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1.  Pro-arrhythmic Effect of Blocking the Small Conductance Calcium Activated Potassium Channel in Isolated Canine Left Atrium 
Background
Small conductance calcium activated potassium channels (SKCa) are voltage insensitive and are activated by intracellular calcium. Genome wide association studies revealed that a variant of SKca is associated with lone atrial fibrillation (AF) in humans. Roles of SKca in atrial arrhythmias remain unclear.
Objective
The purpose of this study was to determine roles of SKCa in atrial arrhythmias.
Methods
Optical mapping using isolated canine left atrium was performed. The optical action potential duration (APD) and induction of arrhythmia were evaluated before and after the addition of specific SKCa blockers, Apamin or UCL-1684.
Results
SKCa blockade significantly increased APD80 (188±19 ms vs 147±11ms, p< 0.001). The pacing cycle length (PCL) thresholds to induce 2:2 alternans and wave breaks were prolonged by SKCa blockade. Increased APD heterogeneity was observed following SKCa blockade, as measured by the difference between maximum and minimum APD (39±4ms vs 26±5ms, p<0.05), by standard deviation (12.43±2.36ms vs 7.49±1.47ms, p<0.001), or by coefficient of variation (6.68±0.97% vs 4.90±0.84%, p<0.05). No arrhythmia was induced at baseline by S1–S2 protocol. After SKCa blockade, 4 out of 6 atria developed arrhythmia.
Conclusion
Blockade of SKCa promotes arrhythmia and prolongs the PCL threshold of 2:2 alternans and wave breaks in the canine left atrium. The proarrhythmic effect could be attributed to the increased APD heterogeneity in the canine left atrium. This study provides supportive evidence of GWAS studies showing association of KCNN3 and lone AF
doi:10.1016/j.hrthm.2013.01.033
PMCID: PMC3663880  PMID: 23376397
Atrial arrhythmia; SKCa; action potential duration; repolarization; optical mapping
2.  Intracellular Calcium Dynamics, Shortened Action Potential Duration and Late-phase 3 Early Afterdepolarization in Langendorff-Perfused Rabbit Ventricles 
Introduction
To elucidate the mechanism of late-phase 3 early after depolarization (EAD) in ventricular arrhythmogenesis, we hypothesized that intracellular calcium (Cai) overloading and action potential duration (APD) shortening may promote late phase 3 EAD and triggered activity, leading to development of ventricular fibrillation (VF).
Methods and Results
In isolated rabbit hearts, we performed microelectrode recording and simultaneous dual optical mapping of transmembrane potential (Vm) and Cai transient on left ventricular endocardium. An IKATP channel opener, pinacidil, was used to abbreviate action potential duration (APD). Rapid-pacing was then performed. Upon abrupt cessation of rapid pacing with cycle lengths of 60–200 ms, there were APD90 prolongation and the corresponding Cai overloading in the first post-pacing beats. The duration of Cai transient recovered to 50% (DCaT50) and 90% (DCaT90) in the first post-pacing beats was significantly longer than baseline. Abnormal Cai elevation coupled with shortened APD produced late-phase 3 EAD induced triggered activity and VF. In additional 6 preparations, the heart tissues were treated with BAPTA-AM, a calcium chelator. BAPTA-AM significantly reduced the maximal Cai amplitude (26.4±3.5% of the control; p<0.001) and the duration of Cai transients in the mapped region, preventing the development of EAD and triggered activity that initiated VF.
Conclusions
IKATP channel activation along with Cai overloading are associated with the development of late phase 3 EAD and VF. Because acute myocardial ischemia activates the IKATP channel, late phase 3 EADs may be a mechanism for VF initiation during acute myocardial ischemia.
doi:10.1111/j.1540-8167.2012.02400.x
PMCID: PMC3479328  PMID: 22809087
ventricular fibrillation; early afterdepolarization; triggered activity; calcium; APD shortening
3.  Heart Failure Decreases Nerve Activity in the Right Atrial Ganglionated Plexus 
Objective
We tested the hypothesis that heart failure (HF) results in right atrial ganglionated plexus (RAGP) denervation which contributes to sinoatrial node dysfunction.
Background
HF is associated with sinoatrial node dysfunction. However, the detailed mechanisms remain unclear.
Methods
We recorded nerve activity (NA) from the RAGP, right stellate ganglion (SG) and right vagal nerve in 7 ambulatory dogs at baseline and after pacing-induced HF. We also determined the effects of RAGP stimulation in isolated normal and HF canine right atria (RA).
Results
Nerve activities in both the SG and vagal were significantly higher in HF than at baseline. The relationship between 1-min integrated nerve activities of vagal and RAGP showed either a positive linear correlation (Group 1, n=4) or an L-shaped correlation (Group 2, n=3). In all dogs, a reduced heart rate was observed when vagal-NA was associated with simultaneously increased RAGP-NA. On the other hand, when vagal-NA was not associated with increased RAGP-NA, the heart rate was not reduced. The induction of HF significantly decreased RAGP-NA in all dogs (P<0.05). Stimulating the superior RAGP in isolated right atrium significantly reduced the sinus rate in normal but not the HF hearts. Immunohistochemical staining revealed lower densities of tyrosine hydroxylase and choline acetyltransferase -positive nerve tissues in HF RAGP than normal (P<0.001 and P=0.001, respectively).
Conclusions
The RAGP nerve activity is essential for the vagal nerve to counterbalance the stellate ganglion in sinus rate control. In heart failure, RAGP denervation and decreased RAGP nerve activity contribute to the sinus node dysfunction.
doi:10.1111/j.1540-8167.2011.02204.x
PMCID: PMC3331905  PMID: 22035201
heart failure; nervous system; autonomic; sinoatrial node
4.  The Role of the Calcium and the Voltage Clocks in Sinoatrial Node Dysfunction 
Yonsei Medical Journal  2011;52(2):211-219.
