Biology subjects

Dudek, J.

Publications and source records attributed to Dudek, J..

10 recordsLinked to original sources

bioRxiv2025

Enhanced iPSC-Cardiomyocyte Maturation via Combined 3D-Culture and Metabolic Cues

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have become an invaluable tool for disease modelling and drug testing. However, while many etiologies of heart failure involve defects in excitation-contraction coupling, mitochondrial energetics or both, iPSC-CM are limited by the developmental immaturity of these processes. Here, we report a combinatorial strategy to enhance the maturation of human iPSC-CM by integrating three-dimensional (3D) spheroid culture conditions with a defined hormone- and fatty acid-enriched maturation medium (MM). A comprehensive analysis of structural, electrophysiological and Ca2+ handling parameters was performed to evaluate cellular and functional maturation. The iPSC-CM generated under these conditions (3D_MM) exhibit many phenotypic characteristics that resemble those of isolated adult human CM, including (i) a rod-shaped morphology, (ii) cardiac ultrastructural features such as aligned myofilaments, unidirectional organized sarcomeres, and the presence of transverse (t)-tubules, (iii) refined action potential (AP) parameters and Ca2+ handling, and (iv) {beta}-adrenergic responsiveness and a positive force-frequency relationship. Compared with long-term (LT) monolayer cultures of 90 days or the individual cues (3D or MM alone), the 3D_MM protocol achieves mostly superior or at the least non-inferior maturation effects. This systematic investigation further demonstrates that while 3D culturing or MM alone improved specific aspects of maturation, only their synergistic combination produced a comprehensive enhancement of key CM processes, such as excitation-contraction coupling and mitochondrial energetics.

cell biology↗

bioRxiv2025

Peroxisome protein import deficiency causes heart failure in mouse and human

Peroxisomes are ubiquitous cellular organelles with potentially vital roles in lipid and reactive oxygen metabolism. The metabolic demands of the heart are substantial; however, the contribution of peroxisomes to cardiac development, health, and their role in heart failure (HF) remain largely unexplored. We developed and examined a mouse and an engineered human myocardium (EHM) model with a deficiency in cardiac peroxisome biogenesis to investigate the role of peroxisomes in cardiac function and pathology. In the EHM, loss of peroxisome protein import and subsequent peroxisomal metabolic impairment trigger mitochondrial damage and compromise cellular respiration and energy production. Peroxisome dysfunction results in incoherent electrical conduction, defective Ca2+-handling, and ultimately presentation of a HF phenotype with pathological force generation. These phenotypes are mirrored in an orthogonal murine model system with defective cardiac peroxisome biogenesis. Preload-dependent deficits in force generation due to insufficient energy supply are eventually fatal. Thus, peroxisomes play an important role in sustaining normal heart operations. Vice versa, peroxisome maintenance is compromised in pressure overload-induced HF, establishing peroxisomes as potential modulators of pathology and targets of therapy.

cell biology↗

bioRxiv2025

Tafazzin-Mediated Cardiolipin Remodeling Controls Metabolic Stress Response and Effector Function of Inflammatory T Cells

Clonal expansion and effector differentiation of T cells require extensive metabolic reprogramming. This includes the restructuring of the inner mitochondrial membrane (IMM) to enhance respiration by increasing chemiosmotic coupling efficiency. Cardiolipin, a unique phospholipid that is exclusively synthesized and localized in the IMM, modulates the biophysical properties of the electron transport chain (ETC) in tissues with high energy demands, such as cardiomyocytes and skeletal muscle. However, it remains unclear whether cardiolipin is also important for metabolic remodeling during T helper (Th) cell differentiation. In this study, we show that cardiolipin transacylation, catalyzed by the enzyme Tafazzin, supports the clonal expansion and effector function of inflammatory Th1 and Th17 cells in vitro and in models of autoimmune colitis and encephalomyelitis. At the molecular level, we demonstrate that loss of Tafazzin-mediated cardiolipin maturation induces a metabolic and transcriptional stress response in Th cells to compensate for impaired coupling efficiency of the ETC complexes and disrupted cellular redox homeostasis. However, the genetic program that restores cellular homeostasis and mitigates oxidative stress concurrently impairs the effector functions of inflammatory T cells, such as cytokine production. Our findings also provide insights into the complex clinical manifestation of patients with Barth syndrome (BTHS) caused by mutations in the human TAFAZZIN gene. BTHS is characterized by cardiac and skeletal myopathy as well as neutropenia and an increased susceptibility to infections. Although the molecular basis of the immunodeficiency remains poorly understood, our findings suggest that impaired Th cell function contributes to the immunopathology observed in BTHS patients.

