Martin Pešl

Left Ventricular Mechanics in Idiopathic Dilated Cardiomyopathy: Systolic-Diastolic Coupling and Torsion

BACKGROUND In idiopathic dilated cardiomyopathy (IDC), myocardial deformational parameters and their mutual relationships remain incompletely characterized. METHODS Thirty-seven patients with IDC underwent two-dimensional speckle-tracking echocardiography (2D-STE) to assess left ventricular rotation, torsion, and longitudinal, circumferential, and radial systolic and diastolic strains and strain rates. Additionally, 2D-STE was performed in 14 controls. RESULTS All deformational parameters on 2D-STE were significantly lower in patients with IDC compared with controls. Seven patients exhibited opposite basal (positive, counterclockwise) and 11 patients exhibited opposite apical (negative, clockwise) rotation at end-systole. Circumferential, radial, and longitudinal early diastolic strain rates were correlated most strongly with the corresponding spatial components of systolic deformation. CONCLUSION In patients IDC, all torsional, systolic, and diastolic deformational parameters were decreased. Corresponding three-dimensional components of systolic and diastolic deformations were closely coupled. Considerable variation in the direction of basal and apical rotation exists in a subset of patients with IDC.

Estimation of left ventricular filling pressures by speckle tracking echocardiography in patients with idiopathic dilated cardiomyopathy

AIMS the ratio of early diastolic transmitral flow velocity (E) to early diastolic mitral annular velocity (E(a)) is frequently used to predict an increase in left ventricular filling pressure (LVFP). However, this approach has several limitations. The aim of this study was to test whether additional information is gained by new echocardiographic indexes utilizing strain and strain rate (SR) derived from 2-dimensional speckle tracking echocardiography (2D-STE) for the estimation of LVFP. METHODS AND RESULTS fifty-one patients with idiopathic dilated cardiomyopathy (IDC) underwent pulsed-wave tissue Doppler echocardiography and 2D-STE performed simultaneously with right heart catheterization. Receiver operating characteristic analysis showed that circumferential strain and the SR during late diastolic LV filling (0.956 and 0.951, both P = 0.001), E/circumferential SR at early diastolic LV filling (0.949, P = 0.001), and E/circumferential strain at the time of peak E-wave (0.948, P = 0.001) had greater area under the curve than the E/E(a) ratio (0.911, P = 0.001) for the prediction of pulmonary capillary wedge pressure > 12 mmHg. CONCLUSION when compared with the E/E(a) ratio, several 2D-STE-derived parameters better estimated the increase in LVFP in patients with IDC.

Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

Human pluripotent stem cells (hPSCs), namely, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs).

YAP regulates cell mechanics by controlling focal adhesion assembly.

Hippo effectors YAP/TAZ act as on–off mechanosensing switches by sensing modifications in extracellular matrix (ECM) composition and mechanics. The regulation of their activity has been described by a hierarchical model in which elements of Hippo pathway are under the control of focal adhesions (FAs). Here we unveil the molecular mechanism by which cell spreading and RhoA GTPase activity control FA formation through YAP to stabilize the anchorage of the actin cytoskeleton to the cell membrane. This mechanism requires YAP co-transcriptional function and involves the activation of genes encoding for integrins and FA docking proteins. Tuning YAP transcriptional activity leads to the modification of cell mechanics, force development and adhesion strength, and determines cell shape, migration and differentiation. These results provide new insights into the mechanism of YAP mechanosensing activity and qualify this Hippo effector as the key determinant of cell mechanics in response to ECM cues.

The role of exercise echocardiography in the diagnostics of heart failure with normal left ventricular ejection fraction

AIMS Few data are available on the exercise-induced abnormalities of myocardial function in patients with exertional dyspnoea and normal left ventricular ejection fraction (LV EF). The main aims of this study were to determine the prevalence of isolated exercise-induced heart failure with normal ejection fraction (HFNEF) and to assess whether disturbances in LV or right ventricular longitudinal systolic function are associated with the diagnosis of HFNEF. METHODS AND RESULTS Eighty-four patients with exertional dyspnoea and normal LV EF and 14 healthy controls underwent spirometry, NT-proBNP plasma analysis, and exercise echocardiography. Doppler LV inflow and tissue mitral and tricuspid annular velocities were analysed at rest and immediately after the termination of exercise. Of the 30 patients with the evidence of HFNEF, 6 (20%) patients had only isolated exercise-induced HFNEF. When compared with the remaining patients, those with HFNEF had a significantly lower resting and exercise peak mitral annular systolic velocity (Sa) and the mitral annular velocity during atrial contraction, lower exercise peak mitral annular velocity at early diastole, and lower exercise peak systolic velocity of tricuspid annular motion. The multivariate logistic regression analysis including both parameters standardly defining HFNEF and the new Doppler variables potentially associated with the diagnosis of HFNEF revealed that NT-proBNP, LV mass index, left atrial volume index, and Sa significantly and independently predict the diagnosis of HFNEF. CONCLUSION A significant proportion of patients require exercise to diagnose HFNEF. Sa appears to be a significant independent predictor of HFNEF, which may increase the diagnostic value of models utilizing the variables recommended by the European Society of Cardiology guidelines.

Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells.

