Chang, Kelly C.

Trayanova, Natalia A.

Elsevier - Publisher Connector 

Monday, February 9, 2015 267aclusters (~50 nm dia). The mouth of the t-tubules were often very heavily
labelled for 2-3 mm into the cell.
1346-Pos Board B297
SuperresolutionMicroscope Image Reconstruction by Spatiotemporal Ob-
ject Decomposition and Association: Application in Resolving T-Tubule
Structure in Skeletal Muscle
Mingzhai Sun1, Jiaqing Huang2, Filiz Bunyak3, Kristyn Gumpper4,
Gejing De1, Matthew Sermersheim1, George Liu5, Pei-Hui Lin1,
Kannappan Palaniappan3, Jianjie Ma1.
1Davis Heart and Lung Research Institute, The Ohio State University,
Columbus, OH, USA, 2Department of Electrical and Computer Engineering,
The Ohio State University, Columbus, OH, USA, 3Department of Computer
Science, University of Missouri, Columbia, MO, USA, 4Department of
Surgery, The Ohio State University, Columbus, OH, USA, 5Department of
Physics, Princeton University, Princeton, NJ, USA.
One key factor that limits resolution of single-molecule superresolution mi-
croscopy relates to the localization accuracy of the activated emitters, which
is usually deteriorated by two factors. One originates from the background
noise due to out-of-focus signals, sample auto-fluorescence, and camera
acquisition noise; and the other is due to the low photon count of emitters
at a single frame. With fast acquisition rate, the activated emitters can last
multiple frames before they transiently switch off or permanently bleach.
Effectively incorporating the temporal information of these emitters is crit-
ical to improve the spatial resolution. However, majority of the existing
reconstruction algorithms locate the emitters frame by frame, discarding or
underusing the temporal information. Here we present a new image recon-
struction algorithm based on tracklets, short trajectories of the same objects.
We improve the localization accuracy by associating the same emitters from
multiple frames to form tracklets and by aggregating signals to enhance the
signal to noise ratio. We also introduce a weighted mean-shift algorithm
(WMS) to automatically detect the number of modes (emitters) in overlap-
ping regions of tracklets so that not only well-separated single emitters but
also individual emitters within multi-emitter groups can be identified and
tracked. In combination with a maximum likelihood estimator method
(MLE), we are able to resolve low to medium density of overlapping emitters
with improved localization accuracy. We evaluate the performance of our
method with both synthetic and experimental data, and show that the
tracklet-based reconstruction is superior in localization accuracy, particularly
for weak signals embedded in a strong background. Using this method, for
the first time, we resolve the transverse tubule structure of the mammalian
skeletal muscle.
1347-Pos Board B298
Voltage-Gated Calcium Influx Contributes to Sarcoplasmic Reticulum
Calcium Loading in Skeletal Muscle
Gaelle Robin, Bruno Allard.
Physiology, University Lyon 1, Villeurbanne, France.
Muscle activation is known to be associated with a voltage-activated trans-
sarcolemmal Ca2þ influx. The role as well as the pathways involved in this
influx are still being debated. In this study, we used the technique of quench-
ing of fura-2 fluorescence resulting from Mn2þ influx in voltage-controlled
isolated skeletal muscle fibres from mice and zebrafish larvae. Depolarizing
pulses of increasing amplitudes evoked L-type Mn2þ currents associated
with fura-2 quenching signals displaying comparable voltage-dependences.
