Rapid coupling between gravitational forces and the transcriptome in human myelomonocytic U937 cells

Abstract

The gravitational force has been constant throughout Earth's evolutionary history. Since the cell nucleus is subjected to permanent forces induced by Earth's gravity, we addressed the question, if gene expression homeostasis is constantly shaped by the gravitational force on Earth. We therefore investigated the transcriptome in force-free conditions of microgravity, determined the time frame of initial gravitational force-transduction to the transcriptome and assessed the role of cation channels. We combined a parabolic flight experiment campaign with a suborbital ballistic rocket experiment employing the human myelomonocytic cell line U937 and analyzed the whole gene transcription by microarray, using rigorous controls for exclusion of effects not related to gravitational force and cross-validation through two fully independent research campaigns. Experiments with the wide range ion channel inhibitor SKF-96365 in combination with whole transcriptome analysis were conducted to study the functional role of ion channels in the transduction of gravitational forces at an integrative level. We detected profound alterations in the transcriptome already after 20 s of microgravity or hypergravity. In microgravity, 99.43% of all initially altered transcripts adapted after 5 min. In hypergravity, 98.93% of all initially altered transcripts adapted after 75 s. Only 2.4% of all microgravity-regulated transcripts were sensitive to the cation channel inhibitor SKF-96365. Inter-platform comparison of differentially regulated transcripts revealed 57 annotated gravity-sensitive transcripts. We assume that gravitational forces are rapidly and constantly transduced into the nucleus as omnipresent condition for nuclear and chromatin structure as well as homeostasis of gene expression.

Figure 1

Sample lysis scheme. (a) U937 cells were analyzed during the 19^th DLR parabolic flight campaign. In total, four sample groups were lysed at defined g conditions and time points: (1) 1 g in-flight (1 g IF) 5 min before the first parabola, (2) 1.8 g samples at the end of the first parabola after 20 s of the 1.8 g hypergravity phase; these samples also serve as baseline (BL-PFC hyp-g) directly before the microgravity phase, (3) at the end of the first parabola after 20 s of microgravity (μg) phase, and (4) 1 g hardware ground controls (H/W 1 g GC), directly after the flight inside the aircraft. (b) U937 cells were investigated during the TEXUS-49 suborbital ballistic rocket campaign. Overall, five sample groups were lysed at set time points and g conditions: (1) the baseline (BL-TX hyp-g) group monitored the first 75 s of the flight after liftoff including hypergravity and vibrations, (2) microgravity samples (µg) were lysed 375 s post-launch, resulting in 300 s of microgravity time, (3) microgravity samples lysed 375 s post-launch with the cation ion channel inhibitor SKF-96365 (µg SKF), (4) 1 g hardware ground controls (H/W 1 g GC) lysed approximately 15 min after launch and (5) 1 g hardware ground controls with SKF-96365 (H/W 1 g GC SKF) lysed approximately 15 min after launch. (c) Experiment hardware of the parabolic flight (19^th DLR PFC). In-flight experiment system for parabolic flights on board the Airbus A300 ZERO-G. Experiment hardware structure which consists of an incubator rack to store the cell containers at 36.5 °C before the experiment (1), an experiment rack, in which all technical aggregates are accommodated for the execution of the experiment and where the living cells are processed during altered gravity (2), and a cooling rack to store all cell containers at 4 °C after the injection of the lysis solution until landing (3). (d) Experiment hardware of the suborbital ballistic rocket (TEXUS-49) experiments. TEXUS consists of a VSB-30 engine (not shown) and of the payload structure. Sets of three sterile syringes were filled with cell suspension, medium with or without SKF-96365, and lysis buffer connected by a T-piece with small plugs at the outlet ports to prevent premature contact of the fluids. The syringe systems are accommodated in tempered and vacuum-resistant containers (shown).

Figure 2

Differentially regulated transcripts (and annotated transcripts) in human U937 myelomonocytic cells during the 19^th DLR Parabolic Flight Campaign. The different comparisons and resulting intersections for hypergravity and microgravity-sensitive transcripts are displayed. Fold change ≤−1.3 and ≥+1.3, p < 0.05.

Figure 3

Differentially regulated transcripts (and annotated transcripts) in human U937 myelomonocytic cells during the TEXUS-49 suborbital ballistic rocket campaign. (a) The different comparisons and resulting intersections for hypergravity and microgravity-sensitive transcripts are displayed. (b) Analysis of the influence of the cation ion channel blocker SKF-96365 on microgravity-regulated gene expression. Fold change ≤−1.3 and ≥+1.3, p < 0.05.

