Cardiac arrhythmogenesis: roles of ion channels and their functional modification

Cardiac arrhythmias cause significant morbidity and mortality and pose a major public health problem. They arise from disruptions in the normally orderly propagation of cardiac electrophysiological activation and recovery through successive cardiomyocytes in the heart. They reflect abnormalities in automaticity, initiation, conduction, or recovery in cardiomyocyte excitation. The latter properties are dependent on surface membrane electrophysiological mechanisms underlying the cardiac action potential. Their disruption results from spatial or temporal instabilities and heterogeneities in the generation and propagation of cellular excitation. These arise from abnormal function in their underlying surface membrane, ion channels, and transporters, as well as the interactions between them. The latter, in turn, form common regulatory targets for the hierarchical network of diverse signaling mechanisms reviewed here. In addition to direct molecular-level pharmacological or physiological actions on these surface membrane biomolecules, accessory, adhesion, signal transduction, and cytoskeletal anchoring proteins modify both their properties and localization. At the cellular level of excitation–contraction coupling processes, Ca2+ homeostatic and phosphorylation processes affect channel activity and membrane excitability directly or through intermediate signaling. Systems-level autonomic cellular signaling exerts both acute channel and longer-term actions on channel expression. Further upstream intermediaries from metabolic changes modulate the channels both themselves and through modifying Ca2+ homeostasis. Finally, longer-term organ-level inflammatory and structural changes, such as fibrotic and hypertrophic remodeling, similarly can influence all these physiological processes with potential pro-arrhythmic consequences. These normal physiological processes may target either individual or groups of ionic channel species and alter with particular pathological conditions. They are also potentially alterable by direct pharmacological action, or effects on longer-term targets modifying protein or cofactor structure, expression, or localization. Their participating specific biomolecules, often clarified in experimental genetically modified models, thus constitute potential therapeutic targets. The insights clarified by the physiological and pharmacological framework outlined here provide a basis for a recent modernized drug classification. Together, they offer a translational framework for current drug understanding. This would facilitate future mechanistically directed therapeutic advances, for which a number of examples are considered here. The latter are potentially useful for treating cardiac, in particular arrhythmic, disease.

. Requirements for their implantation increases with age, reaching 1:600 at age >65 years.The most common sustained arrhythmia, atrial fibrillation (AF), similarly age-related (Dun and Boyden, 2009;Wakili et al., 2011), affects 1%-2% of the general population (Stewart et al., 2002;Lloyd-Jones et al., 2004;Andrade et al., 2014), predisposing to further major cardiac and cerebrovascular morbidity and mortality (Stewart et al., 2002), including stroke (Wolf et al., 1991).Ventricular arrhythmias, Ion current contributions to basic features of cardiac electrophysiological excitation.Human atrial (A) and human and murine ventricular (B) inward (a) and outward ionic current contributions (b), atiributable to surface membrane ion channels, to human (c) and mouse (d) AP waveforms [from Figure 1 of the work of Huang (2017) and Figure 1 of the work of Huang et al. (2020)].
exemplified by ventricular tachycardia (VT), potentially leading to ventricular fibrillation (VF) and SCD (Rubart and Zipes, 2005;Turakhia and Tseng, 2007), are implicated in >300,000 and ~70,000 deaths/year in the United States (Kannel et al., 1987) and United Kingdom, respectively (Colquitt et al., 2014).Some degree of rhythmic cardiac activity persists with AVN or Purkinje conduction blocks.Of the different extents of AVN conduction block, first-degree block results in electrocardiographic (ECG) complexes showing consistent PR interval prolongations (>0.20 s).Second-degree blocks Mobitz type 1 (Wenckebach) and Mobitz type 2 both manifest as repeated ECG cycles with dropped QRS complexes, The latter are preceded by cycles showing progressively increasing (Figure 1Ch) and constant PR intervals, respectively.Third-degree heart block (Figure 1Ci) contrastingly manifests as complete P wave and QRS complex dissociation.Finally, Purkinje tissue right and left bundle branch blocks result in broadened (>120 ms duration) QRS complexes with abnormal kinetic features varying with right-or left-sided ECG leads and the site of the block.

Normal cardiac activity
Cardiac excitation is initiated and paced by repetitive cycles of sinoatrial node excitation (Lei et al., 2007;Donald and Lakatta, 2023).These trigger periodic waves of APs that then propagate through successive cardiac structures (Figure 1B) (Draper and Weidmann, 1951;Matthews et al., 2013).Each AP begins with rapid phase 0 depolarization from the resting membrane potential.Subsequent completion of the slower recovery over action potential duration (APD) and effective refractory period (ERP) permits further excitation cycles.Atrial APs show triangular waveforms with prominent initial phase 1 recoveries (Figure 2A).Human ventricular APs (Figure 2B) similarly begin with rapid (~400 V s −1 ) phase 0 depolarization from the (~-90 mV) resting potential to a +40 to +60 mV overshoot voltage.The subsequent phase 1 initial rapid repolarization is followed by a prolonged phase 2 plateau, lengthening the APD and ERP.It is terminated by phase 3 repolarization leading to phase 4 electrical diastole (Weidmann, 1951).APs initiate the physiologically important cellular events mediating cardiac systole/ diastole.They also drive cell-to-cell axial currents through cytoplasmic and gap junction resistances.They also possibly activate cell-cell ephaptic couplings.The latter events enable their propagation into hitherto quiescent cells, forming a coherent, advancing electrical wavefront through conducting or myocardial tissue, while leaving a trailing, recovering refractory region.Such excitation wavefronts span the thickness of the atrial wall.Ventricular propagation involves excitation proceeding from the transmural epicardium to endocardium and from the base to apex.

