Board, Philip G.

Casarotto, Marco G.

Dulhunty, Angela F.

Hewawasam, Ruwani P.

Elsevier - Publisher Connector 

Tuesday, March 8, 2011 413arelease dysfunction. Cardiac electrical dysfunction in CPVT is remarkably sim-
ilar to that elicited by digitalis toxicity and a related glycoside, ouabain, has
been identified as an endogenous Ca2þ-mobilising ‘hormone’. Circulating
levels of adrenally-derived ouabain are reportedly elevated in heart failure
and post-exercise. Here we investigated whether physiologic levels of ouabain
could provoke mutant RyR2 channel dysfunction. In single HL-1 cardiomyo-
cytes, ouabain (EC50 = 14.3nM) perturbed Ca2þ homeostasis in a manner
strikingly similar to that determined in cells expressing CPVT-linked
R4497C mutation. Moreover, dose-dependent ouabain-evoked Ca2þ dysfunc-
tion resulted in a left-shifted caffeine activation profile typical of mutant RyR2
channels. LC/MS/MS analysis determined equivalent levels of ouabain in se-
rum obtained from CPVT- and normal subjects (500-800pM) and exercise
stress-testing did not acutely elevate ouabain levels in either cohort. In cells ex-
pressing the R4497C mutation, which exhibited basal Ca2þ dysfunction, addi-
tion of ouabain at physiologic (1nM) and supra-physiologic (10-100nM)
concentrations did not further exacerbate cellular Ca2þ abnormalities. In con-
trast, the addition of serum obtained from exercised normal and CPVT subjects
(100ug protein equivalents; ~2pM ouabain) evoked hallmark mutation-linked
RyR2 dysfunction that was not completely abolished by b-blockade. These
findings confirm that non-catecholaminergic factors contribute to RyR2 mutant
dysfunction, but preclude the possibility that endogenous ouabain is a physio-
logically relevant trigger of CPVT. However, the hypothesis that endogenous
ouabain may induce underlying changes in excitation-contraction coupling
that increase the propensity for mutation-linked RyR2 dysfunction is being
investigated.
2235-Pos Board B221
The Effect of Adrenergic Stimulation on the Calcium Release Channel
Jiao Li, Nicole A. Beard, Angela F. Dulhunty, Dirk F. vanHelden,
Derek R. Laver.
Adrenergic stimulation of the heart involves phosphorylation of many intracel-
lular Ca2þ handling proteins including the ryanodine receptor Ca2þ release
channels in the SR (RyRs). It is known that RyRs can be phosphorylated at
three serine residues at 2808, 2814 and 2030 and that phosphorylation of
RyRs via PKA causes an increase in RyR activity in situ. However, little is
known about how phosphorylation of RyRs in the cell alters their regulation
by intracellular Ca2þ and Mg2þ.
RyRs were isolated from rat hearts, which had been rapidly removed, perfused
with Krebs buffer in a Langendorff apparatus. Some hearts were perfused with
1 mM isoproterenol for sufficient time to increase heart rate by>60% for 1 min
and others without isoproterenol for 5 mins. Hearts were snap frozen in liquid
N2 to capture their state of phosphorylation, thus allowing RyRs to be phos-
phorylated by the physiological signalling processes. RyRs were incorporated
into artificial planar lipid bilayers and their activity was measured using single
channel recording.
The activity of RyRs from isoproterenol stimulated hearts (ISO RyRs, n=25)
was 3-fold higher than control RyRs (n=24) at diastolic [Ca2þ] (100 nM) but
was not significantly different at systolic [Ca2þ] (>1 mM). At diastolic
[Ca2þ], addition of Protein Phosphatase1 (PP1, 5 mins) reduced the activity
of ISO RyRs by 98 5 2.6% (n=4) and control RyRs by 70 5 20 % (n=4)
but this treatment had no effect at systolic [Ca2þ]. ISO RyRs displayed
a 100-fold channel-to-channel variation in activity which was larger than,
and encompassed the range of activity seen either for control RyRs or PP1
treated RyRs. The results indicate that adrenergic stimulation increases RyR2
activity during diastole but not during systole.
2236-Pos Board B222
The Functional Impact of Cis Acting Ryanodine Receptor Type 1
Mutations in a Child with a Fatal Spontaneous MH Event
Linda Groom, Sheila Muldoon, Munkhuu Bayarsaikhan, Saiid Bina,
Hee-Suk Lee, Barbara W. Brandom, Nyamkhishig Sambuughin,
Robert T. Dirksen.
Mutations in the RYR1 gene result in MH, a pharmacogenetic disorder of
skeletal muscle typically triggered by administration of anesthetics. How-
ever, cases of sudden death during exertion, heat challenge, and febrile ill-
ness in the absence of triggering drugs have been reported in individuals
with RYR1 gene variants. We describe the clinical history and genetic anal-
ysis of a child that suffered a fatal, non-anesthetic MH episode associated
with febrile illness who was heterozygous for two novel RYR1 variants
where one variant, R3983C, occurred de-novo and another, D4505H, was in-
herited. Effects of the two variants on RYR1 sensitivity to activation by caf-
feine were assessed following expression in RYR1-null myotubes. The
single (R3983C and D4505H) and double (R3983C-D4505H) variants
were engineered into a full-length rabbit RYR1 cDNA and introduced
into RyR1-null myotubes via nuclear microinjection. Effects of the differentheterotypic expression conditions (WTþR3983C, WTþD4505H,
