Overexpression of Transcripts Coding for Renin-b but Not for Renin-a Reduce Oxidative Stress and Increase Cardiomyoblast Survival under Starvation Conditions

Figures

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  Figure 1. (A) Cell number and (B) percentage of trypan blue negative, viable cells of pIRES. control (n = 15), renin-b (n = 15) and renin-a cell lines (n = 11) overexpressing cytosolic and secretory renin, respectively. Cells were exposed to basal (non-treated) conditions, glucosestarvation alone, anoxia alone, and the combination of glucose starvation and anoxia (OGD) for 24 h. Renin-b overexpression was associated with a basal decrease in cell number without affecting the membrane integrity. Anoxia and OGD exposure resulted in a decrease 12 in cell number only in pIRES and renin-a cells and a reduction of membrane integrity in all three cell lines. However, for renin-b cells this effect was comparatively moderate. Data represent mean values ± SEM of 11–15 experiments. Differences were considered significant using two-way ANOVA with Bonferroni correction (p-values see text). (*): effects of the intervention (medium conditions) within the same cell line; (#): effects of renin-b or renin-a 17 overexpression, respectively, compared with pIRES control cells on same intervention.

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  Figure 2. Renin-b protects H9c2 cells from necrosis induced by ischemia-related conditions. (A) Early and (B) late necrosis of pIRES control, renin-b and renin-a cell lines overexpressing cytosolic and secretory renin, respectively. Cells were exposed to basal (non-treated) conditions, glucose starvation alone, anoxia alone and the combination of glucose starvation and anoxia (OGD) for 24 h. Early necrosis was measured by PI labeling, where early necrotic cells were PI-positive but apoptosis-negative. Late necrosis was determined by the Cytotoxicity Detection Kit as declared. Renin-b overexpression was associated with an increased percentage of early necrotic cells under basal and glucose starvation conditions. OGD exposure resulted in a strong increase of early and late necrosis in pIRES control and renin-a cells, while in renin-b cells the amount of late necrosis increased only moderately. Data represent mean values ± SEM of 5–6 experiments. Differences were considered significant using two-way ANOVA with Bonferroni correction (p-values see text). (*) effects of the intervention (medium conditions) within the same cell line; (#): effects of renin-b or renin-a overexpression,…

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  Figure 3. Renin-b protects H9c2 cells from activation of caspases induced by ischemia-related conditions. (A) Representative histograms of apoptotic cells exhibiting caspase activation (CaspACE marker). Propidium iodide (PI) labeling enabled differentiation between early apoptotic (PI-) and late apoptotic cells (PI+). (B) Mean percentages of total and (C) late apoptotic pIRES control, renin-b and renin-a cells are shown. Cells were exposed to basal (non-treated) conditions, glucose starvation alone, anoxia alone and the combination of glucose starvation and anoxia (OGD) for 24 h. Renin-b overexpression was associated with a protection against anoxia- and ODG-induced activation of caspases, which was observed in pIRES control and renin-a cells. Data represent mean values ± SEM of 6–7 experiments. Differences were considered significant using two-way ANOVA with Bonferroni correction (p-values see text). (*): effects of the intervention (medium conditions) within the same cell line; (#): effects of renin-b or renin-a overexpression, respectively, compared with pIRES control cells on same intervention; (§): effects of renin-a overexpression compared with renin-b cells on same intervention.

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  Figure 4. Renin-b protects H9c2 cells from intrinsically mediated apoptosis induced by ischemia-related conditions. (A) Representative histograms of apoptotic cells exhibiting Annexin V binding. Propidium iodide (PI) labeling enabled differentiation between early apoptotic (PI-) and late apoptotic cells (PI+). (B) Mean percentages of total and (C) late apoptotic pIRES control, renin-b and renin-a cells are shown. Cells were exposed to basal (non-treated) conditions, glucose starvation alone, anoxia alone, and the combination of glucose starvation and anoxia (OGD) for 24 h. Renin-b overexpression was associated with an increased number of total Annexin V-positive cells under basal conditions but also with a protection against glucose starvation- and ODG-induced apoptosis, which was observed in pIRES control and renin-a cells. Data represent mean values ± SEM of 6–7 experiments. Differences were considered significant using two-way ANOVA with Bonferroni correction (p-values see text). (*): effects of the intervention (medium conditions) within the same cell line; (#): effects of renin-b or renin-a overexpression, respectively, compared with pIRES control cells on same intervention; (§):…