Electrophysiological basis of arrhythmogenesis 2.1 Spatial stability and instability in the propagated AP wave
This normal propagation of activation waves through continuous electrically coupled three-dimensional cardiomyocyte networks (Kucera et al., 1998;Kléber and Rudy, 2004) can be disrupted by pro-arrhythmic ectopic triggering events.In contrast, compromised spatial and temporal coherence and stability in the wavefronts generate an arrhythmic substrate.The spatial extent and stability of this excitation depend on its wavelength, λ, within which further premature excitation cannot occur.This normally ensures that only one such generated and propagated wave can occur per heartbeat.The term λ is determined by the propagation velocity θ and recovery ERP or APD: λ = θ×ERP or θ×APD (Huang, 2017).Figure 1Aa illustrates a typical basic cycle length (BCL) containing a murine ventricular AP.It defines its critical temporal properties of latency, APD, and succeeding diastolic interval, DI n , at 90% repolarization (APD 90 and DI 90 ).It then maps these temporal properties onto their corresponding spatial properties, assuming a constant AP conduction velocity θ.In the cardiac tissue through which the AP propagates, the latter form active and resting wavelengths λ′ and λ 0 ', making up the basic cycle distance, BCD' = λ' + λ 0 ', of the propagated AP waveform (Figure 1Ab).
Larger λ reduces the likelihood that areas of depolarization and repolarization collide to produce obstacles causing unidirectional conduction blocks.In the latter event, APs have to take a slow conducting pathway to traverse this non-conducting myocardium.Such blocks result from depolarization abnormalities (Figure 3, path 1; dark gray) (King et al., 2013a).Collision would involve previously excited adjacent regions of the normal myocardium (path 2; white).However, should the latter possess APs with sufficient λ (yellow region), the collision would simply involve refractory tissue, precluding re-excitation in path 2 (Figure 3Aa).Similar outcomes result from impulses from ectopic triggering immediately following the normal AP (Figure 3Ab).The traveling wave then passes undisrupted over the heterogeneity (Weiss et al., 2005).Contrastingly, an AP conducting retrogradely along path 1 entering the beginning of path 2 with λ reduced to values smaller than the dimensions of the available circuits would initiate persistent re-entrant excitation into the recovered myocardial regions (Figure 3Bc) (King et al., 2013a;Huang, 2017).
Decreased λs, thus, increase likelihoods of regenerative wave breakup into multiple wavelets, forming scroll waves (Davidenko et al., 1995;Zaitsev et al., 2000;Pandit and Jalife, 2013) along chaotic conduction pathways (Krogh-Madsen et al., 2012;Matthews et al., 2013;Spector, 2013).Contact multi-electrode (0.5 mm) array isochronal AP mapping techniques visualized the initiation of such a sequence.These studies were performed in right ventricular (RV) epicardia of intact beating flecainide-challenged pro-arrhythmic murine Scn5a+/− hearts (Figures 3Ba-f) (Martin et al., 2011c).Scn5a+/− ventricle APs show slowed conduction and increased activation and recovery dispersions.Here, following a delayed epicardial activation in the last normal beat reflected in the close isochronal contours (Figure 3Ba) (Killeen et al., 2007), a superimposed premature ventricular activation (Figure 3Ca) produces a line of block with AP propagation flowing around it (Figure 3Bb).A ventricular ectopic event then initiates an anticlockwise running circuit (Figure 3Bc) persistent through the following beat (Figure 3Bd).A consequent continually changing line of block now produces a non-stationary vortex (Figures 3Be,f) causing the polymorphic VT, apparent in the accompanying electrocardiographic trace (Figure 3Cb).

Temporal stability and instability of the AP wave
Temporal electrophysiological heterogeneities are typically observed as alternating variations in AP amplitude or APD between successive beats.Such alternans classically presages the breakdown of the regular pattern of electrophysiological activity and appearance of clinical (Nearing et al., 1991;Rosenbaum et al., 1994;Armoundas et al., 1998) or experimental arrhythmias (Pastore et al., 1999).Alternans in APD at a given heart rate, and therefore BCL, is thought to arise from variations in the timecourse of AP recovery or restitution to the resting potential, quantifiable as the APD.This variation, in turn, affects the subsequent diastolic interval (DI) prior to the AP corresponding to the following beat (Nolasco and Dahlen, 1968;Hayashi et al., 2007).The DI, thus, defines the period over which the membrane potential of its preceding AP is repolarized to its resting level.This recovery time, in turn, potentially affects the recovery properties, specifically the APD, of the subsequent AP.For example, shortened recovery times, DI, may preclude full recovery of the ion channel activity that underlies AP generation prior to the following beat.Typically, reductions or increases in DI have been reported to result, respectively, in reductions or increases in the APD of the subsequent AP.This effect follows a relationship termed the A-curve (Figure 4A) (Huang, 2021).However, at a fixed BCL, the resulting variations in APD would, in turn, alter the succeeding DI (Figure 4B).This latter effect is described by a linear D-line given by DI = BCL-APD (Figure 4C).These effects together would drive an interaction between DI and APD through successive cardiac cycles.Thus, the altered DI would, in turn, cause alterations in the APD of the subsequent AP and its DI that follows.This interaction can be described by superimposing the respective A-curve and D-lines.
