Heme oxygenase-1 inhibits pro-oxidant induced hypertrophy in HL-1 cardiomyocytes

Abstract

Aims: Reactive oxygen species (ROS) activate multiple signaling pathways involved in cardiac hypertrophy. Since HO-1 exerts potent antioxidant effects, we hypothesized that this enzyme inhibits ROS-induced cardiomyocyte hypertrophy.

Methods: HL-1 cardiomyocytes were transduced with an adenovirus constitutively expressing HO-1 (AdHO-1) to increase basal HO-1 expression and then exposed to 200 microM hydrogen peroxide (H2O2). Hypertrophy was measured using 3H-leucine incorporation, planar morphometry and cell-size by forward-scatter flow-cytometry. The pro-oxidant effect of H2O2 was assessed by redox sensitive fluorophores. Inducing intracellular redox imbalance resulted in cardiomyocyte hypertrophy through transactivation of nuclear factor kappa B (NF-kappaB).

Results: Pre-emptive HO-1 overexpression attenuated the redox imbalance and reduced hypertrophic indices. This is the first time that HO-1 has directly been shown to inhibit oxidant-induced cardiomyocyte hypertrophy by a NF-kappaB-dependent mechanism.

Conclusion: These results demonstrate that HO-1 inhibits pro-oxidant induced cardiomyocyte hypertrophy and suggest that HO-1 may yield therapeutic potential in treatment of.

Figure 1

Hydrogen peroxide results in perturbed redox imbalance in HL-1 cardiomyocytes. A) Representative images of control (left panel) and 200 μM H₂O₂ treated HL-1 cardiomyocytes (right panel) using the ROS-sensitive green CM-H₂DCFDA fluorescence (×200) and B) mean intensity between two groups. C) Representative images of control (left panel) and 200 μM H₂O₂ treated HL-1 cardiomyocytes (right panel) using the ROS-sensitive red DHE (×200). D) Mean intensity of fluorochrome between two groups. (Values are mean ± SEM; * P < 0.05 vs. Vehicle control; FACS analysis was performed in triplicate with a minimum of >10,000 gated cells used to calculate the average log intensity of each replicate in an experiment; the indicated mean is the calculated mean of average log intensities of four independent experiments; N = 4.)

Figure 2

Redox imbalance in HL-1 cardiomyocytes results in cellular hypertrophy indicated by increased cell area and cell volume, with no appreciable alteration in nuclear volume. A) Control (left panel) and treated with 200 μM H₂O₂ (right panel) showing increased cell surface area (dotted lines) with no change in nuclear size (solid lines). B) Increased quantitative planar morphometry between groups. C) Representative forward-scatter histograms of cardiomyocyte cell volume in control (left panel) and treated with 200 μM H₂O₂ (right panel) values are presented as an increase from control in total Ln-Mean forward scatter. (Values are mean ± SEM; * P < 0.05 vs. Vehicle control; B) N = mean area of replicates, with >100 cells measured per replicate, total independent experiments are N ≥20; D) N = average Ln-forward-scatter FACS analysis of >10,000 live gated cells per sample, prepared in triplicate, as before; total independent experiments are N = 5).

Figure 3

HL-1 cardiomyocytes control (A–C) and treated with 200 μM H₂O₂ (D–F) Hoechst nuclear counterstain (blue A, D) and sarcomeric actin staining by phalloidin (red B, E) with merged images (C, F at ×400) demonstrated abundant protein synthesis of alter phenotype of hypertrophied HL-1 cardiomyocytes. Protein synthesis was further quantified by ³H-leucine incorporation (G) as a standardized quantitative index of cellular growth associated with hypertrophy. (Values are mean ± SD; * P ≤0.05 vs. Vehicle control; G) N = replicates individually normalized to total protein from five independent experiments; N ≥20).

Figure 4

Specific expression and activity of HO-1 is achieved using adenoviral vectors. A) Transduction of HO-1 to HL-1 cardiomyocytes (phase) with adenoviral vectors produced >90% transduction efficiency as assessed by (B) GFP green fluorescence (×200). C) HO-1, HO-2 and Actin Protein expression in untransduced 30MOI AdGFP and AdHO-1 demonstrating high levels of HO-1 protein expression without affecting HO-2. D) HO-1 protein expression corresponds to increased basal HO activity. (Values are mean ± SD, results are replicates of a single same day experiment performed in left to right succession of groups; N = 6; *** P ≤0.001 vs. Control).

