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Improving equity is an emerging priority in climate and energy strategies, but little is known how these strategies would alter inequalities. Regional inequalities such as price, employment and land use are especially relevant in the electricity sector, which must decarbonize first to allow other sectors to decarbonize. Here, we show that a European low-carbon electricity sector in 2035 can reduce but also sustain associated regional inequalities. Using spatially-explicit modeling for 296 sub-national regions, we demonstrate that emission cuts consistent with net-zero greenhouse gas emissions in 2050 result in continent-wide benefits by 2035 regarding electricity sector investments, employment gains, and decreased greenhouse gas and particulate matter emissions. However, the benefits risk being concentrated in affluent regions of Northern Europe, while regions of Southern and Southeastern Europe risk high vulnerabilities due to high adverse impacts and sensitivities, and low adaptive capacities. Future analysis should investigate policy mechanisms for reducing and compensating inequalities.

Given the importance of ion gradients and fluxes in biology, monitoring ions locally at the exterior of the plasma membrane of intact cells in a noninvasive manner is highly desirable but challenging. Classical targeting of genetically encoded biosensors at the exterior of cell surfaces would be a suitable approach; however, it often leads to intracellular accumulation of the tools in vesicular structures and adverse modifications, possibly impairing sensor functionality. To tackle these issues, we generated recombinant fluorescent ion biosensors fused to traptavidin (TAv) specifically coupled to a biotinylated AviTag expressed on the outer cell surface of cells. We show that purified chimeras of TAv and pH-Lemon or GEPII 1.0, Förster resonance energy transfer-based pH and K+ biosensors, can be immobilized directly and specifically on biotinylated surfaces including glass platelets and intact cells, thereby remaining fully functional for imaging of ion dynamics. The immobilization of recombinant TAv–GEPII 1.0 on the extracellular cell surface of primary cortical rat neurons allowed imaging of excitotoxic glutamate-induced K+ efflux in vitro. We also performed micropatterning of purified TAv biosensors using a microperfusion system to generate spatially separated TAv–pH-Lemon and TAv–GEPII 1.0 spots for simultaneous pH and K+ measurements on cell surfaces. Our results suggest that the approach can be greatly expanded by immobilizing various biosensors on extracellular surfaces to quantitatively visualize microenvironmental transport and signaling processes in different cell culture models and other experimental settings.

All optical approaches to control and read out the electrical activity in a cardiac syncytium can improve our understanding of cardiac electrophysiology. Here, we demonstrate optogenetic stimulation of cardiomyocytes with high spatial precision using light foci generated with a ferroelectric spatial light modulator. Computer generated holograms binarized by bidirectional error diffusion create multiple foci with more even intensity distribution compared with thresholding approach. We evoke the electrical activity of cardiac HL1 cells expressing the channelrhodopsin-2 variant, ChR2(H134R) using single and multiple light foci and at the same time visualize the action potential using a calcium sensitive indicator called Cal-630. We show that localized regions in the cardiac monolayer can be stimulated enabling us to initiate signal propagation from a precise location. Furthermore, we demonstrate that probing the cardiac cells with multiple light foci enhances the excitability of the cardiac network. This approach opens new applications in manipulating and visualizing the electrical activity in a cardiac syncytium.

Carnosine (β-alanyl-l-histidine) and anserine (β-alanyl-3-methyl-l-histidine) are abundant peptides in the nervous system and skeletal muscle of many vertebrates. Many in vitro and in vivo studies demonstrated that exogenously added carnosine can improve muscle contraction, has antioxidant activity, and can quench various reactive aldehydes. Some of these functions likely contribute to the proposed anti-aging activity of carnosine. However, the physiological role of carnosine and related histidine-containing dipeptides (HCDs) is not clear. In this study, we generated a mouse line deficient in carnosine synthase (Carns1). HCDs were undetectable in the primary olfactory system and skeletal muscle of Carns1-deficient mice. Skeletal muscle contraction in these mice, however, was unaltered, and there was no evidence for reduced pH-buffering capacity in the skeletal muscle. Olfactory tests did not reveal any deterioration in 8-month-old mice lacking carnosine. In contrast, aging (18–24-month-old) Carns1-deficient mice exhibited olfactory sensitivity impairments that correlated with an age-dependent reduction in the number of olfactory receptor neurons. Whereas we found no evidence for elevated levels of lipoxidation and glycation end products in the primary olfactory system, protein carbonylation was increased in the olfactory bulb of aged Carns1-deficient mice. Taken together, these results suggest that carnosine in the olfactory system is not essential for information processing in the olfactory signaling pathway but does have a role in the long-term protection of olfactory receptor neurons, possibly through its antioxidant activity.