The intersection between the A-curve and D-lines defines the steady-state APD and DI at any given BCL (Figure 4, Figures 4Da, Ea) (Sabir et al., 2008b).However, the interaction between the processes that they represent will vary with the heart rate as the latter, in turn, alters the BCL.Alterations in BCLs shift the D-line (Figures 4Db,Eb).This, in turn, alters the intersection point between the A-curve and D-line and, therefore, the DI corresponding to this intersection.The heart will then transition to a new APD as determined by the projection of this point to the A-curve (Figures 4Dc,Ec).This, in turn, alters the DI of the succeeding AP as given by the subsequent projection to the D-line (Figures 4Dd,Ed).The result is an iterative cycle of oscillating APD through successive heartbeats determined by the alternating projections to and from the A-curve and D-line.
Whether these oscillations converge, resulting in stable, persistent activity, or diverge, giving rise to unstable activity, is determined by the slope of the A-curves around the intersection region.For example, zero A-curve slope at the intersection results in an immediate attainment of a final steady-state APD and DI without oscillations.Where the A-curve slope falls between zero and unity (Figure 4D), the successive projection lines map a convergence back to the set point, giving a transient alternans ultimately stabilizing with a waning in the amplitude of the oscillation.The unity slope corresponding to the critical DI, DI crit , gives projection lines that neither converge nor diverge, yielding stable alternans.Intersections where the A-curve slope exceeds unity result in diverging projection lines.This corresponds to a progressively increasing instability and a waxing oscillation in which the projection lines ultimately veer away from the left-hand limit of the A-curve.This causes a pro-arrhythmic conduction block and potential re-entry (Figure 4E) (Matthews et al., 2010;Matthews et al., 2012;Matthews et al., 2013).
Finally, intrinsic instabilities in the AP waveform itself or following its full recovery to resting potential, if large enough to reach re-excitation threshold, potentially cause isolated triggered beats and, in the presence of arrhythmic substrate, persistent arrhythmia (January and Riddle, 1989).In the ventricles, early afterdepolarizations (EADs) interrupting AP recovery timecourses reflect events continuing from the ventricular phase 2 plateau (Figure 4Fi).Transient, delayed afterdepolarizations (DADs) follow full repolarization (Figure 4F(ii)) (Killeen et al., 2008a).Such abnormal triggering in the pulmonary or the superior caval veins may precipitate AF. 3 Ion channels underlying normal and abnormal rhythmic activity

Ion channels mediating action potential excitation
Membrane-level physiological processes can each be identified with specific surface membrane ion channels and transporters, each constituting disease and potential clinical therapeutic targets.Ion channels function and interact through both their sensitivity to and effects on membrane potential, constructing the electrophysiological events found in normal activity or disease states.Membrane transporters mediating metabolically coupled transport or ion exchanges also exert electrogenic effects dependent on the relative stoichiometry and charges of their translocated ions.These targets variously participate in activating rapid depolarizing (phase 0) (exemplified for human atrial (A) and human and murine ventricular (B) cardiomyocytes in Figures 2Aa, Ba) as well as recovery, early repolarizing (phase 1), plateau and late repolarization (phases 2 and 3), and electrically diastolic phases (phase 4) stages of AP waves (Figures 2Ab,Bb).
First, overall cardiac pacing is normally driven by SAN and in heart block, AVN and Purkinje tissue automaticity.Among inward pacemaker currents (Huang et al., 2016) are inward, hyperpolarization-induced cyclic-nucleotide-activated channel (HCN)-mediated I f and electrogenic 3Na + /1Ca 2+ exchange (NCX) currents driven by sarcoplasmic reticular (SR) Ca 2+ release (Lakatta et al., 2010;Donald and Lakatta, 2023).This results in a timedependent phase 4 depolarization from the background resting potential.This process is modulated by autonomic, adrenergic, or cholinergic stimulation or inhibition.It results in the membrane potential attaining the Ca 2+ (I Ca ) and then the Na + current (I Na ) excitation thresholds.This initiates the SAN AP (Lei et al., 2007).Clinical automaticity abnormalities arise from abnormal ionic current activity or altered background diastolic or resting potentials.They can manifest as sinus node disorder (SND), abnormal AVN or Purkinje tissue pacing, and spontaneous impulses in pathologically partially depolarized atrial and ventricular muscle.

