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.

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.

Interventricular dispersion in repolarization causes bifid T waves in dogs with dofetilide-induced long QT syndrome.

BACKGROUND: Long QT2 (LQT2) syndrome is characterized by bifid (or notched) T waves, whose mechanism is not understood. OBJECTIVE: The purpose of this study was to test whether increased interventricular dispersion of repolarization induces bifid T waves. METHODS: We simultaneously recorded surface ECG and unipolar electrograms at baseline and after dofetilide in a canine model of dofetilide-induced LQT2 (6 male mongrel dogs). Standard ECG variables, T-wave duration, and moments of peaks of bifid T waves (Tp1 and Tp2) were correlated with moments of local repolarization. Epicardial electrograms were recorded over the left ventricular (LV) and right ventricular (RV) anterior walls (11 × 11 electrode grid, 5-mm interelectrode distance). In 5 of the 6 hearts, we also recorded intramural unipolar electrograms (n = 4-7 needles per heart). In each unipolar recording, we determined activation time, repolarization time (RTs), and activation-recovery interval. In addition, we studied RT response to heart rate changes. RESULTS: Dofetilide prolonged QT and QTc, induced bifid T waves in 4 of 6 animals, and prolonged RT heterogeneously in LV and RV, resulting in increased interventricular and LV intraventricular RT dispersion. Dofetilide did not induce a disparate response in activation-recovery interval across the transmural axis. Dofetilide-induced separation of RT across the RV-LV interface concurred with the moments of T-wave peaks. Dofetilide-induced steepening of restitution slopes was larger in LV than RV. CONCLUSION: Dofetilide-induced bifid T waves result from interventricular RT dispersion.

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.

SAP97 and cortactin remodeling in arrhythmogenic Purkinje cells.

Because structural remodeling of several proteins, including ion channels, may underlie the abnormal action potentials of Purkinje cells (PCs) that survive in the 48 hr infarcted zone of the canine heart (IZPCs), we sought to determine the subcellular structure and function of the KV1.5 (KCNA5) protein in single IZPCs. Clustering of the Kv1.5 subunit in axons is regulated by a synapse-associated protein, SAP97, and is linked to an actin-binding protein, cortactin, and an intercellular adhesion molecule, N-cadherin. To understand the functional remodeling of the Kv1.5 channel and its regulation in IZPCs, Kv1.5 currents in PCs were measured as the currents blocked by 10 µM RSD1379 using patch-clamp techniques. Immunocytochemistry and confocal imaging were used for both single and aggregated IZPCs vs normal PCs (NZPCs) to determine the relationship of Kv1.5 with SAP-97, cortactin and N-cadherin. In IZPCs, both the sarcolemma (SL) and intercalated disk (ID) Kv1.5 protein are abundant, and the amount of cytosolic Kv1.5 protein is greatly increased. SAP-97 is also increased at IDs and has notable cytosolic localization suggesting that SAP-97 may regulate the functional expression and stabilization of Kv1.5 channels in IZPCs. Cortactin, which is located with N-cadherin at IDs in NZPCs, remains at IDs but begins to dissociate from N-cadherin, often forming ring structures and colocalizing with Kv1.5 within IZPCs. At the same time, cortactin/Kv1.5 colocalization is increased at the ID, suggesting an ongoing active process of membrane trafficking of the channel protein. Finally, the Kv1.5 current, measured as the RSD1379-sensitive current, at +40 mV did not differ between NZPCs (0.81±0.24 pA/pF, n = 14) and IZPCs (0.83±0.21 pA/pF, n = 13, NS). In conclusion, the subcellular structural remodeling of Kv1.5, SAP97 and cortactin maintained and normalized the function of the Kv1.5 channel in Purkinje cells that survived myocardial infarction.

Electromechanical wave imaging of biologically and electrically paced canine hearts in vivo.

Electromechanical wave imaging (EWI) has been show capable of directly and entirely non-invasively mapping the trans mural electromechanical activation in all four cardiac chambers in vivo. In this study, we assessed EWI repeatability and reproducibility, as well as its capability of localizing electronic and, for the first time, biological pacing locations in closed-chest, conscious canines. Electromechanical activation was obtained in six conscious animals during normal sinus rhythm (NSR) and idioventricular rhythms occurring in dogs with complete heart block instrumented with electronic and biologic pacemakers (EPM and BPM respectively). After atrioventricular node ablation, dogs were implanted with an EPM in the right ventricular (RV) endocardial apex (n = 4) and two additionally received a BPM at the left ventricular (LV) epicardial base (n = 2). EWI was performed trans thoracically during NSR, BPM and EPM pacing, in conscious dogs, using an unfocused transmit sequence at 2000 frames/s. During NSR, the EW originated at the right atrium (RA), propagated to the left atrium (LA) and emerged from multiple sources in both ventricles. During EPM, the EW originated at the RV apex and propagated throughout both ventricles. During BPM, the EW originated from the LV basal lateral wall and subsequently propagated throughout the ventricles. EWI differentiated BPM from EPM and NSR and identified the distinct pacing origins. Isochrone comparison indicated that EWI was repeatable and reliable. These findings thus indicate the potential for EWI to serve as a simple, non-invasive and direct imaging technology for mapping and characterizing arrhythmias as well as the treatments thereof.

