Refining fibroblast-to-cardiomyocyte transdifferentiation protocols to explore emergent self- organization in cardiac cultures

Refining fibroblast-to-cardiomyocyte transdifferentiation protocols to explore emergent self- organization in cardiac cultures | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Refining fibroblast-to-cardiomyocyte transdifferentiation protocols to explore emergent self- organization in cardiac cultures Elena Turchaninova, Sofya Robustova, Sandaara Kovalenko, Vitalii Dzhabrailov, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7186257/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fibrotic scars post-myocardial infarction disrupt cardiac conduction, causing arrhythmias. We developed a minimized 4-component cocktail (CHIR99021/BMP4/Activin A/IWP2) for efficient fibroblast-to-cardiomyocyte transdifferentiation. The use of the developed four-component protocol allows achieving significant electromechanical activity and pronounced expression of cardiomyocyte markers, as evidenced by the 56–83% cells expressing α-actinin. The results show that partial transdifferentiation of fibroblast cells into cardiac ones is sufficient to restore cardiac tissue conductivity, while the efficiency exceeds the critical percolation threshold. Systemic delivery of components is safe, but requires further optimization, which will open up opportunities for localized delivery through smart substrates and combinations with cell therapy. Minimization of the transdifferentiation cocktail is not a compromise, but a strategic advantage that provides an optimal balance between functional efficiency and clinical applicability, including safety, delivery, and manufacturing. transdifferentiation fibrosis arrhythmias cardiomyocytes cardiac tissue Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Cardiovascular diseases (CVDs) remain the principal cause of death globally. Myocardial infarction (MI), the acute and often fatal manifestation of ischemic heart disease (IHD), is a major driver of this burden. The American Heart Association's Heart Disease and Stroke Statistics—2024 Update reports that in the United States alone, someone has a myocardial infarction approximately every 40 seconds (Martin et al. 2024 ). Acute MI results from prolonged myocardial ischemia, triggering extensive cardiomyocyte necrosis. This initiates a maladaptive reparative response characterized by activation of resident cardiac fibroblasts (CFs), excessive deposition of extracellular matrix (ECM) (Hinz & Gabbiani 2003 ), and the formation of a dense, non-contractile, and electrically non-conductive fibrotic scar (Sutton & Sharpe 2000 ). This replacement fibrosis (Hinz & Gabbiani 2003 ) disrupts the myocardial syncytium, severely impairing pump function and creating a substrate for lethal arrhythmias (Nguyen et al. 2014 ), directly contributing to heart failure and sudden cardiac death post-MI. Novel therapeutic strategies for the post-MI fibrotic scar include pharmacological agents targeting neurohumoral activation and inflammation (Ghanem & Megahed 2023; Raziyeva et al. 2022 ), but the main solution is still surgical interventions like revascularization or device implantation (Marrouche et al. 2022 ). While essential, these approaches fail to regenerate lost cardiomyocytes or restore physiological electrical conduction across the scar. Regenerative strategies, particularly cell replacement therapy using cardiomyocytes derived from induced pluripotent stem cells (iPSCs) (Takahashi 2025 ) or mesenchymal stem cells (MSCs) (Fernández-Garza et al. 2023 ), show promise for remuscularization (Jebran et al. 2025 ). However, they carry inherent risks of tumorigenicity (Liu et al. 2013 ) and arrhythmogenicity (Takahashi et al. 2023 ), alongside challenges of scalable cell production and immune rejection. In situ direct reprogramming or transdifferentiation of resident CFs within the scar into induced cardiomyocyte-like cells (iCMs), bypassing pluripotency (Yamakawa & Ieda 2021 ), offers a compelling alternative. CFs are a prime target due to their central role in post-MI scar formation and pathological remodeling (Nagalingam et al. 2016 ) and their high abundance within the infarct zone (Pinto et al. 2016 ). Direct reprogramming circumvents the need for large-scale in vitro cell expansion (e.g., billions of cells per graft (Jebran et al. 2025 )) and avoids immune complications associated with allogeneic cell transplantation (Tongers et al. 2011 ). Multiple approaches exist for fibroblast to cardiomyocyte-like cells reprogramming, including viral delivery of transcription factors (e.g., Oct4, Sox2, Klf4 (Efe et al. 2011 ; Wang et al. 2014 ); Gata4, Mef2c, Tbx5 (GMT) (Mohamed et al. 2017 ); GHMT (Ifkovits et al. 2014 )), mRNA (Jayawardena et al. 2012 ), and cocktails of small molecules. Small molecules modulate key signaling pathways (e.g., TGF-β, Wnt, cAMP) and include inhibitors of epigenetic repressors (tranylcypromine, valproate (Fu et al. 2015 )), TGF-β inhibitors (RepSox, SB431542 (Mohamed et al. 2017 ; Park et al. 2015 ; Cao et al. 2016 )), Wnt modulators (CHIR99021 (Fu et al. 2015 ; Cao et al. 2016 ), XAV939 (Jayawardena et al. 2012 )), and cAMP activators (Forskolin (Fu et al. 2015 ; Park et al. 2015 )). Despite progress, current transdifferentiation protocols, especially purely chemical ones, suffer from critically low efficiency. Viral methods combined with small molecules enhance reprogramming in vivo (Mohamed et al. 2017 ; Qian et al. 2012 ; Song et al. 2012 ) but face translational hurdles due to risks of insertional mutagenesis, immunogenicity, and manufacturing complexity (Garry et al. 2022 ). Purely chemical cocktails offer superior safety and delivery flexibility (e.g., injectable, potentially oral (Huang et al. 2018 )). However, reported conversion rates remain inadequate (e.g., ~ 1% after prolonged multi-dose regimens (Huang et al. 2018 )), limiting functional recovery. Crucially, complete reprogramming of all scar fibroblasts is likely unnecessary. One potential therapeutic strategy for post-MI scar treatment involves enhancing electrical conductivity to enable more synchronized contraction. This can be achieved by reprogramming a critical minimal fraction of fibroblasts into conductive iCMs, establishing interconnected conductive pathways that reach the electrophysiological percolation threshold within the scar tissue (Trayanova et al. 2024 ; Rabinovitch et al. 2021 ). Achieving this threshold allows the electrical wavefront to propagate through the formerly inert scar (Kalinin et al. 2023 ). This study directly addresses the challenge of low efficiency in chemical reprogramming for myocardial scar therapy taking the positives from this fact. We hypothesize that a radically minimized, optimized chemical cocktail can achieve iCM conversion rates sufficient to reach the percolation threshold within the fibrotic scar. Minimization is of paramount importance for clinical translation as it increases the likelihood of applicability of the cocktail. Complex cocktails pose significant challenges for pharmacokinetic profiling, safety assessment (including drug-drug interaction studies), formulation stability, manufacturing sequences, and regulatory approval pathways. Minimizing the number of compounds dramatically simplifies these processes, accelerating clinical development (DiMasi et al. 2016 ; Woodcock et al. 2017). In addition, we plan to use the chemical cocktails we have created for gradual targeted delivery to the myocardium. Implantable biomaterial scaffolds designed for localized, sustained, and temporally controlled release in the rumen are a promising delivery platform. However, incorporating multiple compounds with potentially different release kinetics, stability requirements, and complex cross-linking and interactions is highly complex and often impractical. A minimal cocktail is necessary for the efficient development and performance of such next-generation delivery systems (Ruvinov & Cohen 2016 ; Tibbitt et al. 2015 ). Such chemical cocktails also have the potential for synergistic combination therapies. A simple, effective minimal chemical reprogramming strategy could be easily combined with cell replacement therapies (e.g., iPSC-CM patches) to enhance their efficacy and overcome their differentiation-related drawbacks. Reprogramming a subset of scar fibroblasts in situ could create a more hospitable, conductive microenvironment to improve survival, integration, and electromechanical coupling of grafted cardiomyocytes (Tang et al. 2018 ; Vagnozzi et al. 2020 ). Therefore, we aimed to develop, optimize, and validate a novel, highly minimal chemical reprogramming cocktail specifically designed to efficiently transdifferentiate fibroblasts into functional iCM. Our primary goal was to achieve an efficiency sufficient to establish conductive pathways (above the percolation threshold) in models replicating the fibrotic environment following myocardial infarction, using both animal and human cells to demonstrate robust translational potential. 2. Results 2.1. In vitro transdifferentiation from fibroblast to cardiomyocyte-like cells (iCM) The primary goal of this study section was to develop a minimized small-molecule reprogramming protocol for efficient fibroblast-to-cardiomyocyte transdifferentiation. By minimizing the number of active molecules, we reduce potential immunogenicity and manufacturing complexity, which is important for future clinical applications. As a baseline, we chose a protocol for controlling fibroblast to cardiomyocyte transdifferentiation (Fu et al. 2015 ) that was primarily used for mouse embryonic fibroblasts. This section describes the results of testing modified protocols for fibroblasts-to-cardiomyocytes transdifferentiation. The resulting cells were characterized and the effectiveness of the protocol was assessed. For evaluation several types of analysis were performed during transdifferentiation including: patch-clamp, optical mapping of calcium dynamics and immunostaining for fibrillar actin, nuclei and cardiomyocytes marker α-actinin. 2.1.1. Rat embryonic fibroblasts derived iCM Firstly we start to adopt transdifferentiation protocol for rat embryonic fibroblasts (REF) to iCM. The basic protocol was successfully adapted for REF by varying the concentrations of CHIR99021, Activin A, and BMP4 (Fig. 1 A),, which were chosen as the main components. We compared 4 protocols for REFs with varied concentrations of small molecule CHIR99021 and tested the role of Activin A and BMP-4 additives for better induction of cardiomyocytes. The final protocols for REF-to-cardiomyocyte direct reprogramming are presented on Fig. 1 A. We observed changes in the cell's morphology on day 8–10 (Fig. 1 C). Сlustering and strand formation indicate differentiation process success (Fu et al. 2015 ). Electrophysiological profiling on 24th transdifferentiation day using patch-clamp identified: combined INa/Ca currents using ramp protocol (Fig. 1 B), and an apparent voltage-gated IKv current with amplitude ∼200 pA (Fig. 1 B). Cells after 24 days of transdifferentiation were stained positive for cardiac markers. Some cells after transdifferentiation displayed partially striated α-actinin expression patterns (Fig. 1 D). The percentage ratio of α-actinin expression to f-actin expression was measured on day 24: Protocol № 1 — 73 ± 10%, Protocol № 2 — 75 ± 3%, Protocol № 3 — 83 ± 8%, Protocol № 4 — 79 ± 21%. Particularly noteworthy is Protocol № 3, which achieved significantly higher α-actinin content in cells compared to the other protocols supposedly due to high concentration of CHIR99021. 2.2.2. Rat neonatal fibroblasts derived iCM After testing protocols for REFs we adapted it for neonatal rat fibroblasts (RNF) (Fig. 2 A). For survival enhancement Y-27632, selective ROCK inhibitor, was added to prevent apoptosis. Besides Activin A and BMP4 on early transdifferentiation stages another SMAD activating growth factor TGFβ was applied. SMAD is believed to promote smooth muscle cell differentiation and apoptosis inhibition (Shi et al. 2014 ). From the 24th day of transdifferentiation analogous to human IPSC differentiation to cardiac tissue (Lian et al. 2013 ) the protocol was supplied with an IWP2 small molecular inhibitor of the Wnt pathway. Unlike the protocol for REF, during transdifferentiation of RNF (Fig. 2 A) CRM(P) medium combining CRM with 50% of 2–4 day medium from neonatal cardiomyocytes was used to exploit the paracrine effect (Rangappa et al. 2003 ). The modified protocols for RNF-to-cardiomyocyte direct reprogramming are presented on Fig. 2 A. Clusterization and strand formation were observed on day 12–16 of induction (Fig. 2 B). RNF were stained for cardiac marker α-actinin on different days of transdifferentiation; from 16th day a partially striated pattern of α-actinin expression was observed (Fig. 2 C). During optical mapping with calcium-dependent dye Fluo-4 AM on the 21st day we registered periodical calcium activity in response to electric pacing, confirming the successful induction into cardiomyocytes (Fig. 2 D). Patch-clamp analysis on the 21st day revealed an apparent INa via voltage-gated sodium channels with a mean amplitude of ~ 200 pA, typical kinetic of ICa,L via voltage-gated calcium channels with significantly reduced amplitudes, averaging approximately 100 pA; an IKv via voltage-gated potassium channels reaching ~ 600 pA amplitude (Fig. 2 E). This displays electrophysiological properties of INa and ICa improvement since day 16, when combined INa and ICa currents were recorded and IKv since day 8 (~ 150 pA) day 16 (~ 300 pA). The results of the immunocytochemistry analysis showed an increase in the α-actinin/f-actin expression ratio from 62 ± 12% at day 16 to 77 ± 13% at day 21 of differentiation, indicating that the protocol effectively enhances α-actinin expression over time. 2.2.3. Human atrial fibroblasts derived iCM For human atrial fibroblasts (HAF) primarily RNF protocol was applied with slight modifications (Fig. 3 A). Instead of CRM(P) medium CRM(C) was applied for enhancement of human fibroblast transdifferentiation. The final optimized protocols for HAF-to-cardiomyocyte direct reprogramming are presented on Fig. 3 A. We observed changes in cell`s morpholоogy on days 10–12. Clusterization and strand formation, large size, single appearance may be features of successful differentiation (Fig. 3 B). On 15th day, patch-clamp analysis via ramp protocols recorded composite currents with contributions from both voltage-gated sodium (Na⁺) and calcium (Ca²⁺) channels, while depolarizing steps elicited small-amplitude potassium currents (IKv ≈ 100 pA) (Fig. 3 E). Notably, action potential (AP) was recorded on the 15th day. While the absence of a plateau phase prevented the attainment of adult-like atrial action potential duration, the presence of a normal resting membrane potential ( ~ − 80 mV) confirms development of fundamental excitability mechanisms. Optical mapping with calcium-dependent dye Fluo-4 AM was performed on the 21st and 33d days. As can be seen, the calcium concentration responds to electric pacing, confirming the successful induction into cardiomyocytes (Fig. 3 D). Patch-clamp analysis revealed progressive development of voltage-gated ion channels (Fig. 3 E). By day 21, robust Na⁺ currents (amplitude ~ 4000 pA) disciplaying electrophysiological properties improvement since day 16, when voltage-gated sodium (Na⁺) and calcium (Ca²⁺) currents were recorded. The development of nanoampere-range Na⁺ currents by day 21 confirms efficiency of the differentiation protocol. However, consistent gigaseal formation proved challenging at later stages of transdifferentiation, precluding reliable quantification of Ca²⁺ and delayed rectifier K⁺ currents. Consistent with the electrophysiological study, immunofluorescent analysis on day 33 confirmed that HAFa were positive for cardiomyocyte marker α-actinin, and these cells also displayed partially striated α-actinin expression patterns (Fig. 3 C). Additionally, the α-actinin to f-actin expression ratio in these cells at day 33 of differentiation was measured at 56 ± 24%. 