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expression in myocardial ischemia
Section of Cardiology, Center for Cardiovascular Research, University of Illinois, Chicago, Illinois
Submitted 1 June 2005 ; accepted in final form 28 March 2006
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ABSTRACT |
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(HIF-1) plays a role in a number of cell-protective pathways after ischemia. There are clear sex-related differences in the remodeling process, and hearts from males tend to dilate in response to pathological loads and ischemia to a greater degree than do hearts from females. Thus we hypothesized that there would be a sex-related dimorphic response of HIF-1 to an ischemic event. Male and female rats were euthanized 5 and 24 h after coronary ligation (M-MI and F-MI; MI, myocardial ischemia), and HIF-1 expression was determined by immunohistochemistry, Western blot, and quantitative RT-PCR. Sham-operated male and female animals served as controls (M-SH and F-SH). In the ischemic area, histochemical analysis at 5 h showed that HIF was expressed in 33% of cell nuclei in M-MI and in 55% in F-MI. At 24 h, HIF expression increased to 49% in M-MI and to 82% in F-MI (P < 0.05 vs. SH and also M-MI vs. F-MI). This difference was not only statistically significant between the two sexes at 24 h but also within each sex at 5 and 24 h after ligation. Western blots confirmed that, at 24 h after ischemia, HIF protein increased significantly in both male and female hearts relative to sham-operated animals but that the increase in females was 60% greater than that seen in males. mRNA expression of HIF was significantly increased at 24 h in F-MI versus M-MI and sham-operated animals. Expression of downstream HIF target genes (heme oxygenase and brain natriuretic peptide) was increased in proportion to the levels of HIF expression. These data suggest a novel cellular mechanism to explain the sex-related dimorphic response to ischemia and also the possibility that exogenous modulation of HIF might represent a new therapeutic approach to preventing left ventricular remodeling.
hypoxia-inducible factor-1; gene expression; remodeling
(HIF-1), a transcriptional activator that functions as a master regulator of oxygen homeostasis. To date, more than 245 HIF-1 target genes have been identified that encode proteins that play key roles in a number of myocardial protective pathways and critical developmental and physiological processes, including angiogenesis-vascular remodeling, erythropoiesis, glucose transport, glycolysis, iron transport, and cell proliferation and survival (33).
HIF-1 is a heterodimer composed of an inducibly expressed HIF-1 subunit and a constitutively expressed HIF-1
subunit. HIF- subunits are regulated by a newly discovered signaling mechanism, that is, the oxygen-dependent enzymatic hydroxylation of specific amino-acid residues. Under normoxic conditions, the hydroxylation of conserved prolyl residues in two independent degradation domains in the central region of HIF- promotes interactions with the von Hippel–Lindau ubiquitylation complex, which targets HIF- for degradation by the ubiquitin–proteasome pathway. To date, three HIF prolyl hydroxylases, known as prolyl hydroxylase domain 1-3, and one asparaginyl hydroxylase, known as factor-inhibiting HIF, have been defined. The absolute requirement of the HIF hydroxylases for molecular oxygen conveys oxygen sensitivity. Additional cofactor and cosubstrate requirements for Fe2+, the citric-acid-cycle intermediate 2-oxoglutarate and ascorbate might help these enzymes generate the flexibility that is required for oxygen-sensing function. Under hypoxic conditions, proline hydroxylation is inhibited and HIF-1 degradation is blocked, thus allowing its accumulation and migration to the nucleus, where it activates hypoxia-responsive genes (34, 39).
The role of HIF-1-mediated events in cardiac remodeling after an ischemic insult is unknown, although the prominence of this pathway in other tissues and its availability in cardiac muscle (24, 26, 36) make it a plausible and attractive effector pathway. To explore this question further, we evaluated the HIF response to ischemia in male and female animals. Because there are clear sex-related differences in the cardiac remodeling process (5, 9, 11, 32), we postulated that there might be a parallel sex-related dimorphic response of HIF-1 to an ischemic insult.
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METHODS |
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Coronary ligation. Male and female adult Wistar rats (200–250 g weight matched) were euthanized 5 (n = 16) and 24 h (n = 16) after coronary ligation (M-MI and F-MI; MI, myocardial ischemia). Sham-operated male (n = 16) and female animals (n = 16) served as controls (M-SH and F-SH).