HCN2/SkM1 gene transfer into canine left bundle branch induces stable, autonomically responsive biological pacing at physiological heart rates.

OBJECTIVES: This study sought to test the hypothesis that hyperpolarization-activated cyclic nucleotide-gated (HCN)-based biological pacing might be improved significantly by hyperpolarizing the action potential (AP) threshold via coexpression of the skeletal muscle sodium channel 1 (SkM1). BACKGROUND: Gene-based biological pacemakers display effective in vivo pacemaker function. However, approaches used to date have failed to manifest optimal pacemaker properties, defined as basal beating rates of 60 to 90 beats/min, a brisk autonomic response achieving maximal rates of 130 to 160 beats/min, and low to absent electronic backup pacing. METHODS: We implanted adenoviral SkM1, HCN2, or HCN2/SkM1 constructs into left bundle branches (LBB) or left ventricular (LV) epicardium of atrioventricular-blocked dogs. RESULTS: During stable peak gene expression on days 5 to 7, HCN2/SkM1 LBB-injected dogs showed highly stable in vivo pacemaker activity superior to SkM1 or HCN2 alone and superior to LV-implanted dogs with regard to beating rates (resting approximately 80 beats/min; maximum approximately 130 beats/min), no dependence on electronic backup pacing, and enhanced modulation of pacemaker function during circadian rhythm or epinephrine infusion. In vitro isolated LV of dogs overexpressing SkM1 manifested a significantly more negative AP threshold. CONCLUSIONS: LBB-injected HCN2/SkM1 potentially provides a more clinically suitable biological pacemaker strategy than other reported constructs. This superiority is attributable to the more negative AP threshold and injection into the LBB.

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.

Microtubules and angiotensin II receptors contribute to modulation of repolarization induced by ventricular pacing.

BACKGROUND: Left ventricular pacing (LVP) in canine heart alters ventricular activation, leading to reduced transient outward potassium current (I(to)), loss of the epicardial action potential notch, and T-wave vector displacement. These repolarization changes, referred to as cardiac memory, are initiated by locally increased angiotensin II (AngII) levels. In HEK293 cells in which Kv4.3 and KChIP2, the channel subunits contributing to I(to), are overexpressed with the AngII receptor 1 (AT1R), AngII induces a decrease in I(to) as the result of internalization of a Kv4.3/KChIP2/AT1R macromolecular complex. OBJECTIVE: To test the hypothesis that in canine heart in situ, 2h LVP-induced decreases in membrane KChIP2, AT1R, and I(to) are prevented by blocking subunit trafficking. METHODS: We used standard electrophysiological, biophysical, and biochemical methods to study 4 groups of dogs: (1) Sham, (2) 2h LVP, (3) LVP + colchicine (microtubule-disrupting agent), and (4) LVP + losartan (AT1R blocker). RESULTS: The T-wave vector displacement was significantly greater in LVP than in Sham and was inhibited by colchicine or losartan. Epicardial biopsies showed significant decreases in KChIP2 and AT1R proteins in the membrane fraction after LVP but not after sham treatment, and these decreases were prevented by colchicine or losartan. Colchicine but not losartan significantly reduced microtubular polymerization. In isolated ventricular myocytes, AngII-induced I(to) reduction and loss of action potential notch were blocked by colchicine. CONCLUSIONS: LVP-induced reduction of KChIP2 in plasma light membranes depends on an AngII-mediated pathway and intact microtubular status. Loss of I(to) and the action potential notch appear to derive from AngII-initiated trafficking of channel subunits.

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.

Effect of skeletal muscle Na(+) channel delivered via a cell platform on cardiac conduction and arrhythmia induction.

