Espinosa, Alejandra

Jaimovich, Enrique

Jorquera, Gonzalo

Juretic, Nevenka

Riveros, Nora

Elsevier - Publisher Connector 

Both Membrane Depolarization And IL-6 Induce Calcium-Dependent Hsp70 Expression In Skeletal Muscle Cells 

Sunday, March 1, 2009 121a(8.05 0.91 mM). When SR Ca leak was matched (9.0 mM) we found the SR Ca
load necessary to induce that leak was significantly lower in ISO-treated myo-
cytes (91.65 1.9 mM) vs. those treated with ISO and L-NAME (129.45 16.3
mM) or those left untreated (127.4 5 2.8 mM). This evidence indicates that
NOS activation, and therefore generation of nitric oxide, is necessary for
ISO-dependent activation of RyR by CaMKII.
617-Pos Board B496
Discrete Proteolysis Of Neuronal Calcium Sensor 1 By m-calpain Disrupts
Calcium Binding
Courtney R. Blachford, Andjelka S. Celic, Edward T. Petri, Jr., Barbara
E. Ehrlich.
Yale University, New Haven, CT, USA.
Neuronal Calcium Sensor-1 (NCS-1) is a high-affinity, low-capacity calcium-
binding protein abundantly expressed in many cell types. NCS-1 interacts with
the inositol 1,4,5-trisphosphate receptor (InsP3R) and modulates calcium sig-
naling by enhancing InsP3-dependent InsP3R channel activity and intracellular
calcium transients. NCS-1 was also found to be a novel binding partner with the
chemotherapeutic drug, paclitaxel (taxol), used to treat a variety of tumor types
including ovarian, breast, lung, head, and neck cancers. The immediate re-
sponse of cells to taxol is a further enhancement of the NCS-1 amplification
of InsP3R dependent calcium signaling. Prolonged treatment with taxol triggers
m-calpain dependent proteolysis of NCS-1. Degradation of NCS-1may be a crit-
ical step in the induction of peripheral neuropathy associated with the taxol
treatment. To begin the process of designing a strategy that would protect
NCS-1 during taxol administration, we treated NCS-1 with m-calpain in vitro
and identified the cleavage site by N-terminal amino acid sequencing and
MALDI-mass spectroscopy. Using molecular modeling we found that m-cal-
pain cleavage of NCS-1 occurs within an N-terminal pseudoEF-hand domain.
By sequence analysis this pseudoEF-hand domain should be unable to bind cal-
cium. Nonetheless, our results suggest a role for this pseudoEF hand domain in
forming and stabilizing the three functional EF hand domains within NCS-1.
Using isothermal titration calorimetry (ITC) we found that the loss of the
pseudo EF-hand domain of NCS-1 leads to a markedly decreased affinity for
calcium. The inability of the m-calpain treated NCS-1 to bind calcium may ex-
plain the reduced calcium signaling in the presence of taxol and may suggest
a plausible strategy for therapeutic intervention of peripheral neuropathy in
cancer patients undergoing taxol treatment.
618-Pos Board B497
Effect Of ()-epigallocatechin Gallate (EGCG), A Green Tea Extract, On
Excitation-contraction Coupling Of Murine Cardiomyocytes
Hyun Seok Hwang1, Wei Feng2, Tao Yang1, Isaac N. Pessah2, Bjorn
C. Knollmann1.
1Vanderbilt University Medical Center, Nashville, TN, USA, 2University of
California, Davis, CA, USA.
Background: Polyphenolic compounds, green tea, reportedly have protective
benefit for cardiovascular disease, but the mechanism(s) are unknown. We
have recently found that green tea extracts such as (-)-epigallocatechin galla-
te(EGCG) bind to skeletal and cardiac ryanodine receptor(RyR) Ca2þ-release
channels. Here we examined the effect of EGCG(1mM, 100nM, 10nM) on
cell shortening and Ca2þ kinetics in field-stimulated murine ventricular cardi-
omyocytes loaded with Fura-2AM and on cardiac RyR channels incorporated
in lipid bilayers. Results: EGCG at 10 nM already maximally increased myo-
cytes fractional shortening(%FS): ECGC 5.650.7, N¼19, vs. Vehicle,
2.250.3, N¼25, p<0.01), so 10 nM was used for all myocyte experiments. In-
creased contractility was caused by significantly larger Ca2þ transients in pres-
ence of ECGC(Fura-2 ratio: 0.6450.15 vs. 0.2650.03, p<0.01). Ca2þ tran-