A hierarchy of mechanisms for ion channel modification
The immediate cause of pro-arrhythmic events involves actions of, and interactions between, cell membrane ion channels within individual or between adjacent cardiomyocytes.However, these are influenced by a hierarchy of mechanisms.These extend over a 12decade, logarithmic timescale.This ranges from ns/μs (~10 -6 s) timescales shown by molecular events, through cellular events extending over ms/s within each or multiple cardiac cycles, to hours/days or even weeks/months (~10 6 s) shown by remodeling processes (Figure 5A).Thus, there are (Figure 5B) (a) cell surface membrane ion channels and transporters underlying automaticity and AP excitation, propagation, and recovery and the (b) cellular feedforward and feedback effects of excitation-contraction coupling and its triggering by Ca 2+ .These latter overlap over microsecond/ millisecond time scales.In contrast, the (c) systems-level G-protein signaling-dependent autonomic inputs and their related central nervous system circadian rhythms extend over second/minute/ hour time scales.Finally, (d) longer-term upstream mechanisms related to metabolic feedback and other upstream modulators Mapping of physiological mechanisms, molecular targets, and therapeutic agents related to arrhythmia.(A) Pro-arrhythmic processes taking place over successively longer timescales running from ns/μs, ~10 -6 s timescale, molecular events, through ms/s events within each cardiac cycle or multiple cardiac cycles, to hours/days or even week/month, ~10 6 s, timescales resulting from (B) an interleaving hierarchy of physiological processes involving interactions (a) at the molecular/membrane level, interactions between surface membrane ion channels underlying automaticity and AP excitation, extend over days/weeks/months.Nevertheless, each of these processes can be grouped (Figure 5C) by their participating biomolecules (Figure 5D) (Lei et al., 2018).The scheme complements and prompts extensions of existing modeling attempts that extend to full electromechanical coupling at the systems level.These had integrated local and transmural ventricular myocyte surface electrical (ten Tusscher et al., 2004) with the cytosolic and SR Ca 2+ transport and storage properties outlined below.They also modeled the consequent Ca 2+ -troponin binding to cross-bridge cycling activity and myofilament mechanics (Rice et al., 2008).These finally extended to mechanical activity in realistic two-and three-dimensional ventricular cardiac tissue models (Pathmanathan and Whiteley, 2009;Adeniran et al., 2013;Huang, 2015).
Many clarifications of these relationships arise from monogenically modified murine models permitting single cardiomyocyte, tissue, whole organ, and systems-level experimental study (Huang, 2017).Murine hearts translate to human hearts in their two-sided atrial and ventricular circulations, pacing, or conducting SAN, AVN, and atrioventricular (AV) components.They differ in size, heart rates, some ion channel types (Figures 2Ba, b), and consequent detailed AP waveforms (Figures 2c, d).Nevertheless, they share major, depolarization and conduction, AP features and regional heterogeneities (Huang, 2017).Particular genetic variants recapitulate corresponding human clinical arrhythmic and pharmacological phenotypes.Genetically induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) platforms also show promise as cellular rather than systems models in not recapitulating in vivo conducting, Purkinje, and contractile tissue organization.However, many human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show immature embryonic-like rather than adult atrial/ventricular phenotypes (Lee et al., 2012).Nevertheless, recent reports describe hiPSC-CMs with atrial AP properties and acetylcholine-activated K + current expression (Ahmad et al., 2023).There are recent iPSC models for normal and disease changes in ion channel expression, Ca 2+ homeostasis, neurocardiac interactions, and hypertrophy (Li et al., 2023;Chen et al., 2023;Langa et al., 2023;Zhou et al., 2023).Theoretical reconstructions remain useful in predicting physiological end effects (Alrabghi et al., 2023;Hancox et al., 2023).Ultimate translatability of such results still requires direct human clinical electrophysiological studies with recently innovated electrocardiographic (Van Der Werf and Lambiase, 2021) and electrical mapping capacities (Taggart et al., 2022).

Ion channel modulation at the level of the cell membrane
A number of associated proteins modify function and expression of surface membrane ion channels in short local feedback loops.First, auxiliary β-subunits modulate function in their various associated Na + , Ca 2+ , and K + channel subtypes.Na + channel β-subunits (Navβ) each comprise an amino-terminal immunoglobulin (Ig) connected to a single transmembrane domain and a short largely disordered intracellular region.They enhance channel trafficking to the surface membrane modifying peak I Na , modulate steady-state and kinetic inactivation (Namadurai et al., 2015), and ion channel clustering in the plasma membrane (Salvage et al., 2020).
The Navβ 1 and Navβ 3 isoforms associate non-covalently with αsubunits, mostly with the Ig domain binding to the domain III voltage-sensing module (VSM); the Navβ 2 and Navβ 4 Ig domains covalently associate via a disulfide bond to an extracellular loop region of domain II (Salvage et al., 2020a).However, Nav1.5 constitutes an interesting exception to this rule.Here, the Navβ 1 and Navβ 3 subunit Ig domains cannot bind to domain III VSM because the binding site is likely blocked by glycosylation.Similarly, Navβ 2 and Navβ 4 Ig domains cannot form disulfide bonds because the necessary free cysteine residues are absent in the Nav1.5 α-subunit.Consequently, Nav1.5-associated β-subunit Ig domains may be free to form extended cis and trans cross-links.
Second, ion channels interact with cell adhesion, signal transduction, and cytoskeletal anchoring proteins.Nav1.5 interacting proteins may also function in specific subcellular Nav1.5 localizations producing regional variations in Nav1.5 expression, I Na , density, and kinetics between subcellular microdomains.
