Publications by authors named "Guido Caluori"

21 Publications

  • Page 1 of 1

Local electromechanical alterations determine the left ventricle rotational dynamics in CRT-eligible heart failure patients.

Sci Rep 2021 Feb 5;11(1):3267. Epub 2021 Feb 5.

Department of Cardiology and Structural Heart Disease, Medical University of Silesia, Ziołowa 45-47, Katowice, Poland.

Left ventricle, LV wringing wall motion relies on physiological muscle fiber orientation, fibrotic status, and electromechanics (EM). The loss of proper EM activation can lead to rigid-body-type (RBT) LV rotation, which is associated with advanced heart failure (HF) and challenges in resynchronization. To describe the EM coupling and scar tissue burden with respect to rotational patterns observed on the LV in patients with ischemic heart failure with reduced ejection fraction (HFrEF) left bundle branch block (LBBB). Thirty patients with HFrEF/LBBB underwent EM analysis of the left ventricle using an invasive electro-mechanical catheter mapping system (NOGA XP, Biosense Webster). The following parameters were evaluated: rotation angle; rotation velocity; unipolar/bipolar voltage; local activation time, LAT; local electro-mechanical delay, LEMD; total electro-mechanical delay, TEMD. Patients underwent late-gadolinium enhancement cMRI when possible. The different LV rotation pattern served as sole parameter for patients' grouping into two categories: wringing rotation (Group A, n = 6) and RBT rotation (Group B, n = 24). All parameters were aggregated into a nine segment, three sector and whole LV models, and compared at multiple scales. Segmental statistical analysis in Group B revealed significant inhomogeneities, across the LV, regarding voltage level, scar burdening, and LEMD changes: correlation analysis showed correspondently a loss of synchronization between electrical (LAT) and mechanical activation (TEMD). On contrary, Group A (relatively low number of patients) did not present significant differences in LEMD across LV segments, therefore electrical (LAT) and mechanical (TEMD) activation were well synchronized. Fibrosis burden was in general associated with areas of low voltage. The rotational behavior of LV in HF/LBBB patients is determined by the local alteration of EM coupling. These findings serve as a strong basic groundwork for a hypothesis that EM analysis may predict CRT response.Clinical trial registration: SUM No. KNW/0022/KB1/17/15.
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http://dx.doi.org/10.1038/s41598-021-82793-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865069PMC
February 2021

AC Pulsed Field Ablation Is Feasible and Safe in Atrial and Ventricular Settings: A Proof-of-Concept Chronic Animal Study.

Front Bioeng Biotechnol 2020 3;8:552357. Epub 2020 Dec 3.

International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czechia.

Introduction: Pulsed field ablation (PFA) exploits the delivery of short high-voltage shocks to induce cells death via irreversible electroporation. The therapy offers a potential paradigm shift for catheter ablation of cardiac arrhythmia. We designed an AC-burst generator and therapeutic strategy, based on the existing knowledge between efficacy and safety among different pulses. We performed a proof-of-concept chronic animal trial to test the feasibility and safety of our method and technology.

Methods: We employed 6 female swine - weight 53.75 ± 4.77 kg - in this study. With fluoroscopic and electroanatomical mapping assistance, we performed ECG-gated AC-PFA in the following settings: in the left atrium with a decapolar loop catheter with electrodes connected in bipolar fashion; across the interventricular septum applying energy between the distal electrodes of two tip catheters. After procedure and 4-week follow-up, the animals were euthanized, and the hearts were inspected for tissue changes and characterized. We perform finite element method simulation of our AC-PFA scenarios to corroborate our method and better interpret our findings.

