Tyrosine kinase inhibitors (TKIs), despite efficacy as anti-cancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used patient-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen FDA-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a “cardiac safety index” to assess cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that VEGFR2/PDGFR-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. Using phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Upregulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during co-treatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anti-cancer TKIs, correlating with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.
SUMMARY Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na + ) channels and sarcoplasmic reticulum calcium (Ca 2+ ) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na + channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.
SUMMARY Spotted fever group (SFG) rickettsiae are human pathogens that infect cells in the vasculature. They disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Here we show that SFG species Rickettsia parkeri typically lack actin tails during spread and instead manipulate host intercellular tension and mechanotransduction to promote spread. Using transposon mutagenesis, we identified surface cell antigen 4 (Sca4) as a secreted effector of spread that specifically promotes protrusion engulfment. Sca4 interacts with the cell adhesion protein vinculin and blocks association with vinculin’s binding partner, α-catenin. Using traction and monolayer stress microscopy, we show that Sca4 reduces vinculin-dependent mechanotransduction at cell-cell junctions. Our results suggest that Sca4 relieves intercellular tension to promote protrusion engulfment, which represents a distinctive strategy for manipulating cytoskeletal force generation to enable spread.
Patients with COVID19 present a broad spectrum of clinical presentation. Whereas hypoxemia is the marker of severity, different strategies of management should be customized to five specific individual phenotypes. Many intubated patients present with phenotype 4, characterized by pulmonary hypoxic vasoconstriction, being associated with severe hypoxemia with "normal" (>40 ml/cm H20) lung compliance and likely represents pulmonary microvascular thrombosis. Phenotype 5 is often associated with high plasma procalcitonin, and has low pulmonary compliance, being a result of co-infection or acute lung injury after non-invasive ventilation. Identifying these clinical phenotypes and applying a personalized approach would benefit in optimization of therapies and improving outcomes. Tweeter feed:SARS-CoV-2 infections present different specific individual phenotypes. Applying a personalized approach would benefit in optimization of therapies and outcomes improvement. @COVID19
Cardiac drug discovery is hampered by the reliance on non-human animal and cellular models with inadequate throughput and physiological fidelity to accurately identify new targets and test novel therapeutic strategies. Similarly, adverse drug effects on the heart are challenging to model, contributing to costly failure of drugs during development and even after market launch. Human induced pluripotent stem cell derived cardiac tissue represents a potentially powerful means to model aspects of heart physiology relevant to disease and adverse drug effects, providing both the human context and throughput needed to improve the efficiency of drug development. Here we review emerging technologies for high throughput measurements of cardiomyocyte physiology, and comment on the promises and challenges of using iPSC-derived cardiomyocytes to model disease and introduce the human context into early stages of drug discovery.
Cardiotoxicity has historically been a major cause of drug removal from the pharmaceutical market. Several chemotherapeutic compounds have been noted for their propensities to induce dangerous cardiac-specific side effects such as arrhythmias or cardiomyocyte apoptosis. However, improved preclinical screening methodologies have enabled cardiotoxic compounds to be identified earlier in the drug development pipeline. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to screen for drug-induced alterations in cardiac cellular contractility, electrophysiology, and viability. We previously established a novel ‘cardiac safety index’ (CSI) as a metric that can evaluate potential cardiotoxic drugs via high-throughput screening of hiPSC-CMs. This metric quantitatively examines drug-induced alterations in CM function, using several in vitro readouts, and normalizes the resulting toxicity values to the in vivo maximum drug blood plasma concentration seen in preclinical or clinical pharmacokinetic models. In this ~1-month-long protocol, we describe how to differentiate hiPSCs into hiPSC-CMs and subsequently implement contractility and cytotoxicity assays that can evaluate drug-induced cardiotoxicity in hiPSC-CMs. We also describe how to carry out the calculations needed to generate the CSI metric from these quantitative toxicity measurements.
Leukocyte transmigration across vessel walls is a critical step in the innate immune response. Upon their activation and firm adhesion to vascular endothelial cells (VECs), leukocytes preferentially extravasate across junctional gaps in the endothelial monolayer (paracellular diapedesis). It has been hypothesized that VECs facilitate paracellular diapedesis by opening their cell-cell junctions in response to the presence of an adhering leukocyte. However, it is unclear how leukocytes interact mechanically with VECs to open the VEC junctions and migrate across the endothelium. In this study, we measured the spatial and temporal evolution of the 3D traction stresses generated by the leukocytes and VECs to elucidate the sequence of mechanical events involved in paracellular diapedesis. Our measurements suggest that the contractile stresses exerted by the leukocytes and the VECs can separately perturb the junctional tensions of VECs to result in the opening of gaps before the initiation of leukocyte transmigration. Decoupling the stresses exerted by the transmigrating leukocytes and the VECs reveals that the leukocytes actively contract the VECs to open a junctional gap and then push themselves across the gap by generating strong stresses that push into the matrix. In addition, we found that diapedesis is facilitated when the tension fluctuations in the VEC monolayer were increased by proinflammatory thrombin treatment. Our findings demonstrate that diapedesis can be mechanically regulated by the transmigrating leukocytes and by proinflammatory signals that increase VEC contractility.
Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-β (Tgf-β) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-β expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-β inhibitor resulted in increased EMCM size. Functionally, Tgf-β signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS.
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