Cross-Talk of Cells in the Heart: Novel Mechanisms of Disease and Arrhythmias

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Fibroblast-Myocyte interactions and their impact on cardiac electrophysiology

The heart is a complex organ composed of multiple cell types working together to maintain its rhythmic and contractile function. Among the key cell types within the heart are myocytes and cardiac fibroblasts. Myocytes are the primary muscle cells responsible for the mechanical contraction of the heart, enabling it to pump blood efficiently throughout the body. Fibroblasts are the most abundant non-myocyte cells in the heart, and they are responsible for producing and maintaining the extracellular matrix, which provides structural and mechanical support for the cardiac tissue. Fibroblasts number further increases with ageing and pathological conditions [1]. Traditionally considered as passive cells providing structural support, research has now unveiled an active and dynamic role for cardiac fibroblasts in modulating cardiac electrophysiology. In addition to forming passive obstacles to electrical conduction, fibroblasts have been proposed to actively affect cardiac conduction, myocyte excitability, contractility, action potential duration and calcium cycling. Mechanisms include heterocellular cell-cell coupling and paracrine communication [2]. Functional fibroblast-myocyte coupling mediated by gap junctions or tunnelling nanotubes has been observed both in vitro and in situ in animal models [3,4,5]. Importantly, in situ this coupling is limited to cells in the sinoatrial node and in the ventricle at the border zone of post-myocardial infarcts [5,6]. Coupling also occurs between senescence fibroblasts and myocytes at the infarct border zone in the aged rabbit heart, promoting significant arrhythmogenic remodelling [7]. Paracrine communication, mediated by cytokines and growth factors released by cardiac fibroblasts, has also been shown to influence myocyte electrophysiology and calcium handling in animal cell cultures [8,9]. Recently, advanced human cell coculture systems mimicking different cell-cell interaction modalities have been employed to assess the contribution of contact versus paracrine interactions. Activated human cardiac fibroblasts exert adverse effects on the electrophysiological and Ca²⁺ handling of human myocytes and downregulate voltage channels (K_V4.3, K_V11.1 and Kir6.2) and SERCA2a calcium pump. Interleukin-6 and connexin43 were identified as paracrine- and contact-mediators driving these effects [10]. Thus, fibroblast-myocyte interactions are complex and dynamic processes that play a crucial role in cardiac electrophysiology. Understanding these interactions can help to elucidate the mechanisms of normal and abnormal cardiac function, as well as to develop novel strategies for preventing and treating cardiac arrhythmias and progression to heart failure. Details of fibroblast-myocyte interactions in disease conditions such as ischemia-reperfusion injury, hypertrophic and dilated cardiomyopathy as well as in the ageing heart are still poorly explored and form a potentially exciting target for further research. Emerging tools and experimental model systems [11,12] will be invaluable to accelerate this research.

Cardiac fibroblast activation and reversal and their interaction with cardiomyocytes

Introduction: Cardiac fibroblasts (cFbs) maintain the extracellular matrix and provide a scaffold for cardiomyocytes (CMs). Following injury, cFbs become activated to myofibroblasts (MyoFbs) to aid in wound healing and repair. In disease, myofibroblasts contribute to pathological remodelling and cardiac fibrosis. This study aimed to investigate the potential for reversal of chronically activated human cardiac fibroblasts in 2D culture. We also evaluated the effects of quiescent and activated human cardiac fibroblasts on cardiomyocyte electrical and contractile function.

Methods: Primary human cFbs and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were assessed for activation following 2-4 weeks of culture on stiff culture plastic (E = ~ 3 GPa) and in the presence of 10 ng/ml TGF-β. Myofibroblast reversal was assessed using TGF-β receptor inhibition (10 μM SB431542). mRNA and protein expression of activation markers α-SMA, collagens 1 and 3 were investigated. α-SMA expression was also assessed using immunofluorescence staining. Quiescent or activated iPSC-cFbs were plated on top of iPSC-CMs for 5 days. Contraction was assessed using MUSCLEMOTION and electrical activity using optical mapping.

Results: Primary cFbs and hiPSC-cFbs activate to myofibroblasts following culture on stiff plastic. In the absence of any chemical stimuli. Long term culture on stiff plastic led to both cell types becoming unresponsive to TGF-β stimulation or TGF-β receptor inhibition. hiPSC-cFbs remained quiescent in the presence of SB431542, while still being able to activate into MyoFbs upon TGF-β exposure (evident through increased α-SMA and collagen 1). Co-culture for five days with either cFb type did not affect CM contraction or calcium transients. TGF-β-exposed CMs showed increased action potential duration 50, 70 and 90 compared to SB431542-treated CMs, but no significant changes occurred with quiescent or activated fibroblasts.

Conclusions: Human cFbs activate independently of any external stimulus during standard 2D culture. Chronically activated human cFbs have a limited potential for reversal to a quiescent phenotype. Activation status of hiPSC-cFbs does not significantly affect contraction or electrical handling of iPSC-CMs.

Small extracellular vesicles released by cardiac fibroblasts from hypertrophic cardiomyopathy patients alter action potential of human cardiomyocytes

Cardiac fibroblasts (CFs) are abundant in the heart, where they play a major role in cardiac homeostasis and disease development (1). Recently, small extracellular vesicles (sEV) released by mouse CFs have been implicated in cardiac hypertrophy (2). However, if human CF sEV have a role in cardiac disease remains unclear. Here we isolate CFs from hypertrophic cardiomyopathy (HCM) patients, purify and characterise their sEV and study the effect of these sEV on Ca²⁺ cycling and action potentials of human cardiomyocytes (CMs).

CFs isolated from HCM patients’ biopsies were characterised by RT-qPCR, flow cytometry and immunohistochemistry. Media conditioned by CFs over 72 hours was concentrated by ultrafiltration. sEV were purified by size exclusion chromatography and characterised by nanoparticle tracking analysis and tetraspanin staining, purity was determined using microBCA. CMs were differentiated from human induced pluripotent stem cells and cultured for 28 days before purification using MACS bead separation. Purified CMs were cultured with sEV from HCM CFs for 48 hours. Action potentials and calcium (Ca²⁺) transients were studied using optical mapping, with calcium dye (Fluo-4) or voltage dye (FluoVolt), during field stimulation at 0.7Hz, 1Hz and 1.5Hz. Signals were analysed using OPTIQ software. Unpaired T-tests were used for statistical analysis of 2 groups.

CFs from HCM patient biopsies had a myofibroblast pheno/genotype determined by the presence of myofibroblast markers: α-SMA, COL1A1, FAP. Pure sEV were found in fractions 7-9 (>2×10¹⁰ particles/µg), had a modal size of 109±3.5nm and were positive for the tetraspanins CD63, CD81 and CD9. On average HCM CFs secreted 104,676 sEV per cell (n=3). CM Ca²⁺transient parameters, time to peak and time to 50/80% Ca²⁺ decay, were unaffected by HCM CF sEV at a concentration of 5×10¹⁰ particles/ml (p>0.05 vs untreated CM, n=4). However, CM action potentials were prolonged after culture with HCM CF sEV at a concentration of 5×10¹⁰ particles/ml with a significant increase in action potential duration (APD)50 at 1Hz (p<0.01 vs untreated CM), 0.7Hz and 1.5Hz (p<0.05); and significant increase in APD80 at 1Hz and 0.7Hz (p<0.01) and 1.5Hz (p<0.05, n=4).

Our results indicate that CFs isolated from HCM patients have a myofibroblast phenotype and secrete a large number sEV. Treating human CMs with highly pure sEV secreted by HCM CFs prolongs CM action potential duration, highlighting a potential role of CF sEV in heart disease.

Calcium signalling in cardiac fibroblast and myocytes in an in vitro model of heart failure with preserved ejection fraction.

Introduction:

Heart Failure with preserved Ejection Fraction [HFpEF] is a pandemic associated with hypertension and diabetes amongst other co-morbidities. It has been established that fibrosis plays a major role in this form of heart failure. However, the functional mechanisms of cardiac fibroblasts (CFs) in HFpEF and more importantly, how these cells interact with cardiomyocytes (CMs) and impact calcium (Ca²⁺) signalling is limited. This limitation is majorly due to a lack of physiologically relevant cellular models.

Aims/Objectives:

To study the CF-CM interaction in an in vitro model of HFpEF, this study aimed to evaluate the Ca²⁺ response of CFs in diabetic, hypertensive, and HFpEF conditions. Furthermore, the electrophysiology of CMs co-cultured with CFs, under these pathological conditions was assessed.