Results: We applied square, 50% duty cycle, AC bursts of 100 μs duration, 100 kHz internal frequency, 900 V for 60 pulses in the atrium and 1500 V for 120 pulses in the septum. The inter-burst interval was determined by the native heart rhythm - 69 ± 9 bpm. Acute changes in the atrial and ventricular electrograms were immediately visible at the sites of AC-PFA - signals were elongated and reduced in amplitude ( < 0.0001) and tissue impedance dropped ( = 0.011). No adverse event (e.g., esophageal temperature rises or gas bubble streams) was observed - while twitching was avoided by addition of electrosurgical return electrodes. The implemented numerical simulations confirmed the non-thermal nature of our AC-PFA and provided specific information on the estimated treated area and need of pulse trains. The postmortem chest inspection showed no peripheral damage, but epicardial and endocardial discolorations at sites of ablation. T1-weighted scans revealed specific tissue changes in atria and ventricles, confirmed to be fibrotic scars via trichrome staining. We found isolated, transmural and continuous scars. A surviving cardiomyocyte core was visible in basal ventricular lesions.

Conclusion: We proved that our method and technology of AC-PFA is feasible and safe for atrial and ventricular myocardial ablation, supporting their systematic investigation into effectiveness evaluation for the treatment of cardiac arrhythmia. Further optimization, with energy titration or longer follow-up, is required for a robust atrial and ventricular AC-PFA.
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http://dx.doi.org/10.3389/fbioe.2020.552357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744788PMC
December 2020

YAP-TEAD1 control of cytoskeleton dynamics and intracellular tension guides human pluripotent stem cell mesoderm specification.

Cell Death Differ 2021 Apr 28;28(4):1193-1207. Epub 2020 Oct 28.

International Clinical Research Center (ICRC) of St Anne's University Hospital, CZ-65691, Brno, Czech Republic.

The tight regulation of cytoskeleton dynamics is required for a number of cellular processes, including migration, division and differentiation. YAP-TEAD respond to cell-cell interaction and to substrate mechanics and, among their downstream effects, prompt focal adhesion (FA) gene transcription, thus contributing to FA-cytoskeleton stability. This activity is key to the definition of adult cell mechanical properties and function. Its regulation and role in pluripotent stem cells are poorly understood. Human PSCs display a sustained basal YAP-driven transcriptional activity despite they grow in very dense colonies, indicating these cells are insensitive to contact inhibition. PSC inability to perceive cell-cell interactions can be restored by tampering with Tankyrase enzyme, thus favouring AMOT inhibition of YAP function. YAP-TEAD complex is promptly inactivated when germ layers are specified, and this event is needed to adjust PSC mechanical properties in response to physiological substrate stiffness. By providing evidence that YAP-TEAD1 complex targets key genes encoding for proteins involved in cytoskeleton dynamics, we suggest that substrate mechanics can direct PSC specification by influencing cytoskeleton arrangement and intracellular tension. We propose an aberrant activation of YAP-TEAD1 axis alters PSC potency by inhibiting cytoskeleton dynamics, thus paralyzing the changes in shape requested for the acquisition of the given phenotype.
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http://dx.doi.org/10.1038/s41418-020-00643-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027678PMC
April 2021

Multiscale Analysis of Extracellular Matrix Remodeling in the Failing Heart.

Circ Res 2021 Jan 27;128(1):24-38. Epub 2020 Oct 27.

International Clinical Research Center, St. Anne's University Hospital Brno, Czech Republic (A.R.P., J.O.-D.L.C., F.M., V.H., G.C., O.P., V.V., S.P., K.K., G.F.).

Rationale: Cardiac ECM (extracellular matrix) comprises a dynamic molecular network providing structural support to heart tissue function. Understanding the impact of ECM remodeling on cardiac cells during heart failure (HF) is essential to prevent adverse ventricular remodeling and restore organ functionality in affected patients.

Objectives: We aimed to (1) identify consistent modifications to cardiac ECM structure and mechanics that contribute to HF and (2) determine the underlying molecular mechanisms.

