Anatoli N. Lopatin

Cationic Nanoparticles Induce Nanoscale Disruption in Living Cell Plasma Membranes

It has long been recognized that cationic nanoparticles induce cell membrane permeability. Recently, it has been found that cationic nanoparticles induce the formation and/or growth of nanoscale holes in supported lipid bilayers. In this paper, we show that noncytotoxic concentrations of cationic nanoparticles induce 30-2000 pA currents in 293A (human embryonic kidney) and KB (human epidermoid carcinoma) cells, consistent with a nanoscale defect such as a single hole or group of holes in the cell membrane ranging from 1 to 350 nm(2) in total area. Other forms of nanoscale defects, including the nanoparticle porating agents adsorbing onto or intercalating into the lipid bilayer, are also consistent; although the size of the defect must increase to account for any reduction in ion conduction, as compared to a water channel. An individual defect forming event takes 1-100 ms, while membrane resealing may occur over tens of seconds. Patch-clamp data provide direct evidence for the formation of nanoscale defects in living cell membranes. The cationic polymer data are compared and contrasted with patch-clamp data obtained for an amphiphilic phenylene ethynylene antimicrobial oligomer (AMO-3), a small molecule that is proposed to make well-defined 3.4 nm holes in lipid bilayers. Here, we observe data that are consistent with AMO-3 making approximately 3 nm holes in living cell membranes.

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.

Cardiac strong inward rectifier potassium channels

Cardiac I(K1) and I(KACh) are the major potassium currents displaying classical strong inward rectification, a unique property that is critical for their roles in cardiac excitability. In the last 15 years, research on I(K1) and I(KACh) has been propelled by the cloning of the underlying inwardly rectifying potassium (Kir) channels, the discovery of the molecular mechanism of strong rectification and the linking of a number of disorders of cardiac excitability to defects in genes encoding Kir channels. Disease-causing mutations in Kir genes have been shown experimentally to affect one or more of the following channel properties: structure, assembly, trafficking, and regulation, with the ultimate effect of a gain- or a loss-of-function of the channel. It is now established that I(K1) and I(KACh) channels are heterotetramers of Kir2 and Kir3 subunits, respectively. Each homomeric Kir channel has distinct biophysical and regulatory properties, and individual Kir subunits often display different patterns of regional, cellular, and membrane distribution. These differences are thought to underlie important variations in the physiological properties of I(K1) and I(KACh). It has become increasingly clear that the contribution of I(K1) and I(KACh) channels to cardiac electrical activity goes beyond their long recognized role in the stabilization of resting membrane potential and shaping the late phase of action potential repolarization in individual myocytes but extends to being critical elements determining the overall electrical stability of the heart.

Dominant-negative suppression of IK1 in the mouse heart leads to altered cardiac excitability

The inward rectifier potassium current in the heart, I(K1), has been suggested to play a significant role in cardiac excitability by contributing to the late phase of action potential (AP) repolarization and the stabilization of resting potential. To further assess the role of I(K1) in cardiac excitability we have produced transgenic mice expressing a dominant-negative subunit of the Kir2.1 channel, a major molecular determinant of I(K1) in the heart, and studied the effects of I(K1) suppression on major potassium currents, APs and the overall electrical activity of the heart. Kir2.1 channel subunits with a mutated signature sequence (AAA for GYG substitution) were expressed in the heart under control of the alpha-myosin heavy chain promoter. Two lines of transgenic mice were established, both expressing high levels of Kir2.1-AAA-GFP (GFP, green fluorescent protein) subunits in all major parts of the heart. In ventricular myocytes isolated from transgenic mice, I(K1) was reduced by 95% in both lines, leading to a significant prolongation of APs. Surface ECG recordings from anesthetized transgenic mice revealed significant changes in key parameters of excitability, including prolongation of QRS complexes and QT intervals. This study confirms the significant role of I(K1) in control of AP repolarization and major ECG intervals in the intact heart.

