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Microgravity induces proteomics changes involved in endoplasmic reticulum stress and mitochondrial protection

View Article: PubMed Central - PubMed

ABSTRACT

On Earth, biological systems have evolved in response to environmental stressors, interactions dictated by physical forces that include gravity. The absence of gravity is an extreme stressor and the impact of its absence on biological systems is ill-defined. Astronauts who have spent extended time under conditions of minimal gravity (microgravity) experience an array of biological alterations, including perturbations in cardiovascular function. We hypothesized that physiological perturbations in cardiac function in microgravity may be a consequence of alterations in molecular and organellar dynamics within the cellular milieu of cardiomyocytes. We used a combination of mass spectrometry-based approaches to compare the relative abundance and turnover rates of 848 and 196 proteins, respectively, in rat neonatal cardiomyocytes exposed to simulated microgravity or normal gravity. Gene functional enrichment analysis of these data suggested that the protein content and function of the mitochondria, ribosomes, and endoplasmic reticulum were differentially modulated in microgravity. We confirmed experimentally that in microgravity protein synthesis was decreased while apoptosis, cell viability, and protein degradation were largely unaffected. These data support our conclusion that in microgravity cardiomyocytes attempt to maintain mitochondrial homeostasis at the expense of protein synthesis. The overall response to this stress may culminate in cardiac muscle atrophy.

No MeSH data available.


Translation is diminished in microgravity.(A) Immunoblot of biotin-tagged AHA, a methionine analog, and loading control FKBP12, together representing the decline in protein synthesis in μg at 120 h (n = 3, p = 0.003). AHA was added to the media for 2 hours at both 0 h and 120 h time points. (B) A bar chart representing the quantification of the densities of the AHA immunoblot. (C) Luciferase activity was markedly reduced in μg (n = 4, p = 0.02). AHA, azidohomoalanine; RLU, relative light unit.
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f6: Translation is diminished in microgravity.(A) Immunoblot of biotin-tagged AHA, a methionine analog, and loading control FKBP12, together representing the decline in protein synthesis in μg at 120 h (n = 3, p = 0.003). AHA was added to the media for 2 hours at both 0 h and 120 h time points. (B) A bar chart representing the quantification of the densities of the AHA immunoblot. (C) Luciferase activity was markedly reduced in μg (n = 4, p = 0.02). AHA, azidohomoalanine; RLU, relative light unit.

Mentions: To evaluate the influence of microgravity on protein synthesis, orthogonal experiments were performed utilizing the methionine analogue azidohomoalanine (AHA) which is incorporated into proteins via methionine tRNA and can be used to tag newly-translated proteins20. To measure protein synthesis across a small time window, cells were exchanged from Met- to AHA-containing media at 120 h, and labeling was performed for 2 h. Using the total protein lysates from each group, proteins were specifically tagged on the azido side chains with biotin and subsequently immunoblotted using HRP streptavidin (n = 3 per condition). AHA incorporation was robust after two hours of labeling, but was dramatically reduced at 120 h in μg relative to 1xg (p = 0.003; Fig. 6A,B). These data demonstrate that protein synthesis, a factor contributing to overall protein turnover, is significantly diminished after cells have been exposed to μg for 120 h, compared to the same cells under normal gravity, and they suggest that the decreased protein turnover, as measured by RIA, reflected this decrease in protein synthesis.


Microgravity induces proteomics changes involved in endoplasmic reticulum stress and mitochondrial protection
Translation is diminished in microgravity.(A) Immunoblot of biotin-tagged AHA, a methionine analog, and loading control FKBP12, together representing the decline in protein synthesis in μg at 120 h (n = 3, p = 0.003). AHA was added to the media for 2 hours at both 0 h and 120 h time points. (B) A bar chart representing the quantification of the densities of the AHA immunoblot. (C) Luciferase activity was markedly reduced in μg (n = 4, p = 0.02). AHA, azidohomoalanine; RLU, relative light unit.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037457&req=5

f6: Translation is diminished in microgravity.(A) Immunoblot of biotin-tagged AHA, a methionine analog, and loading control FKBP12, together representing the decline in protein synthesis in μg at 120 h (n = 3, p = 0.003). AHA was added to the media for 2 hours at both 0 h and 120 h time points. (B) A bar chart representing the quantification of the densities of the AHA immunoblot. (C) Luciferase activity was markedly reduced in μg (n = 4, p = 0.02). AHA, azidohomoalanine; RLU, relative light unit.
Mentions: To evaluate the influence of microgravity on protein synthesis, orthogonal experiments were performed utilizing the methionine analogue azidohomoalanine (AHA) which is incorporated into proteins via methionine tRNA and can be used to tag newly-translated proteins20. To measure protein synthesis across a small time window, cells were exchanged from Met- to AHA-containing media at 120 h, and labeling was performed for 2 h. Using the total protein lysates from each group, proteins were specifically tagged on the azido side chains with biotin and subsequently immunoblotted using HRP streptavidin (n = 3 per condition). AHA incorporation was robust after two hours of labeling, but was dramatically reduced at 120 h in μg relative to 1xg (p = 0.003; Fig. 6A,B). These data demonstrate that protein synthesis, a factor contributing to overall protein turnover, is significantly diminished after cells have been exposed to μg for 120 h, compared to the same cells under normal gravity, and they suggest that the decreased protein turnover, as measured by RIA, reflected this decrease in protein synthesis.

View Article: PubMed Central - PubMed

ABSTRACT

On Earth, biological systems have evolved in response to environmental stressors, interactions dictated by physical forces that include gravity. The absence of gravity is an extreme stressor and the impact of its absence on biological systems is ill-defined. Astronauts who have spent extended time under conditions of minimal gravity (microgravity) experience an array of biological alterations, including perturbations in cardiovascular function. We hypothesized that physiological perturbations in cardiac function in microgravity may be a consequence of alterations in molecular and organellar dynamics within the cellular milieu of cardiomyocytes. We used a combination of mass spectrometry-based approaches to compare the relative abundance and turnover rates of 848 and 196 proteins, respectively, in rat neonatal cardiomyocytes exposed to simulated microgravity or normal gravity. Gene functional enrichment analysis of these data suggested that the protein content and function of the mitochondria, ribosomes, and endoplasmic reticulum were differentially modulated in microgravity. We confirmed experimentally that in microgravity protein synthesis was decreased while apoptosis, cell viability, and protein degradation were largely unaffected. These data support our conclusion that in microgravity cardiomyocytes attempt to maintain mitochondrial homeostasis at the expense of protein synthesis. The overall response to this stress may culminate in cardiac muscle atrophy.

No MeSH data available.