TASK-1 current is inhibited by phosphorylation during human and canine chronic atrial fibrillation.

Atrial fibrillation (AF) is a common arrhythmia with significant morbidities and only partially adequate therapeutic options. AF is associated with atrial remodeling processes, including changes in the expression and function of ion channels and signaling pathways. TWIK protein-related acid-sensitive K+ channel (TASK)-1, a two-pore domain K+ channel, has been shown to contribute to action potential repolarization as well as to the maintenance of resting membrane potential in isolated myocytes, and TASK-1 inhibition has been associated with the induction of perioperative AF. However, the role of TASK-1 in chronic AF is unknown. The present study investigated the function, expression, and phosphorylation of TASK-1 in chronic AF in atrial tissue from chronically paced canines and in human subjects. TASK-1 current was present in atrial myocytes isolated from human and canine hearts in normal sinus rhythm but was absent in myocytes from humans with AF and in canines after the induction of AF by chronic tachypacing. The addition of phosphatase to the patch pipette rescued TASK-1 current from myocytes isolated from AF hearts, indicating that the change in current is phosphorylation dependent. Western blot analysis showed that total TASK-1 protein levels either did not change or increased slightly in AF, despite the absence of current. In studies of perioperative AF, we have shown that phosphorylation of TASK-1 at Thr383 inhibits the channel. However, phosphorylation at this site was unchanged in atrial tissue from humans with AF or in canines with chronic pacing-induced AF. We conclude that phosphorylation-dependent inhibition of TASK-1 is associated with AF, but the phosphorylation site responsible for this inhibition remains to be identified.

Ability to induce atrial fibrillation in the peri-operative period is associated with phosphorylation-dependent inhibition of TWIK protein-related acid-sensitive potassium channel 1 (TASK-1).

Peri-operative atrial fibrillation (peri-op AF) is a common complication following thoracic surgery. This arrhythmia is thought to be triggered by an inflammatory response and can be reproduced in various animal models. Previous work has shown that the lipid inflammatory mediator, platelet-activating factor (PAF), synthesized by activated neutrophils, can induce atrial and ventricular arrhythmias as well as repolarization abnormalities in isolated ventricular myocytes. We have previously shown that carbamylated PAF-induced repolarization abnormalities result from the protein kinase C (PKC) ε-dependent phosphorylation of the two-pore domain potassium channel TASK-1. We now demonstrate that canine peri-op AF is associated with the phosphorylation-dependent loss of TASK-1 current. Further studies identified threonine 383 in the C terminus of human and canine TASK-1 as the phosphorylation site required for PAF-dependent inhibition of the channel. Using a novel phosphorylation site-specific antibody targeting the phosphorylated channel, we have determined that peri-op AF is associated with the loss of TASK-1 current and increased phosphorylation of TASK-1 at this site.

Ca(2+)-stimulated adenylyl cyclase AC1 generates efficient biological pacing as single gene therapy and in combination with HCN2.

BACKGROUND: Biological pacing performed solely via HCN2 gene transfer in vivo results in relatively slow idioventricular rates and only moderate autonomic responsiveness. We induced biological pacing using the Ca(2+)-stimulated adenylyl cyclase AC1 gene expressed alone or in combination with HCN2 and compared outcomes with those with single-gene HCN2 transfer. METHODS AND RESULTS: We implanted adenoviral HCN2, AC1, or HCN2/AC1 constructs into the left bundle branches of atrioventricular-blocked dogs. During steady-state gene expression (days 5-7), differences between AC1, HCN2/AC1, and HCN2 alone were evident in basal beating rate, escape time, and dependence on electronic backup pacing. In HCN2, AC1, and HCN2/AC1, these parameters were as follows: basal beating rate: 50±1.5, 60±5.0, and 129±28.9 bpm (P<0.05 for HCN2/AC1 versus HCN2 or AC1 alone), respectively; escape time: 2.4±0.2, 1.3±0.2, and 1.1±.0.4 seconds (P<0.05 for AC1 and HCN2/AC1 versus HCN2); and percent electronic beats: 34±8%, 2±1%, and 6±2% (P<0.05 for AC1 and HCN2/AC1 versus HCN2). Instantaneous (SD1) and long-term (SD2) heart rate variability and circadian rhythm analyzed via 24-hour Holter recordings showed a shift toward greater sensitivity to parasympathetic modulation in animals injected with AC1 and a high degree of sympathetic modulation in animals injected with HCN2/AC1. CONCLUSION: AC1 or HCN2/AC1 overexpression in left bundle branches provides highly efficient biological pacing and greater sensitivity to autonomic modulation than HCN2 alone.

