Increased cellular ROS levels may be associated with

1
LIPOFUSCIN
ACCUMULATION IN RAT MYOCARDIUM AS AN INDICATOR OF
AGE ASSOCIATED OXIDATIVE DAMAGEMadhukar 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

• Materials and methods
• 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 gt80
  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 gt80
  µm Sections incubated in PBS
• Sections analyzed by confocal microscopy
  (Ex-488nm / Em-522/32 nm band pass filter)

Abstract 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).
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, 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).
Above, black arrow points to lipofuscin granules
in perinuclear space HE preparation (image from
Indiana U. Pathology)
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.
Purpose The purpose of this study is to examine
the age associated changes in rat myocardium.

• References
• Harman (2003). The free radical theory of aging.
  Antioxidant Redox Signal 5, 557-561.
• Brunk, U. Terman, A. (2002). The
  Mitochondrial-lysosomal axis theory of aging.
  Eur. J. Biochem. 269, 1996-2002.
• 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.

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.
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.