hydroxylation of two prolyl residues (Pro402 and Pro564) in HIF-1α by prolyl-hydroxylases (PHDs or EGLNs) [20], [38]. Under the normoxic condition, pVHL ubiquitinates HIF-1α and consequently targets HIF-1α to the 26S proteasome for degradation [39]. However, in hypoxic cells HIF-1α escapes from degradation because of the inactivation of EGLNs [19], [20], [40]. Among those studies reported in the literature that have analyzed pVHL/HIF-1α interactions in hypoxic cells, none has investigated the effect of pVHL change on HIF-1α expression in CMs.

In hypoxic CMs and H9c2 cells, we revealed that microtubular stabilization or breakdown changed the expression of pVHL, which was inversely related to the protein levels of HIF-1α. Moreover, HIF-1α protein levels increased in microtubule altered-hypoxic cells in which the expression of pVHL was low. Accordingly, HIF-1α decreased when pVHL was overexpressed (Figure 2). These results demonstrated that pVHL contributes to the microtubule network alteration-induced regulation of HIF-1α in hypoxic CMs and H9c2 cells, although the affinity of HIF-1α toward the pVHL decreased in hypoxic cells. Recently, del Peso and colleagues [41] showed that the pVHL/HIF pathway regulated the transcription of EGLNs genes in response to low oxygen, suggesting that EGLNs might be involved in the process of microtubule network alteration-induced regulation of HIF-1α.

Interestingly, we found that microtubule alteration induced change in phospho-p38, which was positively related to the levels of pVHL in hypoxic CMs and H9c2 cells. Furthermore, accumulating data indicate that the p38/MAPK pathway is involved in the intracellular signaling events triggered by microtubule-interfering agents [22][24]. Accordingly, we pharmacologically inhibited p38/MAPK with SB203580 and overexpressed an endogenous p38/MAPK activator, MKK6, in order to observe the change of pVHL in hypoxic CMs and H9c2 cells pretreated with microtubules-interfering agents. We found that p38/MAPK inactivation down-regulated the expression of pVHL, while p38/MAPK activation up-regulated it. These findings suggests that the p38/MAPK pathway is involved in the process of pVHL change induced by microtubule network alteration. It has been reported that phosphorylation of pVHL by CK2 and ubiquitin/SUMO modification of pVHL may regulate VHL protein stability [42][44], but it is not clear if p38/MAPK interacts with pVHL directly or indirectly. Future studies are expected to reveal the details of this process.

The microtubule network participates in a multitude of intracellular signal transduction pathways, including the p38/MAPK pathway [22], [24], [45][47]. It has been reported that phospho-p38 co-immunoprecipitates with MAP4 in both nomoxic and hypoxic CMs [5]. We found that phospho-p38 co-immunoprecipitated with α-tubulin, a major component of microtubule, in hypoxic CMs and H9c2 cells. This finding provides a physically interaction for microtubule alteration-induced activation of the p38/MAPK pathway.

In initial experiments, we identified that stabilizing the microtubule network of hypoxic cardiomyocytes elevates the protein contents of HIF-1α, thus promoting glycolysis and ATP levels, which enhanced the survival of hypoxic CMs [7]. In this study, we also found that the microtubule network stability was positively related to the cell viability of hypoxic CMs and H9c2 cells, accompanied by the up-regulation of HIF-1α. Coincidently, pVHL-siRNA transduction or pretreatment with SB203580 significantly increased hypoxic cells survival. In contrast, overexpression of pVHL or MKK6 (Glu) transduction reduced cell viability of hypoxic CMs and H9c2 cells (Figure S2). These results further supported the mechanism of HIF-1α regulation revealed in our present study.

Taken together, we observed that in both hypoxic CMs and H9c2 cells, microtubule network stabilization downregulated pVHL by inactivating the p38/MAPK pathway, which in turn lead to increased levels of HIF-1α. Conversely, microtubule network breakdown upregulated pVHL-mediated degradation of HIF-1α through activation of the p38/MAPK pathway.

