A Different Route for Modulating Inositol 1,4,5-Trisphosphate Receptors “Exciting” Players in Cardiac Excitation-Contraction Coupling?

the cardiac Na + /Ca 2+ exchanger (NCX), in depolarized resting membrane potential and AP prolongation Figure). Hence, Ca 2+ release from InsP 3 R does not contribute to contractile Ca 2+ directly; rather, the enhanced Ca 2+ transients and contractility induced by GPCR ligands and InsP 3 R signaling are the consequence of the InsP 3 -induced modulation of membrane ion fluxes. Moreover, RyR inhibi-tion did not interfere with the InsP 3 -induced AP regulation, yet intracellular Ca 2+ buffering abolished the InsP 3 effects on the membrane potential. These results suggest the existence of local Ca 2+ signaling microdomains of InsP 3 R and plasma membrane ion channels or transporters that are distinct from the dyadic domains involved in ECC (Figure), akin to local InsP 3 -dependent perinuclear Ca 2+ signaling involved in excitation-transcription coupling. 8,9 Evidence for perinuclear Ca 2+ microdomains containing InsP 3 Rs has been identi-fied by Ca 2+ imaging, 8,9 immunofluorescence, 9 and electron

C a 2+ release through ryanodine receptor (RyR) intracellular Ca 2+ release channels plays the leading role in the regulation of myocyte contraction. However, Ca 2+ signals arising from their less abundant inositol 1,4,5-trisphosphate (InsP 3 )-sensitive counterparts (InsP 3 Rs), also localized to intracellular stores, are emerging as an important modulator of Ca 2+ -sensitive processes in cardiac myocytes. In this week's issue of Circulation, Signore et al 1 report that activation of InsP 3 R Ca 2+ release channels by G-protein-coupled receptor (GPCR) signaling regulates the electric properties of human myocardium. Their results provide new insights into how InsP 3 -induced Ca 2+ release modulates cardiomyocyte Ca 2+ handling and contractility through influencing sarcolemmal membrane potential and ionic currents.

Article see p 1286 Ca 2+ Regulation of Myocyte Contractility
An elevation in intracellular Ca 2+ (known as contractile Ca 2+ ) is a critical step in the sequence of events called excitation-contraction coupling (ECC) that couple depolarization of the plasma membrane by the propagating action potential with myocyte contraction. 2 Ca 2+ is released from sarcoplasmic reticulum Ca 2+ stores via RyRs, which are activated by Ca 2+ entering the cell through L-type voltagegated Ca 2+ channels (LTCCs) opened in response to membrane depolarization. The functional coupling between Ca 2+ entry through LTCCs and RyRs is enabled by the proximity of the sarcolemma and sarcoplasmic reticulum, which lie 10 to 15 nm apart in specialized domains called dyads ( Figure). Invaginations of the sarcolemma at ~1.8µm intervals across the myocyte distribute these signaling domains throughout the cell volume, allowing a rapid and homogenous cell-wide Ca 2+ elevation during an action potential (AP). 2

InsP 3 R: A Different Route for Modulating Contractile Ca 2+ in Ventricular Myocytes
Like RyRs, InsP 3 Rs are large, tetrameric, intracellular Ca 2+ release channels located in the sarcoplasmic reticulum/endoplasmic reticulum intracellular Ca 2+ store that are activated by Ca 2+ . 3 Unlike RyRs, InsP 3 Rs also require InsP 3 for activation, thereby making them subject to control by extracellular ligands that engage phospholipase C-activating plasma membrane receptors, including GPCRs and receptor tyrosine kinase. 3 Of the 3 mammalian InsP 3 R isoforms (InsP 3 R1, InsP 3 R2, and InsP 3 R3), InsP 3 R2 predominates in cardiac muscle. 4 Although InsP 3 Rs are much less abundant than RyRs in the heart (<1:50-1:100 of RyRs), 4 substantial evidence exists for InsP 3 Rs and InsP 3 -induced Ca 2+ signaling in atrial myocytes. 5 However, because of their even lower expression in the ventricle, the importance and contribution of InsP 3 Rs to Ca 2+ signals during ECC have been questioned. 5 Signore et al 1 report that human ventricular myocytes express functional InsP 3 R Ca 2+ release channels that are responsible for the modulation of ECC by GPCR ligands such as ATP and endothelin-1. Although a role for InsP 3 in stimulating cardiac Ca 2+ release was described >2 decades ago, 6 Signore et al 1 are the first to show that the InsP 3 /InsP 3 R signaling pathway plays an important role in regulating ECC in human ventricular myocytes. Previous work had suggested that Ca 2+ release from InsP 3 R channels located in dyadic cleft augments RyR-mediated ECC and contractility ( Figure). 7 Using an elegant combination of studies in single ventricular myocytes and intact myocardium from humans and mice, Signore et al 1 demonstrate a new mechanism by which InsP 3 regulates ECC: InsP 3 R Ca 2+ release signals via an unidentified mechanism to membrane ion channels and the cardiac Na + /Ca 2+ exchanger (NCX), resulting in depolarized resting membrane potential and AP prolongation (the Figure). Hence, Ca 2+ release from InsP 3 R does not contribute to contractile Ca 2+ directly; rather, the enhanced Ca 2+ transients and contractility induced by GPCR ligands and InsP 3 R signaling are the consequence of the InsP 3 -induced modulation of membrane ion fluxes. Moreover, RyR inhibition did not interfere with the InsP 3 -induced AP regulation, yet intracellular Ca 2+ buffering abolished the InsP 3 effects on the membrane potential. These results suggest the existence of local Ca 2+ signaling microdomains of InsP 3 R and plasma membrane ion channels or transporters that are distinct from the dyadic domains involved in ECC (Figure), akin to local InsP 3 -dependent perinuclear Ca 2+ signaling involved in excitation-transcription coupling. 8,9 Evidence for perinuclear Ca 2+ microdomains containing InsP 3 Rs has been identified by Ca 2+ imaging, 8,9 immunofluorescence, 9

Circulation
September 17, 2013 microscopy. 10 Whether the same structural domains also couple to sarcolemmal ion channels or whether such signaling occurs in caveolae-rich microdomains with LTCCs that are not involved in ECC 11 remains to be seen. Nevertheless, the functional data provided by Signore et al suggest that in addition to its role in hypertrophic gene regulation 8,9,12, GPCR/InsP 3 signaling also regulates the electrophysiological properties and excitability of cardiac muscle.