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  Figure 5. Renin-b protects H9c2 cells from Fas receptor (FasR)-mediated extrinsic pathway of apoptosis induced by ischemia-related conditions. (A) Representative histograms of apoptotic cells exhibiting FasR expression. Propidium iodide (PI) labeling enabled differentiation between early apoptotic (PI−) and late apoptotic cells (PI+). (B) Mean percentages of total and (C) late apoptotic pIRES control, renin-b and renin-a cells are shown. Cells were exposed to basal (non-treated) conditions, glucose starvation alone, anoxia alone and the combination of glucose starvation and anoxia (OGD) for 24 h. Renin-b overexpression was associated with a protection against anoxia- and ODG-induced apoptosis, which was observed in pIRES control and renin-a cells. Renin-a overexpression was accompanied by an increase in FasR expression during glucose starvation. Data represent mean values ± SEM of 6–7 experiments. Differences were considered significant using two-way ANOVA with Bonferroni correction (p-values see text). (*): effects of the intervention (medium conditions) within the same cell line; (#): effects of renin-b or renin-a overexpression, respectively, compared with pIRES control cells on same…

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  Figure 6. Renin-b protects H9c2 cells from accumulation of mitochondrial superoxides induced by OGD. (A) Representative histograms, (B) mean fluorescence intensities (FLI), and (C) mean percentages of MitoSOX-positive pIRES control, renin-b, and renin-a cells. Cells were exposed to basal (non-treated) conditions, glucose starvation alone, anoxia alone and the combination of glucose starvation and anoxia (OGD) for 24 h. Then, cells were incubated with the MitoSOX fluorophore to detect mitochondrially localized superoxides. Overexpression of renin-b and renin-a was associated with an increase in basal mitochondrial superoxides as detected by an increase in the mean FLI. Under OGD, the degradation of mitochondrial superoxides was inadequate leading to a marked increase in the percentage of MitoSOX-positive cells in pIRES control and renin-a cells. In renin-b cells, the number of MitoSOX-positive cells was also increased, but to a much smaller degree. Data represent mean values ± SEM of 9 experiments. Differences were considered significant using two-way ANOVA with Bonferroni correction (p-values see text). (*): effects of the intervention (medium conditions) within the same cell line; (#):…

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  Figure 7. Renin-b protects H9c2 cells from accumulation of cytosolic reactive oxygen species (ROS) induced by OGD. (A) Representative histograms, (B) mean fluorescence intensities (FLI) and (C) mean percentages of dihydroethidium (DHE)-positive pIRES control, renin-b, and renin-a cells. Cells were exposed to basal (non-treated) conditions, glucose starvation alone, anoxia alone, and the combination of glucose starvation and anoxia (OGD) for 24 h. Then, cells were incubated with the DHE fluorophore to detect cytosolically localized ROS. Under OGD, the ROS content was increased as detected by an increased mean FLI in a subpopulation (DHEhigh-positive cells) of pIRES control cells. In renin-b and renin-a cells exposed to OGD the percentage of DHEhigh-positive cells was reduced compared to pIRES control cells. Data represent mean values ± SEM of 9 experiments. Differences were considered significant using two-way ANOVA with Bonferroni correction (p-values see text). (*): effects of the intervention (medium conditions) within the same cell line; (#): effects of renin-b or renin-a overexpression, respectively, compared with pIRES control cells on same intervention; (§): effects of renin-a…

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  Figure 8. Renin-b protects H9c2 cells from OGD-induced collapse of mitochondrial membrane potential (ΔΨm) and mitigates anoxia- and OGD-induced ATP depletion. pIRES control, renin-b and renin-a cells were exposed to basal (non-treated) conditions, glucose starvation alone, anoxia alone and the combination of glucose starvation and anoxia (OGD) for 24 h. Afterwards, (A) ΔΨm was detected by incubating cells with the JC-1 dye (n = 6–10). Analysis of the ratio of red (disturbed ΔΨm) to green (intact ΔΨm) fluorescence intensity (FLI) of JC-1-positive cells represents the ΔΨm. OGD caused a marked decrease in ΔΨm in pIRES control and renin-a cells from which renin-b cells were completely spared. (B) ATP levels (n = 9) were detected by the CellTiter-Glo® Luminescent Cell Viability Assay (n = 9). ATP levels were decreased in all cell lines during anoxia and OGD but mitigated in renin-b and renin-a cells. Data represent mean values ± SEM of indicated experiments. Differences were considered significant using two-way ANOVA with Bonferroni correction (p-values see text). (*): effects of the intervention (medium conditions) within the same cell line; (#): effects of renin-b or renin-a overexpression,…