Live-Cell Imaging of the Contractile Velocity and Transient Intracellular Ca²⁺ Fluctuations in Human Stem Cell-Derived Cardiomyocytes

Live-cell imaging techniques are essential for acquiring vital physiological and pathophysiological knowledge to understand and treat heart disease. For live-cell imaging of transient alterations of [Ca²⁺]i in human cardiomyocytes, we engineered human-induced pluripotent stem cells carrying a genetically-encoded Ca²⁺-indicator (GECI). To monitor sarcomere shortening and relaxation in cardiomyocytes in real-time, we generated a alpha-cardiac actinin (ACTN2)-copepod (cop) green fluorescent protein (GFP⁺)-human-induced pluripotent stem cell line by using the CRISPR-Cas9 and a homology directed recombination approach. The engineered human-induced pluripotent stem cells were differentiated in transgenic GECI-enhanced GFP⁺-cardiomyocytes and ACTN2-copGFP⁺ -cardiomyocytes, allowing real-time imaging of [Ca²⁺]i transients and live recordings of the sarcomere shortening velocity of ACTN2-copGFP⁺ -cardiomyocytes. We developed a video analysis software tool to quantify various parameters of sarcoplasmic Ca²⁺ fluctuations recorded during contraction of cardiomyocytes and to calculate the contraction velocity of cardiomyocytes in the presence and absence of different drugs affecting cardiac function. Our cellular and software tool not only proved the positive and negative inotropic and lusitropic effects of the tested cardioactive drugs but also quantified the expected effects precisely. Our platform will offer a human-relevant in vitro alternative for high-throughput drug screenings, as well as a model to explore the underlying mechanisms of cardiac diseases

Parabolic, Flight-Induced, Acute Hypergravity and Microgravity Effects on the Beating Rate of Human Cardiomyocytes

Functional studies of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hCMs) under different gravity conditions contribute to aerospace medical research. To study the effects of altered gravity on hCMs, we exposed them to acute hypergravity and microgravity phases in the presence and absence of the β-adrenoceptor isoprenalin (ISO), L-type Ca2+ channel (LTCC) agonist Bay-K8644, or LTCC blocker nifedipine, and monitored their beating rate (BR). These logistically demanding experiments were executed during the 66th Parabolic Flight Campaign of the European Space Agency. The hCM cultures were exposed to 31 alternating hypergravity, microgravity, and hypergravity phases, each lasting 20-22 s. During the parabolic flight experiment, BR and cell viability were monitored using the xCELLigence real-time cell analyzer Cardio Instrument®. Corresponding experiments were performed on the ground (1 g), using an identical set-up. Our results showed that BR continuously increased during the parabolic flight, reaching a 40% maximal increase after 15 parabolas, compared with the pre-parabolic (1 g) phase. However, in the presence of the LTCC blocker nifedipine, no change in BR was observed, even after 31 parabolas. We surmise that the parabola-mediated increase in BR was induced by the LTCC blocker. Moreover, the increase in BR induced by ISO and Bay-K8644 during the pre-parabola phase was further elevated by 20% after 25 parabolas. This additional effect reflects the positive impact of the parabolas in the absence of both agonists. Our study suggests that acute alterations of gravity significantly increase the BR of hCMs via the LTCC

Recent Topics in Gravitational Biology Research at the DLR Institute of Aerospace Medicine

Ground-based facilities (GBFs) such as clinostats, Random Positioning Machines, centrifuges and others were utilized by European researchers during the ESA Ground-based Facility Program and projects and results were recently published. Here, we provide examples for the use of and experiences gained with GBFs at the Institute of Aerospace Medicine (DLR), department of Gravitational Biology. We discuss differences and limitations of various facilities in comparison to each other in respect to their ability to provide altered gravity from the view of the biosystem employed. Our results support the necessity of a ground-based program, which at low cost - in comparison to space flight - provides the opportunity to intensively prepare experiments for space. Finally, ground-based data should ideally be validated in real microgravity, which we performed to some extent using the drop tower in Bremen, parabolic flights and sounding rockets. Furthermore, we give an overview of our current research topics such as mouse neuronal development in hypergravity as well as mouse embryonic stem cell differentiation, demonstrating a distinct influence of (altered) gravity on the underlying mechanisms

Cell death mechanisms of the anti-cancer drug etoposide on human cardiomyocytes isolated from pluripotent stem cells