Achieving current electricity sector targets in Central Europe (Austria, Denmark, France, Germany, Poland and Switzerland) will redistribute regional benefits and burdens at sub-national level. Limiting emerging regional inequalities would foster the implementation success. We model one hundred scenarios of electricity generation, storage and transmission for 2035 in these countries for 650 regions and quantify associated regional impacts on system costs, employment, greenhouse gas and particulate matter emissions, and land use. We highlight tradeoffs among the scenarios that minimize system costs, maximize regional equality, and maximize renewable electricity generation. Here, we show that these three aims have vastly different implementation pathways as well as associated regional impacts and cannot be optimized simultaneously. Minimizing system costs leads to spatially-concentrated impacts. Maximizing regional equality of system costs has higher, but more evenly distributed impacts. Maximizing renewable electricity generation contributes to minimizing regional inequalities, although comes at higher costs and land use impacts., Implementation of Central European electricity targets will redistribute regional benefits and burdens. Here the authors show that the aims of cost-efficiency, regional equality, and renewable electricity generation have vastly different implementation pathways, impacts, and trade-offs.

Spatially-explicit bottom-up energy models with detailed renewable energy representation are increasingly developed. In order to inform such models, we investigate spatial diffusion patterns of solar PV projects in 2′222 Swiss municipalities. Using a dataset of feed-in tariff and one-time subsidy recipients in 2016, we show that PV diffusion was spatially uneven throughout Switzerland in terms of four indicators: the number of PV projects per municipality, per 1′000 inhabitants, per unit of municipal electricity demand, and per unit of municipal land area. Urban-rural divide and exploitable solar PV potential are the key, but not the only predictors of the spatial heterogeneity in PV diffusion. The structure of the municipal economy, socio-demographic characteristics, regional spillover effects, and additional differences in local contexts, such as local policies, matter as well. Spatial diffusion patterns to some extent structurally differ across sub-national regions too, indicating that such empirical investigations are valuable in order to understand what can be generalized. We conclude with recommendations for developing and validating spatially-resolved energy models so that they capture realistic patterns of solar PV diffusion: gather, maintain and analyze spatial data on PV projects and develop robust modelling functions that do not only rely on PV potential.

Control of heart rhythm is vital in the case of arrhythmia. Cardiac optogenetics is a promising technique to replace electrical stimulation in a next generation of pacemakers and defibrillators. Therefore, further research towards optimizing light delivery methods is essential. A major requirement is the uniform stimulation of all cells in the area of interest while reducing side effects such as photodamage. Here, a 2 × 2 blue (470 nm, InGaN-based) light-emitting diode (LED) optoelectronic module for uniform ex vivo cardiac muscle illumination is demonstrated. It satisfies two important requirements in optogenetics, which are high illumination homogeneity and high irradiance. CCD camera images show an average 90% homogeneity over the central illumination area of close to 38 mm2 at 1 cm distance from the light source. The module is used to perform physiological experiments on channelrhodopsin 2-expressing Langendorff-perfused mouse hearts. Successful ventricular pacing is obtained for an optical power density threshold below 2 mW/mm2 with light pulses as short as 1 ms. For 10 ms long pulses, the threshold was below 0.2 mW/mm2. The large homogeneous illumination area enabled optogenetic pacing with less than half the optical power of previous attempts with smaller areas of 2 mm2 and thus, presumably, will result in less phototoxicity.

