Multi-omics integration identifies key upstream regulators of pathomechanisms in hypertrophic cardiomyopathy due to truncating MYBPC3 mutations.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common genetic disease of the cardiac muscle, frequently caused by mutations in MYBPC3. However, little is known about the upstream pathways and key regulators causing the disease. Therefore, we employed a multi-omics approach to study the pathomechanisms underlying HCM comparing patient hearts harboring MYBPC3 mutations to control hearts. RESULTS: Using H3K27ac ChIP-seq and RNA-seq we obtained 9310 differentially acetylated regions and 2033 differentially expressed genes, respectively, between 13 HCM and 10 control hearts. We obtained 441 differentially expressed proteins between 11 HCM and 8 control hearts using proteomics. By integrating multi-omics datasets, we identified a set of DNA regions and genes that differentiate HCM from control hearts and 53 protein-coding genes as the major contributors. This comprehensive analysis consistently points toward altered extracellular matrix formation, muscle contraction, and metabolism. Therefore, we studied enriched transcription factor (TF) binding motifs and identified 9 motif-encoded TFs, including KLF15, ETV4, AR, CLOCK, ETS2, GATA5, MEIS1, RXRA, and ZFX. Selected candidates were examined in stem cell-derived cardiomyocytes with and without mutated MYBPC3. Furthermore, we observed an abundance of acetylation signals and transcripts derived from cardiomyocytes compared to non-myocyte populations. CONCLUSIONS: By integrating histone acetylome, transcriptome, and proteome profiles, we identified major effector genes and protein networks that drive the pathological changes in HCM with mutated MYBPC3. Our work identifies 38 highly affected protein-coding genes as potential plasma HCM biomarkers and 9 TFs as potential upstream regulators of these pathomechanisms that may serve as possible therapeutic targets.

[The Features of Healthy Life-Style Perception by Students of Medical Universities].

The article presents the results of medical sociological study carried out with the purpose of analyzing concepts and motivation attitudes of students of medical universities concerning healthy life-style and its components. The results of study demonstrate that students admitting importance of preservation of one's own health, are committed to unhealthy life-style and more often renounce sport involvement and are inactive in the area of diseases prevention and health promotion. It is emphasized that healthy life-style of students of medical universities is rather exception than prevalent practice.

ß-adrenergic stimulation augments transmural dispersion of repolarization via modulation of delayed rectifier currents IKs and IKr in the human ventricle.

Long QT syndrome (LQTS) is an inherited or drug induced condition associated with delayed repolarization and sudden cardiac death. The cardiac potassium channel, IKr, and the adrenergic-sensitive cardiac potassium current, IKs, are two primary contributors to cardiac repolarization. This study aimed to elucidate the role of ß-adrenergic (ß-AR) stimulation in mediating the contributions of IKr and IKs to repolarizing the human left ventricle (n = 18). Optical mapping was used to measure action potential durations (APDs) in the presence of the IKs blocker JNJ-303 and the IKr blocker E-4031. We found that JNJ-303 alone did not increase APD. However, under isoprenaline (ISO), both the application of JNJ-303 and additional E-4031 significantly increased APD. With JNJ-303, ISO decreased APD significantly more in the epicardium as compared to the endocardium, with subsequent application E-4031 increasing mid- and endocardial APD80 more significantly than in the epicardium. We found that ß-AR stimulation significantly augmented the effect of IKs blocker JNJ-303, in contrast to the reduced effect of IKr blocker E-4031. We also observed synergistic augmentation of transmural repolarization gradient by the combination of ISO and E-4031. Our results suggest ß-AR-mediated increase of transmural dispersion of repolarization, which could pose arrhythmogenic risk in LQTS patients.

Human Organotypic Cultured Cardiac Slices: New Platform For High Throughput Preclinical Human Trials.

Translation of novel therapies from bench to bedside is hampered by profound disparities between animal and human genetics and physiology. The ability to test for efficacy and cardiotoxicity in a clinically relevant human model system would enable more rapid therapy development. We have developed a preclinical platform for validation of new therapies in human heart tissue using organotypic slices isolated from donor and end-stage failing hearts. A major advantage of the slices when compared with human iPS-derived cardiomyocytes is that native tissue architecture and extracellular matrix are preserved, thereby allowing investigation of multi-cellular physiology in normal or diseased myocardium. To validate this model, we used optical mapping of transmembrane potential and calcium transients. We found that normal human electrophysiology is preserved in slice preparations when compared with intact hearts, including slices obtained from the region of the sinus node. Physiology is maintained in slices during culture, enabling testing the acute and chronic effects of pharmacological, gene, cell, optogenetic, device, and other therapies. This methodology offers a powerful high-throughput platform for assessing the physiological response of the human heart to disease and novel putative therapies.

Technical advances in studying cardiac electrophysiology - Role of rabbit models.

