Eef Dries

Stereotactic arrhythmia radioablation (STAR) has emerged as a promising non‐invasive therapy for refractory ventricular tachycardia, yet its biological mechanisms remain poorly defined. While conventional catheter ablation achieves arrhythmia control through structural remodelling, radiotherapy was initially assumed to act similarly. However, STAR reduces ventricular arrhythmia burden within weeks, preceding the development of radiation‐induced structural remodelling. This temporal mismatch indicates that early anti‐arrhythmic effects are probably driven by functional adaptations. Human tissue findings and preclinical large‐ and small‐animal models have provided the first insights into how the myocardium responds to focal high‐dose radiation, yet experimental data on STAR's underlying cellular mechanisms remain limited. Across these studies, a consistent observation is the temporal dissociation between early functional effects and delayed structural remodelling, which provides a key framework for interpretating STAR's anti‐arrhythmic action. In this review, we synthesize current evidence on cell‐type‐specific responses to ionizing radiation by integrating insights from the emerging STAR literature, oncology and radiation heart disease models. By linking time‐resolved cellular responses to clinical observations, we aim to distinguish mechanisms most relevant to early therapeutic effects from those contributing to delayed remodelling, and to define key knowledge gaps for guiding safe and effective clinical implementation. image

Current approaches for the study of arrhythmogenic Ca ²⁺ waves in intact cardiac tissue have limited capacity to study the relationship between single‐cell properties and the tissue environment. Particularly, wide‐field Ca ²⁺ imaging captures large areas but merges signals from multiple cells, while confocal imaging provides (sub)cellular resolution but is limited to a small number of cells. The aim of this study was to develop an imaging and analysis pipeline capable of extracting single‐cell Ca ²⁺ wave dynamics across large fields within the intact multicellular tissue. Living myocardial slices were prepared from left ventricular tissue from pig and human hearts. Ca ²⁺ transients and waves reported by Fluo8 were imaged in regions of 20–100 cells at the slice surface. Following a 2 min conditioning period of 2 Hz pacing under adrenergic stimulation, Ca ²⁺ waves were evident during the rest period. Images were recorded at 200 fps. After image processing, single Ca ²⁺ waves were identified and propagation paths were tracked using TrackMate. From these tracks, Ca ²⁺ wave parameters (number of tracks, coordinates and kinetics) were extracted for quantitative analysis and assigned to individual cardiomyocytes using a maximum fluorescence intensity mask to identify cellular borders. In healthy pig cardiac tissue, we captured and quantified single‐cell Ca ²⁺ wave dynamics, detecting variability within the population and an absence of synchronization. The recording of Ca ²⁺ dynamics across a large cell population and their cell–cell interactions bridges the gap between cell‐ and tissue‐level observations. Future studies of diseased tissue will offer new insights into mechanisms driving aberrant cellular Ca ²⁺ activity and the translation into arrhythmias. image Key points: We developed an approach that allows high frame‐rate imaging of living cardiac tissue, while retaining capacity to identify Ca ²⁺ waves in single cells. In myocardial slice preparations from pig and human hearts, we recorded Ca ²⁺ waves and, using image analysis tools, tracked their paths and properties simultaneously within individual cells and across a large cell population (20–100 cells) within a region of interest. This approach bridges a key gap between single‐cell observations and whole‐tissue behaviour, and can offer deeper insights into how cellular Ca ²⁺ waves could expand and propagate in the heart. Our approach provides a new methodology to study cellular mechanisms that lead to arrhythmias in diseased cardiac tissue.