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Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Deutsche Zentrum für Herz-Kreislauf-Forschung (DZHK) Background Gq signalling plays an imperative role in cardiac physiology as well as pathophysiologies such as arrhythmias, hypertrophy, and heart failure. However, the underlying kinetics and the transition from physiological to pathological signalling is yet to be investigated. Optogenetics provides an unprecedented spatial and temporal precision as well as cell specificity, but up to now, selective control of Gq signalling in the intact heart with light has not been achieved. Aim To prove the potential of human Neuropsin (OPN5) to selectively control Gq signalling in the adult heart. Methods The biophysical behaviour of OPN5 in HEK293 cells was characterized by measuring IP1 accumulation, Ca2+ imaging, and patch-clamp analysis of GIRK current activity. We generated a new transgenic mouse model expressing OPN5 in fusion with eYFP under the control of the chicken-β-actin promotor and analysed the expression pattern in the whole heart after tissue clearing with light sheet microscopy and the expression rate after single cell dissociation. Adult isolated cardiomyocytes were examined to measure the effect of OPN5 activation on contractility with a novel custom-made software for on-line contraction analysis by pixel tracking. In Langendorff-perfused hearts, illumination of the sinus node region with UV light (385 nm, 10 s, 1 mW/mm2) was used to measure the influence on the heart rate in electrogram recordings. The light-induced effects and basal ECG parameters as well as the heart-weight-to-femur-length ratio of OPN5 hearts were compared to hearts from non-expressing siblings as well as from CD1 wild-type. Results We prove the selective activation of the Gq proteins by UV light-induced increase in IP3 production and induction of Ca2+ transients in HEK293 cells because both effects were abolished after applying the Gq protein specific blocker FR900359. Promiscuous activation of Gi proteins was excluded by light-induced inhibition of GIRK channels instead of activation. Within two different mouse lines, we found an OPN5/eYFP expression rate of ∼70% within the cardiomyocytes targeted to the plasmamembrane. UV light induced a significant increase in contraction velocity in ventricular cardiomyocytes, and this effect could be abolished by blocking Gq proteins. In the right atrium HCN4 positive sinus nodal cells expressed OPN5/ eYFP and illumination of this region resulted in OPN5 expressing hearts in an average increase of 5.9 ± 0.97% (n= 20, in founder line 1) in the heart rate, which was significant lager than control hearts (all groups Conclusion We herein prove the specificity of OPN5 for Gq protein activation and demonstrate its use in cardiomyocytes as well as the whole heart. Thus OPN5 is the first optogenetic tool allowing to control Gq signalling in the whole heart precisely.

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On cellular or tissue level, electrophysiology studies during light stimulation with shaped light can potentially enable the design of effective stimulation strategies towards future cardiac therapies. In this work, we explore the use of optogenetics in probing the electrophysiology of a functional cardiac syncytium. A holographic technique with the use of a spatial light modulator allows beam shaping and precise spatiotemporal control of the origin of action potential in a cardiac-like cell line monolayer. Depolarization was performed by using a blue laser (488 nm) incident to a reflective ferroelectric liquid crystal on silicon spatial light modulator (FLCOS-SLM) which generates multiple foci on to cardiomyocyte-like cells (HL1 line) expressing the light-gated protein Channelrhodopsin-2 (ChR2). The calcium-sensitive fluorophore Cal-630 was used to visualize the cellular electrical activity and thus can be combined with ChR2 as an all-optical method to stimulate and visualize electrophysiology of cardiac-like cells in a non-contact manner. We generated single or multiple foci each with spot size of ~5 µm for optogenetic stimulation. The probability of triggering an action potential, which we refer to as excitation probability, is highly dependent on the laser intensity. The threshold intensity for a single beamlet, where approximately 50% of the laser pulses successfully trigger an action potential, is ~4 µW/μm² (n=5 regions, 5 cells analyzed per region, performed in triplicates) at frequency of 1 Hz and pulse duration of 10 ms. We also demonstrate spatial summation of membrane potential in HL1-ChR2. Generated multiple foci can render the excited cells as individual inputs to elicit action potential in a syncytium. We demonstrate the dependence of the excitation probability on the foci gap distance. Two foci, each with sub-threshold intensity of 2 µW/μm² were generated. At spacing of 50 µm, the median excitation probability increases compared to single spot stimulation with the same intensity.

