Research Interest
A. Research Currently
in Progress: Plasticity of gene expression in single adult skeletal
muscle fibers
Adult mammalian skeletal muscle, including
human skeletal muscle, has the ability to adapt to changing work demands
as a result of changes in neural activity. Part
of the long-term response includes changes in gene expression, which
is mediated by changes in the rate of transcription of specific genes,
and ultimately leads to changes in the rate of synthesis of specific
proteins. When a fast-twitch mammalian skeletal
muscle is subjected to chronic electrical stimulation in vivo, it gradually
takes on the physiological and biochemical characteristics of a slow-twitch
muscle.
My research uses a long-term in vitro single
adult muscle fiber culture system subjected to chronic electrical stimulation
to study plasticity of gene expression in single fibers. During
the transformation from a fast-twitch to a slow-twitch fiber phenotype,
activity-dependent changes in gene expression are monitored and quantified
using molecular biological techniques. Of particular interest, is the expression
of structural genes such as slow myosin heavy chain, whose expression
is up regulated during stimulation, and Z-line titin isoforms, whose
expression varies from muscle to muscle. The
specific aims of this research are:
1. To
determine the extent and time course of changes in fiber type-specific
gene expression that occur as a function of chronic electrical stimulation
of isolated skeletal muscle fibers maintained in vitro in long-term
cell culture.
2. Determine
the time course of changes in fiber type-specific gene expression
as a function of various patterns of electrical stimulation. These
patterns simulate the electrical activity that either fast-twitch
or slow-twitch fibers experience in a living cell.
3. To
ultimately correlate these changes in gene expression with changes
in muscle fiber structure.
B. Planned
Research project: Myosin light chain isoform heterogeneity in SFM of
the lobster.
My doctoral work examined the structural and spatial
heterogeneity of the myosin light chains in a crustacean skeletal
muscle fiber. This work
was performed using the lobster superficial flexor muscle (SFM) as
the model invertebrate system. Heterogeneity was found to extend from
the level of the muscle fiber down to the level of the individual
myosin molecule. SDS-PAGE
gels of individual muscle fibers revealed a uniform distribution
of beta myosin light chains within the SFM, but a non-uniform distribution
of the alpha myosin light chains was found. Subsequent
analyses of both the alpha and beta myosin light chains in situ revealed
that not all of the myosin heads within a single myosin filament
contained one of each of the light chains.
Future continuation studies would include the following
specific aims:
- Characterize the alpha and the beta myosin light chains
isoforms.
There are three alpha and two beta myosin
light chain isoforms. Molecular
biological techniques would be used to
obtain sequence information about the
different isoforms of the two types of
light chains. These sequences would then
be used to compare with other invertebrate
skeletal myosin light chains and vertebrate
skeletal muscle myosin light chains.
- Examine the in situ distribution of the individual
myosin light chain isoforms within the
SFM. Antibodies
used in the in situ experiments
were not specific for the individual
isoforms, but were specific for the two
types of light chains. In situ analysis of the isoforms would
determine if their were more than one
isoform of each type of light chain within
the individual fibers and the within
individual myosin heads.
Since the myosin light chains play
an important role in regulating contractile activity within this
muscle system, these studies will provide some insight into the mechanism
for regulating co
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Dr. Lisa Brown