Skeletal Muscle Apoptosis and Physical Performance; Oxidative RNA/DNA Damage and Repair in Aged Human Muscle

Specific Aims:

Specific Aim 1. To determine the extent of muscle apoptosis during aging in young healthy
controls and in high-functioning and low-functioning older subjects. The investigators
hypothesize that the observed level of muscle cell apoptosis is associated with physical
disability. The investigators will recruit a cohort of ten young control subjects (age
20-35). The investigators will also recruit and stratify a cohort of forty elderly (age
70-99 years) into two distinct categories by set criteria based on their level of physical
performance. Subjects with a summary performance score ≥ 11 at the Short Physical
Performance Battery (SPPB) (66) will be considered high-functioning. Conversely, subjects
with a summary performance score ≤ 7 will be considered low-functioning. In addition to the
SPPB, the investigators will also perform the hand grip strength test and the isokinetic
knee extensor test, and will implement 3D magnetic resonance imaging (MRI) to precisely
quantify the quadriceps contractile area. The investigators will obtain a muscle biopsy for
each subject to determine the severity of apoptosis using biochemical and histological
analyses (see Methods). Disability will be assessed using a self-report questionnaire.
Hence, the investigators will determine the extent muscle apoptosis and the investigators
will be able to correlate this to the level of physical disability and muscle mass.

Specific Aim 2. To elucidate the role of Poly(ADP-ribose) polymerase 1 (PARP-1) and
apoptosis-inducing factor (AIF) in the induction of apoptosis in human skeletal muscle. The
investigators hypothesize that the observed cell loss due to apoptosis is mainly related to
a caspase-independent mechanism. The investigators have recently shown that activation of
mitochondrial caspase-independent pathway of apoptosis may play a more important role than
the caspase-dependent pathway in contributing to nuclear cell death (67). Furthermore, the
investigators hypothesize that the PARP-1/AIF pathway of apoptosis might be involved in the
age-related muscle wasting process. The investigators will quantitatively assess PARP-1 and
AIF and correlate these measures with the extent of muscle apoptosis.

Specific Aim 3. To assess the role of mitochondria dysfunction and energy failure in muscle
apoptosis, sarcopenia, and disability. The investigators hypothesize that mitochondrial
dysfunction and the resulting reduced production of adenosine triphosphate (ATP) is causally
related to sarcopenia, possibly by triggering the apoptosis program. It has been estimated
that the mean ATP production in the quadriceps muscle of old human subjects is approximately
50% of that of younger subjects (68), and it has been postulated that energy failure might
trigger apoptosis (58). Consistently with these findings, the investigators have recently
found that in rats' gastrocnemius muscle, ATP content and the rate of ATP production
declined by ~50% with age (59). The investigators will quantitatively assess myocytes' ATP
content along with key mitochondrial metabolic enzymes, namely citrate synthase,
cytochrome-c-oxidase (COX), ATPase, and aconitase. The investigators will correlate these
parameters with the level of apoptosis and with measures of physical performance,
disability, and muscle mass.

Specific Aim 4. To correlate changes in physical function and muscle mass with muscle
apoptosis and biochemical parameters at follow-up (longitudinal study). After 4 years
subjects in the high-performance group will be reevaluated for muscle apoptosis rate, PARP-1
and AIF activity, and energy production. The investigators will correlate these parameters
with measures of physical performance and muscle mass assessed by MRI, as well as with
self-reported disability. Hence, the investigators will be able to directly correlate an
eventual decline in physical performance, muscle mass and functional status at this very old
age with changes in muscle apoptosis and in biochemical parameters.

Research Plan:

Using a cross-sectional design (age 20-35 and 70-99 years) the investigators will quantify
the level of apoptosis in the vastus lateralis muscle in a healthy (see inclusion/exclusion
criteria) population. Apoptosis of irreplaceable post-mitotic cells may be important
mechanism in the development of sarcopenia and has not been investigated in healthy humans
with age. A variety of chronic diseases have documented cases of apoptosis in human skeletal
muscle; i.e., chronic pulmonary diseases (69,70) and patients with heart failure (69,71,72).
However, it is essential to establish baseline levels of apoptosis in relatively healthy
humans before proceeding to more complex conditions, such as disease conditions and frailty.
In addition, the investigators will determine knee extensors strength, the myocyte volume,
extra cellular space and the myonuclear domain (cross-sectional muscle area/nucleus) by
quantifying the number of nuclei within a cross-sectional area of muscle. This specific aim
will establish baseline levels of apoptosis and assess standard measures to assess
sarcopenia.

