Introduction Atrial fibrillation is a highly prevalent cardiac arrhythmia worldwide. Pulsed-Field Ablation (PFA) is a catheter-based ablation strategy using short, high-voltage electric pulses to induce irreversible electroporation and cell death, providing an efficient strategy for restoration and maintenance of sinus rhythm. Although some studies used ECG-timed ablation protocols, the role of the cardiac cycle-related mechanical state of the heart on PFA efficiency is unclear. Objective To assess whether cardiac mechanical state affects the geometry of lesions induced by PFA. Method Langendorff-perfused beating wild-type mouse hearts were stopped in relaxed state or put into contracture by perfusion of solutions containing the mechanical uncoupler BDM (30mM), or LiCl (140mM replacing NaCl) and caffeine (10mM), respectively. Then, 250V biphasic voltages were applied using a PFA generator and a monopolar linear-tip catheter (Stockert GmbH). The ablation protocol was designed to generate not fully transmural lesions, to not be limited in lesion depth measure. Every heart received two epicardial ablations per ventricle, followed by 30min of recovery perfused by physiological solution. Hearts were stained by perfusion of 2% TTC (2,3,5-triphenyltetrazolium chloride) for 15min, before ablated regions were excised and cryo-sectioned at 30μm thickness, perpendicular to the epicardial surface and parallel to the lesion's long axis. Lesion morphology was assessed. Results PFA delivery on perfused hearts generated ellipsoidal lesions, clearly identifiable after TTC staining. In both ventricles, the maximal lesion area of transmural tissue sections was significantly larger when PFA was applied during contracture, compared to relaxed state: 1.4±0.2mm2 vs 0.9±0.1mm2 for the left and 1.3±0.4mm2 vs 0.6±0.1mm2 for the right ventricle (P<0.001 for both). In left ventricle, this was linked to significantly larger long and short axes, whereas these axes were not significantly higher in right ventricle. In both ventricles, no significant differences were observed in lesion depth. Finally, PFA delivery during contracture led to lesion volumes that were 116±18% and 117±39% greater for left and right ventricle (P<0.01 for both) compared to relaxed state. Conclusion PFA generates more than two times larger lesions when delivered during contracture, compared to relaxed hearts. Further studies will assess whether this is seen in beating hearts (physiological contractions), and in BDM-arrested hearts during passive stretch, to explore myocardial tissue tension as a possible amplifier of PFA-induced electroporation.

Introduction Living cardiac slices as models for testing physiological and pharmacological interventions are recognised to offer unique advantages over whole perfused organs or single isolated cardiomyocytes for several reasons. To name just a few, slices allow multiple experimental samples from biopsies; they can be maintained under culture conditions for several days; they consist of mature cells; and multicellularity and intercellular contacts as well as ECM are preserved. Thus, they provide a promising approach for short- and long-term drug (up to several weeks) screening. Objective Our aim is to assess the validity the model by testing the electrophysiological effects of well-known antiarrhythmic drugs: flecainide (class 1c) and dofetilide (class 3) in human slices. Method Cardiac tissue excised from patients was sliced into 400 μm thick sections, mounted in biomimetic chambers (MyoDish, InVitroSys) allowing us to record contraction forces, preloaded and paced electrically at a frequency of 1 Hz. Action potentials (AP) were recorded at 37°C using sharp intracellular microelectrodes pulled from borosilicate glass capillaries (tip resistance 15–25 MΩ when filled with 3 M KCl solution). Observations before and during drug interventions are all paired. All values are reported as mean±standard error. Results Left ventricular slices (N=8, n=27) and right atrial slices (N=7, n=23) had respectively resting membrane potentials (RMP) of −82.5±0.9 and −72.1±1.4mV, AP amplitudes (APA) of 113±1.3 and 90.2±1.9mV, maximum upstroke velocities (dV/dtmax) of 127.6±7.6 and 190.5±13.8V/s, AP durations at 90% of repolarization (APD90) of 452.8±14.3 and 327.4±23.4ms, contraction durations 90% (CD90) of 551.3±18.4 and 231.1±16.6ms. Flecainide significantly decreased dV/dtmax and APA in both left ventricular and right atrial slices, whereas dofetilide significantly increased APD90 in both tissues. Regarding contractions, both drugs induced a significant negative inotropic effect and an increase of CD90 only in left ventricular slices. Conclusion The living cardiac slice model produced consistent results with published results on trabeculae and whole mount tissues concerning AP and contraction parameters. Classic antiarrhythmic drugs produced the expected effects. The results of this study suggest that the slice model is suitable for assessing drug effects on key electrophysiological parameters, making this model promising for assessing novel antiarrhythmic compounds

Abstract Optogenetic defibrillation uses light-gated ion channels to terminate cardiac arrhythmias through targeted illumination. Previous studies assessed the feasibility of using either cation (e.g. ChR2) or anion (e.g. GtACR1) non-selective channels, both of which depolarise resting cardiomyocytes upon photoactivation. In contrast, recently identified light-gated K + -channels (e.g. WiChR) suppress cardiomyocyte activity while maintaining the membrane potential near its resting state. Here, we use biophysically detailed simulations to compare the defibrillation potential of ChR2, GtACR1, and WiChR. Single-cell simulations show that activation of ChR2 and GtACR1 markedly increase diastolic intracellular Ca 2+ concentration (by 42.6% and 52.6%, respectively), whereas WiChR induces only minimal changes (4.0% increase), suggesting a lower pro-arrhythmogenic risk. WiChR activation, however, slightly increases intracellular Na + levels (by 15.1% compared to 0.1% and 3.4% for ChR2 and GtACR), consistent with the residual Na + permeability of all currently available K + -selective channelrhodopsins. Simulations of human ventricles and atria demonstrate that GtACR1 most effectively terminates re-entrant arrhythmias at low light intensities, while WiChR achieves comparable efficacy at light levels ≥5 mW/mm 2 . Complementary tissue-scale simulations reveal that defibrillation is either based on depolarisation within the excitable gap, followed by fast Na + channel inactivation (depolarising variants ChR2 and GtACR1), or based on a reduction in membrane resistance supporting arrhythmia termination at sufficiently high light levels (large-conductance ion channels GtACR1 and WiChR). Overall, our findings identify channelrhodopsin ion selectivity as a key determinant of both arrhythmia termination success and mechanisms underlying defibrillation. Key points summary We use computational simulations to compare non-selective cation (ChR2), anion (GtACR1), and K + -selective channelrhodopsins (WiChR) for optogenetic termination of re-entrant arrhythmia. Single-cardiomyocyte simulations suggest that ChR2 and GtACR1 activation can cause progressive accumulation of intracellular Ca 2+ , which is minimised when using WiChR. Simulations of human left ventricles and atria indicate that GtACR1 is most effective in terminating re-entrant arrhythmia at low light intensities, while WiChR becomes similarly effective at higher intensities. Tissue-scale simulations indicate distinct defibrillation mechanisms: Excitable gap extinction by de-novo action potential initiation followed by inactivation of fast Na + channels for depolarising channelrhodopsins (ChR2, GtACR1), and reduction in membrane resistance for the large-conductance channels (GtACR1, WiChR), effectively clamping the membrane potential at each channel’s reversal potential at high light levels.

Abstract Model systems that mimic human cardiac structure and function are essential for the development of novel diagnostics and effective treatments for cardiovascular diseases. While non‐human vertebrate models, from zebrafish to pig, remain vital to cardiovascular research, the translatability of findings to human patients is often limited. Therefore, animal experiments should be supplemented with human model systems, including human induced pluripotent stem cell‐derived cells, 3D engineered constructs, and last but not least, native tissue preparations and isolated primary cardiomyocytes. However, while human myocardium remains the gold standard, human heart tissue – and particularly tissue from control hearts–remains scarce, and its use in research is generally restricted to settings where tissue has been excised from diseased or failing hearts. While it is in principle possible to use tissue from rejected non‐failing donor hearts that cannot be transplanted, legal hurdles (e.g., in Germany) can restrict the use of non‐transplanted donor organs in research. Given the challenges associated with accessing and using human tissue in biomedical research, an integrated strategy towards combining non‐human vertebrate models, in silico models, and human tissue‐derived models is recommended, enhancing the chances of successful research and development, and helping bridge the gap between preclinical and clinical research.

