Myocardial edema is a common symptom of pathological processes in the heart, causing aggravation of cardiovascular diseases and leading to irreversible myocardial remodeling. Patient-based studies show that myocardial edema is associated with arrhythmias. Currently, there are no studies that have examined how edema may influence changes in calcium dynamics in the functional syncytium. We performed optical mapping of calcium dynamics on a monolayer of neonatal rat cardiomyocytes with Fluo-4. The osmolality of the solutions was adjusted using the NaCl content. The initial Tyrode solution contained 140 mM NaCl (1T) and the hypoosmotic solutions contained 105 (0.75T) and 70 mM NaCl (0.5T). This study demonstrated a sharp decrease in the calcium wave propagation speed with a decrease in the solution osmolality. The successive decrease in osmolality also showed a transition from a normal wavefront to spiral wave and multiple wavelets of excitation with wave break. Our study demonstrated that, in a cellular model, hypoosmolality and, as a consequence, myocardial edema, could potentially lead to fatal ventricular arrhythmias, which to our knowledge has not been studied before. At 0.75T spiral waves appeared, whereas multiple wavelets of excitation occurred in 0.5T, which had not been recorded previously in a two-dimensional monolayer under conditions of cell edema without changes in the pacing protocol.

Cardiac arrhythmias are common disorders that can be fatal. Modern methods of treating bradyarrhythmias include the implantation of pacemakers and cardioverter–defibrillators. However, implantable devices can cause various complications related to the electrodes installed inside the heart, including infection. Less invasive heart rhythm modulation could be beneficial for some cohorts of patients. Here, we demonstrate an alternative approach to heart pacing based on thermogenetics. We used adeno-associated viruses to deliver genetic human transient receptor potential subfamily V member 1 (TRPV1), a heat-sensitive cation channel, into isolated cardiomyocytes and the mouse heart. This allowed us to induce action potentials and control contractility using short heat pulses delivered by infrared laser illumination. Using this approach, we demonstrated the thermogenetic pacing of isolated cardiomyocytes in vitro and in the mouse heart in vivo . Our results demonstrate the potential of thermogenetics for developing therapeutic strategies for heart rhythm modulation.

Introduction: Preservation of isolated hearts in cardioplegic solution at normothermia is a promising strategy for increasing the organ lifespan. Further development of this strategy and search for new products for solution delivery can help optimize the technology of donor heart transportation. Objective: The study was aimed to test the efficacy of device for normothermic constant perfusion flow with innovative lines for the cardioplegic solution delivery (CardioSystemPharma, Khimki, Moscow Region, Russia) according to the Langendorff mode in comparison with a system based on nodes and tubes of Radnoti Langendorff Constant Perfusion Flow Apparatus (ADInstruments, Colorado Springs, USA) in a series of experiments on rat hearts.Methods: The innovative cardioplegic delivery system (CardioSystemPharma) and traditional perfusion system (Radnoti) were compared in experiments on rat hearts at normothermia without cardioplegic solution and using the cardioplegic solution Normacor (Mosfarm, Moscow Region, Russia). The time until complete cardiac arrest and the changes in the NADH coenzyme fluorescence were recorded. After cardiac arrest, immunohistochemical staining of ventricular sections was performed to detect myocardial injury during perfusion.Results: It was shown that the innovative cardioplegic delivery system was not inferior to the traditional perfusion system (Radnoti) and even surpassed it in terms of the time duration for preservation of the isolated heart. It was found that the using of elevated glucose concentration during normothermic perfusion could significantly increase the organ lifespan. The efficacy of normothermic perfusion in the experiment was confirmed by monitoring cellular respiration through recording the NADH fluorescence.Conclusion: Testing showed that long-term preservation under normothermia with usage of an innovative delivery system of Russian manufacture is a promising replacement for Radnoti solution delivery system. Received 16 October 2024. Revised 23 November 2024. Accepted 10 December 2024. FundingThe study was carried out within the framework of the project “Innovative cardioplegic lines for cardiac surgery” under the Agreement on the provision of a grant from the Government of the Moscow Region in the fields of science, technology, engineering and innovation No. 12/04-24 dated 04/27/2024. Conflict of interestThe authors declare no conflict of interest. Contribution of the authorsConception and study design: V.A. Tsvelaya, S.D. Robustova, V.S. Kachan, M.S. Nosov, A.K. Berezhnoy, V.A. Kozlov, K.I. AgladzeData collection and analysis: S.D. Robustova, V.S. Kachan, D.V. Kononova, A.S. Yarovoy, M.A. Gusev, V.V. Litvinenko, V.D. Dzhabrailov, A.A. Brichagina, Z.V. Obrazumova, S.D. Chekalina, S.S. Bakumenko, E.A. Turchaninova, V.A. TsvelayaStatistic analysis: S.D. Robustova, V.S. Kachan, D.V. Kononova, A.S. Yarovoy, M.A. Gusev, V.V. Litvinenko, V.D. Dzhabrailov, A.A. Brichagina, Z.V. Obrazumova, S.D. Chekalina, S.S. Bakumenko, E.A. TurchaninovaDrafting the article: S.D. Robustova, V.S. Kachan, D.V. Kononova, Z.V. Obrazumova, E.A. Turchaninova, V.A. TsvelayaCritical revision of the article: S.D. Robustova, E.A. Turchaninova, V.A. Tsvelaya, K.I. AgladzeFinal approval of the version to be published: S.D. Robustova, V.S. Kachan, D.V. Kononova, A.S. Yarovoy, M.A. Gusev, V.V. Litvinenko, V.D. Dzhabrailov, A.A. Brichagina, Z.V. Obrazumova, S.D. Chekalina, E.A. Turchaninova, S.S. Bakumenko, M.S. Nosov, A.K. Berezhnoy, V.A. Kozlov, V.A. Tsvelaya, K.I. Agladze

