Spatially resolved multiomics of human cardiac niches.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.

Efficient generation of TBX3+ atrioventricular conduction-like cardiomyocytes from human pluripotent stem cells.

Atrioventricular conduction cardiomyocytes (AVCCs) regulate the rate and rhythm of heart contractions. Dysfunction due to aging or disease can cause atrioventricular (AV) block, interrupting electrical impulses from the atria to the ventricles. Generation of functional atrioventricular conduction like cardiomyocytes (AVCLCs) from human pluripotent stem cells (hPSCs) provides a promising approach to repair damaged atrioventricular conduction tissue by cell transplantation. In this study, we put forward the generation of AVCLCs from hPSCs by stage-specific manipulation of the retinoic acid (RA), WNT, and bone morphogenetic protein (BMP) signaling pathways. These cells express AVCC-specific markers, including the transcription factors TBX3, MSX2 and NKX2.5, display functional electrophysiological characteristics and present low conduction velocity (0.07 ± 0.02 m/s). Our findings provide new insights into the understanding of the development of the atrioventricular conduction system and propose a strategy for the treatment of severe atrioventricular conduction block by cell transplantation in future.

In vivo visualization and molecular targeting of the cardiac conduction system.

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against the CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects on CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo, providing a proof of principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging, and arrhythmia management.

Animal Models to Study Cardiac Arrhythmias.

Cardiac arrhythmias are a significant cause of morbidity and mortality worldwide, accounting for 10% to 15% of all deaths. Although most arrhythmias are due to acquired heart disease, inherited channelopathies and cardiomyopathies disproportionately affect children and young adults. Arrhythmogenesis is complex, involving anatomic structure, ion channels and regulatory proteins, and the interplay between cells in the conduction system, cardiomyocytes, fibroblasts, and the immune system. Animal models of arrhythmia are powerful tools for studying not only molecular and cellular mechanism of arrhythmogenesis but also more complex mechanisms at the whole heart level, and for testing therapeutic interventions. This review summarizes basic and clinical arrhythmia mechanisms followed by an in-depth review of published animal models of genetic and acquired arrhythmia disorders.

Paclitaxel mitigates structural alterations and cardiac conduction system defects in a mouse model of Hutchinson-Gilford progeria syndrome.

AIMS: Hutchinson-Gilford progeria syndrome (HGPS) is an ultrarare laminopathy caused by expression of progerin, a lamin A variant, also present at low levels in non-HGPS individuals. HGPS patients age and die prematurely, predominantly from cardiovascular complications. Progerin-induced cardiac repolarization defects have been described previously, although the underlying mechanisms are unknown. METHODS AND RESULTS: We conducted studies in heart tissue from progerin-expressing LmnaG609G/G609G (G609G) mice, including microscopy, intracellular calcium dynamics, patch-clamping, in vivo magnetic resonance imaging, and electrocardiography. G609G mouse cardiomyocytes showed tubulin-cytoskeleton disorganization, t-tubular system disruption, sarcomere shortening, altered excitation-contraction coupling, and reductions in ventricular thickening and cardiac index. G609G mice exhibited severe bradycardia, and significant alterations of atrio-ventricular conduction and repolarization. Most importantly, 50% of G609G mice had altered heart rate variability, and sinoatrial block, both significant signs of premature cardiac aging. G609G cardiomyocytes had electrophysiological alterations, which resulted in an elevated action potential plateau and early afterdepolarization bursting, reflecting slower sodium current inactivation and long Ca+2 transient duration, which may also help explain the mild QT prolongation in some HGPS patients. Chronic treatment with low-dose paclitaxel ameliorated structural and functional alterations in G609G hearts. CONCLUSIONS: Our results demonstrate that tubulin-cytoskeleton disorganization in progerin-expressing cardiomyocytes causes structural, cardiac conduction, and excitation-contraction coupling defects, all of which can be partially corrected by chronic treatment with low dose paclitaxel.