2.3. Testing transdifferentiation cocktail for in vivo application 2.3.1. Safety testing for in vivo application of transdifferentiation cocktail For in vivo testing the protocol for RNF, excluding ROCK inhibitor Y-27632, was adopted (Fig. 4 A). Since the duration of reprogramming is important for cell maturation, and in vitro takes at least 3–4 weeks (Fu et al. 2015 ; Cao et al. 2016 ), we performed repeated injections of components for induction with almost the same intervals as in in vitro protocol for RNFs (Fig. 2 A) i.e. no more than 2 injections a week, that was found safe for control protocol administration in mice (Huang et al. 2018 ). Treatment started 14 days according to the scheme (Fig. 4 A) after myocardial infarction induction via coronary artery ligation (Fig. 4 B). Figure 4 D presents an example of immunohistochemical characterization of heart fibrosis in our infarction model. Primarily to the infarction treatment experiment we observed safety issues of transdifferentiation application in vivo . Toxicity testing performed on intact animals receiving single dosage intramyocardial injection. No adverse effects were seen during the three day period of observation. Clinical blood analysis also showed no significant difference in both leucocyte formulation and hemoglobin related parameters between treated and non-treated animals (Fig. 4 C). Since intramyocardial injections can only be performed under general anesthesia, repeated interventions of this kind are not safe for animals. Thus during transdifferentiation in vivo testing we instead performed intravenous injections as a compromise between targeting and safety. 2.3.2. Illustration of the efficiency of in vivo transdifferentiation in a rat infarction model We compared NADH fluorescence, a marker of ischemia and cellular respiration, in the rat heart during an ex vivo experiment. As is known, the process of NADH fluorescence intensity reduction is associated not only with its photobleaching but also with the activity of glutamate dehydrogenase, which regenerates NADH, thereby slowing the decline in fluorescence intensity (Moreno et al. 2017 ). Thus, by comparing the dynamics of NADH photobleaching, we can perform a qualitative assessment of tissues (Fig. 5 ). We compared the decrease in NADH fluorescence intensity in two regions of a Langendorff-perfused rat heart with myocardial infarction: the infarcted area and the intact area. The closer the NADH photobleaching curves are to each other, the more similar the infarct scar can be considered to healthy tissue. In Fig. 5 A, the curves are quite similar, which is due to the fact that the infarction in this heart was induced just 30 minutes before recording the curves, so the differences between the infarcted and intact regions are minimal. In contrast, Fig. 5 B shows a significant difference in fluorescence decay between the compared regions, indicating that after 14 days of occlusion, the infarcted area undergoes substantial fibrotic changes. However, with therapy (Fig. 5 C), the photobleaching curves become similar again, suggesting that the treatment has a significant effect on the infarcted region of the heart, bringing the cellular respiration characteristics of the fibrotic area closer to those of healthy cardiomyocytes. To characterize functional improvement during therapy we also adopted optical mapping methodology to assess electric propagation in whole hearts during Langendorff perfusion, and observed certain differences in conduction in the infarction and intact zones of hearts (Fig. 6 ). Two applied dyes: action potential sensitive Di-8-ANEPPS (Fig. 6 A) and calcium sensitive Fluo-4 AM (Fig. 6 B) — displayed comparable pattern on the same heart, and since the action potential wave and the subsequent calcium wave in the absence of drugs affecting their coupling have the same distribution patterns (Eisner et al. 2017 ), they can be compared with each other. In the two experimental groups: control (Fig. 6 C) and therapy (Fig. 6 D), the ratio of the conductive area to the total mapped heart area was measured. The results of this rough estimation demonstrate that the transdifferentiation treatment has a somewhat positive effect on cardiac conduction, increasing the proportion of conductive tissue from 71 ± 2% in the control group to 84 ± 3% in the treated group. This increase in the conductive ratio might indicate a relative reduction in the infarction area, suggesting partial restoration of conductivity in fibrotic cardiac tissue following therapy compared to untreated conditions. 3. Discussion Our study demonstrates that a radically minimized chemical cocktail can successfully drive fibroblast-to-cardiomyocyte transdifferentiation (TdCM) in rodent and human models, reaching functional maturity sufficient to establish conduction pathways. We have previously demonstrated that the threshold of conduction cells in cultured cardiomyocytes and in cardiac tissue sufficient for conduction can be around 20–30%, which is the percolation threshold in the myocardium (Kudryashova et al. 2019 ; Naumov et al. 2025 ). Importantly, we show that partial reprogramming (~ 56–83% α-actinin cells⁺) can achieve the electrophysiological percolation threshold in fibrotic tissue. We demonstrated electrical propagation without the need for complete cellular conversion of fibroblasts. This is consistent with our central hypothesis of minimizing the cocktail composition for use in therapies or smart scaffolds. This study demonstrates that a radically minimized chemical cocktail can successfully reprogram fibroblasts into functionally competent iCMs across species (REF, RNF, HAF). Crucially, the resulting iCMs exhibited key functional properties, especially electrophysiological maturation progressed from early IKv currents (~ 100 pA at day 8) to robust INa (~ 4000 pA in HAFs by day 21), confirming excitability development (Fig. 2 E, 3 E). Action potential generation in HAFs at day 15 (resting potential: −80 mV) (Fig. 3 E), despite absent plateau phase—a known hallmark of immaturity linked to CACNB2 splicing defects (Link et al. 2009 ; Ronaldson-Bouchard et al. 2022 ). Calcium handling responded to electrical pacing (Fig. 2 D, 3 D), confirming excitation-contraction coupling. Reduced number of components (compared to conventional protocols (Fu et al. 2015 ; Cao et al. 2016 )) enables feasible integration into implantable biomaterials for localized, sustained release. Complex cocktails with > 5 components face insurmountable challenges in release kinetics, stability, and intra-scaffold cross-talk (Lokwani et al. 2024 ). Our 4-component core (CHIR99021, BMP4, Activin A, IWP2) has optimal physicochemical properties for encapsulation in hydrogel systems (e.g., alginate (Lokwani et al. 2024 )), facilitating gradual paracrine delivery directly to the scar—a paradigm shift from systemic administration. Furthermore minimalist cocktails allow hybrid approaches where reprogrammed fibroblasts provide a conductive “bridge” for iPSC-derived cardiomyocyte grafts (iPSC-CMs). In vitro induced CMs exhibited normal resting potentials (− 80 mV) and progressive maturation of ion channels (INa ~ 4000 pA), confirming their ability to support electromechanical coupling. This addresses a key limitation of autonomous cell therapy: poor graft-host integration due to fibrous isolation (Qian et al. 2012 ; Riegleret al. 2015 ). We focused on these four components when modifying the protocol because they are the key inhibitors and activators of signaling pathways in the process of direct differentiation into cardiomyocytes from mesenchymal stem cells and iPSCs (Burridge et al. 2014 ). CHIR99021 inhibits GSK3 protein-kinase promoting proliferation of cardiomyocytes (Wang et al. 2016 ). Activin A and BMP4 from the TGFβ superfamily activate SMAD signaling that regulates pathological processes (Dituri et al. 2019 ). Activin A also promotes bFGF expression (Xiao et al. 2006 ), enhancing cell self-renewal. BMP4 prevents neurogenic differentiation and induced cardiac mesoderm formation in mice (Tsaytler et al. 2023 ). The reduction of components simplifies pharmacokinetic profiling, safety testing (e.g. drug interactions), and GMP-compliant manufacturing (Soares et al. 2024). Our in vivo safety data (no hematological toxicity; Fig. 4 C) highlight the advantage of biocompatibility compared to viral methods or multidrug regimens (Garry et al. 2022 ). While conversion efficiency (56–83% α-actinin⁺ cells) was modest compared to complex protocols (Fu et al. 2015 ; Cao et al. 2016 ), our approach prioritizes clinical translation over maximal cellular reprogramming. In vivo optical mapping revealed 84% conductive area in treated infarcts (compared to 71% in controls; Fig. 6 D), confirming that sparse iCM networks can restore electrical syncytium via percolation (Rohr et al. 1998 ). NADH fluorescence further demonstrated metabolic recovery in scars (Fig. 5 C), reflecting the physiological possibility of conductance restoration. This supports our paradigm: efficacy depends on achieving a critical conductance density rather than maximal reprogramming. This study has some notable limitations due to its focus. For example, the lack of AP plateau in HAF-iCMs (Fig. 3 E) reflects underexpression of Cav1.2 (Harvey & Hell 2013 ). In future studies, we may add thyroid hormone to the scaffolds to improve maturation (Ruvinov & Cohen 2016 ). At this stage of the study, we were looking for a possible cocktail to integrate into the targeted delivery system. Of course, intravenous injections (used here for safety testing) limit specific targeting to the scar. It should be stressed that currently published studies presenting results of in vivo fibroblast-to-cardiomyocyte direct reprogramming by small molecules also utilize systemic repeated delivery (Mohamed et al. 2017 ; Huang et al. 2018 ), in contrast with direct reprogramming by retroviral transduction, that is performed via multiple intramyocardial injections right after infarction modeling (Qian et al. 2012 ; Song et al. 2012 ). In the future, we plan to develop smart scaffolds for localized release based on and combine the resulting cocktail with cell therapies, such as our previous work. That is, a combination therapy for co-delivery of the cocktail with iPSC-CMs on bifunctional scaffolds is possible (Aitova et al. 2023 ). The next step of our work will also be to test chronic efficacy in animal models of infarction. In this work, we performed about 6 pilot experiments in rats to demonstrate the effect of the selected cocktails, but they are not the main conclusion. So, by focusing on cocktail minimization, we achieve sufficient electrophysiological activity and iCM maturity to partially restore scar conductance at the percolation threshold, as confirmed in human and weight-bearing cells. This strategy bridges the gap between regenerative potential and clinical feasibility, allowing for the next translational step using scaffold-based delivery. 4. Materials and Methods 4.1. Ethical Approval All procedures were carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were conducted in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of M.F. Vladimirsky Moscow Regional Clinical Research Institute (protocol №7 from 18.04.2024) and by the Moscow Institute of Physics and Technology Life Science Center Provisional Animal Care and Research Procedures Committee (Protocol No. A2-2012-09-02). 4.2. Rat Embryonic Fibroblasts cell culture Rat embryonic fibroblasts were isolated from embryonic day 12-14 rat embryos (Greaney et al. 2021). The head and liver were carefully removed from embryos, and then the rest tissues were cut and trypsinized into single-cell suspensions. Rat embryonic fibroblasts were cultured in fibroblast growth medium consisting of DMEM/F-12 (Gibco), 15% Serum Replacement (Gibco), 1% Glutamax (Gibco), 1% non-essential amino acids (NEAA; Gibco), 100 units/ml penicillin and 100 μg/ml streptomycin, 0.1 mM 2-Mercaptoethanol (Amresco), 10 ng/ml FGF-2 (Paneco). 4.3. Rat Neonatal Fibroblasts Rat neonatal fibroblasts were isolated using two-day isolation protocol from Worthington-Biochem for rat neonatal cardiomyocytes and fibroblasts (http://www.worthingtonbiochem.com/NCIS/default.html). In brief, hearts were extracted from rat pups (Rattus norvegicus, Sprague Dawley breed), aged 1–4 days, and immediately placed in Ca2+- and Mg2+-free Hank’s Balanced Solution (Gibco) on ice. Only the tissue of the ventricles was isolated—with approximately 50–60% of the initial heart mass being cut off—which included the sinoatrial node, the atria, and the atrioventricular node. The isolated ventricles were minced into small pieces and then left at 4 °C overnight for trypsinization (Trypsin-EDTA 0.25%, Gibco). On the second day, the cells were placed into a collagenase solution (Collagenase type II, 2.25 μg/mL; Gibco) and stirred for 1 hour at 37 °C. Next, the suspension of the cells was placed into a T75 flask for 1 hour for pre-plating. The cells remaining in flask after pre-plating were considered fibroblasts and were used after purification from cardiomyocytes. 