Recent evidence indicates that the voltage clock (cyclic activation and deactivation of membrane ion channels) and Ca2+ clocks (rhythmic spontaneous sarcoplasmic reticulum Ca2+ release) jointly regulate sinoatrial node (SAN) automaticity. However, the relative importance of the voltage clock and Ca2+ clock for pacemaking was not revealed in sick sinus syndrome. Previously, we mapped the intracellular calcium (Cai) and membrane potentials of the normal intact SAN simultaneously using optical mapping in Langendorff-perfused canine right atrium. We demonstrated that the sinus rate increased and the leading pacemaker shifted to the superior SAN with robust late diastolic Cai elevation (LDCAE) during β-adrenergic stimulation. We also showed that the LDCAE was caused by spontaneous diastolic sarcoplasmic reticulum (SR) Ca2+ release and was closely related to heart rate changes. In contrast, in pacing induced canine atrial fibrillation and SAN dysfunction models, Ca2+ clock of SAN was unresponsiveness to β-adrenergic stimulation and caffeine. Ryanodine receptor 2 (RyR2) in SAN was down-regulated. Using the prolonged low dose isoproterenol together with funny current block, we produced a tachybradycardia model. In this model, chronically elevated sympathetic tone results in abnormal pacemaking hierarchy in the right atrium, including suppression of the superior SAN and enhanced pacemaking from ectopic sites. Finally, if the LDCAE was too small to trigger an action potential, then it induced only delayed afterdepolarization (DAD)-like diastolic depolarization (DD). The failure of DAD-like DD to consistently trigger a sinus beat is a novel mechanism of atrial arrhythmogenesis. We conclude that dysfunction of both the Ca2+ clock and the voltage clock are important in sick sinus syndrome.
doi:10.3349/ymj.2011.52.2.211
PMCID: PMC3051220  PMID: 21319337
Calcium; sinoatrial node; sarcoplasmic reticulum; sick sinus syndrome
5.  Mapping Cardiac Pacemaker Circuits: Methodological Puzzles of the Sino-Atrial Node Optical Mapping 
Circulation research  2010;106(2):255.
Historically, milestones in science are usually associated with methodological breakthroughs. Likewise, the advent of electrocardiography, microelectrode recordings and more recently optical mapping have ushered in new periods of significance of advancement in elucidating basic mechanisms in cardiac electrophysiology. As with any novel technique, however, data interpretation is challenging and should be approached with caution, as it cannot be simply extrapolated from previously used methodologies and with experience and time eventually becomes validated. A good example of this is the use of optical mapping in the sinoatrial node (SAN): when microelectrode and optical recordings are obtained from the same site in myocardium, significantly different results may be noted with respect to signal morphology and as a result have to be interpreted by a different set of principles. Given the rapid spread of the use of optical mapping, careful evaluation must be made in terms of methodology with respect to interpretation of data gathered by optical sensors from fluorescent potential-sensitive dyes. Different interpretations of experimental data may lead to different mechanistic conclusions. This review attempts to address the origin and interpretation of the “double component” morphology in the optical action potentials obtained from the SAN region. One view is that these two components represent distinctive signals from the sinoatrial node and atrial cells, and can be fully separated with signal processing. A second view is that the first component preceding the phase 0 activation represents the membrane currents and intracellular calcium transients induced diastolic depolarization from the SAN. While the consensus from both groups is that ionic mechanisms, namely the joint action of the membrane and calcium automaticity, are important in the SAN function, it is unresolved whether the double-component originates from the recording methodology or represents the underlying physiology. This overview aims to advance a common understanding of the basic principles of optical mapping in complex three-dimensional anatomical structures.
doi:10.1161/CIRCRESAHA.109.209841
PMCID: PMC2818830  PMID: 20133911
sinoatrial node; optical mapping; calcium; sinoatrial exit block
6.  Induction of Atrial Ectopic Beats with Calcium Release Inhibition: Local Hierarchy of Automaticity in the Right Atrium 
Background
Recent evidence indicates that spontaneous sarcoplasmic reticulum (SR) Ca release underlies the mechanism of sinoatrial node (SAN) acceleration during β-stimulation, indicating the importance of Ca clock in SAN automaticity. Whether or not the same mechanism applies to atrial ectopic pacemakers (AEP) remains unclear.
Objective
The purpose of this study was to assess the mechanism of AEP.
Methods
We simultaneously mapped intracellular calcium (Cai) and membrane potential in 12 isolated canine right atria. The late diastolic Cai elevation (LDCAE) was used to detect the Ca clock activity. Pharmacological interventions with isoproterenol (ISO), ryanodine, and ZD7288, a blocker of the If membrane current, were performed.
Results
Ryanodine, which inhibits SR Ca release, reduced LDCAE in SAN, resulting in an inferior shift of the pacemaking site. Cycle length increased significantly in a dose-dependent way. In the presence of 3-10 μmol/L of ryanodine, ISO infusion consistently induces AEPs from the lower crista terminalis. All ectopic beats continuing over 30 seconds were located at the lower crista terminalis. These AEPs were resistant to ryanodine treatment even at high doses. Subsequent blockade of If inhibited the AEP and resulted in profound bradycardia.
Conclusion
Spontaneous SR Ca release underlies ISO-induced increase of superior SAN activity. As compared with SAN, the AEP is less dependent on the Ca clock and more dependent on the membrane clock for its automaticity. AEPs outside the SAN can effectively serve as backup pacemakers when the Ca clock functionality is reduced.
doi:10.1016/j.hrthm.2009.09.068
PMCID: PMC2818257  PMID: 20129292
calcium; sinoatrial node; sarcoplasmic reticulum; If current; ectopic beat; Ca clock
7.  Synergistic Dual Automaticity in Sinoatrial Node Cell and Tissue Models 
Background
The mechanism of sinoatrial node (SAN) automaticity is traditionally attributed to membrane ion currents. Recent evidence indicates spontaneous sarcoplasmic reticulum (SR) Ca2+ cycling also plays an important role.