Feedback actions of consequently altered intracellular Ca 2+ fit its strategic second messenger function through widespread cellular processes and cell types (Figure 6A) (Huang, 2017).Released Ca 2+ itself exerts a short-loop slow RyR inactivation (Laver and Curtis, 1996).Further longer-loop feedback modulation may modify surface membrane channel or transporter activity with pro-or anti-arrhythmic effects (Figures 6B-D), acting on either arrhythmic triggering or substrate (Figure 6E).Of voltagesensitive channels mediating inward depolarizing current, Na + and Ca 2+ channel C-terminal domains contain both direct and indirect Ca 2+ regulatory binding sites in the form of one or more EF hand motifs and an isoleucine-glutamine (IQ) CaM-binding domain, respectively.Their III-IV loops also contain Ca 2+ -CaMbinding sites.The channels additionally possess multiple kinase, including CaM kinase II (CaMKII) and phosphokinase C (PKC), and phosphorylatable regulatory sites.One or more of these sites have been implicated in Ca 2+ -mediated feedback inhibition of Nav1.5 in intact cardiac myocytes and implicated in arrhythmogenic phenotypes in both pharmacological (King et al., 2013b;Valli et al., 2018b) and genetic disease models (King et al., 2013c;Zhang et al., 2013;Salvage et al., 2021;Salvage et al., 2023).Similar mechanisms may operate for skeletal muscle Nav1.4 (Matthews et al., 2019;Sarbjit-Singh et al., 2020;Liu et al., 2021).Ca 2+ , likely through CaMKII, conversely increases I NaL with potential pro-arrhythmic effects (Liu et al., 2023).
Of ion exchange processes, NCX stoichiometry produces inward, potentially pro-arrhythmic I NCX under conditions of elevated [Ca 2+ ] i in polarized membranes (Shattock and Bers, 1989).

Autonomic feedforward and feedback
Autonomic, sympathetic and parasympathetic, modification of pacing, ion current activation, and excitation-contraction coupling involves transmitter and co-transmitter binding to G-proteincoupled receptors (GPCRs).G-protein-linked activation cascades show significant amplification through their constituent signaling molecules exerting multiple inotropic, chronotropic, and lusitropic effects on cardiac function (Bers, 2002).Both levels of and balance between sympathetic and parasympathetic activity may affect arrhythmogenicity: autonomic dysregulation is involved in multiple cardiac arrhythmias.Thus, both increased sympathetic and vagal rebound activity amplify ventricular arrhythmic risk.Its modulation may offer novel therapeutic options.
The sympathetic transmitters adrenaline and noradrenaline are, respectively, released into the circulation by adrenal medullary release and locally by widely distributed cardiac sympathetic nerve terminals.They, respectively, preferentially bind surface membrane β 1 -and β 2 -adrenergic receptors.Cardiomyocytes express β 1 -adrenergic receptors, activating the stimulatory G s protein.Its G α subunit then binds guanosine triphosphate (GTP), dissociates from the GPCR and its βγ-subunit, and activates adenylyl cyclase, enhancing cellular cyclic 3′,5' adenosine monophosphate (cAMP) levels, with diverse compartmented cellular actions (Zaccolo, 2023).Of nerve-cardiomyocyte communication mechanisms under current study, ventricular Dbh + catecholaminergic cardiomyocytes (Dbh + Cate-CMs) expressing dopamine β-hydroxylase occur in close structural relationship with the sympathetic innervation.Their catecholaminergic-type vesicles suggest endocrine functions.They may also be involved in embryonic development and maturation of the cardiac conduction system (Wang et al., 2017).
At the membrane level, sympathetic activity increases heart rate and modifies AP generation and waveform.cAMP binds to and opens SAN HCN, increasing I f and heart rates.It also activates protein kinase A (PKA) whose widespread phosphorylation actions excite Nav1.5, Kv11.1, Kv7.1, and Cav1.2, increasing rapid inward I Na and slow outward I Kr and I Ks and I CaL , increasing ventricular AP amplitudes but shortening the AP plateau durations.It also accelerates SAN pacemaker potentials (Huang and Lei, 2023).In excitation-contraction coupling, sympathetic activity increases I CaL and net cellular Ca 2+ entry increasing rates and force of subsequent muscle contraction.PKA-mediated RyR2 phosphorylation also reduces regulatory FKBP12 ligand binding otherwise stabilizing RyR2 closure, thereby increasing RyR2 Ca 2+ sensitivity and Ca 2+induced Ca 2+ release.It also phosphorylates phospholamban (PLN), relieving its SERCA2 inhibition, enhancing diastolic SR Ca 2+ store.
The parasympathetic, inhibitory, transmitter, acetylcholine (ACh), acts on muscarinic (M 2 ) receptors activating the G i2 protein in the SAN, AVN or atria, in both the presence and absence of, pre-existing adrenergic challenge.It does so in ventricular tissue in the presence of such challenge.Its G α subunit binds GTP and splits off from the receptor and its Gβγsubunit.The latter opens GIRK1 and GIRK4 channel components, increasing inward rectifying I KACh or I KAdo , particularly in supraventricular tissue (Schmitt et al., 2014;Dascal and Kahanovitch, 2015;Lerman, 2015).The dissociated G iα inhibits adenylate cyclase (AC) and, therefore, cAMP production (DiFrancesco, 1993), increasing I CaL and I f in pacemaker cells.G i activation also increases protein phosphatase (PP2A) activity through cell division control protein 42 homolog (Cdc42)/Rasrelated C3 botulinum toxin substrate 2 (rac2) and the cardioprotective p21-activated kinase PAK1.PP2A dephosphorylates PKA-phosphorylated proteins at the same serine/threonine phosphorylation sites, reversing their L-type Ca 2+ channel, RyR2 and PLN effects.Parasympathetic action, thus, slows heart rates and decreases contractile force.
