Acidic calcium stores significantly influence basal calcium transient amplitude and β-adrenergic responses in cardiomyocytes. Atrial myocytes contain atrial granules (AGs), small acidic organelles that store and secrete atrial natriuretic peptide (ANP) and are absent in healthy ventricular myocytes. AGs are known to be acidic and calcium-rich, but their number and location relative to other signalling sites remain unexplored. Labelling of acidic organelles in adult guinea pig cardiomyocytes showed the presence of acidic puncta throughout the cytosol. Atrial myocytes exhibited an increased concentration of acidic organelles at the nuclear poles. Live cell fluorescent studies using 4-phenyl-3-butenoic acid (PBA) to inhibit peptidylglycine α-amidating monooxygenase, a crucial component of AGs membranes, effectively eliminated staining at the nuclear poles and most acidic puncta in atrial cells, but not in ventricular cells. Our immunofluorescent labelling also emphasizes the differences in acidic punctae between atrial and ventricular myocytes by showing minimal co-localization between AG-specific ANP and lysosomal-associated membrane protein. Electron microscopy studies on goat atrial fibrillation (AF) and sham control tissue allowed visualization of AGs. Quantitative analysis revealed that AGs were positioned significantly further away from the nearest sarcoplasmic reticulum and were closer to mitochondria in AF compared to sinus rhythm control tissue. We raise the question whether the positioning of AGs is strategic for communication with other calcium-containing organelles.

BACKGROUND:The role of aprotinin in modern cardiac surgery is not well defined. While licensed for use in isolated coronary artery bypass grafting it is more commonly used for cases deemed to be at an increased risk of bleeding. The relative efficacy, and safety profile, of aprotinin as compared to other antifibrinolytics in these high-risk cases is uncertain. STUDY DESIGN AND METHODS:A retrospective observational study with propensity matching to determine whether aprotinin versus tranexamic acid reduced bleeding or transfusion requirements in patients presenting for surgical repair of type A aortic dissection (TAD). RESULTS:Between 2016 and 2022, 250 patients presented for repair of TAD. A total of 231 patients were included in the final analysis. Bleeding and transfusion were similar between both groups in both propensity matched and unmatched cohorts. Compared to tranexamic acid, aprotinin use did not reduce transfusion requirements for any product. Rates of bleeding in the first 12 h, return to theater and return to intensive care unit with an open packed chest were similar between groups. There was no difference in rates of renal failure, stroke, or death. CONCLUSION:Aprotinin did not reduce the risk of bleeding or transfusion requirements in patients undergoing repair of type A aortic dissections. Efficacy of aprotinin may vary depending on the type of surgery performed and the underlying pathology.

In the heart, endogenous nicotinic acid adenine dinucleotide phosphate (NAADP) triggers lysosomal calcium release to augment sarcoplasmic reticulum (SR) calcium sequestration, producing larger calcium transients. However, the role of lysosomal calcium signals in pacemaker activity, a distinct calcium-operated function of the sino-atrial node (SAN) or atria, a distinct calcium-operated function, has not been investigated. Pharmacological or genetic ablation of the NAADP pathway inhibits spontaneous beating rate response to β-adrenergic stimulation in intact SAN. We found intracellular signalling microdomains between lysosomes and neighbouring SR or mitochondria in mouse, rabbit, goat, and human atrial tissue. The spatial relationship between lysosomes and other calcium-handling organelles are altered in goat and human atrial fibrillation. Furthermore, we demonstrate atrial myocytes produce 3′–5′-cyclic adenosine monophosphate in response to lysosomal signalling, adding a novel trigger for cyclic nucleotide signalling. Our findings support the hypothesis that lysosomal calcium signaling directly increases cardiomyocyte cAMP and modulates pacemaker activity.

Inositol trisphosphate (IP3), a calcium (Ca2+)-mobilizing second messenger, releases Ca2+ from the sarcoplasmic reticulum (SR) via IP3 receptors and modulates adenylyl cyclase (AC) activity in atrial myocytes. Lysosomes participate in Ca2+ homeostasis by mobilising Ca2+ in response to Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP). We postulate that both downstream activation of Ca2+ sensitive AC (AC1 and AC8) and lysosomal Ca2+ signalling in response to IP3R activation contribute to atrial myocyte function and pacemaking. Ectopic application of phenylephrine (PE) increased chronotropy and inotropy and this response was reduced in the presence of NAADP pathway inhibitors (BZ-194 and SAN4825) and Bafilomycin A1. PE increased cAMP activity in neonatal rat atrial myocytes (NRAMs) and this was inhibited by NAADP pathway inhibitors. This inhibition was not observed in neonatal rat ventricular myocytes (NRVMs), revealing specificity of this response to NRAMs. We investigated expression of AC1 and AC8 as a possible explanation to these observations. Genetic perturbation of AC1 and AC8 by double-knockout of Adcy1 and Adcy8 in a mouse model showed a decrease in positive chronotropic and inotropic response upon cumulative dose of PE in atrial tissue, reduced PE stimulated amplitude of Ca2+ transient in isolated atrial myocytes and presented decreased cytosolic cAMP levels in response to PE in neonatal atrial myocytes that was not inhibited by NAADP pathway inhibitors. Our data identifies a link between NAADP and α-adrenergic signalling pathways in atrial myocytes, highlighting that lysosomal Ca2+ is an important component of α-adrenergic stimulation in the cardiac atria and warrants further investigation. ### Competing Interest Statement The authors have declared no competing interest.

Endolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL facilitate the elimination of damaged membranes, protein aggregates, membranous organelles and play an important role in calcium signaling. The specific role of EL in cardiac atrial fibrillation (AF) is not well understood. We isolated atrial EL organelles from AF goat biopsies and conducted a comprehensive integrated omics analysis to study the EL-specific proteins and pathways. We also performed electron tomography, protein and enzyme assays on these biopsies. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates, including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophagic-vacuole formation, irregularly shaped mitochondria, and glycogen deposition. Our results provide molecular information suggesting EL play a role in AF disease process over extended time frames.

Minimally invasive cardiac surgery (MICS) has been used since the 1990s and encompasses a wide range of techniques that lack full sternotomy, including valve and coronary artery graft surgery as well as transcatheter procedures. Due to the potential benefits offered to patients by MICS, these procedures are becoming more common. Unique anaesthetic knowledge and skills are required to overcome the specific challenges presented by MICS, including mastery of transoesophageal echocardiography (TOE) and the provision of thoracic regional analgesia. This review evaluates the relevance of MICS to the anaesthetist and discusses pre-operative assessment, the relevant adjustments to intra-operative conduct that are necessary for these techniques, as well as post-operative care and what is known about outcomes.

Support with VV-ECMO requires anticoagulation with unfractionated heparin to prevent thrombotic complications. This must be monitored due to bleeding risk. A point-of-care (POC) method of testing aPTT and APR was evaluated for agreement with laboratory methods. In a prospective observational study, patients supported on VV-ECMO as a result of severe respiratory failure secondary to Covid-19 infection were given heparin as part of standard therapy. The aPTT was measured (i) at the bedside using the Hemochron Signature Elite device and (ii) at the hospital laboratory. Duplicate results were compared. Agreement between the POC and laboratory tests was poor, as assessed using the Bland-Altman method. The maximum difference between POC and laboratory methods was 133% and the minimum was 0%. Overall bias was 7.3% and limits of agreement were between -43.8% and 58.5%. Correlation increased when results were normalised to platelet count and creatinine. This POC test is insufficiently accurate for use as the primary method of heparin monitoring in patients requiring VV-ECMO for Covid-19. Platelets and renal function may influence the result of this whole blood POC test.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a highly potent calcium-mobilising second messenger which stimulates calcium release from endo-lysosomes via the opening of TPC2 channels. In cardiac ventricular myocytes, lysosomes form nano-junctions with the sarcoplasmic reticulum (SR) and mitochondria and exposure to NAADP (synthesised by CD38 upon beta-adrenergic stimulation), leads to an augmented calcium transient and SR calcium content, and contributes to the hypertrophy and arrhythmogenesis associated with chronic beta-adrenergic stress. We utilised transmission electron microscopy (TEM) and optical mapping in lapine and murine models to investigate endo-lysosomal calcium signalling in the cardiac atria. TEM of fixed, imbedded rabbit atrial myocytes or tissue (n=3 animals) confirmed that lysosomes form nano-junctions with both the SR (median distance 17nm, IQR 13-21nm, n=15 lysosomes) and mitochondria (median distance 13nm, IQR 10-57nm, n=16 lysosomes). These are close enough to have the potential for localised calcium signalling. In intact murine left atria, 10nM isoprenaline caused a 59.3±4.8% increase in calcium transient amplitude (n=6, fluorophore rhod-2). Abolishing endo-lysosomal calcium storage using 5µM Bafilomycin A1 (Baf) significantly reduced the calcium transient response to isoprenaline (to 35.3±2.7%, n=6, P<0.01). In CD38KO mice, left atrial responses were significantly reduced (35.9±2.6%, n=6) in comparison to wild-type controls (59.3±4.9%, n=6, P<0.01) and there was no longer an effect of Baf on isoprenaline response (35.9±2.6% and 33.6±4.0% in CD38KO control and Baf respectively, P>0.05, both n=6). Our data suggest that, endo-lysosomes can form nanojunctions for calcium signalling within cardiac atrial myocytes and that the endo-lysosomal NAADP pathway is functionally relevant during beta-adrenergic stimulation. The changes to and effects of this stereotyped architecture and pathway in atrial disease states are unknown and will be a vital avenue for future research.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 μm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

Myocardial microstructure and its macroscopic materialisation are fundamental to the function of the heart. Despite this importance, characterisation of cellular features at the organ level remains challenging, and a unifying description of the structure of the heart is still outstanding. Here, we optimised diffusion tensor imaging data to acquire high quality data in ex vivo rabbit hearts in slack and contractured states, approximating diastolic and systolic conditions. The data were analysed with a suite of methods that focused on different aspects of the myocardium. In the slack heart, we observed a similar transmural gradient in helix angle of the primary eigenvector of up to 23.6°/mm in the left ventricle and 24.2°/mm in the right ventricle. In the contractured heart, the same transmural gradient remained largely linear, but was offset by up to +49.9° in the left ventricle. In the right ventricle, there was an increase in the transmural gradient to 31.2°/mm and an offset of up to +39.0°. The application of tractography based on each eigenvector enabled visualisation of streamlines that depict cardiomyocyte and sheetlet organisation over large distances. We observed multiple V- and N-shaped sheetlet arrangements throughout the myocardium, and insertion of sheetlets at the intersection of the left and right ventricle. This study integrates several complementary techniques to visualise and quantify the heart’s microstructure, projecting parameter representations across different length scales. This represents a step towards a more comprehensive characterisation of myocardial microstructure at the whole organ level.

Ca(2+)-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca(2+) responses in cardiac myocytes from Tpcn2(-/-) mice, unlike myocytes from wild-type (WT) mice. Ca(2+)/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca(2+) transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2(-/-) mice. Increases in amplitude of L-type Ca(2+) currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2(-/-) mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2(-/-) mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2(-/-) mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼ 25 nm). We propose that Ca(2+)-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias.