Biology subjects

Scardigli, M.

Publications and source records attributed to Scardigli, M..

8 recordsLinked to original sources

bioRxiv2026

Species-Dependent Limitations of Murine Myocardium for Investigating the Combined Inotropic Effects of SERCA2a Stimulation and Myofilament Ca2+ Sensitization

Impaired sarcoplasmic reticulum Ca2+ handling and myofilament function both contribute to contractile dysfunction in heart failure. A previous study in failing human myocardium showed that combining the SERCA2a activator istaroxime (ISTA) with the Ca2+ sensitizer levosimendan (LEVO) produces greater positive inotropy than either drug alone. However, human multicellular preparations did not allow comprehensive investigation of the underlying cellular mechanisms. We therefore assessed contractile performance and Ca2+ dynamics in murine multicellular myocardial preparations and ventricular cardiomyocytes treated with ISTA (20 M), LEVO (0.5 M), or their combination. ISTA improved developed force, contraction-relaxation kinetics, and mechanical restitution. These effects were associated with increased Ca2+ transient amplitude, accelerated Ca2+ rise and decay, and enhanced sarcoplasmic reticulum reloading. Adding LEVO produced no further mechanical benefit and only minor changes in Ca2+ handling. LEVO alone elicited heterogeneous contractile responses and increased myofilament Ca2+ sensitivity in only a minority of cardiomyocytes, while consistently accelerating Ca2+ transient decay. This effect was reproduced by the phosphodiesterase-3 inhibitor cilostamide, after which LEVO caused no significant additional change, consistent with a contribution of downstream cAMP-PKA signaling to the acceleration of Ca2+ removal. Thus, limited murine responsiveness to LEVO-mediated myofilament sensitization prevented mechanistic reconstruction of the additive inotropic effect observed in human myocardium, highlighting a species-dependent limitation of the mouse model for studying this combined therapy.

physiology↗

bioRxiv2025

Probing relaxed myosin states in hypertrophic cardiomyopathy by second harmonic-generation microscopy

This study explores the use of polarized second-harmonic generation (pSHG) to investigate myosin conformation in the relaxed state, differentiating between the actin-available, disordered (ON) state and the energy-conserving, ordered (OFF) state. By shifting the ON/OFF equilibrium using both physical and chemical manipulations, we demonstrate the sensitivity of pSHG in quantifying the ON/OFF ratio in skeletal and cardiac tissues. Comparisons with X-ray diffraction measurements further validate our findings. Applying this approach to a sarcomeric mutation associated with hypertrophic cardiomyopathy, we show that R403Q/MYH7-mutated minipig ventricle tissue exhibits a higher ON fraction compared to controls. This difference is abolished under high concentrations of a myosin activator (2-deoxyATP) and an inhibitor (Mavacamten), indicating structural similarity between R403Q and controls in these two states. ATPase assays reveal increased resting ATPase activity in R403Q samples, which persists even in the presence of 2-deoxyATP, suggesting that the elevated energy consumption in the R403Q mutation is driven by both a population shift toward the ON state and enhanced myosin ATPase activity per motor head.

physiology↗

bioRxiv2023

Segmentation of supragranular and infragranular layers in ultra-high resolution 7T ex vivo MRI of the human cerebral cortex

Accurate labeling of specific layers in the human cerebral cortex is crucial for advancing our understanding of neurodevelopmental and neurodegenerative disorders. Lever-aging recent advancements in ultra-high resolution ex vivo MRI, we present a novel semi-supervised segmentation model capable of identifying supragranular and infragranular layers in ex vivo MRI with unprecedented precision. On a dataset consisting of 17 whole-hemisphere ex vivo scans at 120 {micro}m, we propose a multi-resolution U-Nets framework (MUS) that integrates global and local structural information, achieving reliable segmentation maps of the entire hemisphere, with Dice scores over 0.8 for supra- and infragranular layers. This enables surface modeling, atlas construction, anomaly detection in disease states, and cross-modality validation, while also paving the way for finer layer segmentation. Our approach offers a powerful tool for comprehensive neuroanatomical investigations and holds promise for advancing our mechanistic understanding of progression of neurodegenerative diseases.