Epicardial border zone overexpression of skeletal muscle sodium channel SkM1 normalizes activation, preserves conduction, and suppresses ventricular arrhythmia: an in silico, in vivo, in vitro study.

BACKGROUND: In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel (SCN5A) is largely inactivated, contributing to low action potential upstroke velocity (V(max)), slow conduction, and reentry. We hypothesized that a fast inward current such as the skeletal muscle sodium channel (SkM1) operating more effectively at depolarized membrane potentials might restore fast conduction in epicardial border zones and be antiarrhythmic. METHODS AND RESULTS: Computer simulations were done with a modified Hund-Rudy model. Canine myocardial infarcts were created by coronary ligation. Adenovirus expressing SkM1 and green fluorescent protein or green fluorescent protein alone (sham) was injected into epicardial border zones. After 5 to 7 days, dogs were studied with epicardial mapping, programmed premature stimulation in vivo, and cellular electrophysiology in vitro. Infarct size was determined, and tissues were immunostained for SkM1 and green fluorescent protein. In the computational model, modest SkM1 expression preserved fast conduction at potentials as positive as -60 mV; overexpression of SCN5A did not. In vivo epicardial border zone electrograms were broad and fragmented in shams (31.5 +/- 2.3 ms) and narrower in SkM1 (22.6 +/- 2.8 ms; P=0.03). Premature stimulation induced ventricular tachyarrhythmia/fibrillation >60 seconds in 6 of 8 shams versus 2 of 12 SkM1 (P=0.02). Microelectrode studies of epicardial border zones from SkM1 showed membrane potentials equal to that of shams and V(max) greater than that of shams as membrane potential depolarized (P<0.01). Infarct sizes were similar (sham, 30 +/- 2.8%; SkM1, 30 +/- 2.6%; P=0.86). SkM1 expression in injected epicardium was confirmed immunohistochemically. CONCLUSIONS: SkM1 increases V(max) of depolarized myocardium and reduces the incidence of inducible sustained ventricular tachyarrhythmia/fibrillation in canine infarcts. Gene therapy to normalize activation by increasing V(max) at depolarized potentials may be a promising antiarrhythmic strategy.

Reactive oxygen species decrease cAMP response element binding protein expression in cardiomyocytes via a protein kinase D1-dependent mechanism that does not require Ser133 phosphorylation.

Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser(133) in cardiomyocytes. Although CREB-Ser(133) phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser(133) phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser(133) phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

GENE AND CELL THERAPY FOR LIFE-THREATENING CARDIAC ARRHYTHMIAS.

Gene and cell therapies of cardiac arrhythmias are nascent fields whose raison d'etre derives from (1) the problematic state of arrhythmia treatment today (especially atrial and ventricular tachyarrhythmias for which drugs, devices and ablation remain more stopgaps then optimal interventions), and (2) the opportunity to learn and potentially treat and cure by exploring new technologies. The state of antiarrhythmic therapy and new directions being taken are reviewed.

Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart.

