Inositol 1,4,5-trisphosphate receptors (InsP₃R) – “exciting” players in cardiac excitation-contraction coupling?

Ca²⁺ release through ryanodine receptor (RyR) intracellular Ca²⁺ release channels plays the leading role in regulation of myocyte contraction. However, Ca²⁺ signals arising from their less abundant inositol 1,4,5-trisphosphate sensitive counterparts (InsP₃R), also localised to intracellular stores, is emerging as an important modulator of Ca²⁺-sensitive processes in cardiac myocytes. In this month’s issue of Circulation, Rota et al¹ report that activation of InsP₃R Ca²⁺ release channels by G-protein coupled receptor (GPCR) signalling regulates the electrical properties of human myocardium. Their results provide new insights into how InsP₃ induced Ca²⁺ release (IICR) modulates cardiomyocyte Ca²⁺ handling and contractility through influencing sarcolemmal membrane potential and ionic currents.

Ca²⁺ regulation of myocyte contractility

An elevation in intracellular Ca²⁺ (known as contractile Ca²⁺) is a critical step in the sequence of events, termed excitation contraction coupling (ECC) that couples depolarisation of the plasma membrane by the propagating action potential with myocyte contraction.² Ca²⁺ is released from sarcoplasmic reticulum (SR) Ca²⁺ stores via RyRs, which are activated by Ca²⁺ entering the cell through L-type voltage-gated Ca²⁺ channels (LTCC) opened in response to membrane depolarisation. The functional coupling between Ca²⁺ entry through LTCC and RyRs is enabled by the proximity of the sarcolemma and SR, which lie 10-15 nm apart in specialised domains, termed dyads (Fig. 1). Invaginations of the sarcolemma at 1.8 μ intervals across the myocyte distribute these signalling domains throughout the cell volume allowing a rapid and homogenous cell-wide Ca²⁺ elevation during an action potential.²

Figure 1. Dual control of excitation contraction coupling (ECC) by GPCR/InsP₃ signalling.

The sarcolemma of the cardiac myocyte forming a single t-tubule and a caveolus are shown together with the machinery underlying ECC. These membrane regions express GPCR, LTCC, I_K, I_Na, NCX and NHE. Juxtaposed to the t-tubule and caveolar membrane domains are cisternae of the SR, forming dyadic (red box) and peripheral couplings respectively (purple box). The distance between the sarcolemma and underlying SR in both these membrane domains is between 10 and 15 nm allowing rapid Ca²⁺ diffusion across the dyadic cleft. Based on the model proposed by Rota et al, in which IICR influences NCX and prolongs AP duration (GPCR AP) independently of Ca²⁺ release via RyRs, InsP₃Rs are localised to a junctional coupling devoid of RyRs (purple box). InsP₃Rs have also been proposed to influence Ca²⁺ release via RyRs due a mechanism involving their co-localisation at the dyad (red box). At this location, IICR contributes minimally to regulation of the AP (red box) but could influence NCX activity secondary to RyR activation. InsP₃ is produced downstream of GPCR stimulation of PLC and hydrolysis of PtdInsP₂. PLC hydrolysis of PtdInsP₂ also generates DAG, which can subsequently activate PKC. PKC influences the activity of NCX, NHE, Na⁺, K⁺ and Ca²⁺ channels on the sarcolemma (in grey), which may also affect the electrophysiological properties of the ventricular cardiomyocyte. For clarity, not all proteins involved in ECC are shown in this cartoon.

InsP₃R – a different route for modulating contractile Ca²⁺ in ventricular myocytes

Like RyRs, InsP₃Rs are large tetrameric intracellular Ca²⁺ release channels located in the SR/Endoplasmic Reticulum intracellular Ca²⁺ store that are activated by Ca²⁺.³ Unlike RyRs, InsP₃Rs also require InsP₃ for activation, thereby making them subject to control by extracellular ligands that engage phospholipase C (PLC)-activating plasma membrane receptors, including GPCRs and receptor tyrosine kinase.³ Of the three mammalian InsP₃R isoforms (InsP₃R1, 2 and 3), InsP₃R2 predominates in cardiac muscle.⁴ Although InsP₃Rs are much less abundant than RyRs in the heart (<1:50-1:100 of RyRs),⁴ substantial evidence exists for InsP₃Rs and InsP₃-induced Ca²⁺ signalling in atrial myocytes.⁵ However, due to their even lower expression in the ventricle, the importance and contribution of InsP₃Rs to Ca²⁺ signals during ECC has been questioned.⁵