The rats were initially anesthetized with methoxyflurane, intubated, and placed on positive pressure ventilation with isoflurane (1.0%) to maintain anesthesia. A left thoracotomy was performed, the left main coronary artery was ligated with 6-0 suture, and the heart was replaced in the chest. The pleural cavity was evacuated, and the chest wall was closed in three layers. Animals were allowed to regain consciousness and were then reanesthetized and euthanized 5 and 24 h after surgery. Sham operations were identical to the above procedure except that the coronary artery was not ligated (17). At the end of 5 or 24 h, the animals were anesthetized and their hearts were excised. The atria and great vessels were trimmed, perfusion fixed with methacarn solution (60% methyl alcohol, 30% chloroform, and 10% acetic acid), and paraffin embedded. Hearts were cut into three bread loaf-like sections from the apex to the base of the heart, spanning the infarct zone (sections A, B, and C). A fourth section adjacent to the atria was not studied. Tissue slices (5-µm thickness) were then mounted on frosted slides for histochemical analysis. Analogous sections from male and female animals were examined. On each section stained for HIF, the ischemic and peri-ischemic zones (adjacent) were determined, and in the 5-h group, in the absence of an overt scar, pimonidazole hydrochloride was used as a marker of hypoxia (1). Additional male (n = 8) and female (n = 8) adult Wistar rats were euthanized 24 h after coronary ligation or sham operation and used for Western blot analysis and PCR. These hearts were perfused from the aorta with Evans blue (1%) and sectioned as described above. In each section, we were able to differentiate the ischemic area (not perfused due to the coronary ligation) from the nonischemic area that appeared perfused (blue). The same protocol was followed in the sham group, and each section was separated along an anterior and posterior axis that roughly corresponded to the ischemic area and the nonischemic area, respectively, in the MI group. Tissues were then processed for immunoblotting and RT-PCR, as described below.
Immunohistochemistry.
Embedded sections (n = 3 for each heart) were deparaffinized, and endogenous peroxidase activity was inhibited by treating the sections with 0.3% H2O2 in PBS for 10 min. Sections were incubated in BD Retrievagen A buffer for antigen unmasking. After several washes with PBS, the sections were incubated with 1.5% normal goat serum (Jackson ImmunoResearch) in PBS for 20 min to block nonspecific binding and incubated overnight at 4°C with the purified anti-HIF-1 antibody (Novus Biologicals; working dilutions 1:100). Subsequently, they were incubated with an anti-mouse IgG antibody (5 h postsurgery sections)(Sigma; working solutions 1:5,000) or with an alkaline phophatase-conjugated goat anti-rabbit IgG antibody (24 h postsurgery sections) (1:5,000) (Jackson ImmunoResearch) for 1 h. Signal amplification was achieved by incubating the slides with Vectastain Elite Avidin-Biotin Complex solution (Vectastain ABC Kit, Vector) for 30 min, followed by Vectastain diaminobenzidine (DAB) solution (5 h postsurgery sections) or by Vector Blue solution (24 h postsurgery sections) as the chromagen marker (Vector). The nuclei were counterstained with methyl green solution (5-h sections) or with Vector nuclear fast red solution (24 h sections).
Goat serum was used in place of HIF-1 antibody as the negative staining control (40, 51).
The stained sections were viewed with an electronic microscope with a x63 objective. In each section of each sample, we were able to differentiate the ischemic area and the peri-ischemic area (adjacent). HIF-1 protein expression was evaluated by counting the number of cells containing immunopositive nuclei. Approximately 1,000 nuclei were counted per section (n = 3 sections for each heart) by using a 10 µm x 10 µm eyepiece grid positioned at 20 areas around the LV.
To evaluate HIF-1 expression in cardiomyocytes, the same sections were stained for HIF and for cardiac troponin I (cTnI) with the use of immunofluorescence. Sections were incubated first overnight at 4°C with the anti-HIF-1 antibody (polyclonal from Novus Biologicals; working dilutions 1:100) and with a goat anti-rabbit IgG antibody tetramethylrhodamine isothiocyanate for 60 min at room temperature (Sigma, 1:100). The sections were then stained with the anti-cTnI antibody (1:300, Research Diagnostics) for 2 h at room temperature, with a biotinylated IgG as secondary antibody from a Vector MOM kit for 10 min at room temperature (1:500), and with fluorescein avidin DCS for 10 min at room temperature. Sections were then incubated for 30 min with 0.1% sudan black solution to quench autofluorescence and mounted with Vectashield with 4,6-diamidino-2-phenylindole (DAPI, Vector). In all sections, immunofluorescence was evaluated with a confocal microscope by counting the number of cells with visible nuclei containing immunopositive staining for HIF-1 and for cTnI. Pimonidazole hydrochloride (Hypoxyprobe TM-1 Kit, Chemicon International) was used as a marker of hypoxia with immunofluorescence to determine the extent of ischemic areas in both female and male animals and with immunohistochemistry in the 5-h animal group to evaluate the hypoxic regions in the absence of an overt scar. After coronary ligation and 90 min before being euthanized, animals were injected with pimonidazole hydrochloride (60 mg/kg body wt).