In the past decade, in vivo models for optogenetic applications have gained importance, especially in the fields of cardiology and neuroscience. To reliably evoke the desired reactions while minimizing adverse effects, the stimulation power must be carefully adjusted. The relevant light intensity for cells in deeper layers of scattering tissue is not easily extrapolated from the power threshold of single cells. In this study, we evaluated a model for deep tissue optogenetic stimulation, using a heart-like cell line and tissue phantoms. Phantoms were fabricated from PDMS and titanium dioxide particles and possessed highly reproducible optical properties. Scattering and absorption coefficients were modeled to match those of realistic tissues. Since power of light traveling through tissues decays exponentially with respect to the scattering and absorption coefficients, the required input power was expected to increase exponentially by the same factor. To test this hypothesis, cells were stimulated through tissue phantoms of varying thickness with different modes of illumination. Cellular reactions revealed that the simplified assumptions were not sufficient to predict the input power required to reach the stimulation threshold. We provide a more comprehensive model to assess cellular reactions in scattering tissues a priori. This study has implications for the use of optogenetics in tissue models, organs and in vivo models as the outcomes can be transferred to different types of cells and tissues.

Aims Besides providing mechanical stability, fibroblasts in the heart could modulate the electrical properties of cardiomyocytes. Here, we aim to develop a three-dimensional hetero-cellular model to analyse the electric interaction between fibroblasts and human cardiomyocytes in vitro using selective optogenetic de- or hyperpolarization of fibroblasts. Methods and results NIH3T3 cell lines expressing the light-sensitive ion channel Channelrhodopsin2 or the light-induced proton pump Archaerhodopsin were generated for optogenetic depolarization or hyperpolarization, respectively, and characterized by patch clamp. Cardiac bodies consisting of 50% fibroblasts and 50% human pluripotent stem cell-derived cardiomyocytes were analysed by video microscopy and membrane potential was measured with sharp electrodes. Myofibroblast activation in cardiac bodies was enhanced by transforming growth factor-β1 (TGF-β1)-stimulation. Connexin-43 expression was analysed by qPCR and fluorescence recovery after photobleaching. Illumination of Channelrhodopsin2 or Archaerhodopsin expressing fibroblasts induced inward currents and depolarization or outward currents and hyperpolarization. Transforming growth factor-β1-stimulation elevated connexin-43 expression and increased cell–cell coupling between fibroblasts as well as increased basal beating frequency and cardiomyocyte resting membrane potential in cardiac bodies. Illumination of cardiac bodies generated with Channelrhodopsin2 fibroblasts accelerated spontaneous beating, especially after TGF-β1-stimulation. Illumination of cardiac bodies prepared with Archaerhodopsin expressing fibroblasts led to hyperpolarization of cardiomyocytes and complete block of spontaneous beating after TGF-β1-stimulation. Effects of light were significantly smaller without TGF-β1-stimulation. Conclusion Transforming growth factor-β1-stimulation leads to increased hetero-cellular coupling and optogenetic hyperpolarization of fibroblasts reduces TGF-β1 induced effects on cardiomyocyte spontaneous activity. Optogenetic membrane potential manipulation selectively in fibroblasts in a new hetero-cellular cardiac body model allows direct quantification of fibroblast–cardiomyocyte coupling in vitro.

Optogenetics is an emerging technique enabling optical control of a specific biological function in target cells by expression of light-sensitive proteins. The application of this technique in cardiac research, that is, cardiac optogenetics, mainly concerns the use of light-activated ion channels and pumps to manipulate the transmembrane potential of cardiomyocytes to control their excitability. Such control ranges from tailored stimulation to inhibition and offers novel and unique research possibilities regarding cardiac pacing, resynchronization and arrhythmia termination. Following a general introduction to optotools and gene transfer to the heart, these applications of optogenetics will be discussed in this chapter from mechanistic and translational points of view. Finally, key emerging innovations and future challenges regarding cardiac optogenetics will be outlined with the aim to provide a critical assessment of its clinical perspective.

L-type Ca2+ channels (LTCCs) play a crucial role in excitation-contraction coupling and release of hormones from secretory cells. They are targets of antihypertensive and antiarrhythmic drugs such as diltiazem. Here, we present a photoswitchable diltiazem, FHU-779, which can be used to reversibly block endogenous LTCCs by light. FHU-779 is as potent as diltiazem and can be used to place pancreatic β-cell function and cardiac activity under optical control.

