Publications
346 results found
Brandenburg S, Pawlowitz J, Eikenbusch B, et al., 2019, Junctophilin-2 expression rescues atrial dysfunction through polyadic junctional membrane complex biogenesis, JCI INSIGHT, Vol: 4
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- Citations: 19
Toomer KA, Yu M, Fulmer D, et al., 2019, Primary cilia defects causing mitral valve prolapse, SCIENCE TRANSLATIONAL MEDICINE, Vol: 11, ISSN: 1946-6234
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- Citations: 55
Rog-Zielinska EA, O'Toole ET, Hoenger A, et al., 2019, Mitochondrial deformation during the cardiac mechanical cycle, Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology, Vol: 302, Pages: 146-152, ISSN: 0749-3002
Cardiomyocytes both cause and experience continual cyclic deformation. The exact effects of this deformation on the properties of intracellular organelles are not well characterized, although they are likely to be relevant for cardiomyocyte responses to active and passive changes in their mechanical environment. In the present study we provide three‐dimensional ultrastructural evidence for mechanically induced mitochondrial deformation in rabbit ventricular cardiomyocytes over a range of sarcomere lengths representing myocardial tissue stretch, an unloaded “slack” state, and contracture. We also show structural indications for interaction of mitochondria with one another, as well as with other intracellular elements such as microtubules, sarcoplasmic reticulum and T‐tubules. The data presented here help to contextualize recent reports on the mechanosensitivity and cell‐wide connectivity of the mitochondrial network and provide a structural framework that may aide interpretation of mechanically‐regulated molecular signaling in cardiac cells. Anat Rec, 302:146–152, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.
Noble D, Blundell TL, Kohl P, 2019, Editorial, PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY, Vol: 141, Pages: 1-2, ISSN: 0079-6107
Kopton RA, Baillie JS, Rafferty SA, et al., 2018, Cardiac electrophysiological effects of light-activated chloride channels, Frontiers in Physiology, Vol: 9, ISSN: 1664-042X
During the last decade, optogenetics has emerged as a paradigm-shifting technique to monitor and steer the behavior of specific cell types in excitable tissues, including the heart. Activation of cation-conducting channelrhodopsins (ChR) leads to membrane depolarization, allowing one to effectively trigger action potentials (AP) in cardiomyocytes. In contrast, the quest for optogenetic tools for hyperpolarization-induced inhibition of AP generation has remained challenging. The green-light activated ChR from Guillardia theta (GtACR1) mediates Cl−-driven photocurrents that have been shown to silence AP generation in different types of neurons. It has been suggested, therefore, to be a suitable tool for inhibition of cardiomyocyte activity. Using single-cell electrophysiological recordings and contraction tracking, as well as intracellular microelectrode recordings and in vivo optical recordings of whole hearts, we find that GtACR1 activation by prolonged illumination arrests cardiac cells in a depolarized state, thus inhibiting re-excitation. In line with this, GtACR1 activation by transient light pulses elicits AP in rabbit isolated cardiomyocytes and in spontaneously beating intact hearts of zebrafish. Our results show that GtACR1 inhibition of AP generation is caused by cell depolarization. While this does not address the need for optogenetic silencing through physiological means (i.e., hyperpolarization), GtACR1 is a potentially attractive tool for activating cardiomyocytes by transient light-induced depolarization.
Noble D, Blundell TL, Kohl P, 2018, Progress in biophysics and molecular biology: A brief history of the journal, PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY, Vol: 140, Pages: 1-4, ISSN: 0079-6107
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- Citations: 2
Sierra YAB, Rost BR, Pofahl M, et al., 2018, Potassium channel-based optogenetic silencing, Nature Communications, Vol: 9, ISSN: 2041-1723
Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this ‘PAC-K’ silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.