The transverse-axial tubular system (TATS) enables close structural and functional coupling between plasma membrane and sarcoplasmic reticulum of cardiomyocytes. It supports fast and efficient Ca2+-induced Ca2+ release upon cell depolarisation, crucial for excitation-contraction coupling in the heart. Due to the small diameter and tortuosity of individual tubules, the TATS forms a domain of restricted diffusive transport. It has previously been suggested that, as a consequence of an uneven distribution of Ca2+ influx and efflux pathways in TATS compared to outer surface plasma membrane domains of cardiomyocytes, cyclic electrical activity may lead to a gradual depletion of Ca2+ in the TATS. Here, we show experimentally that in mechanically uncoupled rabbit ventricular cardiomyocytes, electrical stimulation does indeed lead to an L-type Ca2+ channel-dependent gradual depletion of Ca2+ inside TATS, an effect that scales with pacing frequency. Ca2+ depletion was absent in freely contracting cardiomyocytes, presumably as a result of cyclic TATS deformation during cell shortening. This squeezes transverse TATS tubules and adds an advective contribution to, and thereby accelerates the, intra-TATS content exchange with bulk extracellular fluid. Our results reveal a novel mechanism of cardiac mechano-dependent auto-regulation, where the increased propensity for development of intra-TATS Ca2+ gradients at high electrical stimulation rates is mitigated by the coinciding mechanically induced TATS deformation, twice on each cycle in the heart (during diastolic stretch and systolic shortening), which accelerates luminal content exchange. Our study provides first insight into a novel facet of cardiac mechano-biology, whose auto-regulatory benefit may be reduced by TATS remodelling in disease. ### Competing Interest Statement The authors have declared no competing interest. German Heart Research Foundation, https://ror.org/02yjb3a91

Light-gated ion channels (channelrhodopsins; ChRs) can be used to precisely control the electrical activity of genetically targeted cell populations with light. Although nonselective cation ChRs are widely used to elicit action potentials (APs) in excitable cells, the recently identified class of K+-selective ChRs (KCRs) are promising tools for optogenetic AP inhibition. One of the most K+-selective KCRs identified to date is Wobblia lunata inhibitory ChR (WiChR), which—by combining high light sensitivity and prolonged channel opening with efficient expression in neurons and cardiomyocytes—enables reliable suppression of AP firing in response to blue light pulses. However, a detailed understanding of WiChR photoactivation and its conducting states has so far been missing. Here, we introduce the first model of the WiChR photocycle, designed to quantitatively reproduce and predict its photocurrents, as well as resulting changes in membrane voltage. We combined electrophysiological recordings with simultaneous imaging of intracellular K+ concentration under varied light-stimulation protocols that serve as a basis for computational modeling of putative photocycle transitions. We show that WiChR photocurrents can be fully described by a simple unbranched photocycle model, composed of two closed and two open states of near-constant high K+ selectivity, and are further shaped by changes in intracellular K+ concentration during extended illumination. These changes are promoted by the large photocurrent amplitudes observed in WiChR-expressing cells and differ substantially among individual cells and across cell types, underlining the importance of the optogenetically targeted host system. Our model presents a framework for assessing and predicting WiChR photoresponses and will be useful for guiding the design of optimized stimulation protocols for future application of WiChR and other KCRs.

The electrophysiological relevance of interstitial nonmyocytes for cardiac electrophysiology arises from their abundant direct and indirect interactions with cardiac myocytes. This review defines the interstitium, explores biophysical and biochemical mechanisms of interactions between interstitial components and cardiac myocytes, illustrates consequences of these interactions for heart rhythm, and identifies targets for further research in this area.