InsP 3 Signaling: Punching Above Its Weight to Control Cardiomyocyte Function
In ventricular myocardium, InsP 3 Rs are expressed at exceedingly low levels relative to RyR. The Ca 2+ current they conduct is only a quarter of that of RyRs. 13 InsP 3 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 Ca 2+ concentration with distance from the source channel, 15 the low density and Ca 2+ flux through InsP 3 Rs would prevent a significant elevation in intracellular Ca. 2+ How can this channel then contribute so greatly to cardiomyocyte function, in both regulating ECC and promoting hypertrophic gene transcription? A likely mechanism involves localization of the InsP 3 R proximal to its signaling effectors. Analogous to P/Q channels and BK Ca channels in smooth muscle, 16 the target of InsP 3 -mediated Ca 2+ signals is exposed to the high concentration of Ca 2+ at the mouth of the channel. As illustrated in the Figure 7 This mechanism has also been proposed to explain the increased spark frequency in rabbit and rat ventricular exposed to InsP 3 17,18 and may underlie the positive inotropic action of GPCR/InsP 3 signaling in human myocytes. This cross-talk between InsP 3 Rs and RyRs is likely to make a greater contribution to the augmented GPCR/InsP 3 signaling observed in hypertrophic and failing hearts that exhibit significantly elevated InsP 3 R expression levels. 1,18 Interestingly, Signore et al do not find evidence for InsP 3 R-RyR coupling in mouse ventricular myocytes. In mice, the positive inotropic effect of GPCR agonists and InsP 3 appears to be exclusively the consequence of AP prolongation and suggests an alternative model of coupling of InsP 3 R that controls cardiac excitation (Figure). The functional results by Signore et al 1 suggest that in murine myocardium a pool of functional InsP 3 R exists outside the dyad that couples directly to membrane ion channels or transporters membrane. Signore et al 1 demonstrate that GPCR/InsP 3 signaling activates NCX and suggest enhanced NCX inward currents as a culprit responsible for the InsP 3induced AP prolongation and membrane depolarization. However, it appears unlikely that the NCX effect reported by Signore et al 1 is solely responsible because enhanced NCX, while prolonging the AP in the short term, would eventually deplete cellular Ca 2+ stores and result in reduced contractility. Thus, InsP 3 signaling likely modifies other membrane ion channels that either directly prolong the cardiac AP or cause a net increase in Ca 2+ 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 InsP 3 -dependent increase in AP duration, depolarized resting membrane potential, decreased I to , and increased arrhythmic events. 19 Furthermore, there is Figure. Dual control of excitation-contraction coupling (ECC) by G-protein-coupled receptor/inositol 1,4,5-trisphosphate (GPCR/ InsP 3 ) signaling. 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, L-type voltage-gated Ca 2+ channels (LTCCs), I K , I Na , Na + / Ca 2+ exchanger (NCX), and sodium-hydrogen exchanger (NHE). Juxtaposed to the t-tubule and caveolar membrane domains are cisternae of the sarcoplasmic reticulum (SR), forming dyadic (red box) and peripheral couplings, respectively (purple box). The distance between the sarcolemma and underlying SR in both of these membrane domains is between 10 and 15 nm, allowing rapid Ca 2+ diffusion across the dyadic cleft. Based on the model proposed by Signore et al 1 in which InsP 3 -induced Ca 2+ release (IICR) influences NCX and prolongs action potential (AP) duration (GPCR AP) independently of Ca 2+ release via ryanodine receptors (RyRs), InsP 3 receptors (InsP 3 Rs) are localized to a junctional coupling devoid of RyRs (purple box). InsP 3 Rs have also been proposed to influence Ca 2+ release via RyRs as a result of a mechanism involving their colocalization at the dyad (red box). At this location, IICR contributes minimally to the regulation of the AP (red box) but could influence NCX activity secondary to RyR activation. InsP 3 is produced downstream of GPCR stimulation of phospholipase C (PLC) and hydrolysis of PtdInsP 2 (phosphatidylinositol 4,5-bisphosphate). PLC hydrolysis of PtdInsP 2 also generates diacylglycerol (DAG), which can subsequently activate protein kinase C (PKC). PKC influences the activity of NCX, NHE, Na + , K + , and Ca 2+ channels on the sarcolemma (gray), which may also affect the electrophysiological properties of the ventricular cardiomyocyte. For clarity, not all proteins involved in ECC are shown here. by guest on July 22, 2018 http://circ.ahajournals.org/ Downloaded from extensive cross-talk between signaling messengers downstream of GPCR other than InsP 3 that also regulates the activity of LTCCs, NCX, and the sodium-hydrogen exchanger 20 (Figure), which likely are also involved in the regulation of resting membrane potential and AP duration in response to InsP 3 -induced Ca 2+ release.

Sources of Funding
Work in the Roderick laboratory is funded by the BBSRC (Epigenetics ISPG) and the British Heart Foundation. The Knollmann laboratory is supported in part by grants from the US National Institutes of Health and the American Heart Association.