Transcript CSLM of mitochondrial dysfunction in aging rat skeletal muscle

Microscopic evaluation of Age-related mitochondrial dysfunction in rat Skeletal muscle.
Anjaiah Katta1, Madhukar kolli1, Kevin M. Rice1,2 , David Neff1 and Eric R. Blough1,2
1 Department of Biological Sciences, Marshall University 2 Department of Pharmacology, Physiology and Toxicology, Joan C. Edwards School of Medicine, Marshall
University, Huntington WV
INTRODUCTION
Recent studies demonstrate a
progressive
increase
in
rate
of
mitochondrial free radical production
during aging. Mitochondria are therefore
a likely site of oxidative damage, the
severity of which may increase with
age.4-Hydroxy-2-nonenal(HNE),a major
product of lipid peroxidation, thought to
increase with aging, can react with and
inactivate
enzymes,
and
inhibits
mitochondrial respiration in vitro. HNE
modification of mitochondrial protein(s)
might, therefore, be expected to occur
during aging and result in loss in
mitochondrial function.
CONCLUSIONS
4-HNE
4-HNE
4-HNE
Animals: Adult (6 months, n=12), aged (30
months, n=12), and very aged (36 months,
n=12) male F344/NXBN rats were obtained
from the National Institute on Aging.
Muscle preparation: Animals were
anesthetized with a ketamine-xylazine (4:1)
cocktail (50 mg/kg ip) and supplemented as
necessary for reflexive response . EDL and
Soleus muscles were quickly removed, blotted
dry, trimmed of visible fat and tendon
projections, weighed, and immediately frozen
in liquid nitrogen.
Histological imaging & immunolabeling:
Skeletal muscles were serially sectioned (6 μm)
using an IEC Minotome cryostat and collected
on poly-L-lysine (Sigma, St. Louis, MO) coated
slides. Staining was done with 20 nM
Mitotracker Green FM for 15 min to stain the
mitochondria and washed with 1X PBST for 5
min . The tissue sections were also washed in
1X PBST three times for five minutes, then
thoroughly cleaned off. For HNE
immunostaining, the sample was incubated in
4-HNE primary antibody (1:100 dilution) with
3% BSA in PBST, for an hour at room
temperature and then washed three times, five
minutes each, in 1X PBST. Texas Red Antimouse secondary followed in a 1:200 dilution
for one hour in dark. The samples were
washed three times five minutes and. The
cellular alterations and localization of the
mitochondria was studied by imaging and
spectral analysis on a BioRad MRC1024
confocal microscope.
Stain
Texas red
Mitotracker green
ex
568nm
488nm
em
585 lp
522/32
Sol6m
6m20x
20x
Sol
SolSol
6m6m
40x40x
Sol 30m 20x
Sol 30m 40x
Sol 36m 20x
6m 20X
EDL EDL
6m 20X
EDL 6m 40x
EDL
6m
40X
EDL
6m
40X
Sol 36m 40x
Figure 1. Changes in the expression of
4-HNE in with age in Soleus muscle
(representative images).
Sol 30m 20x
Sol 30m 20x
Sol 36m 20x
Sol 36m 20x
Sol 6m 40x
*
Sol 30m 40x
Sol 30m 40x
*
4.0
2.0
EDL 6m 20x
EDL 6m 20X
EDL 30m 20x
EDL 30m 20X
EDL 6m 40x
EDL
EDL6m
6m40X
40X
Sol 36m 40x
Sol 36m 40x
EDL 30m 40x
EDL 30m 40X
EDL 36m 40x
EDL 36m 40X
8.0
EDL
6.0
*
*
4.0
Aging would be associated with changes in
mitochondrial distribution and the quantity and
distribution of HNE modifications. This may
indicate mitochondrial dysfunction in fasttwitch extensor digitorum longus (EDL) and
slow-twitch soleus muscles Fischer
344/NNiaHSD X Brown Norway / BiNia rats.
2.
Lucas et.al., Declines in mitochondrial
respiration during cardiac reperfusion:
age-dependent inactivation of alphaketoglutarate dehydrogenase. Proc
Natl Acad Sci U S A. 1999 Jun
8;96(12):6689-93.
3.
Rice et.al. Aging influences multiple
incidices of oxidative stress in the
aortic media of the Fischer 344/NNiax
Brown Norway /BiNiarat. Free Radic
Res. 2006 Feb;40(2):185-97
30m
36m
Figure 5. Relative changes ( membrane/ cytosolic ratio) in
protein modification by 4-HNE in in the Soleus muscles of 6 m,
30 m and 36 m F344XBN rats. An asterisk indicates significant
difference from 6-month , (p < 0.05).