Methods and Results
We performed computer simulation on SAN cell and 1D tissue model. In the SAN cells, SR Ca2+ cycling broadly modulated sinus rate from 1.74Hz to 3.87Hz. Shortening of the junctional SR refilling time and increase of SR Ca2+ release were responsible for sinus rate acceleration. However, under the fast SR Ca2+ cycling, decreased L-type Ca2+ current (ICaL) resulted in irregular firing. When Ca2+ cycling was suppressed, If and ICaT both acted to stabilize the pacemaker rhythm, but ICaT had less effect than If. At the 1D level, the electrical coupling between neighboring cells had little effect on the earliest pacemaker location. The leading pacemaking site always colocalized with the site with the highest SR Ca2+ cycling rate, but shifted to the site with less inhibited ICaL.
Conclusions
The rate of SR Ca2+ cycling can effectively and broadly modulate the sinus rate. If, ICaL and ICaT play integral roles to guarantee SAN cell rhythmic firing. The leading pacemaker site is determined by intracellular Ca2+ dynamics and membrane currents, indicating the synergistic dual automaticity not only exists in single SAN cells, but also at the tissue level.
PMCID: PMC2952528  PMID: 20679733
8.  Intracellular Calcium Dynamics and the Acceleration of Sinus Rhythm by β-Adrenergic Stimulation 
Circulation  2009;119(6):788-796.
Background
Recent evidence indicates that membrane voltage and Ca2+ clocks jointly regulate sinoatrial node (SAN) automaticity. Here we test the hypothesis that sinus rate acceleration by β-adrenergic stimulation involves synergistic interactions between these clock mechanisms.
Methods and Results
We simultaneously mapped intracellular calcium (Cai) and membrane potential (Vm) in 25 isolated canine right atrium (RA), using previously described criteria of the timing of late diastolic Cai elevation (LDCAE) relative to the action potential (AP) upstroke to detect the Ca2+ clock. Before isoproterenol, the earliest pacemaking site occurred in the inferior SAN, and LDCAE was observed in only 4/25 preparations. Isoproterenol (1 μmol/L) increased sinus rate and shifted pacemaking site to superior SAN, concomitant with the appearance of LDCAE preceding the AP upstroke by 98 ± 31 ms. Caffeine had similar effects, while SR Ca2+ depletion with ryanodine and thapsigargin prevented isoproterenol-induced LDCAE and blunted sinus rate acceleration. Cai transient relaxation time during ISO was shorter in superior SAN (124 ± 34 ms) than inferior SAN (138 ± 24 ms, p = 0.01) or RA (164 ± 33 ms, p = 0.001), and was associated with a lower SR Ca2+ ATPase pump to phospholamban protein ratio in SAN than in RA. If current blockade with ZD 7288 modestly blunted, but did not prevent LDCAE or sinus rate acceleration by isoproterenol.
Conclusions
Acceleration of the Ca2+ clock in the superior SAN plays an important role in sinus acceleration during β-adrenergic stimulation, interacting synergistically with the voltage clock to increase sinus rate.
doi:10.1161/CIRCULATIONAHA.108.817379
PMCID: PMC2735196  PMID: 19188501
calcium; sinoatrial node; sarcoplasmic reticulum; nervous system, sympathetic
9.  Myocardial Repolarization Dispersion and Autonomic Nerve Activity in a Canine Experimental Acute Myocardial Infarction Model 
Background
Evidence from a canine experimental acute myocardial infarction (MI) model shows that until the seventh week after MI the relationship between stellate ganglionic nerve and vagal nerve activities (SGNA/VNA) progressively increases.
Objective
We evaluated how autonomic nervous system activity influences temporal myocardial repolarization dispersion at this period.
Methods
We analyzed autonomic nerve activity as well as QT and RR variability from recordings previously obtained in 9 dogs. From a total 48 short-term electrocardiographic segments, 24 recorded before and 24 seven weeks after experimentally-induced MI, we obtained three indices of temporal myocardial repolarization dispersion: QTe (from q wave T to wave end), QTp (from q wave to T wave peak) and Te (from T wave peak to T wave end) variability index (QTeVI, QTpVI, TeVI). We also performed a heart rate variability power spectral analysis on the same segments.
Results
After MI, all the QT variables increased QTeVI (median [interquartile range]) (from - 1.76[0.82] to −1.32[0.68]), QTeVI (from −1.90[1.01] to −1.45[0.78]) and TeVI (from −0.72[0.67] to −0.22[1.00]), whereas all RR spectral indexes decreased (p<0.001 for all). Distinct circadian rhythms in QTeVI (p<0.05,) QTpVI (p<0.001) and TeVI (p<0.05) appeared after MI with circadian variations resembling that of SGNA/VNA. The morning QTpVI and TeVI acrophases approached the SGNA/VNA acrophase. Conversely, the evening QTeVI acrophase coincided with another SGNA/VNA peak. After MI, regression analysis detected a positive relationship between SGNA/VNA and TeVI (R2: 0.077; β: 0.278; p< 0.001).
Conclusion
Temporal myocardial repolarization dispersion shows a circadian variation after MI reaching its peak at a time when sympathetic is highest and vagal activity lowest.
doi:10.1016/j.hrthm.2013.10.022
PMCID: PMC4078249  PMID: 24120873
10.  Apamin-Sensitive Calcium-Activated Potassium Currents in Rabbit Ventricles with Chronic Myocardial Infarction 
Introduction
Apamin-sensitive small-conductance calcium-activated potassium current (IKAS) is increased in heart failure. It is unknown if myocardial infarction (MI) is also associated with an increase of IKAS.