Methods And Results: We first performed decellularization of human and murine ECM (decellularized ECM) and then analyzed the pathological changes occurring in decellularized ECM during HF by atomic force microscopy, 2-photon microscopy, high-resolution 3-dimensional image analysis, and computational fluid dynamics simulation. We then performed molecular and functional assays in patient-derived cardiac fibroblasts based on YAP (yes-associated protein)-transcriptional enhanced associate domain (TEAD) mechanosensing activity and collagen contraction assays. The analysis of HF decellularized ECM resulting from ischemic or dilated cardiomyopathy, as well as from mouse infarcted tissue, identified a common pattern of modifications in their 3-dimensional topography. As compared with healthy heart, HF ECM exhibited aligned, flat, and compact fiber bundles, with reduced elasticity and organizational complexity. At the molecular level, RNA sequencing of HF cardiac fibroblasts highlighted the overrepresentation of dysregulated genes involved in ECM organization, or being connected to TGFβ1 (transforming growth factor β1), interleukin-1, TNF-α, and BDNF signaling pathways. Functional tests performed on HF cardiac fibroblasts pointed at mechanosensor YAP as a key player in ECM remodeling in the diseased heart via transcriptional activation of focal adhesion assembly. Finally, in vitro experiments clarified pathological cardiac ECM prevents cell homing, thus providing further hints to identify a possible window of action for cell therapy in cardiac diseases.

Conclusions: Our multiparametric approach has highlighted repercussions of ECM remodeling on cell homing, cardiac fibroblast activation, and focal adhesion protein expression via hyperactivated YAP signaling during HF.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317685DOI Listing
January 2021

Nanotechnology and stem cells in vascular biology.

Vasc Biol 2019 24;1(1):H103-H109. Epub 2019 Sep 24.

Interventional Cardiac Electrophysiology Group, International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic.

Nanotechnology and stem cells are one of the most promising strategies for clinical medicine applications. The article provides an up-to-date view on advances in the field of regenerative and targeted vascular therapies describing a molecular design (propulsion mechanism, composition, target identification) and applications of nanorobots. Stem cell paragraph presents current clinical application of various cell types involved in vascular biology including mesenchymal stem cells, very small embryonic-like stem cells, induced pluripotent stem cells, mononuclear stem cells, amniotic fluid-derived stem cells and endothelial progenitor cells. A possible bridging between the two fields is also envisioned, where bio-inspired, safe, long-lasting nanorobots can fully target the cellular specific cues and even drive vascular process in a timely manner.
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http://dx.doi.org/10.1530/VB-19-0021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439937PMC
September 2019

DMD Pluripotent Stem Cell Derived Cardiac Cells Recapitulate Human Cardiac Pathophysiology.

Front Bioeng Biotechnol 2020 19;8:535. Epub 2020 Jun 19.

Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czechia.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by the lack of functional dystrophin. DMD is associated with progressive dilated cardiomyopathy, eventually leading to heart failure as the main cause of death in DMD patients. Although several molecular mechanisms leading to the DMD cardiomyocyte (DMD-CM) death were described, mostly in mouse model, no suitable human CM model was until recently available together with proper clarification of the DMD-CM phenotype and delay in cardiac symptoms manifestation. We obtained several independent dystrophin-deficient human pluripotent stem cell (hPSC) lines from DMD patients and CRISPR/Cas9-generated DMD gene mutation. We differentiated DMD-hPSC into cardiac cells (CC) creating a human DMD-CC disease model. We observed that mutation-carrying cells were less prone to differentiate into CCs. DMD-CCs demonstrated an enhanced cell death rate in time. Furthermore, ion channel expression was altered in terms of potassium (Kir2.1 overexpression) and calcium handling (dihydropyridine receptor overexpression). DMD-CCs exhibited increased time of calcium transient rising compared to aged-matched control, suggesting mishandling of calcium release. We observed mechanical impairment (hypocontractility), bradycardia, increased heart rate variability, and blunted β-adrenergic response connected with remodeling of β-adrenergic receptors expression in DMD-CCs. Overall, these results indicated that our DMD-CC models are functionally affected by dystrophin-deficiency associated and recapitulate functional defects and cardiac wasting observed in the disease. It offers an accurate tool to study human cardiomyopathy progression and test therapies .
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http://dx.doi.org/10.3389/fbioe.2020.00535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325914PMC
June 2020

Cardiovascular progenitor cells and tissue plasticity are reduced in a myocardium affected by Becker muscular dystrophy.

Orphanet J Rare Dis 2020 03 5;15(1):65. Epub 2020 Mar 5.

Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, Brno, 62500, Czech Republic.

We describe the association of Becker muscular dystrophy (BMD) derived heart failure with the impairment of tissue homeostasis and remodeling capabilities of the affected heart tissue. We report that BMD heart failure is associated with a significantly decreased number of cardiovascular progenitor cells, reduced cardiac fibroblast migration, and ex vivo survival.

Background: Becker muscular dystrophy belongs to a class of genetically inherited dystrophin deficiencies. It affects male patients and results in progressive skeletal muscle degeneration and dilated cardiomyopathy leading to heart failure. It is a relatively mild form of dystrophin deficiency, which allows patients to be on a heart transplant list. In this unique situation, the explanted heart is a rare opportunity to study the degenerative process of dystrophin-deficient cardiac tissue. Heart tissue was excised, dissociated, and analyzed. The fractional content of c-kit/CD45 cardiovascular progenitor cells (CVPCs) and cardiac fibroblast migration were compared to control samples of atrial tissue. Control tissue was obtained from the hearts of healthy organ donor's during heart transplantation procedures.

Results: We report significantly decreased CVPCs (c-kit/CD45) throughout the heart tissue of a BMD patient, and reduced numbers of phase-bright cells presenting c-kit positivity in the dystrophin-deficient cultured explants. In addition, ex vivo CVPCs survival and cardiac fibroblasts migration were significantly reduced, suggesting reduced homeostatic support and irreversible tissue remodeling.

Conclusions: Our findings associate genetically derived heart failure in a dystrophin-deficient patient with decreased c-kit/CD45 CVPCs and their resilience, possibly hinting at a lack of cardioprotective capability and/or reduced homeostatic support. This also correlates with reduced plasticity of the explanted cardiac tissue, related to the process of irreversible remodeling in the BMD patient's heart.
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http://dx.doi.org/10.1186/s13023-019-1257-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057505PMC
March 2020

Bipolar ablation with contact force-sensing of swine ventricles shows improved acute lesion features compared to sequential unipolar ablation.

J Cardiovasc Electrophysiol 2020 05 2;31(5):1128-1136. Epub 2020 Mar 2.

Interventional Cardiac Electrophysiology, International Clinical Research Center, St Anne's University Hospital Brno, Brno, Czech Republic.

Introduction: Despite technical progress, ventricular tachycardia (VT) recurrence after unipolar ablation remains relatively high (12%-47%). Bipolar ablation has been proposed as an appealing solution that may overcome limitations associated with unipolar ablation settings. We designed an animal study to compare bipolar (BPA) vs sequential unipolar ablation (UPA) using contact force-sensing technology on both ablation catheters.

Methods: Twenty large white female pigs (6-months-old, 50-60 kg) underwent multiple RF ablations (30 W, 60 seconds, 30 mL/min irrigation) on the ventricular myocardium from the epicardial and endocardial sides. The hearts were fixed and scanned with high-resolution cardiac magnetic resonance imaging. Thermal lesions were located and characterized in volume, depth, width, and transmurality.

Results: Lesion volume was calculated as the sum of epicardial or endocardial conjoined/isolated lesions at one location. Linear dimensions (width and depth) were measured twice for each location, on the endocardial and epicardial side. We evaluated 35 lesions across the intraventricular septum (UPA, N = 17 vs BPA, N = 18). No difference in volume, linear dimensions or impedance drop was observed in this area between UPA and BPA. However, BPA required half RF time and showed an increased transmurality trend. We then analyzed 73 lesions from the endocardial side (UPA, N = 35 vs BPA, N = 38) and 50 from the epicardial side (UPA, N = 11 vs BPA N = 39) of the ventricular free walls. Lesion transmurality was markedly improved by BPA (P = .030, odds ratio, 23.73 [4.71,31.96]). Ventricular BPA lesions were significantly deeper on the epicardial side (P < .0001) and endocardial side (P = .015).