Over longer systems-level timescales, autonomic innervation provides effector pathways for central nervous responses to environmental inputs.Normal circadian ion channel remodeling cycles explain SAN pacemaking changes, resulting in higher background heart rates during wakefulness than during periods of sleep (Jesus et al., 2021).These are likely driven by sympathetic, as opposed to parasympathetic, actions coupled to suprachiasmatic nuclear circadian rhythms.Rather than variations of autonomic transmitter activity (Black et al., 2019), recent evidence attributes these to periodic transcriptional cardiac remodeling cycles varying ion channel abundances and their consequent ionic current densities.The SAN ion channel transcriptome for many important cardiac ion channels, particularly HCN, displays circadian rhythms (Anderson et al., 2023;Black et al., 2019;D'Souza et al., 2021;Wang et al., 2021), sensitive to chronic but not acute pharmacological autonomic blockade (Pitzalis et al., 1996;Black et al., 2019).It possibly involves cAMP response element action on key Per1 and Per2.18 clock genes (Travnickova-Bendova et al., 2002).Critically needed are future studies of autonomic plasticity and age-related properties (Chadda et al., 2018;Takla et al., 2023).
Mitochondrial mass and function are regulated by transcriptional coactivators (Sonoda et al., 2007): peroxisome proliferator-activated receptor (PPAR) γ coactivator-1 (PGC-1) family members are highly expressed in oxidative tissues including the heart.Of these, mitochondrial promoter PGC-1α or PGC-1β expression increases with upstream signals including cold exposure, fasting, and exercise, likely signaling anticipated cellular energy requirements (Figure 7A) (Huang, 2017).PGC-1 proteins exert matching multi-level regulation of cellular mitochondrial function and metabolism.Of their activated nuclear receptor targets, PPARα regulates genes involved in mitochondrial fatty acid oxidation; estrogen-related receptor alpha (ERRα) regulates mitochondrial fatty acid oxidative and oxidative phosphorylative energy transduction pathways (Vega et al., 2000).Additionally, PCG-1α coactivation of nuclear respiratory factor-1 (NRF-1) and -2 (NRF-2) (Wu et al., 1999) modulates nuclear-encoded transcription factor Tfam expression required in replicating, maintaining, and transcribing mitochondrial DNA (Garesse and Vallejo, 2001) and mitochondrial biogenesis (Vega et al., 2000;Huss et al., 2004).These mechanisms may underlie the induction of nuclear genes encoding mitochondrial proteins in energetic pathways, including the tricarboxylic acid cycle, and nuclear and mitochondrial genes encoding electron transport chain and oxidative phosphorylation complex components in cultured cardiomyocytes with forced PGC-1 expression (Lehman et al., 2000).PCG-1s fall in obesity, insulin resistance, type II diabetes mellitus, and aging in parallel with mitochondrial dysfunction (Leone and Kelly, 2011;Dillon et al., 2012).
Mitochondrial dysfunction, whether intrinsic or arising from excessive energetic demand, compromised vascular oxygen supply or pathological energetic disorders, destabilizes inner membrane potentials, driving their electron transport chain.At the cell membrane level, the consequently compromised ATP and/or rising adenosine diphosphate (ADP) increase sarcolemmal K-ATP (sarcKATP) channel opening probabilities (Akar and O'Rourke, 2011).This shortens APDs and ERPs and compromises cell excitability and AP propagation (Akar and O'Rourke, 2011).These events predispose to re-entrant arrhythmia (Fosset et al., 1988;Faivre and Findlay, 1990).
Second, fibrotic changes may contribute to both AF and ventricular arrhythmias.Increased fibrosis-related gene expression accompanying altered surface and Ca 2+ homeostasis ion channel gene expression may contribute to SAN dysfunction in experimental rat pulmonary arterial hypertension (Logantha et al., 2023).Disruption of myocardial bundle continuity and gap junction formation due to increased extracellular matrix associated with fibrotic and/or hypertrophic change reduces and accentuates heterogeneities in AP conduction velocity, disrupting AP propagation wavefronts and promoting re-entry (Davies et al., 2014).Connexin-mediated electrotonic coupling between cardiomyocytes and inexcitable fibroblasts with relatively depolarized (~-30 mV) resting potentials may depolarize cardiomyocyte resting membrane potentials altering myocyte excitability, slow conduction, shorten APD, and induce spontaneous phase 4 depolarization (Nattel, 2018).Additionally, Ca 2+ signaling in fibroblasts employs stretch-sensitive short TRPC3 or melastatin-type TRPM7 transient receptor potential (TRP) channels, both upregulated in AF fibroblasts (Yue et al., 2015).

Mechanistic insights translated into novel anti-arrhythmic therapies
Pharmacological intervention underpins much clinical arrhythmia management.Both modern drug innovation and optimizing existing therapeutic strategies require systematically classified mechanisms of action at identified molecular physiological targets and their rational correlation with single cell, tissue, or organ tissue effects and, in turn, clinical indications and therapeutic actions.This requirement had been addressed by successive historic Singh-Vaughan Williams (VW) (Vaughan Williams, 1975) and updated European Society of Cardiology classifications (Task force of the working group on arrhythmias The European Society of Cardiology, 1991).The most recent modernized classification scheme (Lei et al., 2018) responding to the recent cardiac electrophysiological and pharmacological insights outlined here pragmatically retained but extended the original VW classes.It mapped actions on molecular targets within more recently characterized levels of surface membrane ion channel, excitation-contraction coupling, autonomic function, and longer-term energetic and remodeling changes outlined above.The resulting scheme for cardiac arrhythmic mechanisms thereby offered a basis for understanding existing explorations for novel arrhythmic therapies (Lei et al., 2018), and reviewing drug cardiac safety (Saadeh et al., 2022).
Of the introduced novel drug categories, class 0, bearing on cardiac automaticity, includes the use-dependent SAN inhibitor ivabradine.It reduces heart rate by acting on I f , sparing myocardial contractility and vascular tone, actions distinct from the existing VW classes II and IV.It is clinically approved for reduced ejection fraction under circumstances of cardiac failure, adjunct therapy improving clinical outcomes by reducing heart rate.
It is yet to be established for stable ischemic heart disease.It may also benefit patients with inappropriate sinus tachycardia intolerant of class II or IV agents (Koruth et al., 2017).
Of extended VW classes I-IV, class I now includes class Id drugs acting on I NaL this complements the existing Class Ia-c, which reduce early I Na thereby reducing AP phase 0 slopes and overshoots, and modify APD and ERP.Class Ia drugs classically bind to the Nav1.5 open state with τ ~1-10 s dissociation time constants.They concomitantly block I K , slow AV conduction, and increase ERP and APD.Classes Ib and Ic contrastingly bind preferentially to the inactivated state with rapid τ ~0.1-1.0 s or slow τ > 10 s dissociation.This results in a use-independent and usedependent channel block and a slowed AV conduction.These insights clarify some of flecainide's paradoxical actions.Thus, flecainide exerts anti-arrhythmic actions in gain of Na + channel, particularly I NaL , function associated with LQTS3 and experimental murine Scn5a +/Δkpq .In contrast, it shows pro-arrhythmic actions under post-infarct conditions and clinical and experimental Scn5a+/− Brugada syndrome (Martin et al., 2010;Martin et al., 2011a).Of drugs in the new class Id, ranolazine produces a frequency-dependent block of the pro-arrhythmic I NaL .This shortens APD and increases refractoriness and repolarization reserve in LQTS3, pathological bradycardic and ischemic conditions, and cardiac failure (Belardinelli et al., 2015).Intracellular Na + overload, otherwise arising from such I NaL , increases reverse-mode NCX, promoting pro-arrhythmic intracellular Ca 2+ overload and SR Ca 2+ leak.Ranolazine may also reduce atrial peak I Na and Kv11.1-mediatedI K (Burashnikov et al., 2007;Antzelevitch et al., 2011).
An expanded class III encompassed drugs directed at the large number of more recently discovered, sometimes atrial or ventricular-specific, voltage-sensitive and -insensitive K + channel types.This added to the voltage-gated K + channel blockers originally employed to delay AP phase 3 repolarization, lengthening ERPs.Exemplars, including investigated agents directed at atrial arrhythmia, included those directed at voltage-sensitive I K such as the atrial I Kur , including XEN-D0103 (Ford et al., 2016) and vernakalant, for rapid AF (Wettwer et al., 2013), with the latter producing some I Na and I NaL , and I KACh in addition to I Kur block.New I Kr blockers under development include vanoxerine (Lacerda et al., 2010).Investigational agents directed at voltage-insensitive I K include the SK channel inhibitor AP30663 (Bentzen et al., 2020) and the K2P blocker doxapram (Wiedmann et al., 2022).
Major advances in excitation-contraction coupling have yielded significant progress in class IV drugs beyond the L-type Ca 2+ channel inhibitors introduced to reduce SAN and AVN rates and conduction (Vaughan Williams, 1975).First, investigational non-selective and phenylalkylamine and benzothiazepine inhibitors supplement dihydropyridine Cav1.2 and Cav1.3 surface membrane I CaL and I CaT inhibitors.
Carvedilol has similar dual class IV in addition to β-adrenergic blocking class II actions: its antioxidant actions reduce RyR2 phosphorylation and oxidation, and its open-state channel block decreases RyR2-mediated Ca 2+ release (Zhou et al., 2011).Finally, explorations of SERCA activation by istaroxime also have potential translational implications (Ferrandi et al., 2013;Huang, 2013).
11 Discussion: future therapeutic prospects Research in the physiological sciences has long involved work in successive, mutually reinforcing, interacting cycles between laboratory and clinic.Identified clinical or novel physiological phenomena initiate the development of representative experimental physiological models.These insights, in turn, prompt innovative clinical testing, management, and treatment, themselves prompting further iterations of experimental testing to identify novel physiological targets, investigational new drugs, and interventions (Huang et al., 2020;Huang and Lei, 2023).Future such cycles could add immunotherapeutic approaches, including antibody-based agents with further enhanced target specificities even targeting particular channel isoforms (Presta, 2008) and micro-RNAs/shRNAs knocking down protein expression at the mRNA level provided suitable delivery methods become available (Chakraborty et al., 2017).Within a framework of a modernized drug classification, these could themselves add to understanding arrhythmic events and their modification besides facilitating future clinical development.

