Early adaptive cardiac hypertrophy (EACH) is initially a compensatory process to optimize pump function. We reported the emergence of Orai3 activity during EACH. This study aimed to characterize how inflammation regulates store-independent activation of Orai3-calcium influx and to evaluate the functional role of this influx. Isoproterenol infusion or abdominal aortic banding triggered EACH. TNFα or conditioned medium from cardiac CD11b/c cells activated either in vivo [isolated from rats displaying EACH], or in vitro [isolated from normal rats and activated with lipopolysaccharide], were added to adult cardiomyocytes before measuring calcium entry, cell hypertrophy and cell injury. Using intramyocardial injection of siRNA, Orai3 was in vivo knockdown during EACH to evaluate its protective activity in heart failure. Inflammatory CD11b/c cells trigger a store-independent calcium influx in hypertrophied cardiomyocytes, that is mimicked by TNFα. Pharmacological or molecular (siRNA) approaches demonstrate that this calcium influx, depends on TNFR2, is Orai3-driven, and elicits cardiomyocyte hypertrophy and resistance to oxidative stress. Neutralization of Orai3 inhibits protective GSK3β phosphorylation, impairs EACH and accelerates heart failure. Orai3 exerts a pathophysiological protective impact in EACH promoting hypertrophy and resistance to oxidative stress. We highlight inflammation arising from CD11b/c cells as a potential trigger of TNFR2- and Orai3-dependent signaling pathways.
Cardiac hypertrophy (CH) is initially a compensatory process to optimize cardiac pump function1. However, CH is progressively associated with structural changes that become pathogenic, with cardiomyocyte death, induction of exacerbated inflammatory responses and interstitial fibrosis. These harmful changes ultimately lead to transition to heart failure (HF). Activation of the sympathetic nervous system plays a determinant role in the induction of early adaptive CH (EACH) and further progression to pathological remodeling2,3. HF is a major health issue4 and a better understanding of cellular mechanisms elicited during EACH is needed to prevent the progression to HF or favor recovery5,6.
Altered myocardial calcium (Ca2+) cycling is a hallmark of HF underlying perturbation in excitation-contraction coupling7. Voltage-gated ion channels, the sarcoplasmic reticulum Ca2+ ATPase, the Na+-Ca2+ exchanger, the ryanodine receptor and t-tubule structure became promising targets for therapeutical intervention. In addition, Ca2+ handling remodeling also drives hypertrophic and apoptotic responses. In this context, TRPCs (canonical transient receptor potential channels)-, STIM1 (stromal interaction molecule 1)-, and Orai1-dependent Ca2+ entry are instrumental for pathological left ventricular hypertrophy development8,9,10,11,12,13. As described in the non-excitable cells, TRPCs, STIM1 and Orai1 molecules drive store-operated Ca2+ entry10,11,13,14,15. An alternative Ca2+ entry pathway, independent of store-depletion, involves the key participation of the Orai3 molecule14,15,16,17,18,19,20. Orai3-driven store-independent Ca2+ entry relies on initial arachidonic acid (AA) production, and is selectively activated by AA itself (ARC channels) or its leukotriene C4 (LTC4) metabolite (LRC channels), in all cell types examined to date. Knowledge regarding Orai3 contribution to cardiac remodeling remains scarce. We recently demonstrated the emergence of an Orai3-dependent pathway that drives an AA-dependent Ca2+ influx in hypertrophied cardiomyocytes from rats subjected to abdominal aortic banding12. This study documented the essential role of constitutive Orai3-dependent activity to initiate and maintain early adaptive hypertrophy in response to pressure overload. But pathophysiological triggers and mechanisms leading to Orai3 activation during EACH remained unknown, as well as its direct impact on cardiomyocytes and its functional relevance in HF.
Cardiac remodeling is a complex inflammatory syndrome5, and beneficial or detrimental role of inflammatory signaling during EACH is not fully understood. Growing evidence indicates that inflammatory responses emerging in EACH and HF are different, displaying divergent cytokine profiling21. The pro-inflammatory cytokine TNFα is upregulated in CH and HF. In the 1990’s, the “cytokine hypothesis” argued for the detrimental contribution of an excessive production of TNFα to the pathogenesis of HF22, via binding to the TNFR1 receptor subtype, suggesting that TNFα neutralization would be beneficial. Surprisingly, large clinical trials failed to demonstrate a benefit of anti-TNFα strategies23,24. There is now evidence that TNFα can also improve remodeling and hypertrophy and alleviate inflammation and fibrosis upon binding to the TNFR2 receptor subtype or regulation of TNFR1 signaling, in cardiomyocytes, or indirectly after induction of GM-CSF secretion by endothelial renal cells, or influencing cardiac immune cell phenotypes2,25,26,27,28,29. In this context, we have previously shown that AA mediates dual effect of TNFα on Ca2+ transients and contraction of adult rat myocytes30 and identified TNFR2-dependent activation of the cytosolic phospholipase A2 (cPLA2) activity as a pathway leading to AA production and conferring resistance of adult cardiomyocytes to H2O226. Recent studies suggested the potential adaptive role of TNFα in early cardiac remodeling showing that myocardial gene expression of TNFα is significantly higher in patients with well compensated aortic stenosis than in patients with decompensated stenosis31 and the association of circulating TNFα with concentric left ventricular remodeling32. The present study aimed to investigate the potential regulation of the AA-dependent Orai3 influx by TNFα in early adaptive cardiac remodeling, identify the potential cellular source of such an inflammatory signal, evaluate the impact of TNFα-induced Orai3 regulation on cardiomyocyte hypertrophy and resistance to H2O2, and assess the functional relevance of Orai3 activity in HF.