Methods and Results
We performed Langendorff perfusion and optical mapping in 6 normal hearts and 10 hearts with chronic (5 weeks) MI. An additional 6 normal and 10 MI hearts were used for patch clamp studies. The infarct size was 25% [95% confidence interval, 20 to 31] and the left ventricular ejection fraction was 0.5 [0.46 to 0.54]. The rabbits did not have symptoms of heart failure. The action potential duration measured to 80% repolarization (APD80) in the peri-infarct zone (PZ) was150 [142 to 159] ms, significantly (p=0.01) shorter than in the normal ventricles (158 to 177] ms). The intracellular Ca transient duration was also shorter in the PZ (148 [139 to 157] ms) than in normal ventricles (168 [157 to 180] ms; P=0.017). Apamin prolonged the APD80 in PZ by 9.8 [5.5 to 14.1] %, which is greater than in normal ventricles (2.8 [1.3 to 4.3] %, p=0.006). Significant shortening of APD80 was observed at the cessation of rapid pacing in MI but not in normal ventricles. Apamin prevented postpacing APD80 shortening. Patch clamp studies showed that IKAS was significantly higher in the PZ cells (2.51 [1.55 to 3.47] pA/pF, N=17) than in the normal cells (1.08 [0.36 to 1.80] pA/pF, N=15, p=0.019).
Conclusion
We conclude that IKAS is increased in MI ventricles and contributes significantly to ventricular repolarization especially during tachycardia.
doi:10.1111/jce.12176
PMCID: PMC4207647  PMID: 23718850
action potentials; intracellular calcium; ion channels; repolarization reserve; potassium currents; myocardial infarction
11.  Apamin Induces Early Afterdepolarizations and Torsades de Pointes Ventricular Arrhythmia From Failing Rabbit Ventricles Exhibiting Secondary Rises in Intracellular Calcium 
Heart rhythm : the official journal of the Heart Rhythm Society  2013;10(10):10.1016/j.hrthm.2013.07.003.
Background
A secondary rise of intracellular Ca2+ (Cai) and an upregulation of IKAS are characteristic findings of failing ventricular myocytes. We hypothesize that apamin, a specific IKAS blocker, may induce torsades de pointes (TdP) ventricular arrhythmia from failing ventricles exhibiting secondary rises of Cai.
Objectives
To test the hypothesis that small conductance Ca2+ activated apamin sensitive K+ current (IKAS) maintains repolarization reserve and prevents ventricular arrhythmia in a rabbit model of heart failure (HF).
Methods
We performed Langendorff perfusion and optical mapping studies in 7 hearts with pacing-induced HF and in 5 normal control rabbit hearts. Atrioventricular (AV) block was created by cryoablation to allow pacing at slow rates.
Results
The left ventricular ejection fraction reduced from 69.1 [95% confidence interval 62.3–76.0]% pre-pacing to 30.4 [26.8–34.0]% (N=7, p<0.001) post-pacing. The QTc in failing ventricles was 337 [313–360] ms at baseline and 410 [381–439] ms after applying 100 nmol/L of apamin (p=0.01). Apamin induced early afterdepolarizations (EADs) in 6 ventricles, premature ventricular beats (PVBs) in 7 ventricles and polymorphic ventricular tachycardia consistent with TdP in 4 ventricles. The earliest activation site of the EADs and PVBs always occurred at the site with long APD and large amplitude of the secondary rises of Cai. Apamin induced secondary rises of Cai in 1 non-failing ventricles, but no EAD or TdP were observed.
Conclusion
In HF ventricles, apamin induces EADs, PVBs and TdP from areas with secondary rises of Cai. IKAS is important in maintaining repolarization reserve and preventing TdP in HF ventricles.
doi:10.1016/j.hrthm.2013.07.003
PMCID: PMC3832504  PMID: 23835258
Action potential duration; apamin; optical mapping; potassium channels; torsades de pointes
12.  Chronic Amiodarone Therapy Impairs the Function of the Superior Sinoatrial Node in Patients with Atrial Fibrillation 
Background
The mechanisms underlying amiodarone-induced sinoatrial node (SAN) dysfunction remain unclear, so we used 3-dimensional endocardial mapping of the right atrium (RA) to investigate.
Methods and Results
In a matched-cohort design, 18 patients taking amiodarone before atrial fibrillation (AF) ablation (amiodarone group) were matched for age, sex and type of AF with 18 patients who had undergone AF ablation without taking amiodarone (no-amiodarone group). The amiodarone group had a slower heart rate than the no-amiodarone group at baseline and during isoproterenol infusion. Only the amiodarone group had sick sinus syndrome (n=4, 22%, P=0.03) and abnormal (>550 ms) corrected SAN recovery time (n=5, 29%; P=0.02). The median distance from the junction of the superior vena cava (SVC) and RA to the most cranial earliest activation site (EAS) was longer in the amiodarone group than in the no-amiodarone group at baseline (20.5 vs. 10.6 mm, P=0.04) and during isoproterenol infusion (12.8 vs. 6.3 mm, P=0.03). The distance from the SVC-RA junction to the EAS negatively correlated with the P-wave amplitudes of leads II (r=−0.47), III (r=−0.60) and aVF (r=−0.56) (P<0.001 for all).
Conclusions
In a quarter of the AF patients, amiodarone causes superior SAN dysfunction, which results in a downward shift of the EAS and reduced P-wave amplitude in leads II, III and aVF at baseline and during isoproterenol infusion.
PMCID: PMC4137393  PMID: 23739532
Amiodarone; Atrial fibrillation; Sick sinus syndrome; Sympathetic nervous system
13.  Low-Level Vagus Nerve Stimulation Upregulates Small Conductance Calcium Activated Potassium Channels in the Stellate Ganglion 
Background
Small conductance calcium activated potassium (SK) channels are responsible for afterhyperpolarization that suppresses nerve discharges.
Objectives
To test the hypotheses that low-level vagus nerve stimulation (LL-VNS) leads to the upregulation of SK2 proteins in the LSG.
