Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 µm) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

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

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/cm2, 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.

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

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.

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. 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 Ca2+ 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.