Human model of primary carnitine deficiency cardiomyopathy reveals ferroptosis as a novel mechanism

Primary carnitine deficiency (PCD) is an autosomal recessive monogenic disorder caused by mutations in SLC22A5. This gene encodes for OCTN2, which transports the essential metabolite carnitine into the cell. PCD patients suffer from muscular weakness and dilated cardiomyopathy. Two OCTN2-defective human induced pluripotent stem cell lines were generated, carrying a full OCTN2 knockout and a homozygous OCTN2 (N32S) loss-of-function mutation. OCTN2-defective genotypes showed lower force development and resting length in engineered heart tissue format compared with isogenic control. Force was sensitive to fatty acid-based media and associated with lipid accumulation, mitochondrial alteration, higher glucose uptake, and metabolic remodeling, replicating findings in animal models. The concordant results of OCTN2 (N32S) and -knockout emphasizes the relevance of OCTN2 for these findings. Importantly, genome-wide analysis and pharmacological inhibitor experiments identified ferroptosis, an iron- and lipid-dependent cell death pathway associated with fibroblast activation as a novel PCD cardiomyopathy disease mechanism

Goosecoid and Calponin : two new regulators of the PCP-pathway

Bei der Embryonalentwicklung von Wirbeltieren spielen insbesondere morphogenetische Umgestaltungen wie Zellmigration oder konvergente Extension (CE), bei der sich ein Gewebe streckt, eine wichtige Rolle. Der planare Zellpolaritäts-Signalweg (PCP; ?planar cell polarity?), ein nicht-kanonischer Wnt-Signalweg, gewährleistet die intrazelluläre Polarität der Zellen entlang der Embryonalachsen. Dieser führt über die polarisierte Lokalisierung von Proteinen wie Dishevelled, Vangl2 und Prickle an die Zellmembran zur Aktivierung von kleinen GTPasen wie Rho und Rac und damit zur Ausrichtung des Zytoskeletts. Diese Polarität der Zellen wird für CE, bei der bipolare Zellen interkalieren, während der Gastrulation und während der Neurulation benötigt. CE führt dabei zur Elongation der Chorda und der Neuralplatte, was den Neuralrohrschluss ermöglicht. In Vorarbeiten wurde beschrieben, dass die dorsale Überexpression des Transkriptionsfaktors Goosecoid (Gsc) in der Maus und im Frosch zu Neuralrohrschlussdefekten führt. In der vorliegenden Arbeit wurde durch Funktionsgewinnexperimente in vivo und ex vivo eine Inhibition konvergenter Extension durch Gsc gezeigt. Der Funktionsgewinn von Gsc verhinderte die Membranlokalisierung von Dishevelled in animalen Polkappenexplantaten, was eine Störung des PCP-Signalwegs anzeigt. Gsc-induzierte Fehlbildungen konnten durch Co-Injektion von Kernkomponenten des PCP-Signalwegs wie Vangl2 oder Prickle kompensiert werden. Auch die Überexpression der kleinen GTPase RhoA und des Liganden Wnt11 verhinderte den Effekt des Gsc-Funktionsgewinns. Brachyury, ein transkriptioneller Aktivator von Wnt11 und bekanntes Zielgen von Gsc, konnte die Wirkung von Gsc ebenso partiell umkehren. Diese Experimente legten eine neue Rolle von Gsc als Repressor PCP-induzierter CE nahe. Durch Funktionsverlustexperimente wurde im Frosch die endogene Funktion von Gsc untersucht. Aufgrund der konservierten und lokalisierten Expression von Gsc im Spemann-Organisator und der Induktion von Doppelachsen war eine Funktion von Gsc bei der Spezifizierung dorsaler Gewebe vorhergesagt worden. Die fehlenden Defekte in der Gsc Knock-out Maus standen allerdings dazu im Widerspruch. Die hier beschriebene Repression des PCP-Signalwegs durch Gsc deutete auf eine Funktion bei der Regulierung von Zellbewegungen hin. Dazu passt die Expression von Gsc in den Zellen des frühen Organisators, die während der Gastrulation zuerst einwandern und das prächordale Mesoderm bilden. In den nachfolgenden Zellen der Chorda findet dagegen CE statt. Gsc-Funktionsverlust reduzierte die Prächordalplatte und, als Konsequenz daraus, den Augenabstand. Die Activin-induzierte CE in animalen Polkappenexplantaten wurde durch Gsc-Funktionsverlust zusätzlich verstärkt. Damit konnte gezeigt werden, dass das Organisatorgen Gsc durch Repression des PCP-Signalwegs Zellbewegungen während der frühen Gastrulation reguliert. PCP steuert neben CE auch die gerichtete Wanderung von Neuralleistenzellen. Vorarbeiten zeigten eine Expression von Calponin2 in Neuralleistenzellen. Auch wurde eine Inhibition von Calponin1 durch die Rho-Kinase beschrieben. Ein Myc-Fusionskonstrukt des Aktin-Bindeproteins Calponin2 war in Xenopus in Zellfortsätzen und migrierenden Neuralleistenzellen lokalisiert. Der Funktionsverlust von Calponin2 verhinderte die gerichtete Bildung von Zellfortsätzen in Neuralleistenzellexplantaten und damit die Wanderung der Neuralleistenzellen. Dabei bildeten sich zusätzliche Stressfasern im zentralen Zellbereich auf Kosten des peripheren Aktin-Zytoskeletts. Der PCP-Signalweg aktiviert auf der der Wanderungsrichtung entgegengesetzten Seite RhoA und inhibiert Rac, was zur gerichteten Wanderung der