The 4q25 variant rs13143308T links risk of atrial fibrillation to defective calcium homoeostasis

Aims: Single nucleotide polymorphisms on chromosome 4q25 have been associated with risk of atrial fibrillation (AF) but the exiguous knowledge of the mechanistic links between these risk variants and underlying electrophysiological alterations hampers their clinical utility. Here, we tested the hypothesis that 4q25 risk variants cause alterations in the intracellular calcium homoeostasis that predispose to spontaneous electrical activity. Methods and results: Western blotting, confocal calcium imaging, and patch-clamp techniques were used to identify mechanisms linking the 4q25 risk variants rs2200733T and rs13143308T to defects in the calcium homoeostasis in human atrial myocytes. Our findings revealed that the rs13143308T variant was more frequent in patients with AF and that myocytes from carriers of this variant had a significantly higher density of calcium sparks (14.1¿±¿4.5 vs. 3.1¿±¿1.3 events/min, P¿=¿0.02), frequency of transient inward currents (ITI) (1.33¿±¿0.24 vs. 0.26¿±¿0.09 events/min, P¿<¿0.001) and incidence of spontaneous membrane depolarizations (1.22¿±¿0.26 vs. 0.56¿±¿0.17 events/min, P¿=¿0.001) than myocytes from patients with the normal rs13143308G variant. These alterations were linked to higher sarcoplasmic reticulum calcium loading (10.2¿±¿1.4 vs. 7.3¿±¿0.5¿amol/pF, P¿=¿0.01), SERCA2 expression (1.37¿±¿0.13 fold, P¿=¿0.03), and RyR2 phosphorylation at ser2808 (0.67¿±¿0.08 vs. 0.47¿±¿0.03, P¿=¿0.01) but not at ser2814 (0.28¿±¿0.14 vs. 0.31¿±¿0.14, P¿=¿0.61) in patients carrying the rs13143308T risk variant. Furthermore, the presence of a risk variant or AF independently increased the ITI frequency and the increase in the ITI frequency observed in carriers of the risk variants was exacerbated in those with AF. By contrast, the presence of a risk variant did not affect the amplitude or properties of the L-type calcium current in patients with or without AF. Conclusions: Here, we identify the 4q25 variant rs13143308T as a genetic risk marker for AF, specifically associated with excessive calcium release and spontaneous electrical activity linked to increased SERCA2 expression and RyR2 phosphorylation.Peer ReviewedPostprint (author's final draft

CaMKII-dependent regulation of cardiac Na(+) homeostasis.

Na(+) homeostasis is a key regulator of cardiac excitation and contraction. The cardiac voltage-gated Na(+) channel, NaV1.5, critically controls cell excitability, and altered channel gating has been implicated in both inherited and acquired arrhythmias. Ca(2) (+)/calmodulin-dependent protein kinase II (CaMKII), a serine/threonine kinase important in cardiac physiology and disease, phosphorylates NaV1.5 at multiple sites within the first intracellular linker loop to regulate channel gating. Although CaMKII sites on the channel have been identified (S516, T594, S571), the relative role of each of these phospho-sites in channel gating properties remains unclear, whereby both loss-of-function (reduced availability) and gain-of-function (late Na(+) current, INa L) effects have been reported. Our review highlights investigating the complex multi-site phospho-regulation of NaV1.5 gating is crucial to understanding the genesis of acquired arrhythmias in heart failure (HF) and CaMKII activated conditions. In addition, the increased Na(+) influx accompanying INa L may also indirectly contribute to arrhythmia by promoting Ca(2) (+) overload. While the precise mechanisms of Na(+) loading during HF remain unclear, and quantitative analyses of the contribution of INa L are lacking, disrupted Na(+) homeostasis is a consistent feature of HF. Computational and experimental observations suggest that both increased diastolic Na(+) influx and action potential prolongation due to systolic INa L contribute to disruption of Ca(2) (+) handling in failing hearts. Furthermore, simulations reveal a synergistic interaction between perturbed Na(+) fluxes and CaMKII, and confirm recent experimental findings of an arrhythmogenic feedback loop, whereby CaMKII activation is at once a cause and a consequence of Na(+) loading

Rabbit models of heart disease.