In the presence of external Cd2þ L-type currents were totally abolished
whereas substantial quenching signals remained associated with voltage
pulses. The voltage-dependence and reactivation kinetics of the quenching
signals were comparable in the presence of Cd2þ and in its absence. In zebra-
fish fibres, neither voltage-activated L-type current nor voltage-activated
quenching signal was observed. Taken together, these data suggest that the
L-type current is the only contributor to voltage-activated Ca2þ entry in skel-
etal muscle. Calibration of the quenching signal in mouse fibres indicated that
an influx producing a current of 0.3 A/F developed during a train of action
potentials (AP). Prolongation of AP induced by tetraethylammonium led to
an increase in the quenching signal evoked by a train of AP. A series of ex-
periments using Fluo5N to monitor sarcoplasmic reticulum (SR) Ca2þ
changes in voltage-controlled mouse fibres showed that increasing the
L-type current amplitude by doubling external Ca2þ concentration led to an
acceleration of the recovery phase of SR Ca2þ signals. Addition of external
Mn2þ led to opposite effects suggesting that Mn2þ ions flowing through
L-type channels have access to the SR lumen and quench Fluo5N fluores-
cence. These results strongly suggest that Ca2þ influx through L-type channels
significantly contribute to replenish the SR and in this way helps to preserve
muscle performance during prolonged activity.1348-Pos Board B299
Contribution of L496-L500-W503 Motif of DHPR-b1A Subunit to
Skeletal-Type EC-Coupling
Jose M. Eltit1, Clara Franzini-Armstrong2, Claudio F. Perez3.
1Department of Physiology and Biophysics, Virginia Commonwealth
University, Richmond, VA, USA, 2Department of Cell and Developmental
Biology, University of Pennsylvania, Philadelphia, PA, USA, 3Anesthesia,
Brigham and Women’s Hospital, Boston, MA, USA.
The b1a subunit is a cytoplasmic component of the dihydropyridine receptor
(DHPR) complex that plays an essential role in skeletal muscle excitation-
contraction (EC) coupling. Here we investigate the role of the carboxyl terminal
end of this auxiliary subunit in the functional and structural communication be-
tween theDHPRand theCa2þ release channel (RyR1). Progressive truncation of
the b1a C-terminus showed that deletion of amino acid residues 489Q- to -W503
resulted in loss of depolarization-induced Ca2þ release, severe reduction of
L-type Ca2þ currents and lack of tetrad formation as evaluated by freeze-
fracture analysis. However, deletion of this domain did not affect expression/tar-
geting or density (Qmax) of DHPR-a1S subunit to the plasma membrane.
Within this region are found residues L496, L500 andW503, which are thought
to mediate direct b1a-RyR1 interactions. Disruption of the motif L496-L500-
W503 by a triple alanine substitution weakened EC-coupling but it did not repli-
cate the truncated phenotype. These data demonstrate that an amino acid
segment encompassing sequence 489QVQVLTSLRRNLSFW503 of b1a con-
tains critical determinant(s) for the physical link of DHPR and RyR1, further
confirming a direct correspondence between DHPR positioning and DHPR/
RyR functional interactions. In addition, our data strongly suggest that motif
L496-L500-W503 within the b1a C-terminal tail plays a non-essential role in
bi-directional DHPR/RyR1 signaling that supports skeletal-type EC-coupling.
Supported by NHI Grants 5K01AR054818, R03AR066359 (to CFP), R37-HL-
048093 (to CFA) and Milton Fund/Harvard Medical School (to CFP).
1349-Pos Board B300
FRET-Based Structural Analysis of the Skeletal Muscle DHPR using Biar-
senical Labeling
Mohana Mahalingam, Claudio F. Perez, James D. Fessenden.
Anesthesia, Brigham and Women’s Hospital, Boston, MA, USA.
The a1s skeletal muscle dihydropyridine receptor (DHPR) subunit triggers
muscle contraction during excitation contraction (EC) coupling. The intracel-
lular loops of this voltage-gated calcium channel have diverse functions,
though the spatial organization of these loops relative to each other and to
other DHPR subunits, is poorly understood. We employed a FRET-based
assay to determine the proximity of these intracellular loops relative to the
N-terminus of the a1s DHPR subunit and then determined how these proxim-
ities changed upon binding of the b1a DHPR subunit. Recombinant a1s
DHPRs were expressed in HEK293T cells and then FRET was measured
from YFP fused to the DHPR N-terminus to the biarsenical reagent ReAsH
targeted to a tetracysteine (Tc) tag inserted into each of the DHPR intracel-
lular loops. Energy transfer was highest to ReAsH bound to a Tc tag placed
adjacent to the N-terminally fused YFP (E~40-50%) whereas low FRET
(E~10%) was observed for a negative control construct lacking a Tc tag.