Neuralleistenzellen führt. Dies implizierte eine Interaktion des PCP-Signalwegs und Calponin2 bei der Neuralleistenzellmigration, die durch Epistasisexperimente in vivo und in Neuralleistenzellexplantaten untersucht wurde. Der Funktionsverlust von Calponin2 konnte dabei den Funktionsverlust von nicht-kanonischem Wnt oder der Rho-Kinase retten. Damit gewährleistet das Aktinbindeprotein Calponin2 als Effektor des PCP-Signalwegs die Polarisierung des Aktin-Zytoskeletts in migrierenden Neuralleistenzellen. Zusammenfassend wurden in der vorliegenden Arbeit mit Calponin2 und Gsc zwei neue Regulatoren von PCP und Zellwanderung beschrieben. Daraus ergeben sich neue Ansätze zur Untersuchung der transkriptionellen Kontrolle des Zellwanderungsverhaltens (Gsc) und der nachgeschalteten Modulation des Zytoskeletts (Calponin2).Vertebrate embryogenesis relies on morphogenetic movements such as cell migration and convergent extension (CE). The planar cell polarity (PCP) branch of non-canonical Wnt signaling governs the orientation of cells along embryonic axes. PCP-signaling leads to intracellular polarization of proteins such as Dishevelled, Prickle and Vangl2, resulting in activation of small GTPases such as Rho and Rac, and consequently oriented alignment of the cytoskeleton. This polarity is required for CE, namely for the intercalation of bipolar cells, during gastrulation and neurulation. CE promotes elongation of the notochord and the neural plate, which is a prerequisite of neural tube closure. Previous work had shown that misexpression of the transcription factor Goosecoid (Gsc) in the primitive streak of the mouse and in the dorsal marginal zone of the frog led to neural tube closure defects. The present work demonstrates that misexpression of Gsc inhibits CE in vivo and ex vivo. Gsc gain-of-function (Gsc-GOF) prevented the membrane localization of Dishevelled in the frog animal cap assay, suggesting a disturbance of the PCP pathway. The Gsc-induced phenotypes could be rescued by co-injection of core components of the PCP pathway, Vangl2 and Prickle. Overexpression of RhoA and the non-canonical Wnt11, rescued the effect of Gsc-GOF. Brachyury, a transcriptional activator of Wnt11 and known target of Gsc, was also able to rescue the effect of Gsc-GOF. Gsc thus acted as a repressor of PCP-mediated CE. Furthermore, loss of function experiments in Xenopus were conducted to reveal the endogenous function of Gsc. Due to the conserved and distinct expression of Gsc in Spemann's organizer and the induction of double axes upon injection of Gsc into the ventral marginal zone in Xenopus, a function of Gsc in the specification of dorsal tissue was predicted. The lack of gastrulation defects in the Gsc knock-out mouse, however, questioned an early role of Gsc. The repression of the PCP pathway by Gsc-GOF suggested a novel role of Gsc in the regulation of cell movements. Interestingly, Gsc is expressed in a distinct population of cells in the early organizer, which migrate out of the organizer during early gastrulation to form the prechordal mesoderm. In contrast, the subsequent involuting cells of the notochord undergo CE. Gsc knock-down in the frog reduced the prechordal plate resulting in a narrowing of eye distance. Furthermore, activin-induced CE in animal cap explants was enhanced by Gsc loss-of-function. These findings are consistent with a novel function of the organizer gene Gsc in the regulation of cell movements during early gastrulation, namely the repression of PCP-mediated CE as a prerequisite of active migration of the prechordal mesoderm. The directed migration of neural crest cells represents another embryological process which depends on PCP-signaling. Previous work showed expression of Calponin2 in neural crest cells. Moreover, inhibition of Calponin1 by the Rho-Kinase has been described. In Xenopus, Calponin2 localized to cell protrusion of delaminating and migrating neural crest cells. Loss of function of Calponin2 prevented the polarized outgrowth of cell extensions in neural crest explants and thus migration of neural crest cells. Moreover, additional stress fibers were formed in the central area of neural crest cells at the expense of the peripheral, cortical actin cytoskeleton. The PCP pathway directs migration via the activation of RhoA and inhibition of Rac in the cell compartment opposed to the leading edge. This suggested an interaction of PCP-signaling and Calponin2 during the migration of neural crest cells, which was examined by rescue experiments in vivo and in neural crest explants. Calponin2 knock-down rescued Wnt11 and Rho-Kinase loss-of-function, strongly suggesting that the actin-binding protein Calponin2 acts as an effector of the PCP pathway and directs the polarization of the actin cytoskeleton in migrating neural crest cells. In summary the present work involved two novel regulators of PCP-mediated CE, Gsc at the transcriptional level and Calponin2 as an effector of the actin cytoskeleton

