Background—: After the recent cloning of light-sensitive ion channels and their expression in mammalian cells, a new field, optogenetics, emerged in neuroscience, allowing for precise perturbations of neural circuits by light. However, functionality of optogenetic tools has not been fully explored outside neuroscience, and a nonviral, nonembryogenesis-based strategy for optogenetics has not been shown before. Methods and Results—: We demonstrate the utility of optogenetics to cardiac muscle by a tandem cell unit (TCU) strategy, in which nonexcitable cells carry exogenous light-sensitive ion channels, and, when electrically coupled to cardiomyocytes, produce optically excitable heart tissue. A stable channelrhodopsin2 (ChR2)-expressing cell line was developed, characterized, and used as a cell delivery system. The TCU strategy was validated in vitro in cell pairs with adult canine myocytes (for a wide range of coupling strengths) and in cardiac syncytium with neonatal rat cardiomyocytes. For the first time, we combined optical excitation and optical imaging to capture light-triggered muscle contractions and high-resolution propagation maps of light-triggered electric waves, found to be quantitatively indistinguishable from electrically triggered waves. Conclusions—: Our results demonstrate feasibility to control excitation and contraction in cardiac muscle by light, using the TCU approach. Optical pacing in this case uses less energy, offers superior spatiotemporal control and remote access and can serve not only as an elegant tool in arrhythmia research but may form the basis for a new generation of light-driven cardiac pacemakers and muscle actuators. The TCU strategy is extendable to (nonviral) stem cell therapy and is directly relevant to in vivo applications.

The improvement of preclinical cardiotoxicity testing, discovery of new ion-channel-targeted drugs, and phenotyping and use of stem cell-derived cardiomyocytes and other biologics all necessitate high-throughput (HT), cellular-level electrophysiological interrogation tools. Optical techniques for actuation and sensing provide instant parallelism, enabling contactless dynamic HT testing of cells and small-tissue constructs, not affordable by other means. Here we show, computationally and experimentally, the limits of all-optical electrophysiology when applied to drug testing, then implement and validate OptoDyCE, a fully automated system for all-optical cardiac electrophysiology. We validate optical actuation by virally introducing optogenetic drivers in rat and human cardiomyocytes or through the modular use of dedicated light-sensitive somatic ‘spark’ cells. We show that this automated all-optical approach provides HT means of cellular interrogation, that is, allows for dynamic testing of >600 multicellular samples or compounds per hour, and yields high-content information about the action of a drug over time, space and doses.

Channelrhodospin-2 (ChR2), a light-sensitive ion channel, and its variants have emerged as new excitatory optogenetic tools not only in neuroscience, but also in other areas, including cardiac electrophysiology. An accurate quantitative model of ChR2 is necessary for in silico prediction of the response to optical stimulation in realistic tissue/organ settings. Such a model can guide the rational design of new ion channel functionality tailored to different cell types/tissues. Focusing on one of the most widely used ChR2 mutants (H134R) with enhanced current, we collected a comprehensive experimental data set of the response of this ion channel to different irradiances and voltages, and used these data to develop a model of ChR2 with empirically-derived voltage- and irradiance- dependence, where parameters were fine-tuned via simulated annealing optimization. This ChR2 model offers: 1) accurate inward rectification in the current-voltage response across irradiances; 2) empirically-derived voltage- and light-dependent kinetics (activation, deactivation and recovery from inactivation); and 3) accurate amplitude and morphology of the response across voltage and irradiance settings. Temperature-scaling factors (Q₁₀) were derived and model kinetics was adjusted to physiological temperatures. Using optical action potential clamp, we experimentally validated model-predicted ChR2 behavior in guinea pig ventricular myocytes. The model was then incorporated in a variety of cardiac myocytes, including human ventricular, atrial and Purkinje cell models. We demonstrate the ability of ChR2 to trigger action potentials in human cardiomyocytes at relatively low light levels, as well as the differential response of these cells to light, with the Purkinje cells being most easily excitable and ventricular cells requiring the highest irradiance at all pulse durations. This new experimentally-validated ChR2 model will facilitate virtual experimentation in neural and cardiac optogenetics at the cell and organ level and provide guidance for the development of in vivo tools. Optogenetics, the use of light-sensitive ion channels for stimulation of mammalian cells and tissues, offers specificity and superior precision of control compared to traditional chemical or electrical means of stimulation. In particular, Channelrhodospin-2 (ChR2), a light-sensitive ion channel, originally derived from algae, has found wide-spread application in neuroscience for controlled stimulation of different brain regions. More recently, this work was extended to other organs, including the heart, where it opens the possibility for a new generation of optical pacemakers. The development of new optogenetic tools that allow for more efficient optical stimulation can be guided by computational prediction of the response of different cells and tissues to light. In this report, we provide a new computational model of ChR2 that was empirically validated and can be inserted into different cell types – neurons or heart cells – for virtual optical stimulation and prediction of optimal light-delivery arrangements, minimum energy needs etc. Overall, virtual optogenetics can accelerate the development of new optical stimulation tools for better understanding and control of brain and heart function.