Rog-Zielinska EA, Kong CHT, Zgierski-Johnston CM, et al., 2018, Species differences in themorphology of transverse tubule openings in cardiomyocytes, Europace, Vol: 20, Pages: 120-124, ISSN: 1099-5129
AimsThe ultrastructure of ventricular cardiomyocyte T-tubule connections with the outer cell surface (‘mouth’ regions) has been reported to differ between mice and rabbits. In mice, T-tubule mouths form convoluted narrow spaces filled with electron-dense matter that impedes diffusion between T-tubular lumen and bulk extracellular space. Here, we explore whether T-tubule mouths are also constricted in rat (another murine model used frequently for cardiac research) and whether pig and human T-tubule mouth configurations are structurally more similar to mice or rabbits.Methods and resultsWe used chemically-fixed tissue and high-pressure frozen isolated cardiomyocytes to compare T-tubule mouth architecture using transmission electron microscopy and three-dimensional electron tomography. We find that rat T-tubular mouth architecture is more similar to that of rabbits than mice, lacking the marked tortuosity and electron-dense ground substance that obstructs access to deeper portions of the T-tubular system in mice. Pilot observations in larger mammals (pig, human) suggest that mouse may be the least representative animal model of T-tubule connectivity with the outer cell surface in larger mammals.ConclusionRat T-tubular system architecture appears to be more similar in size and topology to larger mammals than mice. T-tubular mouth topology may contribute to differences in experimental model behaviour, underscoring the challenge of appropriate model selection for research into cell and tissue function.
Andlauer R, Seemann G, Baron L, et al., 2018, Influence of left atrial size on P-wave morphology: differential effects of dilation and hypertrophy, EUROPACE, Vol: 20, Pages: 36-44, ISSN: 1099-5129
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- Citations: 25
Kong CHT, Rog-Zielinska EA, Kohl P, et al., 2018, Solute movement in the t-tubule system of rabbit and mouse cardiomyocytes, Proceedings of the National Academy of Sciences, Vol: 115, Pages: E7073-E7080, ISSN: 0027-8424
Cardiac transverse (t-) tubules carry both electrical excitation and solutes toward the cell center but their ability to transport small molecules is unclear. While fluorescence recovery after photobleaching (FRAP) can provide an approach to measure local solute movement, extraction of diffusion coefficients is confounded by cell and illumination beam geometries. In this study, we use measured cellular geometry and detailed computer modeling to derive the apparent diffusion coefficient of a 1-kDa solute inside the t-tubular system of rabbit and mouse ventricular cardiomyocytes. This approach shows that diffusion within individual t-tubules is more rapid than previously reported. T-tubule tortuosity, varicosities, and the presence of longitudinal elements combine to substantially reduce the apparent rate of solute movement. In steady state, large (>4 kDa) solutes did not freely fill the t-tubule lumen of both species and <50% of the t-tubule volume was available to solutes >70 kDa. Detailed model fitting of FRAP data suggests that solute diffusion is additionally restricted at the t-tubular entrance and this effect was larger in mouse than in rabbit. The possible structural basis of this effect was investigated using electron microscopy and tomography. Near the cell surface, mouse t-tubules are more tortuous and filled with an electron-dense ground substance, previously identified as glycocalyx and a polyanionic mesh. Solute movement in the t-tubule network of rabbit and mouse appears to be explained by their different geometric properties, which impacts the use of these species for understanding t-tubule function and the consequences of changes associated with t-tubule disease.
Ayub S, Ruther P, Paul O, et al., 2018, Invasive Optical Pacing in Perfused, Optogenetically Modified Mouse Heart Using Stiff Multi-LED Optical Probes., Annu Int Conf IEEE Eng Med Biol Soc, Vol: 2018, Pages: 1-4
We present the first invasive use of a stiff, multiLED optical probe for intramural optical stimulation of cardiac tissue. We demonstrate that optical pacing is possible with high spatial and temporal resolution in transgenic mice expressing channelrhodopsin-2. The technical implementation of this study builds on optical probes recently developed and tested ex vivo in cerebral tissue of mice. The probes comprise LEDs integrated on flexible substrates stiffened by silicon-based MEMS structures enabling the successful penetration into the cardiac tissue. The probe technology is extended to allow dual-sided illumination for directional tissue stimulation. Implantation trials affirm the ability to optically pace the isolated perfused heart at stimulation frequencies between 4Hz and 12Hz with experimentally determined emittance levels of 10mW mm-2 Rapid activation of two distant LEDs could reliably be used to induce short runs of ventricular fibrillation, while simultaneous activation of all LEDs allowed termination of re-entrant rhythm disturbances (optical defibrillation). Thus, spatially-resolved intramural pacing and rhythm control of the isolated heart is possible using stiff, multi-LED optical probes.
