Cardiac inflammatory CD11b/c cells exert a protective role in hypertrophied cardiomyocyte by promoting TNFR2- and Orai3- dependent signaling

Figures

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  Figure 1. TNFα activates a store- and voltage-independent Ca2+ influx in hypertrophied but not in control cardiomyocytes. Representative recordings of Fura2 fluorescence ratio (F340/F380) in iso-treated hypertrophied (A,B) or control (C,D) cardiomyocytes subjected to 2 successive measurements of the rate of voltage- and store-independent Ca2+ entry and quantification of rates of Ca2+ entry. Two successive applications of the same 1 mM Ca2+ basal perfusion medium gave rise to similar rates of Ca2+ entry, both in hypertrophied (A) and normal cells (C). Addition of TNFα to the second 1 mM Ca2+ perfusion medium enhanced the rate of Ca2+ entry (2nd slope as compared to 1st slope) in hypertrophied cardiomyocytes (B) but not in normal cardiomyocytes (D). Number of cells analyzed and number of cell isolations (rats) as indicated. Mean ± SEM of cells, Wilcoxon matched-paired tests to examine if the mean of the 2nd rate differs from the 1st one, arbitrarily set as 1, ****p < 0.0001.

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  Figure 2. TNFα activates a store- and voltage-independent Ca2+ influx in hypertrophied cardiomyocytes further identified as Orai3 dependent. Hypertrophied cardiomyocytes were loaded with Fura2 before measurement of voltage- and store-independent Ca2+ influx. (A) Two successive applications of the same TNFα perfusion medium on iso-treated hypertrophied cardiomyocytes gave rise to similar rates of Ca2+ entry. The first TNFα-induced Ca2+ influx was arbitrarily set to 2.2 to allow comparisons with the control conditions (see Fig. 1B). Preincubation with Orai inhibitors, YM58483 or Synta66, prior the second application of TNFα perfusion medium blunted activation of Ca2+ entry by TNFα. (B) Hypertrophied cardiomyocytes isolated from hearts injected with Cy3-tagged scramble or Orai3 siRNAs three days before isolation and iso-treatment. Typical images show Cy3-fluorescence in cardiomyocytes. TNFα activates Ca2+ influx in scramble siRNA-transfected cells but not in siOrai3-transfected cardiomyocytes. Number of cells analyzed and number of cell isolations (rats) as indicated, mean ± SEM of cells, Wilcoxon matched-paired tests to examine if the mean of the 2nd rate differs from the 1st one,…

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  Figure 3. Activation by TNFα relies on binding to TNFR2, stimulation of cPLA2 and potential production of AA metabolites via the lipoxygenase pathway. (A–C) Iso-treated hypertrophied cardiomyocytes were loaded with Fura2 before measurement of voltage- and store-independent Ca2+ influx. (A) Rates of Ca2+ entry in response to TNFα after a 1 hour preincubation with control IGg2A, neutralizing TNFR1-Ab or TNFR2-Ab. (B) TNFα effect is sensitive to the cPLA2 inhibitor, MAFP, but mimicked by cPLA2 activating peptide PLAP. (C) Activation of Ca2+ entry by TNFα is blunted by preincubation with NDGA (lipoxygenase inhibitor), but unaffected by indomethacin (cyclooxygenase inhibitor) or montelukast (leukotriene receptor antagonist) pretreatments. Number of cells analyzed and number of cell isolations (rats) as indicated. Mean ± SEM of cells, Wilcoxon matched-paired tests to examine if the mean of the 2nd rate differs from the 1st one, arbitrarily set as 1, *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 4. TNFα activates Orai-Ca2+ influx after binding to TNFR2 and potential production of AA metabolites via the lipoxygenase pathway in hypertrophied cardiomyocytes from rats with AAB-induced EACH. (A) Schematic representation of the protocol where rats with AAB-induced EACH for 28 days were subjected to echographic analyses. (B–E) Hypertrophied cardiomyocytes isolated from AAB rats were loaded with Fura2 before measurement of voltage- and store-independent Ca2+ influx. TNFα activates Ca2+ entry (B) in a manner sensitive to Orai inhibitor YM58483 (C), neutralizing TNFR2-Ab (D) or lipoxygenase inhibitor NDGA (E). Number of cells analyzed and number of cell isolations (rats) as indicated, mean ± SEM of cells, Wilcoxon matched-paired tests to examine if the mean of the 2nd rate differs from the 1st one, arbitrarily set as 1 (B,D,E) or 2.2 (C) ****p < 0.0001.

