Revue scientifique : Chapter 26 – Store-Operated Ca 2+ Entry (SOCE) pathways – second edition

74

Figure 25 : Schéma illustrant la localisation et le rôle potentiel de la machinerie SOC dans les cardiomyocytes

Ca2+ : ion calcium ; Na+ : ion sodium ; Tubule T : Tubule Transverse ; TRPC : Transient Receptor Potential Canonical Channels ; NCX : Echangeur Na⁺/ Ca²⁺ ; STIM1 : Stromal Interaction Molecule 1 ; PLB : Phospholamban ; pompe SERCA2a :

Sarco/Endoplasmic Reticulum Ca²⁺ ATPase ; RyR2 : Récepteurs de la Ryanodine de type 2 ; pompe PMCA : Plasma membrane Ca²⁺ ATPase ; RIP₃ : Récepteurs à l’inositol triphosphate ; MCU : Mitochondrial Ca²⁺ Uniporter ; CaMKII : Ca²⁺ /calmodulin-dependent protein kinase II ; CaN : Calcineurine : ERK1/2 : Extracellular Signal Regulated Kinases 1 and 2.

4) Revue scientifique : Chapter 26 – Store-Operated Ca

Entry

(SOCE) pathways – second edition

Cardiac Remodeling and Disease: Current Understanding of STIM1/Orai1-Mediated Store-Operated Ca²⁺ Entry in Cardiac Function and Pathology.

Bartoli F. and Sabourin J.

Cardiac Remodeling and Disease:

Current Understanding of

STIM1/Orai1-Mediated Store-Operated Ca

Entry in

Cardiac Function and Pathology

26

Fiona Bartoli and Jessica Sabourin

Abstract

For a long time, Ca²⁺entry into cardiomyocytes was considered the sole domain of the L-type Ca²⁺channel. Recently, STIM1/Orai1-mediated store-operated Ca²⁺ entry has been also reported to participate to Ca²⁺influx in cardiac cells and has emerged as a key player to alter Ca²⁺in the cardiomyocyte. In this review, we will highlight accumulated knowledge about the presence and the potential contribu-tion of STIM1/Orai1-dependent SOCE to cardiac funccontribu-tion and its role in the cardiac pathogenesis. Overall, even if STIM1/Orai1 proteins are present in the heart, contradictory results have been reported regarding their contribution to cardiac physiology and pathology, pointing out the necessity of further investigations, a major challenge over the coming years.

Keywords

Orai1 • STIM1 • Store-operated Ca²⁺entry • Heart • Cardiomyocytes • Cardiac hypertrophy • Heart failure • Arrhythmias

26.1 Introduction

Heart failure (HF) is an increasingly prevalent syndrome in which abnormal cardiac function leads to inadequate supply of blood to tissues and organs for their meta-bolic demands. Various factors contribute to HF pathogenesis such as ischemia and myocardial infarction, hypertension, or genetic cardiomyopathies.

As a leading cardiovascular cause of death, HF has been singled out as an epidemic disease and is a staggering clinical and public health problem associated

F. Bartoli • J. Sabourin (*)

Inserm UMR S1180, University Paris-Sud, Universite´ Paris-Saclay, 92296 Chaˆtenay-Malabry, France

e-mail:fiona.bartoli@u-psud.fr;jessica.sabourin@u-psud.fr

# Springer International Publishing AG 2017

K. Groschner et al. (eds.),Store-Operated Ca²⁺Entry (SOCE) Pathways, Advances in Experimental Medicine and Biology 993, DOI 10.1007/978-3-319-57732-6_26

with significant mortality and morbidity rates, particularly in patients aged 65 years and older. Despite progress in reducing HF-related mortality, hospitalizations for HF remain frequent and rates of readmissions continue to rise. As a multifactorial clinical syndrome, HF still represents an epidemic threat, highlighting the need to deeper understand the cellular mechanisms involved in the pathogenesis in order to develop innovative therapeutic strategies.

A strict spatiotemporal control of intracellular Ca²⁺concentration is essential for many heart functions such as excitation-contraction coupling (ECC), and it is orchestrated by a wealth of ion channels that are the fundaments determining the cardiac electromechanical activity. Indeed, Ca²⁺mishandling as a central cause of contractile dysfunction and arrhythmias is a hallmark of heart disease and has been the subject of a large body of research to understand how cardiac stress alters Ca²⁺ homeostasis in cardiomyocytes. Several groups have asserted that defects in SR Ca²⁺ uptake via the sarco-/endoplasmic reticulum Ca²⁺-ATPase (SERCA), abnormalities in ECC such as L-type Ca²⁺ channel and ryanodine receptor (RyR) dysfunction, and/or reverse mode Na⁺/Ca²⁺exchange participate to the development of the disease (Roe et al.2015). Currently, experimental therapies manipulating SERCA2a pump or cardiac myosin activity are in progress to improve Ca²⁺ homeostasis and cardiac function (Bernardo and Blaxall2016).