Placement of Tc tags in each of the DHPR intracellular loops resulted in in-
termediate FRET efficiencies whereas insignificant energy transfer was
observed between the DHPR N- and C-termini. Addition of purified recombi-
nant DHPR b1a subunit resulted in significant enhancement of energy transfer
between the N-terminal YFP and ReAsH targeted to the DHPR a1s II-III
loop, a major EC coupling determinant. Our data demonstrate that FRET mea-
surements using biarsenical labeling reagents offer an effective approach to
study DHPR structure since disruption of native conformation is minimal.
In addition, these studies should allow for subsequent measurements of
changes in DHPR structure that occur during EC coupling.
Supported by NIH grants R01AR059126 (to JDF) and R03AR066359 (to CFP).
1350-Pos Board B301
Dependency of Ca2D Alternans on Ion Channel Localization in Human
Atrial Cells
Kelly C. Chang, Natalia A. Trayanova.
Institute for Computational Medicine, Johns Hopkins University, Baltimore,
MD, USA.
Introduction: Localization of ion channels to t-tubules (TTs) comprises a crit-
ical part of excitation-contraction coupling in cardiomyocytes. Recent studies
have shown that TT organization varies greatly among atrial cells, which
have different ion channel distribution than ventricular cells. Ca2þ transient
(CaT) alternans are known to occur in atrial cells with fewer TTs, but the
ion channels which contribute to this are unknown.
268a Monday, February 9, 2015Hypothesis:Decreased localization of L-type Ca2þ channels (LTCCs) and rya-
nodine receptors (RyRs) to TTs in atrial cells results in increased propensity for
arrhythmogenic CaT alternans.
Methods: Using a human atrial cell model from Grandi et al. incorporating
chronic AF remodeling, the effects of ion channel redistribution were evaluated
by moving 0-100% of TT ionic currents to the sub-sarcolemmal compartment
(SL). Ryanodine receptor (RyR) redistribution was also tested by moving
0-100% of RyR release from the TTs to the cytosol. In 500 virtual cells, a total
of 12 different ion channels were redistributed stochastically and indepen-
dently. The effect of each ion channel on alternans threshold cycle length
(CL) was evaluated by regression analysis.
Results: The alternans threshold CL in the original model was 431 ms. Redis-
tribution of LTCCs from TTs to the SL had the greatest effect on alternans
threshold CL (regression coefficient of 0.95), with alternans occurring at
slower pacing rates as the fraction of TT-localized LTCCs decreased due to
steepening of the SR Ca2þ release slope. Redistribution of RyRs or Naþ/
Ca2þ exchangers had a smaller and opposite effect on alternans threshold CL
(regression coefficients of 0.16 and 0.072, respectively), with decreased TT
localization of these channels causing alternans to only occur at faster pacing
rates.
Conclusions: Decreased localization of LTCCs to TTs in atrial cells promotes
arrhythmogenic CaT alternans at slower pacing rates.
1351-Pos Board B302
Ageing causes Severe Ultra-Structural Modification of Calcium Release
Units and Mitochondria in Cardiomyocytes
Laura d’Onofrio, Alessia Di Fonso, Feliciano Protasi, Simona Boncompagni.
Ce.S.I. - Center for Research on Ageing & DNICS - Dept. of Neuroscience,
Imaging & Clinical Sciences, University of G. d’Annunzio, Chieti, Italy.