Transgenic upregulation of IK1 in the mouse heart is proarrhythmic

The role of the cardiac current Ik1 in arrhythmogenesis remains highly controversal. To gain further insights into the mechanisms of IK1 involvement in cardiac excitability, we studied the susceptibility of transgenic mice with altered IK1 to arrhythmia during various pharmacological and physiological challenges.Arrhythmogenesis was studied in transgenic mice expressing either dominant negative Kir2.1-AAA or wild type Kir2.1 subunits in the heart, models of IK1 suppression (AAA-TG) and up-regulation (WT-TG), respectively. Under normal conditions, both anesthetized wild type (WT) and AAA-TG mice did not display any spontaneous arrhythmias. In contrast,WT-TG mice displayed numerous arrhythmias of various types. In isolated hearts, the threshold concentration for halothane-induced ventricular tachycardias (VT) was increased to 170 % in the AAA-TG and decreased to 55 % in WT-TG hearts when compared to WT hearts. The number of PVCs induced by AV node ablation combined with hypokalemia was reduced in AAA-TG hearts and increased in WT-TG mice.After AV node ablation AAA-TG hearts were more tolerant, and WT-TG less tolerant to isoproterenol- induced arrhythmias than WT hearts. Analysis of monophasic action potentials in isolated hearts shows a significant reduction in the dispersion of action potential repolarization in mice with suppressed IK1. The data strongly support the hypothesis that in the mouse heart upregulation of IK1 is proarrhythmic, and that under certain conditions IK1 blockade in cardiac myocytes may be a potentially useful antiarrhythmic strategy.

Differential polyamine sensitivity in inwardly rectifying Kir2 potassium channels

Recent studies have shown that Kir2 channels display differential sensitivity to intracellular polyamines, and have raised a number of questions about several properties of inward rectification important to the understanding of their physiological roles. In this study, we have carried out a detailed characterization of steady‐state and kinetic properties of block of Kir2.1–3 channels by spermine. High‐resolution recordings from outside‐out patches showed that in all Kir2 channels current–voltage relationships display a ‘crossover’ effect upon change in extracellular K+. Experiments at different concentrations of spermine allowed for the characterization of two distinct shallow components of rectification, with the voltages for half‐block negative (V11/2) and positive (V21/2) to the voltage of half‐block for the major steep component of rectification (V01/2). While V11/2 and V21/2 voltages differ significantly between Kir2 channels, they were coupled to each other according to the equation V11/2−V21/2= constant, strongly suggesting that similar structures may underlie both components. In Kir2.3 channels, the V21/2 was ∼50 mV positive to V01/2, leading to a pattern of outward currents distinct from that of Kir2.1 and Kir2.2 channels. The effective valency of spermine block (Z0) was highest in Kir2.2 channels while the valencies in Kir2.1 and Kir2.3 channels were not significantly different. The voltage dependence of spermine unblock was similar in all Kir2 channels, but the rates of unblock were ∼7‐fold and ∼16‐fold slower in Kir2.3 channels than those in Kir2.1 and Kir2.2 when measured at high and physiological extracellular K+, respectively. In all Kir2 channels, the instantaneous phase of activation was present. The instantaneous phase was difficult to resolve at high extracellular K+ but it became evident and accounted for nearly 30–50% of the total current when recorded at physiological extracellular K+. In conclusion, the data are consistent with the universal mechanism of rectification in Kir2 channels, but also point to significant, and physiologically important, quantitative differences between Kir2 isoforms.

Resolution of hyposmotic stress in isolated mouse ventricular myocytes causes sealing of t‐tubules

•  What is the central question of this study? The t‐tubules of ventricular myocytes are critical elements in excitation–contraction coupling. They become disorganized or even lost in various cardiac pathologies. However, the mechanisms leading to disruption of t‐tubules are essentially unknown. This study was designed to identify physiologically relevant processes that underlie remodelling of t‐tubules. •  What is the main finding and its importance? We show that the resolution of physiologically relevant hyposmotic swelling, not the application of osmotic shock itself, leads to dramatic t‐tubular remodelling, including the sealing of individual t‐tubules. The results point to an important and probably general mechanism of acute and fast stress‐induced t‐tubular remodelling that may underlie various relevant pathologies of the heart.