Methods
Six dogs (Group 1) underwent 1-wk LL-VNS of the left cervical vagus nerve. Five normal dogs (Group 2) were used as control. SK2 protein levels were examined by western blotting. The ratio between SK2 and glyceraldehydes-3-phosphate-dehydrogenase (GAPDH) levels was used as an arbitrary unit (AU).
Results
We found higher SK2 expression in Group 1 (0.124 ± 0.049 AU) than Group 2 (0.085 ± 0.031 AU, P < 0.05). Immunostaining showed that the density of nerve structures stained with SK2 antibody was also higher in Group 1 (11,546 ± 7,271 μm2/mm2) than in Group 2 (5,321 ± 3,164 μm2/mm2, P < 0.05). There were significantly more ganglion cells without immunoreactivity to TH in Group 1 (11.4 ± 2.3%) than Group 2 (4.9 ± 0.7%; P < 0.05). The TH-negative ganglion cells mostly stained positive for choline acetyltransferase (ChAT) (95.9 ± 2.8% in Group 1 and 86.1 ± 4.4% in Group 2, P = 0.10). Immunofluorescence confocal microscopy revealed a significant decrease in the SK2 staining in the cytosol but an increase in the SK2 staining on the membrane of the ganglion cells in Group 1 compared to Group 2.
Conclusion
Left LL-VNS results in the upregulation of SK2 proteins, increased SK2 protein expression in the cell membrane and the increased TH-negative (mostly ChAT-positive) ganglion cells in the LSG. These changes may underlie the antiarrhythmic efficacy of LL-VNS in ambulatory dogs.
doi:10.1016/j.hrthm.2013.01.029
PMCID: PMC3671581  PMID: 23357541
Autonomic nervous system; Vagus nerve stimulation; Stellate ganglion; Small conductance calcium activated potassium channel; Western blot
14.  Electroanatomic Remodeling of the Left Stellate Ganglion After Myocardial Infarction 
Objectives
The purpose of this study was to evaluate the changes of left stellate ganglionic nerve activity (SGNA) and left thoracic vagal nerve activity (VNA) after acute myocardial infarction (MI).
Background
Whether MI results in remodeling of extracardiac nerve activity remains unclear.
Methods
We implanted radiotransmitters to record the SGNA, VNA, and electrocardiogram in 9 ambulatory dogs. After baseline monitoring, MI was created by 1-h balloon occlusion of the coronary arteries. The dogs were then continuously monitored for 2 months. Both stellate ganglia were stained for growth-associated protein 43 and synaptophysin. The stellate ganglia from 5 normal dogs were used as control.
Results
MI increased 24-h integrated SGNA from 7.44 ± 7.19 Ln(Vs)/day at baseline to 8.09 ± 7.75 Ln(Vs)/day after the MI (p < 0.05). The 24-h integrated VNA before and after the MI was 5.29 ± 5.04 Ln(Vs)/day and 5.58 ± 5.15 Ln(Vs)/day, respectively (p < 0.05). A significant 24-h circadian variation was noted for the SGNA (p < 0.05) but not the VNA. The SGNA/VNA ratio also showed significant circadian variation. The nerve densities from the left SG were 63,218 ± 34,719 μm2/mm2 and 20,623 ± 4,926 μm2/mm2 for growth-associated protein 43 (p < 0.05) and were 32,116 ± 8,190 μm2/mm2 and 16,326 ± 4,679 μm2/mm2 for synaptophysin (p < 0.05) in MI and control groups, respectively. The right SG also showed increased nerve density after MI (p < 0.05).
Conclusions
MI results in persistent increase in the synaptic density of bilateral stellate ganglia and is associated with increased SGNA and VNA. There is a circadian variation of the SGNA/VNA ratio. These data indicate significant remodeling of the extracardiac autonomic nerve activity and structures after MI.
doi:10.1016/j.jacc.2011.11.030
PMCID: PMC3975658  PMID: 22381432
acute myocardial infarction; autonomic nervous system; nerve recordings
15.  Apamin Sensitive Potassium Current Modulates Action Potential Duration Restitution and Arrhythmogenesis of Failing Rabbit Ventricles 
Background
Apamin-sensitive K currents (IKAS) are upregulated in heart failure (HF). We hypothesize that apamin can flatten action potential duration restitution (APDR) curve and reduce ventricular fibrillation (VF) duration in failing ventricles.
Methods and Results
We simultaneously mapped membrane potential and intracellular Ca (Cai) in 7 rabbits hearts with pacing-induced HF and in 7 normal hearts. A dynamic pacing protocol was used to determine APDR at baseline and after apamin (100 nM) infusion. Apamin did not change APD80 in normal ventricles, but prolonged APD80 in failing ventricles at either long (≥300 ms) or short (≤170 ms) pacing cycle length (PCL), but not at intermediate PCL. The maximal slope of APDR curve was 2.03 [95% CI, 1.73 to 2.32] in failing ventricles and 1.26 [95% CI, 1.13 to 1.40] in normal ventricles at baseline (p=0.002). After apamin administration, the maximal slope of APDR in failing ventricles decreased to 1.43 [95% CI, 1.01 to 1.84] (p=0.018) whereas no significant changes were observed in normal ventricles. During VF in failing ventricles, the number of phase singularities (baseline vs apamin, 4.0 vs 2.5), dominant frequency (13.0 Hz vs 10.0 Hz), and VF duration (160 s vs 80 s) were all significantly (p<0.05) decreased by apamin.