Our study points out a novel TNFR2-dependent signaling pathway in cardiomyocytes that triggers Orai3-driven Ca2+ influx enhancing hypertrophy and promoting an increased resistance to oxidative stress. Cardiac CD11b/c cells arise as a potential source of this protective inflammatory stimulus. Neutralization of Orai3 during EACH fosters evolution towards HF.
TNFα triggers activation of Orai3-Ca2+ influx in hypertrophied cardiomyocytes
To investigate the regulation of the Orai3-Ca2+ influx by TNFα, we first used adult cardiomyocytes isolated from normal rats and incubated or not with isoproterenol (iso) (100 nM overnight) to elicit in vitro hypertrophy, as demonstrated by an increased cell area (2256 ± 37 vs. 2541 ± 53 µm2, n = 308 control vs. 313 Iso cells, p < 0.0001, Mann Whitney U test). We performed Ca2+-imaging experiments in Fura2-loaded cardiomyocytes to directly evaluate the impact of inflammation on Orai-dependent Ca2+ influx. After electrical stimulation as a quality test, cells were placed in a medium appropriate for the measurement of voltage- and store-independent Ca2+ influx containing diltiazem and ryanodine where Na+ was replaced by the large organic ion N-methyl-D-glucamine, as previously reported12 (Fig. S1). After equilibration in the absence of Ca2+, 1 mmol/L Ca2+ was added into the extracellular medium and the resultant initial increase in Fura2 fluorescence (1st slope) was taken as an index of initial rate of Ca2+ influx. This protocol was routinely applied a second time (2nd slope) to allow paired comparison, either between two identical perfusion conditions and to ascertain reproducibility of measurements (Fig. 1A,C), or between two different perfusion conditions (Fig. 1B,D). Two successive applications of the same “basal” medium gave rise to similar rates of Ca2+ influx, either in control or hypertrophied cardiomyocytes (Fig. 1A,C). In contrast, addition of TNFα to the second incubation medium induced a significant increase in the rate of Ca2+ influx (2nd slope) as compared to basal (1st slope). Importantly, this effect was selectively detected in hypertrophied cardiomyocytes (Fig. 1B) but not in control ones (Fig. 1D). These data show that TNFα selectively induces Ca2+ influx in hypertrophied cardiomyocytes.
We then asked whether Orai3 drives this TNFα-activated store- and voltage-independent Ca2+ influx. In in vitro iso-treated hypertrophied cells, two successive applications of identical TNFα containing medium gave rise to similar rates of Ca2+ influx (Fig. 2A left). However, preincubation for 10 min with Orai pharmacological inhibitors, YM58483 or Synta66, reduced the rate of Ca2+ influx observed in response to a second challenge with TNFα, to a value similar to basal (Fig. 2A middle and right). Moreover, in vivo molecular knockdown of Orai3, via intramyocardial injection with Cy3-tagged Orai3 siRNA, also blunted the TNFα effect on Ca2+ influx that was still observed in cells isolated from scramble siRNA-injected hearts (Fig. 2B). Orai3 neutralization was performed as previously reported12 and demonstrated by quantitative RT-PCR and Western-blot (Fig. 2C) and by detection of Cy3 positive cells (Fig. 2B). Of note, knockdown of Orai3 by siRNA injection in normal rats did not modify the cardiomyocyte size (Table S1). In contrast, both siScramble and siOrai3 cardiomyocytes had a tendency to be bigger after in vitro post-treatment with iso and displayed similar sizes. This suggested that the in vitro iso-hypertrophic response was not altered in siOrai3 cardiomyocyte, in contrast to TNFα/Orai3 signaling.
Complementary experiments were performed in hearts from rats subjected to chronic iso-infusion for 14 days (1.5 mg/kg/day) to elicit in vivo hypertrophy (Table 1). Iso-induced EACH remodeling was confirmed by an increase in end-diastolic and end-systolic interventricular septum (IVSd and IVSs), posterior wall thicknesses (PWd and PWs) and concentric hypertrophy (h/r, diastolic wall thickness to radius ratio) in iso-treated rats, relative to control rats, associated with an increased heart rate (HR) and a better cardiac function assessed by fractional shortening measurements (FS).
Iso-induced hypertrophy was also attested by an increased heart weight (HW) to body weight (BW) ratio (0.84 ± 0.12 vs. 0.62 ± 0.11, n = 8 Iso vs. 8 control rats, p < 0.01, Mann Whitney U test) and cell area (3128 ± 63 vs. 2538 ± 54 µm2, n = 214 Iso vs. 223 control cells, p < 0.0001, Mann Whitney U test).
Ca2+-imaging experiments confirmed that TNFα also activated Orai3-dependent Ca2+ influx in in vivo hypertrophied cardiomyocytes isolated from rats implanted with iso-pump and injected or not with siRNAs three days before cardiomyocyte isolation (Fig. 2D). Of note, efficient Orai3 knockdown in in vivo hypertrophied cardiomyocytes was attested by a 40.0 ± 14.6% decrease in Orai3 mRNA expression as compared to cardiomyocytes isolated from scramble siRNA injected rats (p < 0.05, Mann-Whithney U test, n = 3 Orai3 and n = 6 Scramble siRNA injected rats). Moreover, in vivo reduction in Orai3 expression in iso-infused rats decreased the mean cardiomyocyte area (3384 ± 81 vs. 4311 ± 135 µm2, n = 116 vs. 120 cells from Orai3 vs. Scramble siRNA injected rat hearts at day 3 post injection, p < 0.0001, Mann Whitney U test). These results highlighted the role of Orai3-dependent Ca2+ influx as a target of TNFα in in vitro and in vivo iso-hypertrophied cardiomyocytes.