Ageing is associated to a dramatic increase in the incidence of heart failure,
even if the existence of a real age-related cardiomyopathy remains controver-
sial. In this study we performed a morphological study of cardiac cells in
hearts from adult and old mice (4 and R 24 months respectively) using
confocal and electron microscopy. Our results indicates that the cross
sectional areas (CSA) of cardiomyocytes is on the average increased in old
hearts (adults: 1915105 mm2; old: 232 5 147 mm2), with a greater vari-
ability in size indicating also the presence of several atrophic cells. The
increased CSA may be the result of an increased presence of amorphic (appar-
ently empty) cytoplasmic space between myofibrils (adults: 2.2 5 3.1; old:
9.7 5 5.7).
As effective contraction and relaxation of cardiomyocytes also depends on
Ca2þ supply to myofibrils (handled by calcium release units (CRUs) and sarco-
plasmic reticulum (SR) and on efficient ATP production (provided by mito-
chondria), we have also analyzed in detail these intracellular organelles. The
analysis of CRUs indicates that SR/transverse-tubules (TT) couplons become
shorter with age and that the number of CRUs/50 mm2 is decreased of about
24% (adults: 5.153.3; old: 3.952.6). Also mitochondria present structural
modifications, with a significant increase in the percentage of organelles pre-
senting severe alterations (3.5% vs. 16.5%, respectively in adult vs. old).
Importantly, both CRUs and mitochondria undergo a spatial re-organization
with respect to sarcomeres/myofibrils: CRUs are may be miss-oriented (longi-
tudinal) or miss-placed (found at the A band instead of being correctly placed in
proximity of Z-lines), while mitochondria are often grouped in an abnormal
fashion.
These age-related ultra-structural changes may underlie an inefficient supply of
Ca2þ and ATP to contractile elements, providing a possible structural explana-
tion for heart dysfunction.
1352-Pos Board B303
The Effect of Aging on Calcium Transients in Rat Cardiomyocytes: Impact
of NOX Inhibition
Daniel R. Gonzalez, Guillermo B. Barrios.
Ciencias Basicas, Universidad de Talca, Talca, Chile.
Cardiac aging is characterized by alterations in contractility and calcium
handling. It has been suggested that oxidative stress may be involved in
this process. The superoxide generating system NADPH oxidase (NOX) is
expressed in the heart (NOX2 and 4). We and others have reported that
in cardiac failure, the NOX-derived superoxide is increased, with a
negative impact on calcium and contractility. We tested the hypothesis that
calcium transients and contractility in aged rat cardiomyocytes are disturbed
by NOX.
Cardiomyocytes were obtained form adult (5 months-old) and aged (20
months-old) Sprague-Dawley rats. Aged myocytes were treated with apocynin
(50 mmol/L). Cells were field-stimulated from 0.5 to 4 Hz. Contractility was
evaluated as sarcomere shortening and [Ca2þ]i was monitored with fura-2.Sarcomere shortening was similar in adult and aged myocytes. [Ca2þ]i tran-
sients amplitude was increased in aged cardiomyocytes (p<0.005) and was
further increased by apocynin treatment. Time-50 to peak [Ca2þ]i was
increased in aged myocytes (p<0.05), suggesting impairment in the ryanodine
receptor RyR2, and was improved by apocynin treatment. Time-50 to maximal
relaxation was increased in aged myocytes (p<0.05) and reduced towards
normal by NOX inhibition.
Using thapsigargin to block the sarcoplasmic reticulum calcium pump
SERCA2, we submitted myocytes to tetanic stimulation to evaluate the myofil-
aments Ca2þ sensitivity. By comparing the amplitude of the tetanic contraction
achieved, with the level of [Ca2þ]i evoked, we found that myofilaments Ca2þ
sensitivity was reduced in aged myoctes (p<0.05).
In conclusion, contractility was preserved in aged myocytes, but at a higher
[Ca2þ]i level, as a result of diminished myofilaments Ca2þ sensitivity. NOX in-
hibition increased [Ca2þ]i transients amplitude and improved Ca2þ kinetics,
without changes in contractility. These results suggest a NOX-dependent effect
in aged myocytes at the level of RyR2 SERCA2 and myofilaments.