Sections (n = 3 for each heart) were incubated with hypoxyprobe1-MAb1 overnight at 4°C (working dilutions 1:50) and with a goat anti-mouse IgG antibody FITC for 60 min (Sigma) (for immunofluorescence) or with a goat anti-mouse biotinylated IgG antibody for 10 min (for immunohistochemistry). Sections for immunofluorescence were then incubated for 30 min with 0.1% sudan black solution to quench autofluorescence and mounted with Vectashield with DAPI (Vector). In all sections, immunofluorescence was evaluated with a confocal microscope by counting the number of cells with visible nuclei containing immunopositive cytoplasmatic staining. In the 5-h postsurgery sections, signal amplification was achieved by incubating the slides with Vectastain Elite avidin-biotin complex solution (Vectastain ABC kit, Vector) for 30 min, followed by Vectastain DAB solution as the chromagen marker (Vector). The nuclei were counterstained with methyl green solution, and the stained sections were viewed with an electronic microscope with a x63 objective.
Western blot analysis.
LV tissue samples were washed in PBS solution, and nuclei were then extracted according to an established protocol (nuclear fractionation kit from BioVision). Nuclear extract proteins were quantified by using Bio-Rad protein assay. Proteins were then mixed with Laemmli sample buffer, heated at 65° for 10 min, loaded (20 µg for each sample), separated by sodium dodecyl sulfate-polyacrylamide gel (7.5%) electrophoresis under denaturing conditions, and electroblotted to nitrocellulose membranes. The nitrocellulose membranes were blocked by incubation in blocking buffer (1% BSA in Tris-buffered saline-0.1% Tween 20), incubated with anti-HIF-1 antibody (1:500 polyclonal; Bethyl), washed, and incubated with anti-rabbit peroxidase-conjugated secondary antibody (1:10,000; Sigma). Signals were visualized by chemiluminescent detection. Blots were quantified by using Quantity One software from Bio-Rad, and HIF expression after coronary ligation (peak intensity) was normalized to values in the sham group. Equal loading of samples was verified by Coomassie blue staining of simultaneously run gels (
Fig. 5B). Gels were run four times, and the images shown are representative.
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(forward 5'-AGTCAGCAACGTGGAAGG-3' and reverse 5'-TCTTCTCTCTGCTGTTTGTG-3'), heme oxygenase (forward 5'-CACGCATATACCCGCTACCT-3' and reverse 5'-AAGGCGGTCTTAGCCTCTTC-3'), and brain natriuretic peptide (forward 5'-GCTGGAGCTGATAAGAGAAA-3' and reverse 5'-GGAATTTCGAAGTCTCTCCT-3'). The second derivative maximum (log-linear phase) for each amplification curve was determined and plotted against the standard curve to calculate the amount of product. Samples were normalized against GAPDH expression (forward 5'-ATTGCTCTCAATGACAACTT-3' and reverse 5'-GAACTTTATTGATGGTATTCG-3') to ensure equal loading as previously described (18).
Statistical analysis.
The results are presented as means ± SD unless otherwise stated. Group differences were established by one-way ANOVA, and multiple comparisons were made by using a post hoc Newman-Keuls analysis. A value of P 
0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Immunofluorescence analysis showed that after coronary ligation pimonidazole was present in 60% of the cells in the LV of females and in 55% of the cells of male animals (Fig. 1C). The difference was not statistically significant. Pimonidazole staining was minimal in both the male and female sham animals, and the staining that was seen was present in a narrow ring along the epicardial surface of the heart, consistent with rupture of the pericardium during the sham procedure (data not shown).
HIF-1 protein expression after ischemia.
Immunohistochemical analysis revealed that HIF-1 accumulated in the nuclei of myocytes in both male and female animals after regional ischemia compared with sham-operated rats. However, the magnitude and extent of HIF-1 expression were significantly greater in the females (Fig. 2 and Fig. 3). In fact, histochemical analysis 5 h after surgery showed that HIF-1 was expressed in 24% of cell nuclei in M-SH and 36% of F-SH, and this increased to 33% in M-MI and 55% in F-MI in the ischemic region (P < 0.05 vs. matched SH and also M vs. F) (Fig. 4A). In the same sections, in the region adjacent to the ischemic area (peri-ischemic area), HIF-1 was expressed in 23% of cell nuclei in M-SH and 26% of F-SH, and even in this region, HIF-1 expression was increased to 32% in M-MI and 45% in F-MI (Fig. 4B).