Etoposide (ETP) and anthracyclines are applied for wide anti-cancer treatments. However, the ETP-induced cardiotoxicity remains to be a major safety issue and the underlying cardiotoxic mechanisms are not well understood. This study is aiming to unravel the cardiotoxicity profile of ETP in comparison to anthracyclines using physiologically relevant human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Using xCELLigence real-time cell analyser (RTCA), we found that single high dose of ETP induces irreversible increase in hPSC-CMs beating rate and decrease in beating amplitude. We also identified 58 deregulated genes consisting of 33 upregulated and 25 downregulated genes in hPSC-CMs after ETP treatment. Gene ontology (GO) and pathway analysis showed that most upregulated genes are enriched in GO categories like positive regulation of apoptotic process, regulation of cell death, and mitochondria organization, whereas most downregulated genes were enriched in GO categories like cytoskeletal organization, muscle contraction, and Ca2+ ion homeostasis. Moreover, we also found upregulation in 5 miRNAs (has-miR-486-3p, has-miR-34c-5p, has-miR-4423-3p, has-miR-182-5p, and has-miR-139-5p) which play role in muscle contraction, arginine and proline metabolism, and hypertrophic cardiomyopathy (HCM). Immunostaining and transmission electron microscopy also confirmed the cytoskeletal and mitochondrial damage in hPSC-CMs treated with ETP, as well as noticeable alterations in intracellular calcium handling and mitochondrial membrane potential were also observed. The apoptosis inhibitor, Pifithrin-alpha, found to protect hPSC-CMs from ETP-induced cardiotoxicity, whereas hPSC-CMs treated with ferroptosis inhibitor, Liproxstatin-1, showed significant recovery in hPSC-CMs functional properties like beating rate and amplitude after ETP treatment. We suggest that the damage to mitochondria is a major contributing factor involved in ETP-induced cardiotoxicity and the activation of the p53-mediated ferroptosis pathway by ETP is likely the critical pathway in ETP-induced cardiotoxicity. We also conclude that the genomic biomarkers identified in this study will significantly contribute to develop and predict potential cardiotoxic effects of novel anti-cancer drugs in vitro

Live-Cell Mikroskopie unter veränderten Schwerkraftbedingungen

Einleitung: In der Gravitationsbiologie verwenden wir Klinostaten und Zentrifugen, um veränderte Schwerkraftbedingungen zu simulieren und zu erzeugen und die Reaktionen zellulärer Systeme darauf auszuwerten. Standardmäßig werden Proben nach der Exposition aus den Geräten entnommen und chemisch fixiert, wodurch sich mehrere Nachteile ergeben. Zum einen vergeht einige Zeit zwischen Entnahme der Proben und der Fixierung, in der die Zellen wieder normaler 1g Erdbeschleunigung ausgesetzt sind und readaptieren können. Zum anderen kann eine Probe immer nur zu dem einen fixen Zeitpunkt betrachtet werden. Wir haben es uns folglich zur Aufgabe gemacht, Live-Cell Mikroskopie in Hypergravitation und simulierter Mikrogravitation zu etablieren. Fragestellung: Moderne Mikroskope können vollautomatisch über Remote-Verbindungen angesteuert und bedient werden und eignen sich somit für Anwendungen bei denen man nicht direkt auf sie zugreifen kann, wie zum Beispiel auf Zentrifugen oder Klinostaten. Hypergravitation auf der Zentrifuge oder die Drehung um die eigene Achse im Klinostatenprinzip stellen außergewöhnliche Beanspruchungen an die mikrometergenaue Feinmechanik eines Mikroskops dar. Welche Herausforderungen gibt es bei der Anpassung eines Live-Cell Mikroskops und wie haben wir diese gemeistert? Methodik: Für Live-Cell Mikroskopie in Hypergravitation haben wir ein Zeiss Axio Observer Mikroskop mit einer kleinen Inkubationseinheit sowie motorisiertem Tisch und Definite Focus Modul ausgestattet. Für dieses Mikroskop wurde eine ausschwingende Plattform gebaut und auf einen Arm der SAHC1 (Short Arm Human Centrifuge) im DLR :envihab platziert. Vom Zentrifugenkontrollraum aus lässt sich das Mikroskop in Echtzeit bedienen und lebende Zellen bei bis zu 2g Hypergravitation betrachten. Das neue Klinostatenmikroskop hat als zentrales Element ein Keyence Fluoreszenzmikroskop, welches horizontal gelagert wird und bei 60 Umdrehungen pro min um seine eigene Achse rotiert, wobei der Strahlengang des Mikroskops und damit auch der Objektträger genau auf der Drehachse liegt, um die Probe minimalen Restbeschleunigungen auszusetzen und damit in simulierter Mikrogravitation beobachten zu können. Die komplette Mikroskopeinheit ist von einer Inkubationskammer ummantelt, so dass auch Zellkulturen bei 37 Grad C untersucht werden können. Ergebnisse: Durch das Betrachten von Zellen und Geweben in Echtzeit oder in Zeitrafferaufnahmen lassen sich vorher nie dagewesene Einblicke in Dynamiken von Zellen und deren Reaktion auf veränderte Schwerkraftbedingungen gewinnen. Die Analyse der Migrationsgeschwindigkeiten von Astrozyten (ein Typ Gliazelle im Gehirn) über 24 Stunden bei 2g Hypergravitation deutet auf kritische Zeitfenster in der Adaptation dieser Zellen auf die erhöhte Schwerkraft hin. Im Klinostatenmikroskop konnten wir u.a. die Schlaggeschwindigkeit von aus Stammzellen abgeleiteten Herzmuskelzellen in simulierter Mikrogravitation messen. Schlussfolgerungen: Durch die Kombination von Mikroskopie und Bodenanlagen können wir nun neue Einblicke in (intra-)zelluläre Dynamiken und Reizantworten unter veränderten Gravitationsbedingungen gewinnen

Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells

Application of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is limited by the challenges in their efficient differentiation. Recently, the Wingless (Wnt) signaling pathway has emerged as the key regulator of cardiomyogenesis. In this study, we evaluated the effects of cyclooxygenase inhibitors on cardiac differentiation of hPSCs. Cardiac differentiation was performed by adherent monolayer based method using 4 hPSC lines (HES3, H9, IMR90, and ES4SKIN). The efficiency of cardiac differentiation was evaluated by flow cytometry and RT-qPCR. Generated hPSC-CMs were characterised using immunocytochemistry, electrophysiology, electron microscopy, and calcium transient measurements. Our data show that the COX inhibitors Sulindac and Diclofenac in combination with CHIR99021 (GSK-3 inhibitor) efficiently induce cardiac differentiation of hPSCs. In addition, inhibition of COX using siRNAs targeted towards COX-1 and/or COX-2 showed that inhibition of COX-2 alone or COX-1 and COX-2 in combination induce cardiomyogenesis in hPSCs within 12 days. Using IMR90-Wnt reporter line, we showed that inhibition of COX-2 led to downregulation of Wnt signalling activity in hPSCs. In conclusion, this study demonstrates that COX inhibition efficiently induced cardiogenesis via modulation of COX and Wnt pathway and the generated cardiomyocytes express cardiac-specific structural markers as well as exhibit typical calcium transients and action potentials. These cardiomyocytes also responded to cardiotoxicants and can be relevant as an in vitro cardiotoxicity screening model

Modulation of Differentiation Processes in Murine Embryonic Stem Cells Exposed to Parabolic Flight-Induced Acute Hypergravity and Microgravity

Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of short-term altered gravity on embryonic development processes, we exposed mouse embryonic stem cells (mESCs) to phases of hypergravity and microgravity and studied the differentiation potential of the cells using wide-genome microarray analysis. During the 64th European Space Agency's parabolic flight campaign, mESCs were exposed to 31 parabolas. Each parabola comprised phases lasting 22 s of hypergravity, microgravity, and a repeat of hypergravity. On different parabolas, RNA was isolated for microarray analysis. After exposure to 31 parabolas, mESCs (P31 mESCs) were further differentiated under normal gravity (1 g) conditions for 12 days, producing P31 12-day embryoid bodies (EBs). After analysis of the microarrays, the differentially expressed genes were analyzed using different bioinformatic tools to identify developmental and nondevelopmental biological processes affected by conditions on the parabolic flight experiment. Our results demonstrated that several genes belonging to GOs associated with cell cycle and proliferation were downregulated in undifferentiated mESCs exposed to gravity changes. However, several genes belonging to developmental processes, such as vasculature development, kidney development, skin development, and to the TGF-β signaling pathway, were upregulated. Interestingly, similar enriched and suppressed GOs were obtained in P31 12-day EBs compared with ground control 12-day EBs. Our results show that undifferentiated mESCs exposed to alternate hypergravity and microgravity phases expressed several genes associated with developmental/differentiation and cell cycle processes, suggesting a transition from the undifferentiated pluripotent to a more differentiated stage of mESCs