BACKGROUND:Stereotactic arrhythmia radioablation (STAR) is a novel, non-invasive treatment for therapy-refractory ventricular tachycardia (VT). In STAR, a high dose of radiation is used to non-invasively target and treat the VT substrate. Initial studies indicate promising VT burden reduction, but comprehensive efficacy and safety evaluations remain limited. METHODS:A systematic review (Preferred Reporting Items for Systematic Reviews and Meta-Analyses/Meta-analysis Of Observational Studies in Epidemiology guidelines) included studies on STAR for monomorphic VT identified up to 30 June 2024 via MEDLINE and EMBASE. Outcomes assessed were freedom of VT, percentage reduction in VT episodes and implantable cardioverter-defibrillator (ICD) shocks per month, survival and adverse events (AEs). Meta-analyses included prospective and retrospective studies only, using random-effects models with double arcsine transformation. Subgroup analyses by study design and planning target volume (PTV) were performed. AEs were qualitatively analysed and classified by organ system, severity and causality. RESULTS:The meta-analysis included 215 patients from 22 studies (age 66.0±4.4 years, 85.9% men, left ventricular ejection fraction 29.8±5.0%, 52.2% ischaemic cardiomyopathy, mean follow-up of 11.9±6.6 months). The overall survival was 69.6% (95% CI 62.6% to 76.2%). VT episodes and ICD shocks/month reduced by 81.5% (95% CI 64.2% to 94.8%) and 84.7% (95% CI 65.1% to 98.1%), respectively. However, only 23.1% (95% CI 10.7% to 37.7%) were VT-free at the end of follow-up. There were no significant differences in clinical outcomes between prospective and retrospective studies, nor between studies with high PTV and low PTV. A total of 352 AEs were reported in 280 patients, with a mean of 1.26 AE per patient. Of these AEs, 50.6% were classified as severe, though only 9.7% were likely STAR-related. CONCLUSIONS:STAR significantly reduces VT episodes and ICD shocks, offering symptomatic relief. However, high recurrence rates and severe AEs underscore the need for protocol optimisation and multidisciplinary collaboration to improve STAR's safety and efficacy in VT management.

Background Spontaneous Ca2+ 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 Ca2+ waves trigger tissue-wide depolarization in the intact heart is scarce, particularly in human HF. We characterized Ca2+ 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. Ca2+ transients and waves were recorded by wide-field imaging of Fluo-8. Ca2+ waves in relation to single-cell structures within each LMS were identified using custom algorithms. Computational modelling assessed whether experimentally observed HF Ca2+ waves dynamics can lead to focal excitation in tissue models. Results Following pacing, early onset Ca2+ waves, initiating within the first 2 seconds, were more frequent in HF compared to non-HF, and HF cardiomyocytes had more foci, where Ca2+ waves originate, than non-HF. Spatial mapping showed that early onset waves in HF occurred frequently in clusters of neighboring cells. Although early onset Ca2+ 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 Ca2+ propagation, correlating with increased connexin 43 distribution to lateral membranes. Furthermore, HF LMS exhibited more local and global triggered Ca2+ activities compared to non-HF LMS, correlating with local tissue depolarization. Simulations of HF Ca2+ wave dynamics in remodeled tissue demonstrated a greater capacity to elicit focal excitation. Conclusions In human HF, a higher incidence of early onset Ca2+ waves combines with altered intercellular connectivity to create synchrony in clusters of nearby cells that can overcome the current sink, thereby increasing arrhythmia susceptibility. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. * AP : Action potential Cx43 : Connexin43 DAD : Delayed afterdepolarization HF : Heart failure ID : Intercalated disc ISO : Isoproterenol LMS : Living myocardial slices NCX : Na+/Ca2+ exchanger Non-HF : Non-failing heart PVC : Premature ventricular complex RyR : Ryanodine receptor SD : Standard deviation SRF : Spontaneous release function [1]: pending:yes

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.