BACKGROUND: Biological pacemaking has been performed with viral vectors, human embryonic stem cells, and adult human mesenchymal stem cells (hMSCs) as delivery systems. Only with human embryonic stem cells are data available regarding stability for >2 to 3 weeks, and here, immunosuppression has been used to facilitate survival of xenografts. The purpose of the present study was to determine whether hMSCs provide stable impulse initiation over 6 weeks without the use of immunosuppression, the "dose" of hMSCs that ensures function over this period, and the catecholamine responsiveness of hMSC-packaged pacemakers. METHODS AND RESULTS: A full-length mHCN2 cDNA subcloned in a pIRES2-EGFP vector was electroporated into hMSCs. Transfection efficiency was estimated by GFP expression. I(HCN2) was measured with patch clamp, and cells were administered into the left ventricular anterior wall of adult dogs in complete heart block and with backup electronic pacemakers. Studies encompassed 6 weeks. I(HCN2) for all cells was 32.1+/-1.3 pA/pF (mean+/-SE) at -150 mV. Pacemaker function in intact dogs required 10 to 12 days to fully stabilize and persisted consistently through day 42 in dogs receiving > or =700,000 hMSCs (approximately 40% of which carried current). Rhythms were catecholamine responsive. Tissues from animals killed at 42 days manifested neither apoptosis nor humoral or cellular rejection. CONCLUSIONS: hMSCs provide a means for administering catecholamine-responsive biological pacemakers that function stably for 6 weeks and manifest no cellular or humoral rejection at that time. Cell doses >700,000 are sufficient for pacemaking when administered to left ventricular myocardium.

HCN212-channel biological pacemakers manifesting ventricular tachyarrhythmias are responsive to treatment with I(f) blockade.

BACKGROUND: A potential concern about biological pacemakers is their possible malfunction, which might create ventricular tachycardias (VTs). OBJECTIVE: The purpose of this study was to test our hypothesis that should VTs complicate implantation of HCN-channel-based biological pacemakers, they would be suppressed by inhibitors of the pacemaker current, I(f). METHODS: We created a chimeric channel (HCN212) containing the N- and C-termini of mouse HCN2 and the transmembrane region of mouse HCN1 and implanted it in HEK293 cells. Forty-eight hours later, in whole-cell patch clamp recordings, mean steady state block induced by 3 microM ivabradine (IVB) showed HCN1 = HCN212 > HCN2 currents. The HCN212 adenoviral construct was then implanted into the canine left bundle branch in 11 dogs. Complete AV block was created via radiofrequency ablation, and a ventricular demand electronic pacemaker was implanted (VVI 45 bpm). Electrocardiogram, 24-hour Holter monitoring, and pacemaker log record check were performed for 11 days. RESULTS: All dogs developed rapid VT (>120 bpm, maximum rate = 285 +/- 37 bpm) at 0.9 +/- 0.3 days after implantation that persisted through 5 +/- 1 days. IVB, 1 mg/kg over 5 minutes, was administered during rapid VT, and three dogs received a second dose 24 hours later. While VT terminated with IBV in all instances within 3.4 +/- 0.6 minutes, no effect of IVB on sinus rate was noted. CONCLUSION: We conclude that (1) I(f)-associated tachyarrhythmias-if they occur with HCN-based biological pacemakers-can be controlled with I(f)-inhibiting drugs such as IVB; (2) in vitro, IVB appears to have a greater steady state inhibiting effect on HCN1 and HCN212 isoforms than on HCN4; and (3) VT originating from the HCN212 injection site is suppressed more readily than sinus rhythm. This suggests a selectivity of IVB at the concentration attained for ectopic over HCN4-based pacemaker function. This might confer a therapeutic benefit.

Regenerative therapies in electrophysiology and pacing.

The prevention and treatment of cardiac arrhythmias conferring major morbidity and mortality is far from optimal, and relies heavily on devices and drugs for the partial successes that have been seen. The greatest success has been in the use of electronic pacemakers to drive the hearts of patients having high degree heart block. Recent years have seen the beginnings of attempts to use novel approaches available through gene and cell therapies to treat both brady- and tachyarrhythmias. By far the most successful approaches to date have been seen in the development of biological pacemakers. However, the far more difficult problems posed by atrial fibrillation and ventricular tachycardia are now being addressed. In the following pages we review the approaches now in progress as well as the specific methodologic demands that must be met if these therapies are to be successful.

Endogenous adipose tissue as a hemostatic: use in microsurgery.

