Guido Caluori

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.

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.

Phenotypic assays for analyses of pluripotent stem cell-derived cardiomyocytes

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.

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

Calcium ions act like ubiquitous second messengers in a wide amount of cellular processes. In cardiac myocytes, Ca2+ handling regulates the mechanical contraction necessary to the heart pump function. The field of intracellular and intercellular Ca2+ 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.

Irreversible electroporation-Let's keep it cool: CALUORI et al.

We thank Dr. Futyma for his attention to our manuscript 1 and the interesting discussion he provides in his letter 2 . This gives us the opportunity to further highlight the proposed non-thermal character of irreversible electroporation (IRE). Displacement of ohmic currents inside a tissue, a medium with an intrinsic impedance, will anytime, to some extent, produce a local temperature increase by Joule heating. This issue exists whether IRE is elicited by DC pulses or AC bursts. IRE ablation is considered non-thermal only when finely tuned to induce non- necrotic selective cellular death. As we highlighted in our review, the border is not clearly defined, but sparing adjacent heat-sensitive structures or tissues (especially extracellular matrix and blood vessels) is a relevant aim. Thermal damage will occur in muscle immediately, at temperatures above 50C 3 . This value is considered a maximal threshold to avoid thermal damage in tumor IRE ablation, as reported in in vitro 4 and in vivo 5 studies. Discussing the detailed work by Faroja et al 6 , this shows that elevated temperature levels can be generated due to IRE energy application on liver. Nevertheless, even in this study there is an identified non-thermal working range (measured temperature between 34-42C), detected by peaking of caspase 3, a known apoptotic marker. The study of Meyer et al. 7 is mentioned, as this shows that the use of defibrillating shocks produces skin erythema, with 5 applications at 360J. The application of defibrillating shocks is heavily dependent on skin electrode contact and energy delivered can be over two orders of magnitude stronger than a single IRE pulse. Considering, simplistically, an initial tissue-electrode resistive impedance of 100 Ohm 8 , a 3000 V DC application for 100 µs will cause an energy delivery of 9 J, requiring 200 applications to deliver the same amount of energy delivered in the mentioned study. In a recent work by Neven et al., a 200J single defibrillating shock was delivered from a decapolar circular catheter, using an external reference patch to ablate pulmonary veins sleeves. In this scenario, only mild intimal hyperplasia was reported, together with successful isolation 9 . Whether this proliferation is induced by hybrid thermo-electric effect is not clear, yet it does not hamper the therapeutic effects. It must be taken into consideration that IRE energy delivery, given the same electrical parameter, is also affected by the active electrode area and the proximity between active and reference electrode, as these parameters affect the tissue- electrode impedance. Therefore, it is clear how fundamental is to tune the IRE application parameters (e.g., voltage, pulse width and inter-pulse distance) and physical specifications (e.g., electrode area, reference proximity) to achieve the optimal efficacy and benefits of this approach, among the others providing a “cool” non-thermal method to overcome the limits of purely thermal-based ablation methods.

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

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 Youngs 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 in vitro.