Wenwen Li

Resolution of Established Cardiac Hypertrophy and Fibrosis and Prevention of Systolic Dysfunction in a Transgenic Rabbit Model of Human Cardiomyopathy Through Thiol-Sensitive Mechanisms

Background— Cardiac hypertrophy, the clinical hallmark of hypertrophic cardiomyopathy (HCM), is a major determinant of morbidity and mortality not only in HCM but also in a number of cardiovascular diseases. There is no effective therapy for HCM and generally for cardiac hypertrophy. Myocardial oxidative stress and thiol-sensitive signaling molecules are implicated in pathogenesis of hypertrophy and fibrosis. We posit that treatment with N-acetylcysteine, a precursor of glutathione, the largest intracellular thiol pool against oxidative stress, could reverse cardiac hypertrophy and fibrosis in HCM. Methods and Results— We treated 2-year-old β-myosin heavy-chain Q403 transgenic rabbits with established cardiac hypertrophy and preserved systolic function with N-acetylcysteine or a placebo for 12 months (n=10 per group). Transgenic rabbits in the placebo group had cardiac hypertrophy, fibrosis, systolic dysfunction, increased oxidized to total glutathione ratio, higher levels of activated thiol-sensitive active protein kinase G, dephosphorylated nuclear factor of activated T cells (NFATc1) and phospho-p38, and reduced levels of glutathiolated cardiac α-actin. Treatment with N-acetylcysteine restored oxidized to total glutathione ratio, normalized levels of glutathiolated cardiac α-actin, reversed cardiac and myocyte hypertrophy and interstitial fibrosis, reduced the propensity for ventricular arrhythmias, prevented cardiac dysfunction, restored myocardial levels of active protein kinase G, and dephosphorylated NFATc1 and phospho-p38. Conclusions— Treatment with N-acetylcysteine, a safe prodrug against oxidation, reversed established cardiac phenotype in a transgenic rabbit model of human HCM. Because there is no effective pharmacological therapy for HCM and given that hypertrophy, fibrosis, and cardiac dysfunction are common and major predictors of clinical outcomes, the findings could have implications in various cardiovascular disorders.

The role of dynamic instability and wavelength in arrhythmia maintenance as revealed by panoramic imaging with blebbistatin vs. 2,3-butanedione monoxime.

Unlike other excitation-contraction uncouplers, blebbistatin has few electrophysiological side effects and has gained increasing acceptance as an excitation-contraction uncoupler in optical mapping experiments. However, the possible role of blebbistatin in ventricular arrhythmia has hitherto been unknown. Furthermore, experiments with blebbistatin and 2,3-butanedione monoxime (BDM) offer an opportunity to assess the contribution of dynamic instability and wavelength of impulse propagation to the induction and maintenance of ventricular arrhythmia. Recordings of monophasic action potentials were used to assess effects of blebbistatin in Langendorff-perfused rabbit hearts (n = 5). Additionally, panoramic optical mapping experiments were conducted in rabbit hearts (n = 7) that were sequentially perfused with BDM, then washed out, and subsequently perfused with blebbistatin. The susceptibility to arrhythmia was investigated using a shock-on-T protocol. We found that 1) application of blebbistatin did not change action potential duration (APD) restitution; 2) in contrast to blebbistatin, BDM flattened APD restitution curve and reduced the wavelength; and 3) incidence of sustained arrhythmia was much lower under blebbistatin than under BDM (2/123 vs. 23/99). While arrhythmias under BDM were able to stabilize, the arrhythmias under blebbistatin were unstable and terminated spontaneously. In conclusion, the lower susceptibility to arrhythmia under blebbistatin than under BDM indicates that blebbistatin has less effects on arrhythmia dynamics. A steep restitution slope under blebbistatin is associated with higher dynamic instability, manifested by the higher incidence of not only wave breaks but also wave extinctions. This relatively high dynamic instability leads to the self-termination of arrhythmia because of the sufficiently long wavelength under blebbistatin.

