Macro and micro physiological models: Spaceflight Radiation and Senescence

Introduction: Microgravity and radiation are inescapable hazards in spaceflight. The National Aeronautics and Space Administration (NASA) plans to extend human exploration in space well beyond low earth orbit (LEO) and for durations much greater than ever experienced. The work presented here aims to address the biological concerns facing future astronauts through the development of microphysiological systems (MPSs) for scalable, high throughput testing of radiation and microgravity countermeasures. Through exploration in animal models and existing spaceflight omics data we have built an understanding of spaceflight factors at the organism level and are transitioning these findings to inform the generation of a multi-organ chip platforms. The unifying result from our ground-based in vivo spaceflight simulations, validated by other’s observations in true spaceflight, is the activation of cellular senescence pathways in response to spaceflight factors. Specifically, the action of cell cycle arrest molecule, CDKN1a/p21, is noted in our previous work and others as a spaceflight biomarker. Given the shared features between spaceflight and aging: expression of p21, muscle and bone atrophy, and mitochondrial dysfunction, we used aging, disuse, and radiation models to probe the function of CDKN1a/p21-induced senescence in multiple systems. We hypothesize that p21 has tissue specific functions related to proliferation/differentiation, regeneration, and inflammation, thus analysis of individual tissues and inter-organ crosstalk will facilitate our understanding of tissue-specific responses to both spaceflight and aging. To answer this hypothesis we evaluated muscle, liver, immune, and skeletal responses to both spaceflight factors and aging.

Materials and

Methods: This work used the CDKN1a/p21 knockout (KO) and B6129SF2/J (WT) mice to investigate the role of CDKN1a/p21 in aging. KO and WT mice were aged to 24 months and hindlimb muscles, liver, bone (femur/tibia) and bone marrow were collected for analysis. In aged animals, we evaluated fiber dynamics, such as force and power, in the extensor digitorum longus (EDL) and soleus. A portion of liver was PFA fixed for histological analysis of lipid accumulation with Oil Red O stains. The remainder of liver tissue was dissociated to isolate resident macrophages (CD45-, F4/80+, CD11b-), bone marrow derived macrophages (CD45+, F4/80-, CD11b+), and inflammatory monocytes (CD45+, F4/80-, CD11b+, Ly6C+). These populations were quantified using flow cytometry. Bone marrow flushed from the femur in aged animals was cultured in vitro and osteoblast mineral formation was evaluated over the course of 30 days. Additionally, we used a spaceflight relevant hindlimb unloading (HU) model to induce muscle and bone atrophy in conjunction with radiation. A subset of HU and normally loaded mice were subjected to an acute 1 Gy gamma radiation (Cs137) exposure. We collected the EDL, soleus, liver, and liver resident macrophages as described above. Muscle tissues were weighed post euthanasia to determine extent of muscle loss due to HU and radiation. Additionally in this experiment we specifically isolated MerTK+ macrophages in the liver to perform transcriptomic analysis.

Results, Conclusions, and Discussions: Aging:
As expected, aged mice demonstrated increased body mass compared to younger mice. However, we found KO mice to have significantly lower mass. Removal of p21 did not rescue age related muscle atrophy in the EDL or soleus as seen in relative muscle weights. Removal of p21 also did not influence slow twitch fibers (Type I), seen in the minimal isometric tension produced. and had negative effects in faster fibers (Type IIB), regarding force production (Figure 1). Type I fibers were negatively affected by age compared to fast fiber types. However, p21 KO animals demonstrated increased muscle speed in type IIA fibers indicating that p21 may be required for maintaining fast twitch muscle function. On the other hand, osteoblasts derived from 24-month-old KO animals retained significant proliferation and mineralization properties compared to their WT counterparts. Further, liver histology revealed p21 KO conferred resistance to lipid accumulation compared to the WT age matched cohort. This effect was seen only up to 18 months of age and was lost at 24 months (Figure 2).

Spaceflight Factors:
Radiation exposure did not induce muscle loss nor significantly contribute to muscle atrophy in HU. However, HU alone significantly decreased muscle mass in the EDL and soleus. Previous research has demonstrated that radiation induces p21, thereby reducing proliferation but not directly causing atrophy, which explains our finding. Our results also indicated that radiation exposure up-regulates p21 in macrophages isolated from the liver (Figure 3). Expression of p21 halts proliferation and prevents apoptosis in response to DNA damage, suggesting important radio-resistance in this population.

Together these investigations highlight the role of p21 in tissue-specific responses to aging and spaceflight. This provides crucial data for our ongoing work in developing multi-organ tissue chips enabling future investigation of tissue-specific responses and inter-organ communication following spaceflight and aging.

We have already generated human iPSC-derived liver organoids and are currently extending the longevity of these to enable sustained study of aging and exposure to chronic radiation. Our overall goal is to generate a multi-organ platform to rapidly study radiation response, metabolic demands, and the role of senescence in critical-interconnected organ systems.

Acknowledgements (Optional): NASA Grant #80NSSC20K0289
NIGMS T32 Training Grant #5T32GM141865-02