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Project importance
and goals: Calcium is a key
regulator of intracellular signaling and mitochondrial function (Griffiths et
al., 2009). We have recently demonstrated that nanomolar calcium levels
increase the ability to use the membrane potential and result in increased ATP
production (Fink et al., 2017). Higher calcium levels in the micromolar range
were found to result in the inhibition of this ability (Fink et al., 2017). The
mechanisms behind this effect of calcium on production of ATP are not known, nor
has this effect been studied in a whole cell model. For the long term goals,
this project aims to gain a better understanding of how nanomolar calcium
affects aspects of ATP production and mitochondrial function, as well as to
investigate the possibilities for increasing ATP production output of cells
when under unfavorable conditions such as hypoxia. Through this study, we can
gain a more clear understanding of energy production and mitochondrial
function, both of which are vital processes to the cells and tissues of our
bodies. Further down the road, this research has the potential to be applied to
clinical areas that are concerned with the energy output of muscles and
tissues, such as sports medicine, physical rehabilitation, and into
investigations for improved treatments and possible cures for different
mitochondrial disorders.

Production and
function of ATP: Adenosine
triphosphate (ATP) is the main energy currency of all cells. As the final
product of oxidative phosphorylation in eukaryotes, ATP is used for energy both
inside and outside the cell (Stagg et al., 2010). Due to the high energy
phosphate bond, ATP can be used in numerous reactions to provide energy through
ATP hydrolysis (Knowles, 1980). In eukaryotes, it is produced through several
processes such as glycolysis, the Krebs cycle, and oxidative phosphorylation
involving the electron transport chain (ETC). It is the latter that produces
the highest amount of ATP (Rich, 2003). The ETC generates a high proton (H+)
concentration in the intermembrane space of the mitochondria, which is then
used by complex V, or ATP synthase, to drive the phosphorylation of ADP to ATP
(Abrahams et al., 1994). ATP synthase consists of two subunits: the F0
and F1 subunits. The F1 subunit is on the matrix side of
the inner mitochondrial membrane and is responsible for ADP phosphorylation
(Fernandez et al., 1964). The F0 subunit is a lipophilic
transmembrane ring protein that rotates when H+ travels through and
provides the power needed by the F1 subunit to convert ADP to ATP
(Velours et al., 2000). Once made, the ATP can leave the mitochondria through the
adenine nucleotide translocase (ANT), and provide energy for a variety of other
cellular processes and signalling pathways (Hanoune et al., 2001). Some
examples include intracellular signaling, synthesis of DNA and RNA, protein
synthesis, and ATP binding cassette transporter (Hanoune et al., 2001).

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Calcium signaling
in mitochondria: The stimulating
effect of calcium on oxygen dependent metabolism in mitochondria has been well
accepted as vital for maintaining ATP synthesis homeostasis (Tarasov et al.,
2012). Intra-mitochondrial calcium is used for the regulation of the reactions
of the Krebs cycle and the ETC to ensure that synthesis of ATP is closely
matched to the energy demands of the cell/tissue at that time (Tarasov et al.,
2012). Mammalian mitochondria have the ability to house large amounts of
calcium and while once thought to be intracellular reservoirs of calcium
(Lehninger et al., 1967), it has been shown that the mitochondria increase
calcium uptake when signaled by external factors, such as hormones or
neurotransmitters, and when increased ATP synthesis is required by that cell (Hansford
et al., 1981). There are several enzyme complexes in the mitochondria that are
sensitive to calcium (Tarasov et al., 2012). In relation to this project
proposal, calcium has been shown to bind directly to ATP synthase and modulate
its activity (Hubbard et al., 1996). Calcium has also been shown to act on ATP
synthase through post-translational modifications and -subunit
phosphorylation which are sensitive to physiological calcium levels (Hopper et
al., 2006). Finally, a study that was recently done found a protein that
increases the capacity of ATP synthase for ATP production and binds to the
complex in a calcium-dependent manner (Boerries et al., 2007). Just to mention
a few others, FAD-GPDH is regulated by physiological calcium through a calcium
binding site on the cytosol face (MacDonald et al., 1996). The rate-limiting
pyruvate dehydrogenase (PDH) complex is dependent on calcium to associate with
its catalytic subunit in order to activate the protein (Turkan et al., 2004).
Several other mitochondrial enzyme targets for calcium signaling are cytochrome
C oxidase, malate-aspartate shuttle, NAD+-isocitrate dehydrogenase,
and a few others, which demonstrates the significant importance of calcium on
ATP production and overall mitochondrial function (Tarasov et al., 2012).