Enhanced transmural fiber rotation and connexin 43 heterogeneity are associated with an increased upper limit of vulnerability in a transgenic rabbit model of human hypertrophic cardiomyopathy

Human hypertrophic cardiomyopathy, characterized by cardiac hypertrophy and myocyte disarray, is the most common cause of sudden cardiac death in the young. Hypertrophic cardiomyopathy is often caused by mutations in sarcomeric genes. We sought to determine arrhythmia propensity and underlying mechanisms contributing to arrhythmia in a transgenic (TG) rabbit model (&bgr;-myosin heavy chain–Q403) of human hypertrophic cardiomyopathy. Langendorff-perfused hearts from TG (n=6) and wild-type (WT) rabbits (n=6) were optically mapped. The upper and lower limits of vulnerability, action potential duration (APD) restitution, and conduction velocity were measured. The transmural fiber angle shift was determined using diffusion tensor MRI. The transmural distribution of connexin 43 was quantified with immunohistochemistry. The upper limit of vulnerability was significantly increased in TG versus WT hearts (13.3±2.1 versus 7.4±2.3 V/cm; P=3.2e−5), whereas the lower limits of vulnerability were similar. APD restitution, conduction velocities, and anisotropy were also similar. Left ventricular transmural fiber rotation was significantly higher in TG versus WT hearts (95.6±10.9° versus 79.2±7.8°; P=0.039). The connexin 43 density was significantly increased in the mid-myocardium of TG hearts compared with WT (5.46±2.44% versus 2.68±0.77%; P=0.024), and similar densities were observed in the endo- and epicardium. Because a nearly 2-fold increase in upper limit of vulnerability was observed in the TG hearts without significant changes in APD restitution, conduction velocity, or the anisotropy ratio, we conclude that structural remodeling may underlie the elevated upper limit of vulnerability in human hypertrophic cardiomyopathy.

Panoramic imaging reveals basic mechanisms of induction and termination of ventricular tachycardia in rabbit heart with chronic infarction: implications for low-voltage cardioversion.

BACKGROUND Sudden cardiac death due to arrhythmia in the settings of chronic myocardial infarction (MI) is an important clinical problem. Arrhythmic risk post-MI continues indefinitely even if heart failure and acute ischemia are not present due to the anatomic substrate of the scar and border zone (BZ) tissue. OBJECTIVE The purpose of this study was to determine mechanisms of arrhythmia initiation and termination in a rabbit model of chronic MI. METHODS Ligation of the lateral division of the left circumflex artery was performed 72 +/- 29 days before acute experiments (n = 11). Flecainide (2.13 +/- 0.64 microM) was administered to promote sustained arrhythmias, which were induced with burst pacing or a multiple shock protocol (four pulses, 140-200 ms coupling interval). RESULTS Panoramic optical mapping with blebbistatin (5 microM) revealed monomorphic ventricular tachycardia (VT) maintained by a single mother rotor (cycle length [CL] = 174.7 +/- 38.4 ms) as the primary mechanism of arrhythmia. Mother rotors were anchored to the scar or BZ for 16 of the 19 rotor locations recorded. Cardioversion thresholds (CVTs) were determined at various phases throughout the VT CL from external shock electrodes. CVTs were found to be phase dependent, and the maximum versus minimum CVT was 7.8 +/- 1.9 vs. 4.1 +/- 1.6 V/cm, respectively (P = .005). Antitachycardia pacing was found to be effective in only 2.7% of cases in this model. CONCLUSIONS These results indicate that scar and BZ tissue heterogeneity provide the substrate for VT by attracting and stabilizing rotors. Additionally, a significant reduction in CVT may be achieved by appropriately timed shocks in which the shock-induced virtual electrode polarization interacts with the rotor to destabilize VT.

Multiple Monophasic Shocks Improve Electrotherapy of Ventricular Tachycardia in a Rabbit Model of Chronic Infarction