Why Is Only Type 1 Electrocardiogram Diagnostic of Brugada Syndrome? Mechanistic Insights From Computer Modeling.

BACKGROUND: Three types of characteristic ST-segment elevation are associated with Brugada syndrome but only type 1 is diagnostic. Why only type 1 ECG is diagnostic remains unanswered. METHODS: Computer simulations were performed in single cells, 1-dimensional cables, and 2-dimensional tissues to investigate the effects of the peak and late components of the transient outward potassium current (Ito), sodium current, and L-type calcium current (ICa,L) as well as other potassium currents on the genesis of ECG morphologies and phase 2 reentry (P2R). RESULTS: Although a sufficiently large peak Ito was required to result in the type 1 ECG pattern and P2R, increasing the late component of Ito converted type 1 ECG to type 2 ECG and suppressed P2R. Increasing the peak Ito promoted spiral wave breakup, potentiating the transition from tachycardia to fibrillation, but increasing the late Ito prevented spiral wave breakup by flattening the action potential duration restitution and preventing P2R. A sufficiently large ICa,L conductance was needed for P2R to occur, but once above the critical conductance, blocking ICa,L promoted P2R. However, selectively blocking the window and late components of ICa,L suppressed P2R, countering the effect of the late Ito. Blocking either the peak or late components of sodium current promoted P2R, with the late sodium current blockade having the larger effect. As expected, increasing other potassium currents potentiated P2R, with ATP-sensitive potassium current exhibiting a larger effect than rapid and slow component of the delayed rectifier potassium current. CONCLUSIONS: The peak Ito promotes type 1 ECG and P2R, whereas the late Ito converts type 1 ECG to type 2 ECG and suppresses P2R. Blocking the peak ICa,L and either the peak or the late sodium current promotes P2R, whereas blocking the window and late ICa,L suppresses P2R. These results provide important insights into the mechanisms of arrhythmogenesis and potential therapeutic targets for treatment of Brugada syndrome. Graphic Abstract: A graphic abstract is available for this article.

Deletion of Sphingosine 1-Phosphate receptor 1 in cardiomyocytes during development leads to abnormal ventricular conduction and fibrosis.

Sphingosine 1-Phosphate receptor 1 (S1P1 , encoded by S1pr1) is a G protein-coupled receptor that signals in multiple cell types including endothelial cells and cardiomyocytes. Cardiomyocyte-specific deletion of S1pr1 during mouse development leads to ventricular noncompaction, with 44% of mutant mice surviving to adulthood. Adult survivors of embryonic cardiomyocyte S1pr1 deletion showed cardiac hypertrabeculation consistent with ventricular noncompaction. Surprisingly, systolic function in mutant mice was preserved through at least 1 year of age. Cardiac conduction was abnormal in cardiomyocyte S1pr1 mutant mice, with prolonged QRS intervals in mutants as compared with littermate control mice. Immunostaining of hearts from S1pr1 mutant embryos displayed a zone of intermediate Connexin 40 (Cx40) expression in the trabecular myocardium. However, we observed no significant differences in Cx40 and Connexin 43 immunostaining in hearts from adult survivors of embryonic cardiomyocyte S1pr1 deletion, which suggests normalized development of the ventricular conduction system in mutant mice. By contrast, the adult survivors of embryonic cardiomyocyte S1pr1 deletion showed increased cardiac fibrosis as compared with littermate controls. These results demonstrate that ventricular hypertrabeculation caused by embryonic deletion of cardiomyocyte S1pr1 correlates with cardiac fibrosis, which contributes to abnormal ventricular conduction. These results also reveal conduction abnormalities in the setting of hypertrabeculation with normal systolic function, which may be of clinical relevance in humans with ventricular hypertrabeculation.

Neural cell adhesion molecule is required for ventricular conduction system development.

