The overwhelming majority of clinical heart failure cases are the consequence of primary myocardial dysfunction with depressed cardiac output (1). A reduced blood flow in coronary arteries may cause hypoxia in tissues downstream of the lesion (2). The progression to heart failure caused by reasons such as myocardial infarction is associated with a decline in the activity of mitochondrial respiratory pathways leading to diminished capacity for adenosine triphosphate (ATP) production (3, 4). Mitochondrial oxidative phosphorylation is the primary energy source in the myocardial cell. The molecular mechanism of mitochondrial damage and dysfunction to cardiomyocytes and its contribution to the outcome after injury to the heart is not fully known (5, 6). Mitochondria are often referred as the power house of a cell, as its main function is the production of energy in the form of ATP through the process of oxidative phosphorylation. Furthermore, decreased bioenergetics may compromise organ system functional reserve predisposing to increased probability of adverse outcome (5-8). In addition, reduced capacity for energy transduction leads to secondary dysregulation of cellular processes critical for cardiac pump function, including Ca2+ handling and contractile function, which intensifies increased energy demand and diminished function (3, 9).
Alterations in the function of mitochondria are not solely caused by changes in transcripts encoded only in the mitochondrial genome, most of the genetic information for the mitochondrial biogenesis and function resides in the nuclear genome (2, 3, 10). To study mitochondrial gene alterations in health and disease, we developed a mitochondrial gene chip, RoMitoChip (Rodent Mitochochondrial gene Chip), a focused microarray. The exact number of mammalian mitochondrial proteins is not known, although those derived from mitochondrial DNA (mtDNA) are well defined. Indirect evidences suggest approximately 1,200 proteins in this organelle (11). A recent proteomic study has identified approximately 600 mitochondrial proteins in the mouse, and another study identified approximately 700 in the rat (11, 12). Although most of the mitochondrial genes encoded in the nuclear genome are represented in the Affymetrix Mouse 430 2.0 and Affymetrix Rat 230 2.0 GeneChip, the lack of representation of genes of mtDNA in these chips and the presence of several thousand probe sets, and hence the need to analyze several thousand genes that demands time and computing power, prompted us to develop the RoMitoChip. This chip was developed on an Affymetrix platform, and in this article, we report the hypoxia-responsive genes in mouse cardiomyocytes determined using the RoMitoChip.
Mouse cardiomyocytes and hypoxic exposure
Mouse cardiomyocytes isolated from neonatal C57BL/6 mice were obtained from a commercial source (Sciencell Research Laboratories, Carlsbad, Calif) as a custom product. The cells were cultured for 4 days, and the batches that passed the strict quality control were shipped frozen for the investigations. The frozen vials were thawed and were cultured in cardiac myocyte medium (Sciencell Research Laboratories). After 4 days in culture, the cells were exposed to normoxia or 1% hypoxia in a Queue Systems incubator (Parkersburg, WV) for 8 or 24 h. RNA was isolated, and gene expression profiles were determined using the RoMitoChip developed in our laboratory as described in this communication.
Microarray: gene selection
The genes represented in the mitochondrial and nuclear genome of the rat and the mouse that contribute to mitochondrial structure and function were identified. Mitochondrial genome consists of a circular DNA (mtDNA) with about 16,000 bases and 37 transcripts. Of these 37 transcripts, 13 code for proteins, two code for ribosomal RNAs (12 and 16S), and 22 code for tRNAs. The 13 proteins on the mtDNA are seven subunits of NADH dehydrogenase, cytochrome b, three cytochrome c oxidase subunits (1, 2, and 3), and ATP synthases 6 and 8. We placed both mouse and rat mitochondrial genes on the same chip.
The gene sequences representing the mouse mitochondrial genome included in the gene chip were composed of the published mtDNA sequences of the strains C57BL/6J (accession no. AY172335), NZB/B1NJ (accession no. L07095), Balb/cJ (accession no. AJ512208), AKR/J (accession no. AB042432), and C3H/He (accession no. AB049357). The 37 transcripts were represented by a total of 46 different probe sets for mouse mtDNA.
