Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P 2 breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P 2 breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P 2 depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrincoated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P 2 in clathrin coat dynamics and Arp2/3-dependent actin regulation.actin ͉ dynamin ͉ epsin ͉ phosphoinositides ͉ rapamycin P hosphorylation-dephosphorylation of the inositol ring of inositol phospholipids plays a major role in cell regulation. Their phosphorylated head groups interact with a variety of cytosolic protein modules and thus mediate the recruitment and regulation of proteins at the membrane-cytosol interface (1-7). A direct interaction with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], a phosphoinositide concentrated at the plasma membrane, has been demonstrated for all of the endocytic clathrin adaptors and for several of their accessory factors, for the GTPase dynamin, and for many actin regulatory proteins (8-16). The physiological significance of these interactions has been supported by cell biological (9, 15, 17-21) and pharmacological (22) studies in living cells and by forward and reverse genetics. For example, mutations in genes encoding inositol 5-phosphatases, primarily synaptojanin family members, were shown to cause endocytic and actin defects in a variety of organisms (16,(23)(24)(25)(26)(27)(28). However, genetic manipulations that result in long-lasting changes in cellular levels of PI(4,5)P 2 may act indirectly or trigger compensatory mechanisms that can complicate the interpretation of results.Conclusive evidence for the importance of PI(4,5)P 2 in events that occur at the plasma membrane will come from experiments involving the acute and specific manipulation of its levels. Recently, a technique for the rapid, conditional recruitment of proteins to membranes has been used to deliver 5-phosphatase modules to the cell surface (20,(29)(30)(31)(32). In this method, the genes encoding two proteins that can be cross-linked by the add...

The combination of computational models and biophysical simulations can help to interpret an array of experimental data and contribute to the understanding, diagnosis and treatment of complex diseases such as cardiac arrhythmias. For this reason, three-dimensional (3D) cardiac computational modelling is currently a rising field of research. The advance of medical imaging technology over the last decades has allowed the evolution from generic to patient-specific 3D cardiac models that faithfully represent the anatomy and different cardiac features of a given alive subject. Here we analyse sixty representative 3D cardiac computational models developed and published during the last fifty years, describing their information sources, features, development methods and online availability. This paper also reviews the necessary components to build a 3D computational model of the heart aimed at biophysical simulation, paying especial attention to cardiac electrophysiology (EP), and the existing approaches to incorporate those components. We assess the challenges associated to the different steps of the building process, from the processing of raw clinical or biological data to the final application, including image segmentation, inclusion of substructures and meshing among others. We briefly outline the personalisation approaches that are currently available in 3D cardiac computational modelling. Finally, we present examples of several specific applications, mainly related to cardiac EP simulation and model-based image analysis, showing the potential usefulness of 3D cardiac computational modelling into clinical environments as a tool to aid in the prevention, diagnosis and treatment of cardiac diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12938-015-0033-5) contains supplementary material, which is available to authorized users.

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Rule‐based methods are often used for assigning fiber orientation to cardiac anatomical models. However, existing methods have been developed using data mostly from the left ventricle. As a consequence, fiber information obtained from rule‐based methods often does not match histological data in other areas of the heart such as the right ventricle, having a negative impact in cardiac simulations beyond the left ventricle. In this work, we present a rule‐based method where fiber orientation is separately modeled in each ventricle following observations from histology. This allows to create detailed fiber orientation in specific regions such as the endocardium of the right ventricle, the interventricular septum, and the outflow tracts. We also carried out electrophysiological simulations involving these structures and with different fiber configurations. In particular, we built a modeling pipeline for creating patient‐specific volumetric meshes of biventricular geometries, including the outflow tracts, and subsequently simulate the electrical wavefront propagation in outflow tract ventricular arrhythmias with different origins for the ectopic focus. The resulting simulations with the proposed rule‐based method showed a very good agreement with clinical parameters such as the 10 ms isochrone ratio in a cohort of nine patients suffering from this type of arrhythmia. The developed modeling pipeline confirms its potential for an in silico identification of the site of origin in outflow tract ventricular arrhythmias before clinical intervention.

Anatomically based procedures to ablate atrial fibrillation (AF) are often successful in terminating paroxysmal AF. However, the ability to terminate persistent AF remains disappointing. New mechanistic approaches use multiple-electrode basket catheter mapping to localize and target AF drivers in the form of rotors but significant concerns remain about their accuracy. We aimed to evaluate how electrode-endocardium distance, far-field sources and inter-electrode distance affect the accuracy of localizing rotors. Sustained rotor activation of the atria was simulated numerically and mapped using a virtual basket catheter with varying electrode densities placed at different positions within the atrial cavity. Unipolar electrograms were calculated on the entire endocardial surface and at each of the electrodes. Rotors were tracked on the interpolated basket phase maps and compared with the respective atrial voltage and endocardial phase maps, which served as references. Rotor detection by the basket maps varied between 35–94% of the simulation time, depending on the basket’s position and the electrode-to-endocardial wall distance. However, two different types of phantom rotors appeared also on the basket maps. The first type was due to the far-field sources and the second type was due to interpolation between the electrodes; increasing electrode density decreased the incidence of the second but not the first type of phantom rotors. In the simulations study, basket catheter-based phase mapping detected rotors even when the basket was not in full contact with the endocardial wall, but always generated a number of phantom rotors in the presence of only a single real rotor, which would be the desired ablation target. Phantom rotors may mislead and contribute to failure in AF ablation procedures.