Conclusions
Apamin prolongs APD at long and short, but not at intermediate PCL in failing ventricles. IKAS upregulation may be antiarrhythmic by preserving the repolarization reserve at slow heart rate, but is proarrhythmic by steepening the slope of APDR curve which promotes the generation and maintenance of VF.
doi:10.1161/CIRCEP.111.000152
PMCID: PMC3678988  PMID: 23420832
ventricular fibrillation; optical mapping; experimental models heart failure; electrophysiology
16.  Carvedilol Analogue Inhibits Triggered Activities Evoked by Both Early and Delayed Afterdepolarizations 
BACKGROUND
Carvedilol and its analogues suppress delayed afterdepolarizations (DADs) and catecholaminergic polymorphic ventricular tachycardias by direct action on the cardiac ryanodine receptor (RyR2).
OBJECTIVE
We tested a hypothesis that carvedilol analogue may also prevent triggered activities (TAs) through the suppression of early afterdepolarizations (EADs).
METHODS
Intracellular Ca2+ and membrane voltage were simultaneously recorded using optical mapping technique in Langendorff-perfused mouse and rabbit hearts to study the effect of carvedilol analogue, VK-II-36 that does not have significant beta-blocking effects.
RESULTS
Spontaneous intracellular Ca2+ elevations (SCaEs) during diastole was induced by rapid ventricular pacing and isoproterenol infusion in intact rabbit ventricles. Systolic and diastolic SCaEs were simultaneously noted in Langendorff-perfused RyR2 R4496+/− mouse hearts after creating atrioventricular block. VK-II-36 effectively suppressed SCaEs and eliminated TAs observed in both mouse and rabbit ventricles. We tested the effect of VK-II-36 on EADs using a rabbit model of acquired long QT syndrome in which phase-2 and phase-3 EADs were observed in association with systolic SCaEs. VK-II-36 abolished the systolic SCaEs and phase-2 EADs, and greatly decreased the dispersion of repolarization and the amplitude of phase-3 EADs. VK-II-36 completely prevented EAD-mediated TAs in all ventricles studied.
CONCLUSION
A carvedilol analogue, VK-II-36 inhibits ventricular tachyarrhythmias in intact mouse and rabbit ventricles by suppression of SCaEs, independent of beta-blocking activity. The RyR2 may be a potential target for treating focal ventricular arrhythmias triggered by either EADs or DADs.
doi:10.1016/j.hrthm.2012.09.006
PMCID: PMC3534812  PMID: 22982970
afterdepolarization; intracellular calcium; long-QT syndrome
17.  Imaging Arrhythmogenic Calcium Signaling in Intact Hearts 
Pediatric cardiology  2012;33(6):968-974.
Protein complex of the cardiac junctional sarcoplasmic reticulum (SR) membrane formed by type 2 ryanodine receptor, junction, triadin, and calsequestrin is responsible for controlling SR calcium (Ca) release. Increased intracellular calcium (Cai) activates the electrogenic sodium–Ca exchanger current, which is known to be important in afterdepolarization and triggered activities (TAs). Using optical-mapping techniques, it is possible to simultaneously map membrane potential (Vm) and Cai transient in Langendorff-perfused rabbit ventricles to better define the mechanisms by which Vm and Cai interactions cause early afterdepolarizations (EADs). Phase 3 EAD is dependent on heterogeneously prolonged action potential duration (APD). Electrotonic currents that flow between a persistently depolarized region and its recovered neighbors underlies the mechanisms of phase 3 EADs and TAs. In contrast, “late phase-3 EAD” is induced by APD shortening, not APD prolongation. In failing ventricles, upregulation of apamin-sensitive Ca-activated potassium (K) channels (IKAS) causes APD shortening after fibrillation-defibrillation episodes. Shortened APD in the presence of large Cai transients generates late-phase 3 EADs and recurrent spontaneous ventricular fibrillation. The latter findings suggest that IKAS may be a novel antiarrhythmic targets in patients with heart failure and electrical storms.
doi:10.1007/s00246-012-0236-5
PMCID: PMC3586331  PMID: 22349681
Triggered activity; After depolarization; Ventricular fibrillation; Calcium dynamics; Optical mapping
18.  Amiodarone Inhibits Apamin-Sensitive Potassium Currents 
PLoS ONE  2013;8(7):e70450.
Background
Apamin sensitive potassium current (IKAS), carried by the type 2 small conductance Ca2+-activated potassium (SK2) channels, plays an important role in post-shock action potential duration (APD) shortening and recurrent spontaneous ventricular fibrillation (VF) in failing ventricles.
Objective
To test the hypothesis that amiodarone inhibits IKAS in human embryonic kidney 293 (HEK-293) cells.
Methods
We used the patch-clamp technique to study IKAS in HEK-293 cells transiently expressing human SK2 before and after amiodarone administration.
Results
Amiodarone inhibited IKAS in a dose-dependent manner (IC50, 2.67±0.25 µM with 1 µM intrapipette Ca2+). Maximal inhibition was observed with 50 µM amiodarone which inhibited 85.6±3.1% of IKAS induced with 1 µM intrapipette Ca2+ (n = 3). IKAS inhibition by amiodarone was not voltage-dependent, but was Ca2+-dependent: 30 µM amiodarone inhibited 81.5±1.9% of IKAS induced with 1 µM Ca2+ (n = 4), and 16.4±4.9% with 250 nM Ca2+ (n = 5). Desethylamiodarone, a major metabolite of amiodarone, also exerts voltage-independent but Ca2+ dependent inhibition of IKAS.
Conclusion
Both amiodarone and desethylamiodarone inhibit IKAS at therapeutic concentrations. The inhibition is independent of time and voltage, but is dependent on the intracellular Ca2+ concentration. SK2 current inhibition may in part underlie amiodarone's effects in preventing electrical storm in failing ventricles.
doi:10.1371/journal.pone.0070450
PMCID: PMC3726612  PMID: 23922993
19.  Ryanodine receptor inhibition potentiates the activity of Na channel blockers against spontaneous calcium elevations and delayed afterdepolarizations in Langendorff-perfused rabbit ventricles 
BACKGROUND
Na channel blockers are effective in suppressing delayed afterdepolarizations (DADs) in isolated Purkinje fibers. However, in isolated mouse ventricular myocytes lacking calsequestrin, only those Na channel blockers that also inhibit type 2 ryanodine receptor channels were effective against spontaneous Ca elevation (SCaE) and DADs.
