Non-voltage-gated Ca2+ entry pathways in the heart: the untold STOrai?

This editorial refers to ‘Emergence of Orai3 activity during cardiac hypertrophy’ by Y. Saliba et al., pp. 248–259, this issue. Our understanding of cardiomyocyte Ca2+ handling is primarily based on the regulation of voltage-gated Ca2+ entry, via L-type Ca2+ channels (LTCCs), and the resulting Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) required for excitation–contraction coupling. Ca2+ is widely recognized as playing a key signalling role in all cells, and in cardiomyocytes the most commonly accepted pathway involves Ca2+ release from intracellular stores such as the endoplasmic reticulum (ER)/SR and the nuclear envelope. However, in non-excitable cells, agonist-mediated increases in intracellular Ca2+ are known to occur as a result of Ca2+ entry across the plasma membrane. The most widely studied of these pathways is store-operated Ca2+ entry (SOCE) as described by Putney1 where Inositol 1, 4, 5 Triphosphate (IP3)-induced release of Ca2+ from ER/SR stores triggered a subsequent influx of extracellular Ca2+, which was required both for subsequent activation of downstream signalling pathways and also for refilling of ER/SR. Such Ca2+ signals have been shown to play a key role in the regulation of diverse cellular responses, including metabolism, transcription, and differentiation. One of the earliest reports demonstrating that SOCE contributed to Ca2+ signalling in cardiomyocytes was in 2002 where Marchase and colleagues2 showed that activation of nuclear factor of activated T-cells (NFAT) by phenylephrine or angiotensin and the resulting cardiomyocyte hypertrophy was SOCE-dependent. At that time the proteins responsible for regulating SOCE were unknown; however, in 2005 with the identification of stromal interaction molecule (STIM) and Orai protein families, the molecular mediators of SOCE were finally elucidated. It is now generally accepted that the coupling of the ER/SR membrane protein STIM1 with the plasma membrane protein Orai1 is a major mechanism facilitating SOCE. While the most recent studies on non-voltage-gated Ca2+ signalling and cardiomyocyte hypertrophy have focused on STIM and Orai-mediated pathways, it should be noted that transient receptor potential channels have also been implicated; however, it has been suggested that this may involve Ca2+ entry via LTCC.3 Clearly, further studies are needed to understand the relative importance of non-voltage-gated and voltage-gated Ca2+ signalling in the heart. In addition to SOCE, there is also growing evidence of another non-voltage-gated Ca2+ entry pathway, store-independent Ca2+ entry (SICE), primarily characterized as an endogenous arachidonic acid-regulated Ca2+ (ARC) channel, which has current/voltage characteristics similar to SOCE, but does not require store depletion. Interestingly, ARC channel activation has been reported to involve a heteromeric assembly of Orai1 and Orai34–6 as well as coupling with plasma membrane localized STIM1.7 It is of note that while STIM and Orai protein families are highly conserved, with invertebrates having only a single Orai protein and vertebrates two Orai proteins, Orai3 appears to be specific for mammalian cells.6 While there have been a number of studies supporting a role of STIM1 and SOCE in mediating cardiomyocyte hypertrophy, little is known about SICE or ARC activity in the heart. Consequently, the study by Saliba et al.,8 which focuses on the interaction between STIM1 with Orai1 and Orai3 and their role in Ca2+ influx in cardiomyocytes, is both highly novel and timely. They demonstrated for the first time the involvement of Orai3 and ARC channels in non-voltage-gated Ca2+ entry and hypertrophic signalling in rat cardiomyocytes. They found that Orai1, Orai3 and STIM1 were expressed at variable levels in both normal and hypertrophied rat cardiomyocytes; importantly, however, they showed that in response to hypertrophy the levels of STIM1and Orai1 that were immunoprecipitated with Orai3 were increased significantly. Using selective knockdown of Orai1 and Orai3 proteins in vivo, they found that not only was Orai3 rather than Orai1 primarily responsible for interacting with STIM1, but also that Orai3 appeared to regulate SOCE. An earlier study from the same group identified an unknown Ca2+ current in hypertrophic cardiomyocytes;9 here, they have shown that this is an Orai3–STIM1–ARC-mediated current. In hypertrophied cardiomyocytes, they observed an increased rate of Orai3-dependent SOCE as well as an increase in ARC channel current, both of which are consistent with the increased association of Orai3 with Orai1 and STIM1. While Wolkowicz et al.11 implicated Orai1 and Orai3 in cardiac arrhythmogenesis, the in vivo loss-of-function studies by Saliba et al. are the first to demonstrate a functional role for STIM1–Orai3 in regulating Ca2+ signalling in cardiomyocytes.8 Indeed, even in non-excitable cells, our knowledge on STIM1 function has been primarily in the context of STIM1–Orai1 coupling in the regulation of SOCE; consequently, our knowledge of Orai3 function is fairly limited. However, Orai3 has been shown to be responsible for SOCE in breast cancer cells and also permits SOCE in conditions of increased oxidative stress in leukaemia;12,13 in light of the report by Saliba and colleagues, further studies clearly are needed to better understand the function of Orai3 in the heart. It should be noted that while present at low levels in the heart, Orai2 could also be involved in voltage-independent Ca2+ signalling mechanisms; this is especially important since some of the knockdown strategies used by Saliba et al.8 targeted all Orai proteins. This is potentially relevant since Voelkers et al.10 showed that knockdown of Orai1 in neonatal rat ventricular myocytes resulted in an increase in Orai2 rather than Orai3. Moreover, Saliba et al.8 concluded that SOCE and SICE were independent of voltage-dependent Ca2+ entry pathways; however, given reports that both STIM and Orai proteins interact with LTCC14 and sarcoplasmic/endoplasmic reticulum calcium ATPase,15 further studies are clearly required to examine such interactions in the heart. This study by Saliba and colleagues is part of a growing number of reports demonstrating not only the presence but also showing a clear functional role of STIM and Orai proteins in the heart.9,10,16–18 For example, hearts from mice with cardiomyocyte-specific deletion of STIM1 exhibited ER stress, mitochondrial dysfunction, and dilated cardiomyopathy,16 demonstrating that STIM1 is essential for cardiac homeostasis. In addition, Orai1 knockdown in zebrafish resulted in the development of cardiac contractile abnormalities17 and mice lacking Orai1 subjected to transaortic constriction develop an accelerated dilated cardiomyopathy.18 Molkentin and colleagues19 have shown that that the IP3-Receptor plays a significant role in cardiac hypertrophic signalling via calcineurin through IP3-mediated Ca2+ release and importantly in cardiomyocytes the IP3–Ca2+ signalling cascade, activated by agonists such as angiotensin II and phenylephrine, has been linked to SOCE. Moreover, as noted above, Ca2+-dependent activation of NFAT in cardiomyocytes has also been shown to be SOCE-dependent.2 Consequently, it is increasingly clear that we need to expand our current models of cardiomyocyte Ca2+ signalling and homeostasis to include not only SOCE, but also SICE, pathways. This will also require a re-evaluation of well-defined Ca2+ signalling pathways such as the calcineurin–NFAT and the MAPK-dependent pathways to identify the downstream components of SOCE and SICE. The development of a more comprehensive and broader-based understanding of cardiomyocyte Ca2+ signalling networks will open up entirely new avenues for understanding the molecular underpinnings of cardiac hypertrophy and other diseases. Conflict of interest: none declared. J.C.C. is supported by a National Heart, Lung and Blood Institute grant (R21-HL-110366). H.E.C. is supported by a University of Alabama At Birmingham Comprehensive Cardiovascular Center William W. Featheringill Postdoctoral Fellowship.