Methods:

Adult human male CFs (HcFb) (Promocell) at Passage 3 were seeded at a density of 1×10⁴cells per well in a 96-well plate and grown for 24 hours in fibroblast growth medium 3 (FGM3) supplemented with 10% Foetal Calf Serum (FCS) and 1% Penicillin/Streptomycin (P/S). Cells were serum starved for 24 hours prior to treatment. For CFs Ca²⁺ signalling, fibroblasts were treated for 72 hours with high glucose (22mM) to simulate diabetes, high Angiotensin-II (Ang-II) (200nM) to simulate hypertension, or both conditions to simulate HFpEF. Cells were stained with 2µM Cal520 AM calcium indicator and the Ca²⁺ response induced by the addition of 20nM Endothelin-1 was measured by confocal microscopy. For co-culture, adult male rabbit CMs were freshly isolated following all ethical requirements by the UK Home Office Licence. CMs were cultured with(out) CFs in either Medium 199 (M199) or FGM3, using treatment conditions similar to the above. Following treatment, CMs were harvested, stained, and electrically stimulated at 1Hz to measure single cell Ca²⁺ transients.

Results:

The Ca²⁺ transient amplitude of CMs was significantly reduced when cultured in FGM3 or co-cultured with HcFb in M199, compared to M199 (M199: 0.766±0.112 versus FGM3: 0.145±0.0266; co-cultured myocytes in M199: 0.347±0.0385, N=1 rabbit and n≥5 cells per group; p< 0.001, one-way ANOVA). Endothelin-1-induced Ca²⁺ response in CFs was significantly enhanced with high glucose when compared to control, hypertensive and HFpEF conditions (Control: 3.67±1.32 versus Diabetic: 13.7±1.34; Hypertensive: 6.29±1.04; HFpEF: 7.14±1.27, N=2 biological replicates with n=3 technical replicates each; p< 0.05, one-way ANOVA). Using the same treatment conditions, similar effects on Ca²⁺ transient amplitude were observed in electrically stimulated co-cultured CMs. (Control: 0.294±0.0164 versus Diabetic: 0.716±0.108; Hypertensive: 0.611±0.0935; HFpEF: 0.546±0.0460, N=1 rabbit and n≥5 cells per group; p< 0.05, one-way ANOVA). These results are contrary to existing data that show no significant change in Ca²⁺ transient amplitude of CMs treated with high glucose and increased amplitude with high Ang-II treatment.

Conclusions:

Ultimately, these findings suggest different regulation of Ca²⁺ by CFs during hypertension and HFpEF when compared with that seen during diabetes. Furthermore, data indicate a notable influence of CFs on CM Ca²⁺ transient amplitude. Ongoing work is exploring the role and mode of intercellular communication by both cell types in cardiomyopathy.

Chronic myocardial infarction attenuates transmural heterogeneities but alters the response to endothelin and ï¢-adrenergic stimulation in porcine ventricles

Increased susceptibility to ventricular arrhythmias is well documented after myocardial infarction (MI). There is consistent evidence supporting differences in transmural and apicobasal repolarization gradients as substrates for ventricular tachycardia and arrhythmogenesis. Nonetheless, there is limited direct evidence on how post infarct dynamics may differentially affect transmural regions in the heterogeneous peri-infarct zone. Information on how different cell types contribute to an arrhythmogenic electrophysiological profile is also scarce.

Aim: To assess the effects of chronic MI on centre-to-base and transmural repolarization gradients at baseline and in response to adrenergic stimulation (isoproterenol) and endothelial factors (endothelin-1, ET-1).

Methods: Domestic pigs (n=5) were infarcted by temporal occlusion of the left circumflex (LCx) coronary artery. 7-12 weeks after infarct induction, animals were cardioplegically arrested under deep anaesthesia and sacrificed. Healthy pigs (n=11) were used as controls. All animal procedures conformed to the guidelines from Directive 2010/63/EU and were approved by local authorities. Ventricular slices were produced form transmural tissue blocks obtained from remote and adjacent zones of the infarct area at the base and in the centre of the ventricular wall. Same areas of the ventricle from healthy pigs were taken as controls. Slices from the endocardium, midmyocardium and epicardium were optically mapped to record transmembrane potential. Action Potential Duration (APD) was measured at 80% repolarization at 1Hz pacing frequency at baseline conditions and after the addition of 100 nM isoproterenol or 100 nM ET-1 .

Results: Transmural heterogeneities in APD80 at basal conditions were attenuated in the infarcted tissue. Endocardial and epicardial tissue presented APD80 prolongation at the base of the infarcted hearts, especially in the areas adjacent to the infarct, while the midmyocardial tissue presented a slightly decreased APD80. At the centre, APD80 was decreased in all layers of the transmural wall, but this effect was more pronounced in the midmyocardial areas adjacent to the infarct. ET-1 prolonged APD80 by 38% (endo), 13% (mid) and 20% (endo) at the base and by 28%, 17% and 5% at the centre. Such a response was enhanced at the base in the areas adjacent to the infarct (63%, 40% and 15%) and maintained in the remote areas (14%, 18% and -3%). At the centre, the response to ET-1 was slightly diminished in the adjacent areas (3%, 25% and 5%). Isoproterenol shortened APD80 by 8% (mid) and 5% (epi) at the base and by 14% and 17% at the centre of healthy ventricles. There was a very mild response, however, at the base of the infarcted hearts (remote +1% and -3% and adjacent +5% and +11%), and the response in the centre was inverted (remote +19% and +18% and adjacent +8 and +0%).

Conclusions: Chronic MI by occlusion of LCx in pigs leads to attenuation of transmural heterogeneities, spatially heterogeneous response to adrenergic stimulation and increased sensibility to ET-1. All these factors may indicate increased susceptibility to ventricular arrhythmias. Future studies of the mechanisms underlying the here described transmural heterogeneous remodelling in MI would lead to improved antiarrhythmic therapies.

Investigating the effect of substrate stiffness on iPSC-CM structure and function

Introduction:

Cardiac ageing is characterised by increased stiffness of the myocardium, due to excess deposition of extracellular matrix (ECM) proteins such as collagen. The structural remodelling of the myocardium can consequently impact the behaviour of surrounding cells, such as cardiomyocytes (CMs). Current research using induced pluripotent stem-cell derived cardiomyocytes (iPSC-CMs) may not truly reflect the physiological stiffness of the ECM in healthy or diseased conditions. In order to assess the effect of ECM stiffness on iPSC-CMs structure and metabolic function, polydimethylsiloxane (PDMS) substrates can be used to recapitulate healthy (20kPa) and fibrotic (130kPa) stiffnesses.

Aims:

To investigate differences in structure and function of iPSC-CMs on soft and stiff PDMS substrates.

To explore changes in iPSC-CM metabolism on substrate stiffnesses.

Materials and Methods:

iPSC-CMs were plated onto PDMS gels of 20kPa and 130kPa and harvested between days 20-30 of differentiation. Molecular analyses of iPSC-CMs were investigated using qPCR, western blotting and immunofluorescence to explore cardiac gene expression profiles and structural parameters such as sarcomere length and actin organisation. Contractility of iPSC-CMs on stiffnesses were assessed using MuscleMotion software, which utilised video recordings of cells from a GoPro. Isotope labelled mass spectrometry was conducted on iPSC-CMs on varying stiffnesses to explore changes in metabolic pathways, as well as metabolic gene expression changes.

Results: Softer substrates induce maturation of iPSC-CMs, with significantly higher expression of cardiac maturity markers such as MYH7:MYH6 ratios and MYL2:MYL7 ratio compared to stiffer plastic conditions. Maturation is further displayed on 20kPa PDMS, with iPSC-CMs portraying a higher level of sarcomere alignment compared to plastic and 130kPa. Contractility of iPSC-CMs is altered on softer substrates. Metabolic profiles of iPSC-CM on stiffer gels display higher glycolytic metabolites such as glucose, whereas cells on softer substrates portray TCA cycle metabolites.

Conclusions: Substrate stiffnesses can act as physiological models of healthy and diseased ECM by portraying molecular changes in iPSC-CM structure and maturity, as well as recapitulating switches in iPSC-CM metabolism.

Exploring the role of neuronal dysfunction in inherited arrhythmia syndromes

Background: Inherited arrhythmia syndromes are a leading cause of sudden cardiac death in young and otherwise healthy patients. These are often caused by mutations in genes that encode ion channels or transporters, leading to conditions such as Brugada Syndrome (BrS), Long QT Type 3 (LQT3), and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT).