neuroscience↗

bioRxiv2023

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-coordinated excitation-contraction coupling (ECC) and, thus, homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarisation is active (local generation of action potentials; AP) or passive (following depolarisation of the outer cell surface sarcolemma; SS) has not been experimentally assessed in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesise that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were electrically insulated from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTSO_LICardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, ensuring synchronous and uniform cell contraction. C_LIO_LIIn this study, we investigate whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. C_LIO_LIFor this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of sub-cellular membrane domains. C_LIO_LIOur findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. C_LIO_LIThis active electrical capability may serve as a protective mechanism against voltage drops occurring within the T-tubular network. C_LI

physiology↗

bioRxiv2021

A multimodal imaging and analysis pipeline for creating a cellular census of the human cerebral cortex

Cells are not uniformly distributed in the human cerebral cortex. Rather, they are arranged in a regional and laminar fashion that span a range of scales. Here we demonstrate an innovative imaging and analysis pipeline to construct a reliable cell census across the human cerebral cortex. Magnetic resonance imaging (MRI) is used to establish a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with both traditional immunohistochemistry, to provide a stereological gold-standard, and with a custom-made inverted confocal light-sheet fluorescence microscope (LSFM) for 3D imaging at cellular resolution. Finally, mesoscale optical coherence tomography (OCT) enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system.

neuroscience↗

bioRxiv2021

3D molecular phenotyping of cleared human brain tissues with light-sheet fluorescence microscopy

The combination of optical tissue transparency with immunofluorescence allows the molecular characterization of biological tissues in 3D. However, adult human organs are particularly challenging to become transparent because of the autofluorescence contributions of aged tissues. To meet this challenge, we optimized SHORT (SWITCH - H2O2 - antigen Retrieval - TDE), a procedure based on standard histological treatments in combination with a refined clearing procedure to clear and label portions of the human brain. 3D histological characterization with multiple molecules is performed on cleared samples with a combination of multi-colors and multi-rounds labeling. By performing fast 3D imaging of the samples with a custom-made inverted light-sheet fluorescence microscope (LSFM), we reveal fine details of intact human brain slabs at subcellular resolution. Overall, we proposed a scalable and versatile technology that in combination with LSFM allows mapping the cellular and molecular architecture of the human brain, paving the way to reconstruct the entire organ.

neuroscience↗

bioRxiv2021

Exploring the human cerebral cortex using confocal microscopy

Cover-all mapping of the distribution of neurons in the human brain would have a significant impact on the deep understanding of brain function. Therefore, complete knowledge of the structural organization of different human brain regions at the cellular level would allow understanding their role in the functions of specific neural networks. Recent advances in tissue clearing techniques have allowed important advances towards this goal. These methods use specific chemicals capable of dissolving lipids, making the tissue completely transparent by homogenizing the refractive index. However, labeling and clearing human brain samples is still challenging. Here, we present an approach to perform the cellular mapping of the human cerebral cortex coupling immunostaining with SWITCH/TDE clearing and confocal microscopy. A specific evaluation of the contributions of the autofluorescence signals generated from the tissue fixation is provided as well as an assessment of lipofuscin pigments interference. Our evaluation demonstrates the possibility of obtaining an efficient clearing and labeling process of parts of adult human brain slices, making it an excellent method for morphological classification and antibody validation of neuronal and non-neuronal markers.

neuroscience↗

bioRxiv2020

Fast volumetric mapping of human brain slices

We still lack a detailed map of the anatomical disposition of neurons in the human brain. A complete map would be an important step for deeply understanding the brain function, providing anatomical information useful to decipher the neuronal pattern in healthy and diseased conditions. Here, we present several important advances towards this goal, obtained by combining a new clearing method, advanced Light Sheet Microscopy and automated machinelearning based image analysis. We perform volumetric imaging of large sequentially stained human brain slices, labelled for two different neuronal markers NeuN and GAD67, discriminating the inhibitory population and reconstructing the brain connectivity.

biophysics↗