These findings provide new insights into the molecular mechanism of HIF-1α protein change in CMs regulated by microtubule network alteration during early hypoxia. HIF-1α is a key regulator of the molecular hypoxic response, mediating a wide range of physiological and cellular processes necessary for cells to adapt to conditions of reduced oxygen [48]. It has been demonstrated that up-regulation of HIF-1α in cardiomyocytes is protective against myocardial ischemia in vivo [49], [50]. In addition, Xiao et al. reported that the microtubule stabilizer, taxol, prevents ischemic ventricular arrhythmias in rats [51]. Our results may provide a new therapeutic target for regulating HIF-1α that will help in limiting myocardial injury after myocardial hypoxia.

Materials and Methods

Cell culture

Primary cardiomyocyte culture. Neonatal Sprague-Dawley rats (1–2 days old) were provided by the Animal Center of the Third Military Medical University. The animal-based investigations were designed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85-23, revised 1996), and the entire project was reviewed and approved by the Animal Experiment Ethics Committee of the Third Military Medical University(Permit Number SYXK-CQ-20070002). Rat ventricular muscles were harvested and digested with trypsin and then cultured according to the protocols published previously [52], [53]. Neo-natal rat ventricular CMs were cultured in Dulbecco's Modified Eagle's Medium (DMEM)-F12 (Hyclone) supplemented with 5-bromodeoxyuridine (BrdU; 31 mg/L), 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Gibco), penicillin G (100 U/ml) and streptomycin (100 mg/ml) prior to hypoxia treatment. H9c2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences. The cells were cultured in DMEM (Hyclone) supplemented with 10% FBS, 100 U/ml penicillin G and 100 mg/ml streptomycin.

Hypoxia treatment and microtubule-interference

A hypoxia model was established as described previously [5]. Briefly, hypoxic conditions were achieved by using an anaerobic jar (Mitsubishi, Tokyo, Japan) and vacuum glove box (Chunlong, Lianyungang, China). The serum-free medium was placed in the vacuum glove box filled with a gas mixture of 94% N2, 5% CO2, and 1% O2 overnight and allowed to equilibrate with the hypoxic atmosphere. All experimental groups were treated for 3 h under the same hypoxic conditions at 37°C. The p38/MAPK inhibitor, SB203580(Beyotime) (5 µmol/L), was added to these cultures and allowed to incubated at 37°C for 30 min before hypoxia treatment was commenced.

Microtubule-stabilization was acheived by performing an 8 h pretreatment with 10 µmol/L paclitaxel (Sigma) or transfection of MAP4 overexpression plasmids prior to hypoxia treatment. Microtubule depolymerization was achieved by performing an 8 h pretreatment with 10 µmol/L colchicine (Sigma) or transfection of MAP4 siRNA before hypoxia treatment.

Recombinant adenovirus vectors

PCR-amplified rat MAP4 cDNA or VHL cDNA were subcloned into pShuttle2 or pShuttle3 vectors (Clontech) to generate the recombinant shuttle plasmids pShuttle2-MAP4 or pShuttle3-VHL. The pShuttle2-HK (hexokinase) or pShuttle3-EGFP (enhanced green fluorescent protein) were used as negative controls. MKK6 (Glu) recombinant adenoviruses were generously provided by Dr. Hu (The Third Military Medical University, Chongqing, China).

Knockdown of MAP4 or pVHL with siRNA in cardiomyocyte model

Four RNA interference fragments were designed based on rat MAP4 or pVHL cDNA sequences. The rat MAP4 siRNA plasmid, Tuball-1, was constructed using the pGensil-1 vector, and the rat pVHL siRNA plasmid, Tuball-3, was constructed with the pGensil-2 vector. These plasmids were then used to construct recombinant adenoviruses respectively. GS1-HK and GS3-EGFP plasmids were used as negative controls.

Immunofluorescence assay of microtubules

Immunocytochemical staining was performed as described previously [52], [54]. Primary antibody was mouse anti-α-microtubulin (1∶100;Abcam), and secondary antibody was fluorescein isothiocyanate(FITC) -conjugated goat anti-mouse secondary antibody(1∶1000; Sigma-Aldrich,). The nuclei were stained by 4,6-diamidino-2-phenylindole(DAPI; 0.5 µg/ml; Biotium). The cells were observed and photographed with a Leica TCS SP5 laser confocal scanning microscope.

