Fibrosis is a pivotal player in heart failure development and progression. Measurements of (markers of) fibrosis in tissue and blood may help to diagnose and risk stratify patients with heart ...failure, and its treatment may be effective in preventing heart failure and its progression. A lack of pathophysiological insights and uniform definitions has hampered the research in fibrosis and heart failure. The Translational Research Committee of the Heart Failure Association discussed several aspects of fibrosis in their workshop. Early insidious perturbations such as subclinical hypertension or inflammation may trigger first fibrotic events, while more dramatic triggers such as myocardial infarction
and myocarditis give rise to full blown scar formation and ongoing fibrosis in diseased hearts. Aging itself is also associated with a cardiac phenotype that includes fibrosis. Fibrosis is an extremely heterogeneous phenomenon, as several stages of the fibrotic process exist, each with different fibrosis subtypes and a different composition of various cells and proteins — resulting in a very complex pathophysiology. As a result, detection of fibrosis, e.g. using current cardiac imaging modalities or plasma biomarkers, will detect only specific subforms of fibrosis, but cannot capture all aspects of the complex fibrotic process. Furthermore, several anti‐fibrotic therapies are under investigation, but such therapies generally target aspecific aspects of the fibrotic process and suffer from a lack of precision. This review discusses the mechanisms and the caveats and proposes a roadmap for future research.
Abstract Brain organoids are promising tools for disease modeling and drug development. For proper neuronal network formation excitatory and inhibitory neurons as well as glia need to co-develop. ...Here, we report the directed self-organization of human induced pluripotent stem cells in a collagen hydrogel towards a highly interconnected neuronal network at a macroscale tissue format. Bioengineered Neuronal Organoids (BENOs) comprise interconnected excitatory and inhibitory neurons with supportive astrocytes and oligodendrocytes. Giant depolarizing potential (GDP)-like events observed in early BENO cultures mimic early network activity of the fetal brain. The observed GABA polarity switch and reduced GDPs in >40 day BENO indicate progressive neuronal network maturation. BENOs demonstrate expedited complex network burst development after two months and evidence for long-term potentiation. The similarity of structural and functional properties to the fetal brain may allow for the application of BENOs in studies of neuronal plasticity and modeling of disease.
4D printing recently emerges as an exciting evolution of conventional 3D printing, where a printed construct can quickly transform in response to a specific stimulus to switch between a temporary ...variable state and an original state. In this work, a photocrosslinkable polyethylene–glycol polyurethane ink is synthesized for light‐assisted 4D printing of smart materials. The molecular weight distribution of the ink monomers is tunable by adjusting the copolymerization reaction time. Digital light processing (DLP) technique is used to program a differential swelling response in the printed constructs after humidity variation. Bioactive microparticles are embedded into the ink and the improvement of biocompatibility of the printed constructs is demonstrated for tissue engineering applications. Cell studies reveal above 90% viability in 1 week and ≈50% biodegradability after 4 weeks. Self‐folding capillary scaffolds, dynamic grippers, and film actuators are made and activated in a humid environment. The approach offers a versatile platform for the fabrication of complex constructs. The ink can be used in tissue engineering and actuator applications, making the ink a promising avenue for future research.
Hosseinabadi and coworkers develop a generation of single material (non‐composite) actuators by synthesis of photocrosslinkable polyurethanes. Reaction time is adjusted for tuning molecular weight distribution and properties of 4D/DLP printed constructs. Achieved cellular scaffolds exhibit 90% viability, and 50% biodegradation rate during 4 weeks of culture. Printed actuators can transport objects five times their weight by controlling atmospheric humidity.
Myocardial remuscularization can be achieved by cardiomyocyte implantation. Electromechanical integration and long‐term survival of cardiomyocyte grafts are essential for maximal therapeutic impact. ...Cardiomyocyte application with support material has been instrumental in enhancing cell retention. Co‐administration of pro‐survival factors and immunological matching are additional strategies for increased cell graft survival. Finally, larger cardiomyocyte grafts, although therapeutically desirable, will increase the risk for arrhythmias and, if pluripotent stem cells are used to derive cardiomyocytes, tumour formation. This review introduces major challenges pertaining to myocardial remuscularization (cardiomyocyte retention, arrhythmogenicity and tumourigenicity), discusses studies addressing these challenges, and suggests strategies to overcome remaining challenges for the translation of myocardial remuscularization.
Schematic of tissue engineered heart repair with the aim to remuscularize the left ventricle.
Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high ...content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30-40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5-10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research.
Exosomes released from cancer cells support metastasis and growth of recipient cells and increase their resistance to chemotherapy. Therapeutic targeting of exosomes is a promising area in cancer ...research. Our aim is to test the effect of the mast cell stabilizer ketotifen on exosomes release from cancer cells and how this can modify their response to doxorubicin. Exosomes release from three cancer cell lines (MCF7, HeLa and BT549) was assessed by scan electron microscope and exosomes quantification kit. Doxorubicin export within exosomes was monitored flurometrically and cellular sensitivity to doxorubicin ± ketotifen was measured by sulphorhodamine-B and colony formation assays. The three cell lines release different amounts of exosomes with the highest quantity released from BT549 followed by MCF7 and then HeLa. Ketotifen (10 µmol L
−1
) reduced exosomes release in all three cell lines with different efficiency (HeLa>MCF7>BT549). Doxorubicin export via exosomes was highest in BT549, lower in HeLa and lowest in MCF7 cells. Pretreatment with ketotifen sensitized the cells to doxorubicin (HeLa>MCF7>BT549) with a sensitization factor of 27, 8 and 1.25 respectively. Increased sensitivity of cells to doxorubicin by ketotifen was proportional to its effect on exosomes release. Our data is the first report of ketotifen modulating exosomes release from cancer cells and opens the avenue for exosomes-targeting cancer therapy. The differential effects of ketotifen on doxorubicin exosomal export in the cell lines studied, suggests an opportunity of pharmacological enhancement of doxorubicin anti-tumor activity in some but not all cancer types.
