Biology subjects

Pagliari, S.

Publications and source records attributed to Pagliari, S..

3 recordsLinked to original sources

bioRxiv2024

An iPSC-derived bio-inspired scaffold modelling the structure and the effects of extracellular matrix in cardiac fibrosis

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the differentiation of cardiac fibroblasts (cFbs) into myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-{beta}1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (-SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere alignment, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

cell biology↗

bioRxiv2022

Generation and Maturation of Human iPSC-derived Cardiac Organoids in Long Term Culture

Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating and self-organized human cardiac organoids (hCO) in vitro from hiPSCs that can be cultured for long term. This is achieved by differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated human cardiac organoids (hCOs) containing multiple cell types that physiologically compose the heart, gradually self-organize and beat without external stimuli for more than 50 days. We have shown that 3D hCOs display improved cardiac specification, survival and maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hCOs by their response to cardioactive drugs in long term culture. Furthermore, we demonstrated that hCOs can be used to study chemotherapy-induced cardiotoxicity. This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.

cell biology↗

bioRxiv2021

The mechanical regulation of RNA binding protein hnRNPC in the failing heart

Cardiac pathologies are characterized by intense remodeling of the extracellular matrix (ECM) that eventually leads to heart failure. Cardiomyocytes respond to the ensuing biomechanical stress by re-expressing fetal contractile proteins via transcriptional and post-transcriptional processes, like alternative splicing (AS). Here, we demonstrate that the heterogeneous nuclear ribonucleoprotein C (hnRNPC) is upregulated and relocates to the sarcomeric Z-disk upon ECM pathological remodeling. We show that this is an active site of localized translation, where the ribonucleoprotein associates to the translation machinery. Alterations in hnRNPC expression and localization can be mechanically determined and affect the AS of numerous mRNAs involved in mechanotransduction and cardiovascular diseases, like Hippo pathway effector YAP1. We propose that cardiac ECM remodeling serves as a switch in RNA metabolism by impacting an associated regulatory protein of the spliceosome apparatus. These findings offer new insights on the mechanism of mRNAs homeostasis mechanoregulation in pathological conditions.

pathology↗