Study design. To accomplish our aims, the investigators propose two separates, yet
complementary studies

1. In order to gain insights into changes of the apoptosis potential with age and its
contribution to muscle mass and strength loss, the investigators will quantify the
level of apoptosis in the vastus lateralis muscle in young (age 20-35 years; N =20),
old high-functioning, (age 70-99 years; N = 25), and old low-functioning (age 70-99
years; N = 20) subjects. Muscle biopsy and MRI scan to quantify quadriceps contractile
area will be performed in the study participants. Muscle strength will be assessed by
hand grip strength test and the isokinetic knee extensor test.

2. Preliminary data on the contribution of skeletal muscle apoptosis to sarcopenia,
physical function loss in advanced age will be gathered by quantifying the extent of
muscle apoptosis in the vastus lateralis muscle of old (age 70-99 years),
high-functioning (N = 25) and low-functioning (N = 20) subjects, along with measures of
muscle mass (MRI), muscle strength (hand grip and knee extension test), and overall
physical performance (Short Physical Performance Battery, SPPB (67)). In addition, the
investigators will collect preliminary data regarding the role mitochondrial-driven
apoptosis in muscle wasting and functional loss at old age.

A brief physical exam will be administered to all study participants, and major biological
signs (body weight, standing height, body temperature, blood pressure, and pulse) will be
recorded. A basic questionnaire regarding participants' health history will also be
administered

After four years the old participants will be reevaluated for physical performance,
functional status muscle mass (MRI), muscle apoptosis, and biochemical parameters Disability
will also be assessed using a self-report disability questionnaire. In order to gain
information regarding changes in physical performance and functional status with time and to
maximize retention, an annual visit employing SPPB, hand grip and knee extensor testing,
will be scheduled for the old participants.

Inclusion criteria. The following inclusion criteria will be adopted to select the study
participants:

- males and females aged 20-35 and 70-99 years

- sedentary lifestyle (i.e., the subject has spent less than 20 minutes per week in the
past 2 month performing structured physical activity, such as exercising at a gym
and/or weight training)

- willing and able to give informed consent.

Exclusion criteria:

- history of smoking in the prior 12 months

- active treatment for cancer or history of cancer in the past 3 years

- congestive heart failure New York Heart Association (NYHA) Class III or IV

- previous stroke with upper and/or lower extremities involvement within the last 6
months

- peripheral vascular disease Fontaine Class III/IV

- History of life-threatening cardiac arrhythmias, stroke, severe Parkinson's disease
or severe neurological disorders likely to interfere with physical function

- cognitive impairment (i.e., Mini-Mental State Examination (MMSE) ≤ 23)

- renal disease requiring dialysis

- lung disease requiring steroids

- lower extremity amputation

- severe osteoarthritis that interferes with physical function

- Complicated diabetes

- inflammatory diseases such as active rheumatoid arthritis, vasculitis, autoimmune
disorders, and inflammatory bowel disease

- life-threatening illnesses with an estimated life expectancy less than 1 year

- history of drug or alcohol abuse

- taking growth hormone (GH) and/or estrogen replacement therapy

- Testosterone medication

- Anticoagulant therapy

- involved in active weight loss > 5 kg in prior 3 months

- planning to relocate out of the study area in the next 4 years (ages 70-99 only)

- pregnancy

- SPPB score of 8-10

- Lidocaine allergy

- MRI exclusions, such as pregnancy, claustrophobia, heart pacemaker / defibrillator,
heart valve prosthesis, aneurysm clip, metallic stent, neurostimulation system,
cochlear implants or inner ear prosthesis, insulin pump or other infusion pump, metal
slivers in the orbital area/eye socket

Temporary exclusion criteria:

- recent bacterial infection (< 2 weeks)

- acute febrile illness in prior 2 months

- high blood pressure (i.e., BP ≥ 180/110 mm Hg) at the screening visit (subject will
be referred to his/her physician and reevaluated after appropriated therapy being
instituted)

- major surgery or hip/knee replacement in the past 6 months

Assessment of disability. The uniform measure proposed to use for the assessment of
disability will be a self-report disability questionnaire. This questionnaire was
developed for the Fitness Arthritis in Seniors Trial (FAST) and since then has been widely
used in intervention and observational studies (e.g. ADAPT, REACT-1, OASIS, CHAMP). The
questionnaire is not specific to a certain disease and asks about perceived difficulties
in general activities of daily living during the last month. Respondents answer for each
of the items whether they experience 1) no difficulty, 2) a little difficulty, 3) some
difficulty, 4) a lot of difficulty, 5) unable to do or, 6) did not do for other reasons.
Answers can be averaged across the items in order to receive an indication of the overall
perceived disability burden by a person.