BACKGROUND:Nonmyocytes may contribute to regional adaptive changes during persistent atrial fibrillation (PsAF), favoring its perpetuation. We aimed to investigate the differential features of fibroblast and macrophage populations within individual-specific atrial regions associated with PsAF maintenance. METHODS:The study was conducted in 2 pig models of PsAF with and without infarct-related substrate (N=27 and N=27, respectively) and further validated in humans with PsAF (N=20). Sham-operated pigs (N=9), healthy animals (N=4), and patients in sinus rhythm (N=7) were used as comparative controls. In pigs, in vivo high-density instantaneous frequency modulation maps were used to identify atrial regions associated with PsAF maintenance (drivers). Regional cellular composition and phenotypic states of fibroblast and myeloid lineages were determined using flow cytometry, single-cell RNA sequencing, immunohistochemistry, and proteomic analyses. The functional relevance of driver regions was further studied in patients with symptomatic PsAF undergoing ablation. Flow cytometry and single-cell RNA sequencing analyses were performed in tissue samples of the left atrial appendage in a complementary cohort of patients with PsAF undergoing thoracoscopic-guided ablation. RESULTS:PsAF terminated acutely in 12 of 14 pigs undergoing mapping and ablation of driver regions. In humans, driver ablation was associated with 90% AF-freedom (on/off antiarrhythmic drugs) after 2 years of follow-up. Samples from nonablated pigs revealed a phenotypic shift towards ACTA2 (actin alpha 2)-fibroblasts and PTX3 (pentraxin 3)-fibroblasts during PsAF. Although ACTA2-fibroblasts were highly preserved in human samples, paired comparisons in pig samples showed that PTX3-fibroblasts were enriched only in driver regions. PsAF also showed changes in myeloid cells towards inflammatory profiles. However, regional analysis revealed that, in both humans and pigs with PsAF, driver regions were enriched in cardiac resident macrophages with transcriptomic and proteomic profiles favoring cardiomyocyte homeostasis and cell survival. CONCLUSIONS:PsAF shows differential regional changes in fibroblast and myeloid populations with distinctive gene signatures in areas that drive the overall arrhythmia.

Cardiac excitation–contraction coupling relies on a pancellular network of regular cardiomyocyte surface membrane invaginations, termed the transverse-axial tubular system (TATS). The TATS is ubiquitously present in adult mammalian cardiomyocytes, enabling efficient structural and functional coupling of sarcolemma and intracellular Ca2+ stores. However, TATS ultrastructural characteristics across species, and their relation to cardiomyocyte morphology and physiological parameters such as heart rate, remain largely unexplored. Here, we quantified TATS and cardiomyocyte features in a large confocal microscopy dataset (78 3D volumes) obtained from tissue slices across eight species (mouse, rat, rabbit, pig, horse, elephant, whale, and human). We developed and applied a semi-automated image analysis pipeline to quantify mean cytosolic distances to the nearest TATS (Cyto-TATSmin, a measure inversely related to TATS density), transverse-to-axial tubule ratio, and cardiomyocyte dimensions. Cyto-TATSmin and transverse tubule fraction differed substantially between species, with the lowest Cyto-TATSmin in mouse and highest in human. Within species, except mouse, rat, and horse, Cyto-TATSmin was positively correlated with cardiomyocyte cross-sectional area. Across all species, Cyto-TATSmin correlated with species’ life span and body weight, and was inversely correlated with average resting heart rate. Our findings reveal structural scaling principles within species differences in cardiac cellular ultrastructure and provide a resource for studying TATS organisation in health and disease. As TATS remodelling is a common hallmark of cardiac pathology, awareness of species-differences is needed to guide the design and interpretation of translational research. ### Competing Interest Statement The authors have declared no competing interest. German Research Foundation

