O'Rourke, Brian

Solhjoo, Soroosh

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Inhibition of Mitochondrial Na+/Ca2+ Exchanger Suppresses Ischemia/Reperfusion-Induced Reentry in Monolayers of Cardiomyocytes 

Sunday, February 16, 2014 123a635-Pos Board B390
Inhibition of Mitochondrial NaD/Ca2D Exchanger Suppresses Ischemia/
Reperfusion-Induced Reentry in Monolayers of Cardiomyocytes
Soroosh Solhjoo1,2, Brian O’Rourke2.
1Department of Biomedical Engineering, Johns Hopkins University School
of Medicine, Baltimore, MD, USA, 2Division of Cardiology, Department of
Medicine, Johns Hopkins University School of Medicine, Baltimore, MD,
USA.
Introduction:Mitochondria are important organelles that regulate cytosolic
[Ca2þ] ([Ca2þ]c) in cardiomyocytes and are thought to play a major role in
Ca2þ overload and arrhythmogenesis during ischemia/reperfusion. Previ-
ously, we showed that inhibition of the mitochondrial Naþ/Ca2þ exchanger
(mNCE) using CGP-37157 increases mitochondrial [Ca2þ] ([Ca2þ]m) reten-
tion, and prevents delayed afterdepolarizations and triggered arrhythmias
during Naþ overload induced by Naþ/Kþ ATPase inhibitor, ouabain, in vitro
and in vivo. Here, we studied the dynamics of [Ca2þ]m and [Ca
2þ]c during
ischemia/reperfusion, and investigated the role of mNCE in ischemia/
reperfusion-related reentry in monolayers of neonatal rat ventricular myocytes.
Methods: Sarcolemmal electrical activity was recorded with a 464-photodiode
array using voltage-sensitive dye, di-4-ANEPPS. Changes in [Ca2þ]m and
[Ca2þ]c were observed using the ratiometric genetically-encoded
mitochondrial and cytosolic [Ca2þ] indicator, GEM-GECO, respectively.
Ischemia was induced by covering the central region of the monolayer
with a coverslip and reperfusion was prompted by coverslip removal.
Results: As the coverslip was lowered on the monolayer, amplitude and
conduction velocity of action potentials decreased over time until the
ischemic region became inexcitable. During 1 hour of ischemia, [Ca2þ]m and
[Ca2þ]c increased in a sigmoid fashion. Reperfusion of the monolayers
with Tyrode’s solution sharply lowered [Ca2þ]m back to its initial values in
less than 10 minutes. Wavelets and subsequently reentry occurred
upon reperfusion in 6/7 monolayers. CGP-37157 (1mM), at reperfusion, pre-
vented or shortened the duration of wavelets, reducing the occurrence of
reentry to 2/5 monolayers. Viral overexpression of NCLX, the molecular candi-
date for mNCE, did not alter the incidence of reentry, compared to controls.
Conclusion: The results reveal the kinetics of [Ca2þ] during ischemia/
reperfusion using a novel genetically encoded probe and demonstrate the cardi-
oprotective effects of inhibition of mNCE, as CGP-37157 decreased dispersion
of repolarization and suppressed reentry.Excitation-Contraction Coupling I
636-Pos Board B391
Raptor Ablation in Skeletal Muscle Affects the Structure and Function of
the Excitation-Contraction Coupling Macromolecular Complex
Ruben Lopez1, Barbara Mosca2, Leda Bergamelli2, Markus A. Ruegg3,
Florian C. Bentzinger3, Michael N. Hall3, Susan Treves2,4,
Francesco Zorzato2,1.
1Basel University Hospital, Basel, Switzerland, 2University of Ferrara,
Ferrara, Italy, 3Biozentrum Basel University, Basel, Switzerland, 4Basel
Univesity Hospital, Basel, Switzerland.
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase
regulating a number of biochemical pathways controlling cell growth in a
variety of cell type. mTOR complex 1 is associated to raptor, an regulatory
protein which is essential its kinase activity. Specific ablation of raptor in
skeletal muscle results in several phenotypic changes including decreased
life expectancy, appearance of core-like structures in the centre of muscle
fibres, and increased glycogen deposits. Raptor KO mice (RamKO) also
exhibit a remarkable alterations of the twitch kinetics of slow fibres. The
later effect prompted us to investigate whether this was due to alterations
of the structure and function of the molecular complex involved in
excitation-contraction coupling. 3[H]-ryanodine and 3[H]-PN200-110 equi-
librium binding with total sarcotubular membranes fraction show a ryano-
dine to dihydropyridine receptors ratio of 0.79 and 1.35 for wild type
and RamKO skeletal muscle, respectively. Peak amplitude and the time
to peak of the global calcium transients evoked by supramaximal field
stimulation of single isolated muscle fibres were no different between
wild type and RamKO mice. However, the increase of the RyR to
DHPR ratio is associated with a higher frequency and larger FWHM of
short lasting elementary calcium release events (ECRE) induced by
hyper-osmotic shock in FDB fibres from RamKO. This study shows that
the protein composition and the function of the molecular machinery
involved in skeletal muscle excitation-contraction coupling is affected by
mTORC1 signaling.637-Pos Board B392
Modification of Cardiac Ryanodine Receptor Gating by a Peptide from the
