We establish the first protocol for high-fidelity echocardiography in conscious sheep, overcoming the long-standing barrier of apical view acquisition. This enables advanced indices, including volumetric ejection fraction and tissue Doppler, and provides reproducible reference values. Applied to a tachypacing model, it revealed progressive biventricular and atrial remodeling and an early therapeutic window, offering a powerful and clinically relevant platform for translational heart failure research.

Atrial fibrillation (AF) is the most common sustained arrhythmia globally, contributing to significant morbidity and mortality. Recent studies highlight the importance of calcium (Ca²+) handling disruptions in atrial myocytes as a key factor in AF initiation and persistence. Calcium buffering, a process stabilizing intracellular Ca²+ levels, has shown potential relevance in AF pathophysiology, yet its precise role remains insufficiently explored in large-animal models that closely mimic human physiology. This study aimed to investigate whether enhancing calcium buffering through levosimendan, a calcium-sensitizing agent, would increase AF burden, potentially uncovering a novel mechanism of arrhythmogenesis relevant to AF treatment. Six adult Welsh Mountain sheep underwent a series of atrial pacing protocols combined with electrophysiological mapping, pre and post the administration of levosimendan. The atrial conduction velocity (CV) and effective refractory period (ERP) were assessed to evaluate conventional arrhythmic substrates, while AF burden was quantified by measuring total AF duration, episode frequency, and the longest AF episode duration. Statistical comparisons were made across groups using paired analyses, enabling direct evaluation of the effects of calcium buffering enhancement on arrhythmic activity. Data are shown as mean±SEM. Levosimendan significantly increased AF burden across multiple metrics. Total AF duration escalated more than fivefold, from 51.03±23.62s in controls to 301.8±114.8s with levosimendan (p=0.035). AF frequency similarly increased, rising from 5.33±1.5 episodes in controls to 9.17±1.9 episodes post-treatment (p=0.0028), while the longest single AF episode duration increased from 23.87±17.48s to 235.8±105.5s (p=0.031). Despite these marked changes in AF metrics, this could not be explained by a decrease in CV or ERP (156.7±9.19 ms vs. 161.7±7.49 ms), suggesting that the heightened AF susceptibility stems from intracellular Ca²+ cycling rather than traditional electrophysiological changes in conduction or refractoriness. This study demonstrates that enhancing calcium buffering via levosimendan markedly increases AF burden, highlighting calcium handling as an independent and critical arrhythmogenic pathway in AF. By establishing a direct link between calcium buffering and AF susceptibility, our findings suggest that targeted modulation of calcium buffering may offer a novel therapeutic strategy for AF. Future research should focus on developing interventions that finely balance calcium buffering, with the potential to disrupt arrhythmogenic Ca²+ dynamics while preserving myocardial function, ultimately improving outcomes for AF patients.A: Conduction velocity B: ERP of atria. Atrial arrhythmia burden.

Cardiac ischemia followed by reperfusion results in cardiac cell death, which has been attributed to an increase of mitochondrial Ca2+ concentration, resulting in activation of the mitochondrial permeability transition pore (PTP). Evaluating this hypothesis requires understanding of the mechanisms responsible for control of mitochondrial Ca2+ in physiological conditions and how they are altered during both ischemia and reperfusion. Ca2+ influx is thought to occur through the mitochondrial Ca2+ uniporter (MCU). However, with deletion of the MCU, an increase in mitochondrial Ca2+ still occurs, suggesting an alternative Ca2+ influx mechanism during ischemia. There is less certainty about the mechanisms responsible for Ca2+ efflux, with contributions from both Ca2+/H+ exchange and a Na+-dependent Ca2+ efflux pathway. The molecular details of both mechanisms are not fully resolved. We discuss this and the contributions of both pathways to the accumulation of mitochondrial Ca2+ during ischemia and reperfusion. We further discuss the role of mitochondrial Ca2+ in activation of the PTP.

