Helen C. Lam Email: hclam@mednet.ucla.edu

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