Contractility measurements for cardiotoxicity screening with ventricular myocardial slices of pigs.

AIMS: Cardiotoxicity is one major reason why drugs do not enter or are withdrawn from the market. Thus, approaches are required to predict cardiotoxicity with high specificity and sensitivity. Ideally, such methods should be performed within intact cardiac tissue with high relevance for humans and detect acute and chronic side effects on electrophysiological behaviour, contractility, and tissue structure in an unbiased manner. Herein, we evaluate healthy pig myocardial slices and biomimetic cultivation setups (BMCS) as a new cardiotoxicity screening approach. METHODS AND RESULTS: Pig left ventricular samples were cut into slices and spanned into BMCS with continuous electrical pacing and online force recording. Automated stimulation protocols were established to determine the force-frequency relationship (FFR), frequency dependence of contraction duration, effective refractory period (ERP), and pacing threshold. Slices generated 1.3 ± 0.14 mN/mm2 force at 0.5 Hz electrical pacing and showed a positive FFR and a shortening of contraction duration with increasing pacing rates. Approximately 62% of slices were able to contract for at least 6 days while showing stable ERP, contraction duration-frequency relationship, and preserved cardiac structure confirmed by confocal imaging and X-ray diffraction analysis. We used specific blockers of the most important cardiac ion channels to determine which analysis parameters are influenced. To validate our approach, we tested five drug candidates selected from the Comprehensive in vitro Proarrhythmia Assay list as well as acetylsalicylic acid and DMSO as controls in a blinded manner in three independent laboratories. We were able to detect all arrhythmic drugs and their respective mode of action on cardiac tissue including inhibition of Na+, Ca2+, and hERG channels as well as Na+/Ca2+ exchanger. CONCLUSION: We systematically evaluate this approach for cardiotoxicity screening, which is of high relevance for humans and can be upscaled to medium-throughput screening. Thus, our approach will improve the predictive value and efficiency of preclinical cardiotoxicity screening.

Atrial fibrillation-associated electrical remodelling in human induced pluripotent stem cell-derived atrial cardiomyocytes: a novel pathway for antiarrhythmic therapy development.

AIMS: Atrial fibrillation (AF) is associated with tachycardia-induced cellular electrophysiology alterations which promote AF chronification and treatment resistance. Development of novel antiarrhythmic therapies is hampered by the absence of scalable experimental human models that reflect AF-associated electrical remodelling. Therefore, we aimed to assess if AF-associated remodelling of cellular electrophysiology can be simulated in human atrial-like cardiomyocytes derived from induced pluripotent stem cells in the presence of retinoic acid (iPSC-aCM), and atrial-engineered human myocardium (aEHM) under short term (24 h) and chronic (7 days) tachypacing (TP). METHODS AND RESULTS: First, 24-h electrical pacing at 3 Hz was used to investigate whether AF-associated remodelling in iPSC-aCM and aEHM would ensue. Compared to controls (24 h, 1 Hz pacing) TP-stimulated iPSC-aCM presented classical hallmarks of AF-associated remodelling: (i) decreased L-type Ca2+ current (ICa,L) and (ii) impaired activation of acetylcholine-activated inward-rectifier K+ current (IK,ACh). This resulted in action potential shortening and an absent response to the M-receptor agonist carbachol in both iPSC-aCM and aEHM subjected to TP. Accordingly, mRNA expression of the channel-subunit Kir3.4 was reduced. Selective IK,ACh blockade with tertiapin reduced basal inward-rectifier K+ current only in iPSC-aCM subjected to TP, thereby unmasking an agonist-independent constitutively active IK,ACh. To allow for long-term TP, we developed iPSC-aCM and aEHM expressing the light-gated ion-channel f-Chrimson. The same hallmarks of AF-associated remodelling were observed after optical-TP. In addition, continuous TP (7 days) led to (i) increased amplitude of inward-rectifier K+ current (IK1), (ii) hyperpolarization of the resting membrane potential, (iii) increased action potential-amplitude and upstroke velocity as well as (iv) reversibly impaired contractile function in aEHM. CONCLUSIONS: Classical hallmarks of AF-associated remodelling were mimicked through TP of iPSC-aCM and aEHM. The use of the ultrafast f-Chrimson depolarizing ion channel allowed us to model the time-dependence of AF-associated remodelling in vitro for the first time. The observation of electrical remodelling with associated reversible contractile dysfunction offers a novel platform for human-centric discovery of antiarrhythmic therapies.

CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation.