Overall, these experiments identified TNFα as an activator of the Orai3-dependent Ca2+ influx in hypertrophied cardiomyocytes.
Activation of Orai3-Ca2+ influx by TNFα relies on binding to TNFR2, stimulation of cPLA2 and potential production of AA metabolites via the lipoxygenase pathway
Next we aimed to evaluate the role of TNFα receptors 1 and 2 and of the cPLA2 pathways in TNFα signaling. In in vitro iso-treated hypertrophied cells, stimulation of Ca2+ influx by TNFα was unaffected by the preincubation for 1 hour either with control IGg2A or anti-TNFR1-antibodies (Ab) but was impaired in the presence of neutralizing TNFR2-Ab (Fig. 3A). Preincubation with the cPLA2 inhibitor, methyl arachidonyl fluorophosphonate (MAFP), suppressed TNFα signaling whereas addition of the phospholipase A2 activating peptide (PLAP) mimicked TNFα effect, and stimulated Ca2+ influx (Fig. 3B). TNFα-induced activation was unaffected by the pretreatment with the cyclo-oxygenase inhibitor indomethacin but impaired in the presence of a lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), suggesting the potential requirement of AA metabolism into leukotrienes in this signal transduction (Fig. 3C). TNFα signaling persisted in the presence of the antagonist of leukotriene receptors, montelukast, suggesting an effect independent of binding to external receptors (Fig. 3C). These results indicate that TNFα signals in iso-hypertrophied cardiomyocytes through TNFR2 to activate cPLA2 and produce AA potentially leading to increased leukotriene levels which in turn activate Orai3.
Of note, we recently demonstrated the emergence of an Orai3-dependent pathway that drives an AA-dependent Ca2+ influx in hypertrophied cardiomyocytes from rats subjected to abdominal aortic banding (AAB) for 28 days12. Complementary experiments were performed to examine the regulation of Orai3 by TNFα in this model (protocol in Fig. 4A). EACH remodeling was confirmed in AAB rats, relative to Sham rats, by an increase in IVSd, IVSs, PWd and PWs and concentric hypertrophy (h/r) (Table 2). AAB-induced hypertrophy was also attested by an increased cell area (4718 ± 128 vs. 3311 ± 124 µm2, n = 129 vs. 117 cells, p < 0.0001, Mann Whitney U test). A slight decrease of FS was observed in this model (Table 2).
TNFα also induced activation of Orai3-dependent Ca2+ influx in AAB-induced hypertrophied cardiomyocytes (Fig. 4B,C) that relied on binding to TNFR2 (Fig. 4D) and potential production of AA metabolites via activation of the lipoxygenase pathway (Fig. 4E).
Thus, emergence of a TNFR2-dependent Orai3-driven Ca2+ influx characterized cardiomyocyte hypertrophy triggered by either iso-treatment or pressure overload.
Inflammatory CD11b/c cells trigger TNFR2-dependent activation of Orai3-Ca2+ influx in hypertrophied cardiomyocytes
Next experiments aimed to evaluate the potential cellular source of inflammation in EACH hearts. Immunohistological examination of cardiac sections from iso-induced EACH rats indicated an increased number of TNFα-positive cells as compared to control and 66 ± 4% of TNFα-positive cells were identified as myeloid CD11b/c-positive cells (Fig. S2).
Rat hearts (obtained from rats implanted with iso-pump for 14 days, Table 1) were used to isolate cardiomyocytes, fibroblasts and myeloid CD11b/c cells (denominated as in vivo cell activation) (Fig. 5A,B). Conditioned medium (Cmed) obtained from cardiomyocytes or cardiac fibroblasts were without effect on voltage- and store-independent Ca2+ influx in in vitro hypertrophied cardiomyocytes in contrast to the Cmed obtained from their cardiac CD11b/c counterparts (Fig. 5C). This pointed out the CD11b/c cells as the potential source of the Orai3 inflammatory trigger in EACH hearts. Of note, levels of TNFα detected in Cmed from CD11b/c cells (1.15 ± 0.3 pg/ml, n = 6) were 10 to 20 fold higher than levels measured in Cmed from their cardiac counterparts. CD11b/c-Cmed-induced activation was sensitive to neutralizing TNFR2 antibodies and Orai inhibitor YM58483 (Fig. 5D).
A similar effect was triggered using in vitro CD11b/c-Cmed (obtained from CD11b/c cells isolated from normal rat hearts and in vitro stimulated with lipopolysaccharide (LPS) (10 ng/ml for 2 hours) to induce pro-inflammatory activation) (Fig. 5B,E). When CD11b/c cells were pretreated with the anti-inflammatory drug semapimod (Sema) before LPS stimulation, in vitro CD11b/c-Cmed Sema was without effect on Ca2+ influx (Fig. 5B,E). Anti-inflammatory impact of semapimod was indicated by a reduction in TNFα-positive staining of CD11b/c cells (Fig. S3) and a limited TNFα content in Cmed Sema as compared to Cmed LPS (0.01 ± 0.004 vs. 0.24 ± 0.06 pg/ml, n = 5 vs. 8, p < 0.01, Mann Whitney U test).
These results highlighted the cardiac inflammatory CD11b/c cells as the potential triggers of Orai3-dependent Ca2+ influx in hypertrophied cardiomyocytes.