4.4. Human Atrial Fibroblasts cell culture Human atrial fibroblasts were isolated from atrial appendages provided by surgeons. The appendages were immediately placed in the cardioplegic solution cusdiol after excision and transported to the laboratory on ice. Fibroblast growth medium consist of DMEM (Paneco), 10% FBS (Capricorn), L-glutamin (Paneco), 100 units/ml penicillin and 100 μg/ml streptomycin (Paneco). Atrial appendage was cut into pieces and then dispersed on gelatin-coated (Paneco) 10 cm culture dish containing 2 ml fibroblast growth medium, and additional 8 ml medium was supplemented after 2 hours. Our extraction method is based on a well-known protocol (Emig et al. 2020). 4.5. Transdifferentiation Protocols As a starting point, we chose the protocol for controlling the transdifferentiation of fibroblasts into cardiomyocytes (Fu et al. 2015). All abbreviations and main steps correspond to this protocol. CRM (cardiac reprogramming medium) consisting of DMEM knockout medium (Gibco), 15% FBS (Capricorn), 5% Serum Replacement (Gibco), 0.5% N2 supplement (Paneco), 2% B27 supplement (Gibco), 1% Glutamax supplement (Gibco), 1% NEAA (Gibco), 0.1 μM -mercaptoethanol (Amresco), 100 U/ml penicillin and 100 mg/ml streptomycin and 50 mg/ml vitamin C. CMM (medium for maintenance of cells differentiated into cardiomyocytes) was prepared based on DMEM medium (Paneco) supplemented with 15% FBS (Capricorne), 50 mg/ml vitamin C, 3 μM CHIR99021 and 1 mg/ml insulin. Medium from neonatal cardiomyocytes: DMEM medium (Paneco) supplemented with 10% FBS (Capricorne), described above, after 2-3 days of culturing cardiomyocytes. CRM (P) refers to the combination of 50% CRM with 50% of medium from neonatal cardiomyocytes; аll additives’ concentrations refer to total medium volume. CRM (C) refers to a cultural medium combining 50% of fresh CRM and 50% of 3 day CRM from the same well of the plate; аll additives concentrations refer to total medium volume. Fibroblasts transdifferentiation was started when fibroblasts reached 80% confluence in 24-well plates. On day 0 of each protocol culture medium was changed to CRM with reprogramming factors. Medium was changed each 2-3 days with addition of reprogramming factors. On the 16th or the 20th day CRM was replaced with CMM. The main components that were added to the environments were 2 small molecules: CHIR99021 stock solution 36 mM in DMSO and IWP2 stock solution 5 mM in DMSO. Also growth factors was added: Activin A stock solution 50 μg/ml in 0.1% BSA, BMP4 stock solution 10 μg/ml in 0.1% BSA, TGF-β stock solution 10 μg/ml in 0.1% BSA, FGF2 stock solution 10 μg/ml in 0.1% BSA, Y-27632 (ROCK inhibitor) stock solution 5 mM in PBS 1x. 4.3. Immunofluorescent Staining Cells were fixed for 10 min in 4% paraformaldehyde (Sigma-Aldrich), permeabilized for 10 min in 0.4% Triton-X100. Cells were further incubated for 1 hour in a blocking buffer (1% bovine serum albumin in phosphate-buffered saline, PBS), overnight at 4 °C with primary antibodies and for 1 h at room temperature with secondary antibodies. Cells were washed twice in PBS. Nuclei and F-actin were stained with DAPI (Vector Laboratories) and Alexa Fluor™ 488 Phalloidin (Thermo Fisher Scientific). Samples were analyzed and processed on a Zeiss LSM 710 confocal microscope with Zen black 3.0 software (Carl Zeiss AG). Primary antibodies (working dilutions—1:100): sarcomeric α-actinin mouse (Abcam, Cambridge, UK, Cat# ab9465). Secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA, working dilution—1:400): Alexa Fluor 594 goat anti-mouse IgG (HþL) highly cross adsorbed (A11005). 4.4. Perfusion Heart Experimental Protocol The protocol for isolating the heart and cannulating the aorta began with anesthetizing a lab rat and sacrificing it with a spinal fracture (Glukhov et al. 2010). Next, the heart was carefully removed through the following steps: (1) an incision was made from the xiphoid process to the lateral ends of the edges of the ribs and (2) through the ribs along the left and right anterior axillary lines to provide a cot thoracotomy, and (3) the chest was deviated upward. These steps provided full access to the heart. Sections of the vena cava and aorta completed the procedure for extracting the heart, after which the organ was washed with Tyrode’s salt solution (Sigma-Aldrich Co.) with heparin and transferred to a Petri dish with the same solution for further manipulations. The process of attaching the heart to a cannula (a needle with a soft polymer sheath) through the aorta was performed with several turns of surgical thread. From the moment the heart was removed from the body until the start of perfusion, no more than 10 min passed through the cannula. The perfusion of the heart was carried out using a special installation, according to Langendorf. The setup consisted of two main parts: a perfusion circuit and a recording optical system based on a high-speed imaging setup (Olympus MVX-10 Macro-View fluorescent microscope (Olympus Co., Tokyo, Japan) equipped with a high-speed Andor iXon-3 Camera 897-U (Andor Technology Ltd., Belfast, UK)). The perfusion circuit was prepared for constant circulation (Masterflex L/S Digital Drive, 600 rpm; 115/230 VAC, Masterflex L/S Easy-Load® II Pump Head, SS Rotor; 2-Channel (Cole-Parmer Instrument Company, Vernon Hills, IL, USA)) of a fixed volume of fluid, maintaining the temperature of the perfusate throughout the system (Cole-Parmer Polystat Standard 6.5 L Heated Bath, 150 C, 115 V AC/60 Hz (Cole-Parmer Instrument Company, Vernon Hills, IL, USA)), including the heart chamber, and oxygenating the solution (Cole-Parmer Bubble trap, Water Jacketed Reservoir, Oxygenating Bubbler (Cole-Parmer Instrument Company, Vernon Hills, IL, USA)). The total volume of fluid circulating in the unit was optimized using a compact PDMS polymer heart chamber. The minimum volume that allowed the heart to be perfused with an oxygenated heated solution was reduced to 30 mL. 4.5. Optical Mapping Protocols For cell culture, optical mapping with Fluo4-AM (Lumiprobe, 1892-500ug) and Di-8-ANEPPS (Thermo Fisher Scientific, D3167) was performed in Tyrode’s salt solution (pH 7.25 to 7.4) according to the protocol previously described in (Kudryashova et al. 2017). The signal was recorded with a 128×128 pixels resolution and a sampling frequency of 130 frames per second (Olympus MVX-10 Macro-View fluorescent microscope (Olympus Co., Tokyo, Japan) equipped with high-speed Andor iXon-3 EMCCD Camera (Andor Technology Ltd., Belfast, UK)). In general, for the whole heart the mapping protocol for the whole heart was similar to the protocol for cells (Laughner et al. 2012). Optical mapping with Fluo4-AM (Lumiprobe, 1892-500ug) and Di-8-ANEPPS (Thermo Fisher Scientific, D3167) was also performed in Tyrode’s salt solution (pH 7.25 to 7.4). The signal was recorded with a 128×128 pixels resolution and a sampling frequency of 130 frames per second by the same setting. The only difference in optical mapping was that the heartbeats were removed by BDM (Sigma) adding to the solution in concentration 10 mM when the heart was stained for fluorescence Fluo4-AM (Lumiprobe, 1892-500ug) in concentration 2.8 ug/ml and Di-8-ANEPPS (Thermo Fisher Scientific, D3167) in concentration 15 ug/ml. All experiments were performed at 37°C temperature during the observations. The experiments used electrode stimulation. The electrical stimulation duration was set at 20 ms, with the amplitude adjusted within the range of 5 to 8 V depending on the tissue culture or the excitation threshold of the heart. The stimulation interval varied from 200 to 1000 ms. The stimulus was set using a generator (2MHz USB PC Function Generator, PCGU100 (Velleman, Gavere, Belgium)). Platinum electrodes were used in the work. Data processing was fulfilled using the ImageJ program and the associated plugins (http://rsbweb.nih.gov/ij/ (accessed on 5 May 2023)). ImageJ plugin (time lapse color-coder) was used to build pseudo-3D images and activation maps. Principal analysis was performed in Wolfram Mathematica 12. 4.6. Patch-сlamp Voltage-gated ion channel currents in transdifferentiated cardiomyocytes were recorded using the patch-clamp electrophysiological method in the “perforated whole-cell” configuration. A description of how to install a patch clamp has been provided in another work (Abrasheva et al. 2024). The extracellular solution for recording sodium (INa), calcium (ICa, L ), potassium (IKv) contained 15 мМ HEPES, 150 мМ NaCl, 5,4 мM КCl, 1 мМ MgCl2, 1,8 мМ CaCl2, 15 мМ D-glucose, pH = 7.4 (adjusted with NaOH). The pipette was filled with an intracellular solution containing 150 мМ KCl, 5 мМ NaCl, 2 мМ CaCl2, 5 мМ MgATP, 10 mM HEPES, 5 mM EGTA, pH = 7.2 (adjusted with KOH). Currents from voltage-gated fast sodium channels (INa) as well as combined INa/Ca were obtained via the ramp stimulation protocol, which involved a linear increase in potential from –120 to +50 mV over a duration of 250 ms. Using a step-pulse stimulation protocol, current amplitudes of voltage-gated potassium channels (IKv) were quantified during depolarizing voltage steps (from -30 to 60 mV, each step presented for 2.5 s). The step protocol, containing pre-step (-30 mV) and main step (0 mV for 300 ms), was used to observe L-type calcium currents (ICa, L ). All experiments were conducted at room temperature (22–24°C). Data analysis and processing were performed using Clampfit 10.2 (Molecular Devices, USA) and OriginPro 8.1 (OriginLab Corp., USA) software. 4.8. Infarction Model Male Wistar rats weighing 250-350 g were anesthetized with injection combining “Xyla” xylazine hydrochloride 5 ng per 1 g of animal weight (De Adelaar, Netherlands) and “Zoletil” 4 ng tiletamine hydrochloride and 4 ng zolazepam hydrochloride per 1 g of animal weight (Vibrac, France). Trachea was intubated with intravenous cannula 18 G and connected to the Ambu bag (1 squeeze per second) and the RWD R550 Small Animal Anesthesia Machine combining oxygen 0.5-0.7 liter per minute with isoflurane via tubes. Left anterior descending coronary artery permanent occlusion was performed with monofilament prolene 6/0 (Ahn et al. 2004). After the end of the surgery rats were peritoneally injected with meloxicam 2 μg per 1 g of animal weight (Belkarolin, Belarus). The animal was monitored for several hours until regaining consciousness. 4.9. Injections Injections were performed intramyocardially or intravenously via insulin syringe 30G with total volume 50-100 μl. Intramyocardial injections were performed only on control rats under general isoflurane anesthesia, no more than once with cocktail: CHIR99021 — 10 μM, Activin A — 9 ng/ml, BMP 4 — 5 ng/ml, IWP2 — 2 μM, bFGF — 8 ng/ml, TGFβ — 1 ng/ml per 20 ml of circulating blood volume — or with sterile PBS (Gibco). Intravenous injections into the tail vein for IM model animals were started on the 14th day after the surgery. 4.10. Data Processing and Statistical Analysis Preliminary processing of optical mapping data was performed in ImageJ (v1.54p). To construct activation maps, background subtraction was applied to the videos, followed by the Kalman filtering algorithm for noise removal and Gaussian blurring. The activation map generation algorithm was implemented in Python (v3.11.13) using the following libraries: Matplotlib (v3.10.0), NumPy (v2.0.2), SciPy (v1.15.3), Pandas (v2.2.2). The algorithm is based on increasing the luminescence intensity of each individual pixel by a certain percentage relative to its average intensity. This approach enhances the method's sensitivity compared to the standard technique of applying a uniform absolute threshold to all pixels. For analyzing calcium dynamics, action potential dynamics and NADH photobleaching, plots were generated with preliminary processing in ImageJ (using the Kalman filtering algorithm for noise reduction), followed by data processing in Microsoft Excel and further plot generation and statistical analysis in Python (v3.11.13) with the same libraries. Statistical analysis was performed using the Anderson–Darling test with a 5% significance level. For the analysis of α-actinin expression percentage in cells, photographs of cells after immunofluorescent staining were taken. Based on the expression data in the photographs, the percentage of area occupied by cells expressing alpha actinin was calculated in relation to the area occupied by all cells (f-actin) by ImageJ standard functions. 5. Conclusions Electromechanical wave propagation is essential for cardiac function; its disruption by fibrotic scars after myocardial infarction causes arrhythmias. This study develops minimized chemical cocktails for direct fibroblast-to-cardiomyocyte transdifferentiation (TdCM), bypassing pluripotency to treat fibrosis. Optimized 4-component protocols generated functional induced cardiomyocytes (iCMs) from rat and human fibroblasts, with 56-83% expressing α-actinin. Electrophysiological profiling confirmed maturation: human iCMs exhibited action potentials (−80 mV resting potential) and robust sodium currents (∼4000 pA). Crucially, partial reprogramming sufficed to establish conduction pathways exceeding the percolation threshold (20-30%), enabling electrical propagation. In vivo testing showed no hematological toxicity, while optical mapping revealed 84% conductive area in treated infarcts (vs. 71% controls). Our findings demonstrate that minimized cocktails restore scar conductivity without complete cellular conversion, advancing translatable regenerative strategies. Abbreviations The following abbreviations are used in this manuscript: CVDs Cardiovascular Diseases MI Myocardial Infarction IND Ischemic Heart Disease CFs Cardiac Fibroblasts ECM Extracellular Matrix iPSCs Induced Pluripotent Stem Cells MSCs Mesenchymal Stem Cells iCMs Induced Cardiomyocyte-like Cells REF Rat Embryonic Fibroblasts RNF Rat Neonatal Fibroblasts HAF Human Atrial Fibroblasts TdCM Fibroblast-to-cardiomyocyte Transdifferentiation iPSC-CMs iPSC-derived cardiomyocyte Declarations Supplementary Materials: No Author Contributions: Conceptualization, K.A. and V.T.; methodology, M.S. and V.T.; software, V.D. and M.S.; validation, K.A. and V.T; formal analysis, E.T., S.Rob., V.D. and V.T.; investigation, E.T., S.Rob., S.K., V.D., A.D., S.Rom., A.M. and M.S.; resources, D.Z., M.P., A.R., K.A. and V.T.; data curation, S.F., K.A. and V.T.; writing—original draft preparation, E.T., S.Rob., S.K. and V.D.; writing—review and editing, A.R., K.A. and V.T.; visualization, E.T., S.Rob., V.D. and M.S..; supervision, K.A. and V.T..; project administration, V.T.; funding acquisition, A.R. and V.T. All authors have read and agreed to the published version of the manuscript. Funding: The study was carried out within the framework of project No. 25-65-00037 dated 22.05.2025 with the Russian Science Foundation (RSF) Institutional Review Board Statement: The study was approved by the Ethics Committee of M.F. Vladimirsky Moscow Regional Clinical Research Institute (protocol №7 from 18.04.2024) and by the Moscow Institute of Physics and Technology Life Science Center Provisional Animal Care and Research Procedures Committee (Protocol No. A2-2012-09-02). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Raw data (optical mapping, patch clamp data and confocal microscopy) could be found at Data Repository: https://drive.google.com/drive/folders/1jXv3zR2fuMYOcJ_LV8d2YgG2uK89IJPJ Acknowledgments: The authors thank Anastasia Dubrovskaya for accompanying the experiments. 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Heart J. 2011, 32(10), 1197–1206. https://doi.org/10.1093/eurheartj/ehr018 Trayanova, N.A.; Lyon, A.; Shade, J.; Heijman, J. Computational modeling of cardiac electrophysiology and arrhythmogenesis: toward clinical translation. Physiol. Rev. 2024, 104(3), 1265–1333. https://doi.org/10.1152/physrev.00017.2023 Tsaytler, P.; Liu, J.; Blaess, G.; Schifferl, D.; Veenvliet, J.V.;, Wittler, L.; Timmermann, B.; Herrmann, B.G.; Koch, F. BMP4 triggers regulatory circuits specifying the cardiac mesoderm lineage. Development 2023, 150(10), dev201450. https://doi.org/10.1242/dev.201450 Vagnozzi, R.J.; Maillet, M.; Sargent, M.A.; Khalil, H.; Johansen, A.K.Z.; Schwanekamp, J.A.; York, A.J.; Huang, V.; Nahrendorf, M.; Sadayappan, S.; et al. An acute immune response underlies the benefit of cardiac stem cell therapy. Nature 2020, 577(7790), 405–409. https://doi.org/10.1038/s41586-019-1802-2 Wang, H.; Cao, N.; Spencer, C.I.; Nie, B.; Ma, T.; Xu, T.; Zhang, Y.; Wang, X.; Srivastava, D.; Ding, S. Small molecules enable cardiac reprogramming of mouse fibroblasts with a single factor, Oct4. Cell Rep. 2014, 6(5), 951–960. https://doi.org/10.1016/j.celrep.2014.01.038 Wang, S.; Ye, L.; Li, M.; Liu, J.; Jiang, C.; Hong, H.; Zhu, H.; Sun, Y. GSK-3β Inhibitor CHIR-99021 Promotes Proliferation Through Upregulating β-Catenin in Neonatal Atrial Human Cardiomyocytes. J. Cardiovasc. Pharmacol. 2016, 68(6), 425–432. https://doi.org/10.1097/FJC.0000000000000429 Woodcock, J.; LaVange, L.M. Master Protocols to Study Multiple Therapies, Multiple Diseases, or Both. N. Engl. J. Med. 2017, 377(1), 62–70. https://doi.org/10.1056/NEJMra1510062 Xiao, L.; Yuan, X.; Sharkis, S.J. Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells. Stem cells 2006, 24(6), 1476–1486. https://doi.org/10.1634/stemcells.2005-0299 Yamakawa, H.; Ieda, M. Cardiac regeneration by direct reprogramming in this decade and beyond. Inflamm. Regener. 2021, 41, 20. https://doi.org/10.1186/s41232-021-00168- Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7186257","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492581938,"identity":"e687dfdb-eef1-41e6-838a-f91f370eaab7","order_by":0,"name":"Elena Turchaninova","email":"data:image/png;base64,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","orcid":"","institution":"E. Meshalkin National Medical Research Center of the Ministry of Health of the Russian Federation","correspondingAuthor":true,"prefix":"","firstName":"Elena","middleName":"","lastName":"Turchaninova","suffix":""},{"id":492581939,"identity":"da8658ad-7dad-44a4-89a0-f085030bf80b","order_by":1,"name":"Sofya Robustova","email":"","orcid":"","institution":"Moscow Center for Advanced Studies","correspondingAuthor":false,"prefix":"","firstName":"Sofya","middleName":"","lastName":"Robustova","suffix":""},{"id":492581940,"identity":"928f2652-eb47-41d5-8355-caa31c852c02","order_by":2,"name":"Sandaara Kovalenko","email":"","orcid":"","institution":"E. Meshalkin National Medical Research Center of the Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Sandaara","middleName":"","lastName":"Kovalenko","suffix":""},{"id":492581941,"identity":"c1dbaa1e-ce3d-4340-b021-c0ed1c95ac68","order_by":3,"name":"Vitalii Dzhabrailov","email":"","orcid":"","institution":"E. Meshalkin National Medical Research Center of the Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Vitalii","middleName":"","lastName":"Dzhabrailov","suffix":""},{"id":492581942,"identity":"29512155-0742-4e08-b0b9-f96ebdc8a19a","order_by":4,"name":"Aleria Dolgodvorova","email":"","orcid":"","institution":"Moscow Center for Advanced Studies","correspondingAuthor":false,"prefix":"","firstName":"Aleria","middleName":"","lastName":"Dolgodvorova","suffix":""},{"id":492581943,"identity":"ff937345-102e-448a-a9bd-f7846d472982","order_by":5,"name":"Serafima Romanova","email":"","orcid":"","institution":"Moscow Center for Advanced Studies","correspondingAuthor":false,"prefix":"","firstName":"Serafima","middleName":"","lastName":"Romanova","suffix":""},{"id":492581944,"identity":"2f4c1864-1299-4aec-9183-5861086240dc","order_by":6,"name":"Dmitriy Zybin","email":"","orcid":"","institution":"M.F. Vladimirsky Moscow Regional Clinical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Dmitriy","middleName":"","lastName":"Zybin","suffix":""},{"id":492581945,"identity":"2658a862-10ad-436d-b2db-49c8db2634bb","order_by":7,"name":"Mikhail Popov","email":"","orcid":"","institution":"M.F. Vladimirsky Moscow Regional Clinical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Mikhail","middleName":"","lastName":"Popov","suffix":""},{"id":492581946,"identity":"407336fc-7470-4285-8f3b-28e38a64d6fb","order_by":8,"name":"Alsu Miftakhova","email":"","orcid":"","institution":"Moscow Center for Advanced Studies","correspondingAuthor":false,"prefix":"","firstName":"Alsu","middleName":"","lastName":"Miftakhova","suffix":""},{"id":492581947,"identity":"3c1536c7-9f23-47bf-82ab-a25ea5638bd9","order_by":9,"name":"Sheida Frolova","email":"","orcid":"","institution":"M.F. Vladimirsky Moscow Regional Clinical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sheida","middleName":"","lastName":"Frolova","suffix":""},{"id":492581948,"identity":"000dc5e9-163c-4ed5-b304-2dbd4dfdf791","order_by":10,"name":"Mikhail Slotvitsky","email":"","orcid":"","institution":"E. Meshalkin National Medical Research Center of the Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Mikhail","middleName":"","lastName":"Slotvitsky","suffix":""},{"id":492581949,"identity":"5f77b382-eeb4-4858-a863-842da43c2db0","order_by":11,"name":"Alexander Romanov","email":"","orcid":"","institution":"E. Meshalkin National Medical Research Center of the Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Romanov","suffix":""},{"id":492581950,"identity":"621e40a7-f6cd-4733-bb8c-9061e4d5f7b5","order_by":12,"name":"Konstantin Agladze","email":"","orcid":"","institution":"Moscow Center for Advanced Studies","correspondingAuthor":false,"prefix":"","firstName":"Konstantin","middleName":"","lastName":"Agladze","suffix":""},{"id":492581951,"identity":"df8b50b4-3946-4a96-95ea-fd5bb64876f5","order_by":13,"name":"Valeriya Tsvelaya","email":"","orcid":"","institution":"E. Meshalkin National Medical Research Center of the Ministry of Health of the Russian Federation","correspondingAuthor":false,"prefix":"","firstName":"Valeriya","middleName":"","lastName":"Tsvelaya","suffix":""}],"badges":[],"createdAt":"2025-07-22 11:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7186257/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7186257/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87934897,"identity":"c71584cd-33af-4c07-a6db-e04083b72e8e","added_by":"auto","created_at":"2025-07-30 14:13:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2465100,"visible":true,"origin":"","legend":"\u003cp\u003eResults of rat embryonic fibroblasts (REF) direct reprogramming into cardiomyocytes. \u003cstrong\u003e(A) \u003c/strong\u003eThe scheme of REF-to-cardiomyocyte direct reprogramming with small molecule CHIR99021 varied concentrations 2.25, 4.5, 6.75 μM marked by colour intensity and growth factors: Activin A 100 ng/mL and BMP-4 5 ng/mL. Fibroblasts were cultivated in the cardiac reprogramming medium (CRM) containing the small molecules and growth factors. On the 16th day the medium was changed into cardiomyocyte maintaining medium (CMM). \u003cstrong\u003e(B) \u003c/strong\u003ePatch clamp data of native currents recording on indicated days. IKv day 24 — step protocol recording of delayed rectifier potassium current. INa/Ca day 24 — ramp protocol recording of composite voltage-gated sodium and voltage-gated calcium currents.\u003cstrong\u003e(C) \u003c/strong\u003eMorphology of REF-derived cell clusters on 8th and 14th day of induction.\u003cstrong\u003e \u003c/strong\u003eThe first clusters could be observed on day 8-10. \u003cstrong\u003e(D) \u003c/strong\u003eImmunostaining for cardiac marker α-actinin (red), fibrillar actin (green), nuclei (blue) in clusters generated from REFs on day 24. Scale bars 100 μm.\u003c/p\u003e","description":"","filename":"Figure1.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186257/v1/979f741c155aa4cbd976d655.jpg"},{"id":87934898,"identity":"69b7b1f6-07ce-4c15-acdb-e647b5f4cf09","added_by":"auto","created_at":"2025-07-30 14:13:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12185174,"visible":true,"origin":"","legend":"\u003cp\u003eResults of direct reprogramming of rat neonatal fibroblasts (RNF) into cardiomyocytes. \u003cstrong\u003e(A) \u003c/strong\u003eThe scheme of RNF-to-cardiomyocyte direct reprogramming. Fibroblasts were cultivated in a cardiac reprogramming medium (CRM), from day 3 CRM was replaced with CRM (P), from day 20 — with CMM. Small molecules concentrations: CHIR99021 10.0, 3.0 μM marked by colour intensity, IWP2 2μM, Y-27632 5 μM; growth factors concentrations: BMP4 5.0 ng/ml, Activin A 9.0 ng/mL, bFGF 8.0, 5.0, 1.0 ng/ml marked by colour intensity, TGF-β 1.0 ng/ml. \u003cstrong\u003e(B) \u003c/strong\u003eMorphology of RNF-derived clusters induced by small molecules and growth factors.\u003cstrong\u003e \u003c/strong\u003eThe first clusters were observed on day 12-16. \u003cstrong\u003e(C) \u003c/strong\u003eImmunostaining for cardiac marker α-actinin (red), fibrillar actin (green), nuclei (blue) in clusters generated from RNFs on days 16 and 21. Scale bars 50 μm. \u003cstrong\u003e(D) \u003c/strong\u003eCalcium dynamics in RNF-derived cardiomyocytes on day 21. Left: selected region; right: normalized fluorescence intensity of calcium-dependent dye over time for selected region. Yellow colour indicates the times of peak calcium concentrations. \u003cstrong\u003e(E) \u003c/strong\u003ePatch clamp data of native currents recording. Left column: INa/Ca day 16 — ramp protocol recording of overlapping voltage-gated sodium and voltage-gated calcium currents, INa day 21 — ramp protocol recording of voltage-gated sodium current, ICa,L day 21 — step-protocol recording of L-type calcium current. Right column: IKv day 8, IKv day 16, IKv day 21 — representative current trace of voltage-gated potassium currents recorded via step-pulse stimulation protocol during depolarizing voltage steps on indicated days.\u003c/p\u003e","description":"","filename":"Figure2.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186257/v1/8e7e4b8703f6883be050836d.jpg"},{"id":87934899,"identity":"d4ab52aa-05ff-4093-86f5-e37e9e9b1e36","added_by":"auto","created_at":"2025-07-30 14:13:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13277007,"visible":true,"origin":"","legend":"\u003cp\u003eResults of direct reprogramming of human atrial fibroblasts (HAF) into cardiomyocytes. \u003cstrong\u003e(A) \u003c/strong\u003eThe scheme of HAF-to-cardiomyocyte direct reprogramming with small molecules and growth factors. Fibroblasts were cultivated in CRM containing the small molecules and growth factors; on day 6, it was replaced with modified with cardiomyocyte medium CRM (CRM(C)). On day 9 cells were returned to CRM till day 21, when it was replaced with CMM. Small molecules concentrations: CHIR99021 12.0, 10.0 μM marked by colour intensity, IWP2 2μM, growth factors concentrations: BMP4 5.0 ng/ml, Activin A 9.0 ng/mL, bFGF 8.0, 6.0, 4.0 ng/ml marked by colour intensity, TGF-β 1.0 ng/ml. \u003cstrong\u003e(B) \u003c/strong\u003eMorphology of HAF-derived clusters induced by small molecules and growth factors.\u003cstrong\u003e \u003c/strong\u003eThe first clusters could be observed on day 10-12. \u003cstrong\u003e(C) \u003c/strong\u003eImmunostaining for cardiac marker α-actinin (red), fibrillar actin (green), nuclei (blue) in clusters generated from HAFs on day 33. Scale bars 100 μm (left) and 50 μm (right). \u003cstrong\u003e(D) \u003c/strong\u003eCalcium dynamics on day 33 of the induction. Left: selected region; right: normalized fluorescence intensity of a calcium-dependent dye over time for selected region. Yellow colour indicates the times of peak calcium concentrations. The red dashed line marks the time of electrical pacing. \u003cstrong\u003e(E) \u003c/strong\u003ePatch clamp data of native currents recording. Left column: INa/Ca day 15 — ramp protocol recording of overlapping voltage-gated sodium and voltage-gated calcium currents, Action potential day 15 — resting potential was about -80 mV. Right column: IKv day 15 — step protocol recording of summary potassium currents, INa day 21 — ramp protocol recording of voltage-gated sodium current.\u003c/p\u003e","description":"","filename":"Figure3.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186257/v1/1a0ebb41549096c2cda68ff9.jpg"},{"id":87934904,"identity":"2ef0ef12-0bec-45ce-ae10-3db39fb5f7a0","added_by":"auto","created_at":"2025-07-30 14:13:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10393892,"visible":true,"origin":"","legend":"\u003cp\u003eRat infarction model for transdifferentiation in vivo. Safety testing. \u003cstrong\u003e(A) \u003c/strong\u003eThe scheme of \u003cem\u003ein vivo \u003c/em\u003etransdifferentiation in a rat infarction model. SSmall molecules concentrations: CHIR99021 10.0, 3.0 μM marked by colour intensity, IWP2 2μM; growth factors concentrations: BMP4 5.0 ng/ml, Activin A 9.0 ng/mL, bFGF 8.0, 5.0, 1.0 ng/ml marked by colour intensity, TGF-β 1.0 ng/ml. \u003cstrong\u003e(B)\u003c/strong\u003e Rat heart 30 minutes after ligation, ligation site shown with arrow, ischemic zone highlighted in white. \u003cstrong\u003e(C) \u003c/strong\u003eClinical blood analysis data grouped by treatment type. Control in the intact group refers to single PBS injection and in the infarction group to non-treated animals; treatment refers to reprogramming formulation injection: in the intact group single intramyocardial, in the infarction group repeated intravenous according to the protocol. White blood cells, red blood cells concentrations and red blood cell size shown on boxplots, no statistically significant difference observed. \u003cstrong\u003e(D)\u003c/strong\u003e Immunostaining of cardiac marker α-actinin (red) in damaged zone 1 month after infarction. Green color is f-actin staining. Nuclei were stained with DAPI (blue). White arrow shows the damaged zone. Scale bar represents 200 μm.