BACKGROUND: In depolarized myocardial infarct epicardial border zones, the cardiac sodium channel is largely inactivated, contributing to slow conduction and reentry. We have demonstrated that adenoviral delivery of the skeletal muscle Na(+) channel (SkM1) to epicardial border zones normalizes conduction and reduces induction of ventricular tachycardia/ventricular fibrillation. We now studied the impact of canine mesenchymal stem cells (cMSCs) in delivering SkM1. METHODS AND RESULTS: cMSCs were isolated and transfected with SkM1. Coculture experiments showed cMSC/SkM1 but not cMSC alone and maintained fast conduction at depolarized potentials. We studied 3 groups in the canine 7d infarct: sham, cMSC, and cMSC/SkM1. In vivo epicardial border zones electrograms were broad and fragmented in sham, narrower in cMSCs, and narrow and unfragmented in cMSC/SkM1 (P<0.05). During programmed electrical stimulation of epicardial border zones, QRS duration in cMSC/SkM1 was shorter than in cMSC and sham (P<0.05). Programmed electrical stimulation-induced ventricular tachycardia/ventricular fibrillation was equivalent in all groups (P>0.05). CONCLUSION: cMSCs provide efficient delivery of SkM1 current. The interventions performed (cMSCs or cMSC/SkM1) were neither antiarrhythmic nor proarrhythmic. Comparing outcomes with cMSC/SkM1 and viral gene delivery highlights the criticality of the delivery platform to SkM1 antiarrhythmic efficacy.

SkM1 and Cx32 improve conduction in canine myocardial infarcts yet only SkM1 is antiarrhythmic.

AIMS: Reentry accounts for most life-threatening arrhythmias, complicating myocardial infarction, and therapies that consistently prevent reentry from occurring are lacking. In this study, we compare antiarrhythmic effects of gene transfer of green fluorescent protein (GFP; sham), the skeletal muscle sodium channel (SkM1), the liver-specific connexin (Cx32), and SkM1/Cx32 in the subacute canine infarct. METHODS AND RESULTS: Immediately after ligation of the left anterior descending artery, viral constructs were implanted in the epicardial border zone (EBZ). Five to 7 days later, efficient restoration of impulse propagation (narrow QRS and local electrogram duration) occurred in SkM1, Cx32, and SkM1/Cx32 groups (P< 0.05 vs. GFP). Programmed electrical stimulation from the EBZ induced sustained ventricular tachycardia (VT)/ventricular fibrillation (VF) in 15/22 GFP dogs vs. 2/12 SkM1, 6/14 Cx32, and 8/10 SkM1/Cx32 (P< 0.05 SkM1 vs. GFP). GFP, SkM1, and SkM1/Cx32 had predominantly polymorphic VT/VF, whereas in Cx32 dogs, monomorphic VT predominated (P< 0.05 for Cx32 vs. GFP). Tetrazolium red staining showed significantly larger infarcts in Cx32- vs. GFP-treated animals (P< 0.05). CONCLUSION: Whereas SkM1 gene transfer reduces the incidence of inducible VT/VF, Cx32 therapy to improve gap junctional conductance results in larger infarct size, a different VT morphology, and no antiarrhythmic efficacy.

Implantation of sinoatrial node cells into canine right ventricle: biological pacing appears limited by the substrate.

Biological pacing has been proposed as a physiologic counterpart to electronic pacing, and the sinoatrial node (SAN) is the general standard for biological pacemakers. We tested the expression of SAN pacemaker cell activity when implanted autologously in the right ventricle (RV). We induced complete heart block and implanted electronic pacemakers in the RV of adult mongrel dogs. Autologous SAN cells isolated enzymatically were studied by patch clamp to confirm SAN identity. SAN cells (400,000) were injected into the RV subepicardial free wall and dogs were monitored for 2 weeks. Pacemaker function was assessed by overdrive pacing and IV epinephrine challenge. SAN cells expressed a time-dependent inward current (I(f)) activating on hyperpolarization: density = 4.3 ± 0.6 pA/pF at -105 mV. Four of the six dogs demonstrated >50% of beats originating from the implant site at 24 h. Biological pacemaker rates on days 7-14 = 45-55 bpm and post-overdrive escape times = 1.5-2.5 s. Brisk catecholamine responsiveness occurred. Dogs implanted with autologous SAN cells manifest biological pacing properties dissimilar from those of the anatomic SAN. This highlights the importance of cell and substrate interaction in generating biological pacemaker function.

Biological pacemakers in canines exhibit positive chronotropic response to emotional arousal.