BACKGROUND We previously showed that the cardioversion threshold (CVT) for ventricular tachycardia (VT) is phase dependent when a single monophasic shock (1MP) is used. OBJECTIVE The purpose of this study was to extend these findings to a biphasic shock (1BP) and to compare the efficacy of phase-independent multiple monophasic (5MP) and biphasic shocks (5BP). METHODS Panoramic optical mapping with blebbistatin (5 microM) was performed in postmyocardial infarction rabbit hearts (n = 8). Flecainide (1.64 +/- 0.68 microM) was administered to promote sustained arrhythmias. 5MP and 5BP were applied within one VT cycle length (CL). Results were compared to 1BP and antitachycardia pacing. RESULTS We observed monomorphic VT with CL = 149.6 +/- 18.0 ms. Similar to 1MP, CVTs of 1BP were found to be phase dependent, and the maximum versus minimum CVT was 8.6 +/- 1.7 V/cm versus 3.7 +/- 1.9 V/cm, respectively (P = .0013). Efficacy of 5MP was higher than that of 1BP and 5BP. CVT was 3.2 +/- 1.4 V/cm for 5MP versus 5.3 +/- 1.9 V/cm for 5BP (P = .00027). 5MP versus averaged 1BP CVT was 3.6 +/- 2.1 V/cm versus. 6.8 +/- 1.5 V/cm, respectively (P = .00024). Antitachycardia pacing was found to be completely ineffective in this model. CONCLUSION Maintenance of shock-induced virtual electrode polarization by multiple monophasic shocks over a VT cycle is responsible for unpinning of reentry leading to self-termination. Elimination of virtual electrode polarization by shock polarity reversal during multiple biphasic shocks proved ineffective. A significant reduction in CVT can be achieved by applying multiple monophasic shocks within one VT CL or one single shock at the proper coupling interval.

A novel low-energy electrotherapy that terminates ventricular tachycardia with lower energy than a biphasic shock when antitachycardia pacing fails.

OBJECTIVES The authors sought to develop a low-energy electrotherapy that terminates ventricular tachycardia (VT) when antitachycardia pacing (ATP) fails. BACKGROUND High-energy implantable cardioverter-defibrillator (ICD) shocks are associated with device failure, significant morbidity, and increased mortality. A low-energy alternative to ICD shocks is desirable. METHODS Myocardial infarction was created in 25 dogs. Sustained, monomorphic VT was induced by programmed stimulation. Defibrillation electrodes were placed in the right ventricular apex, and coronary sinus and left ventricular epicardium. If ATP failed to terminate sustained VT, the defibrillation thresholds (DFTs) of standard versus experimental electrotherapies were measured. RESULTS Sustained VT ranged from 276 to 438 beats/min (mean 339 beats/min). The right ventricular-coronary sinus shock vector had lower impedance than the right ventricular-left ventricular patch (54.4 ± 18.1 Ω versus 109.8 ± 16.9 Ω; p < 0.001). A single shock required between 0.3 ± 0.2 J to 5.9 ± 2.5 J (mean 2.64 ± 3.22 J; p = 0.008) to terminate VT, and varied depending upon the phase of the VT cycle in which it was delivered. By contrast, multiple shocks delivered within 1 VT cycle length were not phase dependent and achieved lower DFT compared with a single shock (0.13 ± 0.09 J for 3 shocks, 0.08 ± 0.04 J for 5 shocks, and 0.09 ± 0.07 J for 7 shocks; p < 0.001). Finally, a multistage electrotherapy (MSE) achieved significantly lower DFT compared with a single biphasic shock (0.03 ± 0.05 J versus 2.37 ± 1.20 J; respectively, p < 0.001). At a peak shock amplitude of 20 V, MSE achieved 91.3% of terminations versus 10.5% for a biphasic shock (p < 0.001). CONCLUSIONS MSE achieved a major reduction in DFT compared with a single biphasic shock for ATP-refractory monomorphic VT, and represents a novel electrotherapy to reduce high-energy ICD shocks.

Low-Energy Multistage Atrial Defibrillation Therapy Terminates Atrial Fibrillation With Less Energy Than a Single Shock

Background— Implantable device therapy of atrial fibrillation (AF) is limited by pain from high-energy shocks. We developed a low-energy multistage defibrillation therapy and tested it in a canine model of AF. Methods and Results— AF was induced by burst pacing during vagus nerve stimulation. Our novel defibrillation therapy consisted of 3 stages: stage (ST) 1 (1–4 low-energy biphasic [BP] shocks), ST2 (6–10 ultralow-energy monophasic [MP] shocks), and ST3 (antitachycardia pacing). First, ST1 testing compared single or multiple MP and BP shocks. Second, several multistage therapies were tested: ST1 versus ST1+ST3 versus ST1+ST2+ST3. Third, 3 shock vectors were compared: superior vena cava to distal coronary sinus, proximal coronary sinus to left atrial appendage, and right atrial appendage to left atrial appendage. The atrial defibrillation threshold (DFT) of 1 BP shock was <1 MP shock (0.55±0.1 versus 1.38±0.31 J, P=0.003). Two to 3 BP shocks terminated AF with lower peak voltage than 1 BP or 1 MP shock and with lower atrial DFT than 4 BP shocks. Compared with ST1 therapy alone, ST1+ST3 lowered the atrial DFT moderately (0.51±0.46 versus 0.95±0.32 J, P=0.036), whereas 3-stage therapy (ST1+ST2+ST3) dramatically lowered the atrial DFT (0.19±0.12 versus 0.95±0.32 J for ST1 alone, P=0.0012). Finally, the 3-stage therapy was equally effective for all studied vectors. Conclusions— Three-stage electrotherapy significantly reduces the AF DFT and opens the door to low-energy atrial defibrillation at or below the pain threshold.

