23rd Annual NASA Space Radiation Investigators' Workshop (2012)
8063.pdf
Transcriptional Profiling of Cardiac Cells Reveal an
Immense Complexity of Gene Expression Over One
Month after a Full Body 0.15 Gy 56Fe but Not 0.9 Gy Proton Radiation
T. McDonald2, X. Yan1,3, S. P Sasi1, Y. Yang1, L. Hlatky2,3, Shtifman1,3, D. A. Goukassian1,3
(1) CardioVascular Systems Biology and (2) Center of Cancer Systems Biology, Steward St Elizabeth’s Medical
Center, (3) Tufts University School of Medicine, Boston, MA, USA
Background. Cardiomyocytes are the basic contractile cells within the heart, whose contractile function directly
influences the pathogenesis of heart disease and the development of myocardial failure. Understanding of
complex processes that take place in cardiomyocytes after insults caused by exposure to ionizing radiation, such
as proton and HZE particles, is paramount to our understanding of cardiovascular system function during and
after exploration-type space missions.
Methods. Primary, ventricular cardiac cells were isolated using standard preparation protocol that includes
cannulation of the aorta and collagenase digestion followed by a Ca2+ gradient selection. This procedure
yielded >80% of Ca2+-tollerant cardiomyocytes. The resulting cell suspension could contain a small population
of inflammatory and endothelial cells. Following isolation of cardiomyocytes, RNA was extracted via TRIzol
and run on an Affymetrix GeneChip platform. Data was normalized, corrected for background, which was
followed by log2 transformation. Statistical analysis was performed using a one-way ANOVA and p-values
were adjusted using the Benjamini-Hochberg method. There were 5220 genes with p <0.05. After correction
for the False Discovery Rate (FDR), 184 genes had an adjusted p<0.05. Of these, 51 genes displayed a greater
than or less than 2-fold-change in RNA transcription. Functional analysis was performed using Ingenuity
Systems IPA software. A less stringent filter of data was used to uncover possible biological functions. A filter
using an unadjusted p<0.05 (FDR<0.2), greater than or less than 1.5-fold change compared to control and
average signal greater than 50 (au) resulted in a total of 878 genes entered into IPA.
Results. The analysis of proton-irradiated samples at FDR (p<0.05) did not reveal any changes in RNA
transcription. When, FDR for the same samples was decreased (p<0.1), there were only 21 genes that exhibited
an appreciable increase/decrease in RNA transcription, thus eliminating the need for further bioinformatics
analysis of proton irradiated samples.
I.
Very stringent analysis of 56Fe IR samples after correction for FDR (p<0.05) and 2 or more fold change
in RNA transcription using Genome Studio, IPA, Gather software:
Using the above parameter 51 genes displayed a greater than or less than 2-fold-change in RNA transcription
with biphasic time course in comparison to controls. This change included up-regulation at day 7, complete
down-regulation by day 14 and a second significant up-regulation by day 28 post IR. Among others the upregulated genes included CREB1, Snord15, 61, 35b, 35a, 33, 82, Grap2, ADAM 19, Snora62, 74, Fmod,
Igfbp6, Arntl, Sit1, Tyrobp. Alox5ap. Just to name a few: CREB1 is involved in numerous signaling pathways,
including cardiac hypertrophy, calcium signaling and DNA damage repair, apoptosis etc. Snords are small
nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guide chemical modifications of
other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs. There are two main classes of
snoRNA, the C/D box snoRNAs which are associated with methylation, and the H/ACA box snoRNAs which
are associated with pseudouridylation and control microRNA regulation. Sit1 regulates NFAT, MAPK8, Erk1/2
and p38. ADAM19 is a metalloproteinase that cleaves and activates neuregulins that are essential for
cardiomyocyte survival under stress conditions.
II
Less stringent analysis unadjusted p<0.05, FDR p<0.2 and ±1.5-fold change – Genome Studio, IPA:
The 878 gene list (FDR<0.2) has a clear down-regulation of gene expression on days 1 and 3 compared to
control (150 of 150 genes down-regulated on day 1 and 406 of 433 genes down-regulated on day3) while an
increase is observed on days 7 and 28 relative to control (154 of 187 genes up-regulated on day 7 and 202 of
249 genes up-regulated on day 28). There is a striking relationship to inflammatory genes seen just by looking
at the top 10 up-regulated genes on day 28 which are Cd8b1, Ccr9, Themis, Igkv1-117, Lck, Cd3g, Cd8a, Rag1,
Dntt, and Tcf7 ranging 8.0-fold to 15-fold.
III
Functional pathways analysis – IPA software: Functional analysis of the 878 gene list shows a time
dependent switch from decreased free radical scavenging on day 3 to an increased activity on day 7. The most
striking biological functions related from this data are the time dependent increase in immune/inflammatory
categories on days 7 and 28.
IV
Prediction of transcription factor activation/inhibition analysis – IPA software: In line with the above
function analysis, prediction of the activation or inhibition of transcription factors based on downstream gene
expression in the 878 gene list again shows factors related to immunity and inflammation. NFKB2 is predicted
to be activated on day 14. TCF7 is a known as a T-cell specific transcriptional activator that is important in
lymphocyte differentiation. KLF2 regulates T-cell trafficking. Both are predicted to be activated on day 28.
Summary. Our findings indicate that an immense complexity of RNA transcription, regulation of biological
pathways, included,
but not limited to inflammation, DNA damage/repair, free radical scavenging and immune
trafficking post 56Fe irradiation, but not bioequivalent dose of proton.