Massimiliano Zaniboni

The restriction of diffusion of cations at the external surface of cardiac myocytes varies between species

In cardiac muscle sarcolemmal structures such as T-tubules, caveolae and negatively charged protein-polysaccharides may affect the rate of cation exchange on the external surface of the cells. To test this hypothesis, we examined the rate of external cation exchange in adult rabbit and rat ventricular myocytes using a rapid solution switcher to change the bulk external solution within 4 ms. To assess the rate of diffusion of monovalent cations, we increased [K+]o from 4.4 to 6.6 or 8.8 mM and measured the time required to achieve a stable membrane depolarization. In rat myocytes, the mean time to 90% depolarization (t90) was significantly longer than that in rabbit myocytes (137 and 64 ms, respectively) and the difference in t90 was not associated with the cell size. To assess the time course of exchange of external Ca2+, we rapidly exposed the myocytes to 0 Ca2+-2 mM EGTA solution at specific time points before action potentials or voltage clamp steps, and measured the rate of alteration of the normalized peak [Ca2+]i transient (Fluo-3) or Ca2+ current. Exposure to 0 Ca2+-2 mM EGTA solution caused a decline in the intracellular calcium transient. In rat myocytes, the rate of decline in the [Ca2+]i transient was much slower (t90 > 1500 ms, the time required for 90% decline) than for the rabbit (t90 = 295 ms). Also, the rate of decline in the Ca2+ current was prolonged in rat myocytes (t90 = 910 ms) compared with rabbit myocytes (t90 = 241 ms). These data indicate that there is a restricted space on the external surface of sarcolemma which limits diffusion of divalent cations more markedly than monovalent cations. The extent of this limitation of cation diffusion varies between species, and may have functional significance.

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.

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.

Short-term action potential memory and electrical restitution: A cellular computational study on the stability of cardiac repolarization under dynamic pacing

Electrical restitution (ER) is a major determinant of repolarization stability and, under fast pacing rate, it reveals memory properties of the cardiac action potential (AP), whose dynamics have never been fully elucidated, nor their ionic mechanisms. Previous studies have looked at ER mainly in terms of changes in AP duration (APD) when the preceding diastolic interval (DI) changes and described dynamic conditions where this relationship shows hysteresis which, in turn, has been proposed as a marker of short-term AP memory and repolarization stability. By means of numerical simulations of a non-propagated human ventricular AP, we show here that measuring ER as APD versus the preceding cycle length (CL) provides additional information on repolarization dynamics which is not contained in the companion formulation. We focus particularly on fast pacing rate conditions with a beat-to-beat variable CL, where memory properties emerge from APD vs CL and not from APD vs DI and should thus be stored in APD and not in DI. We provide an ion-currents characterization of such conditions under periodic and random CL variability, and show that the memory stored in APD plays a stabilizing role on AP repolarization under pacing rate perturbations. The gating kinetics of L-type calcium current seems to be the main determinant of this safety mechanism. We also show that, at fast pacing rate and under otherwise identical pacing conditions, a periodically beat-to-beat changing CL is more effective than a random one in stabilizing repolarization. In summary, we propose a novel view of short-term AP memory, differentially stored between systole and diastole, which opens a number of methodological and theoretical implications for the understanding of arrhythmia development.

Suppression of beat to beat repolarization variability by electrical coupling in guinea pig ventricular myocytes

To examine relationships between intracellular coupling and cardiac repolarization, we electrically connected isolated guinea pig ventricular myocytes to one another with a junctional resistance. Because coupling was achieved by an external circuit that supplied coupling current based solely on transmembrane potential difference, cell-to-cell interactions were considered in their simplest form, independent of ionic diffusion via gap junctions or mechanical interactions between cells.