Download Application Note LabImage 1D

John Konhilas
Assistant Professor
Department of Physiology
University of Arizona
Tucson AZ
USA
The effects of environmental and
genetic factors on the energetics of
muscle contraction
We are very interested in how the molecular and
cellular biology of the heart cell impacts the
contractile properties of the intact heart. We know
that a number of factors influence gene expression
and cell signaling in the cardiac myocyte including
diet, exercise, disease and sex/gender (Fig. 1). The
changes induced by these factors will ultimately
affect cardiac contractile function. It is this
functional relationship that we are studying using a
variety of molecular, cellular, and physiological
techniques. We focus on the intermediates
responsible
for
substrate
utilization
and
presentation to the contractile apparatus. We hope
to understand how the physiological factors of diet,
exercise, disease and sex/gender impact substrates
in the cell and how the presentation of these
substrates affects contractile function (Fig. 2).
Fig.1: Schematic representation of VLDL and FFA flux. Women
demonstrate higher rates of FFA release as a result of higher lipolytic
rates when compared to men. This is consistent with elevated rates of
VLDL production and clearance in women. In addition, the molar ratio
between hepatic VLDL-TG and VLDL-apoB-100 was greater in women
compared to men suggesting that women secrete larger, more TG-rich
VLDL particles than men, which may contribute to some of the observed
sex dimorphisms.
Application Note
LabImage 1D
We are also interested in how cardiac disease can
influence peripheral organ systems including
skeletal muscle. We know that humans with cardiac
disease also experience severe skeletal muscle
weakness and fatigue. This cannot be attributed
entirely to poor circulation caused by reduced
contractile function. Instead, we believe that is the
specific alteration of specific muscle factors
responsible for substrate metabolism and delivery
to the muscle cell.
Fig. 2: Schematic representation of estrogen action in the cardiomyocyte.
Estrogen has been shown to impact cellular signaling through a number of
pathways. The traditional genomic action of estrogen is believed to act
through nuclear estrogen receptors (ERs) that transactivate genes.
Estrogen has also been shown to act rapidly through nongenomic
mechanisms by acting directly on growth factor receptors or recruitment
of ERs by growth factor receptors. In addition, estrogen can bind and
activate plasma membrane ERs and/or estrogen binding proteins, thereby
inducing intracellular signaling cascades.
We study cardiac disease at the molecular up to the
whole animal level. Therefore, analysis at the
molecular and cellular level is one of the
cornerstone techniques of our lab. We use
LabImage 1D for the quantitative analysis of SDSPAGE stained with coomassie, silver, or the
phospho-stain Pro Q Diamond (Fig. 3). We also use
LabImage 1D to quantify RNA and DNA gel
electrophoresis.
Finally, we perform immunoblots on a daily basis
with a variety of antibodies. LabImage 1D is our
tool to determine relative expression of specific
cellular components following immunoblot analysis.
Since purchasing LabImage 1D, we have been
able to successfully quantify the resultant data
following gel electrophoresis. Much of the data
quantified using LabImage 1D is driving the
direction of our studies. Although we have not
published these data yet, we are working on at
least 3 manuscripts containing data analyzed
with LabImage 1D.
Given the focus and the breadth of techniques
utilized by our laboratory, LabImage 1D is a
central and vital part of data analysis. We are
able to integrate and incorporate enough
subjectivity while maintaining an objective,
quantitative approach for our scientific needs.
References
Konhilas JP, Leinwand LA. Dec 2007. The effects
of biological sex and diet on the development of
heart failure. Circulation, 116:2747-59
Fig. 3: Screenshot of the LabImage 1D software showing the analysis
of a Pro Q Diamond phosphoprotein stained acrylamide gel (top) and
its complimentary total protein stained with Coomassie Blue
(bottom).
LabImage 1D provides the digital interface in
order to quantitatively or semi-quantitatively
determine the expression of specific cellular
components. As mentioned above, this could be
either specific proteins and/or phosphoproteins,
DNA and RNA with a specific focus on microRNAs.
LabImage 1D provides the tools necessary to
compare the expression of these cellular
components within a given gel and across several
gels. We are also able to determine the relative
expression of specific protein subtypes within a
given sample with accurate results. The utility of
LabImage 1D to simply and uniformly calculate the
optical density of our bands in addition to the
possibility to normalize to standards is highly
beneficial to our lab. Through the use of LabImage
1D we are able to consistently analyze our data in
order to evaluate differences at the cellular and
molecular level.
Watson PA, Reusch JE, McCune SA, Leinwand
LA, Luckey SW, Konhilas JP, Brown DA, Chicco
AJ, Sparagna GC, Long CS, Moore RL. Jul 2007.
Restoration of CREB function is linked to
completion and stabilization of adaptive cardiac
hypertrophy in response to exercise.
Am J Physiol Heart Circ Physiol, 293:H246-59
Konhilas JP, Watson PA, Maass A, Boucek DM,
Horn T, Stauffer BL, Luckey SW, Rosenberg P,
Leinwand LA. Mar 2006. Exercise can prevent
and reverse the severity of hypertrophic
cardiomyopathy. Circ Res, 98:540-8
Stauffer BL, Konhilas JP, Luczak ED, Leinwand
LA. Jan 2006. Soy diet worsens heart disease in
mice. J Clin Invest, 116:209-16
Konhilas JP, Widegren U, Allen DL, Paul AC,
Cleary A, Leinwand LA. Jul 2005. Loaded wheel
running and muscle adaptation in the mouse.
Am J Physiol Heart Circ Physiol, 289:H455-65