High-density lipoprotein protects cardiomyocytes against necrosis induced by oxygen and glucose deprivation through SR-B1, PI3K, and AKT1 and 2

Figures

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  Figure 1.. HDL protects HIVCMs against OGD-induced necrosis. (A) Cell viability measured by the Cell Titer Blue viability assay for HIVCM treated for 30 min without or with human HDL (100 μg protein/ml) and then exposed to OGD for 4 h. The dashed line indicates 100 % viability, determined by the measurement of cell viability in control cells not exposed to OGD. n = 10 wells/group. ***P < 0.001. (B) Quantification and (C, top row) representative images of TUNEL- (green) and DAPI- (blue) stained HIVCM. n = 3 wells/group. (C, bottom row) Representative images of FITC-AxV- (green), PI- (red), and DAPI- (blue) stained HIVCM treated with or without HDL (30 min) and exposed to 4 h of OGD or control normoxic conditions (as indicated). All scale bars = 50 µm. Quantification of OGD-induced cell death by FITC-AxV staining (D) or PI staining (E) in HIVCM treated as above. (F) Quantification of OGD-induced cell death by PI staining in HIVCM pretreated for 30 min with human LDL (100 μg protein/ml) and then exposed to OGD for 4 h. n = 3 wells/group. (G) HIVCM cells were pretreated with the indicated concentrations of human HDL and then either maintained under normoxic conditions (OGD−) or exposed to OGD…

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  Figure 2.. Nec-1s, an inhibitor of necroptosis, does not prevent OGD-induced death of HIVCM. HIVCM cells were treated with or without 10 μM Nec-1s, an inhibitor of necroptosis, 10 min prior to and during exposure to OGD for 4 h (A and B) or (C) 24 h treatment with TNF-α (100 ng/ml). Control cells were treated with an equivalent volume of DMSO solvent (final concentration of 0.1%). (A) Quantification of the proportion of PI-positive cells (A) or AxV-positive cells (B) after 4 h of OGD. (C) Quantification of the proportion of PI-positive cells after 24 h of exposure to TNF-α. Data represent means ± SEM of n = 3 replicates. **P < 0.004; *P = 0.02; ns, not statistically significant (P > 0.99), analyzed by one-way ANOVA.

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  Figure 3.. SR-B1 is required for HDL-mediated protection of NMCM against OGD-induced necrosis. (A) Representative images and (B) quantification of PI staining in SR-B1+/+ and SR-B1−/− NMCM treated for 30 min with or without HDL (100 μg/ml) prior to exposure to OGD for 4 h. Control cells were maintained in normal media under normoxic conditions. In (A), cells were stained prior to fixation with PI (red) or, after fixation, immunostained for cTnT (green) and stained with DAPI (blue). Scale bars = 50 μm. (B) Data represent means ± SEM for n = 3 replicates. ****P < 0.0001; **P < 0.01; ns, not statistically significant (P > 0.99) by one-way ANOVA.

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  Figure 4.. siRNA-mediated knock down of SR-B1 impairs HDL-mediated protection of HIVCM against OGD-induced necrosis. (A) Representative immunoblot and (B) quantification (n = 3) of SR-B1 knockdown by siRNA in HIVCM. UT: untransfected cells; siCntrl: transfected with control siRNA; siSR-B1: transfected with siRNA targeting SR-B1. (C) Quantification of the percentage of OGD-induced cell death in siCntrl- or siSR-B1-transfected HIVCMs treated with or without HDL (100 μg/ml) prior to 4 h of OGD. Cell viability was measured using the Cell Titer Blue assay and the reduction in viability in siCntrl-transfected cells treated without HDL was set as 100% OGD-induced cell death (n = 9). (D) Proportion of PI-stained cells when HIVCMs were transfected with siCntrl or siSR-B1, treated for 30 min with or without HDL, and exposed to OGD for 4 h as above (n = 3). (E) Representative immunoblots of SR-B1, AKT, and β-actin and quantification of (F) SR-B1/β-actin (n = 5, 6, 6) and (G) AKT/β-actin (n = 3), expressed as fold change in arbitrary relative units. ***P = 0.0003; **P < 0.005; *P < 0.03; ns, not statistically significant (P > 0.3) by one-way ANOVA.

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  Figure 5.. siRNA-mediated knock down of SR-B1 impairs HDL-stimulated AKT phosphorylation in HIVCMs. (A) Representative immunoblots of phospho-AKT (pAKT), total AKT (tAKT), and β-actin (loading control) and (B) quantification of the ratio of pAKT/tAKT (n = 3) in HIVCMs incubated with HDL (100 μg protein/ml) for different times. (C) Representative immunoblots of pAKT and tAKT and (D) quantification of the ratio of pAKT/tAKT (n = 3) in HIVCMs incubated with HDL (100 μg protein/ml) for 30 min prior to and during exposure to OGD or maintenance under normal conditions (‘Normoxia’). (E) Representative immunoblots of pAKT, tAKT, SR-B1, and β-actin (loading control) in siCntrl and siSR-B1-transfected HIVCMs incubated with or without HDL for 30 min. (F) Quantification of the ratio of pAKT/tAKT (expressed as fold change relative to untreated; n = 4). Data are means ± SEM. **P < 0.004; *P < 0.05; ns, not statistically significant (P > 0.9) by one-way ANOVA. §P < 0.02 by unpaired Student's t-test, relative to 0 h time point.

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  Figure 6.. PI3K, AKT1, and AKT2 are required for HDL-mediated protection of HIVCM against OGD-induced necrosis. (A) Quantification of OGD-induced PI staining in HIVCM treated with or without HDL (100 μg/ml) and inhibitors of PI3K (10 µM LY294002) or AKT (3 µM AKT Inh. V) prior to 4 h of OGD. Control cells were treated with an equal volume of DMSO (0.1%). (B) Representative immunoblots of AKT1 and β-actin (loading control) and (C) quantification of fold change in AKT1/β-actin protein levels in HIVCMs transfected with the indicated siRNAs. (D) Representative immunoblots of AKT2 and β-actin (loading control) and (E) quantification of fold change in AKT2/β-actin protein levels in HIVCMs transfected with the indicated siRNAs. (F) Quantification of OGD-induced necrosis (% PI-positive cells) in HIVCMs transfected with the indicated siRNAs and treated with or without HDL (100 μg protein/ml) for 30 min prior to 4 h of OGD, as indicated. Data in all panels represent means ± SEM (n = 3 replicates). ****P < 0.0001; **P = 0.009; *P < 0.04; ns, not statistically significant (P > 0.33) by one-way ANOVA.

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  Figure 7.. Proposed pathway for HDL-mediated protection against OGD-induced cardiomyocyte necrosis. HDL binding to SR-B1 leads to activation of AKT1 and AKT2 signaling and induction of pathways protecting cardiomyocytes from necrosis induced by OGD. Activation of signaling pathways may be indirect, as indicated by broken arrows. Pharmacological or genetic inhibition of this pathway (shown by the boxed text) prevents protection by HDL.