Bleeding is a frequent complication of microsurgical repair of small blood vessels and time is spent while hemostasis is accomplished. We studied the hemostatic effect of endogenous adipose tissue on bleeding from rat femoral arterial anastomoses. We measured bleeding time (time from removal of clamps to cessation of active bleeding) and mean arterial blood velocity (using a micro-Doppler system), the latter immediately after anastomosis, and again 7 days post-anastomosis. Bleeding time for vessels with fat applied to the artery was 50% less than when no fat was applied. Blood velocity by day 7 post-anastomosis returned to values equivalent to those for intact arteries. Histological evaluation of the anastomotic site demonstrated no significant differences in inflammatory response between fat-treated and untreated arteries. These data suggest that endogenous adipose tissue may be a useful hemostatic agent devoid of significant effects on small artery blood velocity or histology.

Long-term cardiac memory in canine heart is associated with the evolution of a transmural repolarization gradient.

OBJECTIVE: The contribution of regional electrophysiologic heterogeneity to the T-wave changes of long-term cardiac memory (CM) is not known. We mapped activation and repolarization in dogs after induction of CM and in sham animals. METHODS AND RESULTS: CM was induced by three weeks of AV-sequential pacing at the anterior free wall of the left ventricle (LV), midway between apex and base in 5 dogs. In 4 sham controls a pacemaker was implanted but ventricular pacing was not performed. At 3 weeks, unipolar electrograms were recorded (98 epicardial, 120 intramural and endocardial electrodes) during atrial stimulation (cycle length 450 ms). Activation times (AT) and repolarization times (RT) were measured and activation recovery intervals (ARIs) calculated. CM was associated with 1) deeper T waves on ECG, with no change in QT interval; 2) longer activation time at the site of stimulation in CM (29.7+/-1.0, X+/-SEM) than sham (23.9+/-1.3 ms p<0.01); 3) an LV transmural gradient in repolarization time such that repolarization at the epicardium terminated 12.4+/-2.4 ms later than at the endocardium p<0.01), in contrast to no gradient in shams (2.7+/-4.2 ms); in memory dogs, the repolarization time gradient was greatest at sites around the pacing electrode varying from 13.1+/-2.3 ms to 25.5+/-3.8 ms; 4) more negative left ventricular potentials at the peak of the body surface T wave (-4.9+/-0.8 vs -2.2+/-0.4 mV; p<0.05) but no altered right ventricular epicardial T-wave potentials. ARIs did not differ between groups. Right ventricular activation was delayed but was not associated with altered repolarization because of compensatory shortening of the right ventricular ARIs. CONCLUSION: CM-induced T-wave changes are caused by evolution of transmural repolarization gradients manifested during atrial stimulation that are maximal near the site of ventricular pacing.

Microtubular remodeling and decreased expression of Nav1.5 with enhanced EHD4 in cells from the infarcted heart.

Cardiac Na+ channel remodeling provides a critical substrate for generation of reentrant arrhythmias in border zones of the infarcted canine heart. Recent studies show that Nav1.5 cytoskeletal- and endosomal-based membrane trafficking and function are linked to tubulin, microtubular (MT) networks, and Eps15 homology domain containing proteins like EHD4. AIM: Our objective is to understand the relation of tubulin and EHD4 to Nav1.5 channel protein remodeling observed in border zone cells (IZs) when arrhythmias are known to occur; that is, 3-h, 48-h and 5-day post coronary occlusion. MATERIALS METHODS FINDINGS: Our voltage clamp and immunostaining data show that INa density is decreased in the epicardial border zone cells of the 48 h infarcted heart (IZ48h). Immunostaining studies reveal that in post MI cells the cell surface staining of Nav1.5 was reduced and Nav1.5 distribution changed. However, intense co-staining of Nav1.5 and tubulin occurs in core planes and the perinuclear areas in post MI cells. At the same time, there were marked changes in the subcellular location of the EHD4 protein. EHD4 is co-localized with tubulin protein in discrete intracellular "highway" structures. SIGNIFICANCE: The distribution and expression of the three proteins are altered dynamically in post MI cells. In sum, our work illustrates the spatiotemporal complexity of remodeling mechanisms in the post-infarct myocyte. It will be important in future experiments to further explore direct links between MT, EHD proteins, and cell proteins involved in forward trafficking.

Wild-type and mutant HCN channels in a tandem biological-electronic cardiac pacemaker.