Human heart disease is a major cause of death and disability. A variety of animal models of cardiac disease have been developed to better understand the etiology, cellular and molecular mechanisms of cardiac dysfunction and novel therapeutic strategies. The animal models have included large animals (e.g. pig and dog) and small rodents (e.g. mouse and rat) and the advantages of genetic manipulation in mice have appropriately encouraged the development of novel mouse models of cardiac disease. However, there are major differences between rodent and human hearts that raise cautions about the extrapolation of results from mouse to human. The rabbit is a medium-sized animal that has many cellular and molecular characteristics very much like human, and is a practical alternative to larger mammals. Numerous rabbit models of cardiac disease are discussed, including pressure or volume overload, ischemia, rapid-pacing, doxorubicin, drug-induced arrhythmias, transgenesis and infection. These models also lead to the assessment of therapeutic strategies which may become beneficial in human cardiac disease

The G protein-gated potassium current I(K,ACh) is constitutively active in patients with chronic atrial fibrillation

Background— The molecular mechanism of increased background inward rectifier current (IK1) in atrial fibrillation (AF) is not fully understood. We tested whether constitutively active acetylcholine (ACh)-activated IK,ACh contributes to enhanced basal conductance in chronic AF (cAF). Methods and Results— Whole-cell and single-channel currents were measured with standard voltage-clamp techniques in atrial myocytes from patients with sinus rhythm (SR) and cAF. The selective IK,ACh blocker tertiapin was used for inhibition of IK,ACh. Whole-cell basal current was larger in cAF than in SR, whereas carbachol (CCh)-activated IK,ACh was lower in cAF than in SR. Tertiapin (0.1 to 100 nmol/L) reduced IK,ACh in a concentration-dependent manner with greater potency in cAF than in SR (−logIC50: 9.1 versus 8.2; P<0.05). Basal current contained a tertiapin-sensitive component that was larger in cAF than in SR (tertiapin [10 nmol/L]-sensitive current at −100 mV: cAF, −6.7±1.2 pA/pF, n=16/5 [myocytes/patients] versus SR, −1.7±0.5 pA/pF, n=24/8), suggesting contribution of constitutively active IK,ACh to basal current. In single-channel recordings, constitutively active IK,ACh was prominent in cAF but not in SR (channel open probability: cAF, 5.4±0.7%, n=19/9 versus SR, 0.1±0.05%, n=16/9; P<0.05). Moreover, IK1 channel open probability was higher in cAF than in SR (13.4±0.4%, n=19/9 versus 11.4±0.7%, n=16/9; P<0.05) without changes in other channel characteristics. Conclusions— Our results demonstrate that larger basal inward rectifier K+ current in cAF consists of increased IK1 activity and constitutively active IK,ACh. Blockade of IK,ACh may represent a new therapeutic target in AF

An integrative and translational assessment of altered atrial electrophysiology, calcium handling and contractility in patients with atrial fibrillation

