Spatiotemporally controlled cardiac conduction block using high-frequency electrical stimulation

Background

Methods for the electrical inhibition of cardiac excitation have long been sought to control excitability and conduction, but to date remain largely impractical. High-amplitude alternating current (AC) stimulation has been known to extend cardiac action potentials (APs), and has been recently exploited to terminate reentrant arrhythmias by producing reversible conduction blocks. Yet, low-amplitude currents at similar frequencies have been shown to entrain cardiac tissues by generation of repetitive APs, leading in some cases to ventricular fibrillation and hemodynamic collapse in vivo. Therefore, an inhibition method that does not lead to entrainment – irrespective of the stimulation amplitude (bound to fluctuate in an in vivo setting) – is highly desirable.

Methodology/Principal Findings

We investigated the effects of broader amplitude and frequency ranges on the inhibitory effects of extracellular AC stimulation on HL-1 cardiomyocytes cultured on microelectrode arrays, using both sinusoidal and square waveforms. Our results indicate that, at sufficiently high frequencies, cardiac tissue exhibits a binary response to stimulus amplitude with either prolonged APs or no effect, thereby effectively avoiding the risks of entrainment by repetitive firing observed at lower frequencies. We further demonstrate the ability to precisely define reversible local conduction blocks in beating cultures without influencing the propagation activity in non-blocked areas. The conduction blocks were spatiotemporally controlled by electrode geometry and stimuli duration, respectively, and sustainable for long durations (300 s).

Conclusion/Significance

Inhibition of cardiac excitation induced by high-frequency AC stimulation exhibits a binary response to amplitude above a threshold frequency, enabling the generation of reversible conduction blocks without the risks of entrainment. This inhibition method could yield novel approaches for arrhythmia modeling in vitro, as well as safer and more efficacious tools for in vivo cardiac mapping and radio-frequency ablation guidance applications.     

The cardiac valve interstitial cell

Cardiac valve interstitial cells (ICs) are a heterogeneous and dynamic population of specific cell types that have many unique characteristics. They are responsible for maintaining the extracellular scaffold that provides the mechanical characteristics vital for sustaining the unique dynamic behaviour of the valve. A number of cellular phenotypes can be distinguished: some are sparsely arranged throughout the valve leaflets, whilst others are arranged in thin bundles. These cells express molecular markers similar to those of skeletal, cardiac and smooth muscle cells (SMCs) and in particular, many ICs express smooth muscle (SM) alpha-actin, a marker of myofibroblasts. In this respect, these cells exhibit a profile unlike skin fibroblasts, which may allude to their role in valve function.     

犬双心室多点组合同步起搏的心肌力学效应研究

目的 :探讨多点组合同步心室起搏对犬心肌收缩 /舒张力学效应和心脏作功的影响。方法 :12只犬 ,随机进行 5种组合模式的双心室同步起搏 ,并以自身窦性心律状态 (SNR )作为对照。记录各起搏状态下 :左室内压上升和下降最大数率 (±dp/dtmax)、左室松弛时间常数 (τ)、左 /右室游离壁室壁肌张力 (L/RV tensileforce ,L/RV TF)、每搏量 (SV )、左室每搏功 (LVSW )和右室每搏功 (RVSW )等心肌收缩 /舒张力学和心脏作功参数。结果 :双室cHisB LVPL起搏和RVA LVPL起搏的心肌收缩力学参数 +dp/dtmax和L/RV TF较右室双点cHisB RVA起搏增加 ,前两组的心肌舒张力学参数 dp/dtmax也较cHisB RVA起搏增加 ,而τ值较后者缩短。双室三点cHisB RVA LVPL起搏和cHisB RVA LVA起搏的上述各参数均优于双室cHisB LVPL起搏和RVA LVPL起搏。而cHisB RVA LVPL起搏的 +dp/dtmax和L/RV TF均较cHisB RVA LVA起搏增加。cHisB RVA LVPL起搏 dp/dtmax较cHisB RVA LVA起搏提高 6.0 % ,τ值缩短 3 .7%。cHisB LVPL起搏和RVA LVPL起搏的SV、LVSW和RVSW等心室作功参数均较cHisB RVA起搏增加 ,而HisB RVA LVPL起搏的上述心脏作功各参数 ,亦分别较cHisB RVA LVA起搏和cHisB LVPL起搏有不同程度的增加。结论 :双室三点cHisB RVA LVPL组合同     

Generation of bovine multisite haplotypes using random cosmid clones.

One hundred ten random cosmids were used to probe Southern blots of DNA from nine unrelated cattle digested with 12 restriction enzymes. Although only one-third of the expected fragments were explored, 85% of the cosmids revealed at least one polymorphism. The mean heterozygosity of the generated haplotypes was estimated at 51.9%. A surprisingly high proportion of polymorphisms (approximately 25%) was attributed to insertion-deletion events, compensating for the lower level of nucleotide diversity observed in cattle (pi approximately 0.0007) compared to that in human. The mutation rate at cytosines in the CpG dinucleotide was estimated approximately 10 times higher than that at other nucleotides. When used in linkage studies, the generated markers should cover approximately 50% of the bovine genome.     