sient decay kinetics(a measure of SERCA function) and sarcoplasmic
reticulum(SR) Ca2þ content measured by rapid caffeine application were not
significantly altered by ECGC. As a result, EGCG almost doubled the fraction
of SR Ca2þ content released during each beat(5656 % vs. 3254%, p<0.01),
even though EGCG significantly inhibited L-type Ca2þ current(p<0.01). De-
cay of Ca2þ transients during caffeine application was significantly slower(-
EGCG 2.3150.17 sec vs. Vehicle 1.7250.1 sec, p<0.01), suggesting that
EGCG significantly inhibits Ca2þ extrusion via the NaCa exchanger(NCX).
EGCG(500nM) enhanced RyR2 single channel activity >30-fold prolonging
mean open time 15-fold without altering unitary conductance. EGCG did not
alter SR Ca2þ loading capacity in the presence of RyR channel blocker ruthe-
nium red. Taken together, these data suggest that EGCG enhances contractility
of intact myocytes via its action on RyR channels. SR Ca2þ depletion is pre-
vented by EGCG’s concomitant inhibition of the NCX. Conclusions: EGCG
potently modulates cardiac excitation contraction coupling by acting on RyR
and possibly on L-type Ca2þ-channel and NaCa exchanger. Supported by
R01HL71670, R01HL88635, and R01AR43140619-Pos Board B498
Properties and Functions of Store-Operated Calcium Entry in the Devel-
oping Nervous System
Agila Somasundaram, Murali Prakriya.
Northwestern University, Chicago, IL, USA.
Store-operated calcium channels (SOCs) open in response to depletion of cal-
cium stores in the endoplasmic reticulum. These channels are expressed in a va-
riety of tissues including the immune system, vasculature and hepatocytes. The
most widely studied and characterized SOC is the Calcium Release-Activated
Calcium (CRAC) channel in the immune system. Recent findings indicate that
CRAC channels are activated by local interactions between the ER Ca2þ sen-
sor, STIM1 and the CRAC channel subunit, ORAI1. Calcium influx through
these channels has been shown to play an important role in transcription of in-
flammatory mediators such as interleukins and cytokines, mediated by the tran-
scription factor NFAT1.
The current study aims at characterizing these channels in the developing ner-
vous system. Calcium imaging experiments demonstrate the presence of store-
operated calcium entry (SOCE) with properties similar to that mediated by the
CRAC channel. Calcium influx following store-depletion is blocked by La3þ,
a potent CRAC channel blocker. In addition, 2-APB (0.01–0.02 mM) causes
a transient elevation in intracellular calcium followed by a decrease, consistent
with that observed in immune cells. Functional studies done using NFAT1
tagged to GFP show translocation of this transcription factor to the nucleus
upon calcium entry following store-depletion. Further, we find an increase in
the levels of endogenous NFAT-dependent gene expression using Luciferase
reporter assays. Altogether, these results provide evidence for the existence
of store-operated calcium entry in the developing nervous system and point to-
wards a regulatory role for this pathway in gene-transcription.
620-Pos Board B499
Investigating the Architecture of the CRAC Channel Pore using SCAM
Beth McNally, Murali Prakriya.
Northwestern University, Chicago, IL, USA.
Calcium channels are vital for numerous cellular processes in all organisms.
Among the many classes of calcium channels, the calcium release-activated
Ca2þ (CRAC) channel, a member of the Store-operated channel (SOC) fam-
ily is essential for the proper development and maintenance of the immune
system, mediating critical functions such as T cells proliferation, release of
inflammatory mediators, and motility. This interesting channel has a biophys-
ical fingerprint consisting of an extremely high selectivity for Ca2þ, a narrow
pore size (3.9A˚), a very small unitary conductance, and several modes of
modulation. A single amino acid mutation (R91W) results in a loss of
CRAC channel function also known as severe combined immunodeficiency
(SCID) in human patients. Although the CRAC channel current has been