BACKGROUND: Biological pacemakers (BPM) implanted in canine left bundle branch function competitively with electronic pacemakers (EPM). We hypothesized that BPM engineered with the use of mE324A mutant murine HCN2 (mHCN2) genes would improve function over mHCN2 and that BPM/EPM tandems confer advantage over either approach alone. METHODS AND RESULTS: In cultured neonatal rat myocytes, activation midpoint was -46.9 mV in mE324A versus -66.1 mV in mHCN2 (P < 0.05). mE324A manifested a positive shift of voltage dependence of gating kinetics of activation and deactivation compared with mHCN2 (P < 0.05) in myocytes as well as Xenopus oocytes. In intact dogs in complete atrioventricular block, saline (control), mHCN2, or mE324A virus was injected into left bundle branch, and EPM were implanted (VVI 45 bpm). Twenty-four-hour ECGs were monitored for 14 days. With EPM discontinued, there was no difference in duration of overdrive suppression among groups. However, basal heart rates in controls were less than those in mHCN2, which did not differ from those in E324A (45 versus 57 versus 53 bpm; P < 0.05). When spontaneous rate fell below 45 bpm, EPM intervened at that rate, triggering 83% of beats in control, contrasting (P < 0.05) with 26% (mHCN2) and 36% (mE324A). On day 14, epinephrine (1 microg/kg per minute IV) induced a 50% heart rate increase in all mE324A, one third of mHCN2, and one fifth of control (P < 0.05 mE324A versus control or mHCN2). CONCLUSIONS: mE324A induces faster, more positive pacemaker current activation than mHCN2 and stable, catecholamine-sensitive rhythms in situ that compete with EPM comparably but more catecholamine responsively than mHCN2. BPM/EPM tandems function reliably, reduce the number of EPM beats, and confer sympathetic responsiveness to the tandem.

Dispersion of repolarization in canine ventricle and the electrocardiographic T wave: Tp-e interval does not reflect transmural dispersion.

BACKGROUND: The concept that the interval between the peak (T(peak)) and the end (T(end)) of the T wave (T(p-e)) is a measure of transmural dispersion of repolarization time is widely accepted but has not been tested rigorously by transmural mapping of the intact heart. OBJECTIVES: The purpose of this study was to test the relationship of T(p-e) to transmural dispersion of repolarization by correlating local repolarization times at endocardial, midmural, and epicardial sites in the left and right ventricles with the T wave of the ECG. METHODS: Local activation times, activation-recovery intervals, and repolarization times were measured at 98 epicardial sites and up to 120 midmural and endocardial sites in eight open-chest dogs. In four of the dogs, long-term cardiac memory was induced by 3 weeks of ventricular pacing at 130 bpm because previous data suggest that, in this setting, delayed epicardial repolarization increases transmural dispersion. The other four dogs were sham operated. RESULTS: In sham dogs, T(p-e) was 41 +/- 2.2 ms (X +/- SEM), whereas the transmural dispersion of repolarization time was 2.7 +/- 4.2 ms (not significant between endocardium and epicardium). Cardiac memory was associated with evolution of a transmural gradient of 14.5 +/- 1.9 ms (P <.02), with epicardium repolarizing later than endocardium. The corresponding T(p-e) was 43 +/- 2.3 ms (not different from sham). In combined sham and memory dogs, T(p-e) intervals did not correlate with transmural dispersion of repolarization times. In contrast, dispersion of repolarization of the whole heart (measured as the difference between the earliest and the latest moment of repolarization from all left and right ventricular, endocardial, intramural, and epicardial recording sites) did correlate with T(p-e) (P <.0005, r = 0.98), although the latter underestimated total repolarization time by approximately 35%. The explanation for this finding is that parts of the heart fully repolarize before the moment of T(peak). CONCLUSION: T(p-e) does not correlate with transmural dispersion of repolarization but is an index of total dispersion of repolarization.

I(Kr) contributes to the altered ventricular repolarization that determines long-term cardiac memory.