At 24 h, cells expressing nuclear HIF in the ischemic area increased to 49% in M-MI and 82% in F-MI (P < 0.05 vs. matched SH and also M-MI vs. F-MI). This difference was not only statistically significant between the two sexes at 24 h but also within each sex as a function of time after coronary ligation (Fig. 4A). HIF expression in the peri-ischemic area of the same sections at 24 h showed the same statistical differences between M-MI and F-MI (37% of positive cell nuclei in M-MI and 48% of positive cell nuclei in F-MI) (P < 0.05) (Fig. 4B).
To quantify the expression of HIF- 1 after ischemia, Western blotting was performed. The increase of HIF-1 protein was higher in the female rats compared with the male rats. In fact, at 24 h after coronary ligation, the HIF-1 protein level increased 2.8-fold in the female rats compared with the sham group (P < 0.001), whereas HIF-1 protein increased just 1.8-fold in the males (Fig. 5, A and B; P < 0.01 M vs. F).
HIF-1 gene expression after ischemia.
Quantitative RT-PCR demonstrated that HIF-1 mRNA expression when examined for the whole heart was unchanged at 5 h but was increased at 24 h in the F-MI versus M-MI and sham animals (Fig. 6A). When expression was examined for each of the regions of the heart (sections A, B, and C), a significant increase in HIF-1 mRNA expression was noted in section B (the region adjacent to the apex) of the female rats compared with male rats, 24 h after coronary ligation (Fig. 6B). Subdivision of section B into ischemic and nonischemic areas based on the Evans blue dye revealed that the increase in HIF-1 mRNA expression was not just restricted to the ischemic area but showed a wider regional distribution compared with male rats 24 h after coronary ligation (Fig. 6C). A similar pattern of gene expression was also noted for the downstream genes of HIF-1 (heme oxygenase and brain natriuretic peptide), suggesting that increased HIF-1 expression is associated with the transactivation of a specific gene expression program (Fig. 6, D and E).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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to cardiac ischemia and suggests a novel cellular mechanism to explain the relatively improved clinical response of females to ischemia. In the absence of ischemia, we documented persistent, albeit low, levels of expression of HIF-1 in the hearts of both males and females (which has been suggested to maintain basal expression of HIF-1 dependent genes) (49, 52); however, the levels of expression in this basal state were slightly greater in females. After 5 h of ischemia, HIF-1 expression increased in the ischemic myocardium regardless of sex; however, both the magnitude and extent of expression were considerably greater in females.
Twenty-four hours after coronary ligation, HIF-1 expression increased further, and the sex-related differences persisted and became even more marked. In other tissues, after an ischemic insult, the elevation in the mRNA levels of hypoxia-inducible genes is preceded by the nuclear accumulation of HIF (46, 52), and, in fact, in our study, regional mRNA expression of HIF was unchanged at 5 h but was significantly increased at 24 h in the females, consistent with a temporal progression of protein stabilization and translocation followed by transcriptional transactivation. Coordinate-increased expression of HIF-1-dependent genes (heme oxygenase and brain natriuretic peptide) was also seen at 24 h, confirming the biological relevance of this adaptation.
HIF activation is one of the earliest responses to ischemia in many tissues, including the brain (3, 21, 41) and the vasculature (43, 45). The activation of HIF is followed by and causally linked to the activation of a broad spectrum of hypoxia-responsive genes (42, 43), including angiogenic factors (2, 6, 28, 30, 31), antiapoptotic factors (8, 12, 20), and genes encoding proteins that influence tissue metabolism (33, 42), many of which might plausibly reduce tissue loss and favorably influence tissue remodeling. The mechanism by which HIF responds to changes in oxygen tension has been well characterized and appears to be consistent in several tissues (33, 40, 42, 44). The -subunit protein is constitutively present, whereas the stability of the -subunit and its transcriptional activity are precisely controlled by the intracellular oxygen concentration (13, 22, 23). Under normoxic conditions, the prolyl hydroxylase domain enzymes hydroxylate HIF-1, thus targeting it for interaction with the von Hippel-Lindau E3 ubiquitin ligase complex, leading to rapid proteosomal degradation (10). The prolyl hydroxylase domain enzymes have a requirement for dioxygen and are inactive under hypoxic conditions, which therefore favor stabilization of the multimeric HIF complex, nuclear translocation and activation of downstream pathways, such as those related to angiogenesis and metabolic adaptation. The data presented in the current study, showing elevated levels of HIF protein in hypoxic regions of cardiac muscle before an mRNA response, strongly suggest that this same mechanism of oxygen sensing is present in cardiac tissue as well.