Ventricular tachycardia (VT) is the most common and potentially lethal complication following myocardial infarction (MI). Biological correction of the conduction inhomogeneity that underlies re-entry could be a major advance in infarction therapy. As minimal increases in conduction of infarcted tissue markedly influence VT susceptibility, we reasoned that enhanced propagation of the electrical signal between non-excitable cells within a resolving infarct might comprise a simple means to decrease post-infarction arrhythmia risk. We therefore tested lentivirus-mediated delivery of the gap-junction protein Connexin 43 (Cx43) into acute myocardial lesions. Cx43 was expressed in (myo)fibroblasts and CD45+ cells within the scar and provided prominent and long lasting arrhythmia protection in vivo. Optical mapping of Cx43 injected hearts revealed enhanced conduction velocity within the scar, indicating Cx43-mediated electrical coupling between myocytes and (myo)fibroblasts. Thus, Cx43 gene therapy, by direct in vivo transduction of non-cardiomyocytes, comprises a simple and clinically applicable biological therapy that markedly reduces post-infarction VT.

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.

The standard technique for investigating adrenergic effects on heart function is perfusion with pharmaceutical agonists, which does not provide high temporal or spatial precision. Herein we demonstrate that the light sensitive Gs-protein coupled receptor JellyOp enables optogenetic stimulation of Gs-signaling in cardiomyocytes and the whole heart. Illumination of transgenic embryonic stem cell-derived cardiomyocytes or of the right atrium of mice expressing JellyOp elevates cAMP levels and instantaneously accelerates spontaneous beating rates similar to pharmacological β-adrenergic stimulation. Light application to the dorsal left atrium instead leads to supraventricular extrabeats, indicating adverse effects of localized Gs-signaling. In isolated ventricular cardiomyocytes from JellyOp mice, we find increased Ca2+ currents, fractional cell shortening and relaxation rates after illumination enabling the analysis of differential Gs-signaling with high temporal precision. Thus, JellyOp expression allows localized and time-restricted Gs stimulation and will provide mechanistic insights into different effects of site-specific, long-lasting and pulsatile Gs activation., Studying adrenergic signalling in the heart requires perfusion with receptor agonists, which lacks cell specificity and spatiotemporal control. Here the authors use the light sensitive G-coupled receptor JellyOp to optogenetically control Gs-signaling in cardiomyocytes and intact hearts with high spatiotemporal precision.

Decentralized renewable electricity generation (DREG) has been growing at an unprecedented pace, yet the appropriate spatial allocation and associated regional equity implications remain underinvestigated. In this study, we quantify the trade-offs between cost-efficient (least-cost) and regionally equitable DREG allocation in terms of electricity generation costs, investment needs, and DREG capacity requirements. Using the case of the ambitious and publicly-approved Swiss Energy Strategy 2050, we set up a bottom-up, technology-rich electricity system model EXPANSE with Modeling to Generate Alternatives at a spatial resolution of 2’258 Swiss municipalities. In order to measure regional equity implication, we adapt the concepts of the Lorenz curve and the Gini coefficient. We find a significant trade-off by 2035 in Switzerland: 50% increase in regional equity when allocating DREG to various Swiss regions on the basis of population or electricity demand leads to 18% higher electricity generation costs. Least-cost allocation implies concentrating DREG and associated investments to few most productive locations only. Solar PV is the key technology for increasing regional equity. We conclude that in countries with spatially-uneven DREG resources like Switzerland, any policies that focus on cost efficiency should anticipate regional equity implications in advance and, if desired, minimize them by promoting solar PV.

Ventricular arrhythmias are among the most severe complications of heart disease and can result in sudden cardiac death. Patients at risk currently receive implantable defibrillators that deliver electrical shocks to terminate arrhythmias on demand. However, strong electrical shocks can damage the heart and cause severe pain. Therefore, we have tested optogenetic defibrillation using expression of the light-sensitive channel channelrhodopsin-2 (ChR2) in cardiac tissue. Epicardial illumination effectively terminated ventricular arrhythmias in hearts from transgenic mice and from WT mice after adeno-associated virus-based gene transfer of ChR2. We also explored optogenetic defibrillation for human hearts, taking advantage of a recently developed, clinically validated in silico approach for simulating infarct-related ventricular tachycardia (VT). Our analysis revealed that illumination with red light effectively terminates VT in diseased, ChR2-expressing human hearts. Mechanistically, we determined that the observed VT termination is due to ChR2-mediated transmural depolarization of the myocardium, which causes a block of voltage-dependent Na+ channels throughout the myocardial wall and interrupts wavefront propagation into illuminated tissue. Thus, our results demonstrate that optogenetic defibrillation is highly effective in the mouse heart and could potentially be translated into humans to achieve nondamaging and pain-free termination of ventricular arrhythmia.