Effects of the Delta Opioid Receptor Agonist DADLE in a Novel Hypoxia-Reoxygenation Model on Human and Rat-Engineered Heart Tissue: A Pilot Study

Intermittent hypoxia and various pharmacological compounds protect the heart from ischemia reperfusion injury in experimental approaches, but the translation into clinical trials has largely failed. One reason may lie in species differences and the lack of suitable human in vitro models to test for ischemia/reperfusion. We aimed to develop a novel hypoxia-reoxygenation model based on three-dimensional, spontaneously beating and work performing engineered heart tissue (EHT) from rat and human cardiomyocytes. Contractile force, the most important cardiac performance parameter, served as an integrated outcome measure. EHTs from neonatal rat cardiomyocytes were subjected to 90 min of hypoxia which led to cardiomyocyte apoptosis as revealed by caspase 3-staining, increased troponin I release (time control vs. 24 h after hypoxia: cTnI 2.7 vs. 6.3 ng/mL, **\ua0p\ua0= 0.002) and decreased contractile force (64 ± 6% of baseline) in the long-term follow-up. The detrimental effects were attenuated by preceding the long-term hypoxia with three cycles of 10 min hypoxia (i.e., hypoxic preconditioning). Similarly, [d-Ala2,\ua0d-Leu5]-enkephalin (DADLE) reduced the effect of hypoxia on force (recovery to 78 ± 5% of baseline with DADLE preconditioning vs. 57 ± 5% without,\ua0p\ua0= 0.012), apoptosis and cardiomyocyte stress. Human EHTs presented a comparable hypoxia-induced reduction in force (55 ± 5% of baseline), but DADLE failed to precondition them, likely due to the absence of δ-opioid receptors. In summary, this hypoxia-reoxygenation in vitro model displays cellular damage and the decline of contractile function after hypoxia allows the investigation of preconditioning strategies and will therefore help us to understand the discrepancy between successful conditioning in vitro experiments and its failure in clinical trials

Calponin 2 Acts As an Effector of Noncanonical Wnt-Mediated Cell Polarization during Neural Crest Cell Migration

Neural crest cells (NCCs) migrate throughout the embryo to differentiate into cell types of all germ layers. Initial directed NCC emigration relies on planar cell polarity (PCP), which through the activity of the small GTPases RhoA and Rac governs the actin-driven formation of polarized cell protrusions. We found that the actin binding protein calponin 2 (Cnn2) was expressed in protrusions at the leading edge of migratory NCCs in chicks and frogs. Cnn2 knockdown resulted in NCC migration defects in frogs and chicks and randomized outgrowth of cell protrusions in NCC explants. Morphant cells showed central stress fibers at the expense of the peripheral actin network. Cnn2 acted downstream of Wnt/PCP, as migration defects induced by dominant-negative Wnt11 or inhibition of RhoA function were rescued by Cnn2 knockdown. These results suggest that Cnn2 modulates actin dynamics during NCC migration as an effector of noncanonical Wnt/PCP signaling

Human iPSC-derived cardiomyocytes cultured in 3D engineered heart tissue show physiological upstroke velocity and sodium current density

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising tool for drug testing and modelling genetic disorders. Abnormally low upstroke velocity is a current limitation. Here we investigated the use of 3D engineered heart tissue (EHT) as a culture method with greater resemblance to human heart tissue in comparison to standard technique of 2D monolayer (ML) format. INa was measured in ML or EHT using the standard patch-clamp technique. INa density was ~1.8 fold larger in EHT (-18.5 +/- 1.9 pA/pF; n = 17) than in ML (-10.3 +/- 1.2 pA/pF; n = 23; p < 0.001), approaching densities reported for human CM. Inactivation kinetics, voltage dependency of steady-state inactivation and activation of INa did not differ between EHT and ML and were similar to previously reported values for human CM. Action potential recordings with sharp microelectrodes showed similar upstroke velocities in EHT (219 +/- 15 V/s, n = 13) and human left ventricle tissue (LV, 253 +/- 7 V/s, n = 25). EHT showed a greater resemblance to LV in CM morphology and subcellular NaV1.5 distribution. INa in hiPSC-CM showed similar biophysical properties as in human CM. The EHT format promotes INa density and action potential upstroke velocity of hiPSC-CM towards adult values, indicating its usefulness as a model for excitability of human cardiac tissue

