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LIPOFUSCIN ACCUMULATION IN RAT MYOCARDIUM AS AN INDICATOR OF AGE ASSOCIATED OXIDATIVE DAMAGE
Madhukar Kolli1, Anjaiah Katta1, Kevin M Rice1,2, David Neff1, Eric R Blough1,2
1 Department of Biological Sciences, Marshall university, 2 Department of Pharmacology, Physiology, and Toxicology John C Edward School of Medicine
Abstract:
Materials and methods:
An emerging hypothesis described as the free radical theory of aging suggests that aging occurs through the gradual
accumulation of free radical damage to biomolecules (Harman, 2003). Inherent to this theory is that oxidative stress
increases with advancing age. The causes of age-related increases in oxidative stress are not well understood, however
it is thought to be related to declining mitochondrial function. The factor's which mediate age-associated changes in
mitochondrial function are not well understood however, it is thought that age-related increases in lipofuscin may be
involved since the accumulation of this molecule has been hypothesized to impair mitochondrial turnover (Brunk &
Terman, 2002). Using aging Fischer 344 X Brown Norway (FBN) animals, recent studies from our laboratory suggest that
aging increases reactive oxygen species (ROS) levels in these animals and that chronic acetaminophen (APAP)
treatment (30 mg/kg/day) appears to diminish age related ROS levels while improving age related cardiovascular
function. To test whether chronic acetaminophen may reduce ROS levels by altering mitochondria and lipofuscin bodies,
we examined tissue sections obtained from the left ventricles of 6, 27, 33 month old control, and 33 month old FBN
animals that had undergone APAP treatment for 6 months. Confocal imaging of cardiac preparation demonstrated
increased lipofuscin content with aging and this finding was not altered with APAP intervention. Compared to traditional
histological techniques, confocal mapping of lipofuscin bodies in whole mount tissues appears to be a convenient and
valid technique to examine lipofuscin accumulation with aging. Harman, D. (2003).
6-, 27-, 33-month control and 33-month Fischer 344 X BN rats which had undergone daily
APAP for 6 months (30mg/kg/day)
Method 1:
•Heart sections are taken with razor blade of >80 µm notice all images show ventricular
fibers oriented longitudinally in the plane of section.
•Incubated in Mitotracker G FM 40 nM for 15 min.
•Sections analyzed by confocal microscopy (Ex-488nm / Em-522/32 nm band pass filter)
Method 2:
•Heart sections are taken with razor blade of >80 µm; Sections incubated in PBS
•Sections analyzed by confocal microscopy (Ex-488nm / Em-522/32 nm band pass filter)
Lipofuscin granules increase with aging in the F344XBN heart.
Increased cellular ROS levels may be associated with
age-associated lipofuscin granule accumulation.
Schematic diagram showing lipofuscin formation in post
mitotic cells (left):
Biomolecules are degraded by hydrolytic enzymes within the
lysosomal compartment of the cell. Some macro molecules like
mitochondrial cytochromes and ferritin release low molecular
mass iron when degraded. Lysosomes have a low pH and are rich
in reducing substances such as cysteine. Ferric iron would be
reduced to the redox active ferrous form. Hydrogen peroxide,
which freely diffuses into the lysosomes, may consequently
undergo homolytic cleavage with formation of hydroxyl radicals
which, in turn, induce peroxidative reactions and formation of
lipofuscin from macromolecules undergoing degradation (Brunk
2002).
Above, black arrow points to
Lipofuscin granules in
perinuclear space; H&E
preparation (image from
Indiana U. Pathology)
33m con 40x
6m 40x
6m 40x
6m 40x
27m 40x
27m 40x
27m 40x
27m 40x
33m con 40x
33m con 40x
33m con 40x
33m APAP 40x
33m APAP 40x
33m APAP 40x
Above, TEM of hamster ventricle myocardium.
Dashed line surrounds lipofuscin granule. Arrow
points to miochondrial cristae, dark body labeled
L2 is an active lysosome stained for acid
phosphatase activity. Lipofuscin granules are
believed to be the end product as lysosomes
break down aged mitochondria (image from
Skepper 1987).
Lipofuscin granule accumulation in 33m con
Rat Myocardium (above).
Lipofuscin granule
Intercalated disc
Purpose: The purpose of this study is to examine the age associated changes
in rat myocardium
Results & Conclusions:
See above, confocal imaging of cardiac preparation demonstrated increased lipofuscin content with aging and this
finding was not altered with APAP intervention. In 27, 33 month optical-sections of F344 BN Rat hearts with aging the
perinuclear accumulation of lipofuscin granules occurred when compared to 6 month optical-sections of rat hearts.
Scale bar at top left applies to all images.
References:
Specific Aim: To investigate whether chronic acetaminophen treatment may reduce the
age associated oxidative damage in cardiac muscle as manifested in lipofuscin and
mitochondrial morphometric changes.
Hypothesis:
We hypothesize that chronic acetaminophen treatment may alter the age associated
oxidative damage in rat myocardium as evidenced by changes in lipofuscin
accumulation.
1.Harman (2003). The free radical theory of aging. Antioxidant Redox Signal 5, 557-561.
2.Brunk, U. Terman, A. (2002). The Mitochondrial-lysosomal axis theory of aging. Eur. J. Biochem. 269, 1996-2002.
3.Skeeper, J.N. Navaratnam, V. (1987). Lipofuscin accumulation in the myocardium of juvenile golden hamsters: an
ultrastructural study including staining for acid phosphatase. J. Anat. 150, 155-167.
Acknowledgements:
Grants for this study were supported by McNeil Pharmaceuticals to Dr Eric R Blough & Dr
Ernest M Walker.
Dr. M.L. Norton for maintaining the MBIC imaging facilities.