Atrial fibrillation (AF) is the most prevalent sustained arrhythmia reported in clinical practice, and it is associated with deleterious outcomes such as stroke, that increase patient morbidity and mortality. Previous studies have reported atrial remodelling, including structural and electrophysiological remodelling as well as alterations in Ca2+ handling, as contributors to the initiation and perpetuation of AF. However, the contributory role of these remodelling to the pathophysiology of different forms of AF and their corresponding complications is incompletely understood. Hence, the aims of this study are to (i) assess the role of altered intracellular Ca2+ handling in the atrial contractile dysfunction seen in patients with long-term persistent (‘chronic’) AF (cAF); (ii) investigate the role of abnormal intracellular Ca2+ handling in the arrhythmogenesis of postoperative AF (poAF) and its associated contractile dysfunction; (III) study the alteration in extracellular matrix (ECM) protein secretion and their possible role in poAF and cAF associated contractile dysfunction. In pursuance of these aims, right atrial appendages excised from cardiac surgery patients were obtained. In the first part of this thesis, the role of abnormal Ca2+ handling in the atrial contractile dysfunction associated with cAF was studied. Right atrial myocytes of cAF patients examined by simultaneous measurement of their membrane currents (voltage-clamp), intracellular Ca2+ ([Ca2+]i) and cell fractional shortening, exhibited an impaired contractile response to Ca2+. Expression of cTnC was reduced in cAF patients, which could account for the abnormal contractile response of atrial myocytes from cAF patients. Also, Ca2+ buffering was impaired in cAF myocytes as a consequence of lower Ca2+ buffers which is supported by the reduced cTnC expression observed in cAF patients. Conclusively, the findings in this part of this study suggest that reduced cTnC not only contributes to the atrial contractile dysfunction, but also the impaired buffering seen in cAF patients. In the next chapter, we evaluated the participation of altered intracellular handling of Ca2+ in the development of poAF and its related contractile dysfunction. Analysis of preoperative echocardiography recordings by speckle-tracking revealed diminished left atrial contraction in poAF patients. [Ca2+]i measurements indicated reduced systolic Ca2+ transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca2+ load in myocytes from poAF patients, with a delay in the sequestration the SR due to reduced SERCA2a activity identified as the underlying cause. In consonance, protein expression of SERCA2a was reduced in poAF patients, but the phosphorylation and expression of its regulatory protein phospholamban were unchanged. Atrial myocytes from poAF patients exhibited increased vulnerability to CaT and AP alternans, which is attributed to the reduced SERCA activity, based on computational modelling. In summary, our findings suggest that SERCA mediated impairment in SR Ca2+ uptake contributes majorly to the proarrhythmic mechanisms responsible for the development of poAF as well as the associated impaired preoperative atrial contractile function. Finally, we assessed the alterations in the secretions of ECM proteins in poAF and cAF patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analysis of the secretome of atrial appendages from poAF and cAF patients showed minimal changes in ECM protein secretion in the poAF group, with approximately 6% of identified ECM proteins differentially regulated. In comparison, 40% of ECM proteins were differentially regulated in cAF, demonstrating marked alteration in ECM protein secretion. ECM proteins such as collagen I, microfibrillar associated protein, connective tissue growth factor and several other members of the different ECM were identified to contribute to remodelling in atrial ECM protein secretion, with the pro-fibrotic transforming growth factor β1 (TGFβ1), identified by further analysis as a contributor to the modification in ECM protein secretion seen in both poAF and cAF. Altogether this thesis provides novel mechanistic insight on the role of altered Ca2+ handling in the development of cAF and poAF and their associated contractile dysfunction as well as the characteristic remodelling of ECM protein secretion in both forms of AF.2021-10-2