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  Figure 5. Conditioned medium from CD11b/c cells isolated from EACH heart activates Ca2+ influx in a manner sensitive to TNFR2-Ab and Orai inhibitor YM58483. (A) Schematic representation of the protocol where rats implanted with an iso-pump for fourteen days were subjected to echographic analyses and developed EACH. (B) CD11b/c cells, cardiomyocytes and fibroblasts were isolated from EACH hearts and cultured for 18 hours before recovery and concentration of conditioned media. CD11b/c cells pre-incubated with/without the anti-inflammatory drug, semapimod, prior in vitro LPS application to induce pro-inflammatory activation, were isolated from normal hearts and cultured for 18 hours before recovery and concentration of conditioned media. Iso-treated hypertrophied cardiomyocytes were loaded with Fura2 before measurement of voltage- and store-independent Ca2+ influx, 1st in the absence and 2nd in the presence of these conditioned media (Cmed). (C) Only Cmed from CD11b/c cells isolated from EACH hearts activates a voltage- and store-independent Ca2+ influx (D) in a manner sensitive to TNFR2-Ab and Orai inhibitor YM58483. (E) Cmed from CD11b/c cells isolated from normal hearts and in vitro…

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  Figure 6. TNFR2 and Orai signaling pathways enhance hypertrophy in cardiomyocytes after inflammatory stimulation with TNFα or Cmed from in vitro LPS-activated cardiac CD11b/c cells. (A) CD11b/c cells were isolated from normal hearts and further subjected to in vitro activation by LPS incubation following or not semapimod pretreatment. Conditioned media (recovered after 18 hrs in culture) or TNFα were applied on rat cardiomyocytes after 1.5 hours iso treatment and cell hypertrophy analyzed after 18 hours. (B) TNFα or Cmed in vitro enhance hypertrophy of iso-treated cardiomyocytes in a manner sensitive to semapimod, TNFR2-Ab or YM58483. Mean ± SEM of 3–4 experiments, cardiomyocytes from 2–4 rats, 2 Cmed. Kruskal-Wallis followed by Dunn’s post-hoc test vs. the Iso-treated control (white column), *p < 0.05.

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  Figure 7. TNFR2 and Orai signaling pathways promote resistance to oxidative stress of cardiomyocytes after inflammatory stimulation with TNFα or Cmed from in vitro LPS-activated cardiac CD11b/c cells. (A) CD11b/c cells were isolated from normal hearts and further subjected to in vitro activation by LPS incubation following or not semapimod pretreatment. Conditioned media (recovered after 18 hrs in culture) or TNFα were applied on in vitro iso hypertrophied rat cardiomyocytes before H2O2 treatment and analyses of cell resistance. (B) TNFα or Cmed in vitro increase resistance of hypertrophied cardiomyocytes to H2O2 stress in a manner sensitive to semapimod, TNFR2-Ab or YM58483. Mean ± SEM of 3–5 experiments, cardiomyocytes from 3–5 rats, 2–4 Cmed. Kruskal-Wallis followed by Dunn’s post-hoc test vs. the Iso-treated control (white column), **p < 0.01.

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  Figure 8. Orai3 knockdown at the onset of EACH limits cardiac hypertrophy, accelerates alteration of function and dilation and promotes fibrosis. (A) Schematic representation of the protocol where mice implanted with an iso-pump at day 0 were subjected to a unique ultrasound-guided intramyocardial transthoracic injection of Scramble or Orai3 siRNA at day 8 and submitted to an echocardiographic follow-up. (B) In some experiments, siRNA was added with sulforhodamine: typical in-vivo visualization of intracardiac localization of sulforhodamine at day 1 post-injection using an IVIS spectrum in-vivo imaging system. (C) Echocardiographic parameters (mean ± SEM of mice, n = 8–22 mice/group, see Table 3, ANOVA for repeated measures followed by Dunn-Sidak post-hoc tests). (D,E) Efficient knockdown of Orai3 mRNA and protein levels in cardiac homogenates at d12, d15 and d28 following injection (mean ± SEM of mice, n = 7 mice/group, Kruskal-Wallis followed by Dunn’s post-hoc tests). Full length blots were included in SI. (F,G) SiOrai3 injection induced a decrease in Heart Weight/Body Weight ratio, myocyte area and Myh7 mRNA level, an increase in fibrosis, and an elevation of Col1a1, Col3a1 and Tgfβ…

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  Figure 9. Orai3 enhances hypertrophy, promotes resistance to oxidative stress in adult hypertrophied cardiomyocytes and during EACH limits evolution towards HF. Mechanistically, a TNFα paracrine signaling potentially mediated by the cardiac inflammatory CD11b/c cells promotes TNFR2-dependent activation of store-independent arachidonic acid-dependent Orai3-related Ca2+ influx enhancing hypertrophy and inducing resistance to H2O2. Knockdown of Orai3 at the onset of adaptive hypertrophy suppresses the EACH response and accelerates transition towards HF.