Light as a tool in medical therapy and biological research has been studied extensively and its application is subject to continuous improvement. However, safe and efficient application of light-based methods in photomedicine or optogenetics requires knowledge about the optical properties of the target tissue as well as the response characteristics of the stimulated cells. Here, we used tissue phantoms and a heart-like light-sensitive cell line to investigate optogenetic stimulation through tissue layers. The input power necessary for successful stimulation could be described as a function of phantom thickness. A model of light transmission through the tissue phantoms gives insights into the expected stimulation efficiency. Cell-type specific effects are identified that result in deviations of the stimulation threshold from the modelled predictions. This study provides insights into the complex interplay between light, tissue and cells during deep-tissue optogenetics. It can serve as an orientation for safe implementation of light-based methods in vivo.

Directed cardiac differentiation of human pluripotent stem cells (hPSCs) enables disease modeling, investigation of human cardiogenesis, as well as large-scale production of cardiomyocytes (CMs) for translational purposes. Multiple CM differentiation protocols have been developed to individually address specific requirements of these diverse applications, such as enhanced purity at a small scale or mass production at a larger scale. However, there is no universal high-efficiency procedure for generating CMs both in two-dimensional (2D) and three-dimensional (3D) culture formats, and undefined or complex media additives compromise functional analysis or cost-efficient upscaling. Using systematic combinatorial optimization, we have narrowed down the key requirements for efficient cardiac induction of hPSCs. This implied differentiation in simple serum and serum albumin-free basal media, mediated by a minimal set of signaling pathway manipulations at moderate factor concentrations. The method was applicable both to 2D and 3D culture formats as well as to independent hPSC lines. Global time-course gene expression analyses over extended time periods and in comparison with human heart tissue were used to monitor culture-induced maturation of the resulting CMs. This suggested that hPSC-CMs obtained with our procedure reach a rather stable transcriptomic state after approximately 4 weeks of culture. The underlying gene expression changes correlated well with a decline of immature characteristics as well as with a gain of structural and physiological maturation features within this time frame. These data link gene expression patterns of hPSC-CMs to functional readouts and thus define the cornerstones of culture-induced maturation. Stem Cells 2015;33:1456–1469

Background— Sudden infant death syndrome (SIDS) describes the sudden, unexplained death of a baby during its first year of age and is the third leading cause of infant mortality. It is assumed that ≤20% of all SIDS cases are because of cardiac arrhythmias resulting from mutations in ion channel proteins. Besides ion channels also cardiac gap junction channels are important for proper conduction of cardiac electric activation. In the mammalian heart Connexin43 (Cx43) is the major gap junction protein expressed in ventricular cardiomyocytes. Recently, a novel Connexin43 loss-of-function mutation (Cx43E42K) was identified in a 2-month-old SIDS victim. Methods and Results— We have generated Cx43E42K-expressing mice as a model for SIDS. Heterozygous cardiac-restricted Cx43E42K-mutated mice die neonatally without major cardiac morphological defects. Electrocardiographic recordings of embryonic Cx43+/E42K mice reveal severely disturbed ventricular activation, whereas immunohistochemical analyses show normal localization and expression patterns of gap junctional Connexin43 protein in the Cx43E42K-mutated newborn mouse heart. Conclusions— Because we did not find heterogeneous gap junction loss in Cx43E42K mouse hearts, we conclude that the Cx43E42K gap junction channel creates an arrhythmogenic substrate leading to lethal ventricular arrhythmias. The strong cardiac phenotype of Cx43E42K expressing mice supports the association between the human Cx43E42K mutation and SIDS and indicates that Connexin43 mutations should be considered in future studies when SIDS cases are to be molecularly explained.