R3983CþD4505H, and WTþR3983C-D4505H) on RYR1 activation by caf-
feine were determined in indo-1-loaded myotubes. Compared to WT RYR1
alone (EC50 = 1.5 mM), the caffeine sensitivity of RYR1 Ca
2þ release was
only modestly increased following co-expression of either R3983C (EC50 =
1.3 mM) or D4505H (EC50 = 0.9 mM). Remarkably, co-expression of WT
RYR1 with the double mutant in cis (R3983C-D4505H) produced a much
stronger sensitization of caffeine-induced release (EC50 = 0.3) than that ob-
served following co-expression of the two variants on separate subunits
(EC50 = 0.9). Thus, the R3893C mutation potentiates D4505H-mediated sen-
sitization of caffeine-induced RYR1 Ca2þ release when these mutations are
in cis or present on the same subunit, but not when present on separate sub-
units. These results indicate that allelic segregation can be a critical, and
heretofore unappreciated, pathogenic factor in compound heterozygous
MH individuals.
2237-Pos Board B223
GSTM2 C Terminus Reduces Calcium Release Through RyR2 in Sponta-
neously Contracting and Field Stimulated Ventricular Cardiomyocytes
Ruwani P. Hewawasam, Marco G. Casarotto, Philip G. Board,
Angela F. Dulhunty.
The ryanodine receptor (RyR) is an ion channel that releases Ca2þ from the sar-
coplasmic reticulum and is essential for excitation-contraction coupling and
contraction in striated muscle. Previously we reported that the human muscle
specific glutathione transferase (GSTM2-2) is an inhibitor of cardiac muscle
RyR2 and a weak activator of skeletal muscle RyR1. Single channel experi-
ments and Ca2þ release assays using the C-terminal half of GSTM2-2
(GSTM2C) and the mutants, F157A and Y160A in the C terminal domain con-
firmed the importance of helix 6 in the C-terminal (non-enzymic) fold for in-
hibition of RyR2. The objective of this study was to determine the effect of
GSTM2C on the cardiac myocyte function.
Primary cardiomyocytes were cultured from neonatal rats. Spontaneous or
field- stimulated (1Hz with 3V, 2ms pulses) contraction was recorded in control
and 15mMGSTM2C-treated cells using a JVC video camera KY/F550 attached
to Nikon TE2000-U microscope. Images were analysed using Image Pro plus
6.2 software and percentage cell shortening measured.
Preliminary results showed that spontaneous contraction frequency fell from
42.5/min in the control group to 6.9 /min after GSTM2C treatment
(P<0.001). The number of spontaneously beating cells fell from 6.6% in the
control group to 1.9% after treatment (P<0.001). To determine whether the re-
duced contractions were due to the GSTM2 C terminus affecting action poten-
tials or contraction, shortening was measured. Shortening in spontaneous
contraction fell from 7.551.0% in control to 2.950.6% after GSTM2C treat-
ment (P<0.001). Shortening after field stimulation also fell, from 7.350.8%
before to 4.150.6% after, GSTM2C-treatment (P<0.01). These results are
consistent with GSTM2 C reducing contraction in ventricular cardiomyocytes
by reducing Ca2þ release through RyR2.
2238-Pos Board B224
In Situ Measurement of RyR2-Calmodulin Binding in Permeabilized
Cardiomyocytes
Yi Yang, Asima Chakraborty, Tao Guo, Razvan L. Cornea,
Gerhard Meissner, Donald M. Bers.
Calmodulin (CaM) is a Ca-sensing protein and important regulator to RyR2.
Defective CaM association with and regulation of RyR2 has been implicated
in cardiac hypertrophy, heart failure and Catecholaminergic Polymorphic Ven-
tricular Tachycardia. However, direct measurement of the RyR-CaM binding
properties in myocytes is lacking despite its potential clinical importance.
This is most likely due to the interference from CaM’s multiple binding targets,
such as L-type Ca channel, Calcineurin, and CaMKII. Fluorescence Resonance
Energy Transfer (FRET) was used to measure the RyR2-specific bound CaM in
permeabilized rat and mouse cardiomycytes. FKBP12.6 and CaM bind to RyR2
in close physical proximity so that strong FRET can be detected between fluo-
rescently labeled FKBP12.6 (F-FKBP12.6) and F-CaM. To confirm the speci-
ficity of CaM binding to RyR2 and measure the percentage of CaM visible at
the Z-line is RyR-bound under physiological conditions, we used mutant RyR2
knock-in mice expressing a triple mutation in the RyR2 (RyR2ADA). The mu-
tation prevents CaM binding to RyR2 but should not alter binding of CaM to
other targets. Our data showed that 1) the apparent Kd of CaM to RyR in rat
cardiac myocytes (at 100 nM [Ca]) was 18.25 2nM (n=XX); 2) heterozygous
RyR2ADA/þ mutation decreased the maximal binding sites (on RyR2) by 50%
without appreciably altering affinity in mouse myocytes (Kd =16.95XX nM in
RyR2ADA/þ and 13.45XX nM, inWTmice, p< 0.05); 3) The total CaM bind-
ing visible at the Z-line was reduced by 45%, implying that ~90% of Z-line


GSTM2 C Terminus Reduces Calcium Release Through RyR2 in Spontaneously Contracting and Field Stimulated Ventricular Cardiomyocytes 

file:///data/remote/core/dit/data/elsevier/pdf/1fd/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUxMDAzOTUyNA==.pdf

GSTM2 C Terminus Reduces Calcium Release Through RyR2 in Spontaneously Contracting and Field Stimulated Ventricular Cardiomyocytes

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector