Distinct mechanisms underlying tolerance to intermittent and constant hypoxia in Drosophila melanogaster

Abstract

Background: Constant hypoxia (CH) and intermittent hypoxia (IH) occur during several pathological conditions such as asthma and obstructive sleep apnea. Our research is focused on understanding the molecular mechanisms that lead to injury or adaptation to hypoxic stress using Drosophila as a model system. Our current genome-wide study is designed to investigate gene expression changes and identify protective mechanism(s) in D. melanogaster after exposure to severe (1% O(2)) intermittent or constant hypoxia.

Methodology/principal findings: Our microarray analysis has identified multiple gene families that are up- or down-regulated in response to acute CH or IH. We observed distinct responses to IH and CH in gene expression that varied in the number of genes and type of gene families. We then studied the role of candidate genes (up-or down-regulated) in hypoxia tolerance (adult survival) for longer periods (CH-7 days, IH-10 days) under severe CH or IH. Heat shock proteins up-regulation (specifically Hsp23 and Hsp70) led to a significant increase in adult survival (as compared to controls) of P-element lines during CH. In contrast, during IH treatment the up-regulation of Mdr49 and l(2)08717 genes (P-element lines) provided survival advantage over controls. This suggests that the increased transcript levels following treatment with either paradigm play an important role in tolerance to severe hypoxia. Furthermore, by over-expressing Hsp70 in specific tissues, we found that up-regulation of Hsp70 in heart and brain play critical role in tolerance to CH in flies.

Conclusions/significance: We observed that the gene expression response to IH or CH is specific and paradigm-dependent. We have identified several genes Hsp23, Hsp70, CG1600, l(2)08717 and Mdr49 that play an important role in hypoxia tolerance whether it is in CH or IH. These data provide further clues about the mechanisms by which IH or CH lead to cell injury and morbidity or adaptation and survival.

Figure 1. Over-expressed genes in various biological processes during hypoxia (CH or IH).

(A): Over-expressed genes in biological processes during constant hypoxia. (B): Over-expressed genes in biological processes during intermittent hypoxia. The bars represent over-expressed biological processes in terms of percentage of genes changed in the category as calculated by GENMAPP as shown in table 1. The number in parentheses represents the number of genes measured as shown in Table 1.

Figure 2. Role of Hsp70 and Hsp23 during CH.

(A): The microarray results were clustered using GenMapp and viewed with Treeview. Upregulated genes are shown in red and downregulated genes are shown in green. Heat shock proteins specifically Hsp23, Hsp68 and Hsp70, are significantly over-expressed during constant hypoxia (CH1, CH2 and CH3) as compared to normoxia controls (NC1, NC2 and NC3) and intermittent hypoxia replicates (IH1, IH2 and IH3). (B): Genomic location of P-element for genes Hsp23, Hsp70Aa and Hsp70Bbb. (C): Functional testing of Hsp23 and Hsp70 genes during CH. Percent survival of adult flies after exposure to 1.5% O₂ CH for 7 days. CS and yw serve as controls. Hsp70 and Hsp23 P-element lines showed significantly higher survival than CS, yw controls (P<0.05). In contrast, P-element excision lines (Ex) and Hsp70- lines showed survival similar to or less than controls. Each bar represents the average of at least three tests for each line and error bars represent the standard errors. * with unpaired t-test P<0.05. (D): Real-time PCR analysis to validate upregulation of Hsp70Aa, Hsp70Bbb and Hsp23 in controls and P-element lines after exposure to CH. Hsp-P-element lines showed significantly higher mRNA levels of heat shock proteins than controls after 2.5 hours and 7 days of CH exposure(P<0.05). * With unpaired t-test comparing the fold change of P-element lines with yw normoxia control, P<0.05. (E): Testing for survival of adult F1 flies expressing Hsp70 in various tissues using the GAL4 drivers at 1.5% O₂ CH. The graph represents the percent survival of CS, yw and the F1 progeny obtained by crossing UAS-Hsp70Bbb with Hand(Heart) and c736(Brain)-GAL4 drivers. Over-expression of Hsp70 in cardioblasts, pericardial cells and hemocytes(Hand) leads to remarkable survival as compared to controls (at day 18, P<0.0001). * with unpaired t-test P<0.05. ** P<0.0001.

Figure 3. Role of Mdr49 and l(2)08717 genes during IH.

(A):Over-expression of Mdr 49 and l(2)08717 during intermittent hypoxia as demonstrated by microarray analysis. Upregulated genes are shown in red and downregulated genes are shown in green. IH1, IH2 and IH3 represent replicates exposed to intermittent hypoxia for 2.5 hours. Mdr49 and l(2)08717 are specifically upregulated during IH. NC1, NC2 and NC3 represent the normoxia controls and CH1, CH2 and CH3 represent replicates exposed to constant hypoxia for 2.5 hours. (B): Genomic location of P-element for genes l(2)08717 and Mdr49. (C): Functional testing of l(2)08717, Mdr49 and CG14709 genes during IH. Percent survival of adult flies after exposure to 21%–1.5% O₂ IH for 10 days. CS and yw serve as controls. P{PZ}l(2)08717 and Mi{ET1}Mdr49 lines showed significantly higher survival as compared to controls(CS, yw, P<0.05). Each bar represents the average of at least three tests for each line and error bars represent the standard errors. * With unpaired student t-test comparing the % survival of P-element lines with CS and yw controls, P<0.05. (D): Real-time PCR analysis to validate upregulation of l(2)08717, Mdr49 and CG14709 genes after exposure to IH in controls and P-element lines. After normalizing the values with actin, fold change was calculated in comparison to relative mRNA levels at normoxia. For l(2)08717, Mdr49 and CG14709 genes relative mRNA levels and fold change was measured in CS, yw and the P-element lines after 2.5 hours and 10 days. P-element lines of Mdr49 and CG14709 showed increased expression after 2.5 hours and 10 days as compared to controls, P<0.05. * With unpaired t-test comparing the fold change of P-element lines with yw normoxia control, P<0.05.

Figure 4. CG1600 gene has an important role under both paradigms- CH and IH.

(A): Over-expression of CG1600 during IH as well as CH as demonstrated by microarray analysis. Upregulated genes are shown in red and downregulated genes are shown in green. IH1, IH2 and IH3 represent replicates exposed to intermittent hypoxia for 2.5 hours. NC1, NC2 and NC3 represent the normoxia controls and CH1, CH2 and CH3 represent replicates exposed to constant hypoxia for 2.5 hours. (B): Genomic location of P-elements for gene CG1600. (C): Functional testing of CG1600 gene during CH and IH. Percent survival of adults flies after exposure to 21%–1.5% O₂ IH for 10 days. Percent survival of adult flies after exposure to 1.5% O₂ CH for 7 days CS and yw serve as controls. P-element lines of CG1600 survived much better than controls under both CH and IH (P<0.05). Each bar represents the average of at least three tests for each line and error bars represent the standard errors.* with unpaired t- test P<0.05. (D): Real-time PCR analysis to validate upregulation of CG1600 gene in controls and P-element lines after exposure to IH and CH. After normalizing the values with actin, fold change was calculated in comparison to relative mRNA levels at normoxia. For CG1600 gene relative mRNA levels and fold change was measured in CS, yw and the F1 progeny of EP line x dal-Gal4 and P-element line after 2.5 hours and 10 days for IH and CS, yw and the P-element after 2.5 hours and 7 days for CH. P-element lines of CG1600 genes showed several fold higher expression than controls even after 7 days CH or 10 days IH. * With unpaired t-test comparing the fold change of P-element lines with yw normoxia control, P<0.05.