Karen L. Vikstrom

Mechanisms of Wave Fractionation at Boundaries of High-Frequency Excitation in the Posterior Left Atrium of the Isolated Sheep Heart During Atrial Fibrillation

Background— High-frequency fractionated electrograms recorded during atrial fibrillation (AF) in the posterior left atrium (PLA) and elsewhere are being used as target sites for catheter ablation. We tested the hypothesis that highly periodic electric waves emerging from AF sources at or near the PLA give rise to the most fractionated activity in adjacent locations. Methods and Results— Sustained AF was induced in 8 isolated sheep hearts (0.5 &mgr;mol/L acetylcholine). Endocardial videoimaging (DI-4-ANEPPS) and electric mapping of the PLA enabled spatial characterization of dominant frequencies (DFs) and a regularity index (ratio of DF to total power). Regularity index showed that fractionation was lowest within the area with the maximal DF (DFmax domain; 0.19±0.02) and highest within a band of ≈3 mm (0.16±0.02; P=0.047) at boundaries with lower-frequency domains. The numbers of spatiotemporal periodic episodes (25.9±2.3) and rotors per experiment (1.9±0.7) were also highest within the DFmax domain. Most commonly, breakthrough waves at the PLA traveled toward the rest of the atria (76.8±8.1% outward versus 23.2±8.1% inward; P<0.01). In both experiments and simulations with an atrial ionic model, fractionation at DFmax boundaries was associated with increased beat-to-beat variability of conduction velocity and directionality with wavebreak formation. Conclusions— During stable AF, the PLA harbors regular, fast, and highly organized activity; the outer limit of the DFmax domain is the area where the most propagation pattern variability and fractionated activity occur. These new concepts introduce a new perspective in the clinical use of high-frequency fractionated electrograms to localize sources of AF precisely at the PLA and elsewhere.

Spatial Distribution of Fibrosis Governs Fibrillation Wave Dynamics in the Posterior Left Atrium During Heart Failure

Heart failure (HF) commonly results in atrial fibrillation (AF) and fibrosis, but how the distribution of fibrosis impacts AF dynamics has not been studied. HF was induced in sheep by ventricular tachypacing (220 bpm, 6 to 7 weeks). Optical mapping (Di-4-ANEPPS, 300 frames/sec) of the posterior left atrial (PLA) endocardium was performed during sustained AF (burst pacing) in Langendorff-perfused HF (n=7, 4 &mgr;mol/L acetylcholine; n=3, no acetylcholine) and control (n=6) hearts. PLA breakthroughs were the most frequent activation pattern in both groups (72.0±4.6 and 90.2±2.7%, HF and control, respectively). However, unlike control, HF breakthroughs preferentially occurred at the PLAs periphery near the pulmonary vein ostia, and their beat-to-beat variability was greater than control (1.93±0.14 versus 1.47±0.07 changes/[beats/sec], respectively, P<0.05). On histological analysis (picrosirius red), the area of diffuse fibrosis was larger in HF (23.4±0.4%) than control (14.1±0.6%; P<0.001, n=4). Also the number and size of fibrous patches were significantly larger and their location was more peripheral in HF than control. Computer simulations using 2-dimensional human atrial models with structural and ionic remodeling as in HF demonstrated that changes in AF activation frequency and dynamics were controlled by the interaction of electrical waves with clusters of fibrotic patches of various sizes and individual pulmonary vein ostia. During AF in failing hearts, heterogeneous spatial distribution of fibrosis at the PLA governs AF dynamics and fractionation.

Arrhythmogenic Mechanisms in a Mouse Model of Catecholaminergic Polymorphic Ventricular Tachycardia

Catecholaminergic polymorphic ventricular tachycardia (VT) is a lethal familial disease characterized by bidirectional VT, polymorphic VT, and ventricular fibrillation. Catecholaminergic polymorphic VT is caused by enhanced Ca2+ release through defective ryanodine receptor (RyR2) channels. We used epicardial and endocardial optical mapping, chemical subendocardial ablation with Lugols solution, and patch clamping in a knockin (RyR2/RyR2R4496C) mouse model to investigate the arrhythmogenic mechanisms in catecholaminergic polymorphic VT. In isolated hearts, spontaneous ventricular arrhythmias occurred in 54% of 13 RyR2/RyR2R4496C and in 9% of 11 wild-type (P=0.03) littermates perfused with Ca2+and isoproterenol; 66% of 12 RyR2/RyR2R4496C and 20% of 10 wild-type hearts perfused with caffeine and epinephrine showed arrhythmias (P=0.04). Epicardial mapping showed that monomorphic VT, bidirectional VT, and polymorphic VT manifested as concentric epicardial breakthrough patterns, suggesting a focal origin in the His–Purkinje networks of either or both ventricles. Monomorphic VT was clearly unifocal, whereas bidirectional VT was bifocal. Polymorphic VT was initially multifocal but eventually became reentrant and degenerated into ventricular fibrillation. Endocardial mapping confirmed the Purkinje fiber origin of the focal arrhythmias. Chemical ablation of the right ventricular endocardial cavity with Lugols solution induced complete right bundle branch block and converted the bidirectional VT into monomorphic VT in 4 anesthetized RyR2/RyR2R4496C mice. Under current clamp, single Purkinje cells from RyR2/RyR2R4496C mouse hearts generated delayed afterdepolarization–induced triggered activity at lower frequencies and level of adrenergic stimulation than wild-type. Overall, the data demonstrate that the His–Purkinje system is an important source of focal arrhythmias in catecholaminergic polymorphic VT.

Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers.

In pathological conditions such as ischemic cardiomyopathy and heart failure, differentiation of fibroblasts into myofibroblasts may result in myocyte-fibroblast electrical coupling via gap junctions. We hypothesized that myofibroblast proliferation and increased heterocellular coupling significantly alter two-dimensional cardiac wave propagation and reentry dynamics. Co-cultures of myocytes and myofibroblasts from neonatal rat ventricles were optically mapped using a voltage-sensitive dye during pacing and sustained reentry. The myofibroblast/myocyte ratio was changed systematically, and junctional coupling of the myofibroblasts was reduced or increased using silencing RNAi or adenoviral overexpression of Cx43, respectively. Numerical simulations in two-dimensional models were used to quantify the effects of heterocellular coupling on conduction velocity (CV) and reentry dynamics. In both simulations and experiments, reentry frequency and CV diminished with larger myofibroblast/myocyte area ratios; complexity of propagation increased, resulting in wave fractionation and reentry multiplication. The relationship between CV and coupling was biphasic: an initial decrease in CV was followed by an increase as heterocellular coupling increased. Low heterocellular coupling resulted in fragmented and wavy wavefronts; at high coupling wavefronts became smoother. Heterocellular coupling alters conduction velocity, reentry stability, and complexity of wave propagation. The results provide novel insight into the mechanisms whereby electrical myocyte-myofibroblast interactions modify wave propagation and the propensity to reentrant arrhythmias.

Up‐regulation of the inward rectifier K+ current (IK1) in the mouse heart accelerates and stabilizes rotors

Previous studies have suggested an important role for the inward rectifier K+ current (IK1) in stabilizing rotors responsible for ventricular tachycardia (VT) and fibrillation (VF). To test this hypothesis, we used a line of transgenic mice (TG) overexpressing Kir 2.1–green fluorescent protein (GFP) fusion protein in a cardiac‐specific manner. Optical mapping of the epicardial surface in ventricles showed that the Langendorff‐perfused TG hearts were able to sustain stable VT/VF for 350 ± 1181 s at a very high dominant frequency (DF) of 44.6 ± 4.3 Hz. In contrast, tachyarrhythmias in wild‐type hearts (WT) were short‐lived (3 ± 9 s), and the DF was 26.3 ± 5.2 Hz. The stable, high frequency, reentrant activity in TG hearts slowed down, and eventually terminated in the presence of 10 μm Ba2+, suggesting an important role for IK1. Moreover, by increasing IK1 density in a two‐dimensional computer model having realistic mouse ionic and action potential properties, a highly stable, fast rotor (≈45 Hz) could be induced. Simulations suggested that the TG hearts allowed such a fast and stable rotor because of both greater outward conductance at the core and shortened action potential duration in the core vicinity, as well as increased excitability, in part due to faster recovery of Na+ current. The latter resulted in a larger rate of increase in the local conduction velocity as a function of the distance from the core in TG compared to WT hearts, in both simulations and experiments. Finally, simulations showed that rotor frequencies were more sensitive to changes (doubling) in IK1, compared to other K+ currents. In combination, these results provide the first direct evidence that IK1 up‐regulation in the mouse heart is a substrate for stable and very fast rotors.

Hypertrophy, Pathology, and Molecular Markers of Cardiac Pathogenesis

Increased ventricular expression of several genes, including atrial natriuretic factor (ANF), has been documented in experimental models of cardiac hypertrophy. It remains to be clarified whether altered expression of these genes is a consistent marker of the hypertrophy itself or a marker of some parallel pathogenetic process. Using a transgenic mouse model of hypertrophic cardiomyopathy as a tool, we assessed the relationship between the amount of ventricular ANF gene expression and the degree of hypertrophy as well as the relationship between the cells expressing ANF and tissue pathology. We determined that hypertrophy is not always associated with increased ventricular expression of ANF and that cells expressing ANF are found in regions of tissue pathology. We propose that alteration in the ventricular expression of this gene is a sensitive indicator of cardiac pathogenesis and may result from a number of different stimuli that include, among others, abnormal tissue architecture and hemodynamic load.