Conclusion: Bipolar ablation is more likely to create transmural and epicardial lesions in the ventricle wall. Half the time is needed for the creation of comparably deep and large lesions.
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http://dx.doi.org/10.1111/jce.14407DOI Listing
May 2020

Comparison of atrial fibrillation ablation efficacy using remote magnetic navigation vs. manual navigation with contact-force control.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2020 Dec 10;164(4):387-393. Epub 2019 Oct 10.

International Clinical Research Center, Interventional Cardiac Electrophysiology, St. Anne's University Hospital Brno, Czech Republic.

Aims: This study aims to compare procedural parameters and clinical efficacy of remote magnetic navigation (RMN) vs. manual navigation (MAN) approach for radiofrequency ablation (RFA) in patients with atrial fibrillation (AF).

Methods: 146 patients with AF were enrolled in the study. In the RMN group (n=57), patients were treated with the CARTO® 3 in combination with the Niobe ES system. In the MAN group (n=89), ablation was performed with the EnSite Velocity and TactiCath™ Quartz catheter with direct contact force measurement. Procedural time, ablation time, fluoroscopy time, radiation dose and ablation counts were measured and compared between the groups. Recurrence of AF was evaluated after 6 months of follow-up.

Results: Mean procedure times (236.87±64.31 vs. 147.22±45.19 min, P<0.05), counts of RF applications (74.30±24.77 vs. 49.15±20.33, P<0.05) and total RFA times (4323.39±1426.69 vs. 2780.53±1157.85 s, P<0.05) were all significantly higher in the RMN than in the MAN group, respectively. In the same order, mean X-ray dose (9722.6±7507.4 vs. 8087.9±6051.5 mGy/cm, P=0.12) and mean total X-ray exposure time (8.07±4.20 vs. 9.54±5.47 min, P=0.08) were not statistically different. At 6-month follow-up, freedom from AF was similar in RMN and MAN group for paroxysmal (60.8% and 73%, respectively, P=0.42) and persistent AF (69.6% and 75.0%, respectively, P=0.77).

Conclusions: Due to the fact that mid-term clinical outcomes showed no significant differences in AF recurrences between groups and manual ablation strategy provided more favorable results regarding acute procedural parameters, we can conclude that the remote magnetic navigation is not superior to the manual approach.
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http://dx.doi.org/10.5507/bp.2019.045DOI Listing
December 2020

Remotely Navigated Ablations in Ventricle Myocardium Result in Acute Lesion Size Comparable to Force-Sensing Manual Navigation.

Circ Arrhythm Electrophysiol 2019 10 30;12(10):e007644. Epub 2019 Sep 30.

Interventional Cardiac Electrophysiology (J.J., G.C., T.J., F.L., M.P., T.K., Z.S.), International Clinical Research Center of St Anne's University Hospital, Brno, Czech Republic.

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http://dx.doi.org/10.1161/CIRCEP.119.007644DOI Listing
October 2019

Cell-Laden Hydrogel as a Clinical-Relevant 3D Model for Analyzing Neuroblastoma Growth, Immunophenotype, and Susceptibility to Therapies.

Front Immunol 2019 9;10:1876. Epub 2019 Aug 9.

CNR-IEIIT Institute, National Research Council of Italy, Genoa, Italy.

High risk Neuroblastoma (NB) includes aggressive, metastatic solid tumors of childhood. The survival rate improved only modestly, despite the use of combination therapies including novel immunotherapies based on the antibody-mediated targeting of tumor-associated surface ligands. Treatment failures may be due to the lack of adequate models for studying, in a given patient, the efficacy of potential therapeutics, including those aimed to enhance anti-tumor immune responses. We here propose a 3D alginate-based hydrogel as extracellular microenvironment to evaluate the effects of the three-dimensionality on biological and immunological properties of NB cells. NB cell lines grown within the 3D alginate spheres presented spheroid morphology, optimal survival, and proliferation capabilities, and a reduced sensitivity to the cytotoxic effect of imatinib mesylate. 3D cultured NB cells were also evaluated for the constitutive and IFN-γ-induced expression of surface molecules capable of tuning the anti-tumor activity of NK cells including immune checkpoint ligands. In particular, IFN-γ induced de novo expression of high amounts of HLA-I molecules, which protected NB cells from the attack mediated by KIR/KIR-L matched NK cells. Moreover, in the 3D alginate spheres, the cytokine increased the expression of the immune checkpoint ligands PD-Ls and B7-H3 while virtually abrogating that of PVR, a ligand of DNAM-1 activating receptor, whose expression correlates with high susceptibility to NK-mediated killing. Our 3D model highlighted molecular features that more closely resemble the immunophenotypic variants occurring and not fully appreciated in classical 2D culture conditions. Thus, based on our results, 3D alginate-based hydrogels might represent a clinical-relevant cell culture platform where to test the efficacy of personalized therapeutic approaches aimed to optimize the current and innovative immune based therapies in a very systematic and reliable way.
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http://dx.doi.org/10.3389/fimmu.2019.01876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697063PMC
October 2020

