Francesca Cacciani

Correlation of α‐skeletal actin expression, ventricular fibrosis and heart function with the degree of pressure overload cardiac hypertrophy in rats

We have analysed alterations of α‐skeletal actin expression and volume fraction of fibrosis in the ventricular myocardium and their functional counterpart in terms of arrhythmogenesis and haemodynamic variables, in rats with different degrees of compensated cardiac hypertrophy induced by infra‐renal abdominal aortic coarctation. The following coarctation calibres were used: 1.3 (AC1.3 group), 0.7 (AC0.7) and 0.4 mm (AC0.4); age‐matched rats were used as controls (C group). One month after surgery, spontaneous and sympathetic‐induced ventricular arrhythmias were telemetrically recorded from conscious freely moving animals, and invasive haemodynamic measurements were performed in anaesthetized animals. After killing, subgroups of AC and C rats were used to evaluate in the left ventricle the expression and spatial distribution of α‐skeletal actin and the amount of perivascular and interstitial fibrosis. As compared with C, all AC groups exhibited higher values of systolic pressure, ventricular weight and ventricular wall thickness. AC0.7 and AC0.4 rats also showed a larger amount of fibrosis and upregulation of α‐skeletal actin expression associated with a higher vulnerability to ventricular arrhythmias (AC0.7 and AC0.4) and enhanced myocardial contractility (AC0.4). Our results illustrate the progressive changes in the extracellular matrix features accompanying early ventricular remodelling in response to different degrees of pressure overload that may be involved in the development of cardiac electrical instability. We also demonstrate for the first time a linear correlation between an increase in α‐skeletal actin expression and the degree of compensated cardiac hypertrophy, possibly acting as an early compensatory mechanism to maintain normal mechanical performance.

How different two almost identical action potentials can be: A model study on cardiac repolarization

Spatial heterogeneity in the properties of ion channels generates spatial dispersion of ventricular repolarization, which is modulated by gap junctional coupling. However, it is possible to simulate conditions in which local differences in excitation properties are electrophysiologically silent and only play a role in pathological states. We use a numerical procedure on the Luo-Rudy phase 1 model of the ventricular action potential (AP1) in order to find a modified set of model parameters which generates an action potential profile (AP2) almost identical to AP1. We show that, although the two waveforms elicited from resting conditions as a single AP are very similar and belong to membranes sharing similar passive electrical properties, the modified membrane generating AP2 is a weaker current source than the one generating AP1, has different sensitivity to up/down-regulation of ion channels and to extracellular potassium, and a different electrical restitution profile. We study electrotonic interaction of AP1- and AP2- type membranes in cell pairs and in cable conduction, and find differences in source-sink properties which are masked in physiological conditions and become manifest during intercellular uncoupling or partial block of ion channels, leading to unidirectional block and spatial repolarization gradients. We provide contour plot representations that summarize differences and similarities. The present report characterizes an inverse problem in cardiac cells, and strengthen the recently emergent notion that a comprehensive characterization and validation of cell models and their components are necessary in order to correctly understand simulation results at higher levels of complexity.

Bronchodilator Activity of (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino] carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407), a Potent, Long-Acting, and Selective Muscarinic M3 Receptor Antagonist

The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t½ = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [3H]CHF5407 dissociated quickly from hM2 receptors (t½ = 31 min), whereas [3H]tiotropium dissociated slowly from both hM3 (t½ = 163 min) and hM2 receptor (t½ = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC50 = 9.0–9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED50 value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.

Temporal variability of repolarization in rat ventricular myocytes paced with time-varying frequencies

