Simulated Microgravity Alters Gene Regulation Linked to Immunity and Cardiovascular Disease

Abstract

Microgravity exposure induces a cephalad fluid shift and an overall reduction in physical activity levels which can lead to cardiovascular deconditioning in the absence of countermeasures. Future spaceflight missions will expose crew to extended periods of microgravity among other stressors, the effects of which on cardiovascular health are not fully known. In this study, we determined cardiac responses to extended microgravity exposure using the rat hindlimb unloading (HU) model. We hypothesized that exposure to prolonged simulated microgravity and subsequent recovery would lead to increased oxidative damage and altered expression of genes involved in the oxidative response. To test this hypothesis, we examined hearts of male (three and nine months of age) and female (3 months of age) Long-Evans rats that underwent HU for various durations up to 90 days and reambulated up to 90 days post-HU. Results indicate sex-dependent changes in oxidative damage marker 8-hydroxydeoxyguanosine (8-OHdG) and antioxidant gene expression in left ventricular tissue. Three-month-old females displayed elevated 8-OHdG levels after 14 days of HU while age-matched males did not. In nine-month-old males, there were no differences in 8-OHdG levels between HU and normally loaded control males at any of the timepoints tested following HU. RNAseq analysis of left ventricular tissue from nine-month-old males after 14 days of HU revealed upregulation of pathways involved in pro-inflammatory signaling, immune cell activation and differential expression of genes associated with cardiovascular disease progression. Taken together, these findings provide a rationale for targeting antioxidant and immune pathways and that sex differences should be taken into account in the development of countermeasures to maintain cardiovascular health in space.

Keywords: cardiovascular system; hindlimb unloading; immune response; microgravity; transcriptomics.

Figure 1

Body weights of animals included in this study. NL: normally loaded; HU: hindlimb unloading; Rel: reambulated (reloading). The HU group was compared to their age- and sex-matched NL controls by Student’s t-test. Sample sizes: N = 5–8/group for all groups except for 90D older males, where N = 3. Values depicted are means and standard deviation. * Significant at p < 0.05 by Student’s t-test.

Figure 2

8-hydroxydeoxyguanosine (8-OHdG) levels in left ventricular tissue as measured by ELISA. NL: normally loaded control, HU: hindlimb unloading, 7D Rel: 90D HU + 7D reloading and normally loaded control (NL). The HU groups were compared to their age- and sex-matched NL controls by Student’s t-test. Sample sizes: N = 5–8/group for all groups except for 90D older males, where N = 3. Values depicted are means and standard deviation. * Significant at p < 0.05 by Student’s t-test.

Figure 3

Transcript levels of (A) Nfe2l2, (B) Sod1, (C) Sod2, and (D) Sirt1 in left ventricular wall as measured by qPCR. Values depicted are mean fold changes relative to young male control at 14 days of treatment as determined by the ΔΔCt method. Errors bars show upper and lower ranges. NL: normally loaded control, HU: hindlimb unloading, 90D Rel: 90D HU + 90D reloading and NL control. Sample sizes: N = 3–7/group. * Significant at p < 0.05 by Student’s t-test by comparing HU with age- and sex-matched NL control.

Figure 4

PCA plot of transcriptomic data from older males that underwent 14 days of HU (gray triangles) and corresponding NL controls (black circles). Animal ID is indicated by the letter “R” succeeded by numbers.

Figure 5

Heatmap showing normalized counts of differentially expressed genes from older male 14D NL and HU groups. Each cell corresponds to a gene. Red: Upregulated in 14D HU relative to NL group. Blue: Downregulated in 14D HU relative to NL group. Magnitude of upregulation or downregulation is proportional to the intensity of red or blue. Deepest red: most upregulated; deepest blue: most downregulated.

Figure 6

Top 10 upregulated and downregulated genes in older male 14D HU relative to NL groups. Log2 FC: Log2 fold change. Refer to Table 1 for full list of DEGs.

Figure 7

Select enriched gene ontology (GO) terms for biological processes in older male 14D HU group relative to NL group. Gene count refers to the number of DEGs that matched the GO term. The vertical bar represents the color scale of the FDR with black representing the lowest FDR. Refer to Table S2 for the full list of GO terms.