TNFα or inflammatory cardiac CD11b/c cells trigger TNFR2 and Orai signaling pathways, enhancing hypertrophy and promoting resistance to oxidative stress in hypertrophied cardiomyocytes
We next investigated the potential impact of inflammation on cardiomyocyte hypertrophy and evaluated the role of TNFR2 and Orai signaling pathways. Cardiomyocyte hypertrophy was initially induced by the iso stimulation (100 nM) for 1.5 hours. Cells were then challenged with TNFα or in vitro CD11b/c-Cmed. Cell hypertrophy was measured after 18 hours and attested by an increase in cell area (Fig. 6A). Addition of iso alone triggered a mean 14 ± 2% hypertrophy (Fig. 6B). TNFα enhanced iso-induced hypertrophy (up to 28 ± 3%), in a TNRF2- and YM58483-sensitive manner (hypertrophy reduced to 11 ± 3% and 7 ± 3%, by preincubation with TNFR2-Ab or YM58483, respectively) (Fig. 6B). CD11b/c-Cmed also increased iso-induced hypertrophy (up to 35 ± 2%) (Fig. 6B). Pretreatment of CD11b/c cells with anti-inflammatory semapimod blunted the prohypertrophic effect of Cmed (reduced to 5 ± 3%), as well as treatment with neutralizing TNFR2-Ab or YM58483 (reduced to 14 ± 4% and 7 ± 2%, respectively) (Fig. 6B).
These results indicate that inflammation (TNFα or Cmed from cardiac inflammatory CD11b/c cells) enhances cardiomyocyte hypertrophy via TNFR2- and Orai-dependent signaling pathways.
We also evaluated the potential impact of inflammation on the resistance to oxidative stress using in vitro hypertrophied cardiomyocytes and demonstrated the role of TNFR2 and Orai signaling pathways. Iso-hypertrophied cardiomyocytes (100 nM for 18 hours) were preincubated with TNFα or in vitro CD11b/c-Cmed, and cell resistance was estimated by the number of rod-shaped cells after H2O2-oxidative stress (100 µM H2O2 for 2.5 hours) (Fig. 7A). Counting of resistant cells indicated 21 ± 2% vs. 100% in the presence and in the absence of H2O2, respectively (Fig. 7B). Treatment with TNFα increased resistance of hypertrophied cardiomyocytes from 21 ± 2% to 36 ± 3%, in a TNFR2- and YM58483-sensitive manner (rod-shaped cells reduced to 23 ± 1% and 26 ± 4%, after preincubation with the TNFR2-Ab or YM58483, respectively) (Fig. 7B). CD11b/c-Cmed alleviated the deleterious impact of H2O2, improving the yield of resistant cells to 50 ± 6% (Fig. 7B). Pretreatment of CD11b/c cells with anti-inflammatory semapimod blunted the beneficial impact of Cmed resulting in only 21 ± 4% resistant cells, as well as pretreatment with the TNFR2-Ab or YM58483 (36 ± 3% and 38 ± 2% resistant cells, respectively) (Fig. 7B).
These results demonstrate that inflammation (TNFα or Cmed from cardiac inflammatory CD11b/c cells) improves resistance to H2O2 in hypertrophied cardiomyocytes via TNFR2- and Orai-dependent protective signaling pathways.
Taken together our in vitro results argue for a protective Orai3-driven signal emerging during the phase of EACH and promoting adaptive cardiomyocyte hypertrophy and beneficial resistance to oxidative stress. In order to evaluate their relevance in the pathophysiology of HF, we performed a kinetic analysis of echocardiographic parameters and evaluated tissue remodeling in response to a unique intramyocardial Orai3 siRNA injection applied at the onset of EACH.
Orai3 neutralization during EACH impairs cardiac hypertrophy, fosters alteration of function and dilation and deleterious tissue remodeling
To validate in vivo the protective role of Orai3 activity on EACH, we studied the effect of cardiac Orai3 knockdown during iso infusion in mice. First, as shown in Fig. S4, as compared to previous results obtained in rats (Table 1 and12), we checked that this model displayed similar iso-induced EACH remodeling after a 14 days infusion period in control mice. EACH was characterized by evolution of echocardiographic parameters, increase in heart weight to body weight ratio and cardiomyocyte size, and associated with an increase in Nppa and Tnfα mRNA expressions, but no change in Orai3 mRNA level (Fig. S4 and Table S2). This model was chosen to develop a novel approach of percutaneous intramyocardial injection of siRNA under echographic guidance validated in mice by other groups (i.e.33) in order to avoid potential artefactual consequences of the surgical thoracotomy on tissue remodeling that are susceptible to affect the post-operative echographic surveillance and evolution of cardiac parameters.
Iso-infused mice were subjected to a unique intramyocardial injection of either Scramble or Orai3 siRNA at day 8 following pump implantation (Fig. 8A). Efficient knockdown of Orai3 during EACH was attested at the mRNA and protein levels (Fig. 8D,E). At day 15, hearts from siOrai3-injected mice displayed a decreased hypertrophy (lower PWs), an increased dilation (higher LVs) and an altered cardiac function (lower FS) as compared to hearts from siScramble-injected mice (Fig. 8C and Table 3). At day 28, siOrai3-injected mice still presented with a lower heart weight to body weight ratio, a smaller cardiomyocyte area, a decrease in Myh7 mRNA level, as compared to siScramble-injected mice (Fig. 8F). Orai3 knockdown during EACH also fostered fibrosis attested by histological analysis and increased Col1a1, Col3a1 and Tgfβ mRNA levels (Fig. 8G). Mechanistically, a decrease in the ratio phospho-GSK3β/GSK3β was correlated with the reduction of Orai3 expression (Fig. 8H).