well-characterized, no structural information is known about the channel or
its pore.
The objective of this study is to investigate the architecture of the CRAC chan-
nel pore using the substituted cysteine accessibility method (SCAM), which has
been applied to several ion channel proteins and has provided significant struc-
tural insight from topology to conformational changes. Our initial studies ana-
lyzed the effects of MTS reagents, cysteine-modifying compounds, on the TM1
and the TM1-TM2 extracellular loop region around E106, an important residue
that controls Ca2þ selectivity and ion permeation. We find that the ability of
MTS reagents to block current diminishes as the residues in TM1-TM2 become
more removed from E106. These results suggest that this region of the protein
may form the entrance to the channel pore, thus providing the first insight into
the architecture of the CRAC channel.
621-Pos Board B500
Both Membrane Depolarization And IL-6 Induce Calcium-Dependent
Hsp70 Expression In Skeletal Muscle Cells
Gonzalo Jorquera, Nevenka Juretic, Alejandra Espinosa,
Enrique Jaimovich, Nora Riveros.
Centro de Estudios Moleculares de la Ce´lula, Universidad de Chile, Santiago,
Chile.
Adaptive response of skeletal muscle to challenges imposed by contractile ac-
tivity is associated to changes in specific genes expression. IL-6 and Hsp70 are
proteins involved in the maintenance of skeletal muscle homeostasis during
stress episodes and are markedly expressed in skeletal muscle after physiolog-
ical contraction. Muscle-derived IL-6 has systemic and local effects acting in
a hormone-like fashion, nevertheless the molecular bases of its functional
role on skeletal muscle is poorly understood. We have demonstrated that depo-
larization evoked IP3 mediated slow calcium transients, associated to cell nu-
clei are involved in the up-regulation of IL-6 transcriptional activity in skeletal
muscle cells.
122a Sunday, March 1, 2009The aim of this work was to investigate calcium involvement in Hsp70 expres-
sion in both depolarized and IL-6 treated skeletal muscle cells.
We observed that electrical stimulation of myotubes increases Hsp70 mRNA
level and protein expression. Depolarization performed in the presence of the
intracellular calcium chelator BAPTA-AM resulted in a complete inhibition
of Hsp70 induced expression. Inhibitors of IP3-dependent calcium signals
like 2-aminoethoxydiphenyl borate (2-APB) and LY294002, decreased
Hsp70 mRNA induction and the protein expression in depolarized cells. In ad-
dition we determined that inhibitors of calcium dependent PKC abolished
Hsp70 mRNA induction.
We established that IL-6 treatment of myotubes induced changes in intracellu-
lar calcium and promoted the increase of Hsp70 mRNA levels. Observed cal-
cium transients could be associated to early events of IL-6-mediated Hsp70
expression.
Our results provide evidence for the involvement of slow calcium transients
and PKC in the activation of Hsp70 expression in skeletal muscle cells and sug-
gest that intracellular calcium signals also participate in IL-6 induced Hsp70
expression.
FONDAP 15010006
622-Pos Board B501
Ryanodine receptor 1 signaling in dendritic cells
Mirko Vukcevic1, Giulio C. Spagnoli1, Giandomenica Iezzi1,
Francesco Zorzato2, Susan Treves1.
1Basel University Hospital, Basel, Switzerland, 2University of Ferrara,
Ferrara, Italy.
Dendritic cells express the skeletal muscle ryanodine receptor (RyR1), yet little
is known concerning its physiological role and activation mechanism. In the
present report, we provide evidence that dendritic cells also express the
Cav1.2 subunit of the L-type Ca2þ channel and that release of intracellular
Ca2þ via RyR1 depends on the presence of extracellular Ca2þ and is sensitive
to ryanodine and nifedipine. Interestingly, RyR1 activation causes a very rapid
increase in expression of MHC II molecules on the surface of dendritic cells, an
effect which is also observed upon incubation of mouse BM12 dendritic cells