Figure 5

HO-1 therapy ameliorates redox imbalance and cellular hypertrophy. A) Representative images of ROS accumulation by DHE in vehicle (left panel), AdGFP treated with 200 μM H₂O₂ (center panel) and AdHO-1 treated with 200 μM H₂O₂ (right panel; ×200). B) Mean intensity of fluorochrome; AdHO-1 attenuates ROS accumulation. C) Representative forward scatter histograms of control HL-1 cardiomyocytes (left panel) control vector AdGFP treated with 200 μM H₂O₂ (center panel) and AdHO-1 treated with 200 μM H₂O₂ (right panel); AdHO-1 attenuates 200 μM H₂O₂ induced increase in three dimensional cell size. D) Cell surface area and (E) ³H-Luecine incorporation increased by treatment with 200 μM H₂O₂ in AdGFP is markedly reduced by AdHO-1, respectively. (Values are mean ± SEM; * P ≤0.05, *** P ≤0.001 vs. Vehicle control, # P < 0.05, ### P ≤0.001 vs. AdGFP 200 μM H₂O₂; A–D) N = mean of triplicate samples for each experiment. Total independent experiments are B) N = 4; D) N ≥5; E) Values are mean ± SD, N = replicates individually normalized to total protein from five independent experiments; N ≥20).

Figure 6

Oxidant-induced hypertrophy occurs by an NF-κB transactivation dependent mechanism. A) Four repeats of the NF-κB consensus promoter sequence upstream of a luciferase reporter measured an increase in promoter activation after treatment with 200 μM H₂O₂. B) Nuclear protein extract demonstrated active on-target nuclear binding of NF-κB protein after treatment with 200 μM H₂O₂. C) Increased 3H-Leucine incorporation by 200 μM H₂O₂ is abrogated by inhibition of NF-κB transactivation with an NF-κB translocation inhibitor SN-50. (Values are mean ± SEM; * P ≤0.05, *** P ≤0.001 vs. Vehicle control, # P ≤0.05, ## P ≤0.01 vs. 200 μM H₂O₂; A–B) N = mean of duplicate samples as before for each experiment, total independent experiments are A) N ≥8; B) N ≥6; analysis of total samples in B) were performed same day by ELISA from frozen stored isolates; C) Values are mean ± SD, N = replicates individually normalized to total protein from three independent experiments; N = 9).

Figure 7

Specific expression of HO-1 ameliorates the oxidative stress induced NF-κB transactivation. A) Promoter activity after 200 μM H₂O₂ is reduced by HO-1 treatment. B) Nuclear protein NF-κB binding activity after 200 μM H₂O₂ is also reduced by HO-1 treatment. (Values are mean ± SEM; * P ≤0.05 vs. Vehicle control, # P ≤0.05 vs. 200 μM H₂O₂; N = mean of duplicate samples as before for each experiment, total independent experiments are A) N ≥7; B) N ≥15; analysis of B is from frozen isolates and two ELISA sets of the same lot were performed on alternate days for analysis.)

Figure 8

Model of HO-1 mediated antihypertrophic effect in HL-1 cardiomyocytes. Redox imbalance in cardiomyocytes activates IKK to phosphorylate IkB, causing its dissociation from NF-κB thereby exposing the nuclear localization signal. Translocation of NF-κB results in the activation of the hypertrophic gene program, thus leading to increased protein synthesis, a consequence related to increased sarcomere formation that ultimately causes cardiomyocyte hypertrophy. HO-1 interrupts this pathway by abrogating NF-κB transactivation, in part through a reduction in excessive ROS accumulation. (HO-1, heme oxygenase-1; BVR, biliverdin reductase; ROS, reactive oxygen species; IKK, IkB kinase; IkB, inhibitor of kappa B; NF-κB, nuclear factor kappa B; SN-50 inhibitor of NF-κB translocation).