ACKNOWLEDGEMENTS
Grant support for this study was
provided by NSF Grant 0314742
and NIH AG027103 to Eric
Blough.
2.0
6m
30m
36m
Figure 6. Relative changes ( membrane/ cytosolic ratio) in
protein modification by 4-HNE in in the EDL muscles of 6 m,
30 m and 36 m F344XBN rats. An asterisk indicates significant
difference from 6-month , (p < 0.05).
RESULTS
Hypothesis
Marcineket.al.,Reduced mitochondrial
coupling in vivo alters cellular
energetics in aged mouse skeletal
muscle. JPhysio 2005 Dec 1; 569
(Pt2): 467-73.
Epub 2005 Oct27.
Figure 4. Changes in the localization of
Mitochondria with age in EDL muscle
(representative images).
PURPOSE
To examine the effects of aging on the
Mitochondrial dysfunction in fast-twitch
extensor digitorum longus (EDL) and slowtwitch soleus muscles Fischer 344/NNiaHSD X
Brown Norway / BiNia rats.
1.
EDL 36m
20x
EDL36m
36m20X
40X
EDL
0.0
0.0
6m
REFERENCES
Sol
Sol6m
6m40x
40x
SOLEUS
6.0
EDL 36m 40x
EDL 36m 40X
EDL 6m 20X
Figure 3. Changes in the localization of
Mitochondria with age in Soleus muscle
(representative images).
8.0
EDL EDL
30m 30m
40x 40X
EDL 36m
20x
EDL
36m 20X
Mitotracker G FM
Sol 6m 20x
Sol 6m 20x
Sol 6m 20x
EDL 30m
20x
EDL
30m 20X
Our data suggest that aging is associated
with mitochondrial dysfunction as evident
in mitochondrial derangement and
increasing HNE modification of cellular and
possibly mitochondrial proteins. This effect
seems to occur in a fiber-type dependent
manner . Further elucidating specific
signaling mechanisms in aging skeletal
muscle will lead to better understanding of
the mitochondrial dysfunction.
Figure 2. Changes in the expression of
4-HNE with age in EDL muscle
(representative images).
Mitotracker G FM
Fold change
Aging is multifactor phenomenon characterized
by a time dependent decline in physiological
function. Recent data has suggested that ageassociated declines in skeletal muscle function
may be associated with mitochondrial
dysfunction. The mechanism(s) responsible for
altering mitochondrial function with aging are not
known, however age-associated increases in
membrane lipid peroxidation have been linked to
the production of 4-Hydroxy-2-nonenal (4-HNE)
which is thought to play a causative role in
mediating mitochondrial damage. To examine the
potential role that muscle mitochondria and 4HNE may play in age-associated muscle
dysfunction, we examined how increasing age
affected mitochondria number and structure
along with the amount of 4-HNE reactive protein
in the skeletal muscles of adult (6 month), aged
(30 month), and very aged (36 month) Fischer
344/NNiaHSD X Brown Norway / BiNia rats
(F344XBN). Compared to 6 month animals,
mitochondrial content was decreased in the fasttwitch extensor digitorum longus (EDL) and slowtwitch soleus muscles of 30- and 36-months
animals. Interestingly, these alterations in
mitochondrial number were paralleled with
increased evident of increased muscle 4-HNE
levels in both age groups. Moreover, these
alterations are more predominant in Type1
(oxidative) fibers compared to Type 2 (lower
oxidative function) fibers. Taken together, these
results suggest that aging in F344XBN skeletal
muscle is associated with alterations in
mitochondrial number and increased 4-HNE
levels .
RESULTS
MATERIALS AND
METHODS
Fold change
ABSTRACT
1. Compared to 6 months animals the mitochondrial content was decreased in 30 and 36 month animals in both fast-twitch extensor
digitorum longus (EDL) and slow-twitch soleus muscles (qualitatively determined).
2. The localization of mitochondria was predominant along the subsarcolemma in In 6 and 30 months animals compared to 36 months, where
it appears to be more diffused across the cytoplasm.
3. Compared to 6 months animals the 4-HNE signal was increased in 30 and 36 months animals in both fast-twitch extensor digitorum longus
(EDL) and slow-twitch soleus muscles
Dr. Michael Norton for maintaining
The MBIC Imaging facilities.