Ca2+ transients (CaT) underlying cardiomyocyte (CM) contraction require efficient Ca2+ coupling between sarcolemmal Ca2+ channels and sarcoplasmic reticulum (SR) ryanodine receptor Ca2+ channels (RyR) for their generation; reduced coupling in disease contributes to diminished CaT and arrhythmogenic Ca2+ events. SR Ca2+ release also occurs via inositol 1,4,5-trisphosphate receptors (InsP3R) in CM. While this pathway contributes negligeably to Ca2+ handling in healthy CM, rodent studies support a role in altered Ca2+ dynamics and arrhythmogenic Ca2+ release involving InsP3R crosstalk with RyRs in disease. Whether this mechanism persists in larger mammals with lower T-tubular density and coupling of RyRs is not fully resolved. We have recently shown an arrhythmogenic action of InsP3-induced Ca2+ release (IICR) in end stage human heart failure (HF), often associated with underlying ischemic heart disease (IHD). How IICR contributes to early stages of disease is however not determined but highly relevant. To access this stage, we chose a porcine model of IHD, which shows substantial remodelling of the area adjacent to the infarct. In cells from this region, IICR preferentially augmented Ca2+ release from non-coupled RyR clusters that otherwise showed delayed activation during the CaT. IICR in turn synchronised Ca2+ release during the CaT but also induced arrhythmogenic delayed afterdepolarizations and action potentials. Nanoscale imaging identified co-clustering of InsP3Rs and RyRs, thereby allowing Ca2+-mediated channel crosstalk. Mathematical modelling supported and further delineated this mechanism of enhanced InsP3R-RyRs coupling in MI. Our findings highlight the role of InsP3R-RyR channel crosstalk in Ca2+ release and arrhythmia during post-MI remodelling.

The ryanodine receptor type 2 (RyR) is a key player in Ca2+ handling during excitation-contraction coupling. During each heartbeat, RyR channels are responsible for linking the action potential with the contractile machinery of the cardiomyocyte by releasing Ca2+ from the sarcoplasmic reticulum. RyR function is fine-tuned by associated signalling molecules, arrangement in clusters and subcellular localization. These parameters together define RyR function within microdomains and are subject to disease remodelling. This review describes the latest findings on RyR microdomain organization, the alterations with disease which result in increased subcellular heterogeneity and emergence of microdomains with enhanced arrhythmogenic potential, and presents novel technologies that guide future research to study and target RyR channels within specific microdomains.

Aims Takotsubo syndrome (TTS) is an acute heart failure, typically triggered by high adrenaline during physical or emotional stress. It is distinguished from myocardial infarction (MI) by a characteristic pattern of ventricular basal hypercontractility with hypokinesis of apical segments, and in the absence of culprit coronary occlusion. We aimed to understand whether recently discovered circulating biomarkers miR-16 and miR-26a, which differentiate TTS from MI at presentation, were mechanistically involved in the pathophysiology of TTS. Methods and results miR-16 and miR-26a were co-overexpressed in rats with AAV and TTS induced with an adrenaline bolus. Untreated isolated rat cardiomyocytes were transfected with pre-/anti-miRs and functionally assessed. Ventricular basal hypercontraction and apical depression were accentuated in miR-transfected animals after induction of TTS. In vitro miR-16 and/or miR-26a overexpression in isolated apical (but not basal), cardiomyocytes produced strong depression of contraction, with loss of adrenaline sensitivity. They also enhanced the initial positive inotropic effect of adrenaline in basal cells. Decreased contractility after TTS-miRs was reproduced in non-failing human apical cardiomyocytes. Bioinformatic profiling of miR targets, followed by expression assays and functional experiments, identified reductions of CACNB1 (L-type calcium channel Cavβ subunit), RGS4 (regulator of G-protein signalling 4), and G-protein subunit Gβ (GNB1) as underlying these effects. Conclusion miR-16 and miR-26a sensitize the heart to TTS-like changes produced by adrenaline. Since these miRs have been associated with anxiety and depression, they could provide a mechanism whereby priming of the heart by previous stress causes an increased likelihood of TTS in the future.

The fast transient outward potassium current (Ito,f) plays a key role in phase 1 repolarization of the human cardiac action potential (AP) and its reduction in heart failure (HF) contributes to the loss of contractility. Therefore, restoring Ito,f might be beneficial for treating HF. The coding sequence of a P2A peptide was cloned, in frame, between Kv4.3 and KChIP2.1 genes and ribosomal skipping was confirmed by Western blotting. Typical Ito,f properties with slowed inactivation and accelerated recovery from inactivation due to the association of KChIP2.1 with Kv4.3 was seen in transfected HEK293 cells. Both bicistronic components trafficked to the plasmamembrane and in adenovirus transduced rabbit cardiomyocytes both t-tubular and sarcolemmal construct labelling appeared. The resulting current was similar to Ito,f seen in human ventricular cardiomyocytes and was 50% blocked at ~0.8 mmol/l 4-aminopyridine and increased ~30% by 5 μmol/l NS5806 (an Ito,f agonist). Variation in the density of the expressed Ito,f, in rabbit cardiomyocytes recapitulated typical species-dependent variations in AP morphology. Simultaneous voltage recording and intracellular Ca2+ imaging showed that modification of phase 1 to a non-failing human phenotype improved the rate of rise and magnitude of the Ca2+ transient. Ito,f expression also reduced AP triangulation but did not affect ICa,L and INa magnitudes. This raises the possibility for a new gene-based therapeutic approach to HF based on selective phase 1 modification.

