The inositol trisphosphate receptor can facilitate but does not initiate ventricular arrhythmogenesis

In cardiac muscle, ryanodine receptors (RyRs) contribute to global Ca2+ production from Ca2+-induced Ca2+ release and are thus mainly responsible for cardiac muscle contraction. In addition to RyRs, cardiomyocytes express inositol trisphosphate receptors (InsP3Rs), but how InsP3Rs contribute to cardiomyocyte function remains unclear. Three isoforms of InsP3Rs (InsP3R1–InsP3R3) have been observed in cardiomyocytes, and InsP3R2 is the predominant isoform in heart muscle (Lipp et al., 2000). Neural hormones, such as endothelin-1 (ET-1) and angiotensin II (Ang II), activate G protein-coupled receptor (GPCR) signalling cascades and generate inositol trisphosphate (InsP3), which elicits Ca2+ mobilization via InsP3R. The function of InsP3Rs in atrial, sino-atrial node (SAN) and Purkinje cells has been described in many studies and reviews (Hirose et al., 2008; Hohendanner et al., 2015; Lipp et al., 2000; Yaniv et al., 2015). In normal atrial function, InsP3R activation enhances Ca2+ influx during excitation–contraction coupling (ECC) in the subsarcolemmal area and regions distal to the periphery, thereby leading to an increase in the global Ca2+ transient (Hohendanner et al., 2015). However, InsP3R activation leads to a decrease in sarcoplasmic reticulum (SR) Ca2+ load and reduced Ca2+ transients in atrial cells in heart failure (HF) models. Enhanced InsP3R activation facilitates spontaneous SR Ca2+ release together with an activation of the Na+–Ca2+ exchanger (NCX) inward current, which depolarizes the sarcolemmal membrane towards the threshold for firing an action potential (AP), enhancing the arrhythmogenic potential of atrial cells in HF models (Hohendanner et al., 2015). In SAN cells, InsP3Rs are involved in heart rhythm regulation and heart pacemaking (Yaniv et al., 2015). Activation of InsP3Rs causes spontaneous SR Ca2+ flux to modulate the RyR-mediated Ca2+ cycling that accelerates the spontaneous pacing rate. In Purkinje cells, in which InsP3R1 expression is prominent under the sarcolemma, InsP3R activation leads to wide-ranging spontaneous Ca2+ release events (Hirose et al., 2008). In these cells, both InsP3R1 and RyR2 are responsible in combination for spontaneous Ca2+ waves in the nuclear and subsarcolemmal regions of the cell. In ventricular cardiomyocytes (VMs), InsP3Rs are expressed at relatively lower levels than in atrial, SAN and Purkinje cells, and the effect of InsP3Rs on ECC in the healthy ventricle is considerably smaller than in the atria, SAN and Purkinje cells (Domeier et al., 2008). Previous studies have demonstrated that the influence of InsP3Rs on ECC in the ventricle is not consistent. In the presence of RyR inhibition by tetracaine, the remaining Ca2+ release events are almost completely absent, suggesting a very small proportion of InsP3-induced Ca2+ release events or limited InsP3R activity in the ventricle (Blanch & Egger, 2018). Despite this low expression, however, several studies have shown that HF and hypertrophic remodelling are associated with elevated InsP3R2 expression (Higazi et al., 2009; Ljubojevic et al., 2014). High endogenous levels of InsP3 lead to higher responsiveness and Ca2+ release in the nuclear region of the ventricle, observed as an increase in the frequency of Ca2+ sparks in the nucleus and perinucleus (Higazi et al., 2009). An elevation in basal levels of nuclear Ca2+ also contributes to increased Ca2+-dependent gene expression underlying cardiomyocyte hypertrophy (Ljubojevic et al., 2014). Although many studies have shown that nuclear InsP3 signalling is responsible for transcriptional regulation during cardiac remodelling, the mechanisms by which InsP3Rs located in the SR of VMs contribute to arrhythmogenesis have not been fully established, and evidence has remained somewhat contradictory. I believe that InsP3Rs can facilitate but not initiate ventricular arrhythmogenesis. In cardiac muscle, ‘forward-mode’ NCX extrudes Ca2+ from myocytes in exchange for Na+ influx. It is possible that NCX could extrude Ca2+ released from InsP3Rs, because NCX has low affinity for Ca2+, and InsP3Rs have been reported to localize proximally to NCX-enriched domains in the sarcolemma (Mohler et al., 2005). However, whether InsP3 signalling can trigger the