CONTRACTILE PROTEIN MUTATIONS AND HEART DISEASE

Mutations in several muscle structural proteins (the myosin heavy chain, alpha tropomyosin, cardiac troponin T and myosin binding protein C) result in a genetically dominant heart disease, hypertrophic cardiomyopathy. Biochemical data from studies of mutant myosin suggest a dominant-negative mechanism for inheritance of this disease. The most likely primary defect is sarcomere dysfunction, which is followed by the major clinical symptoms.

Phospholipase C ε Modulates β-Adrenergic Receptor Dependent Cardiac Contraction and Inhibits Cardiac Hypertrophy

Phospholipase C (PLC) &egr; is a recently identified enzyme regulated by a wide range of molecules including Ras family small GTPases, Rho A, G&agr;12/13, and G&bgr;&ggr; with primary sites of expression in the heart and lung. In a screen for human signal transduction genes altered during heart failure, we found that PLC&egr; mRNA is upregulated. Two murine models of cardiac hypertrophy confirmed upregulation of PLC&egr; protein expression or PLC&egr; RNA. To identify a role for PLC&egr; in cardiac function and pathology, a PLC&egr;-deficient mouse strain was created. Echocardiography indicated PLC&egr;−/− mice had decreased cardiac function, and direct measurements of left ventricular contraction demonstrated that PLC&egr;−/− mice had a decreased contractile response to acute isoproterenol administration. Cardiac myocytes isolated from PLC&egr;−/− mice had decreased &bgr;-adrenergic receptor (&bgr;AR)-dependent increases in Ca2+ transient amplitudes, likely accounting for the contractile deficiency in vivo. This defect appears to be independent from the ability of the &bgr;AR system to produce cAMP and regulation of sarcoplasmic reticulum Ca2+ pool size. To address the significance of these functional deficits to cardiac pathology, PLC&egr;−/− mice were subjected to a chronic isoproterenol model of hypertrophic stress. PLC&egr;−/− mice were more susceptible than wild-type littermates to development of hypertrophy than wild-type littermates. Together, these data suggest a novel PLC-dependent component of &bgr;AR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve and loss of PLC&egr; signaling sensitizes the heart to development of hypertrophy in response to chronic cardiac stress.

Left versus right atrial difference in dominant frequency, K+ channel transcripts, and fibrosis in patients developing atrial fibrillation after cardiac surgery

BACKGROUND The development of atrial fibrillation (AF) after cardiac surgery is associated with adverse outcomes; however, the mechanism(s) that trigger and maintain AF in these patients are unknown. OBJECTIVE The purpose of this study was to test our hypothesis that postoperative AF is maintained by high-frequency sources in the left atrium (LA) resulting from ion channel and structural features that differ from the right atrium (RA). METHODS Forty-four patients with no previous history of AF who underwent cardiac surgery consented to LA and RA biopsies. Histologic sections evaluated fatty infiltration, fibrosis, and iron deposition; quantitative reverse transcription-polymerase chain reaction (RT-PCR) assessed ion channel expression. In a subset of 27 patients, LA and RA unipolar recording leads were also placed. In patients who developed AF, the dominant frequency (DF) for each lead was calculated using fast Fourier transform. RESULTS DFs during AF were LA 6.26 +/- 0.8 Hz, RA 4.56 +/- 0.7 Hz (P <.01). RT-PCR revealed LA-to-RA differences in mRNA abundance for Kir2.3 (1.8:1) and Kir3.4 (2.3:1). While LA fibrosis was greater in patients developing AF compared with those remaining in normal sinus rhythm (10.8% +/- 11% vs. 3.8% +/- 3.5%; P = .03), the amount of LA fibrosis inversely correlated with the LA DF. CONCLUSIONS This is the first demonstration of LA-to-RA frequency differences during postoperative AF, which are associated with LA-to-RA differences in mRNA levels for potassium channel proteins and LA fibrosis. These results strongly suggest that sources of AF after cardiac surgery are located in the LA and are stabilized by LA fibrosis.

A role for the aryl hydrocarbon receptor in cardiac physiology and function as demonstrated by AhR knockout mice.

The aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, is the receptor for the polycyclic aromatic hydrocarbons found in tobacco smoke, polychlorinated biphenyls, and the environmental pollutant, dioxin. To better understand the role of the AhR in the heart, echocardiography, invasive measurements of aortic and left ventricular pressures, isolated working heart preparations, as well as morphological and molecular analysis were used to investigate the impact of AhR inactivation on the mouse heart using the AhR knockout as a model. Cardiac hypertrophy is an early phenotypic manifestation of the AhR knockout. Although the knockout animals were not hypertensive at the ages examined, cardiomyopathy accompanied by diminished cardiac output developed. Despite the structural left ventricular remodeling, the hearts of these animals exhibit minimal fibrosis and do not have the expected increases in surrogate molecular markers of cardiac hypertrophy. The anatomic remodeling without typical features of molecular remodeling is not consistent with hypertrophic growth secondary to pressure or volume overload, suggesting that increased cardiomyocyte size may be a direct consequence of the absence of the AhR in this cell type.