The absence of unit gravity may cause physiological changes in the cardiovascular system. For instance, in the absence of Earth's gravity, venous return to the heart may increase due, in pan, to decreased pooling of the blood in the extremities. We hypothesize that this would produce an increase in the heart's work load ultimately resulting in hypertrophy

Brunton, Laurence L.

Lio, Francisco M.

Meszaros, J. Gary

English

NASA Technical Reports Server

 ROaRE REPORT
NASA Grant NAG2-9T/ -----'-
PI: Laurence L. Brunton
IN
BIOCHEMICAL ASSESSMENT OF STRESS
IN CARDIAC TISSUE
RESPONSE TO WEIGI-r'rLESS SPACE TRAVEL
_y J
PERSONNEL
Name
LaurenceL. Brunton, Ph.D.
J. Gary Meszaros, Ph.D.
Francisco M. Lie
UGSD Title
Professor of Pharmacology
and Medicine, UCSD
Postdoctoral fellow
Undergraduate and
MBRS awardee
Role in Grant
Principal Investigator
Investigator
Lab Assistant
HYPOTHESIS
The absence of unit gravity may cause physiological changes in the
cardiovascular system. For instance, in the absence of Earth's gravity, venous return
to the heart may increase due, in pad, to decreased pooling of the blood in the
extremities. We hypothesize that this would produce an increase in the heart's work
load, ultimately resulting in hypertrophy.
ORIGINAL PROPOSAL AND ITS REVISION
Originally, we proposed a two project for approximately $50,000 per year.
NASA awarded $20,000 for one year. This required scaling back the aims. The
original included a menu from which items to be measured would be chosen based on
the most current knowledge. Original proposed measurements centered on
transmembrane signaling (cyclic AMP system) and on inducible genes (los, jun,
HSP70 and others mentioned below). In scaling back to met the exigencies of the
budget, we chose to focus on mRNA expression, since multiple probings could be
.
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made from a single mRNA gel blot, and since a careful examination of the recent
literature convinced us that certain genes were very good markers of stress and
hypertrophy in cardiac tissue.
TESTING THE HYPOTHESIS
As a test of the hypothesis, we have analyzed the mRNA levels of ANP in
cardiac tissue from rats subjected to weightless space travel.
ANP is normally synthesized and secreted by atrial myocytes, however its
expression can be induced in the ventricles during hypertrophy and is widely accepted
as a molecular marker of myocardial growth.
RESULTS
In this study, Sprague-Dawley rats were divided into ground control and flight
specimens, with the latter being subjected to two weeks of space travel, Upon their
return to Earth, both flight specimens and ground controls were sacrificed and atria
and ventricles were isolated. We received the frozen heart tissue from NASA.
We have powdered each tissue, then extracted mRNA from each sample and
analyzed it by Northern analysis using a cDNA probe specific for ANP. The
techniques applied are all standard, although some minor adapting was required for
the tissue and for the qualities of the specific mRNA and cDNA.
ANP mRNA levels were elevated in the flight speCimens, with the most dramatic
increase occurring in the ventricular tissues. Densitometric assessments of
autoradiographs demonstrated a 30% increase of ANP message in atrial tissue and a
230% increase of ANP message in the ventricular tissue of rats subjected to
weightlessness. Ground controls showed no change in expression of ANP in either
ventricular or atrial tissues. The increase of ANP message supports our hypothesis
that prolonged space travel leads to cardiac hypertrophy.
Recently, we have been constructing cDNA probes for HSP70 and SERCA2,
as described below.
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CONTINUATION OF THIS WORK
One of the interesting bonuses of assessing expression of mRNAs by Northern
analysis is that the blots may be probed, stripped and re-probed multiple times. In
addition, we have tissue and mRNA for multiple gels/blots. Thus, we can continue to
study this set of tissues. We are currently using additional cDNA probes and Northern