immunology↗

bioRxiv2025

RBM20-variants induce distinct calcium handling and metabolic phenotypes in patient-specific stem cell models of dilated and non-compaction cardiomyopathy

Background and aimMutations in the splice regulator RBM20 account for [~]3 % of genetic cardiomyopathies. In particular, the highly conserved RS domain is a hotspot for disease-associated mutations. Previously, mutations at same amino acid position 634 in the hotspot RS-domain were found to cause dilated cardiomyopathy (DCM) with left ventricular non-compaction (R634L) or without (R634W), but the pathophysiological mechanisms that govern the heterogeneity in phenotype presentation remained unknown. Here, we identify the molecular events caused by the distinct RBM20 mutations from DCM and left-ventricular non-compaction (LVNC) using patient-specific stem cell models. MethodsWe generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) of one LVNC- and two DCM-patients harboring the RBM20-mutations R634L (LVNC) or R634W (DCM). We investigated alternative splicing activity, RBM20 localization, sarcomeric regularity, cAMP level, kinase-specific phosphorylation of key Ca2+ handling enzymes, physiological cardiac functions as Ca2+ homeostasis, and metabolic activity on a patient-specific cardiomyocyte level. Force generation was analyzed in patient-specific engineered myocardial tissues. Isogenic rescue and mutation insertion lines were generated by CRISPR/Cas9 technology to analyze the direct impact of the RBM20 mutations on the cardiac phenotype. ResultsWe observed common splicing aberrations for LVNC- and DCM-CM in TTN and RYR2, RBM20 cytoplasmatic accumulation and irregular sarcomeric structure. LVNC-CM harboring the RBM20-p.R634L variant show distinct molecular, cellular and functional impairments that manifest in CAMK2D, TRDN and IMMT mis-splicing. Splicing defects in LVNC-CM correlate with elevated systolic Ca2+ and faster Ca2+ kinetics with elevated cAMP levels and PLN-hyperphosphorylation. An increased metabolic activity and mitochondrial membrane potential support the hyperactive LVNC-CM. By contrast, DCM-CM (RBM20-p.R634W) distinctly present with decreased systolic Ca2+ and increased SR Ca2+leak but unchanged Ca2+ kinetics and metabolic activity. Both mutations lead to severely reduced force of contraction in engineered myocardium. CRISPR/Cas9 gene-edited isogenic control lines of both described RBM20 mutations in LVNC and DCM demonstrated the causative nature of the two mutations and their diverging effects. Further, L-type Ca2+ channel blockade by verapamil ameliorates the Ca2+ cycling and leakage phenotypes in LVNC- and DCM-CM. ConclusionWe show the first comparative iPSC-model of splice-defect-associated RBM20-dependent LVNC-p.R634L and DCM-p.R634W. We found shared and variant-specific phenotypes on a patient-specific level. Our data demonstrate that the different RBM20 mutations manifest in distinct molecular aberrations in alternative splicing and RBM20 cytoplasmic accumulation that convey various physiological impairments in structure, Ca2+ handling, metabolism and contractile force.

molecular biology↗

bioRxiv2023

HIF-1α-mediated mitochondrial-glycolytic reprogramming controls the transition of precursor to terminally exhausted T cells