Taken together, these results demonstrated the emergence of a functional protective role of Orai3 during EACH.
Our results highlight the emergence of a protective role for Orai3 in the hypertrophied cardiomyocytes. We identified TNFα as a mechanistic trigger of the TNFR2-dependent activation of Orai3-Ca2+ influx and showed that CD11b/c cells are a potential driving source of this signaling in EACH hearts. This paracrine signaling enhances hypertrophy and promotes resistance to oxidative stress of hypertrophied cardiomyocytes. Furthermore, we show that Orai3 knockdown during EACH fosters HF (Fig. 9).
Cardiac Orai3-dependent Ca2+ influx was previously identified as a prohypertrophic stimulus in AAB-induced CH12. We confirm these results in a model of iso-induced EACH, a model of reproducible progressive concentric hypertrophy. In our study, we found that in vivo reduction in Orai3 expression in iso-infused rats decreased the mean cardiomyocyte area. In keeping with this atrophic impact of Orai3 siRNA, neutralization of Orai3 during EACH in iso-infused mice triggers a rapid and significant decrease in hypertrophic parameters still detectable at day 20 post injection. Furthermore, our in vitro experiments document a direct prohypertrophic effect of Orai3 in isolated iso-treated cardiomyocytes.
Interestingly, our results highlight novel protective properties of Orai3-dependent Ca2+ influx. In line with the reported resistance of Orai3 channel to redox regulation34, we show that Orai3 activation confers resistance to oxidative stress in isolated hypertrophied cardiomyocytes. Furthermore, our in vivo results indicate that efficient cardiac knockdown of Orai3 during EACH inhibits adaptive hypertrophy, alters cardiac function and promotes fibrosis. Mechanistically, recent results from our laboratory indicate that the Orai3-interacting protein STIM1 is essential to tune the Akt/GSK3β prosurvival signaling8,9,10,35,36. Our in vivo results show that neutralization of Orai3 during EACH is associated with a decrease in phospho-GSK3β/GSK3 ratio. In the current absence of published knockout data for Orai3, our study is the first to address its pathophysiological relevance and indicate its essential protective role during EACH.
We have identified the first pathophysiological trigger of Orai3-driven store-independent Ca2+ influx in cardiomyocytes, namely a TNFα/TNFR2-dependent signaling. Interestingly, the regulation of Orai3 by TNFα is detected in hypertrophied cardiomyocytes (both in response to iso treatment or in the AAB-model), but not in normal cardiomyocytes. Of note, iso-hypertrophied and normal cardiomyocytes display similar TNFR2 expression (2.4 ± 0.4 vs. 2.4 ± 0.3 pg TNFR2/mg, respectively, n = 4). However, and as previously reported in AAB-induced CH hearts12, co-immunoprecipitation experiments using STIM1 antibodies indicate enhanced Orai3 recruitment to STIM1 in the iso-induced EACH (Fig. S6). Since Orai3-STIM1 interaction is a prerequisite for Orai3-dependent Ca2+ influx activation14,17, its absence in normal cells and its enhancement in hypertrophied cells constitute a major difference likely to explain the lack of impact of Orai3-dependent Ca2+-influx as well as the absence of TNFα regulation in normal cells. Accordingly, our in vivo and in vitro experiments in normal mice show that the knockdown of Orai3 is without impact on echocardiographic parameters as well as heart or cardiomyocyte size. They argue for a pathophysiological impact of Orai3 exerted during EACH but not under control conditions. Dominant impact of inflammation is currently reported as deleterious in normal hearts, as it is associated with reactive oxygen species production, proapoptotic signaling and a dominant role of TNFR1 pathways overwhelming TNFR2 signaling26. Using TNFR1 and TNFR2 knockout mice implanted with iso-pumps, Prabhu et al. demonstrated that TNFR2 but not TNFR1 signaling prevents the detrimental long-term effects of β-adrenergic receptor stimulation in the heart37. In another study, the group of Prasad proposed that the beneficial effects of TNFR2 signaling in presence of sympathetic overdrive could act through the preferential TNFR2-mediated recruitment of GRK2 to mediate βAR desensitization thus reducing deleterious cardiac signaling and remodeling. In agreement with these studies, our study further suggests the emergence of a protective TNFR2 pathway during EACH development via the stimulation of a novel downstream effector Orai3.
We show here that cardiac CD11b/c cells are a potential source of inflammatory TNFα/TNFR2-dependent signaling leading to Orai3-dependent Ca2+ channel activation. Both Cmed from in vitro and in vivo activated CD11b/c cells stimulate a store-independent Ca2+ influx in hypertrophied cardiomyocytes in a TNFR2-Ab and YM58483 sensitive manner. This suggests the presence of activated inflammatory CD11b/c cells in EACH hearts. We demonstrated that LPS-activated CD11b/c cells enhance hypertrophy and promote resistance of hypertrophied cardiomyocytes to oxidative stress, in a TNFR2-Ab and YM58483 sensitive manner. This protective impact is blunted by preincubation of CD11b/c cells with the anti-inflammatory drug semapimod. Our data strengthen the concept that inflammation arising from cardiac myeloid cells may exert paracrine beneficial impact on cardiomyocytes38,39. Of note, cardiac macrophages are an emerging focus for therapeutic strategies aimed at minimizing cardiomyocyte death, ameliorating pathological cardiac remodeling and for treating HF40.
