Grote, Eric

Holewinski, Ronald J.

Kass, David A.

Kirk, Jonathan A.

Van Eyk, Jennifer E.

Venkatraman, Vidya

Elsevier - Publisher Connector 

Monday, February 9, 2015 273aAPD90 beat-to-beat variability (BVR) was larger in KO and, consistently, the
incidence of early afterdepolarizations was higher in KO than WT (respec-
tively, 66% and 18%). In contrast, KD cells showed a slight prolongation of
APD90 (9657.9 ms), but BVR and EADs incidence were comparable to
WT. While voltage-gated Kþ currents (Ito, IKur and Iss) were strongly downre-
gulated in KO, KD myocytes showed only a small reduction of Ito. ICaL density
was significantly lower in both KO and KD mice at negative potentials only.
Consistently, both KD and KO mice showed a positive shift of both steady-
state activation (DV1/2 vs WT: KO 4.2 mV, KD 5.5 mV) and inactivation
curves (DV1/2 vs WT: KO 3.2 mV, KD 3.4 mV). Addition of isoproterenol
(100 nM) resulted in ICaL enhancement, whose magnitude was larger in KO
compared to KD and WT myocytes (% increase,WT: 5856%,KD:4557%,
KO:95.5512%). These data suggest distinct action related to either kinase
and scaffold functions: while the former has an impact on ICaL biophysical
properties, the latter prevents arrhythmias in murine ventricular myocytes by
preserving Kþ currents.
1373-Pos Board B324
Mass Spectrometry-Based Analysis of the Phospho-Proteome for Cardiac
Dyssynchrony and Resynchronization Therapy
Jonathan A. Kirk1, Ronald J. Holewinski1,2, Vidya Venkatraman2,1,
Eric Grote1, David A. Kass1, Jennifer E. Van Eyk1,2.
1School of Medicine, Johns Hopkins University, Baltimore, MD, USA,
2Department of Medicine, Cedars-Sinai Medical Center, Los Angeles,
CA, USA.
In patients suffering from heart failure, conduction abnormalities that
create cardiac dyssynchrony worsen morbidity and mortality. However, a
pacemaker-based treatment termed Cardiac Resynchronization Therapy
(CRT) can significantly restore function and mortality. Our previous work
implicated post-translational signaling cascades in modulating this recovery.
To further understand the mechanism of recovery, we aimed to catalogue, as
completely as possible, the phospho-proteome in canine models of normal car-
diac function (Con), cardiac dyssynchrony (Dys), and CRT. The models were
generated by implanting pacemakers and pacing 6-weeks dyssynchronously
(Dys), 3-weeks dyssynchronously and 3-weeks CRT (CRT), or not at all
(Con). Each cardiac tissue sample (n¼4 per group) was split into three parts:
myofilament-enriched, myofilament-depleted, and phospho-tyrosine enriched.
Each part was phospho-enriched and run on an Orbitrap Elite. By one-way
ANOVA and Holm-Sidak post-hoc test, there were 104 sites Ser/Thr sites
and 85 Tyr sites that were changed in at least one group. These sites were as-
signed one of three categories based on the pattern of changes: ‘‘Dyssynchrony,
Not Fixed by CRT’’, ‘‘Fixed by CRT’’, or ‘‘CRT Only/Exacerbated by CRT’’.
Next we predicted the kinase for each phosphorylation site (Group-based Pred-
ication System 2.1). Many tyrosine kinases were identified, although no kinase
showed specificity to any category. However, the Ser/Thr kinase CK2 was pre-
dicted to target a majority of the ‘‘Fixed By CRT’’ category, and was highly
specific for this group, as CK2 was not predicted to target sites in any of the
other categories. Thus, CRT appears to act as a broad CK2 activator. In the
future, these sites need to be validated as CK2 targets. Moreover, the effect
of CK2 activation on cardiomyocyte function, sarcomere shortening and
calcium transient, will be explored, considering these are already implicated
in recovery with CRT.
1374-Pos Board B325
Modulation of Action Potential Alternans by IKs in Myocardial Infarction
Bum-Rak Choi1, Tae Yun Kim1, Mayara Grizotte-Lake1, Jean Daley1,
Lorraine Schofield1, Kamana Bist1, Joseph Yammine2, Yukiko Kunitomo1,
Yichun Lu1, Xuwen Peng3, Zhilin Qu4, Gideon Koren1.
1Cardiovascular Research Center, Rhode Island Hospital and Brown Medical
School, Providence, RI, USA, 2BrownMedical School, Providence, RI, USA,
3Department of Comparative Medicine, Pennsylvania State University
College of Medicine, Hershey, PA, USA, 4Department of Medicine,
University of California, Los Angeles, Los Angeles, CA, USA.
Background: Patients with post myocardial infarction (MI) have increased inci-
dence of ventricular arrhythmias. The slowly activating Kþ channels (IKs) has
been reported to be downregulated consistently but in different degrees in the
post-MI hearts. The role of downregulation of IKs in arrhythmogenesis in post
MI remains to be defined.
Hypothesis: We hypothesize that significant suppression of IKs in post MI im-
pairs the rate-dependent and Ca2þ-dependent augmentation of repolarization
reserve resulting in increased alternans and thereby arrhythmias.
Methods: We evaluated the effect of IKs on alternans in post MI using trans-
genic rabbit model of Long QT Type 1 (LQT1) lacking IKs. LQT1 and litter-
mate control (LMC) rabbits underwent MI procedures and optical mapping
was performed to record Vm and Ca2þ simultaneously from the 3 weekpost-MI hearts (n¼9 LQT1-MI, 4 LQT-MI Sham, 10 LMC-MI, 9 LMC-