The most distal portion of the ventricular conduction system (VCS) contains cardiac Purkinje cells (PCs), which are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily of cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling, we discovered two additional highly enriched IgSF-CAMs in the VCS: NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early postnatal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in PC gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid leads to disrupted trafficking of sarcolemmal intercalated disc proteins to junctional membranes and abnormal expansion of the extracellular space between apposing PCs. Taken together, our data provide insights into the complex developmental biology of the ventricular conduction system.

The conduction velocity-potassium relationship in the heart is modulated by sodium and calcium.

The relationship between cardiac conduction velocity (CV) and extracellular potassium (K+) is biphasic, with modest hyperkalemia increasing CV and severe hyperkalemia slowing CV. Recent studies from our group suggest that elevating extracellular sodium (Na+) and calcium (Ca2+) can enhance CV by an extracellular pathway parallel to gap junctional coupling (GJC) called ephaptic coupling that can occur in the gap junction adjacent perinexus. However, it remains unknown whether these same interventions modulate CV as a function of K+. We hypothesize that Na+, Ca2+, and GJC can attenuate conduction slowing consequent to severe hyperkalemia. Elevating Ca2+ from 1.25 to 2.00 mM significantly narrowed perinexal width measured by transmission electron microscopy. Optically mapped, Langendorff-perfused guinea pig hearts perfused with increasing K+ revealed the expected biphasic CV-K+ relationship during perfusion with different Na+ and Ca2+ concentrations. Neither elevating Na+ nor Ca2+ alone consistently modulated the positive slope of CV-K+ or conduction slowing at 10-mM K+; however, combined Na+ and Ca2+ elevation significantly mitigated conduction slowing at 10-mM K+. Pharmacologic GJC inhibition with 30-µM carbenoxolone slowed CV without changing the shape of CV-K+ curves. A computational model of CV predicted that elevating Na+ and narrowing clefts between myocytes, as occur with perinexal narrowing, reduces the positive and negative slopes of the CV-K+ relationship but do not support a primary role of GJC or sodium channel conductance. These data demonstrate that combinatorial effects of Na+ and Ca2+ differentially modulate conduction during hyperkalemia, and enhancing determinants of ephaptic coupling may attenuate conduction changes in a variety of physiologic conditions.

Canagliflozin Suppresses Atrial Remodeling in a Canine Atrial Fibrillation Model.

Background Recent clinical trials have demonstrated the possible pleiotropic effects of SGLT2 (sodium-glucose cotransporter 2) inhibitors in clinical cardiovascular diseases. Atrial electrical and structural remodeling is important as an atrial fibrillation (AF) substrate. Methods and Results The present study assessed the effect of canagliflozin (CAN), an SGLT2 inhibitor, on atrial remodeling in a canine AF model. The study included 12 beagle dogs, with 10 receiving continuous rapid atrial pacing and 2 acting as the nonpacing group. The 10 dogs that received continuous rapid atrial pacing for 3 weeks were subdivided as follows: pacing control group (n=5) and pacing+CAN (3 mg/kg per day) group (n=5). The atrial effective refractory period, conduction velocity, and AF inducibility were evaluated weekly through atrial epicardial wires. After the protocol, atrial tissues were sampled for histological examination. The degree of reactive oxygen species expression was evaluated by dihydroethidium staining. The atrial effective refractory period reduction was smaller (P=0.06) and the degree of conduction velocity decrease was smaller in the pacing+CAN group compared with the pacing control group (P=0.009). The AF inducibility gradually increased in the pacing control group, but such an increase was suppressed in the pacing+CAN group (P=0.011). The pacing control group exhibited interstitial fibrosis and enhanced oxidative stress, which were suppressed in the pacing+CAN group. Conclusions CAN and possibly other SGLT2 inhibitors might be useful for preventing AF and suppressing the promotion of atrial remodeling as an AF substrate.

Two-hit mechanism of cardiac arrhythmias in diabetic hyperglycaemia: reduced repolarization reserve, neurohormonal stimulation, and heart failure exacerbate susceptibility.

AIMS: Diabetic hyperglycaemia is associated with increased arrhythmia risk. We aimed to investigate whether hyperglycaemia alone can be accountable for arrhythmias or whether it requires the presence of additional pathological factors. METHODS AND RESULTS: Action potentials (APs) and arrhythmogenic spontaneous diastolic activities were measured in isolated murine ventricular, rabbit atrial, and ventricular myocytes acutely exposed to high glucose. Acute hyperglycaemia increased the short-term variability (STV) of action potential duration (APD), enhanced delayed afterdepolarizations, and the inducibility of APD alternans during tachypacing in both murine and rabbit atrial and ventricular myocytes. Hyperglycaemia also prolonged APD in mice and rabbit atrial cells but not in rabbit ventricular myocytes. However, rabbit ventricular APD was more strongly depressed by block of late Na+ current (INaL) during hyperglycaemia, consistent with elevated INaL in hyperglycaemia. All the above proarrhythmic glucose effects were Ca2+-dependent and abolished by CaMKII inhibition. Importantly, when the repolarization reserve was reduced by pharmacological inhibition of K+ channels (either Ito, IKr, IKs, or IK1) or hypokalaemia, acute hyperglycaemia further prolonged APD and further increased STV and alternans in rabbit ventricular myocytes. Likewise, when rabbit ventricular myocytes were pretreated with isoproterenol or angiotensin II, hyperglycaemia significantly prolonged APD, increased STV and promoted alternans. Moreover, acute hyperglycaemia markedly prolonged APD and further enhanced STV in failing rabbit ventricular myocytes. CONCLUSION: We conclude that even though hyperglycaemia alone can enhance cellular proarrhythmic mechanisms, a second hit which reduces the repolarization reserve or stimulates G protein-coupled receptor signalling greatly exacerbates cardiac arrhythmogenesis in diabetic hyperglycaemia.

Anatomical evidence of non-parasympathetic cardiac nitrergic nerve fibres in rat.

Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) plays a major role in the neural control of circulation and in many cardiovascular diseases. However, the exact mechanism of how NO regulates these processes is still not fully understood. This study was designed to determine the possible sources of nitrergic nerve fibres supplying the heart attempting to imply their role in the cardiac neural control. Sections of medulla oblongata, vagal nerve, its rootlets and nodose ganglia, vagal cardiac branches, Th1 -Th5 spinal cord segments, dorsal root ganglia of C8 -Th5 spinal nerves, and stellate ganglia from 28 Wistar rats were examined applying double immunohistochemical staining for nNOS combined with choline acetyltransferase (ChAT), peripherin, substance P, calcitonin gene-related peptide, tyrosine hydroxylase or myelin basic protein. Our findings show that the most abundant population of purely nNOS-immunoreactive (IR) neuronal somata (NS) was observed in the nodose ganglia (37.4 ± 1.3%). A high number of nitrergic NFs spread along the vagal nerve and entered its cardiac branches. All nitrergic neuronal somata (NS) in the nucleus ambiguus were simultaneously immunoreactive (IR) to ChAT and composed only a small subset of neurons (6%). In the dorsal nucleus of vagal nerve, biphenotypic nNOS-IR/ChAT-IR neurons composed 7.0 ± 1.0%, while small purely nNOS-IR neurons were scarce. Nitrergic NS were plentifully distributed within the nuclei of solitary tract. In the examined dorsal root and stellate ganglia, a few nitrergic NS were sporadically present. The majority of sympathetic NS in the intermediolateral nucleus were simultaneously immunoreactive for nNOS and ChAT. In conclusion, an abundant population of nitrergic NS in the nodose ganglion implies that neuronal NO is involved in afferent cardiac innervation. Nevertheless, nNOS-IR neurons identified within vagal nuclei may play a role in the transmission of preganglionic parasympathetic nerve impulses.