Hypoxia in
skeletal muscle cells: Hypoxia occurs
when a tissue in the body has an insufficient supple of oxygen (Lundby et al.,
2009). Conditions under which there is absolutely no oxygen supply to the
tissue are referred to as anoxic. A common way people encounter hypoxia is
during periods of prolonged physical exercise (Lundby et al., 2009). Some less
common situations under which our tissues may experience hypoxia are chronic
heart failure, anemia, obstructive pulmonary disease, and during recreational
activities done at high altitudes (Lundby et al., 2009). Every nucleated cell
in our bodies has the ability to sense oxygen levels and respond to any changes
accordingly. The cells do so through the hypoxia inducible factor (HIF) pathway
(Lundby et al., 2009). HIF-1 is an important transcription factor in this
pathway and the HIF-1? subunit is particularly sensitive to changes in oxygen
levels (Lundby et al., 2009). Abnormally high levels of HIF-1? in a cell/tissue
would indicate that the cells are undergoing apoptosis due to prolonged hypoxia
(Lundby et al., 2009). This background will focus on the effects of hypoxia mainly
on muscle cells, and not the muscle tissue as whole since the project proposed
involves the use of cells and organelles; the experiments do not deal with the
whole skeletal muscle tissue. There are several ways in which hypoxia is
harmful to the functioning of cells. The outcomes include decreased cellular
energy (ATP), decreased intra-cellular pH, and an accumulation of toxic
intermediates from anaerobic respiration and reactive oxygen species (ROS) (He
et al., 2015). One of the main consequences of hypoxia is to the ETC. Since
there is not enough oxygen to act as the final electron acceptor, this results
in a diminished, or smaller, proton gradient generated by the ETC, which then
subsequently affects how much ATP can be produced by ATP synthase (He et al.,
2015). Some studies have suggested that when muscle tissue is under hypoxic
conditions and oxygen delivery to the tissue is decreased, ATP supply pathways
are upregulated in an effort to compensate (Hoppeler et al., 2003). This is
done so that homeostasis is maintained between the supply of ATP available and
the ATP demand of the cell/tissue (Hoppeler et al., 2003). Hypoxia also
interferes with vital cellular processes such as signalling pathways,
transcription of genes, and protein synthesis (Lundby et al., 2009). A recent
study found that in plants, exogenous calcium leads to improved metabolism and
transport of ions in the cell (He et al., 2015). One of the gaps that remains
unexplored is the relationship between calcium and hypoxia in skeletal muscle
cells and tissue. Thus, in conjunction with our previous study which showed
that nanomolar levels of calcium result in improved ability to utilize the
mitochondrial membrane potential for increased ATP production (Fink et al.,
2017), we thought it would be interesting to see if nanomolar calcium could
also help improve tolerance of skeletal muscle cells to hypoxia through the
increased production of ATP.

Proposal
Aims

We previously demonstrated that in respiratory states
3 and intermediate states, nanomolar mitochondrial calcium helps to regulate
ATP production, ROS production, and cellular respiration (Fink et al., 2017).
One of the limitations of that study was that the effect of nanomolar calcium
on transport of ADP was not separated from an effect on ATP synthase to result
in increased production of ATP (Fink et al., 2017). I will be continuing with
the use of isolated mitochondrial cultures from mice to delineate this
relationship further since it is an ideal system when looking at mitochondrial
function and function. Another limitation of the previous study was that it did
not take into account the effect of calcium on whole, intact cells in which
calcium is known to be stored in intracellular stores such as the sarcoplasmic
reticulum (Fink et al., 2017). For this I will be working with a murine primary
skeletal muscle cell line which will be efficient as my lab already has access
to mice and testing in whole cells will give a clearer picture of how nanomolar
calcium effects energy production in a cell.  

Question 1: Does
nanomolar calcium act on mitochondrial transport of ADP to increase ATP
production? Previous studies have
shown that calcium has several different effects on mitochondrial function and
ATP production (Griffiths et al., 2009). Despite this, it is not known for sure
if calcium interacts or effects the adenine nucleotide translocase (ANT) but it
is thought to not associate with the transporter (Palmieri). I hypothesize that nanomolar calcium does
not affect mitochondrial ADP transport through the ANT to increase production
of ATP.

Question 2: Does
nanomolar calcium act on ATP synthase to increase production of ATP in
mitochondria? Previous studies have
found some evidence of proteins that modulate ATP synthase activity by binding
to the complex in a calcium-dependent manner (Boerries et al., 2007). Other
studies have even suggested that the F1-b subunit of the ATP synthase complex binds to calcium
directly, resulting in increased production of ATP (Hubbard et al., 1996). I hypothesize that nanomolar calcium will
act to enhance the activity of ATP synthase, thus increasing ATP production.