OBJECTIVE: Cardiac memory (CM) is characterized by an altered T-wave morphology, which reflects altered repolarization gradients. We hypothesized that the delayed rectifier currents, I(Kr) and I(Ks), might contribute to these repolarization changes. METHODS: We studied conscious, chronically instrumented dogs paced from the postero-lateral left ventricular (LV) wall at rates 5-10% faster than sinus rate for 3 weeks. ECGs during sinus rhythm were recorded on days 0, 7, 14 and 21 of pacing. Within 3 weeks, CM achieved steady state, hearts were excised, and epicardial and endocardial tissues and myocytes were studied. RESULTS: In unpaced controls, action potential duration to 50% and 90% repolarization (APD) in epicardium was shorter than in endocardium (P < 0.05); in CM epicardial APD increased at CL > or = 500 ms, while endocardial APD was either unchanged or decreased such that the transmural gradient seen in controls diminished (P < 0.05). A transmural I(Kr) gradient occurred in controls (epicardium>endocardium, P < 0.05) and was reversed in CM. No I(Ks) transmural gradient was found in controls, while in CM endocardial I(Ks) was greater than epicardial at greater than +50 mV. Canine ERG (cERG) mRNA and protein in epicardium > endocardium in controls (P < 0.05), and this difference was lost in CM. Expression levels of KCNQ1 and KCNE1 protein were similar in all groups. CONCLUSIONS: A transcriptionally induced change in epicardial I(Kr) contributes to the altered ventricular repolarization that characterizes CM.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes Provide In Vivo Biological Pacemaker Function.

BACKGROUND: Although multiple approaches have been used to create biological pacemakers in animal models, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have not been investigated for this purpose. We now report pacemaker function of iPSC-CMs in a canine model. METHODS AND RESULTS: Embryoid bodies were derived from human keratinocytes, their action potential characteristics determined, and their gene expression profiles and markers of differentiation identified. Atrioventricular blocked dogs were immunosuppressed, instrumented with VVI pacemakers, and injected subepicardially into the anterobasal left ventricle with 40 to 75 rhythmically contracting embryoid bodies (totaling 1.3-2×106 cells). ECG and 24-hour Holter monitoring were performed biweekly. After 4 to 13 weeks, epinephrine (1 µg kg-1 min-1) was infused, and the heart removed for histological or electrophysiological study. iPSC-CMs largely lost the markers of pluripotency, became positive for cardiac-specific markers. and manifested If-dependent automaticity. Epicardial pacing of the injection site identified matching beats arising from that site by week 1 after implantation. By week 4, 20% of beats were electronically paced, 60% to 80% of beats were matching, and mean and maximal biological pacemaker rates were 45 and 75 beats per minute. Maximum night and day rates of matching beats were 53±6.9 and 69±10.4 beats per minute, respectively, at 4 weeks. Epinephrine increased rate of matching beats from 35±4.3 to 65±4.0 beats per minute. Incubation of embryoid bodies with the vital dye, Dil, revealed the persistence of injected cells at the site of administration. CONCLUSIONS: iPSC-CMs can integrate into host myocardium and create a biological pacemaker. Although this is a promising development, rate and rhythm of the iPSC-CMs pacemakers remain to be optimized.

Defective cardiac ryanodine receptor regulation during atrial fibrillation.

BACKGROUND: Ca2+ leak from the sarcoplasmic reticulum (SR) may play an important role in triggering and/or maintaining atrial arrhythmias, including atrial fibrillation (AF). Protein kinase A (PKA) hyperphosphorylation of the cardiac ryanodine receptor (RyR2) resulting in dissociation of the channel-stabilizing subunit calstabin2 (FK506-binding protein or FKBP12.6) causes SR Ca2+ leak in failing hearts and can trigger fatal ventricular arrhythmias. Little is known about the role of RyR2 dysfunction in AF, however. METHODS AND RESULTS: Left and right atrial tissue was obtained from dogs with AF induced by rapid right atrial pacing (n=6 for left atrial, n=4 for right atrial) and sham instrumented controls (n=6 for left atrial, n=4 for right atrial). Right atrial tissue was also collected from humans with AF (n=10) and sinus rhythm (n=10) and normal cardiac function. PKA phosphorylation of immunoprecipitated RyR2 was determined by back-phosphorylation and by immunoblotting with a phosphospecific antibody. The amount of calstabin2 bound to RyR2 was determined by coimmunoprecipitation. RyR2 channel currents were measured in planar lipid bilayers. Atrial tissue from both the AF dogs and humans with chronic AF showed a significant increase in PKA phosphorylation of RyR2, with a corresponding decrease in calstabin2 binding to the channel. Channels isolated from dogs with AF exhibited increased open probability under conditions simulating diastole compared with channels from control hearts, suggesting that these AF channels could predispose to a diastolic SR Ca2+ leak. CONCLUSIONS: SR Ca2+ leak due to RyR2 PKA hyperphosphorylation may play a role in initiation and/or maintenance of AF.