BrS and LQT3 are associated with mutations in SCN5A (encoding sodium channel, Na_v1.5), whilst CPVT is caused by mutations in RYR2 (encoding ryanodine receptor 2, RyR2). Investigation into the effects of these mutations have exclusively been performed in cardiac cells. However, these ion channels are also present in neuronal tissue, including intracardiac neurons¹ (which modulate cardiac function). Moreover, patients often present with clinical signs of dysfunction of the autonomic nervous system.

Aim: Our novel research line aims to investigate the neuronal phenotype induced by these ion channel mutations, using arrhythmia mouse models to assess functional alterations as well as broader autonomic remodelling. Ultimately, we will assess the contribution of this to arrhythmogenesis.

Results: Using immunohistochemistry approaches, we reveal for the first time that both Na_v1.5 and RyR2 are also expressed in mouse stellate ganglia (a crucial autonomic modulator of cardiac function). Current functional investigations, including patch clamp analysis, are aimed at assessing the functional consequences of Scn5a and RyR2 mutations in stellate and intracardiac ganglia in mouse models of BrS, LQT3 and CPVT. These experiments also allow for investigation of the modulatory effects of pharmacological interventions. In addition, autonomic remodelling in stellate/intracardiac ganglia and (ventricular) myocardium is currently being assessed through RNA sequencing, immunohistochemistry in mouse heart cryosections, and whole-heart neuronal imaging. Findings from these investigations will be presented.

Conclusions: All neurons that modulate heart function (stellate ganglion and intracardiac neurons) express Na_v1.5 and RYR2. Mouse models of BrS/LQTS/CPVT are employed to investigate the consequences of Scn5a/RyR2 mutations on neuronal (dys)function and their potential role in arrhythmogenesis.

Adrenergic modulation of ventricular transmural conduction velocity is a novel factor governing electrical excitability in health and disease.

Introduction – Conduction velocity (CV) is an important determinant of the arrhythmic status of the ventricle. SNS-mediated changes in CV may contribute to the normal ventricular activation sequence. We utilised a custom-made electrode array to record epicardial electrograms at sites in longitudinal, transverse, and transmural directions that incorporates a light guide to allow optical AP measurements from the electrogram recording area (Fig. 1). This set-up allows beat-to-beat assessment of myocyte to myocyte conduction and electrophysiological parameters to examine the magnitude and time course of pharmacologically mediated changes targeting the adrenergic signalling pathway. It has been well established that β-adrenoceptor expression and sympathetic nerve remodelling are pathological features of heart failure. Our approach will resolve CV changes in response to adrenergic modulation initially in the healthy heart which will then form the basis of our comparison to adrenergic signalling post-MI.

Methods – The hearts of adult, male New Zealand White rabbits were rapidly excised following terminal Euthatal administration via marginal ear vein according to UK Home Office approval and legislation. Hearts were Langendorff-perfused at 37°C and perfused with the voltage sensitive dye FluoVolt^TM to record APs. For transmural recordings, an electrode was then inserted via the left atria into the LV and placed against the endocardial surface of the mid LV free wall at a corresponding point to the endocardial electrode to measure transmural conduction velocity (CVtm). The custom endocardial electrode comprised of a pair of stimulating electrodes and two pairs of recording electrodes at a 90^oangle from each other corresponding to the longitudinal (CVl) and transverse (CVt) epicardial fibre orientation. Each CVl and CVt recording electrodes were spaced at a 2mm distance and used to calculate epicardial conduction velocity. To calculate CVtm, the thickness of the LV mid-wall was determined post-experiment.

Results – The high sampling rates (20kHz) enabled us to document small electrophysiological changes including alternans in both AP and CV measurements. Thus far, we have resolved CV and APD changes as small as 1%. 100nM Isoprenaline elicits an increase in CVl (13.2%), CVt (14.4%), and CVtm (12.8%) with a corresponding decrease in APD90 of -16.5% (n=8). Interestingly, increasing intracellular Ca²⁺, a known effect of SNS activation, via infusion of 5mM Ca²⁺ resulted in a reduction in CVl (-13.0%), CVt (-11.0%), and CVtm (-9.0%) with a corresponding decrease in APD90 of -9.1% (n=5). Mimicking SNS activation by raising increasing intracellular cAMP via 15µM Forskolin and 50µM IBMX, a similar pattern emerged of CV and APD changes as Isoprenaline in CVl (11.1%), CVt (12.1%), CVtm (10.66%), and APD90 (-13.20%) (n=8). These findings also suggest that the increase in CV brought about by adrenergic stimulation must also overcome the reduction in CV which is a result of increased intracellular Ca²⁺.

Conclusions – By utilising this novel electrode and light guide system, we have begun to establish the effect of adrenergic signaling on the interplay between increased myocyte to myocyte conduction velocity and action potential characteristics at a resolution not previously achieved.

Development of a human model of parasympathetic neurons and atrial cardiomyocytes

INTRODUCTION: The cardiac autonomic nervous system regulates the heart intrinsic activity (e.g., heart rate), and its dysregulation is involved in a number of cardiac arrhythmias. Dysfunction of parasympathetic neurons has been associated with atrial fibrillation, the most common form of cardiac arrhythmias (2.5% of English population [1]). Unfortunately, the interaction between parasympathetic neurons and atrial cardiomyocytes is particularly difficult to study and, to our knowledge, there are currently no human in vitro models.

AIMS: Our project aims to develop a human in vitro model of the interaction between parasympathetic neurons and atrial cardiomyocytes. It will allow us to investigate the cross-talk between these cells, to manipulate them to study atrial fibrillation (e.g. through genetic manipulation) or to carry out drug screening work.

METHODS: We have generated atrial cardiomyocytes and parasympathetic neurons from induced pluripotent stem cells. We followed published protocols for atrial cardiomyocytes [2] and parasympathetic (vagal) neurons [3]. With a combination of immunostaining and functional recordings (e.g. Ca²⁺ imaging and patch clamp recordings) we characterised the neurons confirming their function and lineage. Using qPCR and immunostaining we compared the expression of atrial and ventricular specific markers between atrial iPSC-derived cardiomyocytes and standard iPSC-derived cardiomyocytes (Burridge protocol [4]). We co-cultured atrial cardiomyocytes and parasympathetic neurons for 5-7 days and assessed the modulation of cardiomyocyte beating rates by the activation of the neurons with nicotine. We also tested whether the effect could be reversed by a neuronal blocker (a selective Nav1.8 antagonist).

RESULTS: Our data confirm the parasympathetic nature of the iPSC-derived neurons, as they express the autonomic lineage marker Phox2b and the cholinergic marker ChAT. They are also responsive to nicotine, as expected. Atrial iPSC-derived cardiomyocytes present a reduction in ventricular specific markers (e.g. MYL2, IRX4 assessed with qPCR) and increased expression of atrial markers with immunostaining (e.g. MYL7).

When we cocultured the two cell types for 7 days, nicotine (20µM) resulted in a significant reduction of cardiomyocyte beating rate in co-cultures compared to mono-cultures (mono-culture: 111.2±2.729; co-culture: 65.42±6.931; p=0.002; Mann-Whitney test; n= 6 two differentiations of the parasympathetic neurons). Interestingly, the activity-dependent Nav1.8 blocker A803467 (100nM) increased the beating rate in co-culture but not in mono-culture (mono-culture=96.28±0.7658; co-culture=130.8±11.09; p=0.0043; Mann-Whitney test; n=6; two differentiations of the parasympathetic neurons) reversing, to some extent, the effect of nicotine.

CONCLUSIONS: Our preliminary data show a potential to reproduce in vitro some key physiological features of parasympathetic neurons-cardiomyocyte interactions. In future studies, we aim to extend our characterisation and employ our system for disease modelling.

SYMPATHETIC NERVOUS REGULATION OF VENTRICULAR FIBRILLATION IN GUINEA PIG HEARTS

Background: Sympathetic nervous stimulation (SNS) exerts critical effects on cardiac electrophysiology. Several reports have investigated whether SNS suppresses or promotes the onset of ventricular fibrillation (VF). However, is not known whether SNS alters the dynamics of VF once it is initiated. Recent optical mapping studies have revealed a wide spectrum of mechanisms that drive and sustain VF. Fibrillation mechanisms have important consequences for treatment options, for example whether targeted ablation or pharmacological treatment is optimal for preventing recurrence in VF survivors.