Question 3:
Through increased ATP production, can nanomolar calcium increase hypoxia
tolerance in mouse skeletal muscle cells? Under
anaerobic and/or hypoxic conditions, skeletal muscle cannot perform to its full
efficiency due to decreased rates of oxidative phosphorylation (Michiels, 2004).
Due to its ability to result in increased ATP production, it may be possible to
utilize calcium to mediate this hypoxic effect and improve tolerance of
skeletal muscle cells to hypoxia. I
hypothesize that nanomolar calcium will increase hypoxia tolerance in skeletal
muscle cells by acting to increase production of ATP.

Experimental
Design

Aim 1: To
determine the effect of nanomolar calcium on mitochondrial transport of ADP
through ANT to produce ATP. The
adenine nucleotide translocase (ANT) has been well characterized but it is not
entirely known if its activity can be modulated with calcium. To study this
aim, I will use isolated mitochondria from the hind-limb skeletal muscle tissue
of adult mice (aged 6-9 weeks) and check for mitochondrial integrity using a
cytochrome C oxidase assay as was done in the previous study (Fink et al., 2017).
Differential and density centrifugation will be used to purify the mitochondria
from other tissue and cellular components. The mitochondria will be depleted of
endogenous calcium following the method outlined in (Territo et al., 2000) with
the modifications indicated in the previous study (Fink et al., 2017). The
mitochondria will be incubated at a concentration of 0.1 mg/ml at 37°C in 2ml
of an ionic respiratory buffer as outlined in our previous study (Fink et al.,
2017).  ADP levels will be clamped at 32
nM using the previous method involving 2-deoxyglucose (2DOG) and hexokinase
(HK) (Fink et al., 2017). 32 nM was chosen because it is known to be sufficient
from our previous study and is also comparable to levels found in the
intermediate respiratory state, which is most similar to the state of
endogenous mitochondria within the body (Korzeniewski et al., 2015). Calcium
levels in this study will be clamped at 500 nM since that is similar to levels
found in actively respiring mitochondria (Contreras, et al., 2010). Ionophore
A23187 at 10 uM will be used to equilibrate calcium across the mitochondrial
membranes and the media (O’Doherty et al., 1982). I will be using the water
soluble drug (Sigma-Aldrich, 1), carboxyatractylate (CATR), to inhibit the
ANT (Palmieri). Previous studies working with liver tissue found 1 uM to be
sufficient at inhibiting the ANT (Shabalina et al., 2006) but I will be doing a
preliminary test to find the most effective concentration for my isolated
mitochondrial system. For this I will have the following treatment groups with
mitochondria: no CATR, 0.01 uM CATR, 0.5 uM CATR, 1 uM CATR, 5 uM CATR, and 10
uM CATR. I will measure ATP production as the output via NMR spectroscopy as
done in our previous study (Fink et al., 2017). For the main study of this aim,
I will use the lowest CATR concentration that is most effective at inhibiting
ADP transport as indicated by decreased ATP production so as to avoid the side
effects of higher doses. I will have the following control groups for the main
study: no CATR & no Ca2+ (for baseline reading), no CATR &
500 nM Ca2+ (positive control), and added CATR & no Ca2+
(negative control). My experimental group will be added CATR and 500 nM Ca2+.
I will be using the same method as our previous study to measure the following
outputs for each treatment: ATP production, ROS production, and membrane
potential.

Aim 2: To
determine the effect of nanomolar calcium on the activity of ATP synthase to
produce ATP. Complex V of the electron
transport chain, or ATP synthase, is not known for sure to interact with or be
affected by mitochondrial calcium. To study this aim, I will be using isolated
mitochondria from the hind-limb skeletal muscle tissue of adult mice (aged 6-9
weeks) obtained via differential and density centrifugation, and check for
integrity using the same method as in Aim 1 above. Mitochondrial cultures will
be kept in the same media, the ionic respiratory buffer, as described for Aim
1. The endogenous calcium will be depleted using the method described in our
previous study with the modifications indicated there (Fink et al. 2017). Same
as in Aim 1, ADP levels will be clamped at 32 nM with 2DOG and HK with the
modification of adding radiolabeled phosphate (32P), also at 32 nM
to be equal to the ADP levels, to certain treatment groups as outlined further
below. This is done to be able to monitor the activity of ATP synthase without
the use of any substance that would interfere with its activity and function. Once
again, 500 nM of calcium will be used along with 10 uM of ionophore A23187 to
equilibrate the calcium in the media with the intermembrane space and matrix of
the mitochondria. The treatment groups for this study will be as follows: no Ca2+
& no 32P (baseline readings), 500 nM Ca2+ &
no 32P (positive control), no Ca2+ & 32 nM 32P
(negative control), and 500 nM Ca2+ & 32 nM 32P
(experimental group). For the outputs, I will be measuring and quantifying ROS
production and membrane potential in the same method as our previous study
(Fink et al. 2017). To measure ATP production, I will be using two different
methods dependent on treatment. For the treatment groups with no radioactive 32P
added, I will measure ATP production using the same method as our previous
study involving 2DOG and HK. For the treatment groups with 32 nM of 32P
added, I will measure ATP production via liquid scintillation counting, which
is a standard method of measuring the radioactivity of any particle that emits
alpha or beta particles (Perkin Elmer, 1). In these treatments, there will be
no HK added to ensure that the ATP is not converted back into ADP causing loss
of the radioactive signal. As the 32P is used up along with ADP by
ATP synthase, it will be converted to radiolabeled ATP (-32P)
(Perkin Elmer, 2). The amount of radioactivity present will be measured using
the Microbeta LumiJET microplate counter (Perkin Elmer, 3). In order to
separate any leftover background 32P from radiolabeled ATP (-32P),
I will use the method outline in (Parsons et al., 1998) which uses 20%
polyacrylamide gels to separate the two different radioactive molecules. This
method will allow me to observe the activity of ATP synthase in response to
nanomolar calcium because the amount of radiolabeled ATP present will reflect
this without interfering with the activity or functioning of ATP synthase.