We observed that TNFα-dependent activation of Orai3-Ca2+ influx relies on cPLA2 activation and is mimicked by a cPLA2 activator. This is in accordance with the reported presence of the lipid interaction site located in the NH2 terminal intracytosolic sequence of Orai320. TNFα-dependent effect is sensitive to NDGA treatment which suggests the potential requirement of a lipoxygenase-dependent AA metabolism to activate Orai3-Ca2+ influx. Accordingly, Trebak et al. previously described LTC4, a lipoxygenase AA-metabolite, as an activator of Orai3 in the vascular smooth muscle cells41. However, this proposal needs to be tempered since NDGA, in addition to inhibit lipoxygenase activity may also exert several off target effects (i.e., PKC inhibition, overall anti-oxidant, ER-Golgi protein shuttling inhibition). Our results further illustrate the dual role of cPLA2-AA signaling in mediating TNFα effects in adult cardiac myocytes. We have identified Orai3 as a novel protective TNFα-cPLA2-AA pathway. Accordingly, the beneficial impact of TNFα-cPLA2-AA pathways has been previously reported on the cardiomyocyte calcium transients and contraction (i.e. by the group of Oceandy42 and our previous results26,30) and on the survival to oxidative stress26. In contrast, the group of Gugiyama reported the deleterious impact of a TNFα-cPLA2 cardiac signaling in a model of ischemia-reperfusion43.
In conclusion, our in vitro and in vivo studies characterize the Orai3 signaling pathway that exerts a direct protective role against HF in hypertrophied cardiomyocytes. Mechanistically, we have identified Orai3 as a novel driver of TNFR2-dependent inflammation instrumental in this protection. Furthermore, we showed a protective Orai3-dependent paracrine role of cardiac myeloid cells leading to adaptive hypertrophy and improved resistance to oxidative stress. In summary, our results are the first to address the functional role of Orai3 signaling in HF that may open new perspectives for patients’ treatments.
Care of the animals and surgical procedures were performed according to the Directive 2010/63/EU of the European Parliament, which had been approved by the Ministry of Agriculture, France, (authorization for surgery C-75-665-R). The project was submitted to the French Ethic Committee CEEA (Comité d’Ethique en Expérimentation Animale) and obtained the authorization Ce5/2012/050 and APAFIS#1729-2015-083114195840v8. All experiments were performed in accordance with relevant named guidelines and regulations.
6 week-old male Wistar rats and 10-11 week-old male C57BL/6JRj mice were purchased from Janvier Labs.
In vivo chronic isoproterenol infusion
Rats and mice anesthetized under isoflurane (Iso-vet®, Piramal, UK) (1–3%) were implanted subcutaneously with an osmotic minipump (Alzet, Charles River) containing either isoproterenol (1.5 mg/kg/day for rat or 30 mg/kg/day for mouse) (iso-pump) or vehicle for 14 or 28 days to develop either EACH or HF, respectively, or as otherwise stated.
Abdominal aortic banding
Rats were anaesthetized by intra-peritoneal injection of ketamine (Imalgene®, Merial, Germany) and xylazine (Rompun®, Bayer, Germany) (75 and 10 mg/kg, respectively). Medial abdominal laparotomy was performed and a clip with an internal opening of 0.58 mm was placed, as previously reported12. Sham-operated rats served as controls.
Measurement of cardiac parameters
Echocardiography was performed on lightly anesthetized animals under isoflurane (0.5–1%) with a probe emitting ultrasounds from 8- to 14-MHz frequency (Vivid7 PRO apparatus; GE Medical System Co). The two-dimensionally guided Time Motion mode recording (parasternal long-axis view) of the left ventricle (LV) provided the following measurements: end-diastolic and end-systolic interventricular septum (IVSd and IVSs), posterior wall thicknesses (PWd and PWs), internal diameter (LVEDD and LVESD), and heart rate (HR). Each set of measurements was obtained from the same cardiac cycle. At least three sets of measurements were registered from three different cardiac cycles. Fractional shortening (FS): [(LVEDD − LVESD)/LVEDD] × 100 and h/r: [left ventricle diastolic wall thickness/radius] were calculated.
In vivo ultrasound mediated Cy3-tagged siRNA delivery in rats
After an intra-peritoneal injection of ketamine and xylazine (75 and 10 mg/kg, respectively), an incision was made in the fourth inter-costal space to expose the heart and Cy3-tagged siRNAs were delivered as previously described44 two weeks after iso-pump implantation and three days before cardiac cell isolation. The siRNA for scramble (Life Technologies) and Orai3, were chosen from12,45,46 (FWsiOrai3: 5′-GUUUAUGGCCUUUGCCCUATT-3′; RVsiOrai3: 5′-UAGGGCAAAGGCCAUAAACTT-3′).
In vivo intramyocardial ultrasound-guided transthoracic siRNA delivery in mice
On-target plus Scramble and Orai3 siRNA (Dharmacon GE healthcare) were injected by ultrasound-guided transthoracic intramyocardial injection, as described in33, at day 8 after iso-pump implantation. Echocardiographic parameters were measured regularly as stated.