Methods and Results: The epicardial ventricular surface of innervated guinea pig hearts (n=8) were optically mapped at high resolution using voltage dye Rh-237. VF was induced by a dynamic S1 pacing protocol with incrementally shortening cycle length, starting at 170ms. VF was induced with and without bilateral SNS by direct stimulation of efferent sympathetic nerves in the spinal cord.

SNS shortened action potential duration, increased dispersion of repolarisation, and increased conduction velocity. Rotational activity during VF was quantified by the detection and tracking of phase singularities (PS). During SNS, the incidence of stable PSs (>2 rotations) increased by 51±13% (P<0.01). SNS also increased the time a stable PS was present during VF from 32±3% to 41±2% (P<0.01). Although SNS increased the number of PSs, it did not alter the dynamics of specific PSs such as the number of rotations, lifetime, or spatial spread. In control and SNS conditions, PSs survived for 275±30ms and 310±44ms respectively.

Conclusion: SNS increases the number of transient rotational drivers during VF. Our findings suggest SNS favours multiple stable wavelets driving fibrillation in cardiac tissue, rather than the presence of one ‘Mother Wave’. With modulation of SNS common in several pathophysiological states and treatment options, these results may hold important consequences for the clinical treatment of VF.

Do plasma cytokine levels correlate to cardiac function in coronary artery disease?

Coronary artery disease (CAD) affects over 20 million people worldwide and is the leading cause of morbidity and mortality¹. In CAD, atherosclerosis narrows the coronary arteries leading to myocardial ischemia thence decreased systolic and diastolic cardiac function. Furthermore, previous studies highlight an association between CAD and atrial fibrillation (AF)². The pathogenic mechanisms are complex, though elevations of inflammatory markers such as cytokines play a key role. However, clinical studies examining this are limited³_.In this preliminary study, we measured plasma levels of cytokines in a CAD patient cohort then correlated those levels to indices of systolic and diastolic cardiac function.

The study was conducted in accordance with local and IRAS ethical approval (IRAS ID: 247341). Preoperative serum samples were collected from patients scheduled for routine coronary revascularisation surgery. Serum interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-1ß (IL-1ß) and tumour necrosis factor alpha (TNF-a) concentrations were measured using high-sensitivity ELISA kits (Abcam, UK & Invitrogen, USA). Statistical significance was taken when p < 0.05 and calculated using Pearson R correlation.

Average IL-6, IL-1ß, IL-10 and TNF-a concentrations were 15.20 ± 1.36, 1.46 ± 0.26, 4.21 ± 0.59 and 2.70 ± 0.61 pg/mL, respectively. We found no significant correlations between the levels of these cytokines and the indices of systolic function measured (EF, LVOT, TASPE, PASP). When cytokine levels were correlated to indices of diastolic function, IL-1ß and IL-10 correlated with peak E-wave velocity (IL-1ß: p = 0.02, r² = 0.21, n = 27. IL-10: p = 0.04, r² = 0.08, n = 53). No correlations between cytokine levels and end diastolic volume were observed.

These preliminary data indicate that levels of IL-1ß and IL-10 correlate to certain indices of cardiac diastolic function so may be of use as a prognostic marker in CAD patients. Further experiments will increase study power and seek to identify other key correlates including risk of arrythmia such as AF.

The effect of MOTS-c on inflammation related cytokines in cardiac and abdominal aortic tissue in abdominal aortic constriction induced cardiac hypertrophy model in rat

Introduction: Cardiovascular diseases (CVDs) are a set of disorders combined with vascular and cardiac functions such as cerebrovascular disease, angina, atherosclerosis, coronary heart disease, myocardial infarction, and heart failure. CVDs are the most important cause of mortality and morbidity in both developing and developed countries. While acute inflammation is a process that protects tissues, it’s becoming chronic and causes various pathologies. CVDs are a chronic inflammatory process. Pro-inflammatory cytokines such as IL-1β, IL-6, IL-17, IL-18, IL-21, IL-33, and TNF-α also trigger pathological disease, however, anti-inflammatory cytokines such as IL-10 and TGF-β reduce inflammation. MOTS-c is a mitochondrial-derived peptide encoded from 12S rRNA in mitochondrial DNA and referred to as the mitochondrial open reading frame of 12S Rna-type c. MOTS-c regulates metabolic pathways such as insulin sensitivity, glucose, and lipid homeostasis. It is stated that MOTS-c has a protective role in the progression of diabetes, cardiovascular disease, inflammation, osteoporosis, aging, and lung injury. However, few studies have investigated the effects of MOTS-c on inflammation.

Objective: We aimed to investigate how MOTS-c affects inflammatory cytokine levels after abdominal aortic constriction (AAC) induced cardiac hypertrophy.

Method: For this purpose, twenty-eight male Wistar albino rats grouped as (aged 2 months ) sham+saline (saline;1 ml/kg, n=6), sham+MOTS-c (MOTS-c;5 mg/kg,n=6), AAC+saline( Saline;1 ml/kg,n=8) and AAC+MOTS-c (MOTS-c;5 mg/kg,n=8). Cardiac hypertrophy models were induced by abdominal aortic ligation by using 4.0 silk sutures (Marano G,et al., 2002). Following the formation of the AAC model for 14 days, treatments were administered intraperitoneally for 21 days. Under ketamine+xylazine (100 mg/kg+15 mg/kg; i.p) anesthesia the rats were sacrificed and serum and tissue samples were taken. Histopathological analyses were performed with hematoxylin-eosin, Masson's trichrome, and Toluidine blue in the thoracic and abdominal aorta and heart tissue. The mRNA expressions of IL-1β, IL-18, TGF-β, and TNF-α genes involved in the inflammatory process in both the left ventricle of the heart and abdominal aorta tissues were determined by qPCR. Data were given as mean ± standard error, statistical analysis was compared with t-tests in independent groups. All experiments were performed in accordance with the "Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals prepared by the Adiyaman University, Animal Ethical Committee"( Turkey (Approved date 04.11.2022, no;2021/030).

Result: Histopathological analyses showed that the abdominal aortic tissue deteriorated in the AAC+saline group, while the vessel wall structure of the AAC+ MOTS-c treatment group was preserved.

mRNA expressions of IL-1β, IL-18, TGF-β, and TNF-α in aortic tissue were not a significant difference between the AAC and sham groups, but IL-1β, IL-18, TGF-β1, and TNF-α were upregulated in the AAC+MOTS-c group compared to the AAC group. Similarly, no difference in gene expression of IL-1β, IL-18, and TNF-α in heart tissue was observed between the AAC and sham groups, however, downregulated TGF-β1 levels. Conversely, IL-18 and TGF-β1 levels were approximately increased 10-fold in the AAC+MOTS-c group compared to the AAC group.

Conclusion: Our study revealed that inflammatory response genes were regulated by MOTS-c peptides. This peptide may have protective effects on cardiovascular damage progression.

Physiological Heterocellular Interactions in vitro: Living Myocardial Slices and Micro Vascularised Human Engineered Cardiac Tissue

Tissue and cellular adaptability and plasticity determine cardiac phenotypes but are difficult to elicit in in vitro models. In this talk, I will present two models where plasticity is accounted for: the living myocardial slices, subjected to electromechanical stimulation (Watson et al., 2017, 2019; Pitoulis et al., 2021), and the micro vascularised engineered myocardium, composed of human iPSC-derived cardiomyocytes and human microvascular endothelial cells (King et al., 2022).

The living myocardial slices are the gold standard to study heterocellular interactions in vitro because they are derived from the whole, native myocardium, containing all the cellular and extracellular components. They can be obtained from hearts of many species, including human failing and non-failing hearts, can be cultured for several days/weeks and respond promptly to environmental changes. We have developed bioreactors to maintain and manipulate the electromechanical status of the slices in physiology and disease. We can specifically manipulate preload and/or afterload in long term cultures and have described methods to study individual cell types within the slices, or after isolation. We are thus able to simulate disease in slices, e.g. mechanical overload (Nunez-Toldra et al., 2022), ischemia-reperfusion injury and focal injury. We can genetically modify living myocardial slices using viral vectors, opening novel and more relevant avenues for investigating cardiac disease.

The micro vascularised, IPSC-derived myocardium allows studying specifically into the cross talk between endothelial cells and cardiomyocytes when both contraction and vascular flow are present. In this model, contractility and flow in the engineered micro vessels can be manipulated and the consequences on the heterocellular interactions studied. This model is a step forward in physiological cardiac tissue engineering and represents a relevant tool to understanding cell-cell interaction in the heart.