Cardiohemodynamic and Arrhythmogenic Effects of the Anti-Atrial Fibrillatory Compound Vanoxerine in Halothane-Anesthetized Dogs.

While vanoxerine (GBR-12909) is a synaptosomal dopamine uptake inhibitor, it also suppresses IKr, INa and ICa,L in vitro. Based on these profiles on ionic currents, vanoxerine has been developed as a candidate compound for treating atrial fibrillation. To investigate electropharmacological profiles, vanoxerine dihydrochloride was intravenously administered at 0.03 and 0.3 mg/kg to halothane-anesthetized dogs (n = 4), possibly providing subtherapeutic and therapeutic concentrations, respectively. The low dose increased the heart rate and cardiac output, whereas it prolonged the ventricular refractoriness. The high dose decreased the heart rate but increased the total peripheral vascular resistance, whereas it delayed the ventricular repolarization and increased the atrial refractoriness in addition to further enhancing the ventricular refractoriness. The extent of increase in the refractoriness in the atrium was 0.8 times of that in the ventricle. The high dose also prolonged the early and late repolarization periods of the ventricle as well as the terminal repolarization period. Meanwhile, no significant change was detected in the mean blood pressure, ventricular contraction, preload to the left ventricle, or the intra-atrial, intra-ventricular or atrioventricular conductions. The high dose can be considered to inhibit IKr, but it may not suppress INa or ICa in the in situ heart, partly explaining its poor atrial selectivity for increasing refractoriness. The prolongation of early repolarization period may reflect enhancement of net inward current, providing potential risk for intracellular Ca2+ overload. Thus, vanoxerine may provide both trigger and substrate toward torsade de pointes, which would make the drug less promising as an anti-atrial fibrillatory drug.

Human influenza A virus causes myocardial and cardiac-specific conduction system infections associated with early inflammation and premature death.

AIMS: Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations. METHODS AND RESULTS: We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase. CONCLUSION: Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death.

NOD Mice Recapitulate the Cardiac Disturbances Observed in Type 1 Diabetes.

This work aimed at testing the hypothesis that NOD/ShiLtJ mice (NOD) recapitulate the cardiac disturbances observed on type 1 diabetes (T1D). NOD mice were studied 4 weeks after the onset of hyperglycemia, and NOR/Lt mice matched as control. Cardiac function was evaluated by echocardiography and electrocardiography (ECG). Action potentials (AP) and Ca2+ transients were evaluated at whole heart level. Heart mitochondrial function was evaluated by high-resolution respirometry and H2O2 release. NOD mice presented a reduction in hearth weight. Mitochondrial oxygen fluxes and H2O2 release were similar between NOD and NOR mice. ECG revealed a QJ interval prolongation in NOD mice. Furthermore, AP duration at 30% of repolarization was increased, and it depicted slower Ca2+ transient kinetics. NOD mice presented greater number/severity of ventricular arrhythmias both in vivo and in vitro. In conclusion, NOD mice evoked cardiac electrical and calcium handling disturbances similar to the observed in T1D. Graphical Abstract .

Heart regeneration: beyond new muscle and vessels.

The most striking consequence of a heart attack is the loss of billions of heart muscle cells, alongside damage to the associated vasculature. The lost cardiovascular tissue is replaced by scar formation, which is non-functional and results in pathological remodelling of the heart and ultimately heart failure. It is, therefore, unsurprising that the heart regeneration field has centred efforts to generate new muscle and blood vessels through targeting cardiomyocyte proliferation and angiogenesis following injury. However, combined insights from embryological studies and regenerative models, alongside the adoption of -omics technology, highlight the extensive heterogeneity of cell types within the forming or re-forming heart and the significant crosstalk arising from non-muscle and non-vessel cells. In this review, we focus on the roles of fibroblasts, immune, conduction system, and nervous system cell populations during heart development and we consider the latest evidence supporting a function for these diverse lineages in contributing to regeneration following heart injury. We suggest that the emerging picture of neurologically, immunologically, and electrically coupled cell function calls for a wider-ranging combinatorial approach to heart regeneration.