BACKGROUND: Biological pacemakers based on the HCN2 channel isoform respond to beta-adrenergic and muscarinic stimulation, suggesting a capacity to respond to autonomic input. OBJECTIVE: The purpose of this study was to investigate autonomic response to emotional arousal in canines implanted with murine HCN2-based biological pacemakers using gene therapy. METHODS: An electronic pacemaker was implanted with its lead in the right ventricular apical endocardium (VVI 35 bpm). An adenoviral HCN2/GFP construct (Ad-HCN2, n = 7) or saline (control, n = 5) was injected into the left bundle branch on day 2 after radiofrequency ablation of the atrioventricular node to induce complete atrioventricular block. Emotional arousal was achieved by presenting food following an overnight fast. Autonomic control was evaluated with Poincaré plots of R-R(N) against R-R(N+1) intervals to characterize heart rate variability (HRV) and with continuous RR interval assessment via 24-hour ambulatory ECG. The 24-hour ECG and Poincaré plot shape were analyzed. RESULTS: During day 1 after biological pacemaker implantation, Poincaré HRV parameters and RR intervals were unchanged with food presentation. However, on day 7, food presentation was accompanied by an increase in HRV (SD1, p < 0.07, and SD2, p < 0.05) and shortening of RR interval (P < .05) in dogs with Ad-HCN2 but not in controls. CONCLUSION: This is the first demonstration that biological pacemakers are capable of responding to natural arousal stimuli to elicit appropriate chronotropic responses, a potential advantage over electronic pacemakers.

Cardiac expression of skeletal muscle sodium channels increases longitudinal conduction velocity in the canine 1-week myocardial infarction.

BACKGROUND: Skeletal muscle sodium channel (Nav1.4) expression in border zone myocardium increases action potential upstroke velocity in depolarized isolated tissue. Because resting membrane potential in the 1-week canine infarct is reduced, we hypothesized that conduction velocity (CV) is greater in Nav1.4 dogs compared with in control dogs. OBJECTIVE: The purpose of this study was to measure CV in the infarct border zone border in dogs with and without Nav1.4 expression. METHODS: Adenovirus was injected in the infarct border zone in 34 dogs. The adenovirus incorporated the Nav1.4- and a green fluorescent protein (GFP) gene (Nav1.4 group, n = 16) or only GFP (n = 18). After 1 week, upstroke velocity and CV were measured by sequential microelectrode recordings at 4 and 7 mM [K(+)] in superfused epicardial slabs. High-density in vivo epicardial activation mapping was performed in a subgroup (8 Nav1.4, 6 GFP) at three to four locations in the border zone. Microscopy and antibody staining confirmed GFP or Nav1.4 expression. RESULTS: Infarct sizes were similar between groups (30.6% +/- 3% of left ventricle mass, mean +/- standard error of the mean). Longitudinal CV was greater in Nav1.4 than in GFP sites (58.5 +/- 1.8 vs. 53.3 +/- 1.2 cm/s, 20 and 15 sites, respectively; P <.05). Transverse CV was not different between the groups. In tissue slabs, dV/dt(max) was higher and CV was greater in Nav1.4 than in control at 7 mM [K(+)] (P <.05). Immunohistochemical Nav1.4 staining was seen at the longitudinal ends of the myocytes. CONCLUSION: Nav1.4 channels in myocardium surviving 1 week infarction increases longitudinal but not transverse CV, consistent with the increased dV/dt(max) and with the cellular localization of Nav1.4.

Determinants of CREB degradation and KChIP2 gene transcription in cardiac memory.

BACKGROUND: Left ventricular pacing (LVP) to induce cardiac memory (CM) in dogs results in a decreased transient outward K current (I(to)) and reduced mRNA and protein of the I(to) channel accessory subunit, KChIP2. The KChIP2 decrease is attributed to a decrease in its transcription factor, cyclic adenosine monophosphate response element binding protein (CREB). OBJECTIVE: This study sought to determine the mechanisms responsible for the CREB decrease that is initiated by LVP. METHODS: CM was quantified as T-wave vector displacement in 18 LVP dogs. In 5 dogs, angiotensin II receptor blocker, saralasin, was infused before and during pacing. In 3 dogs, proteasomal inhibitor, lactacystin, was injected into the left anterior descending artery before LVP. Epicardial biopsy samples were taken before and after LVP. Neonatal rat cardiomyocytes (NRCM) were incubated with H(2)O(2) (50 micromol/l) for 1 hour with or without lactacystin. RESULTS: LVP significantly displaced the T-wave vector and was associated with increased lipid peroxidation and increased tissue angiotensin II levels. Saralasin prevented T-vector displacement and lipid peroxidation. CREB was significantly decreased after 2 hours of LVP and was comparably decreased in H(2)O(2)-treated NRCM. Lactacystin inhibited the CREB decrease in LVP dogs and H(2)O(2)-treated NRCM. LVP and H(2)O(2) both induced CREB ubiquitination, and the H(2)O(2)-induced CREB decrease was prevented by knocking down ubiquitin. CONCLUSION: LVP initiates myocardial angiotensin II production and reactive oxygen species synthesis, leading to CREB ubiquitination and its proteasomal degradation. This sequence of events would explain the pacing-induced reduction in KChIP2, and contribute to altered repolarization and the T-wave changes of cardiac memory.

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.

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.

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.

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.