Figure 4

Distribution of differentially expressed transcripts. (a) Hyper- and microgravity-sensitive transcripts identified for the 19^th DLR Parabolic Flight Campaign. (b) Hyper- and microgravity sensitive transcripts identified for the TEXUS-49 suborbital ballistic rocket campaign, (c). SKF-96365-dependent hyper- and microgravity-sensitive transcripts.

Figure 5

Hyper- and microgravity-double-sensitive transcripts were reversely regulated in the 19^th DLR Parabolic Flight Campaign. (a) Venn diagram showing 1602 hyper- and microgravity-double-sensitive transcripts (1532 annotated transcripts). (b) Graphical display of the 1602 transcripts of the intersection. Fold changes (FCs) are ratios between the averages of linear expression values. If the ratio is <1, FC is calculated as the negative reciprocal of the ratio.

Figure 6

Hyper- and microgravity-double-sensitive transcripts are reversely regulated in the TEXUS-49 suborbital ballistic rocket campaign. (a) Venn diagram showing 3575 hyper- and microgravity-double-sensitive transcripts (3515 annotated transcripts). (b) Graphical display of 3575 transcripts of the intersection, including one single transcript that is regulated in the same direction. Fold changes (FCs) are ratios between the averages of linear expression values. If the ratio is <1, FC is calculated as the negative reciprocal of the ratio.

Figure 7

Hypergravity-sensitive transcripts are reversely regulated when comparing 20 s (19^th DLR Parabolic Flight Campaign) and 75 s (TEXUS-49 suborbital ballistic rocket campaign) exposure times. (a) Venn diagram showing the intersection of hypergravity-sensitive transcripts of both platforms. (b) Graphical display of transcripts (annotated transcripts) showing that transcripts are mostly regulated in the opposite direction. Only 113 from 2192 transcripts were regulated in the same direction. Fold changes (FCs) are ratios between the averages of linear expression values. If the ratio is <1, FC is calculated as the negative reciprocal of the ratio.

Figure 8

Microgravity sensitive transcripts are reversely regulated when comparing 20 s (19^th DLR Parabolic Flight Campaign) and 300 s (TEXUS-49 suborbital ballistic rocket campaign) exposure times. (a) Venn diagram showing the intersection of microgravity-sensitive transcripts of both platforms. (b) Graphical display of transcripts (annotated transcripts) showing that transcripts are mostly regulated in the opposite direction. Only 12 out of 110 transcripts are regulated in the same direction. Fold changes (FCs) are ratios between the averages of linear expression values. If the ratio is <1, FC is calculated as the negative reciprocal of the ratio.

Figure 9

Hyper- and microgravity-double-sensitive transcripts identified for the 19^th DLR Parabolic Flight Campaign and the TEXUS-49 suborbital ballistic rocket campaign. (a) Venn diagram showing the intersection of hyper- and microgravity-double-sensitive transcripts of both platforms. (b) Graphical display of transcripts (annotated transcripts) showing that transcripts are regulated in the opposite direction between altered gravity conditions within one platform and between platforms. Only one transcript (arrow) was regulated in the same direction in both platforms. Fold changes (FCs) are ratios between the averages of linear expression values. If the ratio is <1, FC is calculated as the negative reciprocal of the ratio.

Figure 10

Time course of differential gene expression in hypergravity. Human myelomonocytic U937 cells were exposed to 20 s and 75 s of hypergravity. The numbers of annotated transcripts grouped according to their regulation after the two exposure times are shown. Continuous response means that annotated transcript are either up- or down-regulated at both time points. Adaption is either disappearance of the hypergravity induced effect or regulation in the opposite direction after 75 s. Late response is a regulation that only appears after 75 s. Most transcripts did not respond to hypergravity at both time points.

Figure 11

Time course of differential gene expression in microgravity. Human myelomonocytic U937 cells were exposed to 20 s and 300 s of microgravity. The numbers of annotated transcripts grouped according to their regulation after the two exposure times are shown. Continuous response means that annotated transcript are either up- or down-regulated at both time points. Adaption is either disappearance of the microgravity induced effect or regulation in the opposite direction after 300 s. Late response is a regulation that only appears after 300 s. Most transcripts did not respond to microgravity at both time points.