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

IDH3γ functions as a redox switch regulating mitochondrial energy metabolism and contractility in the heart.

Redox signaling and cardiac function are tightly linked. However, it is largely unknown which protein targets are affected by hydrogen peroxide (H2O2) in cardiomyocytes that underly impaired inotropic effects during oxidative stress. Here, we combine a chemogenetic mouse model (HyPer-DAO mice) and a redox-proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we demonstrate that increased endogenous production of H2O2 in cardiomyocytes leads to a reversible impairment of cardiac contractility in vivo. Notably, we identify the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch, linking its modification to altered mitochondrial metabolism. Using microsecond molecular dynamics simulations and experiments using cysteine-gene-edited cells reveal that IDH3γ Cys148 and 284 are critically involved in the H2O2-dependent regulation of IDH3 activity. Our findings provide an unexpected mechanism by which mitochondrial metabolism can be modulated through redox signaling processes.

Optimized Conditions for the Long-Term Maintenance of Precision-Cut Murine Myocardium in Biomimetic Tissue Culture.

Organotypic heart slices from mice might provide a promising in vitro model for cardiac research because of the vast availability of genetically modified specimens, combined with the unrestricted feasibility of experimental interventions. However, murine heart slices undergo rapid degeneration in culture. Therefore, we developed optimal conditions to preserve their structure and function in culture. Mouse ventricular heart samples were transversely cut into 300 µm thick slices. Slices were then cultured under various conditions of diastolic preload, systolic compliance and medium agitation. Continuous stimulation was performed either by optical stimulation or by electrical field stimulation. Contractility was continuously measured, and cellular survival, structure and gene expression were analyzed. Significant improvements in viability and function were achieved by elastic fixation with the appropriate diastolic preload and the rapid shaking of a ß-mercaptoethanol-supplemented medium. At 1 Hz pacing, mouse heart slices maintained stable contractility for up to 48 h under optogenetic pacing and for one week under electrical pacing. In cultured slices, the native myofibril structure was well preserved, and the mRNAs of myosin light chain, titin and connexin 43 were constantly expressed. Conclusions: Adult murine heart slices can be preserved for one week and provide a new opportunity to study cardiac functions.

Selective optogenetic control of Gq signaling using human Neuropsin.

Gq proteins are universally important for signal transduction in mammalian cells. The underlying kinetics and transformation from extracellular stimuli into intracellular signaling, however could not be investigated in detail so far. Here we present the human Neuropsin (hOPN5) for specific and repetitive manipulation of Gq signaling in vitro and in vivo with high spatio-temporal resolution. Properties and G protein specificity of hOPN5 are characterized by UV light induced IP3 generation, Ca2+ transients and inhibition of GIRK channel activity in HEK cells. In adult hearts from a transgenic animal model, light increases the spontaneous beating rate. In addition, we demonstrate light induced contractions in the small intestine, which are not detectable after pharmacological Gq protein block. All-optical high-throughput screening for TRPC6 inhibitors is more specific and sensitive than conventional pharmacological screening. Thus, we demonstrate specific Gq signaling of hOPN5 and unveil its potential for optogenetic applications.

The RyR2-R2474S Mutation Sensitizes Cardiomyocytes and Hearts to Catecholaminergic Stress-Induced Oxidation of the Mitochondrial Glutathione Pool.

Missense mutations in the cardiac ryanodine receptor type 2 (RyR2) characteristically cause catecholaminergic arrhythmias. Reminiscent of the phenotype in patients, RyR2-R2474S knockin mice develop exercise-induced ventricular tachyarrhythmias. In cardiomyocytes, increased mitochondrial matrix Ca2+ uptake was recently linked to non-linearly enhanced ATP synthesis with important implications for cardiac redox metabolism. We hypothesize that catecholaminergic stimulation and contractile activity amplify mitochondrial oxidation pathologically in RyR2-R2474S cardiomyocytes. To investigate this question, we generated double transgenic RyR2-R2474S mice expressing a mitochondria-restricted fluorescent biosensor to monitor the glutathione redox potential (E GSH). Electrical field pacing-evoked RyR2-WT and RyR2-R2474S cardiomyocyte contractions resulted in a small but significant baseline E GSH increase. Importantly, ß-adrenergic stimulation resulted in excessive E GSH oxidization of the mitochondrial matrix in RyR2-R2474S cardiomyocytes compared to baseline and RyR2-WT control. Physiologically ß-adrenergic stimulation significantly increased mitochondrial E GSH further in intact beating RyR2-R2474S but not in RyR2-WT control Langendorff perfused hearts. Finally, this catecholaminergic E GSH increase was significantly attenuated following treatment with the RyR2 channel blocker dantrolene. Together, catecholaminergic stimulation and increased diastolic Ca2+ leak induce a strong, but dantrolene-inhibited mitochondrial E GSH oxidization in RyR2-R2474S cardiomyocytes.

