single muscle fiber mutation detection in mitochondrial mutator...
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Helen C. Lam Email: [email protected]
Medical Student
David Geffen School of Medicine
UCLA
Helen C. Lam1,2, Jing Liu3, Donald Limbrick3, Nazanin Ahmadieh4, Jonathan Wanagat3
Medical Student Training in Aging Research1; DGSOM2; Department of Medicine, Division of Geriatrics, UCLA3; University of California Los Angeles4
Introduction Sarcopenia, the age-related loss of muscle mass and strength, is a
significant contributor to frailty and other declines of aging. Many
etiologies have been implicated and there is growing evidence that
mitochondria and the mitochondrial genome may play a central role.
Recent studies suggest a series of events linking mitochondrial
mutations, mitochondrial dysfunction, fiber atrophy and loss. Genetic
manipulation of mitochondrial mutation rate is illuminating the role of
these mutations in aging and age-related diseases. We are currently
using a mouse model of increased mitochondrial mutation rate, the
polgamma mutator mouse (PolgAmut/mut ) to evaluate the role of
mitochondrial mutation in the development of sarcopenia. PolgAmut/mut
mice have a premature aging phenotype and have significantly reduced
lifespans.
The mechanism driving the premature aging and loss of muscle mass
and function in PolgAmut/mut is unknown and the type of mutation
causing the progeroid phenotype, whether point mutations or
deletions, remains controversial. In old wild type animals, a small
percentage of muscle fibers lose respiratory chain function and show a
cytochrome c oxidase (Complex IV) negative phenotype. PolgAmut/mut
have increased mitochondrial abnormalities and show myopathic
changes at younger ages. Premature muscle mass loss and
dysfunction may be due to mtDNA deletion accumulation and
development of abnormal cytochrome c oxidase activity. We are
evaluating this hypothesis by studying the muscle fiber sections of a
PolgAmut/mut mice and are attempting to selectively amplify mtDNA from
RRFs isolated from PolgAmut/mut mice muscle sections.
Approach
PolgAmut/mut mice show progeroid features (kyphosis, reduced
body size, hair loss) when compared to WT animals at age 40-45
weeks. PolgAmut/mut mice have also been shown to have a
decreased lifespan (Trifunovic et al. 2004).
By age 13 months, PolgAmut/mut mice have significantly decreased
quadriceps weight compared to WT animals.
PolgAmut/mut mice have increased cytochrome c oxidase negative
fibers in muscle sections.
Sections from 13 mo and 15 mo mice have been stained first for cytochrome
C oxidase (COX), followed by staining for succinate dehydrogenase (SDH).
COX-negative and are shown below as SDH-positive (purple).
Laser capture microdissection (LMD) enables selective
identification and isolation of COX-negative fibers.
Currently we are attempting to amplify mtDNA from single cells to
determine the type of mutation present in these fibers.
Discussion The role of mtDNA mutations in the development of cytochrome c
oxidase and in myopathy has broad implications because
understanding the mechanisms causing loss of muscle function
can lead to therapeutic interventions. Preventing sarcopenia-
related frailty is a worthy goal to prolong geriatric independence
and lessen the financial burden of subsequent need for
institutional care. The role of mitochondria in aging is still being
clarified and the PolgAmut/mut mouse model is valuable in evaluating
the role of mtDNA mutations.
Acknowledgements This work is supported by the MSTAR program and funded by generous
support from the NIH, NIA, American Federation for Aging Research,
and the Lillian R. Gleitsman Medical Student Summer Research
Training in Aging Program.
References Trifunovic, A., A. Wredenberg, et al. (2004). "Premature ageing in mice
expressing defective mitochondrial DNA polymerase." Nature 429(6990):
417-423.
Wanagat, J., Z. Cao, et al. (2001). "Mitochondrial DNA deletion mutations
colocalize with segmental electron transport system abnormalities, muscle
fiber atrophy, fiber splitting, and oxidative damage in sarcopenia." FASEB J
15(2): 322-332.
Single Muscle Fiber Mutation Detection in Mitochondrial
Mutator Mice
0
0.05
0.1
0.15
0.2
0.25
Female Male
Gra
ms
(g)
Quadriceps Weight
WT
PolG
DNA extraction
13mo or 15mo
PolgAmut/mut mice
Muscle isolated &
embedded
Muscle
sectioning
Laser capture
microdissection
PCR