Aim 3: To
determine the ability of nanomolar calcium to increase hypoxia tolerance of
skeletal muscle cells by having increased ATP production. The ETC depends on molecular oxygen as the final
electron acceptor to set up its proton gradient (Sirey et al., 2016).
Conditions of low oxygen, or hypoxia, can decrease the activity of the ETC and
result in a diminished proton gradient (Yang et al., 2015). This can
subsequently affect the amount of ATP that is produced and adding calcium may
be able to help compensate in this scenario. 
To study this aim I will be using primary skeletal muscle cells from
adult mice (aged 6-9 weeks), which my lab already has access to. This will be a
better method that using an immortalized mouse muscle cell line because there
will be a higher amount of mitochondria in the primary cells since those cells
will have come from tissue that was regularly used for activity by the animal (Kaur
et al, 2012). To obtain the primary skeletal muscle cells, I will use the
method described and established by Metzinger et al., 1993. The cells will be
incubated in 199 medium with 10% horse serum at 37°C in 5% CO2 (Metzinger
et al., 1993). Once the cells have gone through a passage to acclimate them to
being in culture, I will use a modular incubator chamber to induce hypoxia at a
level of 1% oxygen (Wu et al., 2011). Once again, as described in Aims 1 and 2,
ADP will be clamped at 32 nM using 2DOG and HK. In certain treatments, calcium
will be clamped at 500 nM using 10 uM of ionophore A23187 to equilibrate extra-
and intra-cellularly. Since this study will be conducted with intact, whole
skeletal muscle cells, endogenous calcium will not be depleted. Thus, I will be
using a calcium indicator, Fluo-5F AM, to track calcium inside the cells (ThermoFisher,
1). This indicator is cell permeant so it can simply be added to the media
during incubation prior to taking measurements and it will enter the cells. The
following treatments will be used: normoxic & no Ca2+ (baseline
reading), normoxic with 500 nM Ca2+ (positive control), hypoxic
& no Ca2+ (negative control), and hypoxic with 500 nM Ca2+
(experimental group). I will be using confocal microscopy to track calcium in
the cells as indicated by the product sheet and was done in other studies
(Iwabuchi et al., 2013). The same method will be used to measure ATP production
and ROS production as in our previous study (Fink et al., 2017). ROS production
is of particular interest in this study because cells will be under hypoxia and
this may result in oxidative stress and changes in ROS levels (Kuo et al.,
2015). Also, to ensure that the cells are under hypoxic conditions but not to
an extreme where they are undergoing apoptosis, I will be conducting a qPCR for
HIF-1?, which is a transcription factor subunit regulating the cellular
response to hypoxia (NCBI Gene, 2017). Levels of HIF-1? will show that the
cells are experiencing stress from being in a hypoxic environment, but they are
not undergoing apoptosis, which would be indicated by extremely high levels of
HIF-1?. For controls, the housekeeping genes GAPDH and ?-tubulin will be
measured in the qPCR since they are expressed at relatively constant levels in
all cells (Kozera et al., 2013).     

Summary

This project aims to elucidate if mitochondrial
nanomolar calcium is acting through ADP transport and/or ATP synthase to
increase ATP production, as well as determining if calcium can be used to
increase hypoxia tolerance in skeletal muscle cells due to causing increased
ATP production. This research will shed new light into mitochondrial function
and will be vital in related research fields as well. It can aid with the
research for more effective novel therapies, treatments, and possible cures for
a variety of mitochondrial disorders, as well as research on muscle performance
and sports medicine. 

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