Opto-electronic feedback control of membrane potential for real-time control of action potentials.

To unlock new research possibilities by acquiring control of action potential (AP) morphologies in excitable cells, we developed an opto-electronic feedback loop-based system integrating cellular electrophysiology, real-time computing, and optogenetic approaches and applied it to monolayers of heart muscle cells. This allowed accurate restoration and preservation of cardiac AP morphologies in the presence of electrical perturbations of different origin in an unsupervised, self-regulatory manner, without any prior knowledge of the disturbance. Moreover, arbitrary AP waveforms could be enforced onto these cells. Collectively, these results set the stage for the refinement and application of opto-electronic control systems to enable in-depth investigation into the regulatory role of membrane potential in health and disease.

Modeling bile duct ischemia and reoxygenation injury in human cholangiocyte organoids for screening of novel cholangio-protective agents.

BACKGROUND: Ischemia of the bile duct is a common feature in liver disease and transplantation, which represents a major cause of morbidity and mortality, especially after liver transplantation. Detailed knowledge of its pathogenesis remains incomplete due to the lack of appropriate in vitro models. METHODS: To recapitulate biliary damage induced by ischemia and reperfusion in vitro, human intrahepatic cholangiocyte organoids (ICOs) were grown at low oxygen levels of 1% up to 72 h, followed by re-oxygenation at normal levels. FINDINGS: ICOs stressed by ischemia and subsequent re-oxygenation represented the dynamic change in biliary cell proliferation, upregulation of epithelial-mesenchymal transition (EMT)-associated markers, and the evocation of phase-dependent cell death programs similar to what is described in patients. Clinical-grade alpha-1 antitrypsin was identified as a potent inhibitor of both ischemia-induced apoptosis and necroptosis. INTERPRETATION: These findings demonstrate that ICOs recapitulate ischemic cholangiopathy in vitro and enable drug assessment studies for the discovery of new therapeutics for ischemic cholangiopathies. FUNDING: Dutch Digestive FoundationMLDS D16-26; TKI-LSH (Topconsortium Kennis en Innovatie-Life Sciences & Health) grant RELOAD, EMC-LSH19002; Medical Delta program "Regenerative Medicine 4D"; China Scholarship Council No. 201706230252.

Sbk2, a Newly Discovered Atrium-Enriched Regulator of Sarcomere Integrity.

BACKGROUND: Heart development relies on tight spatiotemporal control of cardiac gene expression. Genes involved in this intricate process have been identified using animals and pluripotent stem cell-based models of cardio(myo)genesis. Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal line of conditionally immortalized neonatal rat atrial myocytes (NRAMs), which allows toggling between proliferative and differentiated (ie, excitable and contractile) phenotypes in a synchronized and homogenous manner. METHODS: In this study, the unique properties of conditionally immortalized NRAMs (iAMs) were exploited to identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation. RESULTS: Transcriptome analysis of iAM-1 cells at different stages during one cycle of differentiation and subsequent dedifferentiation identified ≈13 000 transcripts, of which the dynamic changes in expression upon cardiomyogenic differentiation mostly opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many with known (lineage-specific) functions in cardiac muscle formation. Filtering for cardiac-enriched low-abundance transcripts, identified multiple genes with an uncharacterized role during cardio(myo)genesis including Sbk2 (SH3 domain binding kinase family member 2). Sbk2 encodes an evolutionarily conserved putative serine/threonine protein kinase, whose expression is strongly up- and downregulated during iAM-1 cell differentiation and dedifferentiation, respectively. In neonatal and adult rats, the protein is muscle-specific, highly atrium-enriched, and localized around the A-band of cardiac sarcomeres. Knockdown of Sbk2 expression caused loss of sarcomeric organization in NRAMs, iAMs and their human counterparts, consistent with a decrease in sarcomeric gene expression as evinced by transcriptome and proteome analyses. Interestingly, co-immunoprecipitation using Sbk2 as bait identified possible interaction partners with diverse cellular functions (translation, intracellular trafficking, cytoskeletal organization, chromatin modification, sarcomere formation). CONCLUSIONS: iAM-1 cells are a relevant and suitable model to identify (lowly expressed) genes with a hitherto unidentified role in cardiomyocyte differentiation as exemplified by Sbk2: a regulator of atrial sarcomerogenesis.

Conditional immortalization of human atrial myocytes for the generation of in vitro models of atrial fibrillation.

The lack of a scalable and robust source of well-differentiated human atrial myocytes constrains the development of in vitro models of atrial fibrillation (AF). Here we show that fully functional atrial myocytes can be generated and expanded one-quadrillion-fold via a conditional cell-immortalization method relying on lentiviral vectors and the doxycycline-controlled expression of a recombinant viral oncogene in human foetal atrial myocytes, and that the immortalized cells can be used to generate in vitro models of AF. The method generated 15 monoclonal cell lines with molecular, cellular and electrophysiological properties resembling those of primary atrial myocytes. Multicellular in vitro models of AF generated using the immortalized atrial myocytes displayed fibrillatory activity (with activation frequencies of 6-8 Hz, consistent with the clinical manifestation of AF), which could be terminated by the administration of clinically approved antiarrhythmic drugs. The conditional cell-immortalization method could be used to generate functional cell lines from other human parenchymal cells, for the development of in vitro models of human disease.

The Effects of Repetitive Use and Pathological Remodeling on Channelrhodopsin Function in Cardiomyocytes.

Aim: Channelrhodopsins (ChRs) are a large family of light-gated ion channels with distinct properties, which is of great importance in the selection of a ChR variant for a given application. However, data to guide such selection for cardiac optogenetic applications are lacking. Therefore, we investigated the functioning of different ChR variants in normal and pathological hypertrophic cardiomyocytes subjected to various illumination protocols. Methods and Results: Isolated neonatal rat ventricular cardiomyocytes (NRVMs) were transduced with lentiviral vectors to express one of the following ChR variants: H134R, CatCh, ReaChR, or GtACR1. NRVMs were treated with phenylephrine (PE) to induce pathological hypertrophy (PE group) or left untreated [control (CTL) group]. In these groups, ChR currents displayed unique and significantly different properties for each ChR variant on activation by a single 1-s light pulse (1 mW/mm2: 470, 565, or 617 nm). The concomitant membrane potential (V m) responses also showed a ChR variant-specific profile, with GtACR1 causing a slight increase in average V m during illumination (V plateau: -38 mV) as compared with a V plateau > -20 mV for the other ChR variants. On repetitive activation at increasing frequencies (10-ms pulses at 1-10 Hz for 30 s), peak currents, which are important for cardiac pacing, decreased with increasing activation frequencies by 17-78% (p < 0.05), while plateau currents, which are critical for arrhythmia termination, decreased by 10-75% (p < 0.05), both in a variant-specific manner. In contrast, the corresponding V plateau remained largely stable. Importantly, current properties and V m responses were not statistically different between the PE and CTL groups, irrespective of the variant used (p > 0.05). Conclusion: Our data show that ChR variants function equally well in cell culture models of healthy and pathologically hypertrophic myocardium but show strong, variant-specific use-dependence. This use-dependent nature of ChR function should be taken into account during the design of cardiac optogenetic studies and the interpretation of the experimental findings thereof.