OBJECTIVE
To test the hypothesis that combined Na channel and type 2 ryanodine receptor channel blocker ((R)-propafenone) is more effective than a Na channel blocker (lidocaine) in suppressing SCaE and DADs in the intact rabbit ventricles.
METHODS
We compared (R)-propafenone (3 μmol/L) with lidocaine (50 μmol/L) on SCaE and DADs by using epicardial optical mapping of intracellular calcium (Cai) and membrane voltage in Langendorff-perfused rabbit hearts. SCaE and DADs were induced by rapid pacing trains and isoproterenol (0.3 μmol/L) infusion. One arbitrary unit is equivalent to the Ca transient amplitude of paced beats.
RESULTS
SCaEs were observed at the cessation of rapid pacing in all hearts at baseline. (R)-Propafenone nearly completely inhibited DADs and SCaE (0.04 arbitrary units [95% confidence interval 0.02–0.06] vs 0.23 arbitrary units [95% confidence interval 0.18–0.28] at baseline; n = 6 hearts; P < .001). Lidocaine also significantly reduced the SCaE but was significantly (P < .05) less effective than (R)-propafenone. Both drugs increased the rise time of action potential upstroke and reduced conduction velocity to a similar extent, suggesting a significant inhibition of INa.
CONCLUSIONS
Both Na channel blockers significantly reduced tachycardia-induced SCaEs in the rabbit ventricles, but (R)-propafenone was significantly more effective than lidocaine. These data suggest that type 2 ryanodine receptor inhibition potentiates the activity of Na channel blockers against SCaE and DADs in the intact hearts.
doi:10.1016/j.hrthm.2012.02.031
PMCID: PMC3572724  PMID: 22387372
Depolarization; Action potentials; Calcium; Antiarrhythmic agents
20.  Neural Control of Ventricular Rate in Ambulatory Dogs with Pacing Induced Sustained Atrial Fibrillation 
Background
We hypothesize that inferior vena cava-inferior atrial ganglionated plexus nerve activity (IVC-IAGPNA) is responsible for the ventricular rate (VR) control during atrial fibrillation (AF) in ambulatory dogs.
Methods and Results
We recorded bilateral cervical vagal nerve activity (VNA) and IVC-IAGPNA during baseline sinus rhythm and during pacing-induced sustained AF in 6 ambulatory dogs. Integrated nerve activities and average VR were measured every 10-s over 24-hour periods. LVNA was associated with VR reduction during AF in 5 dogs (from 211 bpm, 95% confidence interval [CI], 186 to 233 to 178 bpm [95% CI, 145 to 210], p<0.001) and RVNA in 1 dog (208 bpm [95% CI, 197 to 223] to 181 bpm [95% CI, 163 to 200], p<0.01). There were good correlations between IVC-IAGPNA and LVNA in the former 5 dogs, and between IVC-IAGPNA and RVNA in the latter dog. IVC-IAGPNA was associated with VR reduction in all dogs studied. RVNA was associated with baseline sinus rate reduction from 105 bpm (95% CI, 95 to 116) to 77 bpm (95% CI, 64 to 91, p<0.01) in 4 dogs while LVNA was associated with sinus rate reduction from 111 bpm (95% CI, 90 to 1250) to 81 bpm (95% CI, 67 to 103, p<0.01) in 2 dogs.
Conclusions
IVC-IAGPNA is invariably associated with VR reduction during AF. In comparison, right or left VNA was associated with VR reduction only when it co-activates with the IVC-IAGPNA. The vagus nerve that controls VR during AF may be different than that controls sinus rhythm.
doi:10.1161/CIRCEP.111.967737
PMCID: PMC3405983  PMID: 22586260
atrial fibrillation; atrioventricular node; ECG; nervous system, autonomic; ventricular rate
21.  Acute Myocardial Infarction Induces Bilateral Stellate Ganglia Neural Remodeling in Rabbits 
Cardiovascular Pathology  2011;21(3):143-148.
Introduction
Myocardial infarction (MI) results in cardiac nerve sprouting in the myocardium. Whether or not similar neural remodeling occurs in the stellate ganglia (SG) is unknown. We aimed to test the hypothesis that MI induces bilateral SG nerve sprouting.
Methods
Acute MI was created by coronary artery ligation in rabbits (n=12). Serum nerve growth factor (NGF) level was measured by enzyme-linked immunosorbent assay (ELISA). The hearts and bilateral SGs were harvested for immunohistochemistry after 1 week in 6 rabbits, and after 1 month in 6 rabbits. Immunostaining for tyrosine hydroxylase (TH), growth-associated protein 43 (GAP43), cholineacetyltransferase (ChAT) and synaptophysin (SYN) was performed to determine the magnitude of nerve sprouting. Tissues from 6 normal rabbits were used as controls. Nerve density was determined by computerized morphometry.
Results
MI results in increased serum NGF levels at 1 week (1519.8±632.2 ng/ml) that persists to 1 month (1361.2±176.3 ng/ml) as compared to controls (89.6±34.9 ng/ml), (p=.0002, and , p=.0001, respectively). Immunostaining demonstrated nerve sprouting and hyperinnervation in both SGs after MI. The nerve densities (µm2/ganglion cell) in SG 1 week after MI, 1 month after MI and in control groups, respectively, were: GAP43, 278±96, 225±39 and 149±57 (p=.01); SYN, 244±152, 268±115 and 102±60 (p=.02); TH, 233±71, 180±50 and 135±68 (p=.047); ChAT, 244±100, 208±46 and 130±41 µm2/cell (p=.01).
