Microgravity-induced stress mechanisms in human stem cell-derived cardiomyocytes

Abstract

Exposure to outer space microgravity poses a risk for the development of various pathologies including cardiovascular disease. To study this, we derived cardiomyocytes (CMs) from human-induced pluripotent stem cells and exposed them to simulated microgravity (SMG). We combined different "omics" and chromosome conformation capture technologies with live-cell imaging of various transgenic lines to discover that SMG impacts on the contractile velocity and function of CMs via the induction of senescence processes. This is linked to SMG-induced changes of reactive oxygen species (ROS) generation and energy metabolism by mitochondria. Taken together, we uncover a microgravity-controlled axis causing contractile dysfunctions to CMs. Our findings can contribute to the design of preventive and therapeutic strategies against senescence-associated disease.

Keywords: Biological sciences; Cell biology; Molecular biology; Stem cells research.

Graphical abstract

Figure 1

Compartments and topologically associated domains (TADs) in SMG-CMs and in c-CMs and interactions gained in SMG-CMs versus c-CMs (A) Each point represents 100 Kbp regions that belongs to the A or B compartments for the c-CMs (x axis) and SMG-CMs (y axis). This shows the differential compartments between the different experiments. (B) In this plot, on the right you can see the proportion of TADs called in 1 g that changed or maintained its status in SMG. (C and D) Interactions gained in SMG-CMs are proximal while interactions lost are distal. C, Chromosome 8 with interactions gained in SMG-CMs (red contact map) and lost in SMG (blue contact map). (D) Interaction-decay plots for chr20. (E) Upregulated and downregulated genes in SMG lose both proximal and intermediary interactions. The promoter of the gene is in the center of the maps shown by the dashed green lines. In this averaged differential contact maps, blue colors indicate interactions lost in SMG and red colors indicate interactions gained in SMG.

Figure 2

Exposure to SMG does not cause cytoskeletal deteriorations in CMs (A) A representative immunofluorescence staining of sarcomeric α-actinin (ACTN2) and cardiac troponin T (cTnT) in c-CMs and SMG-CMs (scale bar: 10 μm). (B) TEM images of SMG-CMs and c-CMs. Black arrow heads indicate caveolae structures (black arrows show stress fibers (scale bar: 500 nm). (C) Western blot analysis of Caveolin (CAV1) in control and SMG-exposed CMs (molecular weight of 22 kDa). GAPDH (36 kDa) was used as an internal protein loading control.

Figure 3

Effects of 48 h SMG on mitochondrial morphology, ROS production, membrane potential, and ATP synthesis (A) Mitochondria staining of the SMG-CMs and c-CMs with the JC-1 dye to visualize disturbances in mitochondrial membrane potential (MMP). The JC-1 dye is a lipophilic, cationic dye that can be accumulated into the mitochondria to form the J-aggregates. The monomeric JC-1 naturally exhibits green fluorescence, whereas the JC-1 aggregates exhibit red fluorescence. Intact cells exhibit normal mitochondrial membrane potential. In this case, JC-1 dye enters and accumulates more in the negatively charged mitochondria and emits red fluorescent hence mitochondria appear redder as compared to mitochondria emitting green fluorescence. In contrast, JC-1 incorporation into mitochondria is greatly hindered due to the loss of mitochondrial membrane potential resulting in a reduction of J-aggregate formation retaining in its original green fluorescence. Both, monomers (green) and aggregates (red) were captured by using a confocal laser scanning microscope. The images were then processed and analyzed by using the ImageJ software. The ratio of green vs red color represents disturbances of the mitochondrial membrane potential; the greener; the intact mitochondria) (scale bar = 10 μm). (B) Fluorescence lifetime imaging microscopy (FLIM) of DyRed1-E5⁺-CMs (see video record 2 and DyRed1-E5⁺-SMG-CMs (see record 3) (Excitation length 488 nm). Visualization of newly synthesized and non-oxidized MitoTimer was performed setting an emission length of 497–531 nm (green color); Oxidized DyRed1-E5 was visualized at a fluorescence length of 583–695 nm. The ratio of green vs red fluorescence is representative for the overall mitochondrial turnover in the cells (scale bar: 20 μm). (C) ATP level in SMG-CMs and c-CMS. Total intracellular ATP was quantified using the ATPlite Luminescence ATP Detection Assay System. ATP levels were expressed as a per cent of the c-CMs levels (100%) (mean ± SD; n = 12, ∗p < 0.05; two-tailed t-test; three independent experiments; each experiment was performed in triplicate wells). (D) TEM images showing mitochondrial morphology and cristae structures in 48 h SMG-CMs versus control c-CMs (scale bar: 500 nm). The maximum number of mitochondria from 5 images from 3 independent experiments (3 separate slides) was selected. The quantification of the mitochondrial length was performed using the ImageJ software and plotted as a boxplot.