with transgenic T cells whose T cell receptor is specific for the I-Abm12 pro-
tein. Based on the present results, we suggest that activation of the RyR1 sig-
naling cascade may be important in the early stages of infection, providing the
immune system with a rapid mechanism to initiate an early response, facilitat-
ing the presentation of antigens to T cells by dendritic cells prior to their full
maturation.
623-Pos Board B502
Understanding Interval-Force Relations in the Rat Ventricular Myocytes
with a Computational Model
Ryosuke Kadoi1, Sameer Bajikar1, Mohsin S. Jafri1,2.
1George Mason University, Manassas, VA, USA, 2University of Maryland
Biotechnology Institute, Baltimore, MD, USA.
We have developed a model of cardiac excitation-contraction (EC) coupling for
the rat ventricular myocytes based upon the published experimental literature
and our previous modeling work in the guinea pig ventricular myocyte. Due
to the rapid rate of Ca2þ cycling in the rat (resulting from the high heart
rate) there are fundamental differences from the guinea pig myocyte data which
was modeled previously. This requires the reformulation of Ca2þ-handling
mechanisms. The rat has a shorter action potential with different morphology
than the guinea pig. This is thought to be due to the properties of the potassium
channels. We reformulate three potassium currents (Ito, Iss, IK1), which give
strong characteristics of the cardiac AP in rat ventricular myocytes (no plateau
phase and shorter APD) and the Naþ current, based on the experimental liter-
ature. This new formulation simulates the negative force-frequency relation ob-
served in experiments on rat ventricular myocytes with the appropriate changes
in action potential duration and different pacing frequencies. The SR Ca2þ
load decreases with increased frequency in contrast to the guinea pig which
does the opposite and has a dome shaped force-frequency relation. Furthemore,
the action potential duration increases with pacing rate opposite the guinea pig.
The new rat model also simulated mechanical restitution and low levels post-
extrasystolic potentiation levels seen in experiments with rat.
624-Pos Board B503
CaþþT LAB - A Computational Model for Intracellular Calcium Signal-
ing In T Lymphocytes
Rayhan Lal, Jose L. Puglisi, Heike Wulff, Donald M. Bers.
U.C. Davis, Davis, CA, USA.
Calcium signaling is crucial for T cell activation. T cell receptor activation leads
to the down-stream generation of IP3 which opens IP3 receptors on the mem-
brane of the endoplasmic reticulum (ER), resulting in an initial small rise in cy-
toplasmic Ca2þ. T cells do not have sufficiently large Ca2þ stores to sustainCa2þelevation and therefore require additional Ca2þ influx through the voltage-inde-
pendent Ca2þ channel CRAC, which is encoded by the ORAI1 or CRACM1
gene and is activated by STIM1, which ‘‘senses’’ decrease in ER Ca2þ content.
However, CRAC can only bring in Ca2þ at negative membrane potentials. This
potential is generated by the potassium channels Kv1.3 and KCa3.1 which drive
themembrane potential back to -60mV and facilitate Ca2þ entry for the duration
of T cell activation ultimately leading to cytokine production and T cell prolif-
eration.While there have been tremendous strides acquiring biophysical data on
the implicated ion channels this data has not been integrated into a dynamic
model. A computer model has been developed that integrates the available
data on calcium signaling in T lymphocytes and that can be utilized to illustrate
and theoretically probe T cell activation. The design is implemented in Lab-
VIEW and can be run on a PC. The program allows free reign over the cell’s en-
vironment and channel characteristics. The interface provides visualization of
IP3 production, membrane potential, charge flow and open probability of the
aforementioned channels.We suggest this programas a tool for identifying phar-
maceutical targets for intervening with immune cell activation and as a teaching
aid for immunology, physiology and cell signaling.Actin & Actin-binding Proteins