Conclusions
MI increases serum NGF levels and induces nerve sprouting and hyperinnervation in bilateral SGs for at least 1 month after MI. The hyperinnervation includes both postganglionic adrenergic axons and preganglionic cholinergic axons in the SG.
doi:10.1016/j.carpath.2011.08.001
PMCID: PMC3267867  PMID: 22001051
Myocardial Infarction; Ventricular Arrhythmia; Autonomic Nervous System; Stellate Ganglion; Nerve Sprouting; Sudden Cardiac Death
22.  Alternans of Diastolic Intracellular Calcium Elevation as the Mechanism of Bidirectional Ventricular Tachycardia in a Rabbit Model of Andersen-Tawil Syndrome 
Heart Rhythm  2010;9(4):626-627.
doi:10.1016/j.hrthm.2010.12.021
PMCID: PMC3065532  PMID: 21167318
Bidirectional ventricular tachycardia; Andersen-Tawil Syndrome; Alternans
23.  Triggered Firing and Atrial Fibrillation in Transgenic Mice With Selective Atrial Fibrosis Induced by Overexpression of TGF-β1 
Background
Calcium transient triggered firing (CTTF) is induced by large intracellular calcium (Cai) transient and short action potential duration (APD). We hypothesized that CTTF underlies the mechanisms of early afterdepolarization (EAD) and spontaneous recurrent atrial fibrillation (AF) in transgenic (Tx) mice with overexpression of transforming growth factor β1 (TGF-β1).
Methods and Results
MHC-TGFcys33ser Tx mice develop atrial fibrosis because of elevated levels of TGF-β1. We studied membrane potential and Cai transients of isolated superfused atria from Tx and wild-type (Wt) littermates. Short APD and persistently elevated Cai transients promoted spontaneous repetitive EADs, triggered activity and spontaneous AF after cessation of burst pacing in Tx but not Wt atria (39% vs. 0%, P=0.008). We were able to map optically 4 episodes of spontaneous AF re-initiation. All first and second beats of spontaneous AF originated from the right atrium (4/4, 100%), which is more severely fibrotic than the left atrium. Ryanodine and thapsigargin inhibited spontaneous re-initiation of AF in all 7 Tx atria tested. Western blotting showed no significant changes of calsequestrin or sarco/endoplasmic reticulum Ca2+-ATPase 2a.
Conclusions
Spontaneous AF may occur in the Tx atrium because of CTTF, characterized by APD shortening, prolonged Cai transient, EAD and triggered activity. Inhibition of Ca2+ release from the sarcoplasmic reticulum suppressed spontaneous AF. Our results indicate that CTTF is an important arrhythmogenic mechanism in TGF-β1 Tx atria.
PMCID: PMC3593311  PMID: 22447020
Arrhythmia; Atrial fibrillation; Ca2+ triggers; Intracellular calcium; Optical mapping; Transgenic mice models
24.  Neural mechanisms of atrial fibrillation 
Current Opinion in Cardiology  2012;27(1):24-28.
Purpose of review
The autonomic nerve system is a potentially potent modulator of the initiation and perpetuation of atrial fibrillation (AF). This review will briefly summarize the neural mechanisms of AF.
Recent findings
Complex interactions exist between the sympathetic and parasympathetic nervous system on the atrial electrophysiologic properties. Direct autonomic recordings in canine models demonstrated simultaneous sympathovagal discharges are the most common triggers of paroxysmal atrial tachycardia and paroxysmal AF. Also, intrinsic cardiac autonomic nerve can serve as a sole triggering factor for the initiation of AF. Modulation of autonomic nervous system (ANS) by electrical stimulation has been tried as a treatment strategy clinically and experimentally. Recent studies showed that autonomic nervous system modulation can suppress the stellate ganglion nerve activity and reduce the incidence of paroxysmal atrial tachyarrhythmias in ambulatory dogs.
Summary
The autonomic nerve system influences the initiation and perpetuation of AF. Scientific advances toward a better understanding of the complex interrelationships of the various components of the ANS will hopefully lead to improvement of treatments for this common arrhythmia.
doi:10.1097/HCO.0b013e32834dc4e8
PMCID: PMC3279730  PMID: 22139702
Autonomic nerve; atrial fibrillation; vagal nerve stimulation
25.  Restrictive loss of plakoglobin in cardiomyocytes leads to arrhythmogenic cardiomyopathy 
Human Molecular Genetics  2011;20(23):4582-4596.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inheritable myocardial disorder associated with fibrofatty replacement of myocardium and ventricular arrhythmia. A subset of ARVC is categorized as Naxos disease, which is characterized by ARVC and a cutaneous disorder. A homozygous loss-of-function mutation of the Plakoglobin (Jup) gene, which encodes a major component of the desmosome and the adherens junction, had been identified in Naxos patients, although the underlying mechanism remained elusive. We generated Jup mutant mice by ablating Jup in cardiomyocytes. Jup mutant mice largely recapitulated the clinical manifestation of human ARVC: ventricular dilation and aneurysm, cardiac fibrosis, cardiac dysfunction and spontaneous ventricular arrhythmias. Ultra-structural analyses revealed that desmosomes were absent in Jup mutant myocardia, whereas adherens junctions and gap junctions were preserved. We found that ventricular arrhythmias were associated with progressive cardiomyopathy and fibrosis in Jup mutant hearts. Massive cell death contributed to the cardiomyocyte dropout in Jup mutant hearts. Despite the increase of β-catenin at adherens junctions in Jup mutant cardiomyoicytes, the Wnt/β-catenin-mediated signaling was not altered. Transforming growth factor-beta-mediated signaling was found significantly elevated in Jup mutant cardiomyocytes at the early stage of cardiomyopathy, suggesting an important pathogenic pathway for Jup-related ARVC. These findings have provided further insights for the pathogenesis of ARVC and potential therapeutic interventions.
doi:10.1093/hmg/ddr392
PMCID: PMC3209829  PMID: 21880664

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