Contractile Work Contributes to Maturation of Energy Metabolism in hiPSC-Derived Cardiomyocytes

Summary: Energy metabolism is a key aspect of cardiomyocyte biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising tool for biomedical application, but they are immature and have not undergone metabolic maturation related to early postnatal development. To assess whether cultivation of hiPSC-CMs in 3D engineered heart tissue format leads to maturation of energy metabolism, we analyzed the mitochondrial and metabolic state of 3D hiPSC-CMs and compared it with 2D culture. 3D hiPSC-CMs showed increased mitochondrial mass, DNA content, and protein abundance (proteome). While hiPSC-CMs exhibited the principal ability to use glucose, lactate, and fatty acids as energy substrates irrespective of culture format, hiPSC-CMs in 3D performed more oxidation of glucose, lactate, and fatty acid and less anaerobic glycolysis. The increase in mitochondrial mass and DNA in 3D was diminished by pharmacological reduction of contractile force. In conclusion, contractile work contributes to metabolic maturation of hiPSC-CMs. : Ulmer et al. show that 3D-cultured human iPSC-derived cardiomyocytes replicate metabolic aspects of developmental hypertrophy, indicating maturation of hiPSC-derived cardiomyocytes. Keywords: human induced pluripotent stem cell-derived cardiomyocytes, engineered heart tissue, developmental hypertrophy, maturation, metabolis

Embryonic exposure to propylthiouracil disrupts left-right patterning in Xenopus embryos

Antithyroid medications are the preferred therapy for the treatment of Graves' disease during pregnancy. Propylthiouracil (PTU) is favored over methimazole (MMI) due to potential teratogenic concerns with MMI. This study was to determine the teratogenic potential of MMI and PTU using a validated Xenopus tropicalis embryo model. Embryos were exposed to 1 mM PTU (EC(50)=0.88 mM), 1 mM MMI, or vehicle control (water) from stages 2 to 45. Treated embryos were examined for gross morphological defects, ciliary function, and gene expression by in situ hybridization. Exposure to PTU, but not MMI, led to cardiac and gut looping defects and shortening along the anterior-posterior axis. PTU exposure during gastrulation (stage 8-12.5) was identified as the critical period of exposure leading to left-right (LR) patterning defects. Abnormal cilia polarization, abnormal cilia-driven leftward flow at the gastrocoel roof plate (GRP), and aberrant expression of both Coco and Pitx2c were associated with abnormal LR symmetry observed following PTU exposure. PTU is teratogenic during late blastula, gastrulation, and neurulation; whereas MMI is not. PTU alters ciliary-driven flow and disrupts the normal genetic program involved in LR axis determination. These studies have important implications for women taking PTU during early pregnanc

Atrial-like Engineered Heart Tissue: An In Vitro Model of the Human Atrium

Summary: Cardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are under investigation for their suitability as human models in preclinical drug development. Antiarrhythmic drug development focuses on atrial biology for the treatment of atrial fibrillation. Here we used recent retinoic acid-based protocols to generate atrial CMs from hiPSCs and establish right atrial engineered heart tissue (RA-EHT) as a 3D model of human atrium. EHT from standard protocol-derived hiPSC-CMs (Ctrl-EHT) and intact human muscle strips served as comparators. RA-EHT exhibited higher mRNA and protein concentrations of atrial-selective markers, faster contraction kinetics, lower force generation, shorter action potential duration, and higher repolarization fraction than Ctrl-EHTs. In addition, RA-EHTs but not Ctrl-EHTs responded to pharmacological manipulation of atrial-selective potassium currents. RA- and Ctrl-EHTs’ behavior reflected differences between human atrial and ventricular muscle preparations. Taken together, RA-EHT is a model of human atrium that may be useful in preclinical drug screening. : Lemme et al. developed a human, atrial-like engineered heart tissue from hiPSCs that could be used as an in vitro model of the human atrium to evaluate selectivity of novel ion channel blockers for atrial fibrillation. Keywords: hiPSC-CMs, pluripotent stem cells, atrial differentiation, atrial myocytes, atrial-like cells, retinoic acid, engineered heart tissue, cardiac tissue engineering, atrial fibrillatio