Klesen A, Jakob D, Emig R, et al., 2018, Cardiac fibroblasts : Active players in (atrial) electrophysiology?, Herzschrittmacherther Elektrophysiol, Vol: 29, Pages: 62-69
Fibrotic areas in cardiac muscle-be it in ventricular or atrial tissue-are considered as obstacles for conduction of the excitatory wave and can therefore facilitate re-entry, which may contribute to the sustenance of cardiac arrhythmias. Persistence of one of the most frequent arrhythmias, atrial fibrillation (AF), is accompanied by enhanced atrial fibrosis. Any kind of myocardial perturbation, whether via mechanical stress or ischemic damage, inflammation, or irregular and high-frequency electrical activity, activates fibroblasts. This leads to the secretion of paracrine factors and extracellular matrix proteins, especially collagen, and to the differentiation of fibroblasts into myofibroblasts. Excessive collagen production is the hallmark of fibrosis and impairs regular impulse propagation. In addition, direct electrical coupling between cardiomyocytes and nonmyocytes, such as fibroblasts and macrophages, via gap junctions affects conduction. Although fibroblasts are not electrically excitable, they express functional ion channels, in particular K+ channels and mechanosensitive channels, some of which could be involved in tissue remodeling. Here, we briefly review these aspects with special reference to AF.
Scardigli M, Crocini C, Ferrantini C, et al., 2018, Reply to Entcheva: The impact of T-tubules on action potential propagation in cardiac tissue, Proceedings of the National Academy of Sciences of the United States of America, Vol: 115, Pages: E562-E563, ISSN: 0027-8424
Kohl P, 2018, Cardiac Stretch-Activated Channels and Mechano-Electric Coupling, Cardiac Electrophysiology: From Cell to Bedside: Seventh Edition, Pages: 128-139, ISBN: 9780323447331
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- Citations: 4
Schneider-Warme F, Johnston CM, Kohl P, 2018, Organotypic myocardial slices as model system to study heterocellular interactions, CARDIOVASCULAR RESEARCH, Vol: 114, Pages: 3-6, ISSN: 0008-6363
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- Citations: 7
Ayub S, Ruther P, Paul O, et al., 2018, Invasive Optical Pacing in Perfused, Optogenetically Modified Mouse Heart Using Stiff Multi-LED Optical Probes, 40th Annual International Conference of the IEEE-Engineering-in-Medicine-and-Biology-Society (EMBC), Publisher: IEEE, Pages: 4836-4839, ISSN: 1557-170X
Wuelfers EM, Kohl P, Seemann G, 2018, Mathematical Modeling of Non-Selective Channels: Estimating Ion Current Fractions and Their Impact on Pathological Simulations, 45th Computing in Cardiology Conference (CinC), Publisher: IEEE, ISSN: 2325-8861
Weber M, Scherf N, Meyer AM, et al., 2017, Cell-accurate optical mapping across the entire developing heart, eLife, Vol: 6, ISSN: 2050-084X
Organogenesis depends on orchestrated interactions between individual cells and morphogenetically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighboring cells, which is established and fine-tuned during embryonic development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca²⁺-mapping of all cells in the entire electro-mechanically uncoupled heart during the looping stage of live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs.
Peyronnet R, Bollensdorff C, Capel RA, et al., 2017, Load-dependent effects of apelin on murine cardiomyocytes, Progress in Biophysics and Molecular Biology, Vol: 130, Pages: 333-343, ISSN: 0079-6107
The apelin peptide is described as one of the most potent inotropic agents, produced endogenously in a wide range of cells, including cardiomyocytes. Despite positive effects on cardiac contractility in multicellular preparations, as well as indications of cardio-protective actions in several diseases, its effects and mechanisms of action at the cellular level are incompletely understood.Here, we report apelin effects on dynamic mechanical characteristics of single ventricular cardiomyocytes, isolated from mouse models (control, apelin-deficient [Apelin-KO], apelin-receptor KO mouse [APJ-KO]), and rat. Dynamic changes in maximal velocity of cell shortening and relaxation were monitored. In addition, more traditional indicators of inotropic effects, such as maximum shortening (in mechanically unloaded cells) or peak force development (in auxotonic contracting cells, preloaded using the carbon fibre technique) were studied.The key finding is that, using Apelin-KO cardiomyocytes exposed to different preloads with the 2-carbon fibre technique, we observe a lowering of the slope of the end-diastolic stress-length relation in response to 10 nM apelin, an effect that is preload-dependent. This suggests a positive lusitropic effect of apelin, which could explain earlier counter-intuitive findings on an apelin-induced increase in contractility occurring without matching rise in oxygen consumption.