Severe infections like sepsis lead frequently to cardiomyopathy. The mechanisms are unclear and an optimal therapy for septic cardiomyopathy still lacks. The aim of this study is to establish an ...endotoxin-induced inflammatory model using human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (hiPSC-CMs) for mechanistic and therapeutic studies. hiPSC-CMs were treated by lipopolysaccharide (LPS) in different concentrations for different times. ELISA, FACS, qPCR, and patch-clamp techniques were used for the study. TLR4 (Toll-like receptor 4) and its associated proteins, CD14, LBP (lipopolysaccharide binding protein), TIRAP (toll-interleukin 1 receptor domain containing adaptor protein), Ly96 (lymphocyte antigen 96) and nuclear factor kappa B as well as some pro-and anti-inflammatory factors are expressed in hiPSC-CMs. LPS-treatment for 6 hours increased the expression levels of pro-inflammatory and chemotactic cytokines (TNF-a, IL-1ß, IL-6, CCL2, CCL5, IL-8), whereas 48 hour-treatment elevated the expression of anti-inflammatory factors (IL-10 and IL-6). LPS led to cell injury resulting from exaggerated cell apoptosis and necrosis. Finally, LPS inhibited small conductance Ca
-activated K
channel currents, enhanced Na
/Ca
-exchanger currents, prolonged action potential duration, suggesting cellular electrical dysfunctions. Our data demonstrate that hiPSC-CMs possess the functional reaction system involved in endotoxin-induced inflammation and can model some bacterium-induced inflammatory responses in cardiac myocytes.
Cardiac fibrosis is a hallmark of heart failure for which there is no effective pharmacological therapy. By genetic modification and in vivo inhibitor approaches it was suggested that the ...Rho-associated kinases (ROCK1 and ROCK2) are involved in pro-fibrotic signalling in cardiac fibroblasts and that they may serve as targets for anti-fibrotic therapies. We demonstrate that simultaneous inhibition of ROCK1 and ROCK2 strongly interfered with tissue formation and their biomechanical properties in a model of engineered connective tissue (ECT), comprised of cardiac fibroblasts and collagen. These effects were observed with both rat and human ECT. Inhibitors of different chemistries, including the isoquinoline inhibitors Fasudil and H1152P as well as the pyrazol-phenyl inhibitor SR-3677, showed comparable effects. By combined treatment of ECT with TGF-β and H1152P, we could identify ROCK as a mediator of TGF-β-dependent tissue stiffening. Moreover, expression analyses suggested that lysyl oxidase (LOX) is a downstream target of the ROCK-actin-MRTF/SRF pathway and inhibition of this pathway by Latrunculin A and CCG-203971 showed similar anti-fibrotic effects in the ECT model as ROCK inhibitors. In line with the collagen crosslinking function of LOX, its inhibition by β-aminopropionitrile resulted in reduced ECT stiffness, but let tissue compaction unaffected. Finally, we show that ROCK inhibition also reduced the compaction and stiffness of engineered heart muscle tissues. Our results indicate that pharmacological inhibition of ROCK has a strong anti-fibrotic potential which is in part due to a decrease in the expression of the collagen crosslinking enzyme lysyl oxidase.
The sarcomeric troponin-tropomyosin complex is a critical mediator of excitation-contraction coupling, sarcomeric stability and force generation. We previously reported that induced pluripotent stem ...cell-derived cardiomyocytes (iPSC-CMs) from patients with a dilated cardiomyopathy (DCM) mutation, troponin T (TnT)-R173W, display sarcomere protein misalignment and impaired contractility. Yet it is not known how TnT mutation causes dysfunction of sarcomere microdomains and how these events contribute to misalignment of sarcomeric proteins in presence of DCM TnT-R173W. Using a human iPSC-CM model combined with CRISPR/Cas9-engineered isogenic controls, we uncovered that TnT-R173W destabilizes molecular interactions of troponin with tropomyosin, and limits binding of PKA to local sarcomere microdomains. This attenuates troponin phosphorylation and dysregulates local sarcomeric microdomains in DCM iPSC-CMs. Disrupted microdomain signaling impairs MYH7-mediated, AMPK-dependent sarcomere-cytoskeleton filament interactions and plasma membrane attachment. Small molecule-based activation of AMPK can restore TnT microdomain interactions, and partially recovers sarcomere protein misalignment as well as impaired contractility in DCM TnT-R173W iPSC-CMs. Our findings suggest a novel therapeutic direction targeting sarcomere- cytoskeleton interactions to induce sarcomere re-organization and contractile recovery in DCM.
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