\u003c/p\u003e","description":"","filename":"Figure4.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186257/v1/3178ea769caa353208a02aeb.jpg"},{"id":87934900,"identity":"e74d40aa-6525-489e-8323-4ca35e7eb6d9","added_by":"auto","created_at":"2025-07-30 14:13:35","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9045202,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of infarction zone by NADH fluorescence. Left — heart image with selected regions of infarction zone (red) and intact myocardium (green); right — Decrease of NADH autofluorescence intensity in the regions plotted in relative scale. \u003cstrong\u003e(A) \u003c/strong\u003eInfarction after 30 minutes occlusion.\u003cstrong\u003e (B) \u003c/strong\u003eInfarction without treatment on the 14th day after surgery. \u003cstrong\u003e(C) \u003c/strong\u003eInfarction with 10 days treatment, treatment started on the 14th day after surgery.\u003c/p\u003e","description":"","filename":"Figure5.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186257/v1/4deffb1fa07370a5f3df1883.jpg"},{"id":87935177,"identity":"f88ccbe6-274f-403d-ac67-c9b710cfe942","added_by":"auto","created_at":"2025-07-30 14:21:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9810746,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of chronic myocardial infarction in rat hearts on activation maps. The infarction zone is marked by a blue circle, and the stimulation area is indicated by a red circle. Colourful time scale enables spatial resolution of wave propagation. \u003cstrong\u003e(A)\u003c/strong\u003e Action potential propagation (Di-8-ANEPPS staining), \u003cstrong\u003e(B) \u003c/strong\u003eCalcium dynamics (Fluo-4 AM staining) in the same infarcted heart. \u003cstrong\u003e(C)\u003c/strong\u003e Action potential propagation in the infarcted heart without treatment \u003cstrong\u003e(D)\u003c/strong\u003e Calcium dynamics in the infarcted heart following therapy.\u003c/p\u003e","description":"","filename":"Figure6.tiff.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7186257/v1/b7c868192ae35a60804aeaac.jpg"},{"id":89741155,"identity":"0bcb1e97-4d3e-4ded-93a1-55059797714a","added_by":"auto","created_at":"2025-08-23 20:31:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":58078344,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7186257/v1/b69614ff-1570-4bf8-a60f-9237ba74187a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Refining fibroblast-to-cardiomyocyte transdifferentiation protocols to explore emergent self- organization in cardiac cultures","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCardiovascular diseases (CVDs) remain the principal cause of death globally. Myocardial infarction (MI), the acute and often fatal manifestation of ischemic heart disease (IHD), is a major driver of this burden. The American Heart Association's Heart Disease and Stroke Statistics\u0026mdash;2024 Update reports that in the United States alone, someone has a myocardial infarction approximately every 40 seconds (Martin et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Acute MI results from prolonged myocardial ischemia, triggering extensive cardiomyocyte necrosis. This initiates a maladaptive reparative response characterized by activation of resident cardiac fibroblasts (CFs), excessive deposition of extracellular matrix (ECM) (Hinz \u0026amp; Gabbiani \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), and the formation of a dense, non-contractile, and electrically non-conductive fibrotic scar (Sutton \u0026amp; Sharpe \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). This replacement fibrosis (Hinz \u0026amp; Gabbiani \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) disrupts the myocardial syncytium, severely impairing pump function and creating a substrate for lethal arrhythmias (Nguyen et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), directly contributing to heart failure and sudden cardiac death post-MI.\u003c/p\u003e\u003cp\u003eNovel therapeutic strategies for the post-MI fibrotic scar include pharmacological agents targeting neurohumoral activation and inflammation (Ghanem \u0026amp; Megahed 2023; Raziyeva et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but the main solution is still surgical interventions like revascularization or device implantation (Marrouche et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While essential, these approaches fail to regenerate lost cardiomyocytes or restore physiological electrical conduction across the scar. Regenerative strategies, particularly cell replacement therapy using cardiomyocytes derived from induced pluripotent stem cells (iPSCs) (Takahashi \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) or mesenchymal stem cells (MSCs) (Fern\u0026aacute;ndez-Garza et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), show promise for remuscularization (Jebran et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, they carry inherent risks of tumorigenicity (Liu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and arrhythmogenicity (Takahashi et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), alongside challenges of scalable cell production and immune rejection.\u003c/p\u003e\u003cp\u003eIn situ direct reprogramming or transdifferentiation of resident CFs within the scar into induced cardiomyocyte-like cells (iCMs), bypassing pluripotency (Yamakawa \u0026amp; Ieda \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), offers a compelling alternative. CFs are a prime target due to their central role in post-MI scar formation and pathological remodeling (Nagalingam et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and their high abundance within the infarct zone (Pinto et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Direct reprogramming circumvents the need for large-scale in vitro cell expansion (e.g., billions of cells per graft (Jebran et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)) and avoids immune complications associated with allogeneic cell transplantation (Tongers et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Multiple approaches exist for fibroblast to cardiomyocyte-like cells reprogramming, including viral delivery of transcription factors (e.g., Oct4, Sox2, Klf4 (Efe et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2014\u003c/span\u003e); Gata4, Mef2c, Tbx5 (GMT) (Mohamed et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e); GHMT (Ifkovits et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)), mRNA (Jayawardena et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and cocktails of small molecules. Small molecules modulate key signaling pathways (e.g., TGF-β, Wnt, cAMP) and include inhibitors of epigenetic repressors (tranylcypromine, valproate (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)), TGF-β inhibitors (RepSox, SB431542 (Mohamed et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)), Wnt modulators (CHIR99021 (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), XAV939 (Jayawardena et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)), and cAMP activators (Forskolin (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)).\u003c/p\u003e\u003cp\u003eDespite progress, current transdifferentiation protocols, especially purely chemical ones, suffer from critically low efficiency. Viral methods combined with small molecules enhance reprogramming in vivo (Mohamed et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Qian et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) but face translational hurdles due to risks of insertional mutagenesis, immunogenicity, and manufacturing complexity (Garry et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Purely chemical cocktails offer superior safety and delivery flexibility (e.g., injectable, potentially oral (Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)). However, reported conversion rates remain inadequate (e.g., ~\u0026thinsp;1% after prolonged multi-dose regimens (Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)), limiting functional recovery.\u003c/p\u003e\u003cp\u003eCrucially, complete reprogramming of all scar fibroblasts is likely unnecessary. One potential therapeutic strategy for post-MI scar treatment involves enhancing electrical conductivity to enable more synchronized contraction. This can be achieved by reprogramming a critical minimal fraction of fibroblasts into conductive iCMs, establishing interconnected conductive pathways that reach the electrophysiological percolation threshold within the scar tissue (Trayanova et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Rabinovitch et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Achieving this threshold allows the electrical wavefront to propagate through the formerly inert scar (Kalinin et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study directly addresses the challenge of low efficiency in chemical reprogramming for myocardial scar therapy taking the positives from this fact. We hypothesize that a radically minimized, optimized chemical cocktail can achieve iCM conversion rates sufficient to reach the percolation threshold within the fibrotic scar. Minimization is of paramount importance for clinical translation as it increases the likelihood of applicability of the cocktail. Complex cocktails pose significant challenges for pharmacokinetic profiling, safety assessment (including drug-drug interaction studies), formulation stability, manufacturing sequences, and regulatory approval pathways. Minimizing the number of compounds dramatically simplifies these processes, accelerating clinical development (DiMasi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Woodcock et al. 2017).\u003c/p\u003e\u003cp\u003eIn addition, we plan to use the chemical cocktails we have created for gradual targeted delivery to the myocardium. Implantable biomaterial scaffolds designed for localized, sustained, and temporally controlled release in the rumen are a promising delivery platform. However, incorporating multiple compounds with potentially different release kinetics, stability requirements, and complex cross-linking and interactions is highly complex and often impractical. A minimal cocktail is necessary for the efficient development and performance of such next-generation delivery systems (Ruvinov \u0026amp; Cohen \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tibbitt et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSuch chemical cocktails also have the potential for synergistic combination therapies. A simple, effective minimal chemical reprogramming strategy could be easily combined with cell replacement therapies (e.g., iPSC-CM patches) to enhance their efficacy and overcome their differentiation-related drawbacks. Reprogramming a subset of scar fibroblasts in situ could create a more hospitable, conductive microenvironment to improve survival, integration, and electromechanical coupling of grafted cardiomyocytes (Tang et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vagnozzi et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTherefore, we aimed to develop, optimize, and validate a novel, highly minimal chemical reprogramming cocktail specifically designed to efficiently transdifferentiate fibroblasts into functional iCM. Our primary goal was to achieve an efficiency sufficient to establish conductive pathways (above the percolation threshold) in models replicating the fibrotic environment following myocardial infarction, using both animal and human cells to demonstrate robust translational potential.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. In vitro transdifferentiation from fibroblast to cardiomyocyte-like cells (iCM)\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe primary goal of this study section was to develop a minimized small-molecule reprogramming protocol for efficient fibroblast-to-cardiomyocyte transdifferentiation. By minimizing the number of active molecules, we reduce potential immunogenicity and manufacturing complexity, which is important for future clinical applications. As a baseline, we chose a protocol for controlling fibroblast to cardiomyocyte transdifferentiation (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) that was primarily used for mouse embryonic fibroblasts. This section describes the results of testing modified protocols for fibroblasts-to-cardiomyocytes transdifferentiation. The resulting cells were characterized and the effectiveness of the protocol was assessed. For evaluation several types of analysis were performed during transdifferentiation including: patch-clamp, optical mapping of calcium dynamics and immunostaining for fibrillar actin, nuclei and cardiomyocytes marker α-actinin.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\u003ch2\u003e2.1.1. Rat embryonic fibroblasts derived iCM\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFirstly we start to adopt transdifferentiation protocol for rat embryonic fibroblasts (REF) to iCM. The basic protocol was successfully adapted for REF by varying the concentrations of CHIR99021, Activin A, and BMP4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA),, which were chosen as the main components. We compared 4 protocols for REFs with varied concentrations of small molecule CHIR99021 and tested the role of Activin A and BMP-4 additives for better induction of cardiomyocytes. The final protocols for REF-to-cardiomyocyte direct reprogramming are presented on Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e\u003cp\u003eWe observed changes in the cell's morphology on day 8\u0026ndash;10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Сlustering and strand formation indicate differentiation process success (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Electrophysiological profiling on 24th transdifferentiation day using patch-clamp identified: combined INa/Ca currents using ramp protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and an apparent voltage-gated IKv current with amplitude \u0026sim;200 pA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Cells after 24 days of transdifferentiation were stained positive for cardiac markers. Some cells after transdifferentiation displayed partially striated α-actinin expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eThe percentage ratio of α-actinin expression to f-actin expression was measured on day 24: Protocol № 1 \u0026mdash; 73\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, Protocol № 2 \u0026mdash; 75\u0026thinsp;\u0026plusmn;\u0026thinsp;3%, Protocol № 3 \u0026mdash; 83\u0026thinsp;\u0026plusmn;\u0026thinsp;8%, Protocol № 4 \u0026mdash; 79\u0026thinsp;\u0026plusmn;\u0026thinsp;21%. Particularly noteworthy is Protocol № 3, which achieved significantly higher α-actinin content in cells compared to the other protocols supposedly due to high concentration of CHIR99021.