Comparing the incidence of ventricular arrhythmias during epicardial ablation in swine versus canine models.

Pacing Clin Electrophysiol 2019 07 29;42(7):862-867. Epub 2019 Apr 29.

International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic.

Background: Choosing the appropriate animal model for development of novel technologies requires an understanding of anatomy and physiology of these different models. There are little data about the characteristics of different animal models for the study of technologies used for epicardial ablation. We aimed to compare the incidence of ventricular arrhythmias during epicardial radiofrequency ablation between swine and canine models using novel epicardial ablation catheters.

Methods: We conducted a retrospective study using data obtained from epicardial ablation experiments performed on swine (Sus Scrofa) and canine (Canis familiaris) models. We compared the incidence of ventricular arrhythmias during ablation between swine and canine using multivariate regression analysis. Six swine and six canine animals underwent successful epicardial radiofrequency ablation. A total of 103 ablation applications were recorded.

Results: Ventricular arrhythmias requiring cardioversion occurred in 13.11% of radiofrequency ablation applications in swine and 9.75% in canine (relative risk: 117.6%, 95% confidence interval [CI]: 83.97-164.69, animal-based odds ratio [OR]: .55, 95% CI: .23-61.33; P = .184). When adjusting for application position, duration of ablation and power, the odds of developing potentially lethal ventricular arrhythmia in swine increased significantly compared to canine (OR: 3.60, 95% CI: 1.35-9.55; P = .010).

Conclusions: The swine myocardium is more susceptible to developing ventricular arrhythmias compared to canine model during epicardial ablation. This issue should be carefully considered in future studies.
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http://dx.doi.org/10.1111/pace.13698DOI Listing
July 2019

Simultaneous AFM Investigation of the Single Cardiomyocyte Electro-Chemo-Mechanics During Excitation-Contraction Coupling.

Methods Mol Biol 2019 ;1886:355-367

Department of Informatics, Bioengineering, Robotics, and Systems Engineering (DIBRIS), Università degli Studi di Genova, Genova, Italy.

The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.
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http://dx.doi.org/10.1007/978-1-4939-8894-5_21DOI Listing
June 2019

Biomechanical Characterization of Human Pluripotent Stem Cell-Derived Cardiomyocytes by Use of Atomic Force Microscopy.

Methods Mol Biol 2019 ;1886:343-353

Faculty of Medicine, Department of Biology, Masaryk University, Brno, Czech Republic.

Atomic force microscopy (AFM) is not only a high-resolution imaging technique but also a sensitive tool able to study biomechanical properties of bio-samples (biomolecules, cells) in native conditions-i.e., in buffered solutions (culturing media) and stable temperature (mostly 37 °C). Micromechanical transducers (cantilevers) are often used to map surface stiffness distribution, adhesion forces, and viscoelastic parameters of living cells; however, they can also be used to monitor time course of cardiomyocytes contraction dynamics (e.g. beating rate, relaxation time), together with other biomechanical properties. Here we describe the construction of an AFM-based biosensor setup designed to study the biomechanical properties of cardiomyocyte clusters, through the use of standard uncoated silicon nitride cantilevers. Force-time curves (mechanocardiograms, MCG) are recorded continuously in real time and in the presence of cardiomyocyte-contraction affecting drugs (e.g., isoproterenol, metoprolol) in the medium, under physiological conditions. The average value of contraction force and the beat rate, as basic biomechanical parameters, represent pharmacological indicators of different phenotype features. Robustness, low computational requirements, and optimal spatial sensitivity (detection limit 200 pN, respectively 20 nm displacement) are the main advantages of the presented method.
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http://dx.doi.org/10.1007/978-1-4939-8894-5_20DOI Listing
June 2019

Non-invasive electromechanical cell-based biosensors for improved investigation of 3D cardiac models.