Tsushima K, Bugger H, Wende AR, et al., 2017, Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induces Post-Translational Modifications of AKAP121, DRP1 and OPA1 That Promote Mitochondrial Fission., Circulation Research, Vol: 122, Pages: 58-73, ISSN: 0009-7330
Rationale: Cardiac lipotoxicity, characterized by increased uptake, oxidation and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. Objective: To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Methods and Results: Using a transgenic mouse model of cardiac lipotoxicity overexpressing long-chain acyl-CoA synthetase 1 in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes (NRVCs). Palmitate exposure to NRVCs initially activates mitochondrial respiration, coupled with increased mitochondrial membrane potential and adenosine triphosphate (ATP) synthesis. However, long-term exposure to palmitate (> 8h) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of A-kinase anchor protein (AKAP121) leading to reduced phosphorylation of DRP1 at Ser637 and altered proteolytic processing of OPA1. Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Conclusions: Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a novel mech
Ravens U, Kohl P, 2017, Max Lab at heart, PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY, Vol: 130, Pages: 124-125, ISSN: 0079-6107
Johnston CM, Rog-Zielinska EA, Wulfers EM, et al., 2017, Optogenetic targeting of cardiac myocytes and non-myocytes: tools, challenges and utility, Progress in Biophysics and Molecular Biology, Vol: 130, Pages: 140-149, ISSN: 0079-6107
In optogenetics, light-activated proteins are used to monitor and modulate cellular behaviour with light. Combining genetic targeting of distinct cellular populations with defined patterns of optical stimulation enables one to study specific cell classes in complex biological tissues. In the current study we attempted to investigate the functional relevance of heterocellular electrotonic coupling in cardiac tissue in situ. In order to do that, we used a Cre-Lox approach to express the light-gated cation channel Channelrhodopsin-2 (ChR2) specifically in either cardiac myocytes or non-myocytes. Despite high specificity when using the same Cre driver lines in a previous study in combination with a different optogenetic probe, we found patchy off-target ChR2 expression in cryo-sections and extended z-stack imaging through the ventricular wall of hearts cleared using CLARITY. Based on immunohistochemical analysis, single-cell electrophysiological recordings and whole-genome sequencing, we reason that non-specificity is caused on the Cre recombination level. Our study highlights the importance of careful design and validation of the Cre recombination targets for reliable cell class specific expression of optogenetic tools.