In vitro human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes (CMs). Protocols for cardiac differentiation of hESCs and hiPSCs include formation of the three-dimensional cell aggregates called embryoid bodies (EBs). The traditional suspension method for EB formation from clumps of cells results in an EB population heterogeneous in size and shape. In this study we show that forced aggregation of a defined number of single cells on AggreWell plates gives a high number of homogeneous EBs that can be efficiently differentiated into functional CMs by application of defined growth factors in the media. For cardiac differentiation, we used three hESC lines and one hiPSC line. Our contracting EBs and the resulting CMs express cardiac markers, namely myosin heavy chain α and β, cardiac ryanodine receptor/calcium release channel, and cardiac troponin T, shown by real-time polymerase chain reaction and immunocytochemistry. Using Ca2+ imaging and atomic force microscopy, we demonstrate the functionality of RyR2 to release Ca2+ from the sarcoplasmic reticulum as well as reliability in contractile and beating properties of hESC-EBs and hiPSC-EBs upon the stimulation or inhibition of the β-adrenergic pathway.

Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing.

Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in many heart pathologies. Biosensing of these parameters represents an important tool in drug development and disease modeling. Human embryonic stem cells and especially patient specific induced pluripotent stem cell-derived cardiomyocytes are well established as cardiac disease model.. Here, a live stem cell derived embryoid body (EB) based cardiac cell syncytium served as a biorecognition element coupled to the microcantilever probe from atomic force microscope thus providing reliable micromechanical cellular biosensor suitable for whole-day testing. The biosensor was optimized regarding the type of cantilever, temperature and exchange of media; in combination with standardized protocol, it allowed testing of compounds and conditions affecting the biomechanical properties of EB. The studied effectors included calcium , drugs modulating the catecholaminergic fight-or-flight stress response such as the beta-adrenergic blocker metoprolol and the beta-adrenergic agonist isoproterenol. Arrhythmogenic effects were studied using caffeine. Furthermore, with EBs originating from patients stem cells, this biosensor can help to characterize heart diseases such as dystrophies.

HIF-1alpha Deficiency Attenuates the Cardiomyogenesis of Mouse Embryonic Stem Cells

Cardiac cell formation, cardiomyogenesis, is critically dependent on oxygen availability. It is known that hypoxia, a reduced oxygen level, modulates the in vitro differentiation of pluripotent cells into cardiomyocytes via hypoxia inducible factor-1alpha (HIF-1α)-dependent mechanisms. However, the direct impact of HIF-1α deficiency on the formation and maturation of cardiac-like cells derived from mouse embryonic stem cells (mESC) in vitro remains to be elucidated. In the present study, we demonstrated that HIF-1α deficiency significantly altered the quality and quantity of mESC-derived cardiomyocytes. It was accompanied with lower mRNA and protein levels of cardiac cell specific markers (myosin heavy chains 6 and 7) and with a decreasing percentage of myosin heavy chain α and β, and cardiac troponin T-positive cells. As to structural aspects of the differentiated cardiomyocytes, the localization of contractile proteins (cardiac troponin T, myosin heavy chain α and β) and the organization of myofibrils were also different. Simultaneously, HIF-1α deficiency was associated with a lower percentage of beating embryoid bodies. Interestingly, an observed alteration in the in vitro differentiation scheme of HIF-1α deficient cells was accompanied with significantly lower expression of the endodermal marker (hepatic nuclear factor 4 alpha). These findings thus suggest that HIF-1α deficiency attenuates spontaneous cardiomyogenesis through the negative regulation of endoderm development in mESC differentiating in vitro.

Mutation Frequency Dynamics in HPRT Locus in Culture-Adapted Human Embryonic Stem Cells and Induced Pluripotent Stem Cells Correspond to Their Differentiated Counterparts

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

Irreversible electroporation ablation for atrial fibrillation

Atrial fibrillation (AF) is one of the most important problems in modern cardiology. Thermal ablation therapies, especially radiofrequency ablation (RF), are currently “gold standard” to treat symptomatic AF by localized tissue necrosis. Despite the improvements in reestablishing sinus rhythm using available methods, both success rate and safety are limited by the thermal nature of procedures. Thus, while keeping the technique in clinical practice, safer and more versatile methods of removing abnormal tissue are being investigated. This review focuses on irreversible electroporation (IRE), a nonthermal ablation method, which is based on the unrecoverable permeabilization of cell membranes caused by short pulses of high voltage/current. While still in its preclinical steps for what concerns interventional cardiac electrophysiology, multiple studies have shown the efficacy of this method on animal models. The observed remodeling process shows this technique as tissue specific, triggering apoptosis rather than necrosis, and safer for the structures adjacent the myocardium. So far, proposed IRE methodologies are heterogeneous. The number of devices (both generators and applicators), techniques, and therapeutic goals impair the comparability of performed studies. More questions regarding systemic safety and optimal processes for AF treatment remain to be answered. This work provides an overview of the electroporation process, and presents different results obtained by cardiology‐oriented research groups that employ IRE ablation, with focus of AF‐related targets. This contribution on the topic aspires to be a practical guide to approach IRE ablation for cardiac arrhythmias, and to highlight controversial features and existing knowledge, to provide background for future improved experimentation with IRE in arrhythmology.