a72. mitochondrial responses of neonatal rat ventricular myocytes exposed to lps
TRANSCRIPT
lular HSP60 has been shown to be a potent danger signal for
the innate immune system. Here we show that exosomes, small
membrane vesicles that are secreted by various cell types,
contribute to the release of HSP60 from adult rat cardiac
myocytes in both basal and stress-induced (hypoxia 4 h,
reoxygenation 2 h, insufficient to release LDH) states. HSP60
release from the rat myocytes was independent of the common
secretary pathway, as Brefeldin A, an inhibitor of the classical
protein transport pathway, did not block HSP60 release. To
examine whether exosomes contributed to HSP60 release from
myocytes, exosomes were isolated from the media in the basal
state and after minor stress. HSP 60 was found in exosomes in
the basal state, and both the amount of HSP60 and the amount
exosomes released were increased after stress. In addition, we
found that HSP60 release from the myocytes occurred via a
lipid raft-dependent pathway, as treatment with methyl-h-cyclodextrin, a raft disrupting drug, somewhat decreased the
amount of HSP 60 released. Treatment of myocytes with the
exosome inhibitor, dimethyl-amiloride, decreased the amount
of HSP60 released to a much greater extent than the lipid raft
inhibitor, methyl-h-cyclodextrin. Exosomes accounted for
more than 70% of the HSP60 released in the media by
myocytes. HSP60 released in the exosomes was ubiquinated.
In conclusion, HSP60 is actively released from cardiac
myocytes via an exosomal secretory pathway. Supported by
HL077281 and the Department of Veterans Affairs.
doi:10.1016/j.yjmcc.2006.03.318
A44. Properties of Ca spark voids in normal and failing
myocytes revealed by flash photography
Sivan V. Meethal, Timothy J. Kamp, Hector H. Valdivia,
Robert A. Haworth. University of Wisconsin, 600 Highland
Ave, Madison, WI 53792
Excitation-induced Ca gradients were imaged in isolated rat
and dog myocytes by a novel method of flash photography of
fluo-3 fluorescence. In normal rat myocytes loaded with fluo-
3AM, Ca sparks were evident 6msec after stimulation emerging
from around t-tubules, as judged by co-localization with di-8-
ANEPPS staining. Voids in the spark pattern coincided not only
with gaps in di-8-ANEPPS staining but also with points of
vacuolar fluo-3 labeling, visible in unstimulated cells at the z-
lines, previously identified as lysosomes. In a cell at rest that
showed spontaneous Ca spark activity, baseline [Ca] was on
average elevated at sites of vacuolar fluo3 labeling, suggesting a
higher level of Ca spark activity at these locations. These results
suggest that voids in excitation-induced sparks occur at sites of t-
tubule turnover, but these sites may still support spontaneous Ca
sparks. In normal dog myocytes, the beat-to-beat variance of Ca
sparks was very low. Inmyocytes from dogs with failure induced
by rapid pacing, the reduced Ca transient was associated with
greater beat-to-beat spark variance (Ratio of image intensity
variance at ‘‘Spark’’ locations/‘‘No Spark’’ locations: 2.42 T 0.45and 6.05 T 1.08, mean T SD, for control and heart failure
respectively, n = 3 cells/group, P < 0.01). A non-uniform spatial
distribution of Ca sparks was also evident in failing cells, which
resulted in Ca gradients at longer times after stimulation. Ca
gradients rapidly resolved when t-tubular voids were small, as in
normal cells, but persisted when the voids were larger, such as
occurred in the failing cells, along with t-tubule depletion and
derangement. In heart failure this may cause heterogeneous
contraction and contribute to contractile failure.
doi:10.1016/j.yjmcc.2006.03.319
A72. Mitochondrial responses of neonatal rat ventricular
myocytes exposed to lps
Chad Jones, L. Maximilian Buja, Diane L.M. Hickson-Bick.
University of Texas Medical School at Houston, TX 77030
Unlike in the isolated adult ventricular cardiomyocyte,
exposure of neonatal cells to lipopolysaccharide (LPS) does
not induce apoptosis. Neonatal cells exhibit a protected
phenotype towards this bacterial toxin. LPS does induce
significant changes to the mitochondria of neonatal cells. Upon
addition of LPS to culture media, neonatal cells rapidly lose
energized mitochondria as observed with the potential sensitive
dye TMRE. This generalized loss of mitochondrial membrane
potential also exhibits an inverse relationship to a redistribution
of uncoupling protein UCP3 from a perinuclear concentration
into peripheral mitochondria. No changes were observed in the
distribution of UCP2. ATP levels also decrease in these cells.
The observed changes in mitochondrial membrane potential,
UCP3 distribution and ATP levels are reversed over a period of
several hours. The cellular recovery of energizedmitochondria is
the result of mitochondrial biogenesis. Transcription and
replication of mitochondrial DNA (mtDNA) requires TFAM, a
nuclear encoded enhancer protein, which in turn can be
regulated by transcription factors nuclear respiratory factor 1
(NRF-1) and NRF-2. NRF-1 and NRF-2 also act as transcription
factors for nuclear genes that encode respiratory complex
subunits. PGC-1a, a transcriptional co-activator, can also
interact with NRF-1 to induce mitochondrial biogeneseis. In
neonatal rat cardiomyocytes exposed to LPS for several hours,
we have observed a time-dependent increase in PGC-1a protein,
a nuclear translocation of NRF-1 and an up-regulation of TFAM
mRNA. These all indicate that the neonatal cells_ ability to
recover its energy capacity requires a co-ordinated expression of
both nuclear and mitochondrial genes. Supported by grants
DAMD17-01-2-0047 and DAMDW81XWH-04-02-0035.
doi:10.1016/j.yjmcc.2006.03.320
A21. Cardiac excitation-contraction coupling is altered in
ventricular myocytes from aged male but not female mice
S.A. Grandy, S.E. Howlett. Department of Pharmacology,
Dalhousie University, Halifax, NS, Canada B3H 1X5
This study characterized age-related alterations in excitation-
contraction (EC)-coupling in ventricular myocytes and investi-
ABSTRACTS / Journal of Molecular and Cellular Cardiology 40 (2006) 862–918876