Recently, non-voltage-gated Ca²⁺channels such as store-operated Ca²⁺channels (SOCs) have been reported to participate to Ca²⁺influx in cardiac cells and have emerged as potential key players in Ca²⁺deregulation in cardiomyocytes.

Although still under debate, the prime candidate proteins for SOCs encompass the nonselective cation channels, transient receptor potential canonical (TRPC) channels, as well as the Ca²⁺-selective pore-forming unit of the Ca²⁺ release-activated Ca²⁺channel (CRAC), Orai1. Stromal interaction molecule 1 (STIM1) acts as a Ca²⁺ sensor in the endo-/sarcoplasmic reticulum (ER/SR) and oligomerizes when Ca²⁺ stores are depleted. It forms clusters proximal to the plasma membrane to physically interact with and activate Orai1 and TRPC channels allowing store-operated Ca²⁺entry (SOCE) (Prakriya2013; Cheng et al.

2013).

In this review, we will highlight the accumulated knowledge about the presence and the contribution of STIM1/Orai1-dependent SOCE to cardiac function and its potential role in the pathogenesis of HF.

26.2 Presence of SOCE in the Heart

The first evidence of the presence of SOCE in cardiomyocytes was reported in 2002. Hunton et al. showed that in neonatal cardiomyocytes, the depletion of SR Ca²⁺stores with either the SERCA inhibitor (thapsigargin, Tg) or inositol trisphosphate (IP₃ )-generating agonists such as angiotensin (Ang II) or phenylephrine (PE) induces a sustained increase in cytoplasmic Ca²⁺dependent on extracellular Ca²⁺, which is abolished by nonselective SOC inhibitors, glucosamine or SKF-96365 (Hunton et al.

2002). It has been also reported that SOCE is present in cardiomyocytes at embryonic

and neonatal stages and is resistant to inhibitors of voltage-dependent Ca²⁺ channels and Na⁺/Ca²⁺exchanger (Uehara et al.2002). Thereafter, the presence of SOCE activated by Ang II and Tg has been shown in adult rat and mouse ventricular myocytes (Hunton et al.2004; Kojima et al.2012), but its amplitude is lower than in embryonic/neonatal cardiomyocytes, indicating that SOCE regulation depends on the developmental stage of the heart, which is probably linked to the developmental changes in Ca²⁺handling between embryonic and adult hearts. In this line, we recently showed, in adult rat ventricular cardiomyocytes, low functional SOCE, which can be stimulated by Epac signal-ing (Domsignal-inguez-Rodriguez et al.2015). However, the existence of SOCE was really confirmed much later by the identification of SOCE molecular components in cardiac tissue.

Indeed, several groups simultaneously identified STIM1 and Orai1 proteins in embryonic, neonatal, and adult rat ventricular cardiomyocytes (Ohba et al.2009; Voelkers et al.2010; Hulot et al. 2011; Volkers et al.2012; Zhu-Mauldin et al.

2012) and asserted their requirement for SOCE. Luo et al. further showed that neonatal hearts express significant amounts of a splice variant of STIM1 (STIM1L), already described in skeletal muscle, and whose expression decreases with cardiomyocyte maturation (Luo et al. 2012). In accordance with studies on non-excitable cells, it has been shown that SR Ca²⁺depletion leads to the formation of STIM1 puncta (Hulot et al.2011) and its interaction with Orai1 (Zhu-Mauldin et al. 2012) in neonatal cardiomyocytes. It has been also reported that STIM1 colocalizes with key regulators of ECC such as SERCA, phospholamban, and RyR in neonatal and adult rat hearts (Zhu-Mauldin et al. 2012; Correll et al.

2015; Zhao et al.2015). Consistent with earlier findings, we also recently reported a physical interaction between STIM1 and Orai1 elicited by Ca²⁺store depletion in neonatal cardiomyocytes (Sabourin et al.2016). In this in vitro model, one study suggested, using Ca²⁺imaging and Sr²⁺permeability assays, that SOCE is highly selective for Ca²⁺, reflecting that SOCE is mediated by STIM1/Orai1 proteins rather than TRPC channels (Zhu-Mauldin et al. 2012). By contrast, with the whole-cell patch-clamp mode, a method that precisely determines ion selectivity of channels, we recorded a mixed nature ofISOCcarried by a dynamic assembly of TRPC-STIM1-Orai1 (Sabourin et al.2016).