Novel Optics-Based Approaches for Cardiac Electrophysiology: A Review.

Optical techniques for recording and manipulating cellular electrophysiology have advanced rapidly in just a few decades. These developments allow for the analysis of cardiac cellular dynamics at multiple scales while largely overcoming the drawbacks associated with the use of electrodes. The recent advent of optogenetics opens up new possibilities for regional and tissue-level electrophysiological control and hold promise for future novel clinical applications. This article, which emerged from the international NOTICE workshop in 2018, reviews the state-of-the-art optical techniques used for cardiac electrophysiological research and the underlying biophysics. The design and performance of optical reporters and optogenetic actuators are reviewed along with limitations of current probes. The physics of light interaction with cardiac tissue is detailed and associated challenges with the use of optical sensors and actuators are presented. Case studies include the use of fluorescence recovery after photobleaching and super-resolution microscopy to explore the micro-structure of cardiac cells and a review of two photon and light sheet technologies applied to cardiac tissue. The emergence of cardiac optogenetics is reviewed and the current work exploring the potential clinical use of optogenetics is also described. Approaches which combine optogenetic manipulation and optical voltage measurement are discussed, in terms of platforms that allow real-time manipulation of whole heart electrophysiology in open and closed-loop systems to study optimal ways to terminate spiral arrhythmias. The design and operation of optics-based approaches that allow high-throughput cardiac electrophysiological assays is presented. Finally, emerging techniques of photo-acoustic imaging and stress sensors are described along with strategies for future development and establishment of these techniques in mainstream electrophysiological research.

Correction: Rehnelt et al. Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation. Int. J. Mol. Sci. 2017, 18, 2634.

The authors wish to make the following corrections to this paper [...].

Direct optogenetic stimulation of smooth muscle cells to control gastric contractility.

Rationale: Antral peristalsis is responsible for gastric emptying. Its failure is called gastroparesis and often caused by dysfunction of enteric neurons and interstitial cells of Cajal (ICC). Current treatment options, including gastric electrical stimulation, are non-satisfying and may improve symptoms but commonly fail to restore gastric emptying. Herein, we explore direct optogenetic stimulation of smooth muscle cells (SMC) via the light-gated non-selective cation channel Channelrhodopsin2 (ChR2) to control gastric motor function. Methods: We used a transgenic mouse model expressing ChR2 in fusion with eYFP under the control of the chicken-ß-actin promoter. We performed patch clamp experiments to quantify light-induced currents in isolated SMC, Ca2+ imaging and isometric force measurements of antral smooth muscle strips as well as pressure recordings of intact stomachs to evaluate contractile responses. Light-induced propulsion of gastric contents from the isolated stomach preparation was quantified in video recordings. We furthermore tested optogenetic stimulation in a gastroparesis model induced by neuronal- and ICC-specific damage through methylene blue photo-toxicity. Results: In the stomachs, eYFP signals were restricted to SMC in which blue light (460 nm) induced inward currents typical for ChR2. These depolarizing currents led to contractions in antral smooth muscle strips that were stronger than those triggered by supramaximal electrical field stimulation and comparable to those evoked by global depolarization with high K+ concentration. In the intact stomach, panoramic illumination efficiently increased intragastric pressure achieving 239±46% (n=6) of the pressure induced by electrical field stimulation and triggered gastric transport. Within the gastroparesis model, electric field stimulation completely failed but light still efficiently generated pressure waves. Conclusions: We demonstrate direct optogenetic stimulation of SMC to control gastric contractility. This completely new approach could allow for the restoration of motility in gastroparesis in the future.

Optogenetic Stimulation of Gi Signaling Enables Instantaneous Modulation of Cardiomyocyte Pacemaking.

