Cerium Oxide Nanoparticle Administration to Skeletal Muscle Cells under Different Gravity and Radiation Conditions

Aging, disease onset, radiation exposure, and mechanical unloading all cause skeletal muscle tissue modifications such as atrophy, muscular force decline, and a shift in muscle fiber composition. Astronauts subjected to microgravity (also known as "μg") and cosmic radiation during spaceflight experience rapid degeneration of skeletal muscle tissue. For this reason, a variety of strategies for muscular maintenance in vitro and in vivo have also been devised for terrestrial benefit, including physical exercise, mechanical stimulation in the form of vibrations and pressure application, electrical stimulation, exposure to hypergravity, administration of soluble factors (such as activin type IIB receptor, recombinant myokine irisin, and myostatin antibody YN41, and even genetic transduction finalized to the overexpression.

A common approach to skeletal muscle waste caused by mechanical unloading includes the administration of antioxidant compounds such as (-)-epicatechin, lecithin, N-acetylcysteine, complex mixtures of polyphenols associated with other antioxidants (such as vitamin E, selenium, and omega-3 fatty acids), (19), or even seed extracts (from Oenothera odorata). Recent study focuses on the role of oxidative stress (OS) caused by excess reactive oxygen species (ROS) and mitochondrial dysregulation in skeletal muscle degeneration under real or simulated microgravity (s-μg).

As human permanence in low Earth orbit increases in duration and opens the door to interplanetary travel, the understanding of the biological effects of mechanical unloading necessitates deepening by careful consideration of the consequences of exposure to highly energetic cosmic radiations, which to date is largely obscure and appears to follow divergent molecular pathways in comparison to microgravity.

Based on a large body of evidence of the antioxidant and radioprotective properties of cerium oxide nanoparticles (also known as "nanoceria" (NC)) accumulated on the ground, and assimilating the long-standing catalytic activity of this type of inorganic material to that of superoxide dismutase and catalase, we decided to test them for skeletal muscle tissue protection in space. In a recent work, our group investigated the effects of NC on differentiating muscle cell cultures grown aboard the International Space Station (ISS).The obtained transcriptional evidence revealed cellular adaptability to nanoparticle administration and gravitational unloading, as well as the regulation of biological processes such as aging, fat tissue growth, and mesodermal tissue proliferation. Another pilot work conducted by our lab demonstrated that NC reduced cell death and DNA fragmentation while increasing stemness and tissue regeneration in planarian worms exposed to low-dose radiations, indicating possible radioprotective activity relevant to spaceflight.

?Studies sought to confirm NC's potential protection against deleterious space environment effects on proliferating C2C12 mouse skeletal muscle cells cultured on board the ISS in two different experimental configurations, namely exposure to microgravity and cosmic radiations or artificially obtained Earth gravity (1g by centrifugation) and cosmic radiations. Samples collected in space were compared to samples collected later on the ground using the same time and temperature characteristics as spaceflight. On-the-ground investigations using RNA next-generation sequencing (RNA-seq) allowed the identification of differentially expressed genes (DEGs) between paired experimental classes. The results denoted important transcriptional regulation of mitochondrial and nuclear compartments; they demonstrated opposite effects of gravitational unloading and cosmic radiations and evidenced the need for further optimization of NC delivery modes in view of exploitation of their remarkable antioxidant properties in the space environment.

Cerium oxide nanoparticles (NC, Sigma 544841) were disseminated at 10 mg/mL in ultrapure water. The dispersion was sonicated for 1 minute at 8 W using a tip sonicator (Bandelin). Then, 5 mg/mL NC dispersions in 50% fetal bovine serum (FBS, Sigma F4135) were created by incubating them for 1 hour with gentle shaking to promote nanoparticle coating with serum proteins. To generate very uniform dispersions, this method was followed by 5 minutes of sonication using a Branson sonication bath. To characterize the substance, dispersions were made in ultrapure water, 10% FBS solution in ultrapure water, and a full cell culture medium.

Simulated microgravity conditions (s-μg) were applied by operating a random positioning machine (RPM, Airbus 2.0) at 20°/s for studies on nanoparticle stability, and at 8-20°/s (speed change every 5 s) for nanoparticle internalization studies with C2C12 mouse myoblasts. To accomplish this, containers completely filled with liquid media were placed within 22 mm of the RPM center of rotation: microcentrifuge tubes for stability studies and ad hoc-prepared polydimethylsiloxane (silicone) vessels sealed with transparent adhesive films (Bio-Rad MSB1001) for internalization studies. The RPM home frame setting had the sample holder aligned with the Earth gravity vector (both frames were placed perpendicular to it).

