Micropattern platform promotes extracellular matrix remodeling by human PSC-derived cardiac fibroblasts and enhances contractility of co-cultured cardiomyocytes.

In native heart tissue, cardiac fibroblasts provide the structural framework of extracellular matrix (ECM) while also influencing the electrical and mechanical properties of cardiomyocytes. Recent advances in the field of stem cell differentiation have led to the availability of human pluripotent stem cell-derived cardiac fibroblasts (iPSC-CFs) in addition to cardiomyocytes (iPSC-CMs). Here we use a novel 2D in vitro micropatterned platform that provides control over ECM geometry and substrate stiffness. When cultured alone on soft micropatterned substrates, iPSC-CFs are confined to the micropatterned features and remodel the ECM into anisotropic fibers. Similar remodeling and ECM production occurs when cultured with iPSC-CMs in a co-culture model. In addition to modifications in the ECM, our results show that iPSC-CFs influence iPSC-CM function with accelerated Ca2+ transient rise-up time and greater contractile strains in the co-culture conditions compared to when iPSC-CMs are cultured alone. These combined observations highlight the important role cardiac fibroblasts play in vivo and the need for co-culture models like the one presented here to provide more representative in vitro cardiac constructs.

Aligned human cardiac syncytium for in vitro analysis of electrical, structural, and mechanical readouts.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC-CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC-CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two-dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC-CMs.

[Electrophysiological mechanisms of heart rate stability of the hibernating mammals during hypothermia].

Despite more than 100 years of study, the mechanisms of natural resistance of the hibernator heart to cardiac arrhythmias during hypothermia has remained unknown. Renewed optimism in this area of research comes with recent methodological advances which enable to shed light on the hidden secrets of the hibernator's heart. This review discusses basic mechanisms of hypothermic ventricular arrhythmias and highlights some recent findings from the hibernator's heart electrophysiology, which may have an antiarrhythmic potential for the human heart as well.

[Comparative study of cardiac alternans of action potential duration in hypothermia in rabbits and ground squirrels].

Cardiac alternans is a promising predictor of sudden death, yet its role in the mechanism of hypothermic arrhythmia induction is unclear. We aimed to investigate the effect of hypothermia on spatial-temporal characteristics of repolarization pattern in the Langendorff-perfused hearts of summer active (SA, n = 6) and winter hibernating (WH, n = 7) ground squirrels Spermophilus undulatus and rabbits (n = 5), who were immobilized with the excitation-contraction uncoupler BDM (10 mM) and optically mapped using the voltage sensitive dye di-4-ANNEPS and CCD camera (128 x 128 pixels; 500 frames/sec). Action potential duration (APD) restitution was quantified over the posterior epicardial heart surface and estimated using Nolasco-Dahlen criterion. In rabbit hearts, hypothermia resulted in arrhythmogenic overshoots of APD alternans as well as increase of APD restitution curve steepness. In contrast, significant APD alternans were observed in SA hearts at 27 degrees C, and at 17 degrees C in WH hearts. Moreover, slope of APD restitution curve in ground squirrels hearts did not reached arrhythmogenic threshold (43 +/- 9 degrees and 39 +/- 5 degrees for SA and WH respectively). Our results demonstrate different resistance of hibernating and non-hibernating mammals against induction of arrhythmogenic cardiac alternans which is closely associated with adaptive changes in intracellular Ca2+ cycling during hibernation.

[Spatiotemporal characteristics of activation of the heart of hibernating and non-hibernating mammals during hypothermia].

The heart of hibernating mammals is known to demonstrate the nature\'s model of resistance to rhythm disturbances, including ventricular arrhythmias, during hypothermia. However, electrophysiological mechanism of this phenomenon is not completely understood. Using optical mapping technique with voltage-sensitive dye di-4-ANEPPS, we investigated the spatiotemporal characteristics of ventricular activation in Langendorff-perfused hearts of winter hibernating ground squirrels Spermophyllus undulatus and rabbits at temperatures from +37 degrees C to +3 degrees C. In rabbit hearts, reduction of temperature from 37 to 17 degrees C resulted in significant decrease of conduction velocity and increase of conduction anisotropy. Excitation failure was observed in the rabbit heart at 12+/-1 degree C. In contrast, ground squirrels exhibited significantly faster conduction velocity compared with rabbits at all temperatures and insensibility of conduction anisotropy to cooling down to 3C which can protect the hibernator heart against arrhythmias during hypothermia.