Cellular cross-talks in the aging heart

Introduction: Aging is a major risk factor for impaired cardiovascular function. The aging heart is characterized by vascular dysfunction, increased hypertrophy, fibrosis and electrophysiological alterations. Studies show impaired endothelial cell function associates with senescence in aging. Since vessels are aligned with nerves, and this interplay is critical for tissue homeostasis, we investigated whether an impairment of the neuro-vascular interface may contribute to age-associated pathologies in the heart.

Methods and Results: To investigate the innervation of the aging mouse heart, we histologically assessed the cardiac autonomic nervous system in 12-week and 20-month old mice using the pan-neuronal marker Tuj1 and the sympathetic marker tyrosine hydroxylase. Interestingly, sympathetic innervation of the left ventricle, especially around arteries, was reduced by 0.66±0.07-fold with aging (n=9; p<0.05) indicating that aging reduces innervation of the left ventricle. Similar findings were observed in premature aging models of TERT-deficient and endothelial-transgenic progeria mice. Electrophysiological studies confirmed an increased incidence of ventricular arrhythmias and reduced heart rate variability in aged mice.

In order to determine the potential contribution of the neurovascular cellular cross-talk to the reduced innervation, we analyzed gene expression of isolated endothelial cells in the aging mouse heart by bulk RNA sequencing. Aging significantly induced the expression of genes associated with GO-terms related to neuronal cell death and axon injury. Sema3a, a known axon repelling factor, was significantly increased by 1.5±0.2-fold in endothelial cells of aged mice hearts, a finding which was confirmed by qPCR and by histology at protein level (p<0.05). Next we determined the mechanism of Sema3a de-repression. We show that miR-145 acts as an up-stream regulator of Sema3a expression. miR-145-/- mice showed increased Sema3a expression and reduced cardiac innervation.

We observed an increase in acidic β-galactosidase activity as a marker for cellular senescence in the aging heart, particularly in the proximate arterial space. In addition, Sema3a expression was significantly increased in senescent endothelial cells in vitro, thus suggesting a putative role for senescence to impair cardiac innervation. To test whether eliminating senescent cells may restore left ventricular axon density, we treated 18-month old mice with a combination of the two senolytic drugs dasatinib and quercetin, which are known to deplete senescent cells in vivo. After 2 months of senolytic treatment, cellular senescence was reduced by 0.4±0.1-fold (p<0.05) in the aging heart in parallel with reduced expression of endothelial Sema3a. Importantly, senolytic treatment restored ventricular innervation (by 1.8±0.4-fold vs. old-placebo mice), augmented heart rate variability, and normalized electrophysiological abnormalities (all p<0.05).

Conclusion: In conclusion, we show that aging augments axon repelling signals in endothelial cells and reduces left ventricular innervation. The depletion of senescent cells prevented age-induced impairment of innervation and normalized electrophysiological abnormalities suggesting a critical role of senescence-associated axon repulsion in the aging heart.

Signalling between cardiomyocyte InsP3Rs and RyRs elicits arrhythmogenic activity in human heart failure.

Heart failure (HF) is a leading cause of cardiovascular mortality and morbidity. While pump failure is a frequent cause of mortality, up to 50% of deaths are due to sudden cardiac death following lethal arrhythmias. Dysregulation in Ca²⁺ handling has emerged as a key mediator of these arrhythmias. The rhythmic contraction of cardiomyocytes necessary for the efficient pumping of the heart is mediated by the process of excitation contraction coupling (EC) whereby Ca²⁺ entering the cell via L-type Ca²⁺ channels during the action potential activates Ca²⁺ release through Ryanodine Receptor channels (RyR) on the Sarcoplasmic Reticulum (SR) Ca²⁺ store, which then engages the contractile machinery to induce cell contraction. This mechanism is highly dependent upon the synchronized release of Ca²⁺ from clusters of RyRs dispersed throughout the cytosol, predominantly at the Z lines. Ensuring the coupling of Ca²⁺ entry and SR Ca²⁺ release are T-tubular invaginations of the sarcolemma that bring its voltage gated channels into close proximity with RyRs on the SR at cell compartments termed dyads. During heart failure, T-tubules are lost leading to reduced coupling between L-type channels and RyRs, which in turn reduces Ca²⁺ release synchrony as well as increasing arrhythmogenic Ca²⁺ waves. We have identified increased expression of a second SR Ca²⁺ channel, the InsP₃ receptor (InsP₃R), in animal models of CV disease and in human heart failure. This channel is engaged by InsP₃ generated downstream of G-protein coupled receptors such as those liganded by the neurohormones endothelin-1 and Angiotensin, which are also elevated in disease. We find potent effects of InsP₃ on Ca²⁺ handling in voltage clamped cardiomyocytes isolated from failing human hearts in comparison to cardiomyocytes from age-matched non failing hearts. Specifically, in the presence of InsP₃, Ca²⁺ transient amplitude was reduced and frequency of elementary Ca²⁺ release events (Ca²⁺ sparks) via RyRs was increased. RyRs were required for the action of InsP₃, suggesting Ca²⁺ channel crosstalk, an observation that was supported by super resolution microscopy. The effects of InsP₃ were most prominent at RyRs that were not coupled to the T-tubular membrane at dyads. Mathematical modelling provided further evidence supporting the role of crosstalk between InsP₃R and RyRs and the potential for Ca²⁺ release via InsP₃Rs to sensitize RyRs to Ca²⁺. The increased spontaneous Ca²⁺ spark events contributed to the development of Ca²⁺ waves, which through engaging the sodium Ca²⁺ exchanger, led to cell depolarization and action potential generation. This arrhythmogenic activity was further established in a tissue wedge model where increased arrhythmic activity was detected when perfused with AngII to increase InsP₃. The effects of AngII were suppressed by the InsP₃R inhibitor 2-APB. Together, provide a new mechanistic basis for the action of InsP₃mediated Ca²⁺ release in cardiomyocytes, whereby InsP₃Rs in the neighbourhood of RyRs act to tune their sensitivity to Ca²⁺ release. We further identify InsP₃Rs as important players in the pathology of human heart where they promote arrhythmogenic activity and diminished Ca²⁺ transients.

S-nitrosylation of CaMKIIÎ´ can modulate the progression of cardiac arrhythmias in isolated hearts during stress

Introduction: Nitric oxide can regulate cardiac function by targeting calcium-handling protein kinases such as calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ). Recent evidence has shown that during stress response, there is an increase in nitric oxide production which can activate CaMKII through S-nitrosylation. Chronic CaMKII activation, as a result of S-nitrosylation, has been implicated in the progression of arrhythmias. However, CaMKII S-nitrosylation can have protective or detrimental effects on cardiac function depending on the S-nitrosylation site that is modified. In the heart, the Cys 273 site inhibits CaMKII activation while the Cys 290 site enhances autonomous CaMKII activity. The mechanism underlying the role of CaMKII S-nitrosylation sites in the regulation of cardiac function during acute ischemia/reperfusion (I/R) injury has not been fully elucidated.

Aim: To determine if CaMKII S-nitrosylation site was detrimental to the heart during I/R injury.

Methods: Male mouse hearts were isolated and perfused with modified Krebs-Henseleit buffer via the Langendorff perfusion system. Hearts from wild-type C57BL/6 (WT) (n = 5) and C273S knock-in mice (C273S-KI, that lack the Cys 273 site; n = 5) were acutely exposed to 100 nM isoproterenol (ISO), a β-adrenergic receptor agonist before or after 150 µM S-nitrosoglutathione (GSNO), a nitric oxide donor for 10 min. Arrhythmias were counted per heart. For ischemia studies, WT (n = 4 – 6) and C273S-KI (n = 3) hearts were exposed to 100 nM ISO for 5 min prior to 20 min global ischemia, followed by 90 min reperfusion. Duration of arrhythmias was measured for 10 minutes post-ischemia. Data is presented as mean ± standard error of mean and ANOVA was used determine the variance in means.

Results: Treatment of isolated WT hearts with GSNO before ISO reduced arrhythmias compared to control (15.2 ± 4.2 vs 7.6 ± 2.4; p<0.05) while treating isolated hearts with ISO before GSNO promoted arrhythmias compared to control (38.0 ± 14.5 vs 4.6 ± 1.4; p < 0.05). When S-nitrosylation at C273 was not available in C273S-KI hearts, ISO before GSNO significantly increased arrhythmic events and GSNO before ISO had the similar effects (46.2 ± 23.2 vs 5.5 ± 2.9; 46.5 ± 8.9 vs 4.0 ± 1.7; p<0.05). The ischemic WT hearts and C273S-KI hearts had similar % LVDP recovery after ISO treatment. In addition, we did not detect a difference in the duration of arrhythmic events early during reperfusion in WT and C273S-KI hearts before (175.0 ± 41.3 vs 196.7 ± 38.4 secs) and after (277.5 ± 96.0 vs 336.7 ± 132.5 secs) treatment with ISO.