Calcium signaling underlies much of physiology. Almost all the Ca2+ in the cytoplasm is bound to buffers, with typically only ∼1% being freely ionized at resting levels in most cells. Physiological Ca2+ buffers include small molecules and proteins, and experimentally Ca2+ indicators will also buffer calcium. The chemistry of interactions between Ca2+ and buffers determines the extent and speed of Ca2+ binding. The physiological effects of Ca2+ buffers are determined by the kinetics with which they bind Ca2+ and their mobility within the cell. The degree of buffering depends on factors such as the affinity for Ca2+, the Ca2+ concentration, and whether Ca2+ ions bind cooperatively. Buffering affects both the amplitude and time course of cytoplasmic Ca2+ signals as well as changes of Ca2+ concentration in organelles. It can also facilitate Ca2+ diffusion inside the cell. Ca2+ buffering affects synaptic transmission, muscle contraction, Ca2+ transport across epithelia, and the killing of bacteria. Saturation of buffers leads to synaptic facilitation and tetanic contraction in skeletal muscle and may play a role in inotropy in the heart. This review focuses on the link between buffer chemistry and function and how Ca2+ buffering affects normal physiology and the consequences of changes in disease. As well as summarizing what is known, we point out the many areas where further work is required.

The start of the year is an appropriate time to reflect on JGP, particularly as the associate editors and I are beginning our second 3-year term.For many of us, life is now more normal than when I wrote my two previous start-of-year editorials.While still causing infection, COVID-19 has been tamed by effective vaccination programs in many countries.One can only hope that all will be in this position sooner rather than later.The development of COVID-19 vaccines has been quite properly lauded.It is important to remember and, in particular, remind, both politicians and the general public that the ability to respond to this and future pandemics relies on the vitality and therefore the funding of basic science which can then be translated into clinical benefit.Undoubtedly, the major event which has cast a shadow over the world in 2022 is the war in Ukraine.JGP is proud of its authors, reviewers, and readers in that country and saddened by the conditions in which they and their compatriots now live.Membrane physiology in Ukraine was world-leading, contributing enormously to the knowledge of such important areas as the properties of calcium currents (Kostyuk et al., 1979) and ATP-sensitive currents (Krishtal et al., 1988).The history of Ukrainian electrophysiology has been summarized recently in the context of a discussion of what the response of the international community should be to this war (Petersen and Verkhratsky, 2022).JGP has had another successful year, publishing novel, mechanistic, quantitative molecular and cellular physiology writ large and in Special Issues encompassing: Mechanotransduction by Membrane Proteins; and Excitation-Contraction Coupling in Cardiac, Skeletal and Smooth Muscle (Dirksen et al., 2022).

Cardiac myocytes rely on transverse (t)-tubules to facilitate a rapid rise in calcium throughout the cell. However, despite their importance in triggering synchronous Ca2+ release, t-tubules are highly labile structures. They develop postnatally, increase in density during exercise training and are lost in diseases such as heart failure (HF). In the majority of settings, an absence of t-tubules decreases function. Here we show that despite reduced t-tubule density due to immature t-tubules, the newborn atrium is highly specialised to maintain Ca2+ release. To compensate for fewer t-tubules triggering a central rise in Ca2+, Ca2+ release at sites on the cell surface is enhanced in the newborn, exceeding that at all Ca2+ release sites in the adult. Using electron and super resolution microscopy to investigate myocyte ultrastructure, we found that newborn atrial cells had enlarged surface sarcoplasmic reticulum and larger, more closely spaced surface and central ryanodine receptor clusters. We suggest that these adaptations mediate enhanced Ca2+ release at the sarcolemma and aid propagation to compensate for reduced t-tubule density in the neonatal atrium.

Ventricular arrhythmias can cause death in heart failure (HF). A trigger is the occurrence of Ca2+ waves which activate a Na+ -Ca2+ exchange (NCX) current, leading to delayed after-depolarisations and triggered action potentials. Waves arise when sarcoplasmic reticulum (SR) Ca2+ content reaches a threshold and are commonly induced experimentally by raising external Ca2+ , although the mechanism by which this causes waves is unclear and was the focus of this study. Intracellular Ca2+ was measured in voltage-clamped ventricular myocytes from both control sheep and those subjected to rapid pacing to produce HF. Threshold SR Ca2+ content was determined by applying caffeine (10  mM) following a wave and integrating wave and caffeine-induced NCX currents. Raising external Ca2+ induced waves in a greater proportion of HF cells than control. The associated increase of SR Ca2+ content was smaller in HF due to a lower threshold. Raising external Ca2+ had no effect on total influx via the L-type Ca2+ current, ICa-L , and increased efflux on NCX. Analysis of sarcolemmal fluxes revealed substantial background Ca2+ entry which sustains Ca2+ efflux during waves in the steady state. Wave frequency and background Ca2+ entry were decreased by Gd3+ or the TRPC6 inhibitor BI 749327. These agents also blocked Mn2+ entry. Inhibiting connexin hemi-channels, TRPC1/4/5, L-type channels or NCX had no effect on background entry. In conclusion, raising external Ca2+ induces waves via a background Ca2+ influx through TRPC6 channels. The greater propensity to waves in HF results from increased background entry and decreased threshold SR content. KEY POINTS: Heart failure is a pro-arrhythmic state and arrhythmias are a major cause of death. At the cellular level, Ca2+ waves resulting in delayed after-depolarisations are a key trigger of arrhythmias. Ca2+ waves arise when the sarcoplasmic reticulum (SR) becomes overloaded with Ca2+ . We investigate the mechanism by which raising external Ca2+ causes waves, and how this is modified in heart failure. We demonstrate that a novel sarcolemmal background Ca2+ influx via the TRPC6 channel is responsible for SR Ca2+ overload and Ca2+ waves. The increased propensity for Ca2+ waves in heart failure results from an increase of background influx, and a lower threshold SR content. The results of the present study highlight a novel mechanism by which Ca2+ waves may arise in heart failure, providing a basis for future work and novel therapeutic targets.