Aims Optogenetic pacing of the heart has been demonstrated in transgenic animals expressing channelrhodopsin-2 (ChR2). However, for the clinical use of optogenetics to treat cardiac arrhythmias, gene transfer to non-transgenic hearts is required. The aim of this study was to describe a reliable method for gene transfer of ChR2 into a sufficient percentage of cardiomyocytes to overcome the electrical sink of all the coupled non-expressing cardiomyocytes during optical pacing of the whole heart in vivo . Methods and results Adeno-associated virus (AAV) with cardiac tropism for expression of ChR2 in fusion with mCherry was systemically injected into wild-type mouse hearts. Bright mCherry fluorescence was detected in the whole heart 4–10 weeks later. Single-cell dissociation revealed that on average 58% cardiomyocytes were mCherry-positive. These showed light-induced inward currents, action potentials, and contractions. Pulsed illumination of the left ventricle induced ventricular pacing in vivo in 74% of mice, and higher light intensities were required for reduced pulse duration or size of illumination. Non-responding hearts showed low AAV expression, and the threshold for optical pacing was estimated to be 35–40% ChR2-expressing cardiomyocytes. Optical pacing in vivo was stable over extended periods without negative effects on normal sinus rhythm and ECG parameters after termination of stimulation indicating sufficient cardiac output during pacing. Conclusions Gene transfer generates sufficient ChR2 photocurrent for reliable optogenetic pacing in vivo and lays out the basis for future optogenetic pacemaker and pain-free defibrillation therapies.

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

Side effects on cardiac ion channels causing lethal arrhythmias are one major reason for drug withdrawals from the market. Field potential (FP) recording from cardiomyocytes, is a well-suited tool to assess such cardiotoxic effects of drug candidates in preclinical drug development, but it is currently limited to the spontaneous beating of the cardiomyocytes and manual analysis. Herein, we present a novel optogenetic cardiotoxicity screening system suited for the parallel automated frequency-dependent analysis of drug effects on FP recorded from human pluripotent stem cell-derived cardiomyocytes. For the expression of the light-sensitive cation channel Channelrhodopsin-2, we optimised protocols using virus transduction or transient mRNA transfection. Optical stimulation was performed with a new light-emitting diode lid for a 96-well FP recording system. This enabled reliable pacing at physiologically relevant heart rates and robust recording of FP. Thereby we detected rate-dependent effects of drugs on Na+, Ca2+ and K+ channel function indicated by FP prolongation, FP shortening and the slowing of the FP downstroke component, as well as generation of afterdepolarisations. Taken together, we present a scalable approach for preclinical frequency-dependent screening of drug effects on cardiac electrophysiology. Importantly, we show that the recording and analysis can be fully automated and the technology is readily available using commercial products.

Cell replacement in the heart is considered a promising strategy for the treatment of post-infarct heart failure. Direct intramyocardial injection of cells proved to be the most effective application route, however, engraftment rates are very low (

Damage of peripheral nerves results in paralysis of skeletal muscle. Currently, the only treatment option to restore proper function is electrical stimulation of the innervating nerve or of the skeletal muscles directly. However this approach has low spatial and temporal precision leading to co-activation of antagonistic muscles and lacks cell-type selectivity resulting in pain or discomfort by stimulation of sensible nerves. In contrast to electrical stimulation, optogenetic methods enable spatially confined and cell-type selective stimulation of cells expressing the light sensitive channel Channelrhodopsin-2 with precise temporal control over the membrane potential. Herein we summarize the current knowledge about the use of this technology to control skeletal muscle function with the focus on the direct, non-neuronal stimulation of muscle fibers. The high temporal flexibility of using light pulses allows new stimulation patterns to investigate skeletal muscle physiology. Furthermore, the high spatial precision of focused illumination was shown to be beneficial for selective stimulation of distinct nearby muscle groups. Finally, the cell-type specific expression of the light-sensitive effector proteins in muscle fibers will allow pain-free stimulation and open new options for clinical treatments. Therefore, we believe that direct optogenetic stimulation of skeletal muscles is a very potent method for basic scientists that also harbors several distinct advantages over electrical stimulation to be considered for clinical use in the future.