In the current issue of Circulation, Rota et al¹ report that human ventricular myocytes express functional InsP₃R Ca²⁺ release channels that are responsible for the modulation of ECC by GPCR ligands such as ATP and endothelin-1 (ET-1). Although a role for InsP₃ in stimulating cardiac Ca²⁺ release was described over two decades ago,⁶ Rota et al¹ are the first to show that the InsP₃/InsP₃R signalling pathway plays an important role in regulating ECC in human ventricular myocytes. Previous work had suggested that Ca²⁺release from InsP₃R channels located in dyadic cleft augments RyR-mediated ECC and contractility (Fig. 1).⁷ Using an elegant combination of studies in single ventricular myocytes and intact myocardium from human and mouse, Rota et al¹ demonstrate a new mechanism by which InsP₃ regulates ECC: InsP₃R Ca²⁺ release signals via an unidentified mechanism to membrane ion channels and the cardiac Na⁺/Ca²⁺ exchanger (NCX), resulting in depolarized resting membrane potential and action potential (AP) prolongation (Fig. 1). Hence, Ca²⁺ release from InsP₃R does NOT contribute to contractile Ca²⁺ directly but rather, the enhanced Ca²⁺ transients and contractility induced by GPCR ligands and InsP₃R signalling are the consequence of the InsP₃-induced modulation of membrane ion fluxes. Moreover, since RyR inhibition did not interfere with the InsP₃-induced AP regulation, yet intracellular Ca²⁺ buffering abolished the InsP₃ effects on the membrane potential, these results suggest the existence of local Ca²⁺ signalling microdomains of InsP₃R and plasma membrane ion channels or transporters that are distinct from the dyadic domains involved in ECC (Fig. 1), akin to local InsP₃-dependent perinuclear Ca²⁺ signalling involved in excitation-transcription coupling.^{8, 9} Evidence for perinuclear Ca²⁺ microdomains containing InsP₃Rs has been identified by Ca²⁺ imaging,^{8, 9} immunofluorescence⁹ and electronmicroscopy.¹⁰ Whether the same structural domains also couple to sarcolemmal ion channels, or whether such signalling occurs in caveolae-rich microdomains with LTCC that are not involved in ECC¹¹ remains to be seen. Nevertheless, the functional data provided by Rota et al suggest that GPCR/InsP₃ signalling is not only a critical player for hypertrophic gene regulation^{8, 9, 12} but also regulates electrophysiological properties and excitability of cardiac muscle.

InsP₃ signalling, punching above its weight to control cardiomyocyte function

In ventricular myocardium, InsP₃Rs are expressed at exceedingly low levels relative to RyR. The Ca²⁺ current they conduct is only a quarter of that of RyRs.¹³ InsP₃ is generated at a very low level in cardiac myocytes relative to other tissues,¹⁴ thereby limiting the number of channels opened. Together with the exponential decay in Ca²⁺ concentration with distance from the source channel,¹⁵ the low density and Ca²⁺ flux through InsP₃Rs would prevent a significant elevation in intracellular Ca²⁺. How can this channel then contribute so greatly to cardiomyocyte function – both in regulating ECC and promoting hypertrophic gene transcription? A likely mechanism involves localisation of the InsP₃R proximal to its signalling effectors. Analogous to P/Q channels and BK_Ca channels in smooth muscle,¹⁶ the target of InsP₃ mediated Ca²⁺ signals is exposed to the high concentration of Ca²⁺ at the mouth of the channel. As illustrated in Fig. 1, InsP₃Rs localised to the dyad within a few nm of RyRs could “prime” RyRs to activation by Ca²⁺ influx via LTCC. Moreover, association of Ca²⁺ arising from InsP₃Rs with resident cytosolic Ca²⁺ buffers would reduce the buffering capacity of the cytosol in the vicinity of the dyad thereby allowing Ca²⁺ subsequently released via RyRs to have a greater impact upon free Ca²⁺ levels. In support of this InsP₃R-RyR coupling model, block of RyRs with ryanodine, reduces the amplitude of InsP₃-stimulated elementary Ca²⁺ release events in cardiac myocytes.⁷ This mechanism has also been proposed to explain the increase spark frequency in rabbit and rat ventricular exposed to InsP₃,^{17, 18} and may underlie the positive inotropic action of GPCR/InsP₃ signalling in human myocytes. This crosstalk between InsP₃Rs and RyRs is likely to make a greater contribution to the augmented GPCR/InsP₃ signalling observed in hypertrophic and failing hearts that exhibit significantly elevated InsP₃R expression levels.^{1, 18} Interestingly, Rota et al do not find evidence for InsP₃R-RyR coupling in mouse ventricular myocytes. In mice, the positive inotropic effect of GPCR agonists and InsP₃ appears to be exclusively the consequence of AP prolongation and suggests an alternative model of coupling of InsP₃R that controls cardiac excitation (Fig. 1). The functional results by Rota et al¹ suggest that in murine myocardium a pool of functional InsP₃R exists outside the dyad that couple directly to membrane ion channels and/or transporters membrane. Rota et al¹ demonstrate that GPCR/InsP₃ signalling activates NCX and suggest enhanced NCX inward currents as a culprit responsible for the InsP₃-induced AP prolongation and membrane depolarization. However, it appears unlikely that the NCX effect reported by Rota et al¹ is solely responsible, since enhanced NCX, while prolonging the AP in the short term, would eventually deplete cellular Ca²⁺ stores and result in reduced contractility. Thus, InsP₃ signalling likely modifies other membrane ion channels that either directly prolong the cardiac AP or cause a net increase in Ca²⁺ influx into the cell. Consistent with this idea, a profound effect on membrane electrophysiology was observed in mouse ventricular myocytes exposed to Fas ligand.¹⁹ Fas ligand caused an InsP₃-dependent increase in AP duration, depolarized resting membrane potential, decreased I_to and increased arrhythmic events.¹⁹ Furthermore, there is extensive crosstalk between signalling messengers downstream of GPCR other than InsP₃ that also regulate the activity of LTCC, NCX and the sodium hydrogen exchanger (NHE)²⁰ (Fig. 1), which likely are also involved in the regulation of resting membrane potential and AP duration in response to IICR.