Repolarization gradients in the canine left ventricle before and after induction of short-term cardiac memory.

BACKGROUND: Questions remain about the contributions of transmural versus apicobasal repolarization gradients to the configuration of the T wave in control settings and after the induction of short-term cardiac memory. METHODS AND RESULTS: Short-term cardiac memory is seen as T-wave changes induced by altered ventricular activation that persists after restoration of sinus rhythm. We studied cardiac memory in anesthetized, open-chest dogs paced from the ventricle for 2 hours. Unipolar electrograms were recorded from as many as 98 epicardial and 144 intramural sites, and activation times and activation-recovery intervals (ARIs) were measured. In separate experiments, epicardial monophasic action potentials were recorded. We found no appreciable left ventricular intramural gradients in repolarization times (activation time+ARI) in either control conditions or after the induction of memory. In controls, there was a left ventricular apicobasal gradient, with the shortest repolarization times in anterobasal regions and longest repolarization times posteroapically. After induction of memory, repolarization times shortened uniformly throughout the ventricular wall. Monophasic action potential duration at 90% repolarization decreased by approximately 10 ms after induction of memory. CONCLUSIONS: In the intact canine left ventricle at physiological rates, there is no transmural gradient in repolarization. Apicobasal gradients in repolarization time, with shortest repolarization times in anterobasal areas and longest repolarization times in posteroapical regions, are important in the genesis of the T wave. Repolarization times and monophasic action potentials at the 90% repolarization level shorten after the induction of memory. The deeper T wave in the ECG after induction of memory may be explained by the more rapid phase 3 of the action potential.

Cardiac memory is associated with decreased levels of the transcriptional factor CREB modulated by angiotensin II and calcium.

Cardiac memory (CM) has short- (STCM) and long-term (LTCM) components modulated by calcium and angiotensin II. LTCM is associated with reduced Ito and Kv4.3 mRNA levels. Because the cAMP response element binding protein, CREB, contributes to CNS memory transcription, we hypothesized that it might be a transcriptional factor in CM, influenced by calcium and angiotensin II. We studied STCM in dogs that were AV sequentially paced (AVP) for 2 hours or sham-operated. STCM was evaluated with ECG and vectorcardiogram (VCG), and subepicardial biopsies were taken at 5 to 120 minutes and investigated for CREB. LTCM was studied in dogs paced for 3 weeks and in sham controls. At 3 weeks the heart was excised, biopsies obtained, and CRE binding tested. STCM induction occurred in AVP dogs but not in sham or AVP dogs treated with saralasin or nifedipine. Nuclear CREB was significantly decreased at 2 hours in the AVP no-drug group only. LTCM dogs manifested reduced binding of nuclear proteins to CRE, and CRE binding activity in the promoter region of Kv4.3. In conclusion, there is an association between STCM induction and decreased nuclear CREB that is angiotensin-modulated and calcium-dependent. Moreover, the decreased CRE binding after 3 weeks of AVP combined with CRE binding activity in the Kv4.3 promoter can explain the Kv4.3 mRNA and Ito downregulation that characterize LTCM.

Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers.

We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs+-sensitive current (31.1+/-3.8 pA/pF at -150 mV) activating in the diastolic potential range with reversal potential of -37.5+/-1.0 mV, confirming the expressed current as I(f)-like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93+/-16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161+/-4 bpm when hMSCs were expressing both EGFP+mHCN2 (P<0.05). We next injected 10(6) hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45+/-1 bpm, 2 of right-sided origin and 2 of left). In the EGFP+mHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rate=61+/-5 bpm; P<0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.