J Physiol

Cardiac electrophysiology and Ca| handling change rapidly during the fight-or-flight response to meet physiological demands. Despite dramatic differences in cardiac electrophysiology, the cardiac fight-or-flight response is highly conserved across species. In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca| transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca| handling responses between mouse and rabbit hearts, we found notable species differences in spatio-temporal repolarization dynamics during SNS. Species-specific computational models revealed that these electrophysiological differences allowed for enhanced Ca| handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological responses are fine-tuned across species to produce optimal cardiac fight-or-flight responses.|Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca| handling changes underlying chronotropic and inotropic responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca| transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff-perfused rabbit and mouse hearts. Dual optical mapping with voltage- and Ca| -sensitive dyes allowed for analysis of spatio-temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT responses; however, a pronounced biphasic APD response occurred, with initial prolongation (50.9\ua0\ub1\ua05.1\ua0ms at t\ua0=\ua00\ua0s vs. 60.6\ua0\ub1\ua04.1\ua0ms at t\ua0=\ua015\ua0s, P\ua0<\ua00.05) followed by shortening (46.5\ua0\ub1\ua09.1\ua0ms at t\ua0=\ua060\ua0s, P\ua0=\ua0NS vs. t\ua0=\ua00). We determined the biphasic APD response in mouse was partly due to dynamic changes in HR during SNS and was exacerbated by \u3b2-adrenergic activation. Simulations with species-specific cardiac models revealed that transient APD prolongation in mouse allowed for greater and more rapid CaT responses, suggesting more rapid increases in contractility; conversely, the rabbit heart requires APD shortening to produce optimal inotropic responses. Thus, while the cardiac fight-or-flight response is highly conserved between species, the underlying mechanisms orchestrating these effects differ significantly.16GRNT30960054/American Heart Association/K99 HL138160/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United StatesR01 HL111600/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United StatesUC Davis Academic Federation Professional Development Award (LW)/OT2 OD026580/ODCDC CDC HHS/Office of the Director/United StatesOT2 OD026580/ODCDC CDC HHS/Office of the Director/United StatesR01HL131517/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United StatesR01 HL131517/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United States1OT2OD026580/NIH HHS/National Institutes of Health/United States15SDG24910015/American Heart Association/R01 HL131517/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United States1OT2OD023848-01/NIH HHS/National Institutes of Health/United StatesOT2 OD023848/ODCDC CDC HHS/Office of the Director/United States1OT2OD026580-01/NIH HHS/National Institutes of Health/United StatesR01HL141214/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United StatesRG/17/3/32774/British Heart Foundation/United KingdomK99 HL138160/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United StatesR01HL111600/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United StatesR01 HL111600/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United StatesK99HL138160/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United StatesR01 HL141214/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United States2020-08-01T00:00:00Z31215643PMC66756326525vault:3359

CARDIAC CALSEQUESTRIN AS A POSSIBLE TARGET FOR ARRHYTHMIA

The term arrhythmia encapsulates all the conditions which result in the change in rhythm of the heart. It is classified broadly into two classes;tachycardia, with increased and rhythm of heart and bradycardia, with decreased rhythm of the heart. In most cases, the arrhythmias are not seriousand asymptomatic, however, in some cases it leads to heart attack which becomes fatal. The present review focuses on stepwise understanding offunctioning of the heart and its excitation-contraction (EC) coupling followed by the mechanism of arrhythmia and the present line of medications.The systematic flow of blood in and out of the cardiac chambers is due to the rhythmic contraction and relaxation of cardiac muscle, i.e. the ECcoupling of cardiac myocyte. Any disruption in the ion flow during the EC coupling causes arrhythmia. Caions act as the trigger for the excitationcontractioncoupling,;therefore,understanding its movementis an essentialtounderstand the rhythmdisordersofthe heart.Calsequestrin (CASQ)isthe mostabundantcalciumbufferingproteinpresentin the sarcoplasmicreticulumofskeletalandcardiacmuscles. Thisreviewfurther focusesonCASQ;its structureand functions. Moreover,it describestheassociation of CASQwitharrhythmia.Withthecalcium bindingthe CASQattainsitslinearpolymericstructureon the neutralizationof itshighlyelectronegativesurface.The proteinbindscalciumwith highcapacity and intermediateaffinitywhichreleasesanduptakescalciumreversiblyduring theEC coupling.Mutation inCASQgenes hasbeenassociatedwithcatecholaminergicpolymorphicventriculartachycardia,and moreover,therearequitea fewmolecules whichareknowntobindtoCASQand bring about changesinionicbufferingproperties.Thereforeunder suitableoptimizedconditions CASQcouldbechosenas anoveltargetforcardiacarrhythmia.Scrutinizingthescopeof CASQthis reviewpresentsthe first in depth studysuggesting cardiacCASQas a possible targetforarrhythmia.2+ Keywords: Calsequestrin, Arrhythmia, Tachycardia, Bradycardia, Catecholaminergic polymorphic ventricular tachycardia, Excitation-contractioncoupling