Interestingly, Hulot et al. found two separate STIM1-dependent currents in adult rat cardiomyocytes (Hulot et al.2011): one with double rectifying current rapidly activated by Ca²⁺store depletion and the other fully active even when Ca²⁺stores are replete, with predominantly inward rectifying current reflecting Orai1-activated currents. This work therefore joins a growing number of studies that show a more rapid kinetics ofISOCactivation in cardiac myocytes than in non-excitable cells. This more rapid activation and store-independent current may be explained, at least, by the constitutive association of STIM1 and Orai1 at the membrane, as observed in sinoatrial node cells (Zhang et al.2015). In line with the idea, STIM1 appears to form pre-constituted “punctate” structures without the need of Ca²⁺store depletion in neonatal and adult cardiomyocytes (Parks et al.2016). Furthermore, in skeletal muscle, STIM1L has been demonstrated to colocalize with Orai1 channels and to 26 Cardiac Remodeling and Disease: Current Understanding of STIM1/Orai1. . . 525

interact with actin to form permanent clusters, allowing the immediate activation of SOCE (Darbellay et al.2011). Similar experiments with STIM1L may provide new data regarding the kinetics of SOCE activation in cardiomyocytes.

The presence of both STIM1 and Orai1-mediating SOCE tightly coupled to SR Ca²⁺release has also been found in HL-1 cells, an immortal atrial cardiomyocyte cell line (Touchberry et al.2011). Nonetheless, the existence of cardiac STIM1-/ Orai1-dependent SOCE machinery raises the need to understand its underlying physiological role in the heart.

26.3 Physiological Roles of SOCE in the Heart

Firstly, it has been observed that siRNA-mediated knockdown (KD) of STIM1 impacts the cytosolic and SR Ca²⁺handling leading to lower diastolic Ca²⁺levels and a decline in SR Ca²⁺content in neonatal cardiomyocytes, while silencing Orai1 expression did not appreciably alter these parameters (Voelkers et al. 2010). In addition, the frequency of spontaneous Ca²⁺transients is significantly decreased in STIM1 and Orai1-KD cardiomyocytes. These authors also showed that KD of Orai1, but not of STIM1, causes a significant decrease in neonatal cardiomyocytes’ size associated with a reduction of calcineurin (CnA) and Ca²⁺ /calmodulin-depen-dent kinase II (CaMKII) activity under resting conditions. By contrast, at adult stages, genetic manipulation of STIM1 in vitro and in vivo using gain- and loss-of-function strategies greatly influenced cardiac size and loss-of-function (Hulot et al.2011). In HL-1 cells, the KD of Orai1 also decreases SOCE and both cytosolic and SR Ca²⁺ levels (Touchberry et al.2011).

It has been also shown that STIM1 participates to the contractile rhythmicity of HL-1 cells by moderating T-type Ca²⁺channel expression and activity (Nguyen et al.2013). Of note, STIM1 and Orai1 limit the surface expression and activity of the L-type Ca²⁺channels (LTCCs) via physical interaction in A7r5 vascular smooth muscle cells and Ca_v1.2 expressing HEK-293 cells (Wang et al.2010). We and others also demonstrated in embryonic and adult cardiac cells that TRPC channels form macromolecular complexes with the LTCCs, in particular in caveolae (Sabourin et al.2011; Makarewich et al.2014). To date, the interaction between STIM1/Orai1 and LTCCs in adult cardiomyocytes remains to be determined.

In our recent study, we showed a diastolic Ca²⁺overload induced by chronic aldosterone treatment in electrically paced neonatal cardiomyocytes that was totally prevented when SOCE was blocked by BTP2 ( N-[4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-1,2,3-thiadiazole-5-carboxamide) or when Orai1 was specifically blocked by Synta66 (N-(2⁰,50_{-dimethoxy[1,1}0

-biphenyl]-4-yl)-3-fluoro-4-pyridinecarboxamide). This phenomenon was also partially prevented in the presence of the serum- and glucocorticoid-regulated kinase 1 (SGK1) inhibitor GSK, suggesting that Orai1-mediated SOCE plays a role in the regulation of cardiac diastolic Ca²⁺homeostasis, via an SGK1-dependent mechanism (Sabourin et al.2016).