Multiparametric optical mapping of the Langendorff-perfused rabbit heart.

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc. Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

Multistage electrotherapy delivered through chronically-implanted leads terminates atrial fibrillation with lower energy than a single biphasic shock.

OBJECTIVES The goal of this study was to develop a low-energy, implantable device-based multistage electrotherapy (MSE) to terminate atrial fibrillation (AF). BACKGROUND Previous attempts to perform cardioversion of AF by using an implantable device were limited by the pain caused by use of a high-energy single biphasic shock (BPS). METHODS Transvenous leads were implanted into the right atrium (RA), coronary sinus, and left pulmonary artery of 14 dogs. Self-sustaining AF was induced by 6 ± 2 weeks of high-rate RA pacing. Atrial defibrillation thresholds of standard versus experimental electrotherapies were measured in vivo and studied by using optical imaging in vitro. RESULTS The mean AF cycle length (CL) in vivo was 112 ± 21 ms (534 beats/min). The impedances of the RA-left pulmonary artery and RA-coronary sinus shock vectors were similar (121 ± 11 Ω vs. 126 ± 9 Ω; p = 0.27). BPS required 1.48 ± 0.91 J (165 ± 34 V) to terminate AF. In contrast, MSE terminated AF with significantly less energy (0.16 ± 0.16 J; p < 0.001) and significantly lower peak voltage (31.1 ± 19.3 V; p < 0.001). In vitro optical imaging studies found that AF was maintained by localized foci originating from pulmonary vein-left atrium interfaces. MSE Stage 1 shocks temporarily disrupted localized foci; MSE Stage 2 entrainment shocks continued to silence the localized foci driving AF; and MSE Stage 3 pacing stimuli enabled consistent RA-left atrium activation until sinus rhythm was restored. CONCLUSIONS Low-energy MSE significantly reduced the atrial defibrillation thresholds compared with BPS in a canine model of AF. MSE may enable painless, device-based AF therapy.

Low Energy Multi-Stage Atrial Defibrillation Therapy Terminates Atrial Fibrillation with Less Energy than a Single Shock

Background— Implantable device therapy of atrial fibrillation (AF) is limited by pain from high-energy shocks. We developed a low-energy multistage defibrillation therapy and tested it in a canine model of AF. Methods and Results— AF was induced by burst pacing during vagus nerve stimulation. Our novel defibrillation therapy consisted of 3 stages: stage (ST) 1 (1–4 low-energy biphasic [BP] shocks), ST2 (6–10 ultralow-energy monophasic [MP] shocks), and ST3 (antitachycardia pacing). First, ST1 testing compared single or multiple MP and BP shocks. Second, several multistage therapies were tested: ST1 versus ST1+ST3 versus ST1+ST2+ST3. Third, 3 shock vectors were compared: superior vena cava to distal coronary sinus, proximal coronary sinus to left atrial appendage, and right atrial appendage to left atrial appendage. The atrial defibrillation threshold (DFT) of 1 BP shock was <1 MP shock (0.55±0.1 versus 1.38±0.31 J, P=0.003). Two to 3 BP shocks terminated AF with lower peak voltage than 1 BP or 1 MP shock and with lower atrial DFT than 4 BP shocks. Compared with ST1 therapy alone, ST1+ST3 lowered the atrial DFT moderately (0.51±0.46 versus 0.95±0.32 J, P=0.036), whereas 3-stage therapy (ST1+ST2+ST3) dramatically lowered the atrial DFT (0.19±0.12 versus 0.95±0.32 J for ST1 alone, P=0.0012). Finally, the 3-stage therapy was equally effective for all studied vectors. Conclusions— Three-stage electrotherapy significantly reduces the AF DFT and opens the door to low-energy atrial defibrillation at or below the pain threshold.