Transverse (t)-tubules enable close coupling between L-type calcium (Ca2+) channels and ryanodine receptors (RyR) to facilitate triggered Ca2+ release throughout the cell. In heart failure (HF) there is disruption of the t-tubule network that contributes to dyssynchronous Ca2+ release. Despite the importance of t-tubules in triggering Ca2+ release in the atria of large mammals, little is known about Ca2+ release sites and how they are altered in HF.

Rationale: PDE5 (phosphodiesterase 5) inhibition reduces the occurrence of ventricular arrhythmias following myocardial ischemia. However, the mechanisms of the antiarrhythmic effects of PDE5 inhibition are unknown. Diastolic calcium (Ca 2+ ) waves lead to arrhythmias by inducing delayed afterdepolarizations (DADs). Ca 2+ waves are initiated when sarcoplasmic reticulum (SR) Ca 2+ content reaches a threshold level and the SR releases Ca 2+ spontaneously and generates a depolarizing inward sodium-calcium exchange current. Objective: To determine the effects of PDE5 inhibition on the propensity for ventricular arrhythmias in a proarrhythmic large animal model and establish the role of alterations of intracellular Ca 2+ cycling/SR Ca 2+ content. Methods and Results: Arrhythmia burden, monophasic action potentials, and beat-to-beat variability of repolarization were measured in a sheep model using the I Kr inhibitor dofetilide to induce QT prolongation and arrhythmia. Ca 2+ transients, Ca 2+ waves, and SR Ca 2+ content were measured in isolated ventricular myocytes. PDE5 inhibition was achieved using acute application of sildenafil, and PKG (protein kinase G) was inhibited with KT5823. PDE5 inhibition reduced beat-to-beat variability of repolarization and suppressed afterdepolarizations, premature ventricular complexes, and torsade de pointes in vivo. In single cells, dofetilide-induced delayed afterdepolarizations and triggered action potentials were suppressed by PDE5 inhibition. PDE5 inhibition decreased Ca 2+ wave frequency in all cells and abolished waves in 12 of 22 cells. A decrease in SR Ca 2+ uptake, increased trans-sarcolemmal Ca 2+ efflux, and reduced trans-sarcolemmal Ca 2+ influx led to a reduction of SR Ca 2+ content and Ca 2+ wave abolition. These effects were dependent on PKG activation. Conclusions: PDE5 inhibition acutely suppresses triggered ventricular arrhythmias in vivo, and cellular data suggests this occurs via suppression of cellular Ca 2+ waves. These novel antiarrhythmic properties of PDE5 inhibition are mediated by a reduction of SR Ca 2+ content and are PKG dependent.

Sodium glucose transport 2 (SGLT2) inhibitors, such as empagliflozin (EMPA), inhibit glucose re-absorption by the kidney and have been developed to reduce plasma glucose levels in diabetic patients. However, studies have suggested that these drugs have cardioprotective benefits, independent of their effect on plasma glucose 1 . Several possibilities have been proposed to explain these cardioprotective effects including effects on metabolism, weight loss, alterations in EPO and inhibition of plasma membrane sodium-proton exchanger (NHE).

