From our studies the following general concepts were obtained:

1. Protein expression is altered in cardiomyocyte following simulated microgravity. We measured protein-level expression for 848 proteins (6,174 peptides) across all timepoints and gravity conditions. As expected very few proteins are differentially expressed (p<0.05) at 12 hours, and the number grows over time, until nearly 100 proteins are differentially expressed. Proteins were classified into three groups: 1) unchanged over time under either condition, 2) equally changed over time under either condition, and 3) differentially changed over time between microgravity and 1x gravity. The significantly altered proteins were then grouped by biological function; seven functional groups were formed: Muscle contraction/structure (26%); Translation (23%); Metabolism (21%); Protein transport and folding (12%); Calcium handling (5%); and Other.

2. Cardiomyocytes under simulated microgravity have diminished protein turnover. Using the ratio of the heavy-isotope label to the total of heavy and light peptide in the dynamic SILAC labeling component of the experiment, our data strongly suggest that amino acid incorporation and thus protein turnover is diminished in the cardiomyocyte subjected to simulated microgravity. No statistical difference exists between measured turnover at 12 hours, however p<1e-6 at both 48 hr and 120 hr. This data is highly suggestive that protein turnover is drastically decreased under the microgravity condition, and since access to the translational machinery to precursor amino acid did not seem to be different, the difference points to transcriptional or translational control.

3. Protein translation may be slowed in cardiomyocytes under simulated microgravity. Following the independent quantitative and label-incorporation analyses, two features of the data were connected. The protein expression data revealed that many proteins were downregulated following 120 h of microgravity, specifically, a number of translational proteins were diminished. The decrease in translational proteins suggests that protein translation is slowed, which could be mediating the decline in expression of the other significantly reduced proteins. This revelation was strengthened by the markedly reduced protein turnover observed in the label-incorporation data. Because decreased translation is a characteristic that has been observed in microgravity-induced skeletal muscle atrophy, we decided to perform independent experiments that could validate this potential phenomenon: azidohomoalanine (AHA) incorporation, luciferase activity using adenoviral technology, and total protein-to-total RNA ratio.

AHA is a methionine analogue that is incorporated into an individual peptide in a similar fashion as arg or lys. However, AHA was presented to the groups only at 120 h time point and only for 2 h; this design was merely to obtain a snapshot of protein synthesis at the time when the largest separation in protein turnover was observed. AHA incorporation is robust in 0 h controls but is dramatically reduced after 120 h in microgravity relative to 1x gravity.

Adenoviral technology was used to deliver the luciferase gene to each group. This technology allows the gene to be transcribed and translated into protein using the cell’s inherent machinery. For this experiment, the Adenoviral-Luciferase was delivered to all cells 3 h prior to group separation. The adenovirus was removed upon group separation, and the luciferase protein activity was measured at 120 h. Luciferase activity is a direct correlate to luciferase protein content, since no post-translational modifications are necessary to initiate luciferase enzyme function. Light detection revealed that luciferase protein was significantly diminished 7.5 fold in microgravity compared to 1x gravity. Cell pellets were collected from each group at 120 h, and total protein and total RNA were assessed in order to provide a global examination of protein synthesis relative to the amount of RNA. Data revealed that the total protein-to-total RNA ratio was significantly attenuated 4 fold in microgravity relative to 1x gravity.

In all, these independent measures support the SILAC label-incorporation data, indicating that protein turnover in the cardiomyocyte is diminished following 120 h of exposure to simulated microgravity.

4. Indicators of cell damage and protein degradation are not significantly affected in response to simulated microgravity. We next examined indicators of cell damage, death, and protein degradation in attempt to determine if microgravity was inducing damage to the cardiomyocyte, thereby altering the proteome, or enhancing protein degradation, thereby slowing protein turnover. Lactate dehydrogenase is a soluble cytosolic enzyme, and its release is an indication of cell membrane permeability or damage. The media was examined from each group at 0, 1, 2, 3, 4, 8, 12, 24, 48, 96, and 120 h. No statistically significant difference was observed between microgravity and 1x gravity at any time period.

Caspase-3 is a classical end-effector protease involved in apoptosis. An increase in caspase-3 is a surrogate for enhanced cell death. We surveyed caspase-3 activity at 0, 12, 48, and 120 h, and did not observe a statistically significant difference between microgravity and 1x gravity at any time point. Finally, we assessed global ubiquitination of protein lysates at 0 h and from each group at 120 h. Again, no difference was observed between microgravity and 1x gravity.

Together, these data indicate that cardiomyocytes under microgravity are not markedly damaged in response to simulated microgravity. Moreover, because ubiquitination of proteins is a way proteins are tagged for degradation, these data suggest that microgravity is not enhancing protein degradation, which also indicates that protein degradation may not be playing a critical role in the diminished protein turnover induced in microgravity.

Aim 1. To determine the proteomic and phosphoproteomic changes which occur to rat neonatal cardiomyocytes (RNNCM) upon exposure to microgravity. RNNCM will be placed in the NASA bioreactor, also referred to as the Rotating Wall Vessel (RWV). RNNC were chosen for the first aim in this pilot as they are a readily attainable and hardy cell type which can withstand being cultured for weeks, for determining the minimum time needed for observable simulated microgravity effects via proteomics analysis. Initially we planned to evaluate proteomic changes at 1 week, 2 week, and 1 month of (+/-) exposure to simulated microgravity.

Although we proposed to evaluate changes at 1 week, 2 week, and 1 month, our preliminary data (on the poster presented at the HRP Investigators' Workshop, Galveston 2013) indicated that proteomics changes were observed as soon as 12 hours post exposure to simulated microgravity. Therefore in our series of experiments for Aim1 we modified the experiment to evaluate changes at 12, 48, and 120 hours of simulated microgravity. In addition, we modified our experiments to include stable isotope labeling by amino acids in cell culture (SILAC) to evaluate changes in protein synthesis as a consequence of microgravity exposure.

These experiments described above were performed three independent times. The samples were then quantified using mass spectrometry proteomics analysis by the Duke Proteomics Core Facility. Data analysis resulted in over 6,100 peptides and 848 proteins quantified, with 492 proteins having more than 1 peptide to match. There were over 1,400 peptides identified as having incorporated amino acid isotopes. The indicates that incorporation rates can be calculated for many proteins and that we will be able to evaluate protein levels change as a result in protein synthesis or degradation.

We have just begun to analyze the data. The samples (microgravity and 1x gravity) are trending together with time. We plan to begin to analyze the data between time points, to determine what is differentially expressed between for example 48 hr and 12 hr, and 120 hr versus 12 hr. We plan to perform these analyses independently for the microgravity and 1x gravity controls. This type of analysis should provide us with information on the changes occurring to protein expression as a function of time. A second analysis will help confirm these findings but will specifically compare expression at 12, 48, and 120 hr between microgravity and 1x gravity. This should help us to classify quickly the proteins that are changing differently as a function of time, between 1x gravity and microgravity.