Western blot analysis

Cells were washed with ice-cold phosphate-buffered saline (PBS), harvested in 70–200 µL of 1× loading buffer on ice, and homogenized. Lysates were sonicated for 4 s, and separated by centrifugation at 4°C for 2 min at 14000×g. Whole cell extracts were probed with primary antibodies against rat MAP4 (1∶2000;BD Biosciences), pVHL (1∶1000;Cell Signaling Technology), HIF-1α (1∶1000;Chemicon), p38(1∶1000; Cell Signaling Technology, Beverly, MA), phos-pho-p38 (1∶1000, Thr180/Tyr182; Cell SignalingTechnology), and GAPDH (1∶5000;Santa Cruz Biotechnology). GAPDH staining was used as an internal control. Immunocomplexes were visualized and quantified with an enhanced chemiluminescence detection kit (Amersham Pharmacia,Piscataway, NJ), using horseradish peroxidase-conjugated secondary antibodies (1∶2000; Santa Cruz).The results were analyzed with the DOC Gel2000 imaging system (Bio-Rad). Each experiment was repeated twice, and each group was evaluated five times.

Immunoprecipitation

Cells (60 mm dish) were lysed in 300 µl RIPA buffer containing 2 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail. Anti-rat phospho-p38 antibody or anti-rat α-microtubulin antibody was incubated with 150 µl of cell lysate for 6 h at 4°C, then the complexes were precipitated with protein A/G-Sepharose (Santa Cruz Biotechnology) overnight at 4°C. The precipitates were washed three times with PBS at 4°C and probed with anti- phospho-p38 or anti-α-microtubulin antibodies using the Western blotting technique described above.

Statistical analysis

Normal distribution and homoscedasticity tests of the data were confirmed and one-way ANOVA followed by post-hoc tests was performed with SPSS 12.0 software. Results are presented as mean ± standard deviation (SD), and statistical significance was set at P<0.05.

Supporting Information

Figure S1.

Effects of microtubule network alteration on HIF-1α protein levels in normoxic cardiomyocytes. (A) HIF-1α protein expression of normoxic CMs and H9c2 cells under different microtubule interfering treatments. (B) relative protein levels for HIF-1α quantified for each group.

https://doi.org/10.1371/journal.pone.0035017.s001

(TIF)

Figure S2.

CMs and H9c2 cells viability were measured using a cell counting kit (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan). (A–D) shows the CMs and H9c2 cells viability under different experimental treatments. ΔP<0.05 vs. H(hypoxia) group.

https://doi.org/10.1371/journal.pone.0035017.s002

(TIF)

Author Contributions

Conceived and designed the experiments: MT YH. Performed the experiments: MT XJ QZ. Analyzed the data: MT XJ QZ. Contributed reagents/materials/analysis tools: DZ JZ GL. Wrote the paper: MT XJ YH.