Electrical remodeling and cardiotoxicity precedes structural and functional remodeling of mouse hearts under hyperoxia treatment.

Clinical reports suggest a high incidence of ICU mortality with the use of hyperoxia during mechanical ventilation in patients. Our laboratory is pioneer in studying effect of hyperoxia on cardiac pathophysiology. In this study for the first time, we are reporting the sequence of cardiac pathophysiological events in mice under hyperoxic conditions in time-dependent manner. C57BL/6J male mice, aged 8-10 weeks, were treated with either normal air or >90% oxygen for 24, 48, and 72 h. Following normal air or hyperoxia treatment, physical, biochemical, functional, electrical, and molecular parameters were analyzed. Our data showed that significant reduction of body weight observed as early as 24 h hyperoxia treatment, whereas, no significant changes in heart weight until 72 h. Although we do not see any fibrosis in these hearts, but observed significant increase in cardiomyocyte size with hyperoxia treatment in time-dependent manner. Our data also demonstrated that arrhythmias were present in mice at 24 h hyperoxia, and worsened comparatively after 48 and 72 h. Echocardiogram data confirmed cardiac dysfunction in time-dependent manner. Dysregulation of ion channels such as Kv4.2 and KChIP2; and serum cardiac markers confirmed that hyperoxia-induced effects worsen with each time point. From these observations, it is evident that electrical remodeling precedes structural remodeling, both of which gets worse with length of hyperoxia exposure, therefore shorter periods of hyperoxia exposure is always beneficial for better outcome in ICU/critical care units.

Vascular endothelial growth factor promotes atrial arrhythmias by inducing acute intercalated disk remodeling.

Atrial fibrillation (AF) is the most common arrhythmia and is associated with inflammation. AF patients have elevated levels of inflammatory cytokines known to promote vascular leak, such as vascular endothelial growth factor A (VEGF). However, the contribution of vascular leak and consequent cardiac edema to the genesis of atrial arrhythmias remains unknown. Previous work suggests that interstitial edema in the heart can acutely promote ventricular arrhythmias by disrupting ventricular myocyte intercalated disk (ID) nanodomains rich in cardiac sodium channels (NaV1.5) and slowing cardiac conduction. Interestingly, similar disruption of ID nanodomains has been identified in atrial samples from AF patients. Therefore, we tested the hypothesis that VEGF-induced vascular leak can acutely increase atrial arrhythmia susceptibility by disrupting ID nanodomains and slowing atrial conduction. Treatment of murine hearts with VEGF (30-60 min, at clinically relevant levels) prolonged the electrocardiographic P wave and increased susceptibility to burst pacing-induced atrial arrhythmias. Optical voltage mapping revealed slower atrial conduction following VEGF treatment (10 ± 0.4 cm/s vs. 21 ± 1 cm/s at baseline, p < 0.05). Transmission electron microscopy revealed increased intermembrane spacing at ID sites adjacent to gap junctions (GJs; 64 ± 9 nm versus 17 ± 1 nm in controls, p < 0.05), as well as sites next to mechanical junctions (MJs; 63 ± 4 nm versus 27 ± 2 nm in controls, p < 0.05) in VEGF-treated hearts relative to controls. Importantly, super-resolution microscopy and quantitative image analysis revealed reorganization of NaV1.5 away from dense clusters localized near GJs and MJs to a more diffuse distribution throughout the ID. Taken together, these data suggest that VEGF can acutely predispose otherwise normal hearts to atrial arrhythmias by dynamically disrupting NaV1.5-rich ID nanodomains and slowing atrial conduction. These data highlight inflammation-induced vascular leak as a potential factor in the development and progression of AF.