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Age-related fibrosis in the left ventricle (LV) has been mainly studied in animals by assessing collagen content. Using second-harmonic generation microscopy and image processing, we evaluated amount, aggregation and spatial distribution of LV collagen in young to old pigs, and middle-age and elder living donors. All collagen features increased when comparing adult and old pigs with young ones, but not when comparing adult with old pigs or middle-age with elder individuals. Remarkably, all collagen parameters strongly correlated with lipofuscin, a biological age marker, in humans. By building patient-specific models of human ventricular tissue electrophysiology, we confirmed that amount and organization of fibrosis modulated arrhythmia vulnerability, and that distribution should be accounted for arrhythmia risk assessment. In conclusion, we characterize the age associated changes in LV collagen and its potential implications for ventricular arrhythmia development. Consistency between pig and human results substantiate the pig as a relevant model of age-related LV collagen dynamics. This work was supported by Ministerio de Ciencia e Innovacio´ n (Spain) (PID2019-105674RB-I00), by Gobierno de Arago´ n (LMP94_21) and (LMP128_21), by the European Research Council through grant ERC-StG 638284 and by European Social Fund and by Gobierno de Arago´ n (Reference Group BSICoS T39-20R) cofunded by FEDER 2014–2020 ‘‘Building Europe from Aragon‘‘. L.G-M was supported by a predoctoral fellowship from the Departamento de Ciencia, Universidad y Sociedad del Conocimiento from the Gobierno de Arago´ n 2016–2020 cofounded by Programa Operativo del Fondo Social Europeo Arago´ n (C150/2016), EMBO Short-Term fellowship (7710), and Ibercaja-CAI Estancias de Investigacio´ n IT18/18. Authors would like to acknowledge the Experimental Surgery Service of Aragon Health Sciences Institute (CIBA, Zaragoza, Spain), Mercazaragoza slaughterhouse (Zaragoza, Spain), and The Pink Pig slaughterhouse (Zuera, Spain) for their collaboration in the collection of young, adult, and boar tissue specimens, respectively.

Anisotropic environments can drastically alter the spectroscopy and photochemistry of molecules, leading to complex structure-function relationships. We examined this using fluorescent proteins as easy-to-modify model systems. Starting from a single scaffold, we have developed a range of 27 photochromic fluorescent proteins that cover a broad range of spectroscopic properties, including the determination of 43 crystal structures. Correlation and principal component analysis confirmed the complex relationship between structure and spectroscopy, but also allowed us to identify consistent trends and to relate these to the spatial organization. We find that changes in spectroscopic properties can come about through multiple underlying mechanisms, of which polarity, hydrogen bonding and presence of water molecules are key modulators. We anticipate that our findings and rich structure/spectroscopy dataset can open opportunities for the development and evaluation of new and existing protein engineering methods. We are grateful to Gerrit Groenhof (University of Jyvaskyla), Jeremy Harvey (KU Leuven), Raffaele Vitale (Universite de Lille), and Dominique Bourgeois (Institut de Biologie Structurale) for critical insights and discussion. E.D.Z. and L.V.M. thank the beamline staff from X06DA at the Swiss Light Source (Villigen, Switzerland), Proxima1 and Proxima2A at synchrotron Soleil (Gif-sur-Yvette, France), XRD1 at Elettra (Trieste, Italy) and I03 at Diamond Light Source (Oxfordshire, UK) for assistance during X-ray diffraction data collection. E.D.Z., S.H., and S.D. thank the Research Foundation Flanders (FWO) for a doctoral fellowship and postdoctoral fellowships (12X7919N and 12R2817N). This work was supported through funding from the Research Foundation Flanders through grants 1514319 N, G090819N, G0B8817N, and the European Research Council through grant 714688 NanoCellActivity. Dedecker, P (corresponding author), Katholieke Univ Leuven, Dept Chem, Celestijnenlaan 200G Box 2403, B-3001 Leuven, Belgium. peter.dedecker@kuleuven.be