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