The gene sequences representing the rat mitochondrial genome included in the gene chip encompassed published mtDNA sequences of 10 different inbred strains: WKY/NCrl (Wistar Kyoto, accession no. DQ673907); BN/NHsdMcwi (Brown Norway, accession no. AC_000022); F344/NHsd (Fisher 344, accession no. DQ673909); ACI/Eur (August × Copenhagen Irish, accession no. DQ673908); FHH/Eur (fawn hooded hypertensive, accession no. DQ673910); GK/Swe (accession no. DQ673913); GK/Far (accession no. DQ673912); T2DN/Mcwi (accession no. DQ673915); GH/OmrMcwi (accession no. DQ673911); and SS/JrHsdMcwi (Dahl Salt-sensitive, accession no. DQ673914) (13). These specific strains were chosen because complete sequences of mtDNA were available for each of them. Each of the genes from these 10 different strains was aligned, and 48 different probe sets were created to represent all the 37 transcripts from these 10 strains.
We used four different databases to identify and extract nuclear genes important in the structure and function of mitochondria. These were the mouse genome database of the Jackson Laboratory, the Mitop2 database, the Rat Genome database from Wisconsin, and the NetAffyx. In addition, some relevant publications and the NCBI database were also used.
Probe sets corresponding to these genes were placed on an 11-μm chip as antisense probe sets, on Affymetrix platform. The chip contained a total of 1,088 probe sets representing genes from mitochondrial and nuclear DNA from the mouse and 419 from the rat. The experiments described in this communication harness the mouse probe sets on this chip. Each probe set consists of 11 oligonucleotide probes directed against the same target transcript, along with 11 control probes with single base pair mismatches; these function as a combined unit to assess transcript levels and determine the background hybridization for each probe, thereby establishing internal controls for the hybridization signals of each gene (14).
RNA was isolated from snap-frozen cardiomyocytes, and RNA integrity was checked by resolving on an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, Calif). One hundred nanograms of total RNA was amplified from each RNA sample and labeled using the Affymetrix Whole-Transcript Assay Sense Target Labeling Protocol. Ribosomal RNA was not removed. RoMitoChip gene arrays were hybridized with 11 μg of labeled sense DNA. To compare the results obtained using the RoMitoChip to that of a gold standard, Affymetrix Gene 1.0 ST chips were also hybridized with the same pool of labeled sense DNA. The hybridized chips were washed using the Affymetrix fluidics station 450, stained, and scanned on the 3000 7G scanner as described by the manufacturer (Affymetrix, Santa Clara, Calif). GeneChip hybridizations were carried out in the Gene Expression Shared Facility of the Comprehensive Cancer Center at the University of Alabama at Birmingham. Four chips were used for each treatment group.
Gene expression data were normalized using RMA (15) and quantile normalization methods. After normalization, ANOVA was applied to compare gene expression level changes for three time points. P values for pairwise comparisons among three time points were adjusted using Tukey method in ANOVA analysis. The final P values were adjusted using the FDR method of Hochberg and Benjamini (16). We used statistical software R and SAS v9.13 for data analysis.
Real-time polymerase chain reaction (PCR) was carried out using FAM-labeled Taqman real-time PCR primers for Pdk1, Bnip3, and β-actin (ABI, Foster City, Calif). The template cDNA was prepared by random priming from RNA isolated from cardiomyocytes subjected to normoxia or hypoxia. The results were expressed in relation to β-actin expression. The PCR reaction was carried out in an ABI 7500 thermal cycler (ABI).
Protein expression of Bnip3 and Pdk1 were analyzed by Western Blot as described (17). Briefly, total proteins in cardiomyocyte lysates were resolved using 4% to 12% Nupage gel (Invitrogen, Carlsbad, Calif) and transferred to polyvinylidene fluoride membranes. The membranes were saturated with blocking buffer (10 mM Tris, 150 mM NaCl, and 0.05% Tween-20 supplemented with 5% dry milk) for 1 h at room temperature and incubated with the respective primary antibodies: Bnip3 (Abcam, Inc, Cambridge, Mass), Pdk1 (Cell Singling Technology, Beverly, Calif), and β-actin (Abcam, Inc). The membranes were then washed five times with Tris-buffered saline supplemented with 0.05% Tween-20 followed by incubation with an appropriate secondary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif) conjugated with horseradish peroxidase for 1 h at room temperature. The membranes were again washed five times with Tris-buffered saline supplemented with 0.05% Tween-20 and probed using enhanced chemiluminescence (Amersham, Piscataway, NJ) and autoradiographed.