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Rat neonatal fibroblasts derived iCM\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAfter testing protocols for REFs we adapted it for neonatal rat fibroblasts (RNF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). For survival enhancement Y-27632, selective ROCK inhibitor, was added to prevent apoptosis. Besides Activin A and BMP4 on early transdifferentiation stages another SMAD activating growth factor TGFβ was applied. SMAD is believed to promote smooth muscle cell differentiation and apoptosis inhibition (Shi et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). From the 24th day of transdifferentiation analogous to human IPSC differentiation to cardiac tissue (Lian et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) the protocol was supplied with an IWP2 small molecular inhibitor of the Wnt pathway. Unlike the protocol for REF, during transdifferentiation of RNF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) CRM(P) medium combining CRM with 50% of 2\u0026ndash;4 day medium from neonatal cardiomyocytes was used to exploit the paracrine effect (Rangappa et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe modified protocols for RNF-to-cardiomyocyte direct reprogramming are presented on Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Clusterization and strand formation were observed on day 12\u0026ndash;16 of induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). RNF were stained for cardiac marker α-actinin on different days of transdifferentiation; from 16th day a partially striated pattern of α-actinin expression was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eDuring optical mapping with calcium-dependent dye Fluo-4 AM on the 21st day we registered periodical calcium activity in response to electric pacing, confirming the successful induction into cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Patch-clamp analysis on the 21st day revealed an apparent INa via voltage-gated sodium channels with a mean amplitude of ~\u0026thinsp;200 pA, typical kinetic of ICa,L via voltage-gated calcium channels with significantly reduced amplitudes, averaging approximately 100 pA; an IKv via voltage-gated potassium channels reaching\u0026thinsp;~\u0026thinsp;600 pA amplitude (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This displays electrophysiological properties of INa and ICa improvement since day 16, when combined INa and ICa currents were recorded and IKv since day 8 (~\u0026thinsp;150 pA) day 16 (~\u0026thinsp;300 pA).\u003c/p\u003e\u003cp\u003eThe results of the immunocytochemistry analysis showed an increase in the α-actinin/f-actin expression ratio from 62\u0026thinsp;\u0026plusmn;\u0026thinsp;12% at day 16 to 77\u0026thinsp;\u0026plusmn;\u0026thinsp;13% at day 21 of differentiation, indicating that the protocol effectively enhances α-actinin expression over time.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. Human atrial fibroblasts derived iCM\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor human atrial fibroblasts (HAF) primarily RNF protocol was applied with slight modifications (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Instead of CRM(P) medium CRM(C) was applied for enhancement of human fibroblast transdifferentiation.\u003c/p\u003e\u003cp\u003eThe final optimized protocols for HAF-to-cardiomyocyte direct reprogramming are presented on Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. We observed changes in cell`s morpholоogy on days 10\u0026ndash;12. Clusterization and strand formation, large size, single appearance may be features of successful differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). On 15th day, patch-clamp analysis via ramp protocols recorded composite currents with contributions from both voltage-gated sodium (Na⁺) and calcium (Ca\u0026sup2;⁺) channels, while depolarizing steps elicited small-amplitude potassium currents (IKv\u0026thinsp;\u0026asymp;\u0026thinsp;100 pA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Notably, action potential (AP) was recorded on the 15th day. While the absence of a plateau phase prevented the attainment of adult-like atrial action potential duration, the presence of a normal resting membrane potential (\u0026thinsp;~\u0026thinsp;\u0026minus;\u0026thinsp;80 mV) confirms development of fundamental excitability mechanisms.\u003c/p\u003e\u003cp\u003eOptical mapping with calcium-dependent dye Fluo-4 AM was performed on the 21st and 33d days. As can be seen, the calcium concentration responds to electric pacing, confirming the successful induction into cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Patch-clamp analysis revealed progressive development of voltage-gated ion channels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). By day 21, robust Na⁺ currents (amplitude\u0026thinsp;~\u0026thinsp;4000 pA) disciplaying electrophysiological properties improvement since day 16, when voltage-gated sodium (Na⁺) and calcium (Ca\u0026sup2;⁺) currents were recorded. The development of nanoampere-range Na⁺ currents by day 21 confirms efficiency of the differentiation protocol. However, consistent gigaseal formation proved challenging at later stages of transdifferentiation, precluding reliable quantification of Ca\u0026sup2;⁺ and delayed rectifier K⁺ currents.\u003c/p\u003e\u003cp\u003eConsistent with the electrophysiological study, immunofluorescent analysis on day 33 confirmed that HAFa were positive for cardiomyocyte marker α-actinin, and these cells also displayed partially striated α-actinin expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Additionally, the α-actinin to f-actin expression ratio in these cells at day 33 of differentiation was measured at 56\u0026thinsp;\u0026plusmn;\u0026thinsp;24%.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Testing transdifferentiation cocktail for in vivo application\u003c/h2\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1. Safety testing for \u003cem\u003ein vivo\u003c/em\u003e application of transdifferentiation cocktail\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor \u003cem\u003ein vivo\u003c/em\u003e testing the protocol for RNF, excluding ROCK inhibitor Y-27632, was adopted (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Since the duration of reprogramming is important for cell maturation, and in vitro takes at least 3\u0026ndash;4 weeks (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), we performed repeated injections of components for induction with almost the same intervals as in \u003cem\u003ein vitro\u003c/em\u003e protocol for RNFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) i.e. no more than 2 injections a week, that was found safe for control protocol administration in mice (Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Treatment started 14 days according to the scheme (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) after myocardial infarction induction via coronary artery ligation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD presents an example of immunohistochemical characterization of heart fibrosis in our infarction model.\u003c/p\u003e\u003cp\u003ePrimarily to the infarction treatment experiment we observed safety issues of transdifferentiation application \u003cem\u003ein vivo\u003c/em\u003e. Toxicity testing performed on intact animals receiving single dosage intramyocardial injection. No adverse effects were seen during the three day period of observation. Clinical blood analysis also showed no significant difference in both leucocyte formulation and hemoglobin related parameters between treated and non-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eSince intramyocardial injections can only be performed under general anesthesia, repeated interventions of this kind are not safe for animals. Thus during transdifferentiation in vivo testing we instead performed intravenous injections as a compromise between targeting and safety.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2. Illustration of the efficiency of \u003cem\u003ein vivo\u003c/em\u003e transdifferentiation in a rat infarction model\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe compared NADH fluorescence, a marker of ischemia and cellular respiration, in the rat heart during an ex vivo experiment. As is known, the process of NADH fluorescence intensity reduction is associated not only with its photobleaching but also with the activity of glutamate dehydrogenase, which regenerates NADH, thereby slowing the decline in fluorescence intensity (Moreno et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, by comparing the dynamics of NADH photobleaching, we can perform a qualitative assessment of tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe compared the decrease in NADH fluorescence intensity in two regions of a Langendorff-perfused rat heart with myocardial infarction: the infarcted area and the intact area. The closer the NADH photobleaching curves are to each other, the more similar the infarct scar can be considered to healthy tissue. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, the curves are quite similar, which is due to the fact that the infarction in this heart was induced just 30 minutes before recording the curves, so the differences between the infarcted and intact regions are minimal.\u003c/p\u003e\u003cp\u003eIn contrast, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB shows a significant difference in fluorescence decay between the compared regions, indicating that after 14 days of occlusion, the infarcted area undergoes substantial fibrotic changes. However, with therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), the photobleaching curves become similar again, suggesting that the treatment has a significant effect on the infarcted region of the heart, bringing the cellular respiration characteristics of the fibrotic area closer to those of healthy cardiomyocytes.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo characterize functional improvement during therapy we also adopted optical mapping methodology to assess electric propagation in whole hearts during Langendorff perfusion, and observed certain differences in conduction in the infarction and intact zones of hearts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Two applied dyes: action potential sensitive Di-8-ANEPPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) and calcium sensitive Fluo-4 AM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) \u0026mdash; displayed comparable pattern on the same heart, and since the action potential wave and the subsequent calcium wave in the absence of drugs affecting their coupling have the same distribution patterns (Eisner et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), they can be compared with each other.\u003c/p\u003e\u003cp\u003eIn the two experimental groups: control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) and therapy (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), the ratio of the conductive area to the total mapped heart area was measured. The results of this rough estimation demonstrate that the transdifferentiation treatment has a somewhat positive effect on cardiac conduction, increasing the proportion of conductive tissue from 71\u0026thinsp;\u0026plusmn;\u0026thinsp;2% in the control group to 84\u0026thinsp;\u0026plusmn;\u0026thinsp;3% in the treated group. This increase in the conductive ratio might indicate a relative reduction in the infarction area, suggesting partial restoration of conductivity in fibrotic cardiac tissue following therapy compared to untreated conditions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eOur study demonstrates that a radically minimized chemical cocktail can successfully drive fibroblast-to-cardiomyocyte transdifferentiation (TdCM) in rodent and human models, reaching functional maturity sufficient to establish conduction pathways. We have previously demonstrated that the threshold of conduction cells in cultured cardiomyocytes and in cardiac tissue sufficient for conduction can be around 20\u0026ndash;30%, which is the percolation threshold in the myocardium (Kudryashova et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Naumov et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Importantly, we show that partial reprogramming (~\u0026thinsp;56\u0026ndash;83% α-actinin cells⁺) can achieve the electrophysiological percolation threshold in fibrotic tissue. We demonstrated electrical propagation without the need for complete cellular conversion of fibroblasts. This is consistent with our central hypothesis of minimizing the cocktail composition for use in therapies or smart scaffolds.\u003c/p\u003e\u003cp\u003eThis study demonstrates that a radically minimized chemical cocktail can successfully reprogram fibroblasts into functionally competent iCMs across species (REF, RNF, HAF). Crucially, the resulting iCMs exhibited key functional properties, especially electrophysiological maturation progressed from early IKv currents (~\u0026thinsp;100 pA at day 8) to robust INa (~\u0026thinsp;4000 pA in HAFs by day 21), confirming excitability development (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Action potential generation in HAFs at day 15 (resting potential: \u0026minus;80 mV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), despite absent plateau phase\u0026mdash;a known hallmark of immaturity linked to CACNB2 splicing defects (Link et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ronaldson-Bouchard et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Calcium handling responded to electrical pacing (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), confirming excitation-contraction coupling.\u003c/p\u003e\u003cp\u003eReduced number of components (compared to conventional protocols (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e)) enables feasible integration into implantable biomaterials for localized, sustained release. Complex cocktails with \u0026gt;\u0026thinsp;5 components face insurmountable challenges in release kinetics, stability, and intra-scaffold cross-talk (Lokwani et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our 4-component core (CHIR99021, BMP4, Activin A, IWP2) has optimal physicochemical properties for encapsulation in hydrogel systems (e.g., alginate (Lokwani et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e)), facilitating gradual paracrine delivery directly to the scar\u0026mdash;a paradigm shift from systemic administration. Furthermore minimalist cocktails allow hybrid approaches where reprogrammed fibroblasts provide a conductive \u0026ldquo;bridge\u0026rdquo; for iPSC-derived cardiomyocyte grafts (iPSC-CMs). In vitro induced CMs exhibited normal resting potentials (\u0026minus;\u0026thinsp;80 mV) and progressive maturation of ion channels (INa\u0026thinsp;~\u0026thinsp;4000 pA), confirming their ability to support electromechanical coupling. This addresses a key limitation of autonomous cell therapy: poor graft-host integration due to fibrous isolation (Qian et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Riegleret al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWe focused on these four components when modifying the protocol because they are the key inhibitors and activators of signaling pathways in the process of direct differentiation into cardiomyocytes from mesenchymal stem cells and iPSCs (Burridge et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). CHIR99021 inhibits GSK3 protein-kinase promoting proliferation of cardiomyocytes (Wang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Activin A and BMP4 from the TGFβ superfamily activate SMAD signaling that regulates pathological processes (Dituri et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Activin A also promotes bFGF expression (Xiao et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), enhancing cell self-renewal. BMP4 prevents neurogenic differentiation and induced cardiac mesoderm formation in mice (Tsaytler et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe reduction of components simplifies pharmacokinetic profiling, safety testing (e.g. drug interactions), and GMP-compliant manufacturing (Soares et al. 2024). Our in vivo safety data (no hematological toxicity; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) highlight the advantage of biocompatibility compared to viral methods or multidrug regimens (Garry et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile conversion efficiency (56\u0026ndash;83% α-actinin⁺ cells) was modest compared to complex protocols (Fu et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), our approach prioritizes clinical translation over maximal cellular reprogramming. In vivo optical mapping revealed 84% conductive area in treated infarcts (compared to 71% in controls; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), confirming that sparse iCM networks can restore electrical syncytium via percolation (Rohr et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). NADH fluorescence further demonstrated metabolic recovery in scars (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), reflecting the physiological possibility of conductance restoration. This supports our paradigm: efficacy depends on achieving a critical conductance density rather than maximal reprogramming.\u003c/p\u003e\u003cp\u003eThis study has some notable limitations due to its focus. For example, the lack of AP plateau in HAF-iCMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) reflects underexpression of Cav1.2 (Harvey \u0026amp; Hell \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In future studies, we may add thyroid hormone to the scaffolds to improve maturation (Ruvinov \u0026amp; Cohen \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). At this stage of the study, we were looking for a possible cocktail to integrate into the targeted delivery system. Of course, intravenous injections (used here for safety testing) limit specific targeting to the scar. It should be stressed that currently published studies presenting results of in vivo fibroblast-to-cardiomyocyte direct reprogramming by small molecules also utilize systemic repeated delivery (Mohamed et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), in contrast with direct reprogramming by retroviral transduction, that is performed via multiple intramyocardial injections right after infarction modeling (Qian et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the future, we plan to develop smart scaffolds for localized release based on and combine the resulting cocktail with cell therapies, such as our previous work. That is, a combination therapy for co-delivery of the cocktail with iPSC-CMs on bifunctional scaffolds is possible (Aitova et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The next step of our work will also be to test chronic efficacy in animal models of infarction. In this work, we performed about 6 pilot experiments in rats to demonstrate the effect of the selected cocktails, but they are not the main conclusion.\u003c/p\u003e\u003cp\u003eSo, by focusing on cocktail minimization, we achieve sufficient electrophysiological activity and iCM maturity to partially restore scar conductance at the percolation threshold, as confirmed in human and weight-bearing cells. This strategy bridges the gap between regenerative potential and clinical feasibility, allowing for the next translational step using scaffold-based delivery.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cp\u003e4.1. Ethical Approval\u003c/p\u003e\n\u003cp\u003eAll procedures were carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were conducted in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of M.F. Vladimirsky Moscow Regional Clinical Research Institute (protocol №7 from 18.04.2024) and by the Moscow Institute of Physics and Technology Life Science Center Provisional Animal Care and Research Procedures Committee (Protocol No. A2-2012-09-02).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.2. \u0026nbsp;Rat Embryonic Fibroblasts cell culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRat embryonic fibroblasts were isolated from embryonic day 12-14 rat embryos (Greaney et al. 2021). The head and liver were carefully removed from embryos, and then the rest tissues were cut and trypsinized into single-cell suspensions. Rat embryonic fibroblasts were cultured in fibroblast growth medium consisting of DMEM/F-12 (Gibco), 15% Serum Replacement (Gibco), 1% Glutamax (Gibco), 1% non-essential amino acids (NEAA; Gibco), 100 units/ml penicillin and 100 \u0026mu;g/ml streptomycin, 0.1 mM 2-Mercaptoethanol (Amresco), 10 ng/ml FGF-2 (Paneco).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.3. Rat Neonatal Fibroblasts\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRat neonatal fibroblasts were isolated using two-day isolation protocol from Worthington-Biochem for rat neonatal cardiomyocytes and fibroblasts (http://www.worthingtonbiochem.com/NCIS/default.html). In brief, hearts were extracted from rat pups (Rattus norvegicus, Sprague Dawley breed), aged 1\u0026ndash;4 days, and immediately placed in Ca2+- and Mg2+-free Hank\u0026rsquo;s Balanced Solution (Gibco) on ice. Only the tissue of the ventricles was isolated\u0026mdash;with approximately 50\u0026ndash;60% of the initial heart mass being cut off\u0026mdash;which included the sinoatrial node, the atria, and the atrioventricular node. The isolated ventricles were minced into small pieces and then left at 4 \u0026deg;C overnight for trypsinization (Trypsin-EDTA 0.25%, Gibco). On the second day, the cells were placed into a collagenase solution (Collagenase type II, 2.25 \u0026mu;g/mL; Gibco) and stirred for 1 hour at 37 \u0026deg;C. Next, the suspension of the cells was placed into a T75 flask for 1 hour for pre-plating. The cells remaining in flask after pre-plating were considered fibroblasts and were used after purification from cardiomyocytes.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.4. Human Atrial Fibroblasts cell culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHuman atrial fibroblasts were isolated from atrial appendages provided by surgeons. The appendages were immediately placed in the cardioplegic solution cusdiol after excision and transported to the laboratory on ice. Fibroblast growth medium consist of DMEM (Paneco), 10% FBS (Capricorn), L-glutamin (Paneco), 100 units/ml penicillin and 100 \u0026mu;g/ml streptomycin (Paneco). Atrial appendage was cut into pieces and then dispersed on gelatin-coated (Paneco) 10 cm culture dish containing 2 ml fibroblast growth medium, and additional 8 ml medium was supplemented after 2 hours. Our extraction method is based on a well-known protocol (Emig et al. 2020).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.5. Transdifferentiation Protocols\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs a starting point, we chose the protocol for controlling the transdifferentiation of fibroblasts into cardiomyocytes\u0026nbsp;(Fu et al. 2015). All abbreviations and main steps correspond to this protocol. CRM (cardiac reprogramming medium) consisting of DMEM knockout medium (Gibco), 15% FBS (Capricorn), 5% Serum Replacement (Gibco), 0.5% N2 supplement (Paneco), 2% B27 supplement (Gibco), 1% Glutamax supplement (Gibco), 1% NEAA (Gibco), 0.1 \u0026mu;M -mercaptoethanol (Amresco), 100 U/ml penicillin and 100 mg/ml streptomycin and 50 mg/ml vitamin C. CMM (medium for maintenance of cells differentiated into cardiomyocytes) was prepared based on DMEM medium (Paneco) supplemented with 15% FBS (Capricorne), 50 mg/ml vitamin C, 3 \u0026mu;M CHIR99021 and 1 mg/ml insulin. Medium from neonatal cardiomyocytes: DMEM medium (Paneco) supplemented with 10% FBS (Capricorne), described above, after 2-3 days of culturing cardiomyocytes. CRM (P) refers to the combination of 50% CRM with 50% of medium from neonatal cardiomyocytes; аll additives\u0026rsquo; concentrations refer to total medium volume. CRM (C) refers to a cultural medium combining 50% of fresh CRM and 50% of 3 day CRM from the same well of the plate; аll additives concentrations refer to total medium volume.\u003c/p\u003e\n\u003cp\u003eFibroblasts transdifferentiation was started when fibroblasts reached 80% confluence in 24-well plates. On day 0 of each protocol culture medium was changed to CRM with reprogramming factors. Medium was changed each 2-3 days with addition of reprogramming factors. On the 16th or the 20th day CRM was replaced with CMM.\u003c/p\u003e\n\u003cp\u003eThe main components that were added to the environments were 2 small molecules: CHIR99021 stock solution 36 mM in DMSO and IWP2 stock solution 5 mM in DMSO. Also growth factors was added: Activin A stock solution 50 \u0026mu;g/ml in 0.1% BSA, BMP4 stock solution 10 \u0026mu;g/ml in 0.1% BSA, TGF-\u0026beta; stock solution 10 \u0026mu;g/ml in 0.1% BSA, FGF2 stock solution 10 \u0026mu;g/ml in 0.1% BSA, Y-27632 (ROCK inhibitor) stock solution 5 mM in PBS 1x.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.3. Immunofluorescent Staining\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCells were fixed for 10 min in 4% paraformaldehyde (Sigma-Aldrich), permeabilized for 10 min in 0.4% Triton-X100. Cells were further incubated for 1 hour in a blocking buffer (1% bovine serum albumin in phosphate-buffered saline, PBS), overnight at 4 \u0026deg;C with primary antibodies and for 1 h at room temperature with secondary antibodies. Cells were washed twice in PBS. Nuclei and F-actin were stained with DAPI (Vector Laboratories) and Alexa Fluor\u0026trade; 488 Phalloidin (Thermo Fisher Scientific). Samples were analyzed and processed on a Zeiss LSM 710 confocal microscope with Zen black 3.0 software (Carl Zeiss AG). Primary antibodies (working dilutions\u0026mdash;1:100): sarcomeric \u0026alpha;-actinin mouse (Abcam, Cambridge, UK, Cat# ab9465). Secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA, working dilution\u0026mdash;1:400): Alexa Fluor 594 goat anti-mouse IgG (H\u0026thorn;L) highly cross adsorbed (A11005).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.4. Perfusion Heart Experimental Protocol\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol for isolating the heart and cannulating the aorta began with anesthetizing a lab rat and sacrificing it with a spinal fracture (Glukhov et al. 2010). Next, the heart was carefully removed through the following steps: (1) an incision was made from the xiphoid process to the lateral ends of the edges of the ribs and (2) through the ribs along the left and right anterior axillary lines to provide a cot thoracotomy, and (3) the chest was deviated upward. These steps provided full access to the heart. Sections of the vena cava and aorta completed the procedure for extracting the heart, after which the organ was washed with Tyrode\u0026rsquo;s salt solution (Sigma-Aldrich Co.) with heparin and transferred to a Petri dish with the same solution for further manipulations.\u003c/p\u003e\n\u003cp\u003eThe process of attaching the heart to a cannula (a needle with a soft polymer sheath) through the aorta was performed with several turns of surgical thread. From the moment the heart was removed from the body until the start of perfusion, no more than 10 min passed through the cannula.\u003c/p\u003e\n\u003cp\u003eThe perfusion of the heart was carried out using a special installation, according to Langendorf. The setup consisted of two main parts: a perfusion circuit and a recording optical system based on a high-speed imaging setup (Olympus MVX-10 Macro-View fluorescent microscope (Olympus Co., Tokyo, Japan) equipped with a high-speed Andor iXon-3 Camera 897-U (Andor Technology Ltd., Belfast, UK)). The perfusion circuit was prepared for constant circulation (Masterflex L/S Digital Drive, 600 rpm; 115/230 VAC, Masterflex L/S Easy-Load\u0026reg; II Pump Head, SS Rotor; 2-Channel (Cole-Parmer Instrument Company, Vernon Hills, IL, USA)) of a fixed volume of fluid, maintaining the temperature of the perfusate throughout the system (Cole-Parmer Polystat Standard 6.5 L Heated Bath, 150 C, 115 V AC/60 Hz (Cole-Parmer Instrument Company, Vernon Hills, IL, USA)), including the heart chamber, and oxygenating the solution (Cole-Parmer Bubble trap, Water Jacketed Reservoir, Oxygenating Bubbler (Cole-Parmer Instrument Company, Vernon Hills, IL, USA)). The total volume of fluid circulating in the unit was optimized using a compact PDMS polymer heart chamber. The minimum volume that allowed the heart to be perfused with an oxygenated heated solution was reduced to 30 mL.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.5. Optical Mapping Protocols\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFor cell culture, optical mapping with Fluo4-AM (Lumiprobe, 1892-500ug) and Di-8-ANEPPS (Thermo Fisher Scientific, D3167) was performed in Tyrode\u0026rsquo;s salt solution (pH 7.25 to 7.4) according to the protocol previously described in (Kudryashova et al. 2017). The signal was recorded with a 128\u0026times;128 pixels resolution and a sampling frequency of 130 frames per second (Olympus MVX-10 Macro-View fluorescent microscope (Olympus Co., Tokyo, Japan) equipped with high-speed Andor iXon-3 EMCCD Camera (Andor Technology Ltd., Belfast, UK)).