Few studies on HIF-1 in response to ischemia have been reported in cardiac myocytes, although the presence of the protein complex in adult cardiocytes has been acknowledged for some time (36). Recently, an increase in expression of HIF-1 protein was described in the ischemic human myocardium (24), and HIF-1 and VEGF proteins were detected by immunohistochemical staining in biopsy specimens obtained from ischemic or infarcted myocardium, whereas they were undetectable in specimens of nonischemic myocardium (28). Further investigations have shown that HIF-1 plays an important role in the induction of VEGF in mechanically stressed myocardium (26, 30, 31). A more recent study (53) demonstrated that HIF-1 mediates the angiogenic response to hypoxia by upregulating the expression of multiple angiogenic factors, including VEGF, PDGF, and angiopoietins (4, 6). In addition, several studies (8, 12, 20) in isolated cardiocytes have provided evidence in support of the hypothesis that HIF-1 is involved in preventing ischemic cell death by engaging antiapoptotic pathways. Hydralazine, used clinically for the treatment of heart failure, has been shown to activate the HIF pathway through inhibition of prolyl hydroxylase activity and to initiate a proangiogenic phenotype (27). Finally, a recently characterized transgenic mouse expressing a constitutively active HIF-1 transgene in the heart was shown to have attenuated cardiac dysfunction and an increase in capillary density and iNOS expression after myocardial infarction (25). All these data suggest that HIF may function as a critical and common mediator of a spectrum of myocardial and vascular adaptations to hypoxic stress (as in other tissues) and could represent a potential target for treatment of ischemic disease.
Although the influence of sex on ventricular remodeling has been well established both in animal models and in clinical trials (29, 32), the cellular mechanisms responsible have remained speculative. A surprising number of diverse biological processes have been implicated, including sex hormone-mediated effects on myocyte hypertrophy and contractility (7, 14), the relative resistance of cardiocytes from female hearts to undergo hypoxia related apoptosis (48), changes in interstitial collagen and elastin deposition (14), shifts in substrate utilization, and altered collateral vessel growth (35). The richness and diversity of this biological response suggest that a single process is not sufficient to impart relative protection to ischemia but instead that a coordinated set of adaptations occur in response to the initial insult. HIF activation is linked through its effect as a master transcriptional transactivator to all of the above cell processes (23), and the sex-related dimorphic and early response to ischemia suggests that HIF-1 stabilization, translocation, and transcriptional transactivation might be a primary event.
Although the phenomenon of increased HIF-1 expression in hearts from females both under normoxic and, more strikingly, under hypoxic conditions is clear, the precise mechanism underlying this response is as yet purely speculative. There are several plausible hypotheses, including differential activation of prolyl hydroxylase domain or ubiquitin ligase enzymes or expression of a HIF-1 variant (as has been described during development in other organs) (19, 47, 50), which might result in a more stable protein. Future studies, perhaps using animals subjected to ovariectomy and/or hormone replacement or animals with defined haplotypes, may shed light on this question.
Regardless, our study suggests not only a novel cellular mechanism to explain the relatively improved clinical response of females to ischemia but also the possibility that exogenous modulation of HIF might represent a new therapeutic approach to preventing LV remodeling in general. In fact, our laboratory and others have already shown that prolylhydroxylase inhibition can improve cardiac function after myocardial infarction (37, 38), presumably by inhibiting proteosomal degradation of HIF-1.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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overexpression alters myofilament properties and composition during the progression of heart failure. Circ Res 95: 424–432, 2004.. J Biol Chem 275: 6922–6927, 2000. deletion alters vascularization, energy availability, calcium flux, and contractility in the normoxic heart. FASEB J 18: 1138–1140, 2004. in response to mechanical stresses: regulation by stretch-activated channels and the phosphatidylinositol 3-kinase signaling pathway. Circ Res 90: 25e-33, 2002., vascular endothelial growth factor, and angiogenesis by inhibition of prolyl hydroxylases. Circ Res 95: 162–169, 2004.This article has been cited by other articles:
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  K. M. Shioura, D. L. Geenen, and P. H. Goldspink Sex-related changes in cardiac function following myocardial infarction in mice Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R528 - R534. [Abstract] [Full Text] [PDF]
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0.05 female (n = 4) vs. male (n = 4).
P < 0.05 F-MI vs. M-MI; 
P < 0.05 F-MI vs. F-SH and M-MI vs. M-SH.
P < 0.001 F-MI vs. M-MI and vs. sham group.