Biosens Bioelectron 2019 Jan 16;124-125:129-135. Epub 2018 Oct 16.

Department of Informatics, Bioengineering Robotics and Systems Engineering, University of Genova, Via All'Opera Pia, 13, 16145 Genova, Italy. Electronic address:

Cardiomyocytes (CM) placed on microelectrode array (MEA) were simultaneously probed with cantilever from atomic force microscope (AFM) system. This electric / nanomechanical combination in real time recorded beating force of the CMs cluster and the triggering electric events. Such "organ-on-a-chip" represents a tool for drug development and disease modeling. The human pluripotent stem cells included the WT embryonic line CCTL14 and the induced dystrophin deficient line reprogrammed from fibroblasts of a patient affected by Duchenne Muscular Dystrophy (DMD, complete loss of dystrophin expression). Both were differentiated to CMs and employed with the AFM/MEA platform for diseased CMs' drug response testing and DMD characterization. The dependence of cardiac parameters on extracellular Ca was studied. The differential evaluation explained the observed effects despite variability of biological samples. The β-adrenergic stimulation (isoproterenol) and antagonist trials (verapamil) addressed ionotropic and chronotropic cell line-dependent features. For the first time, a distinctive beating-force relation for DMD CMs was measured on the 3D cardiac in vitro model.
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http://dx.doi.org/10.1016/j.bios.2018.10.021DOI Listing
January 2019

Advanced and Rationalized Atomic Force Microscopy Analysis Unveils Specific Properties of Controlled Cell Mechanics.

Front Physiol 2018 17;9:1121. Epub 2018 Aug 17.

International Clinical Research Center of the St. Anne's University Hospital Brno (FNUSA-ICRC), Center for Translational Medicine, Brno, Czechia.

The cell biomechanical properties play a key role in the determination of the changes during the essential cellular functions, such as contraction, growth, and migration. Recent advances in nano-technologies have enabled the development of new experimental and modeling approaches to study cell biomechanics, with a level of insights and reliability that were not possible in the past. The use of atomic force microscopy (AFM) for force spectroscopy allows nanoscale mapping of the cell topography and mechanical properties under, nearly physiological conditions. A proper evaluation process of such data is an essential factor to obtain accurate values of the cell elastic properties (primarily Young's modulus). Several numerical models were published in the literature, describing the depth sensing indentation as interaction process between the elastic surface and indenting probe. However, many studies are still relying on the nowadays outdated Hertzian model from the nineteenth century, or its modification by Sneddon. The lack of comparison between the Hertz/Sneddon model with their modern modifications blocks the development of advanced analysis software and further progress of AFM promising technology into biological sciences. In this work, we applied a rationalized use of mechanical models for advanced postprocessing and interpretation of AFM data. We investigated the effect of the mechanical model choice on the final evaluation of cellular elasticity. We then selected samples subjected to different physicochemical modulators, to show how a critical use of AFM data handling can provide more information than simple elastic modulus estimation. Our contribution is intended as a methodological discussion of the limitations and benefits of AFM-based advanced mechanical analysis, to refine the quantification of cellular elastic properties and its correlation to undergoing cellular processes .
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http://dx.doi.org/10.3389/fphys.2018.01121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107778PMC
August 2018

Simultaneous study of mechanobiology and calcium dynamics on hESC-derived cardiomyocytes clusters.

J Mol Recognit 2019 02 6;32(2):e2760. Epub 2018 Aug 6.

Department of Biology, Masaryk University, Faculty of Medicine, Brno, Czech Republic.