Burton RAB, Rog-Zielinska EA, Corbett AD, et al., 2017, Caveolae in rabbit ventricular myocytes: distribution and dynamic diminution after cell isolation, Biophysical Journal, Vol: 113, Pages: 1047-1059, ISSN: 0006-3495
Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae unde
Quinn TA, Jin H, Lee P, et al., 2017, Mechanically induced ectopy via stretch-activated cation-nonselective channels is caused by local tissue deformation and results in ventricular fibrillation if triggered on the repolarization wave edge (Commotio Cordis), Circulation: Arrhythmia and Electrophysiology, Vol: 10, Pages: 1-32, ISSN: 1941-3084
Background—External chest impacts (commotio cordis) can cause mechanically induced premature ventricular excitation (PVEM) and, rarely, ventricular fibrillation (VF). Because block of stretch-sensitive ATP-inactivated potassium channels curtailed VF occurrence in a porcine model of commotio cordis, VF has been suggested to arise from abnormal repolarization caused by stretch activation of potassium channels. Alternatively, VF could result from abnormal excitation by PVEM, overlapping with normal repolarization-related electric heterogeneity. Here, we investigate mechanisms and determinants of PVEM induction and its potential role in commotio cordis–induced VF.Methods and Results—Subcontusional mechanical stimuli were applied to isolated rabbit hearts during optical voltage mapping, combined with pharmacological block of ATP-inactivated potassium or stretch-activated cation-nonselective channels. We demonstrate that local mechanical stimulation reliably triggers PVEM at the contact site, with inducibility predicted by local tissue indentation. PVEM induction is diminished by pharmacological block of stretch-activated cation-nonselective channels. In hearts where electrocardiogram T waves involve a well-defined repolarization edge traversing the epicardium, PVEM can reliably provoke VF if, and only if, the mechanical stimulation site overlaps the repolarization wave edge. In contrast, application of short-lived intraventricular pressure surges neither triggers PVEM nor changes repolarization. ATP-inactivated potassium channel block has no effect on PVEM inducibility per se, but shifts it to later time points by delaying repolarization and prolonging refractoriness.Conclusions—Local mechanical tissue deformation determines PVEM induction via stretch-activation of cation-nonselective channels, with VF induction requiring PVEM overlap with the trailing edge of a normal repolarization wave. This defines a narrow, subject-specific vulnerable window
Scardigli M, Crocini C, Ferrantini C, et al., 2017, Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy, Publisher: ELSEVIER SCI LTD, Pages: 16-16, ISSN: 0022-2828
Kong CHT, Rog-Zielinska EA, Orchard CH, et al., 2017, Sub-microscopic analysis of t-tubule geometry in living cardiac ventricular myocytes using a shape-based analysis method, JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, Vol: 108, Pages: 1-7, ISSN: 0022-2828
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- Citations: 16
Scardigli M, Crocini C, Ferrantini C, et al., 2017, Quantitative assessment of passive electrical properties of the cardiac T-tubular system by FRAP microscopy, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 114, Pages: 5737-5742, ISSN: 0027-8424
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- Citations: 30
Kroll KT, Zhou Q, Kohl P, 2017, Finding the culprit: who is turning hearts to stone?, Stem Cell Investigation, Vol: 4, Pages: 33-33, ISSN: 2306-9759
Hulsmans M, Clauss S, Xiao L, et al., 2017, Macrophages facilitate electrical conduction in the heart, Cell, Vol: 169, Pages: 510-522, ISSN: 0092-8674
Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.
Casero R, Siedlecka U, Jones ES, et al., 2017, Transformation diffusion reconstruction of three-dimensional histology volumes from two-dimensional image stacks, Medical Image Analysis, Vol: 38, Pages: 184-204, ISSN: 1361-8423
Traditional histology is the gold standard for tissue studies, but it is intrinsically reliant on two-dimensional (2D) images. Study of volumetric tissue samples such as whole hearts produces a stack of misaligned and distorted 2D images that need to be reconstructed to recover a congruent volume with the original sample's shape. In this paper, we develop a mathematical framework called Transformation Diffusion (TD) for stack alignment refinement as a solution to the heat diffusion equation. This general framework does not require contour segmentation, is independent of the registration method used, and is trivially parallelizable. After the first stack sweep, we also replace registration operations by operations in the space of transformations, several orders of magnitude faster and less memory-consuming. Implementing TD with operations in the space of transformations produces our Transformation Diffusion Reconstruction (TDR) algorithm, applicable to general transformations that are closed under inversion and composition. In particular, we provide formulas for translation and affine transformations. We also propose an Approximated TDR (ATDR) algorithm that extends the same principles to tensor-product B-spline transformations. Using TDR and ATDR, we reconstruct a full mouse heart at pixel size 0.92 µm × 0.92 µm, cut 10 µm thick, spaced 20 µm (84G). Our algorithms employ only local information from transformations between neighboring slices, but the TD framework allows theoretical analysis of the refinement as applying a global Gaussian low-pass filter to the unknown stack misalignments. We also show that reconstruction without an external reference produces large shape artifacts in a cardiac specimen while still optimizing slice-to-slice alignment. To overcome this problem, we use a pre-cutting blockface imaging process previously developed by our group that takes advantage of Brewster's angle and a polarizer to capture the outline of
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