References

  1. 1. Kyriakides ZS, Kremastinos DT, Michelakakis NA, Matsakas EP, Demovelis T, et al. (1991) Coronary collateral circulation in coronary artery disease and systemic hypertension. Am J Cardiol 67: 687–690.ZS KyriakidesDT KremastinosNA MichelakakisEP MatsakasT. Demovelis1991Coronary collateral circulation in coronary artery disease and systemic hypertension.Am J Cardiol67687690
  2. 2. Horwitz LD, Fennessey PV, Shikes RH, Kong Y (1994) Marked reduction in myocardial infarct size due to prolonged infusion of an antioxidant during reperfusion. Circulation 89: 1792–1801.LD HorwitzPV FennesseyRH ShikesY. Kong1994Marked reduction in myocardial infarct size due to prolonged infusion of an antioxidant during reperfusion.Circulation8917921801
  3. 3. Huang YS, Li ZQ, Yang ZC (2003) Roles of ischemia and hypoxia and the molecular pathogenesis of post-burn cardiac shock. Burns 29: 828–833.YS HuangZQ LiZC Yang2003Roles of ischemia and hypoxia and the molecular pathogenesis of post-burn cardiac shock.Burns29828833
  4. 4. Huang YS, Zhang JP, Li XH (2007) A serial studies on post-burn shock heart. Burns 33: S14–S15.YS HuangJP ZhangXH Li2007A serial studies on post-burn shock heart.Burns33S14S15
  5. 5. Hu JY, Chu ZG, Han J, Dang YM, Yan H, et al. (2010) The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells. Cellular and Molecular Life Sciences 67: 321–333.JY HuZG ChuJ. HanYM DangH. Yan2010The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells.Cellular and Molecular Life Sciences67321333
  6. 6. Iwai K, Hori M, Kitabatake A, Kurihara H, Uchida K, et al. (1990) Disruption of microtubules as an early sign of irreversible ischemic injury. Immunohistochemical study of in situ canine hearts. Circulation Research 67: 694–706.K. IwaiM. HoriA. KitabatakeH. KuriharaK. Uchida1990Disruption of microtubules as an early sign of irreversible ischemic injury. Immunohistochemical study of in situ canine hearts.Circulation Research67694706
  7. 7. Teng M, Dang YM, Zhang JP, Zhang Q, Fang YD, et al. (2010) Microtubular stability affects cardiomyocyte glycolysis by HIF-1 alpha expression and endonuclear aggregation during early stages of hypoxia. American Journal of Physiology-Heart and Circulatory Physiology 298: H1919–H1931.M. TengYM DangJP ZhangQ. ZhangYD Fang2010Microtubular stability affects cardiomyocyte glycolysis by HIF-1 alpha expression and endonuclear aggregation during early stages of hypoxia.American Journal of Physiology-Heart and Circulatory Physiology298H1919H1931
  8. 8. Mabjeesh NJ, Escuin D, LaVallee TM, Pribluda VS, Swartz GM, et al. (2003) 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell 3: 363–375.NJ MabjeeshD. EscuinTM LaValleeVS PribludaGM Swartz20032ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF.Cancer Cell3363375
  9. 9. Jung YJ, Isaacs JS, Lee S, Trepel J, Neckers L (2003) Microtubule disruption utilizes an NFkappa B-dependent pathway to stabilize HIF-1alpha protein. J Biol Chem 278: 7445–7452.YJ JungJS IsaacsS. LeeJ. TrepelL. Neckers2003Microtubule disruption utilizes an NFkappa B-dependent pathway to stabilize HIF-1alpha protein.J Biol Chem27874457452
  10. 10. Sato H, Nagai T, Kuppuswamy D, Narishige T, Koide M, et al. (1997) Microtubule stabilization in pressure overload cardiac hypertrophy. J Cell Biol 139: 963–973.H. SatoT. NagaiD. KuppuswamyT. NarishigeM. Koide1997Microtubule stabilization in pressure overload cardiac hypertrophy.J Cell Biol139963973
  11. 11. Fang YD, Xu X, Dang YM, Zhang YM, Zhang JP, et al. (2011) MAP4 Mechanism that Stabilizes Mitochondrial Permeability Transition in Hypoxia: Microtubule Enhancement and DYNLT1 Interaction with VDAC1. Plos One 6: e28052.YD FangX. XuYM DangYM ZhangJP Zhang2011MAP4 Mechanism that Stabilizes Mitochondrial Permeability Transition in Hypoxia: Microtubule Enhancement and DYNLT1 Interaction with VDAC1.Plos One6e28052