FIGURE 3
FIGURE 3 Spatial instabilities in the genesis of re-entrant arrhythmia.(A) Pro-arrhythmic mechanisms involving the conducting pathway with compromised conduction [dark gray path 1, (a)] and a second normally conducting pathway [white path 2, (a)].A normal AP conduction with velocity θ and ERP has wavelength λ = θ × ERP (yellow region) in path 2 (b) (blue arrow).If the latter initiates a slow-conducting AP, this travels along path 1 (a) but under normal circumstances (b), this collides with refractory tissue within path 2 and then cannot re-enter the circuit (b).Thus, an abnormal triggered impulse immediately following the normal AP (a) cannot enter the refractory path 2 (b).However, a retrogradely conducting AP with reduced wavelength Λ shorter than the dimensions of the propagation pathways, due to reduced θ and/or ERP, along path 1 (a) entering the beginning of path 2 (b) causes selfperpetuating re-entrant excitation (c).(B) Isochronal AP propagation multi-electrode array mapping of re-entrant circuit initiation of ventricular tachycardia (VT) in the right ventricular epicardium of isolated Langendorff-perfused murine Scn5a+/− hearts following flecainide challenge.Thick black lines denote conduction block.Thin arrows denote lines of propagation.(a) Crowded isochronal lines in the last sinus beat, demonstrating area of conduction slowing.(b) Superimposed premature ventricular beat leads to line of block with impulse propagation flowing around it.(c) A second ventricular ectopic event causes the formation of a re-entrant circuit.(d) The circuit continues into the next beat (e, f) to initiate VT.Migration of line of block causing non-stationary vortex resulting in polymorphic arrhythmia should be noted.(C) Premature ventricular ectopic beat occurring as (a) isolated monophasically recorded event and (b) resulting in the initiation of electrocardiographically recorded sustained polymorphic VT [(A) Reproduced from King et al. (2013a), Figure 3; licensed under CC-BY 4.0 (B), (Cb) Adapted from Martin et al. (2011c), Figure 4; (Ca) Reproduced from Killeen et al. (2007), Figure 5C].

FIGURE 4
FIGURE 4 Arrhythmic substrate arising from temporal electrophysiological instabilities.(A) The nth action potential in a series has duration APD n dependent on its preceding diastolic interval DI n−1 along an A-curve.Symbols clarified in (B) showing action potential with a BCL made up of the APD and succeeding diastolic interval DI. (C) D-line showing the linear effect of APD n variations on DI n−1 at different BCLs: DI = BCL-APD.(D, E) Intersection between the A-curve and D-line gives steady-state APD and DI at any BCL (a).Increasing heart rate decreases the BCL and alters the D-line (b).This alters the APD (c) and, consequently, the succeeding DI (d).(D) A converging cycle resulting in declining oscillations results where APD n depends on DI n−1 with less than unity slope.The cycle remains constant with a unity slope and (E) diverges with a greater than unity slope.APD giving unstable waxing oscillations.(F) Transient instabilities in the form of (i) early and (ii) delayed afterdepolarizations in the course and following full recovery of the AP waveform [(A-E) from Figure 14.4 of the work of Huang (2021)].

FIGURE 5
FIGURE 5 propagation and recovery.(b) At the cellular level, excitation contraction coupling processes mediate Ca 2+ signaling.(c) At the systems level, autonomic inputs and their related G-protein signalling, modulated by central nervous system circadian rhythms.(d) Longer-term electrophysiological and structural remodeling effects related to metabolic feedback and other upstream modulators.These map onto (C) recently reclassified (a) anti-arrhythmic drugs that, in turn, act upon (b-e) the molecular targets mediating these physiological processes.These include (b) surface membrane ion channels contributing inward depolarizing hyperpolarization-activated cyclic nucleotide gate (HCN); cardiac Na + (Nav1.5)and Ca 2+ (Cav1.2) channel, outward K + channel (Kvx.x), and conducting connexin (CX) mediated currents.(c) Excitation contraction coupling-related Cav1.2, cardiac ryanodine receptor type 2 (RyR2) and transient receptor potential channel (TRPx), and their regulatory calcium/calmodulin kinase II (CaMKII); (d) autonomic signaling through inhibitory Gi, and stimulatory G proteins, Gs, involving PKA signaling.Longer-term modulation involves (e) energetic and modeling processes.

FIGURE 7
FIGURE 7 Pro-arrhythmic feedback relationships between energetic dysfunction and surface membrane ionic channel activity.Simplified scheme in which (A) energetic dysfunction in ischemia, cardiac failure, aging, or diabetes is reflected in (B) mitochondrial dysfunction causing ROS production, altered NAD + / NADH, and ATP/ADP.This leads to increased (C) RyR2-mediated SR Ca 2+ release and increased [Ca 2+ ] i NCX and consequent DAD triggering activity.(D) Altered Na + and K + channel function affects AP excitation, propagation, and recovery.The trigger/substrate combination (C, D) causes (E) sustained arrhythmia [from Figure 20 of the work of Huang (2017)].