published or not.The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

The death of Edward Carmeliet has robbed us of one of the founders of modern cardiac electrophysiology. Here we summarize his contributions and, equally importantly, consider the current situation of the fields which he helped to develop. It would, however, be remiss not to point out that from 1980 to 1987, he was a member of the Editorial Board of The Journal of Physiology, one of the many roles in which he contributed to helping others develop their science. The Journal has also been an important forum for his work and was his publication of choice for the full reports of his mechanistic studies of cardiac electrophysiology. The dazzling variety of topics which he contributed to is also noteworthy and ranges from the basic properties of Ca2+ and K+ currents, the control of action potential duration, excitation–contraction coupling, to translational aspects such as the effects of ischaemia, and antiarrhythmic therapy. Such breadth was perhaps more common in days gone by than today. Edward Carmeliet studied Cl− movements while working in Berne in Silvio Weidmann's laboratory. His work was published as a single-author paper (Carmeliet, 1961) in which he simply thanked Weidmann for ‘valuable help and criticism’. One wonders whether today's culture might have resulted in a longer author list. He found that Cl− removal had no effect on either the maximum diastolic potential or the resting potential, a result which differed markedly from findings which had been recently published in skeletal muscle (Hodgkin & Horowicz, 1959). Carmeliet returned to Cl− currents 30 years later (Sipido et al. 1993) showing the existence of a Ca2+-activated Cl− current in the heart in rabbit Purkinje cells. This current activates and deactivates quickly during release of calcium from the sarcoplasmic reticulum, and adds to the early repolarization of the action potential. When the sarcoplasmic reticulum is emptied with caffeine, an additional slow component is observed, consistent with a fast activation by local calcium followed by activation by the bulk cytosolic calcium (Papp et al. 1995). While the molecular basis of the Ca2+-activated Cl− current is now known (for recent review, see Varró et al. 2021), there is still a lack of clarity about its function. It is present in the ventricle of sheep and pig, but absent in rat and mouse where fast early repolarization results from activation of K+ currents. It is unclear what the situation is in the human and pharmacological evidence suggests little role (Verkerk et al. 2003). In species that do possess this current, the contribution to the fast phase of repolarization may increase the driving force for Ca2+ entry as has been shown for K+ currents (Sah et al. 2002). Edward Carmeliet made major contributions to our understanding of calcium signalling in the heart. He demonstrated that Ca2+ release from the SR inactivated the L-type Ca2+ current and suggested that recovery from this Ca2+-dependent inhibition might underlie abnormal early afterdepolarizations (EADs) (Sipido et al. 1995a). This work led to a comprehensive study of the Ca2+-dependent membrane currents underlying the so-called transient inward current, Iti. Under calcium-overload conditions, this current occurs at diastolic potentials resulting in a delayed afterdepolarization (Lederer & Tsien, 1976), responsible for many cardiac arrhythmias. At the time there were several calcium-dependent candidates for this current: Na+/Ca2+ exchange (NCX), the non-specific cation current, and Ca2+-activated Cl− current. Carmeliet and colleagues demonstrated that NCX was the major contributor (Sipido et al. 1995b). It is still a puzzle as to why the non-specific cation current, which was one of the first single channels to be identified with the then novel patch clamp technique (Colquhoun et al. 1981), does not contribute more to cardiac electrophysiology. Interestingly, however, there was a significant contribution from calcium-dependent inactivation of the L-type Ca2+ current confirming its potential role in early afterdepolarizations. Even today, it is unclear exactly what the mechanisms are that underlie EADs. To what extent are they, as was originally suggested, a product of reactivation of the L-type Ca2+ current and, if so, is this due to recovery from voltage- as opposed to calcium-dependent inactivation? Alternatively, does the EAD (like the delayed afterdepolarization; DAD) result from Ca2+ release from the SR activating inward membrane currents? In the presence of high sympathetic drive, one of the major triggers for arrhythmias, probably both mechanisms contribute to EADs, which then are also seen together with DADs (Volders et al. 2000). Carmeliet also made important contributions to the concept of microdomains near the membrane where ion concentrations differ from those in the bulk cytosol. This was triggered by his observations on activation of a Na+-dependent K+ current, and of gradients created by the Na+/K+ pump (Carmeliet, 1992a). This became a controversial area in the 1990s when it was proposed that local Na+ increase was sufficient for Ca2+ influx through the Na+/Ca2+ exchanger to trigger release of calcium from the sarcoplasmic reticulum (Leblanc & Hume, 1990). The work generated much discussion on appropriate experimental conditions and Carmeliet encouraged further studies (Sipido et al. 1995c). Lack of proper tools has hampered a resolution but, with the availability of new methods such as