Cardiovascular research has made a major contribution to an unprecedented 10 year increase in life expectancy during the last 50 years: most of this increase due to a decline in mortality from heart disease and stroke. The majority of the basic cardiovascular science discoveries, which have led to this impressive extension of human life, came from investigations conducted in various small and large animal models, ranging from mouse to pig. The small animal models are currently popular because they are amenable to genetic engineering and are relatively inexpensive. The large animal models are favored at the translational stage of the investigation, as they are anatomically and physiologically more proximal to humans, and can be implanted with various devices; however, they are expensive and less amenable to genetic manipulations. With the advent of CRISPR genetic engineering technology and the advances in implantable bioelectronics, the large animal models will continue to advance. The rabbit model is particularly poised to become one of the most popular among the animal models that recapitulate human heart diseases. Here we review an array of the rabbit models of atrial and ventricular arrhythmias, as well as a range of the imaging and device technologies enabling these investigations.

Arrhythmogenic and metabolic remodelling of failing human heart.

Heart failure (HF) is a major cause of morbidity and mortality worldwide. The global burden of HF continues to rise, with prevalence rates estimated at 1-2% and incidence approaching 5-10 per 1000 persons annually. The complex pathophysiology of HF impacts virtually all aspects of normal cardiac function - from structure and mechanics to metabolism and electrophysiology - leading to impaired mechanical contraction and sudden cardiac death. Pharmacotherapy and device therapy are the primary methods of treating HF, but neither is able to stop or reverse disease progression. Thus, there is an acute need to translate basic research into improved HF therapy. Animal model investigations are a critical component of HF research. However, the translation from cellular and animal models to the bedside is hampered by significant differences between species and among physiological scales. Our studies over the last 8 years show that hypotheses generated in animal models need to be validated in human in vitro models. Importantly, however, human heart investigations can establish translational platforms for safety and efficacy studies before embarking on costly and risky clinical trials. This review summarizes recent developments in human HF investigations of electrophysiology remodelling, metabolic remodelling, and ß-adrenergic remodelling and discusses promising new technologies for HF research.

Structure-function relationship in the sinus and atrioventricular nodes.

Recently published optical mapping studies of larger mammals, including humans, have identified functionally discrete sinoatrial exit pathways of activation. This is in line with earlier mapping studies of the dog and the human but in contrast with findings in the mouse and the rabbit, wherein a propagation wave front pattern of activation has been described. It underpins the complex three-dimensional (3D) organization of the cardiac pacemaking and conduction system in larger species, wherein sinoatrial and atrioventricular nodal physiologies both demonstrate identifiable activation pathways, which coincide with anatomic landmarks and histologic architecture, so that in addition to muscle fiber orientation and cell coupling, these intrinsic factors act to determine excitation pathways. This complex 3D organization increases the effect of source-to-sink mismatch both by greater variability in the space constant of tissue and by the 3D projection of this effect in all directions. Mathematical modeling provides a means to study these interactions, and newer models should incorporate these additional factors and their effect into the 3D structure of large mammal physiology.

Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): standardised reporting for model reproducibility, interoperability, and data sharing.

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.

Rabbit-specific ventricular model of cardiac electrophysiological function including specialized conduction system.

The function of the ventricular specialized conduction system in the heart is to ensure the coordinated electrical activation of the ventricles. It is therefore critical to the overall function of the heart, and has also been implicated as an important player in various diseases, including lethal ventricular arrhythmias such as ventricular fibrillation and drug-induced torsades de pointes. However, current ventricular models of electrophysiology usually ignore, or include highly simplified representations of the specialized conduction system. Here, we describe the development of an image-based, species-consistent, anatomically-detailed model of rabbit ventricular electrophysiology that incorporates a detailed description of the free-running part of the specialized conduction system. Techniques used for the construction of the geometrical model of the specialized conduction system from a magnetic resonance dataset and integration of the system model into a ventricular anatomical model, developed from the same dataset, are described. Computer simulations of rabbit ventricular electrophysiology are conducted using the novel anatomical model and rabbit-specific membrane kinetics to investigate the importance of the components and properties of the conduction system in determining ventricular function under physiological conditions. Simulation results are compared to panoramic optical mapping experiments for model validation and results interpretation. Full access is provided to the anatomical models developed in this study.

Molecular architecture of the human specialised atrioventricular conduction axis.