inward NCX current in the resting potential of VMs has remained unclear. Signore et al. (2013) have shown that dialysis of InsP3 stimulates ∼0.5–0.6 pA/pF inward NCX current at −70 to −80 mV and causes a reverse potential right shift; simultaneously, application of ATP and ET-1 into VMs could initiate ∼0.5 pA/pF inward current in resting potential. In the dorsal root ganglion, InsP3Rs localize proximally to their signalling effectors, Ca2+-activated Cl− channels (ANO1) (Jin et al., 2013). High concentrations of Ca2+ released in synchrony from stores lead to large Ca2+-activated Cl– currents, and fluorescence imaging has revealed that Ca2+ signalling is strong and transient when InsP3Rs are stimulated. In cardiac myocytes, rapid application of caffeine produces an inward NCX current as bulk cytosolic Ca2+ is released from the SR and, simultaneously, NCX rapidly extrudes Ca2+ from the cleft. Fluorescence imaging has shown that caffeine induces a rapid rise in [Ca2+]i (time constant of dozens of milliseconds) that occurs as propagating waves (Trafford et al., 1995). However, given that Ca2+ released from InsP3Rs has a low unitary Ca2+ flux rate and non-synchronized openings (vs. RyRs), ET-1 does not induce either a detectable global increase or a local [Ca2+]i increase in VMs (Wu et al., 2006). It has been noted that InsP3 might bind InsP3Rs asynchronously and slowly owing to dialysis. I have observed that caffeine diffuses slowly into cardiac myocytes and cannot induce NCX current. It is also contradictory that Signore et al. (2013) have found that InsP3 causes Ca2+ to elevate to a stable level and not return to the base level, while ‘forward-mode’ NCX extrudes Ca2+ out of the cell to decrease [Ca2+]i rapidly. Therefore, I would argue that the inward currents from InsP3/ATP/ET-1 are not authentic NCX currents and are more similar to leaky currents (with slow activation and deactivation and plateaus without peaks). In contrast, we have observed no significant effect of InsP3 on inward NCX current and reverse potential in pig sham and post-myocardial infarction (post-MI) VMs following flash photolysis of InsP3 (Jin et al., 2023). Another study showed that the GPCR agonist Ang II, which produces InsP3 via phospholipase C, did not induce an NCX current (or induced only a very small one) in cardiac myocytes (Ronchi et al., 2018). It is believed that spontaneous RyR2-triggered SR Ca2+ release requires SR overload. Given that the SR Ca2+ exceeds the store overload and that Ca2+ release occurs spontaneously, Ca2+ propagates across myocytes in the form of intercellular Ca2+ waves, which activate the sarcolemmal NCX. Extrusion of Ca2+ via NCX during diastole generates transient inward currents, resulting in delayed after-depolarizations (DADs). These DADs, if large enough to exceed a threshold, can become spontaneous APs and eventually trigger arrhythmia. Signore et al. (2013) have found that ET-1 and ATP can trigger abnormal monophasic action potentials (MAPs) in human myocardium; however, it is unclear how they applied ET-1 and ATP to trigger the APs. Nevertheless, their fluorescence imaging results and those of other studies do not support the theory that InsP3/ATP/ET-1 produce a Ca2+ wave or that the SR store is overloaded (the Ca2+ rise is small and slow) (Domeier et al., 2008; Signore et al., 2013). In addition, Signore et al. (2013) have shown that ATP/ET-1 decreases Ca2+ content in normal mouse VMs. In contrast, we have found that the administration of Ang II or InsP3 does not increase waves or DADs in pig and human VMs (Jin et al., 2022, 2023). In addition, MAP results have shown that Ang II administration does not increase MAPs in human myocardium (Jin et al., 2022). Of note, when β-adrenergic receptor stimulation (by isoprenaline) produced an SR Ca2+ load, InsP3 induced a significant increase in Ca2+ waves in human HF and post-MI VMs, which was not observed in similarly treated non-HF (NF) human or sham pig VMs (Jin et al., 2022, 2023). Inositol trisphosphate combined with isoprenaline induced a significant increase in APs in both NF and HF VMs. The combined action of InsP3 and isoprenaline was greater in HF VMs than in NF VMs (Jin et al., 2022). Monophasic action potentials also showed that co-application of Ang II with isoprenaline significantly increased the incidence of both DADs and APs