Sham hearts).
Results: The dispersion of APDwas enhanced in LQT1-MI compared to LQT1-
Sham and LMC-MI (aAPDmax-min¼ 2854 ms vs. 2053 ms and 2756 ms).
APD alternans was more pronounced in LQT1-MI, accompanied with large
amplitude Ca2þ alternans, and often spatially discordant (see traces and resti-
tution curves in panel A&B). Spatially discordant alternans in LQT1-MI was
highly dynamic, preceded by sudden appearance of Ca2þ nodal lines near the
MI border zone (panel C, dark lines), associated with high VF induction
(n¼9/9 LQT1-MI vs. 1/4 LQT1-sham and 7/10 LMC-MI).
Conclusion: Our results demonstrate the importance of IKs as a repolarization
reserve in MI and that significant downregulation of IKs may promote spatially
discordant Ca2þ and APD alternans and VF induction.
1375-Pos Board B326
A Guinea Pig Model for Heart Failure-Associated Sudden Cardiac Death
Ting Liu, Deeptankar Demazumder, Brian O’Rourke.
The Johns Hopkins University, Baltimore, MD, USA.
Sudden cardiac death (SCD) is the leading cause of mortality in patients with
heart failure (HF); However, the mechanisms underlying SCD are incom-
pletely understood. In the present study, we developed a guinea pig model
for HF-associated SCD. Male adult guinea pigs underwent ascending aortic
constriction (AC) in addition to isoproterenol (iso) challenge by daily i.p. in-
jection of iso at 1 mg/kg in week1 and 2 mg/kg after week 1. Iso treatment at
the selected doses for 4 weeks had no significant effects on cardiac function
and geometry in sham control, however, iso treatment worsened cardiac func-
tion in AC animals. Animal with AC and iso challenge (ACi) displayed earlier
decompensation and higher incidence of SCD compared to AC group. ~60%
of ACi animals died without overt symptom of HF within 4 weeks. Electro-
cardiography (ECG) indicated QT prolongation and high incidence of prema-
ture ventricular beat (PVB) and ventricular fibrillation (VF) in ACi animals.
Electrophysiological study, performed in isolated ventricular myocytes from
ACi and sham hearts in week4, revealed prolonged action potential (AP)
(APD90: 279ms515 in ACi vs 203ms517 in sham) and reduced potassium cur-
rents (Ik1 at 150mV was 28.4pA/pF50.8 in ACi vs 47.6pA/pF52.3 in
sham; Tail current of Ikr and Iks at þ50mV were 0.28pA/pF50.03 and
0.3050.03 in ACi vs 0.55pA/pF50.02 and 0.54pA/pF50.03 in sham, respec-
tively) in ACi animals. Our results demonstrated that the combination of AC and
iso challenge produces high incidence of arrhythmia and SCD in guinea pig with
features of prolonged QT and reduced repolarization reserve, thus providing a
novel model for HF-associated arrhythmia and SCD.
1376-Pos Board B327
Iron Overload Promotes Arrhythmias via ROS Production and Mitochon-
drial Membrane Potential Depolarization
Richard Gordan, Nadezhda Fefelova, Judith Gwathmey, Lai-Hua Xie.
Cell Biology and Molecular Medicine, Rutgers Biomedical and Health
Sciences, Newark, NJ, USA.
Genetic disorders or frequent blood transfusions often cause iron overload
which can lead to arrhythmias. However, the underlying mechanism is not
well known. In the present study, we assess the hypothesis that iron-
overload promotes arrhythmias via reactive oxygen species (ROS) production
and mitochondrial membrane potential (DJm) depolarization, opening the
mitochondrial permeability transition pore (mPTP) and promoting Ca waves.
ROS levels were measured by DCF fluorescence. Perfusion with Fe2þ (1 – 10
mM) significantly increased relative ROS levels. For example, 1 mM Fe2þ
raised ROS levels 465 5 14% compared to the control (p <0.01). TMRM
fluorescence decrease was used as an index of DJm depolarization. Fe
2þ
significantly decreased TMRM fluorescence (1 mM: 8.1 5 2.3%; 10 mM:
94.85 2.9 %), compared to the baseline in myocytes, indicating DJm depo-
larization by Fe2þ. Intracellular Ca2þ was imaged with Fluo-4-AM. Sponta-
neous Ca waves were induced in high external Ca2þ (4 mM). Furthermore,
Fe2þ significantly increased the rate of Ca waves: 1 mM Fe2þ significantly
increased the rate from 23.95 1.7 to 39.85 7.6 min1 (p < 0.05). Fe2þ-pro-
moted Ca waves were significantly reduced by the mPTP inhibitor cyclo-
sporine A (CsA; 1 mM). ECGs were recorded from Langedorff-perfused
hearts. Arrhythmia induction testing was conducted through programmed
S1-S2 stimulation. Hearts treated with Fe
2þ (1 mM) alone significantly
increased arrhythmia scores compared to hearts pretreated with CsA prior
to Fe2þ (p < 0.05), indicating mPTP inhibition can ameliorate the proarrhyth-
mic effects of iron-overload. These observation were confirmed in the cyclo-
philin D knockout (CypD KO) mice, in which mPTP opening is impaired. In
conclusion, our results suggest that exogenous Fe2þ treatment increases ROS
production, depolarizes the DJm, opens the mPTP, and promotes Ca waves
and arrhythmogenesis.


Mass Spectrometry-Based Analysis of the Phospho-Proteome for Cardiac Dyssynchrony and Resynchronization Therapy 

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Mass Spectrometry-Based Analysis of the Phospho-Proteome for Cardiac Dyssynchrony and Resynchronization Therapy

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