Abstract: Fibrosis is associated with aging and many cardiac pathologies. It is characterized both by myofibroblast differentiation and by excessive accumulation of extracellular matrix proteins. Fibrosis-related tissue remodeling results in significant changes in tissue structure and function, including passive mechanical properties. This research area has gained significant momentum with the recent development of new tools and approaches to better characterize and understand the ability of cells to sense and respond to their biophysical environment. We use a novel hydrogel, termed CyPhyGel, to provide an advanced in vitro model of remodeling-related changes in tissue stiffness. Based on light-controlled dimerization of a Cyanobacterial Phytochrome, it enables contactless and reversible tuning of hydrogel mechanical properties with high spatial and temporal resolution. Human primary atrial fibroblasts were cultured on CyPhyGels. After 4 days of culturing on stiff (~4.6 kPa) or soft (~2.7 kPa) CyPhyGels, we analyzed fibroblast cell area and stiffness. Cells grown on the softer substrate were smaller and softer, compared to cells grown on the stiffer substrate. This difference was absent when both soft and stiff growth substrates were combined in a single CyPhyGel, with the resulting cell areas being similar to those on homogeneously stiff gels and cell stiffnesses being similar to those on homogeneously soft substrates. Using CyPhyGels to mimic tissue stiffness heterogeneities in vitro, our results confirm the ability of cardiac fibroblasts to adapt to their mechanical environment, and suggest the presence of a paracrine mechanism that tunes fibroblast structural and functional properties associated with mechanically induced phenotype conversion toward myofibroblasts. In the context of regionally increased tissue stiffness, such as upon scarring or in diffuse fibrosis, such a mechanism could help to prevent abrupt changes in cell properties at the border zone between normal and diseased tissue. The light-tunable mechanical properties of CyPhyGels and their suitability for studying human primary cardiac cells make them an attractive model system for cardiac mechanobiology research. Further investigations will explore the interactions between biophysical and soluble factors in the response of cardiac fibroblasts to spatially and temporally heterogeneous mechanical cues

Abstract: Aims The 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 results We 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. Conclusion Rat 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

Abstract: Objectives Examination of lymph nodes is one of the most common indications for imaging in the head and neck region. The purpose of this study is to evaluate whether multispectral optoacoustic tomography can be used to observe chromophore differences between benign and malignant neck lymph nodes. Materials and methods Proof-of-concept ex vivo study of resected cervical lymph nodes from 11 patients. The examination of lymph nodes included imaging with hybrid ultrasound and multispectral tomography system followed by spectral unmixing to separate signals from the endogenous chromophores water, lipid, hemoglobin and oxygenated hemoglobin; calculation of semi-quantitative parameters (total hemoglobin and relative oxygenation of hemoglobin). Comparison of the results from the hybrid measurement with the histopathological results. Results Most patients suffered from squamous cell carcinoma (n = 7), also metastasis from salivary gland adenocarcinoma and papillary thyroid carcinoma, were included. The comparison between benign cervical lymph nodes and metastases showed significant differences for the absorbers water, lipid, hemoglobin and oxygenated hemoglobin and total hemoglobin. Conclusions Our ex vivo study suggests that multispectral optoacoustic tomography can be used to detect differences between reactive lymph nodes and metastases. The measurement of endogenous chromophores can be used for this purpose. The examinations are non-invasively and thus potentially improve diagnostic prediction. However, potential influences from the ex vivo setting must be considered

Abstract: 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

Abstract: The mechanical environment of cardiac cells changes continuously and undergoes major alterations during diseases. Most cardiac diseases, including atrial fibrillation, are accompanied by fibrosis which can impair both electrical and mechanical function of the heart. A key characteristic of fibrotic tissue is excessive accumulation of extracellular matrix, leading to increased tissue stiffness. Cells are known to respond to changes in their mechanical environment, but the molecular mechanisms underlying this ability are incompletely understood. We used cell culture systems and hydrogels with tunable stiffness, combined with advanced biophysical and imaging techniques, to elucidate the roles of the stretch-activated channel Piezo1 in human atrial fibroblast mechano-sensing. Changing the expression level of Piezo1 revealed that this mechano-sensor contributes to the organization of the cytoskeleton, affecting mechanical properties of human embryonic kidney cells and human atrial fibroblasts. Our results suggest that this response is independent of Piezo1-mediated ion conduction at the plasma membrane, and mediated in part by components of the integrin pathway. Further, we show that Piezo1 is instrumental for fibroblast adaptation to changes in matrix stiffness, and that Piezo1-induced cell stiffening is transmitted in a paracrine manner to other cells by a signaling mechanism requiring interleukin-6. Piezo1 may be a new candidate for targeted interference with cardiac fibroblast function