Dysregulated intracellular Ca2+ handling involving altered Ca2+ release from intracellular stores via RyR channels underlies both arrhythmias and reduced function in heart failure (HF). Mechanisms linking RyR dysregulation and disease are not fully established. Studies in animals support a role for InsP3 receptor Ca2+ channels (InsP3R) in pathological alterations in cardiomyocyte Ca2+ handling but whether these findings translate to the divergent physiology of human cardiomyocytes during heart failure is not determined. Using electrophysiological and Ca2+ recordings in human ventricular cardiomyocytes, we uncovered that Ca2+ release via InsP3Rs facilitated Ca2+ release from RyR and induced arrhythmogenic delayed after depolarisations and action potentials. InsP3R–RyR crosstalk was particularly increased in HF at RyR clusters isolated from the T-tubular network. Reduced SERCA activity in HF further facilitated the action of InsP3. Nanoscale imaging revealed co-localisation of InsP3Rs with RyRs in the dyad, which was increased in HF, providing a mechanism for augmented Ca2+ channel crosstalk. Notably, arrhythmogenic activity dependent on InsP3Rs was increased in tissue wedges from failing hearts perfused with AngII to promote InsP3 generation. These data indicate a central role for InsP3R–RyR Ca2+ signalling crosstalk in the pro-arrhythmic action of GPCR agonists elevated in HF and the potential for their therapeutic targeting.

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.

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.

BACKGROUND Sympathetic activation in ischemic heart disease can cause lethal arrhythmias. These often are preceded by premature ventricular complexes (PVCs), which at the cellular level could result from delayed afterdepolarizations. OBJECTIVE The purpose of this study was to identify and map vulnerable areas for arrhythmia initiation after myocardial infarction (MI) and to explore the link between PVCs and cellular events. METHODS Anterior-septal wall MI was induced by 120 minutes of coronary occlusion followed by reperfusion (27 MI and 16 sham pigs). After 4 weeks, EnSiteTM electroanatomic mapping combined with imaging was performed to precisely locate PVC sites of origin and subsequently record monophasic action potentials. Cardiomyocytes were isolated from different regions to study regional cellular remodeling. Isoproterenol was used as a surrogate for adrenergic stimulation both in vivo and in cardiomyocytes. RESULTS PVCs originated from the MI border zone (BZ) and

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.

Pulmonary hypertension is a complex disorder characterized by pulmonary vascular remodeling and right ventricular hypertrophy, leading to right heart failure. The mechanisms underlying this process are not well understood. We hypothesize that the structural remodeling occurring in the cardiomyocytes of the right ventricle affects the cytosolic Ca 2+ handling leading to arrhythmias. After 12 days of monocrotaline-induced pulmonary hypertension in rats, epicardial mapping showed electrical remodeling in both ventricles. In myocytes isolated from the hypertensive rats, a combination of high-speed camera and confocal line-scan documented a prolongation of Ca 2+ transients along with a higher local Ca 2+ -release activity. These Ca 2+ transients were less synchronous than in controls, likely due to disorganized transverse-axial tubular system. In fact, following pulmonary hypertension, hypertrophied right ventricular myocytes showed significantly reduced number of transverse tubules and increased number of axial tubules; however, Stimulation Emission Depletion microscopy demonstrated that the colocalization of L-type Ca 2+ channels and RyR2 (ryanodine receptor 2) remained unchanged. Finally, Stimulation Emission Depletion microscopy and super-resolution scanning patch-clamp analysis uncovered a decrease in the density of active L-type Ca 2+ channels in right ventricular myocytes with an elevated open probability of the T-tubule anchored channels. This may represent a general mechanism of how nanoscale structural changes at the early stage of pulmonary hypertension impact on the development of the end stage failing phenotype in the right ventricle.