Adaptation of action potential duration (APD) to pacing cycle length (CL) has been previously characterized in isolated cardiomyocytes for sudden changes in constant CL and for pre‐/postmature stimuli following constant pacing trains. However, random fluctuations characterize both physiological sinus rhythm (up to 10% of mean CL) and intrinsic beat‐to‐beat APD at constant pacing rate. We analysed the beat‐to‐beat sensitivity of each APD to the preceding CL during constant–sudden, random or linearly changing pacing trains in single patch clamped rat left ventricular myocytes, in the absence of the autonomic and electrotonic effects that modulate rate dependency in the intact heart. Beat‐to‐beat variability of APD at −60 mV (APD−60 mV), quantified as s.d. over 10‐beat sequences, increased with corresponding mean APD. When measured as coefficient of variability (CV), APD−60 mV variability was inversely proportional to pacing frequency (from 1.2% at 5 Hz to 3.2% at 0.2 Hz). It was increased, at a basic CL (BCL) of 250 ms, by 55% by the L‐type calcium current (ICaL) blocker nifedipine, and decreased by 23% by the transient‐outward potassium current (Ito) blocker 4‐aminopyridine. Variability of APD at BCL of 250 ms prevented the detection of random changes of CL smaller than ∼5%. Ten per cent random changes in CL were detected as a 40% increase in CV of APD and tended to correlate with it (r= 0.43). Block of ICaL depressed this correlation (r= 0.23), whereas block of Ito significantly increased it (r= 0.67); this was similar with linearly changing CL ramps (ranging ±10% and ±20% of 250 ms). We conclude that beat‐to‐beat APD variability, a major determinant of the propensity for development of arrhythmia in the heart, is present in isolated myocytes, where it is dependent on mean APD and pacing rate. Action potential duration shows a beat‐to‐beat positive correlation with preceding randomly/linearly changing CL, which can be pharmacologically modulated.

Beat-to-Beat Cycle Length Variability of Spontaneously Beating Guinea Pig Sinoatrial Cells: Relative Contributions of the Membrane and Calcium Clocks

The heartbeat arises rhythmically in the sino-atrial node (SAN) and then spreads regularly throughout the heart. The molecular mechanism underlying SAN rhythm has been attributed by recent studies to the interplay between two clocks, one involving the hyperpolarization activated cation current If (the membrane clock), and the second attributable to activation of the electrogenic NaCa exchanger by spontaneous sarcoplasmic releases of calcium (the calcium clock). Both mechanisms contain, in principle, sources of beat-to-beat cycle length variability, which can determine the intrinsic variability of SAN firing and, in turn, contribute to the heart rate variability. In this work we have recorded long sequences of action potentials from patch clamped guinea pig SAN cells (SANCs) perfused, in turn, with normal Tyrode solution, with the If inhibitor ivabradine (3 µM), then back to normal Tyrode, and again with the ryanodine channels inhibitor ryanodine (3 µM). We have found that, together with the expected increase in beating cycle length (+25%), the application of ivabradine brought about a significant and dramatic increase in beat-to-beat cycle length variability (+50%). Despite the similar effect on firing rate, ryanodine did not modify significantly beat-to-beat cycle length variability. Acetylcholine was also applied and led to a 131% increase of beating cycle length, with only a 70% increase in beat-to-beat cycle length variability. We conclude that the main source of inter-beat variability of SANCs firing rate is related to the mechanism of the calcium clock, whereas the membrane clock seems to act in stabilizing rate. Accordingly, when the membrane clock is silenced by application of ivabradine, stochastic variations of the calcium clock are free to make SANCs beating rhythm more variable.

Vulnerability to ventricular arrhthmias and heterogeneity of action potential duration in normal rats

In normal rats, we analysed the arrhythmogenic role of intrinsic action potential duration (APD) heterogeneity. In each animal, ventricular arrhythmic events (VAEs) occurring spontaneously and during the exposure to an acute social challenge were telemetrically recorded. Action potentials were recorded from isolated left ventricular myocytes, at a pacing rate of 5 Hz (patch clamp: current‐clamp mode). APDs were measured at −20 mV, −30 mV, −40 mV, −50 mV and −60 mV. The difference between the shortest and the longest APD was also computed, as an index of individual APD heterogeneity. Animals predisposed to stress‐induced arrhythmias showed higher values of APD and APD heterogeneity as compared with the remaining rats. We concluded that, in the normal heart, a large intrinsic APD heterogeneity resulting from specific electrophysiological properties of ventricular myocytes is not in itself arrhythmogenic, but can predispose towards arrhythmia development under certain conditions, such as autonomic activation.

Effects of the alpha2-adrenergic/DA2-dopaminergic agonist CHF-1024 in preventing ventricular arrhythmogenesis and myocyte electrical remodeling, in a rat model of pressure-overload cardiac hypertrophy.

Cardiac hypertrophy induces morpho-functional myocardial alterations favoring arrhythmogenesis, especially under specific conditions such as sympathetic stimulation. We analyzed whether the dopaminergic agent CHF-1024, given its effect in decreasing adrenergic drive and collagen deposition in hypertrophied hearts, can also reduce arrhythmia vulnerability. Eighty-one male Wistar rats with intrarenal aortic coarctation and 18 control animals were studied. Fifty-eight banded animals were treated with CHF-1024 at four different doses (6, 2, 0.67, or 0.067 mg/Kg/die). One month after aortic ligature, spontaneous and sympathetic-induced ventricular arrhythmic events (VAEs) were telemetrically recorded in conscious animals. After sacrifice, membrane capacitance (Cm) and action potential duration (APD) were measured in isolated left ventricular myocytes (patch-clamp). In all groups, spontaneous VAEs were negligible whereas they significantly increased during sympathetic activation (stress exposure). Banded untreated animals showed a higher number of stress-induced VAEs, longer action potentials, and larger values of Cm and cell width as compared with control group. The treatment with CHF-1024 exhibited an antiarrhythmic effect, abolished APD prolongation, and reduced cell width at all doses. The lowest dose also prevented Cm increase. In conclusion, we demonstrated that in this model of pressure-overload hypertrophy CHF-1024 reduces arrhythmogenesis and causes a recovery of cell excitable properties toward a normal phenotype.

Chronotropic Modulation of the Source-Sink Relationship of Sinoatrial-Atrial Impulse Conduction and Its Significance to Initiation of AF: A One-Dimensional Model Study

Initiation and maintenance of atrial fibrillation (AF) is often associated with pharmacologically or pathologically induced bradycardic states. Even drugs specifically developed in order to counteract cardiac arrhythmias often combine their action with bradycardia and, in turn, with development of AF, via still largely unknown mechanisms. This study aims to simulate action potential (AP) conduction between sinoatrial node (SAN) and atrial cells, either arranged in cell pairs or in a one-dimensional strand, where the relative amount of SAN membrane is made varying, in turn, with junctional resistance. The source-sink relationship between the two membrane types is studied in control conditions and under different simulated chronotropic interventions, in order to define a safety factor for pacemaker-to-atrial AP conduction (SASF) for each treatment. Whereas antiarrhythmic-like interventions which involve downregulation of calcium channels or of calcium handling decrease SASF, the simulation of Ivabradine administration does so to a lesser extent. Particularly interesting is the increase of SASF observed when downregulation G Kr, which simulates the administration of class III antiarrhythmic agents and is likely sustained by an increase in I CaL. Also, the increase in SASF is accompanied by a decreased conduction delay and a better entrainment of repolarization, which is significant to anti-AF strategies.

Instantaneous current-voltage relationships during the course of the human cardiac ventricular action potential: new computational insights into repolarization dynamics.

AIMS To adopt a novel three-dimensional (3D) representation of cardiac action potential (AP) to compactly visualize dynamical properties of human cellular ventricular repolarization. METHODS AND RESULTS We have recently established a novel 3D representation of cardiac AP, which is based on the iterative measurement of instantaneous ion current-voltage profiles during the course of an AP. Such an approach has been originally developed on real patch-clamped ventricular cells, and subsequently improved in silico on several cardiac ventricular AP models of different mammals, and on models of different AP types of the human heart. We apply it here on two different models of human ventricular AP, and show that it compactly provides further insights into repolarization dynamics. The 3D representation of the AP includes equilibrium points during repolarization, and can be screened in terms of what we have shown to be a region, during late repolarization, when membrane conductance becomes negative and repolarization therefore auto-regenerative. We have called this time window auto-regenerative-repolarization-phase (ARRP). CONCLUSION In addition to previous findings obtained through the same procedure, we show here that 3D current-voltage-time representations of human ventricular AP allow compact visualization of dynamical properties, which are relevant for the physiology and pathology of ventricular repolarization. In particular, we suggest that the volume under the current surface corresponding to the ARRP might be used as a predictor of safety of repolarization, in single cells and during AP conduction in cell pairs.