Mouse cardiomyocytes were subjected to hypoxia (1% oxygen) for 24 h or normoxia, and quantitative determination of total ATP was performed by a bioluminescence assay (ATP determination kit; Invitrogen). The assay used a recombinant firefly luciferase and its substrate d-luciferin. Briefly, reaction solution containing luciferase and luciferin are plated, and background luminescence was measured. The ATP standard solution or sample containing ATP was added to respective wells, and luminescence was measured. After subtracting the background luminescence, ATP concentration was deduced from the standard curve and normalized to total protein concentration.
In the present study, we examined the alteration of mitochondrial gene expression in cardiomyocytes in an in vitro hypoxia model using a custom-made microarray method. Mouse cardiomyocytes were subjected to normoxia or hypoxia for 8 or 24 h, and the alteration in mitochondrial gene expression was assessed using the custom mitochondrial gene chip, RoMitoChip. Hypoxia was confirmed by reduction in protein-normalized ATP level (18) to 56% after 24 h of hypoxia. When the best-matched probes of Gene ST 1.0 chip were compared with that of RoMitochip, we obtained a good correlation (0.89) (data not shown). The mitochondrial gene chip contained genes from the nuclear as well as the mitochondrial DNA, which were reported to be important in the structure and function of the mitochondria.
Gene expression changes after hypoxia
The probe sets representing 483 of the 1,088 mouse probe sets demonstrated significant changes (P < 0.05) in expression levels after hypoxia at least at one of the three time intervals tested (0 h versus 8 h, 0 h versus 24 h, or 8 h versus 24 h) (See Table, Supplemental Digital Content 1, http://links.lww.com/SHK/A35). When the expression levels of these genes at 8 h were compared with that at normoxia, there were 263 probe sets with decreased expression and 220 probe sets showed an increase (see Table, Supplemental Digital Content 1, http://links.lww.com/SHK/A35). At 24 h, when compared with normoxia, 201 probe sets showed increased expression whereas 282 registered a decrease. When the genes with the most altered expression levels were sorted, the expression of 27 genes were upregulated by 2-fold or more (Table 1) when 21 genes were downregulated by at least 50% at 24 h after hypoxia (Table 2). Among the top five genes that demonstrated significant upregulation at 24 h were pyruvate dehydrogenase kinase, isoenzyme 1 (Pdk1); cytochrome c oxidase subunit IV isoform 2 (Cox4i2); bcl2/adenovirus e1b-interacting protein 1 (Bnip3); adenylate kinase 3α-like 1 (Ak3l1); and aldolase 3 (Aldoc).
The expression patterns of several genes were dynamic during the 24-h period. There were 24 such genes, the expressions of which were significantly changed (P < 0.05) at all three different time intervals tested, 0 h (normoxia) versus 8 h, 0 h versus 24 h, and 8 h versus 24 h. Among these genes were Vdac1, Bnip3, and MT-CO3 (Fig. 1). Vdac1 and Bnip3 showed an increased expression, 2- and 10-fold, respectively, at 24 h after hypoxia. MT-CO3 demonstrated a decrease in expression at 8 h. The increased expression of a subset of genes, Bnip3 and pdk1, were further confirmed by real-time PCR (Fig. 2A, B) and western blot (Fig. 2C).
Alteration of genes on the mtDNA after hypoxia
When the expression profile of genes on the mtDNA was analyzed, two clusters of genes were evident, those that increased at 24 h and those that decreased at 24 h. The expression of 26 of 46 probe sets representing the 37 mtDNA transcripts changed significantly after the hypoxic exposure. These 26 probe sets represented 23 genes, considering the duplicate probe sets for Tn, Te, and Tw (transcripts for asparagine, glutamic acid, and tryptophan tRNA). The 23 genes included both protein-coding mRNA as well as several tRNAs. As seen in Figure 3, when almost all the tRNAs were increased at 24 h, the levels of most of the protein-coding transcripts were decreased from the basal level.