Calcium ions act like ubiquitous second messengers in a wide amount of cellular processes. In cardiac myocytes, Ca handling regulates the mechanical contraction necessary to the heart pump function. The field of intracellular and intercellular Ca handling, employing in vitro models of cardiomyocytes, has become a cornerstone to understand the role and adaptation of calcium signalling in healthy and diseased hearts. Comprehensive in vitro systems and cell-based biosensors are powerful tools to enrich and speed up cardiac phenotypic and drug response evaluation. We have implemented a combined setup to measure contractility and calcium waves in human embryonic stem cells-derived cardiomyocyte 3D clusters, obtained from embryoid body differentiation. A combination of atomic force microscopy to monitor cardiac contractility, and sensitive fast scientific complementary metal-oxide-semiconductor camera for epifluorescence video recording, provided correlated signals in real time. To speed up the integrated data processing, we tested several post-processing algorithms, to improve the automatic detection of relevant functional parameters. The validation of our proposed method was assessed by caffeine stimulation (10mM) and detection/characterization of the induced cardiac response. We successfully report the first simultaneous recording of cardiac contractility and calcium waves on the described cardiac 3D models. The drug stimulation confirmed the automatic detection capabilities of the used algorithms, measuring expected physiological response, such as elongation of contraction time and Ca cytosolic persistence, increased calcium basal fluorescence, and transient peaks. These results contribute to the implementation of novel, integrated, high-information, and reliable experimental systems for cardiac models and drug evaluation.
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http://dx.doi.org/10.1002/jmr.2760DOI Listing
February 2019

Irreversible electroporation-Let's keep it cool.

J Cardiovasc Electrophysiol 2018 07 22;29(7):E12. Epub 2018 May 22.

International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic.

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http://dx.doi.org/10.1111/jce.13619DOI Listing
July 2018

Irreversible electroporation ablation for atrial fibrillation.

J Cardiovasc Electrophysiol 2018 04 6;29(4):643-651. Epub 2018 Mar 6.

International Clinical Research Center, St. Anne's University Hospital Brno, Brno, Czech Republic.

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.
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http://dx.doi.org/10.1111/jce.13454DOI Listing
April 2018

YAP regulates cell mechanics by controlling focal adhesion assembly.

Nat Commun 2017 05 15;8:15321. Epub 2017 May 15.

International Clinical Research Center (ICRC), St Anne's University Hospital, CZ-65691 Brno, Czech Republic.

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.
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http://dx.doi.org/10.1038/ncomms15321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5440673PMC
May 2017

Phenotypic assays for analyses of pluripotent stem cell-derived cardiomyocytes.

J Mol Recognit 2017 06 20;30(6). Epub 2016 Dec 20.

Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

Stem cell-derived cardiomyocytes (CMs) hold great hopes for myocardium regeneration because of their ability to produce functional cardiac cells in large quantities. They also hold promise in dissecting the molecular principles involved in heart diseases and also in drug development, owing to their ability to model the diseases using patient-specific human pluripotent stem cell (hPSC)-derived CMs. The CM properties essential for the desired applications are frequently evaluated through morphologic and genotypic screenings. Even though these characterizations are necessary, they cannot in principle guarantee the CM functionality and their drug response. The CM functional characteristics can be quantified by phenotype assays, including electrophysiological, optical, and/or mechanical approaches implemented in the past decades, especially when used to investigate responses of the CMs to known stimuli (eg, adrenergic stimulation). Such methods can be used to indirectly determine the electrochemomechanics of the cardiac excitation-contraction coupling, which determines important functional properties of the hPSC-derived CMs, such as their differentiation efficacy, their maturation level, and their functionality. In this work, we aim to systematically review the techniques and methodologies implemented in the phenotype characterization of hPSC-derived CMs. Further, we introduce a novel approach combining atomic force microscopy, fluorescent microscopy, and external electrophysiology through microelectrode arrays. We demonstrate that this novel method can be used to gain unique information on the complex excitation-contraction coupling dynamics of the hPSC-derived CMs.
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http://dx.doi.org/10.1002/jmr.2602DOI Listing
June 2017