Conclusion: Our findings suggest that CaMKII S-nitrosylation at Cys 273 may provide some protection against ISO-induced arrhythmias. Our preliminary ischemia experiments showed no significant benefit of Cys 273 presence on post-ischemic LVDP recovery or early arrhythmias, without prior GSNO treatment. We are still testing the hypothesis that pre-treatment with GSNO before ischemia will provide protection in WT, but not in the C273S knock-in mouse. If that hypothesis is supported, S-nitrosylation of CaMKIIδ at Cvs 273 may be a more generally protective event under β-adrenergic and ischemia-reperfusion stress.

Targeting Runx1 prevents regional heterogeneity of impaired calcium handling post-MI

Following myocardial infarction (MI), dead cardiomyocytes are replaced by collagenous scar and result in a heterogenous transition zone between the scar and healthy myocardium. The region of myocardium bordering an infarct (border zone, BZ) undergoes many transcriptional changes, some of which may drive adverse remodelling. Master regulator transcription factor Runx1 is increased in the BZ as early as 1 day post-MI. Previously, we have shown that cardiomyocyte-specific Runx1 deficient (Runx1^Δ/Δ) mice demonstrate remarkably preserved contractility 1 day post-MI and that Runx1 modulates cardiac sarcoplasmic reticulum (SR) calcium uptake and contractile function (McCarroll et al. 2018). Ventricular arrhythmias and sudden cardiac death are major complications over the days and weeks following MI, the BZ region being most vulnerable site for arrhythmogenesis. (Qin et al. 1996). As such, we hypothesised that changes in Runx1 may contribute to arrhythmogenicity in the BZ region post-MI.

It is well understood that both heterogenous tissue electrophysiology and impaired calcium handling can result in an increased propensity for arrhythmia (Smaill et al. 2013). Thus, calcium measurements were performed on BZ and remote zone (RZ) cardiomyocytes loaded with a calcium-sensitive fluorophore (5.0 μmol/L Fura-4F AM, Invitrogen) from Runx1^Δ/Δ and flox control (Runx1^fl/fl) mice.

Overall, we found that at 1-day post-MI, BZ cardiomyocytes from Runx1^fl/fl mice had impaired calcium handling compared to RZ cardiomyocytes, whereas there were no regional differences in Runx1^Δ/Δ mice. Specifically, in BZ cardiomyocytes, calcium transient peak was 64% of the RZ peak, whereas in Runx1^Δ/Δ hearts, calcium transient peak was not different in the BZ compared to the RZ. Calcium transient amplitude in the BZ was 47% of that observed in RZ cardiomyocytes Runx1^fl/fl mice, whereas it was preserved in Runx1^Δ/Δ hearts. This pattern was also consistent for caffeine-induced calcium transient amplitude, representing SR calcium content, was 71% of that observed in RZ cardiomyocytes in Runx1^fl/fl mice but again not different regionally in Runx1^Δ/Δ hearts and SERCA activity, quantified by the rate constant of decay of the caffeine-induced calcium transient, which was 48% of RZ cardiomyocytes in the BZ of Runx1^fl/fl mice but not different between Runx1^Δ/ΔBZ and RZ cardiomyocytes.

Further, in ventricular cardiomyocytes isolated from C57BL/6 mice and incubated with Runx1 small molecule inhibitor Ro5-3335 or with vehicle control DMSO, we utilised a burst pacing protocol followed by a two-minute period of rest and measured spontaneous calcium events as a measure of arrhythmogenicity. We found that cells treated with Ro5-335 had significantly fewer spontaneous events in the rest period compared cells incubated in DMSO (0.5 ± 0.02 vs. 26 ± 8, p = 0.017, n = 9). Overall, these results demonstrate the potential mechanistic contribution of Runx1 to arrhythmogenicity in the BZ post-MI.

The entrainment of action potential duration and contractile amplitude by recapitulation of respiratory sinus arrhythmia in isolated cardiac myocytes requires a functioning sarcoplasmic reticulum

Respiratory sinus arrhythmia (RSA) is the oscillation of heart rate in phase with ventilation and represents the high frequency component of heart rate variability. RSA is prevalent in the young and the endurance-trained and its loss in heart failure is considered a prognostic indicator for sudden death. The recapitulation of RSA improves cardiac function in animal models of heart failure (O'Callaghan et al., 2020; Shanks et al., 2022). However, the precise mechanisms by which RSA affects cardiac function are unknown. We have previously shown that recapitulation of RSA through oscillatory pacing (OP), in which cycle length (CL) was set to 20% below and 20% above the average basic CL in a 2-short, 2-long stimulus train, entrained action potential duration (APD), Ca²⁺ transient amplitude (CaT) and sarcomere shortening and reduced the incidence of alternans in comparison to pacing at constant CL in guinea pig isolated ventricular myocytes (Carpenter et al., 2021; Carpenter et al., 2022). The OP entrainment of CaT and sarcomere shortening persisted in voltage clamp experiments with fixed APD (i.e. AP clamp), suggesting that APD is not an essential component of entrainment. Here, we examine the hypothesis that OP in isolated ventricular myocytes affects the stimulus-interval dependence of APD and sarcomere shortening through mechanisms involving the sarcoplasmic reticulum (SR). Animal procedures were approved by the University of Bristol Animal Welfare and Ethics Review Board and conducted according to UK legislation. Hearts were excised from adult male guinea pigs under general anaesthesia (140 mg/kg bodyweight euthatal, i.p.) and left ventricular myocytes isolated following enzymatic digestion. Isolated myocytes were superfused with Tyrode’s solution at 37 °C for perforated patch whole-cell current clamp recording and action potentials and sarcomere length measured simultaneously. Sample sizes are presented as n (number of cells) from N (number of animals) and nested hierarchical analysis applied using a generalized linear mixed model (SPSS, IBM). The limit of statistical confidence was P<0.05. Using a three-step S₁S₂S₃ protocol to examine the dependence of APD and sarcomere shortening on both preceding (CL_n-1) and anteceding (CL_n-2) stimulus intervals, both APD and sarcomere shortening were found to depend directly on the preceding cycle length (CL_n-1, P<0.001). In contrast, sarcomere shortening depended inversely on the anteceding CL (CL_n-2, P<0.001), while APD₉₀ did not depend significantly on CL_n-2 (n/N = 24/4). This is consistent with the entrainment by OP, the effect of CL_n-1 on sarcomere shortening being akin to post-extrasystolic potentiation (Yue et al., 1985). Following treatment of the cells with caffeine (50 µM) to sensitise ryanodine receptors, both APD and sarcomere shortening in response to the CL_n-1 stimulus were markedly suppressed, and the inverse dependence of sarcomere shortening on anteceding CL_n-2 was lost (P<0.001, n/N = 6/3). These data are consistent with an obligatory role for the SR in the entrainment of APD, CaT and sarcomere shortening and the suppression of alternans by the recapitulation of RSA. Further work is required on the effects of OP in intact hearts to determine the potential for a protective effect of RSA recapitulation against ventricular tachyarrhythmias.

Interactions between MARCKS, cav1.2 and PIP2 in cardiac myocytes, a potential novel pathway in heart failure and arrhythmogenesis

Introduction:

Cardiovascular disease (CVD) is a leading public health problem in the UK, with over 7 million people affected and at a cost of more than 150,000 deaths and £10 billion annually. Such a major cause of morbidity, mortality and economic burden reveals a huge medical need for novel therapeutic targets to treat CVD. It is well established that stimulation of CaV1.2 voltage-gated Ca²⁺ channels (VGCCs) plays a fundamental role in cardiac cell function, regulating both rate and rhythm and contractility of the heartbeat. Therefore, understanding novel regulatory mechanisms of CaV1.2 VGCCs may reveal new targets to treat CVD such as heart failure and arrhythmogenesis. We recently showed that myristoylated alanine-rich C kinase substrate (MARCKS) acts as phosphatidylinositol 4,5-bisphosphate (PIP₂) buffer to regulate the activity of CaV1.2 VGGCs and vasoconstriction of mesenteric arteries (Jahan et al. 2020).