Central Domain of the RyR2
Andrea Faltinova1,2, Alexandra Zahradnikova1,2.
1Department of Muscle Cell Research, Institute of Molecular Physiology and
Genetics, Slovak Academy of Sciences, Bratislava, Slovakia, 2Department of
Biochemistry and Structural Biology, Institute of Molecular Biology,
Slovak Academy of Sciences, Bratislava, Slovakia.
The effect of a domain peptide DPCPVTc from the central region of the RYR2 on
ryanodine receptors isolated from rat heart was examined in planar lipid bilayers.
At a zero holding potential and at 100 nM cytosolic and 8mM luminal Ca2þ con-
centration, DPCPVTc induced concentration-dependent activation of the ryano-
dine receptor that led up to 20-fold increase of open probability at saturating
DPCPVTc concentrations.The effect of thepeptide appearedwithin 30 s after addi-
tion to the experimental chamber. At all DPCPVTc concentrations RyR2 channels
displayed large variability in open probability, open time and opening frequency.
DPCPVTc prolonged RyR2 openings up to 8 and increased RyR2 opening fre-
quency by up to 100%. With increasing DPCPVTc concentration, the fraction of
high open probability records increased up to 5, and their open time increased
up to 4. The closed times did not depend on DPCPVTc concentration either in
low- or high-open probability records. The DPCPVTc concentration dependence
of all gating parameters had EC50 of 20 mM and a Hill slope of 2. Comparison
of the effects of DPCPVTc with those of ATP [1] and cytosolic Ca
2þ [2] suggests
that activation does not involve luminal feed-through and is not caused by mod-
ulation of the cytosolic activationA-site. The data suggest that although ‘‘domain
unzipping’’ by DPCPVTc occurs in both modes of RyR activity, it affects RyR
gating only when the channel resides in the H-mode of activity.
Supported in part by the European Union Contract No. LSHM-CT-2005-
018802/CONTICA and by grants APVV-LPP-0441-09, APVV-0628-10 and
VEGA 2/0197/11.
[1] Tencerova B, Zahradnikova A, Gaburjakova J, Gaburjakova M. J Gen
Physiol 140: 93, 2012.
[2] Gaburjakova J, Gaburjakova M. J Membr Biol 212: 17, 2006.
638-Pos Board B393
Ultrastructural Quantification of Electron-Dense Strings in the
Sarcoplasmic Reticulum of Rat Heart Cells
Lin-Lin Li, Xue-Xin Fan, Shi-Qiang Wang.
Peking University, Beijing, China.
Ca2þ release from the ryanodine receptors (RyRs) in the sarcoplasmic reticu-
lum (SR), in the form of Ca2þ sparks, is the fundamental events underlying
excitation-contraction. Recent structural studies of calsequestrin has character-
ized the molecular size, assembly and Ca2þ binding capacity of this Ca2þ bind-
ing protein in the SR. In order to link the molecular properties of calsequestrin
to the in situ behavior of RyR Ca2þ sparks, we studied the ultrastructural orga-
nization of junctional SR contents in rat heart cells using transmission electron
microscopy. We found that the junctional SR lumen is usually ~20 nm thick,
with the assembled calsequestrin chains visualized as electron-dense strings
(EDS) residing at the midline of the SR lumen. Between the EDS and the SR
membrane of each side, there was a symmetric electron-transparent space of
5~7 nm. Quantitative comparison between the EDS and the molecular geome-
try of crystallized calsequestrin suggested that cardiac SR lumen contained
only a single layer of calsequestrin network. Morphometric analysis predicted
that the number of calsequestrin molecules per 1000 nm2 dyadic area (roughly
the luminal area beneath a single RyR) is no more than 30~50. By comparing
spark rising phase to standard Ca2þ sparklets of single L-type Ca2þ channels,
we determined that a typical Ca2þ spark represents a 2~4 pA Ca2þ release
flux lasting for ~10 ms. Based on above structural quantification, this flux
would be supplied by 2000~6000 calsequestrin molecules within an 0.04~0.2
mm2 dyadic area, agreeing well with the size of a single dyad (or the size of
40~200 inter-linked RyRs). Our results indicated that the calsequestrin was
dyad-widely well organized for firing a single Ca2þ spark. The number and
organization of calsequestrin molecules should be important factors in shaping
Ca2þ sparks in healthy and disease conditions.
639-Pos Board B394
Resolving the Calcium Release Machinery of Mammalian Fast- and
Slow-Twitch Skeletal Muscle
Isuru D. Jayasinghe1, Michelle Munro2, David Baddeley3,
Bradley S. Launikonis4, Christian Soeller1.
1College of Physics, University of Exeter, Exeter, United Kingdom,
2Department of Physiology, University of Auckland, Auckland, New
Zealand, 3Department of Cell Biology, Yale University, New Haven, CT,
USA, 4School of Biomedical Sciences, University of Queensland, Brisbane,
Australia.


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Inhibition of Mitochondrial Na+/Ca2+ Exchanger Suppresses Ischemia/Reperfusion-Induced Reentry in Monolayers of Cardiomyocytes

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