Functional exhaustion of T cells in cancer and persistent infections is characterized by the upregulation of inhibitory receptors, the progressive decline in cytokine secretion and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex) with phenotypic features of memory T cells and a stem-like capacity to self-renew. However, the metabolic principles of Tpex maintenance and the regulatory circuits that control the exhaustion of their progeny remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics and metabolomic analyses, we here show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the T cell exhaustion program. At the molecular level, we found that diminished mitochondrial respiration and metabolic remodeling cause oxidative stress, which inhibits the proteasomal degradation of hypoxia inducible factor 1 alpha (HIF-1) in Tpex cells. HIF-1 mediates the transcriptional-glycolytic reprogramming of Tpex cells as an initial step towards terminal differentiation and functional exhaustion. Finally, we show that enhancing respiration by limiting the glycolytic activity of CAR T cells is a feasible metabolic intervention strategy to preserve the stemness of Tpex cells during chronic viral infection and cancer immunotherapy.

immunology↗

bioRxiv2023

Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model

Acral burning pain triggered by fever, thermal hyposensitivity, and skin denervation are hallmarks of small fibre neuropathy in Fabry disease, a life-threatening X-linked lysosomal storage disorder. Variants in the gene encoding alpha-galactosidase A may lead to impaired enzyme activity with cellular accumulation of globotriaosylceramide (Gb3). To study the underlying pathomechanism of Fabry-associated small fibre neuropathy, we generated a neuronal in vitro disease model using patient-derived induced pluripotent stem cells from three Fabry patients and one healthy control. We further generated an isogenic control line via CRISPR/Cas9 gene editing. We subjected iPSC to targeted peripheral neuronal differentiation and observed intra-lysosomal Gb3 accumulations in somas and neurites of Fabry sensory neurons using super-resolution microscopy. At functional level, patch-clamp analysis revealed a hyperpolarizing shift of voltage-gated sodium channel steady-state inactivation kinetics in Fabry cell lines as compared to the healthy control. Moreover, we demonstrate a drastic increase in Fabry sensory neuron Ca2+ levels at 39{degrees}C mimicking clinical fever (p < 0.001). This pathophysiological phenotype was accompanied by thinning of neurite calibres in sensory neurons obtained from Fabry patients compared to healthy control cells (p < 0.001). Linear-Nonlinear cascade models fit to spiking responses revealed that Fabry cell lines exhibit altered single neuron encoding properties relative to control. We further observed jam of mitochondrial trafficking at sphingolipid accumulations within Fabry sensory neurites utilizing a click-chemistry approach together with mitochondrial dysmorphism compared to healthy control cells. We pioneer insights into the cellular mechanisms contributing to pain, thermal hyposensitivity, and denervation in Fabry small fibre neuropathy, and pave the way for further mechanistic in vitro studies in Fabry disease and the development of novel treatment approaches.

neuroscience↗

bioRxiv2022

Proteomics identifies substrates and a novel component in hSnd2-dependent ER protein targeting

Protein import into the endoplasmic reticulum (ER) is essential for about 30% of the human proteome. It involves targeting of precursor proteins to the ER and insertion into or translocation across the ER membrane. Furthermore, it relies on signals in the precursor polypeptides and components, which read the signals and facilitate their targeting to a protein-conducting channel in the ER membrane, the Sec61 complex. Compared to the SRP- and TRC-dependent pathways, little is known about the SRP-independent/SND pathway. Our aim was to identify additional components and characterize the client spectrum of the human SND pathway. The established strategy of combining depletion of the central hSnd2-component from HeLa cells with proteomic- and differential protein abundance-analysis was used. The SRP and TRC targeting pathways were analyzed in comparison. TMEM109 was characterized as hSnd3. Unlike SRP but similar to TRC, the SND clients are predominantly membrane proteins with N-terminal, central, or C-terminal targeting signals.