625-Pos Board B504
Actin Branching Is Affected by Local Bending of the Mother Filament
Viviana Risca1, Ovijit Chaudhuri1, JiaJun Chia2, Daniel A. Fletcher1.
1University of California, Berkeley, Berkeley, CA, USA, 2The Harker
School, San Jose, CA, USA.
Actin filaments serve as structural elements of the cytoskeleton subject to me-
chanical forces and provide binding sites for actin-binding proteins (ABPs).
Structural studies have established that actin filaments can adopt several differ-
ent twist structures that are stabilized by ABPs such as ADF/cofilin or scruin.
We sought to investigate whether strain on the actin filament due to bending
affects the binding or function of ABPs and chose the Arp2/3 complex, which
is involved in the assembly of force-generating actin networks, as our initial tar-
get. The Arp2/3 complex nucleates the growth of actin branches from pre-ex-
isting filaments, making it easy to detect where on the mother filament it has
bound. To test the effect of bending on branching, biotinylated and rhoda-
mine-phalloidin-stabilized actin filaments were bound to a streptavidin-coated
glass surface, immobilized in a distribution of bent conformations. These teth-
ered mother filaments were then incubated with actin, Arp2/3, and its activator.
Growth of branches was stabilized with green fluorescent phalloidin. Branches
originating from highly curved sections (radius of curvature < 1.5um) of actin
filaments were more likely to grow from the extended side of the filament (pos-
itive strain) than from the compressed side (negative strain), with a statistically
significant (P < 0.05) difference. To elucidate structural distortions that may
give rise to this effect, we used Monte Carlo simulations based on a coarse-
grained model of the actin filament to estimate the changes in inter-monomer
spacing that occur for the experimentally probed range of filament curvatures.
We conclude that mother filament bend strain is sufficient to alter actin branch-
ing and may play an important role in the organization of actin networks grow-
ing under load. Similar bend-induced effects may be important for other ABPs
and provide a mechanism for mechanotransduction in cells.
626-Pos Board B505
Mechanics of Biophysical Networks with Flexible Cross-links
Goran Zagar, Patrick R. Onck, Erik Van der Giessen.
University of Groningen, Groningen, Netherlands.
Various mechanical properties and functions of euchariotic cells largely origi-
nate from the cytoskeleton. The predominant cytoskeletal constituent is the bio-
polymer filamentous actin (F-actin). In the presence of various cross-linking
proteins, F-actins can comprise two rather different structures: isotropic orthog-
onal networks or bundled fibers. Actin bundles are formed mostly by short and
stiff cross-linking proteins (like a-actinin and scruin), while large and flexible
cross-linkers, such as filamin, lead to an orthogonal network. Orthogonal net-
works can also be formed at lower concentrations of short cross-linking pro-
teins, but rheological experiments of in vitro F-actin networks showed that
the mechanical response of such networks is different from that of networks
cross-linked with filamin. Moreover, atomic force microscope stretching exper-
iments on filamin demonstrated the possibility of force-induced domain unfold-
ing, characterized by a sawtooth-like pattern in the force-displacement curve.
Here we present a 3D discrete model of F-actin networks that extends our pre-
vious, rigidly cross-linked network model by incorporating a flexible cross-
linking model for human filamin A (hsFLNa). The implemented hsFLNa ele-
ment has a highly nonlinear response to stretching, incorporating the transition
to a softer response that characterizes filamin domain unfolding. Simple shear


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Both Membrane Depolarization And IL-6 Induce Calcium-Dependent Hsp70 Expression In Skeletal Muscle Cells

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