Simulated microgravity studies (with average g values ranging from 0.020 to 0.008 over a 6-48 hour timeframe) revealed that nanoparticles were internalized by cell cultures but, as shown in NC, poorly co-localized with the signal from both internalization markers at each time point and under various gravity levels. Quantitative data from confocal microscopy images revealed modest nanoparticle internalization via caveolin-1 and clathrin-mediated routes, respectively. Singh et al. found that nanoparticle internalization is influenced by size and shape, with passive internalization for nanoparticles under 5 nm and active internalization for larger nanoparticles. The heterogeneous size of the NC utilized in this study may have resulted in passive ingestion of ～5 nm nanoparticles, whereas bigger particles may have undergone different internalization mechanisms (such as micropinocytosis). Quantitative data from ICP-OES show that nanoparticles are internalized by cell cultures under normal gravity (Ce: 22.5 ± 0.4 ppm) and simulated microgravity (Ce: 1.3 ± 0.3 ppm), though to a lesser extent than under normal gravity (～6% of the amount internalized under 1 g).

Consistent with previous data from different??group, comparisons examining the effects of NC show a relatively modest number of DEGs. The 17 genes identified as responding to NC on land (E vs F) do not exhibit substantial enrichment in GO keywords. The analogous experiments in space (A vs B) and in space with gravity (C vs D) generate 32 and 1 DEGs, respectively, with no significant GO terms. These sets have no genes in common. The current investigation's moderately lower yield for NC could be explained by its more difficult experimental design, specifically the deployment of actively growing cells.

Previously, downregulation was the dominating dynamic among DEGs upon NC administration. However, in our study, over 75% of DEGs are overexpressed when NC is provided (～77% for E vs F, ～72% for A vs B). Space had a clear impact on cell transcriptome, with upregulation being slightly more prevalent and DEGs exhibiting surprisingly coherent behavior, which is consistent with our previous findings. Two exceptions are comparisons assessing the influence of microgravity (C vs A and D vs B): in these, the majority of DEGs are downregulated, and those in common with comparisons examining space radiations show a systematically non-coherent trend. In other words, microgravity and space radiations appear to produce opposing responses from a single metabolic circuit, despite the fact that no linked GO enrichment was discovered. When genes appear at the junction of a comparison for microgravity and others exploring space in general, coherence is restored. However, it should be noted that all space samples in our study were subjected to ISS space radiations; as a result, microgravity is only evaluated by removing space radiations from space, that is, with space radiations as part of the experimental environment.

When assessing the sole effects of space radiations, the comparison (E vs C) is exceptionally rich in DEGs. In agreement with a prior study often indicate enhanced catabolic activity in response to an external stimulus. Intersecting with which is the overall comparative study space, the set isolates a group of 281 DEGs that indicate short-listed probable radiation-responsive genes. Twenty-six genes are at the intersection of four comparisons that examine space in distinct ways. Overall, they do not have a substantial relationship with any GO term, although they may be good candidates for validation research. The most notable subgroup of the Venn diagram for the effects of space on NC-treated samples may be the 98 genes at the intersection, which suggest another group of probable radiation-sensitive DEGs. The existence of several references to eyesight in GO graphs investigating space radiations is noteworthy. Rhodopsin (Rho) and its molecular switch guanine nucleotide-binding protein G(t) subunit α-1 (Gnat1) are included in the 26-gene group of space genes, specifically in the subgroup of five downregulated genes. One possible reason is that the exposure to damaging electromagnetic radiations may be slightly indicative of light hitting photoexcitable cells.

The transcriptional evidence collected by applying an experimental protocol involving antioxidant nanoparticle administration to muscle cell cultures during spaceflight (i.e., exposure to different gravity levels and cosmic radiations) demonstrated that space effects outweighed antioxidant effects. Under experimental settings, space radiations in particular emerged as a force influencing muscle cell transcriptome. Previous research into synergies between microgravity and space radiations yielded slightly contradictory results. The results?obtain show that the two stressors work through partially overlapping molecular components, eliciting opposing reactions, and that there may be a parallel between photoreception and response to space radiations that merits additional molecular investigation. Along with earlier findings, the new work supports the notion of OS as a crucial dynamic in space pathology. The results ?found support Ucp2's involvement as a critical mediator of cell responsiveness to space, whether alone or in conjunction with MAPK/ERK signaling pathway components. Future research will focus on optimizing antioxidant administration in space and completely understanding the role of Ucp2 as a possible molecular target for treating spaceflight-induced tissue waste.