  12. 12. Turcotte S, Desrosiers RR, Beliveau R (2004) Hypoxia upregulates von Hippel-Lindau tumor-suppressor protein through RhoA-dependent activity in renal cell carcinoma. Am J Physiol Renal Physiol 286: F338–348.S. TurcotteRR DesrosiersR. Beliveau2004Hypoxia upregulates von Hippel-Lindau tumor-suppressor protein through RhoA-dependent activity in renal cell carcinoma.Am J Physiol Renal Physiol286F338348
  13. 13. Kamura T, Sato S, Iwai K, Czyzyk-Krzeska M, Conaway RC, et al. (2000) Activation of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. Proc Natl Acad Sci U S A 97: 10430–10435.T. KamuraS. SatoK. IwaiM. Czyzyk-KrzeskaRC Conaway2000Activation of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex.Proc Natl Acad Sci U S A971043010435
  14. 14. Iwai K, Yamanaka K, Kamura T, Minato N, Conaway RC, et al. (1999) Identification of the von Hippel-lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex. Proc Natl Acad Sci U S A 96: 12436–12441.K. IwaiK. YamanakaT. KamuraN. MinatoRC Conaway1999Identification of the von Hippel-lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex.Proc Natl Acad Sci U S A961243612441
  15. 15. Mole DR, Maxwell PH, Pugh CW, Ratcliffe PJ (2001) Regulation of HIF by the von Hippel-Lindau tumour suppressor: implications for cellular oxygen sensing. IUBMB Life 52: 43–47.DR MolePH MaxwellCW PughPJ Ratcliffe2001Regulation of HIF by the von Hippel-Lindau tumour suppressor: implications for cellular oxygen sensing.IUBMB Life524347
  16. 16. Sufan RI, Moriyama EH, Mariampillai A, Roche O, Evans AJ, et al. (2009) Oxygen-independent degradation of HIF-alpha via bioengineered VHL tumour suppressor complex. EMBO Mol Med 1: 66–78.RI SufanEH MoriyamaA. MariampillaiO. RocheAJ Evans2009Oxygen-independent degradation of HIF-alpha via bioengineered VHL tumour suppressor complex.EMBO Mol Med16678
  17. 17. Miro-Murillo M, Elorza A, Soro-Arnaiz I, Albacete-Albacete L, Ordonez A, et al. (2011) Acute Vhl Gene Inactivation Induces Cardiac HIF-Dependent Erythropoietin Gene Expression. Plos One 6: e22589.M. Miro-MurilloA. ElorzaI. Soro-ArnaizL. Albacete-AlbaceteA. Ordonez2011Acute Vhl Gene Inactivation Induces Cardiac HIF-Dependent Erythropoietin Gene Expression.Plos One6e22589
  18. 18. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, et al. (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399: 271–275.PH MaxwellMS WiesenerGW ChangSC CliffordEC Vaux1999The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.Nature399271275
  19. 19. Yu F, White SB, Zhao Q, Lee FS (2001) HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci U S A 98: 9630–9635.F. YuSB WhiteQ. ZhaoFS Lee2001HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation.Proc Natl Acad Sci U S A9896309635
  20. 20. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, et al. (2001) Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292: 468–472.P. JaakkolaDR MoleYM TianMI WilsonJ. Gielbert2001Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.Science292468472
  21. 21. Gundersen GG, Cook TA (1999) Microtubules and signal transduction. Curr Opin Cell Biol 11: 81–94.GG GundersenTA Cook1999Microtubules and signal transduction.Curr Opin Cell Biol118194
  22. 22. Yang Y, Zhu X, Chen Y, Wang X, Chen R (2007) p38 and JNK MAPK, but not ERK1/2 MAPK, play important role in colchicine-induced cortical neurons apoptosis. Eur J Pharmacol 576: 26–33.Y. YangX. ZhuY. ChenX. WangR. Chen2007p38 and JNK MAPK, but not ERK1/2 MAPK, play important role in colchicine-induced cortical neurons apoptosis.Eur J Pharmacol5762633