\u003c/p\u003e\n\u003cp\u003eIn general, for the whole heart the mapping protocol for the whole heart was similar to the protocol for cells (Laughner et al. 2012). Optical mapping with Fluo4-AM (Lumiprobe, 1892-500ug) and Di-8-ANEPPS (Thermo Fisher Scientific, D3167) was also performed in Tyrode\u0026rsquo;s salt solution (pH 7.25 to 7.4). The signal was recorded with a 128\u0026times;128 pixels resolution and a sampling frequency of 130 frames per second by the same setting. The only difference in optical mapping was that the heartbeats were removed by BDM (Sigma) adding to the solution in concentration 10 mM when the heart was stained for fluorescence Fluo4-AM (Lumiprobe, 1892-500ug) in concentration 2.8 ug/ml and Di-8-ANEPPS (Thermo Fisher Scientific, D3167) in concentration 15 ug/ml. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll experiments were performed at 37\u0026deg;C temperature during the observations. The experiments used electrode stimulation. The electrical stimulation duration was set at 20 ms, with the amplitude adjusted within the range of 5 to 8 V depending on the tissue culture or the excitation threshold of the heart. The stimulation interval varied from 200 to 1000 ms. The stimulus was set using a generator (2MHz USB PC Function Generator, PCGU100 (Velleman, Gavere, Belgium)). Platinum electrodes were used in the work.\u003c/p\u003e\n\u003cp\u003eData processing was fulfilled using the ImageJ program and the associated plugins (http://rsbweb.nih.gov/ij/ (accessed on 5 May 2023)). ImageJ plugin (time lapse color-coder) was used to build pseudo-3D images and activation maps. Principal analysis was performed in Wolfram Mathematica 12.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.6. Patch-сlamp\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eVoltage-gated ion channel currents in transdifferentiated cardiomyocytes were recorded using the patch-clamp electrophysiological method in the \u0026ldquo;perforated whole-cell\u0026rdquo; configuration. A description of how to install a patch clamp has been provided in another work (Abrasheva et al. 2024). The extracellular solution for recording sodium (INa), calcium (ICa,\u003cem\u003eL\u003c/em\u003e), potassium (IKv) contained 15 мМ HEPES, 150 мМ NaCl, 5,4 мM КCl, 1 мМ MgCl2, 1,8 мМ CaCl2, 15 мМ D-glucose, pH = 7.4 (adjusted with NaOH). The pipette was filled with an intracellular solution containing 150 мМ KCl, 5 мМ NaCl, 2 мМ CaCl2, 5 мМ MgATP, 10 mM HEPES, 5 mM EGTA, pH = 7.2 (adjusted with KOH). Currents from voltage-gated fast sodium channels (INa) as well as combined INa/Ca were obtained via the ramp stimulation protocol, which involved a linear increase in potential from \u0026ndash;120 to +50 mV over a duration of 250 ms. Using a step-pulse stimulation protocol, current amplitudes of voltage-gated potassium channels (IKv) were quantified during depolarizing voltage steps (from -30 to 60 mV, each step presented for 2.5 s). The step protocol, containing pre-step (-30 mV) and main step (0 mV for 300 ms), was used to observe L-type calcium currents (ICa,\u003cem\u003eL\u003c/em\u003e). All experiments were conducted at room temperature (22\u0026ndash;24\u0026deg;C). Data analysis and processing were performed using Clampfit 10.2 (Molecular Devices, USA) and OriginPro 8.1 (OriginLab Corp., USA) software.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.8. Infarction Model\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMale Wistar rats weighing 250-350 g were anesthetized with injection combining \u0026ldquo;Xyla\u0026rdquo; xylazine hydrochloride 5 ng per 1 g of animal weight (De Adelaar, Netherlands) and \u0026ldquo;Zoletil\u0026rdquo; 4 ng tiletamine hydrochloride and 4 ng zolazepam hydrochloride per 1 g of animal weight (Vibrac, France). Trachea was intubated with intravenous cannula 18 G and connected to the Ambu bag (1 squeeze per second) and the RWD R550 Small Animal Anesthesia Machine combining oxygen 0.5-0.7 liter per minute with isoflurane via tubes. Left anterior descending coronary artery permanent occlusion was performed with monofilament prolene 6/0 (Ahn et al. 2004). After the end of the surgery rats were peritoneally injected with meloxicam 2 \u0026mu;g per 1 g of animal weight (Belkarolin, Belarus). The animal was monitored for several hours until regaining consciousness.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.9. Injections\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eInjections were performed intramyocardially or intravenously via insulin syringe 30G with total volume 50-100 \u0026mu;l. Intramyocardial injections were performed only on control rats under general isoflurane anesthesia, no more than once with cocktail: CHIR99021 \u0026mdash; 10 \u0026mu;M, Activin A \u0026mdash; 9 ng/ml, \u0026nbsp;BMP 4 \u0026mdash; 5 ng/ml, IWP2 \u0026mdash; 2 \u0026mu;M, bFGF \u0026mdash; 8 ng/ml, TGF\u0026beta; \u0026mdash; 1 ng/ml per 20 ml of circulating blood volume \u0026mdash; or with sterile PBS (Gibco). Intravenous injections into the tail vein for IM model animals were started on the 14th day after the surgery. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4.10. Data Processing and Statistical Analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePreliminary processing of optical mapping data was performed in ImageJ (v1.54p). To construct activation maps, background subtraction was applied to the videos, followed by the Kalman filtering algorithm for noise removal and Gaussian blurring. The activation map generation algorithm was implemented in Python (v3.11.13) using the following libraries: Matplotlib (v3.10.0), NumPy (v2.0.2), SciPy (v1.15.3), Pandas (v2.2.2). The algorithm is based on increasing the luminescence intensity of each individual pixel by a certain percentage relative to its average intensity. This approach enhances the method\u0026apos;s sensitivity compared to the standard technique of applying a uniform absolute threshold to all pixels.\u003c/p\u003e\n\u003cp\u003eFor analyzing calcium dynamics, action potential dynamics and NADH photobleaching, plots were generated with preliminary processing in ImageJ (using the Kalman filtering algorithm for noise reduction), followed by data processing in Microsoft Excel and further plot generation and statistical analysis in Python (v3.11.13) with the same libraries. Statistical analysis was performed using the Anderson\u0026ndash;Darling test with a 5% significance level.\u003c/p\u003e\n\u003cp\u003eFor the analysis of \u0026alpha;-actinin expression percentage in cells, photographs of cells after immunofluorescent staining were taken. Based on the expression data in the photographs, the percentage of area occupied by cells expressing alpha actinin was calculated in relation to the area occupied by all cells (f-actin) by ImageJ standard functions.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eElectromechanical wave propagation is essential for cardiac function; its disruption by fibrotic scars after myocardial infarction causes arrhythmias. This study develops minimized chemical cocktails for direct fibroblast-to-cardiomyocyte transdifferentiation (TdCM), bypassing pluripotency to treat fibrosis. Optimized 4-component protocols generated functional induced cardiomyocytes (iCMs) from rat and human fibroblasts, with 56-83% expressing \u0026alpha;-actinin. Electrophysiological profiling confirmed maturation: human iCMs exhibited action potentials (\u0026minus;80 mV resting potential) and robust sodium currents (\u0026sim;4000 pA). Crucially, partial reprogramming sufficed to establish conduction pathways exceeding the percolation threshold (20-30%), enabling electrical propagation. In vivo testing showed no hematological toxicity, while optical mapping revealed 84% conductive area in treated infarcts (vs. 71% controls). Our findings demonstrate that minimized cocktails restore scar conductivity without complete cellular conversion, advancing translatable regenerative strategies.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eThe following abbreviations are used in this manuscript:\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"524\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCVDs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eCardiovascular Diseases\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eMyocardial Infarction\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eIND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eIschemic Heart Disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eCFs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eCardiac Fibroblasts\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eECM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eExtracellular Matrix\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eiPSCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eInduced Pluripotent Stem Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eMSCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eMesenchymal Stem Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eiCMs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eInduced Cardiomyocyte-like Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eREF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eRat Embryonic Fibroblasts\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eRNF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eRat Neonatal Fibroblasts\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eHAF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eHuman Atrial Fibroblasts\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eTdCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eFibroblast-to-cardiomyocyte Transdifferentiation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eiPSC-CMs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 461px;\"\u003e\n \u003cp\u003eiPSC-derived cardiomyocyte\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Materials:\u0026nbsp;\u003c/strong\u003eNo\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, K.A. and V.T.; methodology, \u0026nbsp;M.S. and V.T.; software, V.D. and M.S.; validation, K.A. and V.T; formal analysis, E.T., S.Rob., V.D. and V.T.; investigation, E.T., S.Rob., S.K., V.D., A.D., S.Rom., A.M. and M.S.; resources, D.Z., M.P., A.R., K.A. and V.T.; data curation, S.F., K.A. and V.T.; writing\u0026mdash;original draft preparation, E.T., S.Rob., S.K. and V.D.; writing\u0026mdash;review and editing, A.R., K.A. and V.T.; visualization, E.T., S.Rob., V.D. and M.S..; supervision, K.A. and V.T..; project administration, V.T.; funding acquisition, A.R. and V.T. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The study was carried out within the framework of project No. 25-65-00037 dated 22.05.2025 \u0026nbsp;with the Russian Science Foundation (RSF)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe study was approved by the Ethics Committee of M.F. Vladimirsky Moscow Regional Clinical Research Institute (protocol №7 from 18.04.2024) and by the Moscow Institute of Physics and Technology Life Science Center Provisional Animal Care and Research Procedures Committee (Protocol No. A2-2012-09-02).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Raw data (optical mapping, patch clamp data and confocal microscopy) could be found at Data Repository: https://drive.google.com/drive/folders/1jXv3zR2fuMYOcJ_LV8d2YgG2uK89IJPJ\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors thank Anastasia Dubrovskaya for accompanying the experiments. Also, we would like to thank Applied Genetics Resource Facility of MIPT for providing the necessary equipment for the experiments. We would like to express special gratitude to the administration of the MIPT, M.F. Vladimirsky Moscow Regional Clinical Research Institute, E.Meshalkin National Medical Research Center of the Ministry of Health of the Russian Federation. The study was carried out within the framework of project No. 25-65-00037 of 22.05.2025 \u0026nbsp;with the Russian Science Foundation (RSF)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbrasheva, V.O.; Kovalenko, S.G.; Slotvitsky, M.; Romanova, S.А.; Aitova, A.A.; Frolova, S.; Tsvelaya, V.; Syunyaev, R.A. Human sodium current voltage-dependence at physiological temperature measured by coupling a patch-clamp experiment to a mathematical model. J. 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Regener. 2021, 41, 20. https://doi.org/10.1186/s41232-021-00168-\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"transdifferentiation, fibrosis, arrhythmias, cardiomyocytes, cardiac tissue","lastPublishedDoi":"10.21203/rs.3.rs-7186257/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7186257/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFibrotic scars post-myocardial infarction disrupt cardiac conduction, causing arrhythmias. We developed a minimized 4-component cocktail (CHIR99021/BMP4/Activin A/IWP2) for efficient fibroblast-to-cardiomyocyte transdifferentiation. The use of the developed four-component protocol allows achieving significant electromechanical activity and pronounced expression of cardiomyocyte markers, as evidenced by the 56\u0026ndash;83% cells expressing α-actinin. The results show that partial transdifferentiation of fibroblast cells into cardiac ones is sufficient to restore cardiac tissue conductivity, while the efficiency exceeds the critical percolation threshold. Systemic delivery of components is safe, but requires further optimization, which will open up opportunities for localized delivery through smart substrates and combinations with cell therapy. Minimization of the transdifferentiation cocktail is not a compromise, but a strategic advantage that provides an optimal balance between functional efficiency and clinical applicability, including safety, delivery, and manufacturing.\u003c/p\u003e","manuscriptTitle":"Refining fibroblast-to-cardiomyocyte transdifferentiation protocols to explore emergent self- organization in cardiac cultures","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-30 14:13:30","doi":"10.21203/rs.3.rs-7186257/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"71049d12-3332-44dd-b0b7-0c667001a340","owner":[],"postedDate":"July 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-23T20:23:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-30 14:13:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7186257","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7186257","identity":"rs-7186257","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}