Vagal stimulation suppresses ischemia-induced myocardial interstitial norepinephrine release

Although electrical vagal stimulation exerts beneficial effects on the ischemic heart such as an antiarrhythmic effect, whether it modulates norepinephrine (NE) and acetylcholine (ACh) releases in the ischemic myocardium remains unknown. To clarify the neural modulation in the ischemic region during vagal stimulation, we examined ischemia-induced NE and ACh releases in anesthetized and vagotomized cats. In a control group (VX, n = 8), occlusion of the left anterior descending coronary artery increased myocardial interstitial NE level from 0.46+/-0.09 to 83.2+/-17.6 nM at 30-45 min of ischemia (mean+/-SE). Vagal stimulation at 5 Hz (VS, n = 8) decreased heart rate by approximately 80 beats/min during the ischemic period and suppressed the NE release to 24.4+/-10.6 nM (P < 0.05 from the VX group). Fixed-rate ventricular pacing (VSP, n=8) abolished this vagally mediated suppression of ischemia-induced NE release. The vagal stimulation augmented ischemia-induced ACh release at 0-15 min of ischemia (VX: 11.1+/-2.1 vs. VS: 20.7+/-3.9 nM, P < 0.05). In the VSP group, the ACh release was not augmented. In conclusion, vagal stimulation suppressed the ischemia-induced NE release and augmented the initial increase in the ACh level. These modulations of NE and ACh levels in the ischemic myocardium may contribute to the beneficial effects of vagal stimulation on the heart during acute myocardial ischemia.     

New method of cardiac output measurement using ultrasound velocity dilution in rats.

The aim of this study was to validate a new technique for the measurement of cardiac output (CO) based on ultrasound and dilution (COUD) in anesthetized rats. A transit time ultrasound (TTU) probe was placed around the rat carotid artery, and ultrasound velocity dilution curves were generated on intravenous injections of saline. CO by COUD were calculated from the dilution curves for normal and portal hypertensive rats in which CO was known to be increased. COUD was compared with the radiolabeled microsphere method and with direct aortic TTU flowmetry for baseline CO and drug-induced CO variations. CO in direct aortic TTU flowmetry was the ascending aorta blood flow measured directly by TTU probe (normal use of TTU flowmetry). The reproducibility of COUD within the same animal was also determined under baseline conditions. COUD detected the known CO increase in portal hypertensive rats compared with normal rats. CO values by COUD were correlated with those provided by microsphere technique or direct aortic TTU flowmetry (adjusted r = 0.76, P < 10(-4) and r = 0.79, P < 0.05, respectively). Baseline CO values and terlipressin-induced CO variations were detected by COUD and the other techniques. Intra- and interobserver agreements for COUD were excellent (intraclass r = 0.99 and 0.98, respectively). COUD was reproducible at least 10 times in 20 min. COUD is an accurate and reproducible method providing low-cost, repetitive CO measurements without open-chest surgery. It can be used in rats as an alternative to the microsphere method and to direct aortic flowmetry.     

Kinetic analysis of multisite phosphorylation using analytic solutions to Michaelis-Menten equations

Phosphorylation-induced expression or modulation of a functional protein is a common signal in living cells. Many functional proteins are phosphorylated at multiple sites and it is frequently observed that phosphorylation at one site enhances or suppresses phosphorylation at another site. Therefore, characterizing such cooperative phosphorylation is important. In this study, we determine a temporal progress curve of multisite phosphorylation by analytically integrating the Michaelis-Menten equations in time. Using this theoretical progress curve, we derive the useful criterion that an intersection of two progress curves implies the presence of cooperativity. Experiments generally yield noisy progress curves. We fit the theoretical progress curves to noisy progress curves containing 4% Gaussian noise in order to determine the kinetics of the phosphorylation. This fitting correctly identifies the sites involved in cooperative phosphorylation.     

Bombesin-induced stimulation of cardiac parasympathetic innervation

The central nervous system effects of bombesin on cardiovascular function were examined in conscious, freely-moving rats. Intracerebroventricular administration of bombesin elevated mean arterial pressure, reduced heart rate and inhibited cold-induced tachycardia. Adrenalectomy prevented bombesin-induced elevations of mean arterial pressure. In contrast, bombesin-induced bradycardia was neither adrenal-dependent nor a baroreceptor-mediated reflex response to increased arterial pressure. Systemic atropine methyl nitrate treatment attenuated bombesin-induced bradycardia, suggesting that bombesin acts within the central nervous system to stimulate cardiac vagal activity.     