  23. 23. Subbaramaiah K, Hart JC, Norton L, Dannenberg AJ (2000) Microtubule-interfering agents stimulate the transcription of cyclooxygenase-2. Evidence for involvement of ERK1/2 AND p38 mitogen-activated protein kinase pathways. J Biol Chem 275: 14838–14845.K. SubbaramaiahJC HartL. NortonAJ Dannenberg2000Microtubule-interfering agents stimulate the transcription of cyclooxygenase-2. Evidence for involvement of ERK1/2 AND p38 mitogen-activated protein kinase pathways.J Biol Chem2751483814845
  24. 24. Seidman R, Gitelman I, Sagi O, Horwitz SB, Wolfson M (2001) The role of ERK 1/2 and p38 MAP-kinase pathways in taxol-induced apoptosis in human ovarian carcinoma cells. Experimental Cell Research 268: 84–92.R. SeidmanI. GitelmanO. SagiSB HorwitzM. Wolfson2001The role of ERK 1/2 and p38 MAP-kinase pathways in taxol-induced apoptosis in human ovarian carcinoma cells.Experimental Cell Research2688492
  25. 25. Khandrika L, Lieberman R, Koul S, Kumar B, Maroni P, et al. (2009) Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1alpha levels contribute to emergence of an aggressive phenotype in prostate cancer. Oncogene 28: 1248–1260.L. KhandrikaR. LiebermanS. KoulB. KumarP. Maroni2009Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1alpha levels contribute to emergence of an aggressive phenotype in prostate cancer.Oncogene2812481260
  26. 26. Caretti A, Morel S, Milano G, Fantacci M, Bianciardi P, et al. (2007) Heart HIF-1alpha and MAP kinases during hypoxia: are they associated in vivo? Exp Biol Med (Maywood) 232: 887–894.A. CarettiS. MorelG. MilanoM. FantacciP. Bianciardi2007Heart HIF-1alpha and MAP kinases during hypoxia: are they associated in vivo?Exp Biol Med (Maywood)232887894
  27. 27. Raingeaud J, Whitmarsh AJ, Barrett T, Derijard B, Davis RJ (1996) MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Molecular & Cellular Biology 16: 1247–1255.J. RaingeaudAJ WhitmarshT. BarrettB. DerijardRJ Davis1996MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway.Molecular & Cellular Biology1612471255
  28. 28. Risinger AL, Giles FJ, Mooberry SL (2009) Microtubule dynamics as a target in oncology. Cancer Treatment Reviews 35: 255–261.AL RisingerFJ GilesSL Mooberry2009Microtubule dynamics as a target in oncology.Cancer Treatment Reviews35255261
  29. 29. Hescheler J, Meyer R, Plant S, Krautwurst D, Rosenthal W, et al. (1991) Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circulation Research 69: 1476–1486.J. HeschelerR. MeyerS. PlantD. KrautwurstW. Rosenthal1991Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart.Circulation Research6914761486
  30. 30. Zordoky BN, El-Kadi AO (2007) H9c2 cell line is a valuable in vitro model to study the drug metabolizing enzymes in the heart. J Pharmacol Toxicol Methods 56: 317–322.BN ZordokyAO El-Kadi2007H9c2 cell line is a valuable in vitro model to study the drug metabolizing enzymes in the heart.J Pharmacol Toxicol Methods56317322
  31. 31. Cassimeris L (1999) Accessory protein regulation of microtubule dynamics throughout the cell cycle. Curr Opin Cell Biol 11: 134–141.L. Cassimeris1999Accessory protein regulation of microtubule dynamics throughout the cell cycle.Curr Opin Cell Biol11134141
  32. 32. Ke QD, Costa M (2006) Hypoxia-inducible factor-1 (HIF-1). Molecular Pharmacology 70: 1469–1480.QD KeM. Costa2006Hypoxia-inducible factor-1 (HIF-1).Molecular Pharmacology7014691480
  33. 33. Semenza GL (2000) HIF-1 and human disease: one highly involved factor. Genes Dev 14: 1983–1991.GL Semenza2000HIF-1 and human disease: one highly involved factor.Genes Dev1419831991
  34. 34. Thomas SL, Zhong D, Zhou W, Malik S, Liotta D, et al. (2008) EF24, a novel curcumin analog, disrupts the microtubule cytoskeleton and inhibits HIF-1. Cell Cycle 7: 2409–2417.SL ThomasD. ZhongW. ZhouS. MalikD. Liotta2008EF24, a novel curcumin analog, disrupts the microtubule cytoskeleton and inhibits HIF-1.Cell Cycle724092417
  35. 35. Di Cristofano C, Minervini A, Menicagli M, Salinitri G, Bertacca G, et al. (2007) Nuclear expression of hypoxia-inducible factor-1 alpha in clear cell renal cell carcinoma is involved in tumor progression. American Journal of Surgical Pathology 31: 1875–1881.C. Di CristofanoA. MinerviniM. MenicagliG. SalinitriG. Bertacca2007Nuclear expression of hypoxia-inducible factor-1 alpha in clear cell renal cell carcinoma is involved in tumor progression.American Journal of Surgical Pathology3118751881
  36. 36. Frew IJ, Krek W (2007) Multitasking by pVHL in tumour suppression. Current Opinion in Cell Biology 19: 685–690.IJ FrewW. Krek2007Multitasking by pVHL in tumour suppression.Current Opinion in Cell Biology19685690
  37. 37. Czyzyk-Krzeska MF, Meller J (2004) von Hippel-Lindau tumor suppressor: not only HIF's executioner. Trends in Molecular Medicine 10: 146–149.MF Czyzyk-KrzeskaJ. Meller2004von Hippel-Lindau tumor suppressor: not only HIF's executioner.Trends in Molecular Medicine10146149
  38. 38. Ivan M, Kondo K, Yang H, Kim W, Valiando J, et al. (2001) HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292: 464–468.M. IvanK. KondoH. YangW. KimJ. Valiando2001HIFalpha targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing.Science292464468
  39. 39. Haase VH (2006) The VHL/HIF oxygen-sensing pathway and its relevance to kidney disease. Kidney Int 69: 1302–1307.VH Haase2006The VHL/HIF oxygen-sensing pathway and its relevance to kidney disease.Kidney Int6913021307
  40. 40. Min JH, Yang H, Ivan M, Gertler F, Kaelin WG Jr, et al. (2002) Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling. Science 296: 1886–1889.JH MinH. YangM. IvanF. GertlerWG Kaelin Jr2002Structure of an HIF-1alpha -pVHL complex: hydroxyproline recognition in signaling.Science29618861889
  41. 41. del Peso L, Castellanos MC, Temes E, Martin-Puig S, Cuevas Y, et al. (2003) The von Hippel Lindau/hypoxia-inducible factor (HIF) pathway regulates the transcription of the HIF-proline hydroxylase genes in response to low oxygen. J Biol Chem 278: 48690–48695.L. del PesoMC CastellanosE. TemesS. Martin-PuigY. Cuevas2003The von Hippel Lindau/hypoxia-inducible factor (HIF) pathway regulates the transcription of the HIF-proline hydroxylase genes in response to low oxygen.J Biol Chem2784869048695
  42. 42. Cai Q, Verma SC, Kumar P, Ma M, Robertson ES (2010) Hypoxia inactivates the VHL tumor suppressor through PIASy-mediated SUMO modification. Plos One 5: e9720.Q. CaiSC VermaP. KumarM. MaES Robertson2010Hypoxia inactivates the VHL tumor suppressor through PIASy-mediated SUMO modification.Plos One5e9720
  43. 43. Cai QL, Robertson ES (2010) Ubiquitin/SUMO Modification Regulates VHL Protein Stability and Nucleocytoplasmic Localization. Plos One 5: e12636.QL CaiES Robertson2010Ubiquitin/SUMO Modification Regulates VHL Protein Stability and Nucleocytoplasmic Localization.Plos One5e12636
  44. 44. Ampofo E, Kietzmann T, Zimmer A, Jakupovic M, Montenarh M, et al. (2010) Phosphorylation of the von Hippel-Lindau protein (VHL) by protein kinase CK2 reduces its protein stability and affects p53 and HIF-1alpha mediated transcription. Int J Biochem Cell Biol 42: 1729–1735.E. AmpofoT. KietzmannA. ZimmerM. JakupovicM. Montenarh2010Phosphorylation of the von Hippel-Lindau protein (VHL) by protein kinase CK2 reduces its protein stability and affects p53 and HIF-1alpha mediated transcription.Int J Biochem Cell Biol4217291735