BACKGROUND:The heart's mechanical state feeds back to its electrical activity, potentially contributing to arrhythmias. Mechano-arrhythmogenesis has been mechanistically explained during electrical diastole, when cardiomyocytes are at their resting membrane potential. During electrical systole, cardiomyocytes are refractory right after the onset of depolarization, while during repolarization in physiological conditions, they seem to be protected from systolic mechano-arrhythmogenesis by near-simultaneous restoration of resting membrane potential and cytosolic calcium concentration ([Ca2+]i): repolarization-relaxation coupling (RRC). Yet, late-systolic mechano-arrhythmogenesis has been reported in ischemic myocardium, with unclear underlying mechanisms. We hypothesize that ischemia-induced alteration of RRC gives rise to a vulnerable period for mechano-arrhythmogenesis. METHODS:Acute left ventricular regional ischemia was induced by coronary artery ligation in Langendorff-perfused rabbit hearts, with mechanical load controlled by an intraventricular balloon. Mechanical activity was assessed by echocardiography and arrhythmia incidence by ECG. Single left ventricular cardiomyocytes were exposed to simulated ischemia or pinacidil (ATP-sensitive potassium channel opener). Stretch was applied in diastole or late systole using carbon fibers. Stretch characteristics and arrhythmia incidence were assessed by sarcomere length measurement. In both models, RRC was assessed by simultaneous voltage-[Ca2+]i fluorescence imaging and mechano-arrhythmogenesis mechanisms were pharmacologically tested. RESULTS:In whole hearts, acute regional ischemia leads to systolic stretch and disturbed RRC at the ischemic border. These electro-mechanical changes were associated with waves of arrhythmias, which could be reduced by mechanical unloading, electro-mechanical uncoupling, or buffering of [Ca2+]i. In left ventricular cardiomyocytes, physiological RRC is associated with a low incidence of systolic mechano-arrhythmogenesis, while a vulnerable period emerged by prolonged RRC during ischemia. The increase in systolic mechano-arrhythmogenesis was reduced by restoring RRC, chelating [Ca2+]i, blocking mechano-sensitive TRPA1 (transient receptor potential ankyrin 1) channels, or buffering reactive oxygen species levels. CONCLUSIONS:Prolonged RRC allows for late-systolic mechano-arrhythmogenesis in acute ischemia, involving contributions of elevated [Ca2+]i, TRPA1 activity, and reactive oxygen species, which represent potential antiarrhythmic targets.

Reticulated platelets are newly formed, RNA-rich platelets with heightened reactivity. Although elevated levels are observed after myocardial ischemia/reperfusion injury, their functional contributions to postischemic pathology remains poorly defined. We aimed to determine whether reticulated platelets actively contribute to inflammation and repair following myocardial ischemia and reperfusion, rather than serving solely as biomarkers of platelet turnover. We generated Pf4-Cre:RiboTag mice, in which hemagglutinin-tagged ribosomal proteins are selectively expressed in megakaryocytes and platelets. Using hemagglutinin-based flow cytometry, we identified reticulated platelets without relying on nucleic acid dyes. Surface marker expression and agonist responsiveness were evaluated ex vivo. Bulk RNA sequencing was performed on sorted reticulated and non-reticulated platelets 48 hours after ischemia/reperfusion injury. Hemagglutinin-based detection revealed a time-dependent increase in circulating reticulated platelets after myocardial ischemia/reperfusion, confirmed by conventional dye-based methods. These platelets exhibited higher baseline expression of glycoprotein Ibα and greater agonist-induced activation of glycoprotein IIb/IIIa and P-selectin. Transcriptomic profiling demonstrated enrichment of genes associated with platelet activation, cytoskeletal reorganization, and wound healing. Ligand-receptor analysis suggested interactions between reticulated platelets and cardiac endothelial cells, fibroblasts, and macrophages. In conclusion, reticulated platelets constitute a transcriptionally distinct, hyperreactive platelet subset that may modulate post-ischemia/reperfusion inflammation and tissue remodeling. This genetic model provides a platform for mechanistic studies and may inform therapeutic strategies targeting platelet-mediated responses in cardiovascular disease.