Aim: In the present study, we have investigated whether similar mechanisms are present in cardiomyocytes.

Methods:

All animal procedures were carried out in accordance with guidelines laid down by St George's, University of London Animal Welfare Committee and conform with the principles and regulations described by the Service Project Licence: 70/8512 Freshly isolated heart lysates and cardiomyocytes from hearts of male Wistar rats were studied through a wide range of techniques including western blotting, immunocytochemistry, proximity ligation assay (PLA), and whole-cell patch clamp electrophysiology.

Results:

In western blot studies, expression levels of MARCKS, CaV1.2, and PIP₂ were similar in right atrium, right ventricle, left atrium, and left ventricle (n= 7, pËƒ0.05, one-way ANOVA). When heart samples were incubated with wortmannin (20 μM), PIP₂ levels were significantly supressed in all heart chambers (P<0.0001, one-way ANOVA). The functionality of MARCKS on VGCCs was then investigated using the MARCK activator, MANS peptide (100 μM), which significantly increased whole-cell Ba²⁺ currents from 3.19 ± 0.34 pA/pF to 6.05 ± 0.43 pA/pF at 0 mV (n= 7, p<0.001, t-test). This effect of MANS peptide was abolished when cells were preincubated with wortmannin (20 μM). Immunocytochemistry and PLA studies demonstrated that MARCKS and PIP₂ colocalise significantly at resting condition. However, upon stimulation with MANS peptide, this colocalization of MARCKS and PIP₂ was reduced and CaV1.2 was shown to colocalise with PIP₂(n= 6, P<0.0001, t-test).

Conclusion:

These preliminary findings reveal the expression and interaction between MARCKS, CaV1.2 and PIP₂ in cardiomyocytes, which likely controls cardiomyocytes calcium homeostasis. These molecular complexes may play an essential role in regulating cardiac function, and may be altered in cardiac dysfunction, thus providing a novel target for treating CVD.

Epicardial Adipose Tissue as a Modulator of Cardiac Arrhythmogenesis

Background: Obesity is a significant risk factor for arrhythmic cardiovascular death. Interactions between Epicardial Adipose Tissue (EAT) and myocytes are thought to play a key role in the development of arrhythmias. Cross talk between EAT and the heart has various component. First, adipose tissue infiltration within the myocardium constitutes an anatomical obstacle to cardiac excitation. It causes activation delay and increases the risk of arrhythmias. Intercellular electrical coupling between cardiomyocytes and adipocytes can further slow conduction and increase the risk of block, favouring reentry and arrhythmias. Finally, EAT secretes multiple substances that influence cardiomyocyte electrophysiology either by modulating ion currents and electrical coupling, or by stimulating fibrosis. However, the effect of EAT secretome (EATs) on cardiac electrophysiology is largely unknown. We investigated the arrhythmogenicity of EAT secretome and its underlying molecular and electrophysiological mechanisms.

Methods: We collected atrial EAT and subcutaneous adipose tissue (SAT) from 30 patients with atrial fibrillation (AF), and EAT from 3 donors without AF. The secretome was collected after a 24-hour incubation of the adipose tissue explants. We cultured neonatal rat ventricular myocytes (NRVMs) with EATs, SAT secretome (SATs) and cardiomyocytes conditioned medium (CCM) for 72H. We implemented the electrophysiological changes observed after EATs incubation into a model of human left atrium and tested in silico arrhythmia inducibility.

Results: Cardiomyocytes incubated with EATs showed reduced conduction velocity and increased conduction heterogeneity compared to SATs and CCM. This was associated with a decreased expression of the potassium channel subunit Kcnj2 by 26% and correspondingly reduced the inward rectifier K+ current (IK1) by 35% in comparison to incubation with CCM, and resulted in a depolarized resting membrane of cardiomyocytes. EATs caused decreased expression of connexin43 (29% mRNA, 46% protein) in comparison to CCM. Cells incubated with SATs showed no significant differences in Kcnj2 nor Gja1 expression in comparison to CCM, and their resting membrane was not depolarized. In silico modeling of human left atrium revealed that the electrophysiological changes induced by EATs promote sustained reentrant arrhythmias if EAT partially covers the myocardium.

Conclusion: EAT slows conduction, depolarizes the resting membrane, alters electrical cell-cell coupling and facilitates reentrant arrhythmias.

Characterising a novel cardiac phenotype in Niemann-Pick Disease Type C

Introduction: Niemann-Pick disease Type C (NPC) is an autosomal recessive disease that is caused by mutations in two genes, NPC1 or NPC2 that encode lysosomal proteins. Mutations in either gene result in the accumulation of cholesterol, sphingomyelin and glycosphingolipids in lysosomes[1]. The principal clinical manifestations include hepatosplenomegaly, ataxia, seizures and neurodegeneration. No in-depth studies have been performed on cardiac function in these patients. Recent studies have shown that Ca²⁺ released from acidic stores such as lysosomes can take a part in cytosolic Ca²⁺ transients, enhancing contraction amplitude and speed[2]. NPC cells have reduced levels of lysosomal Ca²⁺[3], thus, it is possible that altered Ca²⁺ signalling in cardiomyocytes can affect the lysosome mediated Ca²⁺ signalling cascade and may be potentially arrhythmogenic.

Aims: To characterise the NPC mouse heart both structurally and at the molecular level, and to investigate its susceptibility to arrhythmia.

Methods: All experiments complied with the United Kingdom Home Office Guide on the Operation of Animal (Scientific Procedures) Act of 1986. Wild-type (WT) and Npc1^-/- mice were bred from heterozygous BALBc/cNctr- Npc1^m1n/J. Masson’s trichrome and picrosirius red staining was performed on heart sections to assess the level of fibrosis and further validated by western blotting of the fibrosis marker collagen I and vimentin. Npc1^-/-heart electrophysiology (ECG) was performed on a Langendorff perfusion system using electrical (burst pacing) and pharmacological (50nM isoprenaline) protocols. We also performed a transcriptomic analysis on Npc1^-/-hearts at 3, 7 and 9 weeks of age to investigate the molecular mechanism involved in Npc1^-/- cardiac pathophysiology development at the transcriptional level.

Result: Histological staining revealed significantly increased fibrosis in Npc1^-/- heart sections at 7 weeks of age (WT 17.1%±0.3 vs NPC 19.4%±0.28, p<0.0001, n=3). Western blot for collagen I and vimentin supported the histology results, with increased levels of collagen I and vimentin in NPC tissue (p=0.0261 or 0.0435 respectively, n=6). Compared to WT, Npc1^-/- hearts had significantly pro-longed QT interval for 11.7 ms (p=0.0029, n=7), suggesting a change in ion channel function. Also, Npc1^-/- hearts presented ECG abnormalities including arrhythmias under pharmacological stress. Transcriptomic analysis found 3240, 2190 and 5300 differential expressed genes (DEGs) comparing hearts at 3, 7 and 9 weeks of age (p<0.05, n=5). Several significantly altered cellular pathways were identified including immune/inflammation response, lysosome, calcium signalling, arrhythmogenic right ventricular cardiomyopathy, and adrenergic signalling, which support our hypothesise that Npc1^-/-heart is more susceptible to arrhythmia.

Conclusion: Our studies highlight in Npc1-/- hearts fibrotic remodelling, inflammation and ion signalling alterations. We provide the first evidence that NPC hearts have a higher tendency to display structural and functional disturbances including arrhythmias as a consequence of lysosomal Ca²⁺ signalling defects in the heart.

Intramyocardially implanted engineered heart tissues show intermittent entrainment during the acute post-implantation phase

The therapeutic effect of engineered heart tissues (EHTs) is only at its full potential once the EHTs integrate into the native myocardium, and thus electrically and/or mechanically couple. However, the electromechanical coupling of EHTs with the host remains a challenge. The electromechanical cues of the heart might be crucial for EHTs to mechano-electrically couple and this might be especially true for the first couple of hours post-implantation (acute phase), which, to our knowledge, has not been studied.

The aim is to investigate whether small EHTs show signs of mechanical and/or electrical coupling during the acute implantation phase.