  45. 45. Bacus SS, Gudkov AV, Lowe M, Lyass L, Yung Y, et al. (2001) Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene 20: 147–155.SS BacusAV GudkovM. LoweL. LyassY. Yung2001Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53.Oncogene20147155
  46. 46. Suomalainen M, Nakano MY, Boucke K, Keller S, Greber UF (2001) Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-mediated nuclear targeting of virus. EMBO J 20: 1310–1319.M. SuomalainenMY NakanoK. BouckeS. KellerUF Greber2001Adenovirus-activated PKA and p38/MAPK pathways boost microtubule-mediated nuclear targeting of virus.EMBO J2013101319
  47. 47. Cvetkovic I, Miljkovic D, Vuckovic O, Harhaji L, Nikolic Z, et al. (2004) Taxol activates inducible nitric oxide synthase in rat astrocytes: the role of MAP kinases and NF-kappaB. Cellular and Molecular Life Sciences 61: 1167–1175.I. CvetkovicD. MiljkovicO. VuckovicL. HarhajiZ. Nikolic2004Taxol activates inducible nitric oxide synthase in rat astrocytes: the role of MAP kinases and NF-kappaB.Cellular and Molecular Life Sciences6111671175
  48. 48. Weidemann A, Johnson RS (2008) Biology of HIF-1 alpha. Cell Death and Differentiation 15: 621–627.A. WeidemannRS Johnson2008Biology of HIF-1 alpha.Cell Death and Differentiation15621627
  49. 49. Czibik G, Martinov V, Ruusalepp A, Sagave J, Skare O, et al. (2009) In vivo remote delivery of DNA encoding for hypoxia-inducible factor 1 alpha reduces myocardial infarct size. Clin Transl Sci 2: 33–40.G. CzibikV. MartinovA. RuusaleppJ. SagaveO. Skare2009In vivo remote delivery of DNA encoding for hypoxia-inducible factor 1 alpha reduces myocardial infarct size.Clin Transl Sci23340
  50. 50. Czibik G, Gravning J, Martinov V, Ishaq B, Knudsen E, et al. (2011) Gene therapy with hypoxia-inducible factor 1 alpha in skeletal muscle is cardioprotective in vivo. Life Sci 88: 543–550.G. CzibikJ. GravningV. MartinovB. IshaqE. Knudsen2011Gene therapy with hypoxia-inducible factor 1 alpha in skeletal muscle is cardioprotective in vivo.Life Sci88543550
  51. 51. Xiao J, Cao H, Liang D, Liu Y, Zhang H, et al. (2011) Taxol, a microtubule stabilizer, prevents ischemic ventricular arrhythmias in rats. J Cell Mol Med 15: 1166–1176.J. XiaoH. CaoD. LiangY. LiuH. Zhang2011Taxol, a microtubule stabilizer, prevents ischemic ventricular arrhythmias in rats.J Cell Mol Med1511661176
  52. 52. Vandroux D, Schaeffer C, Tissier C, Lalande A, Bes S, et al. (2004) Microtubule alteration is an early cellular reaction to the metabolic challenge in ischemic cardiomyocytes. Molecular and cellular biochemistry 258: 99–108.D. VandrouxC. SchaefferC. TissierA. LalandeS. Bes2004Microtubule alteration is an early cellular reaction to the metabolic challenge in ischemic cardiomyocytes.Molecular and cellular biochemistry25899108
  53. 53. Zhang JP, Liang WY, Luo ZH, Yang ZC, Chan HC, et al. (2007) Involvement of p38 map kinase in burn-induced degradation of membrane phospholipids and upregulation of cPLA(2) in cardiac myocytes. Shock 28: 86–93.JP ZhangWY LiangZH LuoZC YangHC Chan2007Involvement of p38 map kinase in burn-induced degradation of membrane phospholipids and upregulation of cPLA(2) in cardiac myocytes.Shock288693
  54. 54. Webster DR (1997) Regulation of post-translationally modified microtubule populations during neonatal cardiac development. Journal of molecular and cellular cardiology 29: 1747–1761.DR Webster1997Regulation of post-translationally modified microtubule populations during neonatal cardiac development.Journal of molecular and cellular cardiology2917471761