The information on molecular changes occurring in mitochondria of the cardiomyocytes after conditions such as hypoxia is important in understanding the molecular basis of the pathobiology associated with low-oxygen conditions (1, 2, 19). Studies have shown that alterations in activity or expression of transcription factors acting on nuclear DNA mediate the shifts in energy production and substrate utilization (11, 20, 21). In one recent investigation of quantitative relationships between gene expression and left ventricular hypertrophy, metabolic genes involved in various mitochondrial energy production mechanisms were reported to be reduced with increased left ventricular size (22). Therefore, the nuclear-mitochondrial cross-talk is an essential factor in maintaining energy balance and mitochondrial function within the cell.
It is important to note that genes on the H or L strand of mtDNA are transcribed as a single polycistronic RNA, which is then cleaved and polyadenylated. As extensive studies have shown functional autonomy for H and L strand transcriptions, the well-studied cis-acting elements and less known trans-acting (nuclear encoded) elements that affect mtDNA transcription can influence relative transcription of each strand. In addition, polycistronic transcription of each strand does not ensure equimolar quantities of mRNA species to be derived from the respective strands (23, 24). This is because of varying known and unknown factors that control the generation and stability of each mRNA through systematic cleavage and polyadenylation. Such factors vary with cellular compartments, organisms, and the environment (23, 24). This is reaffirmed by experimental observations as reported by Piechota et al. (25) of up to 10-fold difference in the steady state levels among mitochondrial mRNA transcripts and their widely varying decay rates.
When the gene symbols for the 483 probe sets, the expressions of which were significantly altered after hypoxia in cardiomyocytes, were analyzed using DAVID (National Institutes of Health, Bethesda, Md) to cluster them in functional pathways, among the most represented pathways were apoptosis (43 genes), oxidative phosphorylation (40 genes), followed by those involved in fatty acid oxidation (20 genes). Among the most upregulated genes after 24-h hypoxia were hypoxia-regulated genes such as Bnip3 (10-fold at 24 h and 3-fold at 8 h), Aldoc (5.6-fold at 24 h), and Pdk1 (21-fold at 24 h and 3.6-fold at 8 h). The significant augmentation of Bnip3 is consistent with previous findings by Kubasiak et al. (26) that chronic hypoxia induced the expression and accumulation of Bnip3 mRNA, and overexpression of Bnip3 activated cardiac myocyte death (26). Consistent with these was the observation of a set of 43 apoptosis-related genes to be significantly altered after 24 h of hypoxia (Fig. 4). Furthermore, it is also suggested that autophagy after hypoxic insult may be mediated by Bnip3 (27). Therefore, the 10-fold increased expression of Bnip3 transcript after hypoxic exposure of cardiomyocytes is an indication of its role in cell death under low-oxygen conditions. Bnip3 is reported to be able to kill cardiomyocytes through caspase-dependent apoptosis, as well as in a manner that does not require caspase activation but involve mitochondrial permeability transition (1). Bnip3 is also reported to interact with survival protein Bcl2, suggesting that Bcl2 could suppress the death-inducing activity of Bnip3 (28). In our experiments, Bcl2 was also significantly (P < 0.05) upregulated by 2- and 1.4-fold, respectively, after 8 and 24 h hypoxic exposure (see Table, Supplemental Digital Content 1, http://links.lww.com/SHK/A35). The interaction of Bnip3 with other genes as reported in literature was created using the program Pathway Architecture and is shown in Figure 5. As seen in this illustration, sp1 transcription factor, which may function independent of hypoxia-inducible factor 1, α subunit (HIF-1α), is downregulated after hypoxia in cardiomyocytes and is consistent with the lack of upregulation of Akt, the upregulation of which is dependent on sp1.
The Bnip3-induced cell death is also characterized by increased production of reactive oxygen species (ROS). The enzyme superoxide dismutase (Sod) plays a key role in scavenging the ROS generated. Both the cytosolic (Sod1) and the mitochondrial (Sod2) isoforms of Sod were significantly decreased (P < 0.05) at the end of 24 h of hypoxic exposure of cardiomyocytes. We used gene-specific knockout mice heterozygous for Sod2 mutation (Sod2+/−) and their wild-type littermates to test mitochondrial gene alterations and observed the aberrant expression of only a few genes in the left ventricles of these mice (data not shown). It may be noted that although a mitochondrial deficiency is reported in the Sod2 heterozygous mice, these mice do not exhibit any pathology, whereas the Sod2 null mice do not survive.