cell biology↗

bioRxiv2022

Epigenetic modulators link mitochondrial redox homeostasis to cardiac function

Excessive production of reactive oxygen species (ROS) is characteristic of numerous diseases, but most studies in this area have not considered the impact of endogenous antioxidative defenses. Here, utilizing multi-omics, we demonstrate that in cardiomyocytes mitochondrial isocitrate dehydrogenase (IDH2) constitutes a major antioxidant defense. In both male and female mice and humans the paradoxical reduction in expression of IDH2 associated with heart failure is compensated for by an increase in the enzymes activity. We describe extensive mutual regulation of the antioxidant activities of IDH2 and NRF2 by a network involving 2-oxoglutarate and L2-hydroxyglutarate and mediated in part through unconventional hydroxymethylation of cytosine residues present in introns. Conditional targeting of ROS in a murine model of heart failure improves cardiac function. Together, these insights may explain why previous attempts to treat heart failure with antioxidants have been unsuccessful and open new approaches to personalizing and, thereby, improving such treatment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/485908v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@1032ebeorg.highwire.dtl.DTLVardef@1f1c7bforg.highwire.dtl.DTLVardef@70f6a0org.highwire.dtl.DTLVardef@4fafbc_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIParadoxical downregulation of mitochondrial isocitrate dehydrogenase (IDH2) in response to oxidative stress leads to the discovery of a robust antioxidative defense in the heart. C_LIO_LIAn antioxidative loop involving IDH2 coordinates other antioxidative defenses, such as NRF2. C_LIO_LIThis loop produces epigenetic modifications that link oxidative stress to mitochondrial function. C_LIO_LIThe conclusion that enhancing antioxidative capacity improves cardiac function only when the endogenous capacity is insufficient opens new approaches to individualized treatment of patients with heart failure. C_LI

molecular biology↗

bioRxiv2021

Hydroxyapatite pellets as versatile model surfaces for systematic studies on enamel

Research into materials for medical application draws inspiration from naturally occurring or synthesized surfaces, just like many other research directions. For medical application of materials, particular attention has to be paid to biocompatibility, osseointegration and bacterial adhesion behavior. To understand their properties and behavior, experimental studies with natural materials such as teeth are strongly required. The results, however, may be highly case-dependent because natural surfaces have the disadvantage of being subject to wide variations, for instance in their chemical composition, structure, morphology, roughness, and porosity. A synthetic surface which mimics enamel in its performance with respect to bacterial adhesion and biocompatibility would, therefore, facilitate systematic studies much better. In this study, we discuss the possibility of using hydroxyapatite (HAp) pellets to simulate the surfaces of teeth and show the possibility and limitations of using a model surface. We performed single-cell force spectroscopy with single Staphylococcus aureus cells to measure adhesion-related parameters such as adhesion force and rupture length of adhesins binding to HAp and enamel. We also examine the influence of blood plasma and saliva on the adhesion properties of S. aureus. The results of these measurements are matched to water wettability, elemental composition of the samples and the change in the macromolecules adsorbed over time. We found that the adhesion properties of S. aureus were similar on both samples under all conditions: Significant decreases in adhesion strength were found equally in the presence of saliva or blood plasma on both surfaces. We therefore conclude that HAp pellets are a good alternative for natural dental material. This is especially true when slight variations in the physicochemical properties of the natural materials may affect the experimental series.

biophysics↗

bioRxiv2019

Proteomics identifies signal peptide features determining the substrate specificity in human Sec62/Sec63-dependent ER protein import

In mammalian cells one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61-channel. While the Sec61-complex facilitates ER-import of most precursor polypeptides, the Sec61-associated Sec62/Sec63-complex supports ER-import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63-complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER-import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified twenty-two novel Sec62/Sec63-substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides with comparatively longer but less hydrophobic H-region and lower C-region polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly-gating signal peptide and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62-/Sec63-requirement. In the case of ERj3, these features were found to be responsible for an additional BiP-requirement and to correlate with sensitivity towards the Sec61-channel inhibitor CAM741. Thus, the human Sec62/Sec63-complex may support Sec61-channel opening for precursor polypeptides with slowly-gating signal peptides by direct interaction with the cytosolic amino-terminal peptide of Sec61 or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked Polycystic Liver Disease. DatabasesThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data under the DOI:10.17632/6s5hn73jcv.1 (http://dx.doi.or/10.17632/6s5hn73jcv.1).

biochemistry↗