analysis to assess the effects of weightless flight on other markers of growth and stress
in cardiac tissue, including SERCA,?. and HSP70.
SERCA2. is the protein that regulates the accumulation of Ca ++ back into the
sarcoplasmic reticulum to permit (or cause) relaxation of the heart. We have evidence
that the SERCA2 gene is regulated by cAMP and the PI/PKC pathways and that the
mRNA for SERCA2 is down-regulated by the hormone endothelin, which is also a
cardiac growth factor and positive inotropic agent. Down regulation of the
sarcoplasmic reticulum calcium ATPase (SERCA2) is associated with cardiac
dysfunction in the failing heart. Plasma levels of the growth peptide endothelin (ET-1)
levels are elevated in cardiac hypertrophy and failure. Investigating the capacity of
ET-1 to regulate SERCA2 gene expression in cardiac myocytes, we used Northern
analysis studies of mRNA of both adult and neonatal cardiac rnyocytes. The data
indicate that after a 24 hour treatment with EF-1, SERCA,?. expression is decreased
by 30% and 50%, respectively. In transfection experiments using the SERCA,?.
promoter region linked to CAT activity, Er inhibits basal SERCA2. promoter activity as
well as thyroid hormone stimulated SERCA2 promoter activity. Preliminary data
indicate that the signaling pathways by which ET down-regulates SERCA2 expression
require ET-1 activation of both Gq and Gi linked pathways and the lowering of
intracellular cAMP levels (1).
Based on this knowledge, we believe that probing the cardiac tissue of the
NASA "astronaut rats" will be exciting. We have produced a cDNA probe for SERCA2
and will shortly begin working out conditions for probing the mRNA blots for the
message for SERCA2..
We are also trying to set up a collaboration with colleagues at UCSD who study
the capacity of cardiac fibroblasts to "re-model" the extracellular matrix and who study
angiogenesis. Our thought here is that alteration of the extracellular matrix could be a
prelude to hypertrophic responses in the heart and that expression of different
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collagens may alter the mechanical properties of the ventricles. Similarly, expression
of VEGF (Vascular Endothelial Growth Factor) is an early marker for an angiogenic
response that is often observed in stressed cardiac tissue when 02 demand exceeds
02 supply. Whether this gene is activated by weightless space flight is not known but
readily determined and we are preparing to obtain the cDNA probe.
CAVEAT
A complete analysis cannot be made until NASA sends us the full key to the
sample code. Much of the system of labeling is apparent but we need to be certain
that we have the key and that we interpret Our results correctly according to the
protocol NASA used. We have requested the key and are currently filing a second
request.
PUBLICATIONS SUPPORTED BY THIS GRANT
° LiD FM, Meszaros JG, Brunton LL. (1997). Induction of mRNA for Atrial
Natriuretic Peptide (ANP) in Hearts of Rats Subjected to Weightless Space
Travel. Abstract for National Minority Biomedical Research Symposium, New
Orleans, October, 1997.
. A manuscript based on the above work is being prepared but will not be
completed until NASA sends the full key to the labeling of the samples.
, Meszaros JG, Brunton LL and Bloor CM. "Animal Models of Angiogenesis in
Cardiovascular Tissues" in, Anaioaenesis in C_lr_iovascular Disease, ed., JA
Ware and M Simons, Oxford University Press, for late 19971early 1998.
- 4-
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REFERENCES
. Hilal-Dandan R, Brunton LL and Dillmann W (1996). Endothelin Regulates
Sarcoplasmic Reticular Calcium ATPase (SERCA2) Gene Expression in Rat
Cardiac Myocytes. Circulation (abstracts for AHA Meetings, Fall, 1996)
. Martin J L, Mestril R, HilaI-Dandan R, Brunton LL and Dillmann WH (1997).
Small heat shock proteins and protection against ischemic injury in
cardiomyocytes. Circulation, in press.
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Biochemical Assessment of Stress in Cardiac Tissue in Response to Weightless Space Travel

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