Glycolysis versus mitochondrial oxidation
Under hypoxic conditions, cells augment glycolysis to compensate for the reduced ATP synthesis, the observed increase of aldolase (Aldoc) and another key enzyme in the glycolytic pathway, hexokinase 1 (HK1), further establishes the promotion of glycolysis (Table 1). The increased glycolytic flux demands an increased expression of genes involved in glycolysis. In fact, seven of 10 genes involved in the glycolytic process and altered significantly in our experiments were upregulated after hypoxia (Table 3). Furthermore, increased Pdk1 inactivates pyruvate dehydrogenase (Pdh) enzyme complex by phosphorylation. Pyruvate dehydrogenase converts pyruvate to acetyl CoA, and therefore, its inhibition slows down mitochondrial oxidation, preventing excess ROS production (29). In addition, among the most upregulated genes, Aldoc, Bnip3, HK1, and Pdk1 are known to be directly regulated by HIF-1α, and their expression levels continued to remain increased even after 24 h. The HIF-1 is an oxygen-sensing transcription factor that initiates transcription of an array of genes during hypoxic insult (30, 31). These results clearly indicate the effect of hypoxia in promoting glycolysis while reducing ATP production through mitochondrial oxidation in cardiomyocytes subjected to hypoxia. This metabolic adaptation is critical for the function of cardiomyocytes in low oxygen states as observed in conditions involving reduced blood flow in coronary arteries. The results previously described also demonstrate the inhibition of pyruvate catabolism and respiration in cardiomyocytes after hypoxia, as observed in other cell types (29). In a B-cell line, it has been shown that active suppression of the tricarboxylic acid cycle and shunting of pyruvate to lactate via inactivation of Pdh by Pdk1 are required for cell survival under prolonged hypoxic conditions (29). Forced expression of Pdk1 by the same investigators inhibited hypoxia-induced apoptosis in the absence of HIF1. These experiments demonstrate the critical role that Pdk1 plays under hypoxic conditions in conserving cellular energy and considering the significant upregulation of this gene in cardiomyocytes subjected to hypoxia, we speculate a similar function for Pdk1 in the heart.
The expression pattern of the transcripts originating from mtDNA was unexpected and not observed so far. Although the reason for the increase of the levels of tRNAs of mtDNA is not clear, it is likely caused by altered translational regulation after hypoxia. It is known that hypoxia can alter translational regulation, enhancing the translation of specific genes (32). In response to hypoxic conditions, cells reduce their overall rate of mRNA translation; but individual mRNA species are affected to highly varying degrees, with some even translationally stimulated under these conditions (32, 33). The elevated levels of tRNAs is likely complementing the increased translation of specific genes that are needed for cell survival as well as allowing cells to function at a low-energy supply state. When almost all the tRNAs located on mtDNA were increased and the expression levels were significantly altered at 24 h, the transcript levels of most of the mtDNA-derived protein-coding mRNA were decreased (Fig. 3). However, at 24 h, the expression levels of the tRNAs began decreasing from the elevated levels at 8 h. Therefore, when the expression profiles of genes for the mtDNA were analyzed, two clusters of genes were evident, those that increased at 24 h and those that decreased at 24 h. Further studies on the molecular basis of the dichotomy in changes in the expression of transcripts of mtDNA origin would be useful in further understanding this novel observation after hypoxia.
The hypoxia-induced expression profile of mitochondria-associated genes clearly demonstrates the activation of both death and survival pathways in cardiomyocytes. The novel observation on the differential expression profile of tRNA transcripts versus protein-coding transcripts of mtDNA may be attributed to transcription-translation regulation to enable the survival of the cells.
The authors thank Dr Ambalavanan Namasivayam, Department of Pediatrics, UAB, for providing the hypoxia incubator facility (R01 HL092906). The microarray experiments were carried out in the Heflin Center for Genomic Science by Dr Michael Crowley.
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