In the present work, we investigated the synchronization of electrical activity in cultured cardiac cells of different origin put in direct contact. In the first set of experiments synchronization was studied in the primary culture cells of neonatal rats taken at different developmental ages, and in the second – in the neonatal rat cardiomyocytes and HL-1 cells. The electrical excitation of cells was recorded using the calcium transient marker Fluor-4. In the confluent cell layers created with the aid of a specially devised mask, the excitation waves and their propagation between areas occupied by cells of different origin were observed. On the level of individual cells, their contact and synchronization was monitored with the aid of scanning fluorescence microscopy. It was found that populations of cultured cells of different origin are able to synchronize, suggesting the formation of electrical coupling between them. The results obtained may be considered as a proof of concept that implanted alien grafted cells are able to create electrical coupling with the host cardiac tissue.

Aims The primary goal in the treatment of symptomatic atrial fibrillation/flutter (AF) is to restore sinus rhythm by cardioversion. Electrical shocks are highly effective, but have to be applied under analgo-sedation and can further harm the heart. In order to develop a novel pain-free and less harmful approach, we explored herein the optogenetic cardioversion by light-induced depolarization. Methods and results Hearts from mice expressing Channelrhodopsin-2 (ChR2) and the AF-promoting loss-of-function Connexin 40 Ala96Ser mutation were explanted and perfused with low K+ Tyrode's solution and an atrial KATP-channel activator. This new protocol shortened atrial refractoriness as well as slowed atrial conduction and thereby enabled the induction of sustained AF. AF episodes could be terminated by epicardial illumination of the atria with focussed blue light (470 nm, 0.4 mW/mm2) with an efficacy of ∼97% (n = 17 hearts). In > 80% of cases, light directly terminated the AF episode with onset of illumination. Because similar illumination intensity was able to locally inhibit atrial activity, we propose that a light-induced block of electrical activity is responsible for reliable AF termination. The success rate was strongly depending on the illuminated area, applied light intensity and duration of illumination. Importantly, we were also able to demonstrate optogenetic termination of AF in vivo, using epicardial illumination through the open chest (n = 3 hearts). To point towards a translational potential, we systemically injected an adeno-associated virus to express ChR2 in wild type hearts. After 6-8 months, we found robust ChR2 expression in the atria, enabling light-mediated AF termination in six of seven mice tested. Conclusion We provide the first evidence for optogenetic termination of atrial tachyarrhythmia in intact hearts from transgenic as well as wild type mice ex and in vivo. Thus, this report could lay the foundation for the development of implantable devices for pain-free termination of AF.

L-type Ca

KEY POINTS: The present study demonstrates, by in vitro and in vivo analyses, the novel concept that signal transmission between sympathetic neurons and the heart, underlying the physiological regulation of cardiac function, operates in a quasi‐synaptic fashion. This is a result of the direct coupling between neurotransmitter releasing sites and effector cardiomyocyte membranes. ABSTRACT: Cardiac sympathetic neurons (SNs) finely tune the rate and strength of heart contractions to match blood demand, both at rest and during acute stress, through the release of noradrenaline (NE). Junctional sites at the interface between the two cell types have been observed, although whether direct neurocardiac coupling has a role in heart physiology has not been clearly demonstrated to date. We investigated the dynamics of SN/cardiomyocyte intercellular signalling, both by fluorescence resonance energy transfer‐based imaging of cAMP in co‐cultures, as a readout of cardiac β‐adrenergic receptor activation, and in vivo, using optogenetics in transgenic mice with SN‐specific expression of Channelrhodopsin‐2. We demonstrate that SNs and cardiomyocytes interact at specific sites in the human and rodent heart, as well as in co‐cultures. Accordingly, neuronal activation elicited intracellular cAMP increases only in directly contacted myocytes and cell–cell coupling utilized a junctional extracellular signalling domain with an elevated NE concentration. In the living mouse, optogenetic activation of cardiac SNs innervating the sino‐atrial node resulted in an instantaneous chronotropic effect, which shortened the heartbeat interval with single beat precision. Remarkably, inhibition of the optogenetically elicited chronotropic responses required a high dose of propranolol (20–50 mg kg(–1)), suggesting that sympathetic neurotransmission in the heart occurs at a locally elevated NE concentration. Our in vitro and in vivo data suggest that the control of cardiac function by SNs occurs via direct intercellular coupling as a result of the establishment of a specific junctional site.