The atrioventricular conduction axis, located in the septal component of the atrioventricular junctions, is arguably the most complex structure in the heart. It fulfils a multitude of functions, including the introduction of a delay between atrial and ventricular systole and backup pacemaking. Like any other multifunctional tissue, complexity is a key feature of this specialised tissue in the heart, and this complexity is both anatomical and electrophysiological, with the two being inextricably linked. We used quantitative PCR, histology and immunohistochemistry to analyse the axis from six human subjects. mRNAs for ~50 ion and gap junction channels, Ca(2+)-handling proteins and markers were measured in the atrial muscle (AM), a transitional area (TA), inferior nodal extension (INE), compact node (CN), penetrating bundle (PB) and ventricular muscle (VM). When compared to the AM, we found a lower expression of Na(v)1.5, K(ir)2.1, Cx43 and ANP mRNAs in the CN for example, but a higher expression of HCN1, HCN4, Ca(v)1.3, Ca(v)3.1, K(ir)3.4, Cx40 and Tbx3 mRNAs. Expression of some related proteins was in agreement with the expression of the corresponding mRNAs. There is a complex and heterogeneous pattern of expression of ion and gap junction channels and Ca(2+)-handling proteins in the human atrioventricular conduction axis that explains the function of this crucial pathway.

[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.

[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.

[Ten-year outcomes of lymphogranulomatosis treatment according to the protocol MOPP-ABVD+radiotherapy].

AIM: To analyse overall recurrence-free survival of lymphogranulomatosis (LGM) patients given polychemotherapy (PCT) MOPP (mustargen-caryolisin, vincristine, natulan, prednisolone) - ABVD (adriamycin, bleomycin, vinblastin, dacarbasin) in combination with radiotherapy (RT) for 10 years. MATERIAL AND METHODS: The trial included 211 LGM patients admitted to Hematological Research Center in 1990-1996 from other hospitals without random selection. The patients were examined by the standard program including biopsy of the affected organ or lymph node, bilateral trephine biopsy. Splenectomy was performed in 17 patients, 83 patients received PCT in other hospitals, 128 untreated patients received MOPP-ABVD therapy (3 courses of MOPP and 3 courses of ABVD). Forty one patients had defects in PCT, 16 of them rejected PCT and RT. The latter was performed 4 weeks after the 6th course, contraceptives were not prescribed to women. At LGM stage II-III RT was performed by the subradical program (no radiation to ilioinguinal lymph nodes) in doses 40-44 Gy on the foci and 32-36 Gy preventively, on massive and residual foci after PCT - 5-10 Gy additionally. RESULTS: Ten-year overall and recurrence-free survival in the untreated group reached 83 and 80%, respectively, for pretreated patients - 46 and 36%, respectively. Causes of death of 26 patients were LGM progression, infection (tuberculosis, as a rule), secondary tumors and acute myeloblastic leukemia (AML). After remission 25 women gave birth to a healthy child and 12 healthy children were born to 9 males. CONCLUSION: MOPP-ABVD plus radiotherapy program according to subradical and radical variants was in the past effective but invalidating rescue therapy. Present-day programs consider the histological variant, stage and prognostic factors allowing an individual therapeutic approach with step-by-step reduction of RT in the treatment of LGM patients. Involvement of the bone marrow in primary patients had no influence on the treatment results. This refers this affection not to a generalized stage IV, but to stage III along with involvement of the lymph nodes and the spleen.

Connexins in the sinoatrial and atrioventricular nodes.

The sinoatrial node (SAN) and the atrioventricular node (AVN) are specialized tissues in the heart: the SAN is specialized for pacemaking (it is the pacemaker of the heart), whereas the AVN is specialized for slow conduction of the action potential (to introduce a delay between atrial and ventricular activation during the cardiac cycle). These functions have special requirements regarding electrical coupling and, therefore, expression of connexin isoforms. Electrical coupling in the center of the SAN should be weak to protect it from the inhibitory electrotonic influence of the more hyperpolarized non-pacemaking atrial muscle surrounding the SAN. However, for the SAN to be able to drive the atrial muscle, electrical coupling should be strong in the periphery of the SAN. Consistent with this, in the center of the SAN there is no expression of Cx43 (the principal connexin of the working myocardium) and little expression of Cx40, but there is expression of Cx45 and Cx30.2, whereas in the periphery of the SAN Cx43 as well Cx45 is expressed. In the AVN, there is a similar pattern of expression of connexins as in the center of the SAN and this is likely to be in large part responsible for the slow conduction of the action potential.

4D embryonic cardiography using gated optical coherence tomography.

Simultaneous imaging of very early embryonic heart structure and function has technical limitations of spatial and temporal resolution. We have developed a gated technique using optical coherence tomography (OCT) that can rapidly image beating embryonic hearts in four-dimensions (4D), at high spatial resolution (10-15 mum), and with a depth penetration of 1.5 - 2.0 mm that is suitable for the study of early embryonic hearts. We acquired data from paced, excised, embryonic chicken and mouse hearts using gated sampling and employed image processing techniques to visualize the hearts in 4D and measure physiologic parameters such as cardiac volume, ejection fraction, and wall thickness. This technique is being developed to longitudinally investigate the physiology of intact embryonic hearts and events that lead to congenital heart defects.

[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.