in comparison to perfusion with isoprenaline alone in NF, whereas only the frequency of APs was increased in HF (Jin et al., 2022). The InsP3R inhibitor 2-apb abrogated this pro-arrhythmic action. Likewise, Ronchi et al. (2018) have reported that stimulation of Ang II receptors might facilitate the occurrence of spontaneous Ca2+ release events induced by β-adrenergic stimulation (isoprenaline); thus, these authors have suggested that InsP3/InsP3Rs could be co-factors for SR instability. The resting membrane potential (RMP) is determined by the movement of several ions across ion channels and transporters in the plasma membrane. According to the Nernst equation, sodium and potassium have a dominant influence on RMP. Signore et al. (2013) have shown that ATP and ET-1 decrease the RMP and prolong the action potential duration in human cardiomyocytes; however, no results have demonstrated that short-term application of ATP and ET-1 influences the number of leakage channels or pumps in the membrane or changes the concentrations of various ion types outside the cell. It is possible that NCX extrudes Ca2+ and permits Na+ influx, while Squecco et al. (2016) have ruled out a substantial role for NCX in determining the RMP. We have shown that InsP3 signalling has no effect on the RMP and action potential duration of human and pig VMs (Jin et al., 2022, 2023); likewise, Zhang et al. (2020) have demonstrated that InsP3R2 deficiency does not affect the features of the APs of cardiomyocytes derived from human embryonic stem cells. The role of InsP3Rs in Ca2+ signalling during ECC has been inconsistent among studies to date, owing to the lower expression of InsP3Rs in the ventricle. These differences might be attributable to diversity in the expression, intracellular localization and downstream cascades of different receptors. Signore et al. (2013) have reported that ATP or ET-1 can increase the Ca2+ transient amplitude and contractility in human VMs, an effect mediated by InsP3Rs, and they have also shown that ATP or ET-1 can decrease Ca2+ transient amplitude in normal mouse VMs. We have found that direct application of InsP3 has no effect on the Ca2+ transient properties in NF, normal and post-MI VMs (Jin et al., 2022, 2023). Zhang et al. (2020) have reported that inactivation of the gene encoding InsP3R2 does not significantly affect Ca2+ transients in cardiomyocytes derived from human embryonic stem cells. Of note, we have found that InsP3 reduces the Ca2+ transient amplitude in HF VMs, correlating with decreased SR Ca2+-ATPase activity and Ca2+ content (Jin et al., 2022). Mijares et al. (2020) also found that the InsP3R blocker Xest-C could partly restore the SR Ca2+ content and improve contractile dysfunction in cardiomyocytes from patients with Chagas disease. Therefore, both Ca2+ leakage from InsP3Rs and a reduction in SR Ca2+-ATPase activity might lead to depletion of SR Ca2+ stores and a reduction in the Ca2+ transient amplitude. Ryanodine receptors play a key role in ventricular arrhythmias, because many arrhythmias are initiated by spontaneous diastolic SR Ca2+ release events via RyR2. Sufficient Ca2+ release can activate a potentially arrhythmogenic, depolarizing inward NCX current, which can cause DADs and trigger ventricular arrhythmias. In contrast, recent results have demonstrated that Ca2+ release from InsP3Rs cannot trigger inward NCX current but can enhance the amplitude of DADs triggered by spontaneous Ca2+ release from RyRs, thus providing an additional arrhythmogenic stimulus (Jin et al., 2022, 2023). It is likely that InsP3R expression levels and their induced calcium dynamics in VMs vary with respect to those of RyRs. It is believed that cardiomyocyte InsP3R expression levels are lower than those of RyRs (typically, 50 times less abundant) (Moschella & Marks, 1993). Previous studies have shown that InsP3Rs can trigger ventricular arrhythmias alone (Signore et al., 2013); however, our results (Jin et al., 2022, 2023) have demonstrated that InsP3Rs cannot initiate ventricular arrhythmogenesis but might act as auxiliary factors in promoting or facilitating the activation of RyRs in the development of ventricular arrhythmias (Figure 1). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. No conflicts of interest, financial or otherwise, are declared by the author. Sole author. None.