Rhythmic increases in intracellular Ca2+ concentration underlie the contractile function of the heart. These heart muscle-wide changes in intracellular Ca2+ are induced and coordinated by electrical depolarization of the cardiomyocyte sarcolemma by the action potential. Originating at the sinoatrial node, conduction of this electrical signal throughout the heart ensures synchronization of individual myocytes into an effective cardiac pump. Ca2+ signaling pathways also regulate gene expression and cardiomyocyte growth during development and in pathology. These fundamental roles of Ca2+ in the heart are illustrated by the prevalence of altered Ca2+ homeostasis in cardiovascular diseases. Indeed, heart failure (an inability of the heart to support hemodynamic needs), rhythmic disturbances, and inappropriate cardiac growth all share an involvement of altered Ca2+ handling. The prevalence of these pathologies, contributing to a third of all deaths in the developed world as well as to substantial morbidity makes understanding the mechanisms of Ca(2+ )handling and dysregulation in cardiomyocytes of great importance.

Cardiac fast transient outward potassium current (Ito,f) underlies the early repolarization phase of the human action potential (AP) and its functional expression varies transmurally and with heart disease. Human cardiac Ito,f channels are composed of Kv4.3 pore-forming α-subunit plus the auxiliary KChIP2 β-subunit. Currently, there is no animal model featuring a typical human Ito,f phenotype. Here, we designed a P2A peptide-based bicistronic transgene to deliver a stoichiometric expression of Kv4.3/KChIP2.1 and examined its effect on AP waveforms of rabbit left ventricular myocytes. Western blot of HEK293 cells overexpressing the bicistronic transgene confirmed successful ribosomal skipping mediated by the P2A sequence and efficient co-expression of Kv4.3 and KChIP2.1. When delivered to rabbit left ventricular myocytes in which the native Ito,f is small, the bicistronic transgene conferred an Ito,f similar to that reported in human ventricular cardiomyocytes, featuring slowed inactivation at positive Vms and rapid recovery from inactivation. Introducing a moderate level of the Ito,f to rabbit ventricular myocytes produced a prominent phase I repolarization. Increasingly larger Ito,f densities initially resulted in a ‘spike-and-dome’ AP waveform similar to that in dogs and then dramatically shortened the repolarization phase to triangular murine-like AP morphology. Simultaneous current-clamp recording and intracellular Ca2+ imaging further demonstrated that moderate augmentation of phase I repolarization improved the time course and amplitude of the initial phase of the Ca2+ transient. Collectively, manipulating magnitude of Ito,f by stoichiometric (1:1) expression of Kv4.3/KChIP2.1 effectively shapes the trajectory of phase 1 repolarization and influences excitation-contraction coupling.

Introduction: Spontaneous Ca2+ release events (SCRE) can trigger left ventricular (LV) arrhythmias during disease remodeling. Intrinsic transmural heterogeneities in Ca2+ handling may differentially modulate SCRE, leading to regional-dependent arrhythmia susceptibility. The geometry of the LV wall further complicates the transmural study of SCRE. Using an in vitro approach mimicking disease remodeling in intact cardiac tissue slices, we aimed to dissect transmural heterogeneities of SCRE. Methods: LV myocardial slices were prepared from adult rats (established protocol by our group). Cryoinjury was induced by applying a frozen probe on the surface of the tissue slice. After 24h-culture, SCRE were studied following pro-arrhythmia pacing using Fluo8AM and high-resolution imaging in regions adjacent to the cryo-injury in endocardial (ENDO, ≤600 µm endo-surface) and epicardial (EPI, ≤600 µm epi-surface) slices. Results: Cryo-injured slices showed local remodeling with reduced T-tubule density and Ca2+ transient amplitudes in the region adjacent to the cryo-injury. After pro-arrhythmic conditioning, injured ENDO slices showed more Ca2+ foci (ENDO: 4.54±0.40 vs. EPI: 3.29±0.29 #foci/mm²/s), which are more closely distributed (ENDO: 67.7±2.7 vs. EPI: 77.9±3.7 µm) with reduced latency times to induce SCRE (ENDO: 222±48 ms vs. EPI: 601±160 ms) compared to injured EPI slices. Inhibition of CaMKII using AIP reduced the number of Ca2+ foci (61% in ENDO vs. 29% in EPI) and increased the distance between Ca2+ foci (40% in ENDO vs. 20% in EPI) more effectively in injured ENDO vs. EPI slices, without affecting the Ca2+ transient amplitude. Conclusions: Our novel in vitro model allows for studies of transmural heterogeneity upon disease remodeling. The current data reveals increased arrhythmogenic susceptibility in the injured endocardium and highlight regional-specific targeting in therapies.