Functional consequences of Kir2.1/Kir2.2 subunit heteromerization

Kir2 subunits form channels that underlie classical strongly inwardly rectifying potassium currents. While homomeric Kir2 channels display a number of distinct and physiologically important properties, the functional properties of heteromeric Kir2 assemblies, as well as the stoichiometries and the arrangements of Kir2 subunits in native channels, remain largely unknown. Therefore, we have implemented a concatemeric approach, whereby all four cloned Kir2 subunits were linked in tandem, in order to study the effects of Kir2.1 and Kir2.2 heteromerization on properties of the resulting channels. Kir2.2 subunits contributed stronger to single-channel conductance than Kir2.1 subunits, and channels containing two or more Kir2.2 subunits displayed conductances indistinguishable from that of a Kir2.2 homomeric channel. In contrast, single-channel kinetics was a more discriminating property. The open times were significantly shorter in Kir2.2 channels compared with Kir2.1 channels and decreased nearly proportionally to the number of Kir2.2 subunits in the heteromeric channel. Similarly, the sensitivity to block by barium also depended on the proportions of Kir2.1 to Kir2.2 subunits. Overall, the results showed that Kir2.1 and Kir2.2 subunits exert neither a dominant nor an anomalous effect on any of the properties of heteromeric channels. The data highlight opportunities and challenges of using differential properties of Kir2 channels in deciphering the subunit composition of native inwardly rectifying potassium currents.

Cardiac-directed parvalbumin transgene expression in mice shows marked heart rate dependence of delayed Ca2+ buffering action

Relaxation abnormalities are prevalent in heart failure and contribute to clinical outcomes. Disruption of Ca2+ homeostasis in heart failure delays relaxation by prolonging the intracellular Ca2+ transient. We sought to speed cardiac relaxation in vivo by cardiac-directed transgene expression of parvalbumin (Parv), a cytosolic Ca2+ buffer normally expressed in fast-twitch skeletal muscle. A key feature of Parvs function resides in its Ca2+/Mg2+ binding affinities that account for delayed Ca2+ buffering in response to the intracellular Ca2+ transient. Cardiac Parv expression decreased sarcoplasmic reticulum Ca2+ content without otherwise altering intracellular Ca2+ homeostasis. At high physiological mouse heart rates in vivo, Parv modestly accelerated relaxation without affecting cardiac morphology or systolic function. Ex vivo pacing of the isolated heart revealed a marked heart rate dependence of Parvs delayed Ca2+ buffering effects on myocardial performance. As the pacing frequency was lowered (7 to 2.5 Hz), the relaxation rates increased in Parv hearts. However, as pacing rates approached the dynamic range in humans, Parv hearts demonstrated decreased contractility, consistent with Parv buffering systolic Ca2+. Mathematical modeling and in vitro studies provide the underlying mechanism responsible for the frequency-dependent fractional Ca2+ buffering action of Parv. Future studies directed toward refining the dose and frequency-response relationships of Parv in the heart or engineering novel Parv-based Ca2+ buffers with modified Mg2+ and Ca2+ affinities to limit systolic Ca2+ buffering may hold promise for the development of new therapies to remediate relaxation abnormalities in heart failure.

Ca2+ homeostasis in sealed t-tubules of mouse ventricular myocytes

We have recently shown that in mouse ventricular myocytes, t-tubules can be quickly and tightly sealed during the resolution of hyposmotic shock of physiologically relevant magnitude. Sealing of t-tubules is associated with trapping extracellular solution inside the myocytes but the ionic homeostasis of sealed t-tubules and the consequences of potential transtubular ion fluxes remain unknown. In this study we investigated the dynamics of Ca(2+) movements associated with sealing of t-tubules. The data show that under normal conditions sealed t-tubules contain Ca(2+) at concentrations below 100μM. However, blockade of voltage-dependent Ca(2+) channels with 10μM nicardipine, or increasing extracellular concentration of K(+) from 5.4mM to 20mM led to several fold increase in concentration of t-tubular Ca(2+). Alternatively, the release of Ca(2+) from sarcoplasmic reticulum using 10mM caffeine led to the restoration of t-tubular Ca(2+) towards extracellular levels within few seconds. Sealing of t-tubules in the presence of extracellular 1.5mM Ca(2+) and 5.4mM extracellular K(+) led to occasional and sporadic intracellular Ca(2+) transients. In contrast, sealing of t-tubules in the presence of 10mM caffeine was characterized by a significant long lasting increase in intracellular Ca(2+). The effect was completely abolished in the absence of extracellular Ca(2+) and significantly reduced in pre-detubulated myocytes but was essentially preserved in the presence of mitochondrial decoupler dinitrophenol. This study shows that sealed t-tubules are capable of highly regulated transport of Ca(2+) and present a major route for Ca(2+) influx into the cytosol during sealing process.