G-protein signaling pathways are central in the regulation of cardiac function in physiological and pathophysiological conditions. Their functional analysis through optogenetic techniques with selective expression of opsin proteins and activation by specific wavelengths allows high spatial and temporal precision. Here, we present the application of long wavelength-sensitive cone opsin (LWO) in cardiomyocytes for activation of the Gi signaling pathway by red light. Murine embryonic stem (ES) cells expressing LWO were generated and differentiated into beating cardiomyocytes in embryoid bodies (EBs). Illumination with red light (625 nm) led to an instantaneous decrease up to complete inhibition (84-99% effectivity) of spontaneous beating, but had no effect on control EBs. By using increasing light intensities with 10 s pulses, we determined a half maximal effective light intensity of 2.4 µW/mm2 and a maximum effect at 100 µW/mm2. Pre-incubation of LWO EBs with pertussis toxin completely inhibited the light effect proving the specificity for Gi signaling. Frequency reduction was mainly due to the activation of GIRK channels because the specific channel blocker tertiapin reduced the light effect by ~80%. Compared with pharmacological stimulation of M2 receptors with carbachol with slow kinetics (>30 s), illumination of LWO had an identical efficacy, but much faster kinetics (<1 s) in the activation and deactivation demonstrating the temporal advantage of optogenetic stimulation. Thus, LWO is an effective optogenetic tool for selective stimulation of the Gi signaling cascade in cardiomyocytes with red light, providing high temporal precision.

Carnosine synthase deficiency is compatible with normal skeletal muscle and olfactory function but causes reduced olfactory sensitivity in aging mice.

Carnosine (ß-alanyl-l-histidine) and anserine (ß-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.

Selective optogenetic stimulation of fibroblasts enables quantification of hetero-cellular coupling to cardiomyocytes in a three-dimensional model of heart tissue.

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-ß1 (TGF-ß1)-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-ß1-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-ß1-stimulation. Illumination of cardiac bodies prepared with Archaerhodopsin expressing fibroblasts led to hyperpolarization of cardiomyocytes and complete block of spontaneous beating after TGF-ß1-stimulation. Effects of light were significantly smaller without TGF-ß1-stimulation. CONCLUSION: Transforming growth factor-ß1-stimulation leads to increased hetero-cellular coupling and optogenetic hyperpolarization of fibroblasts reduces TGF-ß1 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.

Towards the clinical translation of optogenetic skeletal muscle stimulation.

Paralysis is a frequent phenomenon in many diseases, and to date, only functional electrical stimulation (FES) mediated via the innervating nerve can be employed to restore skeletal muscle function in patients. Despite recent progress, FES has several technical limitations and significant side effects. Optogenetic stimulation has been proposed as an alternative, as it may circumvent some of the disadvantages of FES enabling cell type-specific, spatially and temporally precise stimulation of cells expressing light-gated ion channels, commonly Channelrhodopsin2. Two distinct approaches for the restoration of skeletal muscle function with optogenetics have been demonstrated: indirect optogenetic stimulation through the innervating nerve similar to FES and direct optogenetic stimulation of the skeletal muscle. Although both approaches show great promise, both have their limitations and there are several general hurdles that need to be overcome for their translation into clinics. These include successful gene transfer, sustained optogenetic protein expression, and the creation of optically active implantable devices. Herein, a comprehensive summary of the underlying mechanisms of electrical and optogenetic approaches is provided. With this knowledge in mind, we substantiate a detailed discussion of the advantages and limitations of each method. Furthermore, the obstacles in the way of clinical translation of optogenetic stimulation are discussed, and suggestions on how they could be overcome are provided. Finally, four specific examples of pathologies demanding novel therapeutic measures are discussed with a focus on the likelihood of direct versus indirect optogenetic stimulation.

No light without the dark: Perspectives and hindrances for translation of cardiac optogenetics.

Over the last decade, optogenetic stimulation of the heart and its translational potential for rhythm control attracted more and more interest. Optogenetics allows to stimulate cardiomyocytes expressing the light-gated cation channel Channelrhodopsin 2 (ChR2) with light and thus high spatio-temporal precision. Therefore this new approach can overcome the technical limitations of electrical stimulation. In regard of translational approaches, the prospect of pain-free stimulation, if ChR2 expression is restricted to cardiomyocytes, is especially intriguing and could be highly beneficial for cardioversion and defibrillation. However, there is no light without shadow and cardiac optogenetics has to surmount critical hurdles, namely "how" to inscribe light-sensitivity by expressing ChR2 in a native heart and how to avoid side effects such as possible immune responses against the gene transfer. Furthermore, implantable light devices have to be developed which ensure sufficient illumination in a highly contractile environment. Therefore this article reviews recent advantages in the field of cardiac optogenetics with a special focus on the hindrances for the potential translation of this new approach into clinics and provides an outlook how these have to be carefully investigated and could be solved step by step.