Effect of cardiac lymph flow obstruction on cardiac collagen synthesis and interstitial fibrosis

The effect of chronic cardiac lymphatic obstruction on the myocardial synthesis of collagen type I and III was investigated in a rabbit model. In the lymphatic obstruction group (n=16), plasma C-terminal propeptide type I procollagen (PICP) and N-terminal propeptide type III procollagen (PIIINP) were elevated at 7, 14 and 30 days after the operation (p<0.05). The elevated PICP and PIIINP returned to the pre-operation values 60 days after the operation. The myocardial expression of collagen type I and III mRNA were also enhanced in the lymphatic flow obstruction group. Plasma PICP, PIIINP and myocardial collagen type I and III mRNA remained unchanged in the control group (n=16). We concluded that chronic obstruction of cardiac lymph flow leads to enhanced myocardial collagen synthesis in rabbits. The enhanced collagen synthesis starts within seven days after lymphatic obstruction and subsides after 60 days.     

Control of cardiac response under aversive stimulation

The relative heart rate effects of biofeedback training, deep muscle relaxation, and a no-feedback/music procedure were compared during two criterion situations. The first consisted of a 25-min training period during which subjects received the assigned treatments. The second consisted of the pre- to posttraining reductions in heart rate reactivity to a series of aversive tone-shock trials. On the first criterion, the heart rate decreases of the feedback and no-feedback/music groups were not clearly distinguishable; however, both groups fell significantly below the muscle-relaxation group. By contrast, on the second criterion, the three groups were clearly distinguishable, with feedback subjects evidencing the most heart rate control, followed by the muscle-relaxation and no-feedback/music groups, respectively. On the segment of the posttraining aversive trials conducted in the absence of the feedback signal, transfer of heart rate control was incomplete for feedback subjects, but still remained below the level of the other two groups. Training effects were more pronounced on tonic than on phasic heart rate changes. The difference between the two criterion situations suggests the possible need for and feasibility of employing a situational arousal methodology in evaluating the extent and limitation of physiological training procedures.     

Echocardiographic measurement of cardiac output in rats

The systematic evaluation of different transthoracic echocardiographic (TTE) methods to determine cardiac output (CO) and the effect of changes in intravascular volume on echocardiographically determined indexes of cardiovascular structure in the rat has not been documented. With the use of 11 Wistar rats, simultaneous echocardiographic and thermodilution measurements of CO were compared at baseline and after blood withdrawal or transfusion at 43 different levels of intravascular volume and using 10 different echocardiographic approaches. The best correlation (r = 0.93; P < 0.0001), least bias (-3 ml/min), and best precision (16 ml/min) between thermodilution and echocardiographic methods were obtained at the level of aortic annulus using pulsed Doppler. In conclusion, CO could be accurately assessed in rats using TTE and pulsed Doppler at the level of the aortic annulus. This annulus was demonstrated to remain stable, but pulmonary annulus, thoracic aorta, mitral valve, and left ventricular diameters were found to be more modifiable during volumic changes.     

Tropomyosin dynamics in cardiac thin filaments: a multisite forster resonance energy transfer and anisotropy study

Cryoelectron microscopy studies have identified distinct locations of tropomyosin (Tm) within the Ca²⁺-free, Ca²⁺-saturated, and myosin-S1-saturated states of the thin filament. On the other hand, steady-state Förster resonance energy transfer (FRET) studies using functional, reconstituted thin filaments under physiological conditions of temperature and solvent have failed to detect any movement of Tm upon Ca²⁺ binding. In this investigation, an optimized system for FRET and anisotropy analyses of cardiac tropomyosin (cTm) dynamics was developed that employed a single tethered donor probe within a Tm dimer. Multisite FRET and fluorescence anisotropy analyses showed that S1 binding to Ca²⁺ thin filaments triggered a uniform displacement of cTm toward F-actin but that Ca²⁺ binding alone did not change FRET efficiency, most likely due to thermally driven fluctuations of cTm on the thin filament that decreased the effective separation of the donor probe between the blocked and closed states. Although Ca²⁺ binding to the thin filament did not significantly change FRET efficiency, such a change was demonstrated when the thin filament was partially saturated with S1. FRET was also used to show that stoichiometric binding of S1 to Ca²⁺-activated thin filaments decreased the amplitude of Tm fluctuations and revealed a strong correlation between the cooperative binding of S1 to the closed state and the movement of cTm.     

The geometry of multisite phosphorylation

Reversible protein phosphorylation on multiple sites is a key regulatory mechanism in most cellular processes. We consider here a kinase-phosphatase-substrate system with two sites, under mass-action kinetics, with no restrictions on the order of phosphorylation or dephosphorylation. We show that the concentrations of the four phosphoforms at steady state satisfy an algebraic formula—an invariant—that is independent of the other chemical species, such as free enzymes or enzyme-substrate complexes, and holds irrespective of the starting conditions and the total amounts of enzymes and substrate. Such invariants allow stringent quantitative predictions to be made without requiring any knowledge of site-specific parameter values. We introduce what we believe are novel methods from algebraic geometry—Gröbner bases, rational curves—to calculate invariants. These methods are particularly significant because they make it possible to treat parameters symbolically without having to specify their numerical values, and thereby allow us to sidestep the parameter problem. We anticipate that this approach will have much wider applications in biological modeling.