Figure 4

Effects of SMG on ROS, RNS production, and β-galactosidase activity in CMs (A) Fluorescent images showing an increase in DHE staining in SMG-CMs in comparison to c-CMs. Red staining in nuclei indicates a net increase in the ROS production (Nuclei: DAPI) (Scale bar: 20 μm). Quantification was performed using ImageJ software (red: nuclear DHE; blue: DAPI). Five images of c-CMs (in total n = 71 cells) and SMG-CMs (in total n = 53 cells) from tree independent experiments (3 slides) were selected and all the cells in each image were counted for positive and negative red nuclear staining. Then, the percent positive staining was calculated and represented in the graph as % DHE staining (Mean ± SD, p < 0.05). (B) ROS quantification in c-CMs and SMG-CMs by the fluorescence DCFH-DA test. The fluorescence intensity was measured using Softmax Pro M5e 96-well plate reader with excitation and emission wavelengths of 485 and 535 nm, respectively. All the fluorescence intensities are normalized with control c-CMs (100%) (Mean ± SD, p < 0.05, n = 9, three independent experiments, performed in triplicates). (C) Reactive nitrogen species (RNS) production in SMG-CMs and c-CMs. Fluorescence was quantified in a fluorometric plate reader at 480 nm/530 nm (expressed as percent of the absolute values of RNS in c-CMs (mean ± SD, p < 0.05, n = 9, two-tailed t-test; three independent experiments, performed in triplicates). (D) Bright filter and fluorescent images showing increase β-galactosidase staining in SMG-CMs and c-CMs (Scale bar = 50 μM) in IMR90-and iCell-CMs. Quantification of the GFP intensity from 35 CMs from each condition (c-CMs and SMG-CMs, 3 independent experiments) was determined by the ImageJ software. Then, the raw intensity data were plotted as a box plot.

Figure 5

Effects of SMG on Ca²⁺ handling of CMs (A–D) SMG-CMs and c-CMs were loaded with FLUO4-AM and line scans were performed using a FV1000 Olympus microscope with a 60× oil immersion objective and the 488 nm argon line for excitation. After capturing the [Ca²⁺] transients, background correction was applied before performing the line scan data analysis using Sigma Plot (Version 8.04, SPSS) and a self-made Excel macro. The calculated [Ca²⁺] transient parameters were (A) the relative amplitude (F/F0 where F0 is the resting fluorescence intensity in arbitrary units), (B) the time-to-peak (TTP), (C) the maximum slope of the rise in ΔF ([ΔF/ΔT]_max) and (D) the time for the transient to decay to 10% of the F/F0 peak (T90%). For both SMG and c-CMs, we recorded seventy independent readings to perform a robust statistical analysis. Values in A–D represent the mean ± SD (∗p < 0.05, n = 70 ± 5; two-tailed t-test). E, Voltage-gated calcium currents in SMG-CMs (orange) and c-CMs (black). Current densities (pA/pF) were determined by the patch-clamp technique in the whole-cell configuration. Barium was used as a charge carrier (Mean ± SD, n = 8 for c-CMs and n = 6 for SMG-CMs, ∗p < 0.05; two-tailed t-test. F, effect of SMG-CMs on the expression of RYR2 and phospholamban protein.

Figure 6

Live imaging of contraction and relaxation velocity activity of ACTN2-eGPF⁺-CMs and ACTN2-eGPF⁺-SMG-CMs (A and B) Fluorescence microscopy shows the Z-discs of ACTN2-eGPF⁺-CMs in c-CMs and ACTN2-eGPF⁺-SMG-CMs (ACTN2 is enriched in Z-discs) immediately after 48 h SMG. ACTN2 Video record 4 and 5 shows the contractile activity of the c-CMs and SMG-CMs, respectively. Fluorescence microscopy has been performed using the Olympus FluoView1000 confocal system C–D, Contractile velocity of c-CMs and SMG-CMs (representative experiments; see Videos S3 and S4, respectively) was determined by analyzing the video records 4 and 5 (50fps), respectively by using the VA1.9 software. E–G, Diagrams show the duration of contraction, the duration of relaxation, and the TTP values and of the c-CMs and SMG-CMs, respectively (mean ± SD, n = 6,∗p < 0.05; two-tailed t-test; 3 independent experiments). H, Diagram shows the ΔV/ΔT)_max ratio in SMG-CMs versus c-CMs ((mean ± SD, n = 6,∗p < 0.05; two-tailed t-test; 6 independent experiments). I, beating frequency of SMG-CMs versus c-CMs (mean ± SD, n = 11,∗p < 0.05; 3; two-tailed t-test; independent experiments).