1353-Pos Board B304
Optogenetic Manipulation of Ca2D Transients and Contraction in a Math-
ematical Model of Cardiomyocyte Function
Yasser Aboelkassem, Stuart G. Campbell.
Biomedical Engineering, Yale University, New Haven, CT, USA.
The cardiac optogenetics ‘toolbox’ combines optical physics and genetic engi-
neering technology to provide a means of manipulating heart cell function with
high specificity. For instance, recent studies have shown that light irradiance at
specific wavelengths can perturb the processes of excitation, transmembrane
potential propagation, and beating frequency in ChR2-expressing cardiac cells.
We hypothesized that optogenetics could be used to manipulate not just beating
frequency of heart cells, but also the intensity of contractions.
In this study, we present an integrative optogenetics-electromechanical math-
ematical model that uses an engineered light-electric impulse to stimulate
genetically modified cardiomyocytes. This hybrid excitation protocol simu-
lates the excess inward ionic current produced by brief, high-intensity light
flashes and demonstrates that when properly timed, these pulses can be
used to manipulate the cellular action potential duration, intracellular calcium
release, and myofilament twitch dynamics. The model is composed of three
coupled modules; a light controlled ion channel (ChR2) module, an electrical
cellular model with enhanced calcium induced calcium released process
(CICR), and a myofilament (MF) mechanical component. The cell membrane
connects activity of ChR2 with other time- and voltage-dependent ionic cur-
rents in the cell as well as CICR. The Ca2þ transient produced by CICR then
links ChR2 activity to the local cardiac MF dynamics causing muscle to con-
tract. The model is expected to be useful for designing many different types of
in vitro cardiomyocytes studies where beat-to-beat manipulation of contrac-
tility is desirable.
1354-Pos Board B305
MG29/SYPL2 Contributes to Dysregulation of Lipid Composition and
Store Operated Ca2D Entry in Aging Skeletal Muscle
Julian A. Vallejo1, Liubov V. Gushchina2, Sainath Kotha2, Leticia Brotto1,
Narasimham Parinandi2, Noah Weisleder2, Marco Brotto1.
1Muscle Biology Research Group-MUGIG, School of Nursing & Health
Studies and the School of Medicine, University of Missouri-Kansas City,
Kansas City, MO, USA, 2Department of Physiology and Cell Biology, Ohio
State University, Columbus, OH, USA.
We proposed over one decade ago that age-related muscle weakness and not the
loss of muscle size is the principal contributor to disability. In searching for
cellular and molecular explanations for this weakness that exceeds the loss
of muscle mass, we found that elements of contractile force generation in skel-
etal muscle are dependent on store-operated Ca2þ entry (SOCE) and that this
capacity is lost in aging skeletal muscle. This altered SOCE is directly associ-
ated with reduced content of mitsugumin 29 (MG29), a synaptophysin family
protein (SYPL2) containing a MARVEL domain involved in cholesterol bind-
ing and formation of oligomers of these proteins. MG29 is essential for the for-
mation of the transverse-tubule system and efficient SOCE, creating a link
between reduced MG29 expression in aged muscle and the development of
muscle dysfunction in sarcopenia as our previous data demonstrate that aspects
of aged skeletal muscle are present in young mg29-/-muscle. Here we test how
the loss of MG29 affects the phospholipid composition of skeletal muscle fibers
and if producing similar changes in muscle fibers can influence SOCE activity.
Lipidomic analysis of youngmg29-/- skeletal muscle showed decreased choles-
terol content and altered levels of various phospholipid species compared to
young control muscle. To simulate the reduced cholesterol content observed
in mg29-/- muscle we partially extracted it from mature C2C12 myotubes


Dependency of Ca2+ Alternans on Ion Channel Localization in Human Atrial Cells 

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Dependency of Ca2+ Alternans on Ion Channel Localization in Human Atrial Cells

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