BACKGROUND:Mechanical stretch of the myocardium is proarrhythmic and alters cellular Ca2+ handling, potentially involving cation nonselective mechano-sensitive ion channels. This study aimed to assess the presence and mechanisms of stretch-induced increase in Ca2+-spark rate (SiS) in isolated atrial cardiomyocytes. METHODS:Freshly isolated rabbit, pig, and human left atrial cardiomyocytes were stretched axially using glass microrods. Free cytosolic Ca2+ concentration was monitored using confocal microscopy at resting sarcomere length (≈1.79 μm) and during severe (≈12%) increase in sarcomere length. RESULTS:Diastolic stretch provoked SiS, which was prevented by disrupting microtubules with colchicine, but unaffected by inhibition of NADPH oxidase 2 or scavenging of reactive oxygen species. SiS was absent in Na+- and Ca2+-free external solution, suggesting that it requires transsarcolemmal influx of Na+ or Ca2+. Activation of Piezo1 increased baseline spark rate, which was further increased by stretch. TRPA1 (transient receptor potential ankyrin 1) activation also increased baseline spark rate, with no further change upon stretch. SiS was not detectable in the presence of streptomycin (a blocker of nonselective mechano-sensitive ion channels), and HC-030031 and A-967079 (selective blockers of TRPA1), even when Piezo1 was activated. SiS was also observed in pig and human atrial cardiomyocytes. CONCLUSIONS:In atrial cardiomyocytes, diastolic stretch enhances Ca2+-spark rate through a mechanism that requires microtubular integrity and TRPA1 but that is independent of redox signaling. TRPA1 emerges as a key regulator of stretch-induced Ca2+ handling in atrial cells, with potential implications for arrhythmogenesis.

Schwann cells (SC) are crucial for physiological impulse conduction in peripheral nerves. They produce myelin, provide axonal metabolic support, and contribute to reparatory processes after nerve injury. During aging, peripheral nerves acquire myelin structural anomalies and are characterized by a lower fraction of SC and a higher fraction of senescent cells. All these changes correlate with impaired electrical conduction and consequently altered function of target tissues including skeletal muscle weakness and cardiac arrhythmia. To characterize and compare cardiac and sciatic nerve SC, as well as to explore age-related differences in SC abundance and their properties, we analyzed two TdTomato reporter mouse strains to isolate Sox10 or Plp1 expressing SC. We performed RNA-sequencing on sorted TdTomato-positive cells from the heart and sciatic nerve and validated transcriptomic findings at the protein level using immunofluorescence. Our data reveal a pro-angiogenic profile in cardiac SC when compared to sciatic SC. In addition, higher levels of neural-death associated genes and lower gene and protein expression levels of the fatty acid co-transporter Fabp4/FABP4 are detected in cardiac SC from old compared to young mice, suggesting an aging-related impairment of fatty acid transport. Finally, sciatic SC activate collagen remodeling and increased pro-inflammatory signaling including TNFα. Thus, cardiac and musculoskeletal SC have different expression profiles, and undergo different changes during aging, which may contribute to impaired nerve function in both organ systems.

Cardiac non-myocytes are increasingly recognized as active contributors to cardiac electrophysiology. Fibroblasts have been shown to form connexin-based electrotonic connections with cardiomyocytes (CM) in situ, and more recently, macrophages have also been found to engage in electrotonic interactions with CM. This growing evidence requires a conceptual reassessment of cardiac electrophysiology. However, studying heterocellular coupling in situ remains challenging. These experimental uncertainties define a scope for computational modelling and simulation. In this review, we provide an overview of computational models of heterocellular coupling across multiple spatial scales, from single-cell interactions to whole-organ dynamics. We start by presenting the rationale for studying cardiac heterocellular coupling that is based on clinical and experimental evidence, followed by an overview of computational modelling studies, and conclude with an outlook to future research directions.