Recent in vivo studies have proved the contribution of Orai1 and Orai3 activity on the regulation of myocardial electromechanical stability (Wolkowicz et al.2011; Wang et al.2012) and of contractile development machinery (Horton et al.2014). Collins et al. have demonstrated that STIM1 is essential for normal cardiac homeo-stasis through modulation of ER and mitochondrial functions (Collins et al.2014). Altogether, we and others proposed that STIM1/Orai1-mediated SOCE, pre-dominantly expressed in developing cardiomyocytes, plays a role in maintaining diastolic Ca²⁺homeostasis over time under unstressed conditions and that SOCE is crucial for normal postnatal cardiac growth.

26.4 Pathological Roles of SOCE in the Heart

Despite an uncertain role of SOCE in normal cardiac physiology, notably in adults, several studies reported strong evidence that deregulation of SOCE pathway is an important contributor to the development and progression of cardiac pathologies such as cardiac hypertrophy, atrial and ventricular arrhythmias, and dilated cardio-myopathy leading to heart failure.

26.4.1 Cardiac Hypertrophy

Cardiac hypertrophy is the primary mechanism by which the heart responds to cardiac stressors such as myocardial infarction or hypertension-induced pressure overload, to preserve the pump function. Hypertrophy from different pathologic stimuli is associated with sustained increases in intracellular Ca²⁺which activate signaling pathways such as CnA/NFAT (calcineurin/nuclear factor of activated T cells) and CaMKII/HDACs (Ca²⁺/calmodulin-dependent protein kinase II/histone deacetylases) signaling leading to hypertrophic transcription programs, pathologi-cal growth, and cardiac remodeling, in fine altering cardiac function. Several studies proposed a key role for STIM1/Orai1-mediated SOCE in altered Ca²⁺ signaling underlying the development of cardiac hypertrophy, by changing the fetal gene program governed by CnA/NFAT signaling.

The first study on neonatal cardiomyocytes showed that a treatment with IP₃ -generating agonists such as PE or Ang II for 48 h produced an increased intracellu-lar Ca²⁺concentration and an increased cell area, and led to the nuclear transloca-tion of NFAT. All these parameters were prevented in the presence of nonselective SOC inhibitors, glucosamine or SKF-96365, and, to a lesser extent, in the presence of LTCC inhibitor (Hunton et al.2002). Subsequently, similar results were found on neonatal cardiomyocytes where KD of STIM1 and Orai1 suppressed enhanced SOCE and increased NFAT activation and cell size induced by 48 h treatment with endothelin 1 (ET-1) or PE (Ohba et al.2009; Voelkers et al.2010; Hulot et al.

2011). Conversely, neonatal cardiomyocytes overexpressing STIM1 are signifi-cantly larger and display enhanced NFAT activity, which are prevented in the presence of SKF-96365 (Hulot et al.2011). Furthermore, both STIM1 and Orai1 26 Cardiac Remodeling and Disease: Current Understanding of STIM1/Orai1. . . 527

KD completely abrogated PE-mediated hypertrophic neonatal myocyte growth, by inhibiting CaMKII and ERK1/2 (extracellular regulated kinases 1/2) signal-ing pathway, while only Orai1 KD prevented PE-mediated CnA-dependent prohypertrophic signaling (Voelkers et al.2010).

Later, STIM1L has been identified as predominant at neonatal stages, but its expression decreases in adults and only reemerges with hypertrophic agonist- or afterload-induced cardiac stress. Indeed, mice subjected to transverse aortic con-striction (TAC) during 3 weeks to trigger ventricular hypertrophy exhibit an increase in STIM1L mRNA and protein levels associated with robust SOCE compared to sham animals, consistent with the fact that reactivation of STIM1L expression increases SOCE during hypertrophic process (Luo et al.2012). A similar induction of STIM1L was observed on isolated adult cardiomyocytes stimulated with PE (Luo et al.2012). More recently, it has been shown that treatment of human embryonic stem cell-derived cardiomyocytes (hESC-CM) with PE during 48 h causes a marked hypertrophy, accompanied with an upregulation of Orai1 protein. The development of hypertrophy is suspended when Orai1 expression is silenced using siRNA or using dominant-negative construct of Orai1^G98Aor by nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) via protein kinase G (PKG) activation. Nevertheless, these anti-hypertrophic effects are lost when Orai1 is mutated on serine 34, suggesting that NO, cGMP, and PKG inhibit hypertrophy of hESC-CM cells via PKG-mediated phosphorylation of Orai1 channels on serine 34 (Wang et al.2015).