membrane-targeted probes (Shang et al. 2014), this may change. He also provided evidence for microdomains of calcium concentration by showing that the Ca2+-activated Cl− current decayed much more quickly than the measured change of bulk calcium concentration ([Ca2+]i), an effect attributed to local gradients of [Ca2+]i (see also Papp et al. 1995; Trafford et al. 1995). In 1955, as a medical student in the laboratory of Physiology, Edward Carmeliet published his first single author paper on ‘Influence of rhythm on the duration of the action potential’ (Carmeliet, 1955). This interest would remain throughout his life, with an authoritative book that brings together insights in the modulation of voltage-dependent channels, the influence of changes in calcium handling and the autonomic modulation of ion channels and calcium (Carmeliet & Vereecke, 2002). Changes in rhythm are the consequence of changes in autonomic drive, acting on the sinus node. Carmeliet made important contributions to our understanding of pacemaker mechanisms and the properties of the acetylcholine-sensitive K+ channel (Callewaert et al. 1984; Carmeliet & Mubagwa, 1986). This was a foundation for later work, which demonstrated the importance of this current in atrial fibrillation (Dobrev et al. 2001). Here downregulation of the channel reduces muscarinic regulation, further contributing to the pathology. Carmeliet also studied the α-adrenergic modulation of the action potential through noradrenaline, fuelled by an interest in ischaemia and the associated catecholamine release. The effects were different in atrium and ventricle and between species, related to differences in receptor types and distribution. This complexity was unravelled and clearly presented by Carmeliet in his comprehensive review of ionic currents during ischaemia (Carmeliet, 1999). Yet, still today, the α-adrenergic modulation in human ventricular myocytes is not as well understood as β-adrenergic modulation. Carmeliet started his studies of K+ currents using isotope flux measurements in Purkinje fibres and in embryonic chick hearts (Carmeliet et al. 1976). He next used the two-microelectrode voltage clamp of thin Purkinje fibres and direct current measurements (Coraboeuf & Carmeliet, 1982). Work in these multicellular tissues was hampered by changes of extracellular ion concentrations in intercellular spaces. Later experiments in isolated myocytes removed some of the complexity, and with the patch clamp technique a new era of direct K+ current recordings started (Carmeliet et al. 1987). Carmeliet had a strong interest in K+ channels and studied many members of this large family. His studies were of direct relevance to drug development. K+ currents are a prime target to modulate action potential duration, and the delayed rectifier current a strong candidate (Carmeliet, 1992b). His work on modulation of K+ and Na+ currents was at the heart of his involvement with a translational and multi-stakeholder task force on anti-arrhythmic drug development, which first met in Taormina, Sicily. After this first meeting, the group published a viewpoint, the Sicilian Gambit (Task Force of the Working Group on Arrhythmias of the European Society of Cardiology, 1991), proposing a new approach to anti-arrhythmic drugs after the devastating results of the CAST study where flecainide increased rather than decreased mortality (Echt et al. 1991). The concept was that the use and development of anti-arrhythmic drugs should be based on insights in arrhythmia mechanisms, in ion channel properties and in the mechanisms of action of drugs that modulate ion currents. The group convened regularly and published several papers over the next 10 years. These concepts still stand today and are important in guiding therapy and drug development (Rosen & Janse, 2010). What has, however, remained elusive is effective and safe anti-arrhythmic drugs for ventricular arrhythmias. Patients at risk for ventricular tachycardia and fibrillation after myocardial infarction form the largest population in need of preventive drug therapy. The best available treatment today is implantation of an ICD, with or without ablation and supportive therapy, with very limited anti-arrhythmic drug options (Priori et al. 2015; Dan et al. 2018). This article has focused on Edward Carmeliet's scientific contributions. His colleagues were also fortunate to receive much timeless advice from him including the following. (1) It is important to revisit unresolved questions with new technologies, for example as shown by the refinement of his work on Na+ channels from two-microelectrode voltage clamp, to patch clamp, and finally transgenic technology. (2) Keep an open mind and be suspicious and critical of one's observations, using data to test rather than confirm hypotheses. It is noteworthy that in early papers in The Journal of Physiology, the abstract does not contain conclusions, only observations. (3) Embrace curiosity. Gathering data without preconceived ideas, often derided as a ‘fishing trip’, may prove its value at a time when we have enormous potential for unbiased big data. This allows curiosity-driven research and encourages us to pursue the unexpected. Finally, Edward Carmeliet showed us that science and curiosity are a powerful antidote for ageing. At a meeting in 2018 he explained how he had studied the biophysics and working of his hearing aid, which he now could tune optimally for lectures as well as for conversations in a crowd. He was a great scientist, mentor and friend.