Cardiac fibrosis is a key factor in electrical conduction disturbances, yet its specific impact on conduction remains unclear, hindering predictive insight of cardiac electrophysiology and arrhythmogenesis. Among the different cardiac disorders, arrhythmogenic cardiomyopathy (ACM) is known to be associated with massive fibrotic remodelling of the myocardium, and it accounts for most cases of stress-related arrhythmic sudden death. To explore ACM further, we employed a Desmoglein-2-mutant mouse model and developed a correlative imaging approach to integrate macro-scale cardiac electrophysiology with 3D micro-scale reconstructions of the ventricles, to characterise the dynamics of conduction wavefronts and relate them to the underlying structural substrate. Our findings confirm that this ACM model shows localised replacement of cardiomyocytes with collagen and non-myocytes, contributing to electrical dysfunction. Moreover, we observed that conduction through fibrotic tissue areas shows a frequency-dependent behaviour, where conduction fails at high stimulation frequencies, promoting re-entrant arrhythmias, even in regions that were electrophysiologically inconspicuous at lower stimulation rates. Using a computational model, informed by high-resolution structural data, we found that frequency-dependent conduction through fibrotic tissue cannot be explained solely by collagen deposition or cardiomyocyte re-organisation. Indeed, fibrotic areas feature electrophysiological remodelling which acts as a low-pass filter for conduction, which can be quantitatively explained by electrotonic coupling of cardiomyocytes with non-myocytes. Collectively, our study provides a novel structure-function mapping pipeline and describes a previously unrecognised pro-arrhythmogenic mechanism in ACM, underscoring the need for dynamic assessment of functional conduction block in fibrotic myocardium using multiple diagnostic pacing protocols.

Segmentation of cardiomyocytes in microscopic 3D volumes is key to our understanding of cardiac (patho-)physiology; however, it poses substantial experimental and analytical challenges. Therefore, researchers often resort to inferring 3D information from 2D segmentations, which can lead to biased and incorrect conclusions. Deep learning-based methods are showing promise with respect to robustly segmenting objects in volumes acquired using various imaging modalities; yet, they have not been applied to high-resolution 3D cardiomyocyte segmentations, and suitable open-source tools and datasets are lacking. Here, we present a deep learning-enabled toolkit for segmentation of individual cardiomyocytes in 3D confocal microscopy volumes. We include a dataset of 73 volumes with expert annotations, covering seven species, including mouse, human, and elephant, and containing samples generated under different experimental conditions, such as post-myocardial infarction and ex vivo slice cultures. The toolkit additionally contains an image restoration workflow to address imaging-related artefacts, such as spatially varying blur. Our automatic cardiomyocyte segmentation workflow achieved an adapted Rand error of 0.063 ± 0.034 (∼94% voxel-pair agreement) on the test set. Our semi-automatic workflow reached a throughput of 3 cells min-1 on a challenging, previously unseen dataset. The toolkit and data are open-source and accessible through a dedicated graphical user interface. In summary, we provide an accessible toolkit enabling researchers to extract quantitative data on cardiomyocyte microstructure from 3D confocal image stacks of cardiac tissue. Given the size and diversity of our dataset, we expect our methods to perform well across species and experimental conditions, facilitating high-quality 3D reconstructions of large numbers of individual cardiomyocytes. KEY POINTS: 3D cardiomyocyte microstructure is a key determinant of cardiac function in health and disease. However, reliable extraction and quantification of 3D cardiomyocyte cytoarchitecture pose significant experimental and computational challenges. We present an effective experimental protocol and a deep learning-enabled toolkit for sample preparation and 3D analysis of cardiomyocyte morphology in ventricular myocardium. Our method is validated across seven species (mouse to human) and in samples prepared in diverse experimental conditions from a range of models, including myocardial infarction and ex vivo tissue culture, highlighting the robustness and versatility of our workflow. Our open-source dataset and toolkit enable large-scale analyses and extraction of realistic 3D geometries of ventricular microstructure. These can be used to explore a host of research questions and provide a new resource for modelling cardiac function at the cellular level.