Cardiac cell isolation and culture
Rat cardiac myocytes were isolated either from chronically iso-infused hearts or from normal hearts, after injection or not with Cy3-tagged siRNA, three days before, when stated. Rats were administered a sodium pentobarbital (Ceva Sante Animale, France) intra-peritoneal injection (200 mg/kg). Hearts were harvested and kept in ice-cold Krebs-Henseleit (KH) solution supplemented with 10 mmol/L taurine and 0.5 mmol/L EGTA, then rapidly canulated and mounted on the Langendorff apparatus. The hearts were retrogradely perfused through the aorta, first with a Krebs-Henseleit (KH) solution supplemented with 10 mmol/L taurine for 5 minutes, then with enzymatic solution for 20 minutes. The KH solution contained (in mmol/L): 100 NaCl, 4 KCl, 5.5 NaHCO3, 1 KH2PO4, 1.7 MgCl2, 10 D-glucose, 15 2,3-butanedione monoxime, 22 Hepes, pH = 7.4 with NaOH. The enzyme solution was supplemented with 1 mg/ml collagenase A (Roche Applied Science, Meylan, France) and 5 mg/ml bovine serum albumin BSA (Sigma, Lyon, France). All chemicals were from Sigma (Lyon, France. Cell suspension was then used to differentially isolate ventricular cardiomyocytes, cardiac fibroblasts and cardiac CD11b/c cells.
Ventricular cardiomyocytes from iso-pump rats (in vivo hypertrophy) were plated onto laminin-coated glasses and maintained overnight in M199 medium (Life technologies). Cells isolated from normal hearts or from saline-pump rats were added with 100 nM isoproterenol plus 100 µM ascorbic acid (Sigma), when stated, to induce in vitro hypertrophy, three hours after plating, and let overnight in M199 medium.
Cardiac fibroblasts were isolated by centrifugation, plated onto 12-well plates and maintained in DMEM medium (Life technologies).
Cardiac CD11b/c cells were isolated by centrifugation, enriched using an anti-CD11b/c antibody coupled to magnetic beads (MiltenyiBiotec) and maintained overnight in RPMI medium (Life technologies) supplemented with 10 mmol/L Hepes. When stated, CD11b/c cells were in vitro polarized towards a pro-inflammatory phenotype upon incubation for 2 hours with 10 ng/ml lipopolysaccharide (LPS) before overnight incubation with a new LPS-free medium.
All cardiac cells were cultured for 18 hours following plating. Conditioned media from cardiac cells were concentrated on Amicon 10 kDa centrifugal filter.
Fura-2 AM calcium imaging
Isolated rat ventricular myocytes were loaded with Fura2-AM (Molecular Probes, Life Technologies) as reported26. Transfected cells (detected by fluorescence imaging as cells positive for Cy3-tagged siRNA) or non-transfected cells, rhythmically beating in response to electrical stimulation (square waves, 0.5 Hz, as previously described26), were analyzed. Measurements were recorded on a Zeiss Platform equipped with an Axio Observer Z1 microscope, a DGA plus illuminator and a camera Coolsnap HQ2 (workstation Carl Zeiss).
Cells were first paced for few cycles and Ca2+ transients were recorded to ensure viability and functionality of the cell. Cells were incubated in tyrode buffer (1.8 mmol/L Ca2+) to check the stability of basal cytosolic calcium level and then switched to appropriate store- and voltage-independent Ca2+-free buffer. Store-independent Ca2+ entry was then measured upon readdition of 1 mmol/L Ca2+. Ca2+off/Ca2+on protocols repeated twice allowed paired comparison between two similar (for reproducibility assessment) or distinct perfusion conditions.
Tyrode buffer contained (in mmol/L): 135 NaCl, 4 KCl, 1 MgCl2, 10 D-glucose, 20 Hepes, pH = 7.4 with NaOH. Store- and voltage-independent buffer contained 1 µM ryanodine, 20 µM diltiazem and 135 mmol/L N-methyl D-glucamine (NMDG) instead of NaCl. All chemicals, excepted ryanodine (Tocris), were from Sigma.
Data analysis was performed using the Zen Software (2012, blue edition). The rates of Ca2+ entry were estimated by the slope of the first minute of initial increase in Fura-2 fluorescence ratios in response to the re-addition of Ca2+. 10–100 myocytes isolated from 2–16 animals were analyzed per experimental condition (as stated in Figs).
Measurement of cell hypertrophy
Cardiomyocyte hypertrophy was estimated after 18 hours in culture in the presence of isoproterenol. After an initial incubation with iso alone (100 nM) for 1.5 hours, cardiomyocytes were then treated or not for 1 hour with TNFR2-Ab or YM58483 before addition of control medium, TNFα or CD11b/c Cmed. Cardiomyocytes were visualized using brightfield at x20 magnification and cell area was measured in at least 300 cells per condition per experiment. Results were the mean of at least three different experiments performed on two cell isolations (using at least two different in vitro CD11b/c-Cmed).
Measurement of cell resistance to H2O2
Cell resistance experiments were performed as previously described26. In vitro hypertrophied cardiomyocytes were preincubated for 1 hour with or without TNFR2-Ab or for 10 minutes with or without YM58483. Then, TNFα or Cmed from in vitro activated CD11b/c cells, or control medium were added for 10 minutes before subsequent treatment or not with H2O2 (100 µM, (Sigma)) for 2.5 hours, that was the time corresponding to a mean 75% injury in response to H2O2 for control. Cardiomyocytes were visualized using brightfield at x100 magnification, and resistance was estimated by counting rod-shaped cells in 12 random microscopic fields. At least 300 cells were counted in each dish, and results were the mean of at least three different experiments performed on three different cell isolations (using at least two different in vitro CD11b/c-Cmed).
Rat total RNA was isolated with the RNeasy Mini kit (Qiagen). RNA reverse transcriptase-PCR analysis was performed using the Absolute QPCR SYBR green mix (ABgene) on an MX3005P QPCR system (Stratagene, Agilent Technologies). Primer sequences are listed below in Table 4. Transcript levels were normalized to the RPL32 (rat) mRNA levels.