[Effect of nibentan on dispersion of repolarization of ventricular myocardium in the rabbit].

Among factors determining development of polymorphic ventricular tachycardia torsades de pointes under influence of class III antiarrhythmic drugs great value is attributed to enhanced heterogeneity of repolarization of ventricular myocardium which can cause functional conduction blocks and development of re-entry of excitation. In this work with the help of optical mapping of electrical activity of the heart we investigated effect of nibentan (0.3 and 1 mM) on chronotopography of repolarization of epicardial surface of ventricles of isolated after Langendorf rabbit heart (n=5). For assessment of heterogeneity of repolarization we measured the following parameters: standard deviation of mean action potential duration (APDm) along mapped region (SD-APD), dispersion index (DI=1000 SD-APDm), maximal dispersion (Dmax=APDmax-APDmin). Nibentan in concentrations 0.3 and 1 mM increased APD at the level of 90% repolarization (APD90%) from 231 +/- 12 to 277 +/- 7 ms (p < 0.05) 318 +/- 7 ms (p < 0.001), respectively, but in concentration 1 mM it practically did not affect parameters of heterogeneity of repolarization (SD-APD: from 4.4 +/- 0.9 to 5.7 +/- 1.1 ms, p=0.4; DI: from 19.1 +/- 4.2 to 18.0+3.5, p=0.8; Dmax: from 16.6 +/- 2.5 to 24.8 +/- 4.3 ms, < 0.05). These results show that nibentan in the range of clinically used doses does not effect heterogeneity of ventricular myocardium. This can explain low proarrhythmic effect of nibentan.

[The effect of hypothermia on the wavelength and vulnarability to ventricular arrhythmias in mammals].

Arrhythmias developing in isolated Langendorff-perfused heart following the cooling of the perfusion solution from +37 to +3 degrees C were studied in rats and winter hibernating ground squirrels Citellus undulatus with application of no drugs. In rats, hypothermia significantly increased the probability of ventricular arrhythmias (from 22 +/- 6 % at 37 degrees C to 56 +/- 14 % at 17 degrees C). Excitation failure was observed in the rat hearts below 10 +/- 1 degrees C. The appearance of arrhythmias was closely correlated with a decrease in the wavelength which strongly suggests a reentrant mechanism of the hypothermic arrhythmias. In contrast, ground squirrels showed insensibility of the wavelength to cooling and were resistant to arrhythmias during hypothermia.

[Pattern of excitation in isolated heart of hibernator ground squirrel Citellus undulatus].

Aim of our study was to measure conduction velocity and pattern of excitation during hypothermia in hearts of ground squirrels Citellus undulatus, known to be most resilient hibernators. We imaged electrical conduction in intact isolated hearts of summer active and winter hibernating ground squirrels at temperatures varying from +37 degrees C to +3 degrees C. Electrical activity was mapped using CCD camera (500 frames/sec) and voltage-sensitive dye di-4-ANEPPS during normal sinus rhythm and ventricular pacing. No spontaneous tachyarrhythmia was observed in all hearts at any temperature. Hearts were able to maintain spontaneous sinus rhythm and normal pattern of epicardial excitation throughout the whole range of studied temperatures. Despite responsiveness to pacing in all hearts ventricular conduction velocity was significantly reduced (about 10-fold) at low temperatures +3 degrees C. Our data provides the first direct demonstration that isolated heart of the summer active and winter hibernating ground squirrel Citellus undulatus is able to maintain normal excitation pattern in a range of temperatures from +37 degrees C to +3 degrees C.

[Anticholinergic Effect of a New Antiarrhythmic Class III Drug RG-2]. / Kholinoliticheskaia aktivnost' antiaritmicheskogo preparata III klassa RG-2.

In experiments on isolated rat and rabbit right atrium, a new class III antiarrhythmic drug RG-2 (0,01-1 microM) was shown to have anticholinergic action competing with 0.2-1 mM carbachol. RG-2 (0.1-1 microM) produced dose-dependent increase of APD90% in rat and rabbit atrial cells and had no effects on other action potential parameters. In presence of carbachol RG-2 produced significantly greater increase of APD90% and caused significant increase of APD50%. Thus RG-2 exerts anticholinergic action, which can take important part in RG-2 antiarrhythmic activity.