Most of the mechanistic insights into atrial fibrillation (AF) pathophysiology have been reported on cardiomyocytes, and it is commonly assumed that they apply in the same manner to different regions of the atria. This study aimed to investigate the differential regional features of non-myocyte populations in the atria of pigs and patients with persistent AF (PsAF) based on their functional relevance for AF maintenance. We developed a porcine model resembling clinical PsAF without underlying structural heart disease (N=19), and a second PsAF model with infarct-related atrial cardiomyopathy (MI-PsAF, N=14). Animals with MI-PsAF underwent 3-hour ischaemia-reperfusion in the proximal circumflex artery 2 months prior to the initiation of the AF protocol. The latter aimed to resemble a common clinical scenario with underlying coronary artery disease. After long-lasting self-sustained PsAF, all animals underwent in vivo electroanatomical mapping to identify individual-specific atrial regions associated with AF maintenance (i.e., driver regions), which were further characterized in ex vivo studies by flow cytometry, single-cell RNA transcriptomics (Figure 1), proteomics, and immunolabeling. The results in animals were validated on atrial samples from patients with driver regions in the left atrial appendage (N=8). In a subset of 7 and 6 animals with PsAF and MI-PsAF, respectively, radiofrequency ablation at driver regions effectively terminated the arrhythmia in 100% and 67% of the cases. In patients, ablation of driver regions achieved 90% AF-free survival at 2-years of follow-up. Atrial remodeling during PsAF showed differential adaptative changes in non-myocyte populations, which depended on the functional relevance to sustain the overall arrhythmia. Both in animals and patients, driver regions showed overt compositional shifts in fibroblasts and myeloid populations. More specifically, driver regions were characterized by a phenotypic shift towards cardiac resident macrophages with an associated transcriptomic and proteomic profile favoring cardiomyocyte homeostasis and cell survival within a substrate prone to re-entry (Figure 2). In fibroblasts, PTX3 represented a transcriptional hallmark exclusively present in driver regions, which supports their role on modulating regional specific changes during PsAF. The results reported herein provide a new perspective on the critical role of regional atrial differences in non-myocyte populations that contribute to sustain AF in the long-term.Figure 1 Figure 2

Cardiac fibrosis contributes to electrical conduction disturbances, yet its specific impact on conduction remains unclear, hindering predictive insight into cardiac electrophysiology and arrhythmogenesis. Arrhythmogenic cardiomyopathy is associated with fibrotic remodeling, and it accounts for most cases of stress-related arrhythmic sudden death. Here we develop a correlative imaging approach to integrate macroscale cardiac electrophysiology with three-dimensional microscale reconstructions of the ventricles. We apply this tool to a desmoglein-2 mutant mouse model to characterize the dynamics of conduction wavefronts and relate them to the underlying structural substrate. We observed that conduction through fibrotic tissue areas shows a frequency-dependent behavior, where conduction fails at high stimulation frequencies; this promotes reentrant arrhythmias, even in regions that were electrophysiologically inconspicuous at lower stimulation rates. We found that fibrotic areas undergo electrophysiological remodeling that acts as a low-pass filter for conduction, quantitatively explained by computational models informed by structural data. Collectively, our study provides a structure–function mapping pipeline and describes a pro-arrhythmogenic mechanism in arrhythmogenic cardiomyopathy. Giardini et al. present an imaging method that combines quantitative measurements of cardiac electrophysiology with high-resolution three-dimensional structural reconstructions, enabling the detection of arrhythmogenic electrical coupling between cardiomyocytes and non-myocytes in murine hearts.

The heart is one of the least regenerative organs in humans, and ischemic heart disease is the leading cause of death worldwide. Understanding the cellular and molecular processes that occur during cardiac wound healing is an essential prerequisite to reducing health burden and improving cardiac function after myocardial tissue damage. Here, by integrating single-cell RNA sequencing with high-resolution spatial transcriptomics, we reconstruct the spatiotemporal dynamics of the fibrotic niches after cardiac injury in adult mice. We reveal a complex multicellular network that regulates cardiac repair, including fibroblast proliferation silencing by Trem2high macrophages to prevent excessive fibrosis. We further discovered a rare population of progenitor-like cardiomyocytes after lesion, promoted by myeloid and lymphoid niche signals. Culturing non-regenerative mouse cardiomyocytes or human heart tissue with these niche factors reactivated progenitor gene expression and cell cycle activity. In summary, this spatiotemporal atlas provides valuable insights into the heterocellular interactions that control cardiac repair.