Mice total RNA was isolated using TRIzol (invitrogen). RNA reverse transcriptase-PCR analysis was performed using Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix (Agilent Technologies) on an LightCycler® 480 Real-Time PCR System (Roche). Primer sequences are listed below in Table 5. Transcript levels were normalized to the RPL13 (mice) mRNA levels.
Cardiac myocytes were lysed in 150 mM NaCl, 50 mM Tris pH = 7.5, EDTA 5 mM, 0.5% NP40, 1% triton, protease and phosphatase inhibitors cocktail (Sigma, Lyon, France) and samples isolated by centrifugation. Forty µg of proteins in a final volume of thirty µL of Laemmli loading buffer were heated to 70 °C for 10 minutes. Samples were run on a 4–12% Nu-PAGE gel (Life technologies), transferred to Hybond-C PVDF membrane according to the manufacturer protocol (Amersham Biosciences, GE Healthcare). Membrane was incubated with rabbit anti-Orai3 (1/500, ProsciInc 4117), or rabbit anti-GAPDH (1/2500, Cell Signaling 2118) followed by anti-rabbit HRP (1/5000, Amersham Biosciences, GE Healthcare). Detection was performed using the ECL Western Blotting Substrate (Pierce) and signals were recorded using a Camera LAS 4000.
Cardiac tissue was lysed in 150 mM NaCl, 50 mM Tris pH = 7.5, EDTA 5 mM, 0.5% NP40, 1% triton, protease and phosphatase inhibitors cocktail (Sigma, Lyon, France) and samples isolated by centrifugation. Eighty µg of proteins in a final volume of fifteen µL of Laemmli loading buffer were heated to 70 °C for 10 minutes. Samples were run on a 4–12% Nu-PAGE gel (Life technologies), transferred to Trans-Blot Turbo Mini-size nitrocellulose membrane according to the manufacturer protocol (Bio-Rad). Membrane was incubated with rabbit anti-Orai3 (1/500, ProsciInc 4117), rabbit anti-Pi-GSK3β (1/1000, Cell signaling mAb#9323), rabbit anti-GSK3β (1/1000, Cell signaling mAb#9315), or mouse-GAPDH (1/1000, Santa Cruz sc-365062) followed by anti-rabbit HRP (1/10000, Abcam 6721), or anti-mouse HRP (1/5000, NA931 GE Healthcare). Detection was performed using the Clarity Western ECl Substrate (Bio-Rad) and signals were recorded using a Camera LAS 4000.
Quantification of TNFα and TNFR2 expression
TNFα and TNFR2 protein expression was quantified as previously reported using Elisa R&D kits26.
Quantification of cardiomyocyte area and tissue fibrosis
Frozen sections fixed in paraformaldehyde were labeled with Wheat Germ Agglutinin (WGA)-Alexa 647 (1/500 dilution, Molecular Probes). Tissue sections were analyzed with a Zeiss Axio Observer Z1 microscope using ImageJ software. A low vs. high threshold allowed quantification of cardiomyocyte area or tissue fibrosis, respectively, as previously reported47. Results were quantified from 7 mice/group (12 images/animal).
Isoproterenol (Sigma) and TNFα (R&D) were respectively used at 100 nM and 50 ng/mL, as previously reported26. Neutralizing anti-TNFR1 or anti-TNFR2 antibodies (R&D) were preincubated 1 hour at 37 °C at a final concentration of 2.5 µg/mL. All pharmacological inhibitors were preincubated for 10 min. Orai pharmacological inhibitors YM58483 (Tocris) and Synta66 (Servier) were added at 1 µM. PLA2 activating peptide (R&D) and the cPLA2 inhibitor, methyl arachidonylfluorophosphonate (MAFP) (Sigma), were respectively used at 20 µg/mL and 4 µg/mL. Lipoxygenase and cycloxygenase inhibitors, nordihydroguaiaretic acid (NDGA) and indomethacin (Sigma), were used at 1 µM. Montelukast (Sigma) was used at 2 µM. To explore ventricular myocyte resistance to oxidative stress, we applied 100 µM of H2O2 (Sigma). Semapimod (MedKoo) was used in vitro on CD11b/c cells at 10 µM.
Quantitative data are represented using means and error bars indicating standard error of the mean (SEM) and were analyzed using XLStat 2014 (Addinsoft, New York, USA). Non-parametric Mann-Whitney U or Wilcoxon matched-paired tests were used for comparisons between two groups, when appropriate. Non-parametric Kruskal-Wallis test was used for comparisons between more than two groups. ANOVA for repeated measures followed by Dunn-Sidak post-hoc test was used to analyze differences in echocardiographic parameters over time. All values with p < 0.05 were considered significant.
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The authors would like to thank Bocar Kane for his assistance in animal care and surgical procedures, Pr. Philippe Ménasché and Dr. Anaïs Kervadec for their help in ultrasound directed transthoracic injections, Dr. Adeline Beuriot and Dr. Dario Melgari for their help in cardiac cell isolation, and Dr. Ewa Ninio for critical reading of the manuscript. This study was supported by an Agence Nationale de la Recherche grant to A.M.L. (Cardiosoc project), by two Institute of Cardiometabolism and Nutrition innovative seeding project grants to C.P., and by the National Institutes of Health grant R01HL113497 to J.S.H. Part of the work was also funded by a Leducq Foundation grant to J.S.H.