Small EHTs consisted of a recombinant collagen hydrogel disc (6 mm x 300 µm thick) with commercial human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) (ICell² CDI) seeded on top (<50.000 cells). Hearts of healthy male NZW rabbits were excised under anaesthesia (0.5 ml/kg euthatal with 500U heparin) and prepared for Langendorff perfusion. Blebbistatin was used to block contraction before implanting Cal520-AM loaded EHTs underneath the epicardium. To measure the effect of ventricular contraction, a balloon was inserted into the left ventricle (LV) and hearts were divided into two groups: 1) constant contraction block (CB) using blebbistatin (N=9 hearts) and 2) graded levels of contraction (GC) achieved by washing off the blebbistatin (N=6). Calcium traces (CaT) were recorded alongside the ECG for approximately 30 seconds every 25 mins using a lightguide and traces were categorized into three timepoints post-implantation: Start (T=0), Early (T=20-50mins) and Late (T=70-100mins). The Rayleigh test (1,2) was used to detect potential entrainment based on the distribution of the phase differences between ECG R-wave and CaT. In recordings with significant tendency towards a single phase, runs of 5 or more CaT with delay from the previous R-wave within a 18ms window were considered entrained. Then, entrained parts of the trace were analysed on RR interval, CaT interval, time from the QRS to the CaT upstroke and the ECG:CaT ratio.

CaT could be recorded for multiple hours post-implantation. Contraction was 1.0±0.3% (mean±SD) in the presence of blebbistatin and increased after wash-out to 34.3±20.3% and 85.2±13.3% in the Early and Late groups, respectively (GC group only). The Rayleigh test showed significant result for some traces (12/65 [18.5%] for CB and 11/35 [31.4%] for GC) and this was equally divided between Early and Late timepoints. Furthermore, it appeared that entrainment occurred intermittently (Figure 1). Further analysis of entrained trace selections did not show a 1:1 coupling with the heart but often 1:3 or 1:4 and occasionally 1:2, indicating that the human EHTs could not follow the fast intrinsic rate of the rabbit heart.

These data indicate that EHTs show periodic entrainment when engrafted into myocardium in the first hours post-implantation. Entrainment was seen in absence of contraction and therefore contraction seemed not essential. One potential underlying mechanism could be ephaptic coupling and this hypothesis is currently being tested using a mathematical model. The early phase coupling indicated here may be important for the long-term survival and therapeutic potential of EHTs.

Cardiac ion channel expression profiles in the pulmonary vein of the racing equine athlete with atrial fibrillation

Background: Atrial fibrillation (AF) is one of the most common causes of cardiac arrhytmia in the heart (1). A relationship between AF and exercise is controversial as exercise seems to protect the heart, in contrast, extremely vigorous long-term exercise could increase cardiac problems (2). AF causes poor performance in horses, and knowledge about the molecular initiation of AF of the pulmonary vein (PV) (3) in the athletic horse is still (4) unknown. Ion channels, responsible for cardiac action potential generation and electrical propagation are target-study of AF causes, in addition to this, the interest in investigating them is because they seem to interact with AF treatments (3). Therefore, this study aims to investigate the molecular ion channel expression pattern in the PV of the athletic horse to get a global gene map expression of this condition.

Methods: Heart samples were obtained from ten horses. Five horses were healthy, and the other five clinically reported suffering AF condition. All ten horses were humanly euthanized due to cardiac affections, and the healthy horses were selected because of musculoskeletal problem- related. Horses belonged to the Hong Kong Jockey Club and were clinically evaluated by veterinarians. Heart tissue biopsies were taken from the left and right atria and ventricle respective sides. Myocardial sleeves from the PV were dissected from the heart. RNA from heart tissue was analysed by RNA sequencing; protein and immunohistochemistry were performed in the samples of healthy and AF horses.

Differential expression of a selection of ion channels and electrophysiological proteins were evaluated. We analysed results by hierarchical clustering and left atrium and PV sequenced samples. PV gene expressions were compared with paired Mann-Whitney-Wilcoxon test. Bioinformatics data and statistics analysis were performed with R software. Results from PV and left atrium samples were clustered separately based on their gene expression. Seven ion channels were significantly (p<0.05) increased while six ion channels were significantly under- expressed (p<0.05) in the PV sleeves of horses. Gene expression of potassium ion channels such as KCNA4, KCNQ1, KCNH2, KCND3, KCNJ2, KCNN1, KCNN2, and KCNN3 were positively expressed. In addition, calcium-handling proteins (NCX1 and PLN), connexins (CX40), and HCN4 channel ion genes were expressed. Genes in the PV sleeves and left atrium coincide with the differences in the electrophysiological characteristics and conduction velocities. Variations in KCNQ1 and NCX1 genes showed the repolarization patterns of the potassium and calcium homeostasis in the PV sleeves and left atrium. The left atrium and the PV sleeves gene expression showed a specific ion channel pattern of the pulmonary sleeves in the horse.

Conclusion: Our findings show that PV sleeves in athletic horses could initiate AF, this was observed by gene and molecular expression patterns. Therefore, the study of the PV in racing horses using bioinformatics tools analyses could help us clarify relevant biological data and provide us with an understanding location gene map. This study focuses on cardiac dysrhythmias as AF in athlete horses will provide knowledge in human athlete’s studies due to similarities in ion channel expression patterns.

Registration opens	01 March 2023
Abstract submission opens	01 May 2023
Abstract submission closes	31 May 2023
Early bird registration deadline	17 July 2023
Late breaking poster submission deadline	28 July 2023
Conference Attendance Award deadline	31 July 2023
Registration deadline	21 August 2023
Meeting dates	11 – 12 September 2023

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Neurophysiological Bases of Human Movement 2025

Monday 11 September - Tuesday 12 September 2023

Cross-Talk of Cells in the Heart: Novel Mechanisms of Disease and Arrhythmias

Date

Location

10:00 SA01 Fibroblast-myocyte interactions and their impact on cardiac electrophysiology

10:30 SA02 Cardiac fibroblast activation and reversal and their interaction with cardiomyocytes

11:00 C03 Small extracellular vesicles released by cardiac fibroblasts from hypertrophic cardiomyopathy patients alter action potential of human cardiomyocytes

11:15 C04 Calcium signalling in cardiac fibroblast and myocytes in an in vitro model of heart failure with preserved ejection fraction

11:30 C05 Chronic myocardial infarction attenuates transmural heterogeneities but alters the response to endothelin and β-adrenergic stimulation in porcine ventricles

11:45 C06 Investigating the effect of substrate stiffness on iPSC-CM structure and function

13:00 SA03 Exploring the role of neuronal dysfunction in inherited arrhythmia syndromes

13:30 C07 Adrenergic modulation of ventricular transmural conduction velocity is a novel factor governing electrical excitability in health and disease

13:45 C08 Development of a human model of parasympathetic neurons and atrial cardiomyocytes

14:00 C09 A novel electrophysiological approach to investigate sympathetic autonomic ganglia in Parkinson's Disease Animal Model: a loose-patch clamp

14:15 C10 Sympathetic nervous regulation of ventricular fibrillation in guinea pig hearts

15:00 SA04 Towards new therapeutics in atrial fibrillation

15:30 C11 Do plasma cytokine levels correlate to cardiac function in coronary artery disease?

15:45 C12 The effect of MOTS-c on inflammation related cytokines in cardiac and abdominal aortic tissue in abdominal aortic constriction induced cardiac hypertrophy model in rat

09:40 SA05 Physiological heterocellular interactions in vitro: Living myocardial slices and micro vascularised human engineered cardiac tissue

10:00 SA06 Multi-scale modelling of cardiac heterogeneity from nanodomain to the whole heart

10:20 SA07 From microscale origin to whole heart manifestation: the journey of spontaneous Ca2+ release

10:40 SA08 Cellular cross-talks in the aging heart

11:30 SA09 Signalling between cardiomyocyte IP3Rs and RyRs elicits arrhythmogenic activity in human heart failure

12:00 C19 S-nitrosylation of CaMKIIδ can modulate the progression of cardiac arrhythmias in isolated hearts during stress

12:15 C20 Targeting Runx1 prevents regional heterogeneity of impaired calcium handling post-MI

12:30 C21 The entrainment of action potential duration and contractile amplitude by recapitulation of respiratory sinus arrhythmia in isolated cardiac myocytes requires a functioning sarcoplasmic reticulum

12:45 C22 Interactions between MARCKS, cav1.2 and PIP2 in cardiac myocytes, a potential novel pathway in heart failure and arrhythmogenesis

14:45 SA10 Epicardial adipose tissue as a modulator of cardiac arrhythmogenesis

15:15 C23 Characterising a novel cardiac phenotype in Niemann-Pick disease type C

15:30 C01 Intramyocardially implanted engineered heart tissues show intermittent entrainment during the acute post-implantation phase

15:45 C02 Cardiac ion channel expression profiles in the pulmonary vein of the racing equine athlete with atrial fibrillation

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