Background: Heart rhythm disturbances remain a serious problem in modern cardiology. Traditional pacemakers have certain limitations including invasiveness, risk of infection, mechanical complications, and a limited service life. Advances in bioengineering and optogenetics technologies offers new prospects for the production of minimally invasive, biocompatible, and controllable cardiac pacing systems. The combination of cell therapy and optogenetics enables to create a photo-controlled biological pacemaker, free from the key drawbacks of traditional devices. Objective: The aim of this study was to produce photosensitive cellular patches and to further investigate their functionality as an optogenetic tissue-engineered pacemaker in an ex vivo rat heart model. Methods: We engineered a cell-based construct using either human cardiomyocytes derived from induced pluripotent stem cells or neonatal rat cardiomyocytes expressing channelrhodopsin-2. These cells were seeded onto fibrous scaffolds made of poly-L-lactic acid and collagen, coated with fibronectin. The testing model was an isolated, temporarily maintained ex vivo rat heart. Optical mapping of calcium activity was used to record cardiac electrophysiology. Results: Functional coupling between the implanted patch and the host myocardium was observed 35 minutes after implantation. Photostimulation reliably increased the heart rate, which was confirmed by stochastic dominance analysis. The experiments in vitro on cell cultures demonstrated the operational capacity of channelrhodopsin-2 upon illumination with 470 nm light. Conclusion: This study successfully demonstrates a complete technology cycle, from the genetic modification of cells to the control of contractions in a whole organ. It represents a significant step towards developing targeted and safe methods for future temporary cardiac pacing. Our results confirm the fundamental feasibility of a hybrid optogenetic approach and lay the groundwork for further research into creating safe, controllable, and biocompatible next-generation pacemaker systems.

Background: Visualization of reduced nicotinamide adenine dinucleotide (NADH) fluorescence is a promising method for assessing the metabolic status of the myocardium during ischemia. However, its widespread clinical application is hindered by the fundamental ambiguity in signal interpretation, as it depends both on the metabolic concentration of this coenzyme and the illumination conditions that prevents reliable distinction of ischemia state from technical artifacts. Objective: The study was aimed to develop and validate a method for unambiguous interpretation of the fluorescence signal to differentiate ischemia from artifacts, based on the combined analysis of fluorescence intensity and the rate of NADH photolysis. Methods: A series of experiments was conducted on isolated rat hearts (n = 8) perfused using the Langendorff technique. In 4 experiments, ischemia was modeled due to cessation of perfusion; in the other 4 cases, a technical artifact was induced by change in power and position of the ultraviolet source. For each pair of recordings, namely before and after intervention, the relative changes in fluorescence intensity (dF) and the rate of photolysis (dN), calculated by approximating the kinetics with a mathematical model, were assessed. The primary endpoint was the accuracy of classifying the intervention type based on the combined analysis of dF and dN. Results: In induced ischemia, a significant increase in dF (294 ± 89 a.u.) was revealed with no change in dN (1.31 ± 1.17 a.u., p > 0.05). In the case of the artifact assessing, an increase in dF was also recorded (289 ± 83 a.u.) but it was accompanied by a significant increment in dN (17.46 ± 5.25 a.u., p < 0.01). The combined analysis of dF and dN enabled to accurately classify technical artifacts across the entire test sample (n = 8). Conclusion: The combined analysis of NADH fluorescence and photolysis eliminates signal interpretation ambiguity and reliably differentiates myocardial ischemia state from technical artifacts. The proposed method, which does not require a reference recording or prolonged scanning, could facilitate the adoption of metabolic imaging to intraoperative monitoring and the assessment of graft viability.

A critical obstacle in cardiac cell therapy is the unpredictable and poorly understood initial electrophysiological integration of grafted cardiomyocytes into the host tissue, a process that dictates therapeutic success and arrhythmic risk. Current models fail to capture the earliest stages of functional coupling formation. Here, we employed a tailored bioengineering platform, where single cardiomyocytes were stabilized on minimalist electrospun polycaprolactone (PCL) nanofibers, to model the “graft–host” interface and study the dynamics of excitation wave transmission in real-time. Using high-speed optical mapping enhanced by a custom SUPPORT neural network, we achieved the first quantitative insights into the efficiency of nascent intercellular contacts. We determined that within the first 3 h, these initial connections are 39–44 times less effective at conducting excitation than mature contacts within the native monolayer, explaining the observed partial (46%) synchronization of grafted cells. This work provides the first direct measurement of the functional deficit during the initial minutes and hours of graft integration. It establishes that simple, inert polymer fibers can act as a catalytic scaffold to enable this fundamental biological process, offering a powerful strategy to deconstruct and ultimately control the integration of engineered tissues (or cells) for safer cell therapies.