RESUMO
Side effects on cardiac ion channels are one major reason for new drugs to fail during preclinical evaluation. Herein we propose a simple optogenetic screening tool measuring extracellular field potentials (FP) from paced cardiomyocytes to identify drug effects over the whole physiological heart range, which is essential given the rate-dependency of ion channel function and drug action. Human induced pluripotent stem cell-derived cardiomyocytes were transduced with an adeno-associated virus to express Channelrhodopsin2 and plated on micro-electrode arrays. Global pulsed illumination (470 nm, 1 ms, 0.9 mW/mm2) was applied at frequencies from 1 to 2.5 Hz, which evoked FP simultaneously in all cardiomyocytes. This synchronized activation allowed averaging of FP from all electrodes resulting in one robust FP signal for analysis. Field potential duration (FPD) was ~25% shorter at 2.5 Hz compared to 1 Hz. Inhibition of hERG channels prolonged FPD only at low heart rates whereas Ca2+ channel block shortened FPD at all heart rates. Optogenetic pacing also allowed analysis of the maximum downstroke velocity of the FP to detect drug effects on Na+ channel availability. In principle, the presented method is well scalable for high content cardiac toxicity screening or personalized medicine for inherited cardiac channelopathies.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Optogenética/métodos , Células Cultivadas , Channelrhodopsins/análise , Channelrhodopsins/genética , Dependovirus/genética , Genes Reporter , Vetores Genéticos , Humanos , Transdução GenéticaRESUMO
PURPOSE: Magnetic nanoparticles (MNPs) and magnets can be used to enhance gene transfer or cell attachment but gene or cell delivery to confined areas has not been addressed. We therefore searched for an optimal method to simulate and perform local gene targeting and cell delivery in vitro. METHODS: Localized gene transfer or cell positioning was achieved using permanent magnets with newly designed soft iron tips and MNP/lentivirus complexes or MNP-loaded cells, respectively. Their distribution was simulated with a mathematical model calculating magnetic flux density gradients and particle trajectories. RESULTS: Soft iron tips generated strong confined magnetic fields and could be reliably used for local (~500 µm diameter) gene targeting and positioning of bone marrow cells or cardiomyocytes. The calculated distribution of MNP/lentivirus complexes and MNP-loaded cells concurred very well with the experimental results of local gene expression and cell attachment, respectively. CONCLUSION: MNP-based gene targeting and cell positioning can be reliably performed in vitro using magnetic soft iron tips, and computer simulations are effective methods to predict and optimize experimental results.
Assuntos
Marcação de Genes , Técnicas de Transferência de Genes , Magnetismo , Modelos Teóricos , Nanopartículas , Animais , Linhagem Celular , Células Cultivadas , Técnicas de Transferência de Genes/instrumentação , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismoRESUMO
The 7SK small nuclear RNA (snRNA) is a key player in the regulation of polymerase (pol) II transcription. The 7SK RNA was long believed to be specific to vertebrates where it is highly conserved. Homologs in basal deuterostomes and a few lophotrochozoan species were only recently reported. On longer timescales, 7SK evolves rapidly with only few conserved sequence and structure motifs. Previous attempts to identify the Drosophila homolog thus have remained unsuccessful despite considerable efforts. Here we report on the discovery of arthropod 7SK RNAs using a novel search strategy based on pol III promoters, as well as the subsequent verification of its expression. Our results demonstrate that a 7SK snRNA featuring 2 highly structured conserved domains was present already in the bilaterian ancestor.