All these in vitro data clearly demonstrate that STIM1-/Orai1-dependent Ca²⁺ entry is instrumental for pathological cardiac hypertrophy development. However, its contribution in adults and in an integrated in vivo model remains limited.

Hulot and colleagues were the first in 2011 to demonstrate an overexpression of STIM1 protein and enhanced SOC current in the in vivo model of pressure overload-induced left ventricular hypertrophy in rat. By contrast, STIM1 gene silencing results in less SOCE and protects the heart from the development of hypertrophy by reducing the CnA/NFATc3 signaling cascade (Hulot et al.2011). A more recent report also found that STIM1 deletion protects the heart from pressure overload-induced cardiac hypertrophy in mice (Parks et al.2016). For Orai1, only one study observed an upregulation of Orai1 expression at mRNA and protein levels in mice after pressure overload-induced cardiac hypertrophy, without further functional investigation (Volkers et al.2012).

Overall, these studies strongly support a role for STIM1-mediated SOCE as playing a key role in regulating cardiac hypertrophy and further suggest that increased STIM1 protein that occurs in response to hypertrophic stimuli is respon-sible for fetal gene program activation, a characteristic of stressed cardiomyocytes. However, additional studies are clearly necessary to determine the relative impor-tance and the role of Orai1 in mediating this response.

26.4.2 Dilated Cardiomyopathy and Heart Failure

Cardiac hypertrophy with normal cardiac function tends to progress over time to depressed cardiac function, described as “hypertrophy decompensation,” clinically resulting in the syndrome of HF. The patients with HF are highly exposed to sudden cardiac death (SCD) due to electrical and mechanical disorders of the heart. The decompensation phase is associated with progressive ventricular dilatation and systolic dysfunction, and one of the processes involved in is the loss of cardiomyocytes and their replacement by fibrous tissue leading to a decrease in cardiac contractile performance. Deregulation of Ca²⁺homeostasis is important in this pathological process. Recent studies tried to show the involvement of SOCE in the transition from cardiac hypertrophy to HF.

Importantly, it has been described that Orai1-/STIM1-deficient patients or Orai1-/STIM1-deficient mice exhibit immunodeficiencies, muscular hypotonia, ectodermal dysplasia, autoimmunity, and lymphoproliferative diseases, but no obvious cardiac muscle-related phenotypes and no prejudice to the cardiovascular function (Feske 2010), consistent with the idea that the SOCE pathway is not crucial during cardiac muscle development and/or contractility under normal phys-iological conditions. Alteration of heart function related to defect in SOCE might appear with aging, stressful stimuli, or enduring exercise, but could not be revealed due to early death of patients or deficient mice.

However, in a zebrafish model, the inactivation of Orai1 results in the develop-ment of HF and the progressive loss of skeletal muscle myofiber integrity. Indeed, 48 h post fertilization, Orai1-deficient embryos spontaneously develop severe HF associated with bradycardia, decreased blood circulation, and blood congestion at the inflow tract and have reduced skeletal muscle force generation without affecting early cardiogenesis and cardiomyocyte differentiation (Volkers et al.2012). Orai1 appears to be essential for sarcomere growth and function by regulating the locali-zation of calsarcins, a family of striated muscle-specific proteins, at the sarcomeric Z-disk domain. The absence of a comparable cardiac deficit in human patients may be attributed to the presence of Orai3 in mammals.

Similarly, the global Orai1-deficient mice submitted to TAC during 8 weeks (Horton et al.2014) show a significantly reduced survival rate, a much earlier loss of cardiac function and greater dilatation of the left ventricle compared to WT mice, suggesting that Orai1 deficiency accelerates the pathology and leads rapidly to dilated cardiomyopathy and HF. The maximum rate of hypertrophy is similarly reached in both groups, but Orai1-deficient mice are not able to compensate the overload, leading to the development of dilated cardiomyopathy. Thus, Orai1 may play an important role in the progression of this pathology (Horton et al.2014). The authors explained these differences by significant apoptosis without major differences in cellular hypertrophy and hypertrophic markers and fibrosis. How-ever, the difference in amount of apoptosis observed in WT and Orai1^+/mice is not clearly established. Of interest, these clinical symptoms seem contradictory to in vitro studies where KD of Orai1 is associated with protection from hypertrophic phenotype. Therefore, cardiac protective or deleterious effect of Orai1 is still under 26 Cardiac Remodeling and Disease: Current Understanding of STIM1/Orai1. . . 529