[The role of stretching the sinus node artery in initiation of atrial fibrillation]. / Rol' rastiazheniia arterii sinusovogo uzla v mekhanizme vozniknoveniia fibriliatsii predserdii.

In anaesthetised dogs with open chest (n = 10), against the background of the Gd3+ (a blocking agent of mechano-sensitive ion channels), acetylcholine perfusion induced no significant changes either in probability of atrial fibrillation occurrence or in the paroxysm duration. The Gd3+ did not alter the deceleration of the sinus rhythm either. Therefore the mechanism of spontaneous occurrence of atrial cholinergic fibrillation seems not to be associated with the trigger activity induced by an increased blood pressure in the right atrium.

[The role of atrial pressure in spontaneous initiation of atrial fibrillation in the dog.]. / Vliianie vnutripredserdnogo davleniia na kharakter vozniknoveniia vagotonicheskoi fibrilliatsii predserdii u sobak.

Atrial fibrillation (AF) frequently occurred under conditions associated with atrial dilatation (stretch) or vagal hyperactivity. To study possible role of atrial stretch in spontaneous initiation of vagal AF we compared changes of right atrial pressure (RAP) and activation patterns during AF beginning. In anesthetized open-chest dogs (n=45) AF was induced by stimulation of vagal nerves (VS) (30-60 Hz, 5-10 s train). VS resulted in sinus node arrest (4.7+/-0.7 sec) with subsequent AF initiation in 153 of 229 cases. In 41% of cases of AF initiation the first atrial wave (A(1)) was closely related to ventricular activation (V) with V-A(1) interval of 94+/-5 ms (<> AF). This ventricular excitation induced acute short increase of RAP from 6.6.+/-0.6 to 12.9+/-1.1 mmHg (p<0.00l). Whereas other cases of AF initiation (59%) had no relation to ventricular activation (A(1)-V interval of 1382+/-173 ms) (<> AF). Atrial activation mapping (224 unipolar electrodes) showed that interval A(1)-A(2) of <> AF was significantly shorter than of <>. These data indicate that atrial stretch induced by elevation of RAP may facilitate the induction of AF but do not play a significant role in the mechanism of spontaneous AF initiation in this animal model.

[Effects of verapamil on atrial fibrillation spontaneous initiation in the intact canine heart]. / Vliianie verapamila na spontannoe vozniknovenie fibrilliatsii predserdii u sobak.

L-type Ca(2+) current (I(Ca,L)) has been shown to play a crucial role in initiation of early after depolarization (EAD) in cardiomyocytes. To study the possible role of EAD in spontaneous initiation of atrial fibrillation (AF), we tested the effects of L-type Ca(2+) channel blocker verapamil in canine models of cholinergic-dependent AF. In anesthetized open-chested dogs (n=13) spontaneous AF was induced by two methods: (1) perfusion with acetylcholine (ACh) in normal Tyrode solution at 9 ml/min into the sinus node artery (SNA) and (2) tonic stimulation of the right cervical vagus nerve (5 sec train). In the control, AF was induced in all dogs by perfusion with ACh (2.9+/-0.8 microM, mean+/-SEM) in 96+/-4% of attempts and by vagal stimulation (VS, 59+/-8 Hz) in 74+/-9% of attempts. Verapamil (0.2 mg/kg i.v.) did not alter the AF inducibility both during ACh perfusion and during VS (93+/-4% and 77+/-13%, NS, respectively) in dogs that retained sinus rhythm (n=8). However, verapamil significantly decreased AF inducibility to 50+/-4% and 21+/-13%, respectively, in dogs that passed to AV rhythm (n=5). Verapamil increased duration of both ACh- and vagally-mediated AF from 15+/-2 sec and 15+/-2 sec to 34+/-6 sec and 23+/-4 see (p<0.05 vs. control), respectively. The activation mapping (112 unipolar electrodes) during the initiation of AF did not reveal a difference in epicardial activation patterns before and after verapamil treatment. Inhibiting the I(Ca,L) by verapamil resulted in significant (p<0.05 vs. control) decrease in systolic and diastolic blood pressure, PQ interval prolongation and slowing down of the sinus rate. Verapamil did not affect atrial effective refractory period (AERP) and conduction velocity in the right atria. The reduction of AERP and the deceleration of heart rate by VS (8 Hz) remained unchangeable after verapamil treatment in comparison to control. Thus, the data suggest that the mechanism of spontaneous AF initiation during increased cholinergic activity is not related to EAD in atria.