Background: Spontaneous Ca ²⁺ release events and waves are frequent in isolated ventricular cardiomyocytes from failing hearts (HF) and are proposed to initiate arrhythmias in the intact heart. However, evidence supporting whether single-cell Ca ²⁺ waves trigger tissue-wide depolarization in the intact heart is scarce, particularly in human HF. We characterized Ca ²⁺ waves at single-cell resolution within the multicellular network of the intact heart and identified propagating dynamics and mechanisms facilitating arrhythmogenesis at tissue level. Methods: Living myocardial slices (LMS) from HF and non-HF human hearts were prepared from left ventricular tissue and paced at 2 Hz under adrenergic stimulation. Ca ²⁺ transients and waves were recorded by wide-field imaging of Fluo-8. Ca ²⁺ waves in relation to single-cell structures within each LMS were identified using custom algorithms. Computational modelling assessed whether experimentally observed HF Ca ²⁺ waves dynamics can lead to focal excitation in tissue models. Results: Following pacing, early onset Ca ²⁺ waves, initiating within the first 2 seconds, were more frequent in HF compared to non-HF, and HF cardiomyocytes had more foci, where Ca ²⁺ waves originate, than non-HF. Spatial mapping showed that early onset waves in HF occurred frequently in clusters of neighboring cells. Although early onset Ca ²⁺ waves propagated similar distances in HF and non-HF cardiomyocytes, they more frequently crossed cell boundaries in HF. Particularly, HF LMS exhibited more side-to-side Ca ²⁺ propagation, correlating with increased connexin 43 distribution to lateral membranes. Furthermore, HF LMS exhibited more local and global triggered Ca ²⁺ activities compared to non-HF LMS, correlating with local tissue depolarization. Simulations of HF Ca ²⁺ wave dynamics in remodeled tissue demonstrated a greater capacity to elicit focal excitation. Conclusions: In human HF, a higher incidence of early onset Ca ²⁺ waves combines with altered intercellular connectivity to create synchrony in clusters of nearby cells that can overcome the current sink, thereby increasing arrhythmia susceptibility. GRAPHICAL

Rad is an emerging key Cav1.2 modulator. In the present issue of JGP, Elmore, Ahern et al. examine how the Rad C-terminus affects its subcellular distribution and Cav1.2 regulation.

Ischemic heart disease is the most common cause of lethal ventricular arrhythmias and sudden cardiac death (SCD). In patients who are at high risk after myocardial infarction, implantable cardioverter defibrillators are the most effective treatment to reduce incidence of SCD and ablation therapy can be effective for ventricular arrhythmias with identifiable culprit lesions. Yet, these approaches are not always successful and come with a considerable cost, while pharmacological management is often poor and ineffective, and occasionally proarrhythmic. Advances in mechanistic insights of arrhythmias and technological innovation have led to improved interventional approaches that are being evaluated clinically, yet pharmacological advancement has remained behind. We review the mechanistic basis for current management and provide a perspective for gaining new insights that centre on the complex tissue architecture of the arrhythmogenic infarct and border zone with surviving cardiac myocytes as the source of triggers and central players in re-entry circuits. Identification of the arrhythmia critical sites and characterisation of the molecular signature unique to these sites can open avenues for targeted therapy and reduce off-target effects that have hampered systemic pharmacotherapy. Such advances are in line with precision medicine and a patient-tailored therapy.

Spontaneous Ca²⁺ release (SCR) can cause triggered activity and initiate arrhythmias. Intrinsic transmural heterogeneities in Ca²⁺ handling and their propensity to disease remodeling may differentially modulate SCR throughout the left ventricular (LV) wall and cause transmural differences in arrhythmia susceptibility. Here, we aimed to dissect the effect of cardiac injury on SCR in different regions in the intact LV myocardium using cryoinjury on rat living myocardial slices (LMS). We studied SCR under proarrhythmic conditions using a fluorescent Ca²⁺ indicator and high-resolution imaging in LMS from the subendocardium (ENDO) and subepicardium (EPI). Cryoinjury caused structural remodeling, with loss in T-tubule density and an increased time of Ca²⁺ transients to peak after injury. In ENDO LMS, the Ca²⁺ transient amplitude and decay phase were reduced, while these were not affected in EPI LMS after cryoinjury. The frequency of spontaneous whole-slice contractions increased in ENDO LMS without affecting EPI LMS after injury. Cryoinjury caused an increase in foci that generates SCR in both ENDO and EPI LMS. In ENDO LMS, SCRs were more closely distributed and had reduced latencies after cryoinjury, whereas this was not affected in EPI LMS. Inhibition of CaMKII reduced the number, distribution, and latencies of SCR, as well as whole-slice contractions in ENDO LMS, but not in EPI LMS after cryoinjury. Furthermore, CaMKII inhibition did not affect the excitation–contraction coupling in cryoinjured ENDO or EPI LMS. In conclusion, we demonstrate increased arrhythmogenic susceptibility in the injured ENDO. Our findings show involvement of CaMKII and highlight the need for region-specific targeting in cardiac therapies.

Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic Ca²⁺ release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic Ca²⁺ release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel Ca²⁺ entry and coupling to Ca²⁺ release microdomains at the intercalated disc, resulting in enhanced Ca²⁺ dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and Ca²⁺ release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure.