Heme oxygenase-1 regulates mitochondrial quality control in the heart

Abstract

The cardioprotective inducible enzyme heme oxygenase-1 (HO-1) degrades prooxidant heme into equimolar quantities of carbon monoxide, biliverdin, and iron. We hypothesized that HO-1 mediates cardiac protection, at least in part, by regulating mitochondrial quality control. We treated WT and HO-1 transgenic mice with the known mitochondrial toxin, doxorubicin (DOX). Relative to WT mice, mice globally overexpressing human HO-1 were protected from DOX-induced dilated cardiomyopathy, cardiac cytoarchitectural derangement, and infiltration of CD11b+ mononuclear phagocytes. Cardiac-specific overexpression of HO-1 ameliorated DOX-mediated dilation of the sarcoplasmic reticulum as well as mitochondrial disorganization in the form of mitochondrial fragmentation and increased numbers of damaged mitochondria in autophagic vacuoles. HO-1 overexpression promotes mitochondrial biogenesis by upregulating protein expression of NRF1, PGC1α, and TFAM, which was inhibited in WT animals treated with DOX. Concomitantly, HO-1 overexpression inhibited the upregulation of the mitochondrial fission mediator Fis1 and resulted in increased expression of the fusion mediators, Mfn1 and Mfn2. It also prevented dynamic changes in the levels of key mediators of the mitophagy pathway, PINK1 and parkin. Therefore, these findings suggest that HO-1 has a novel role in protecting the heart from oxidative injury by regulating mitochondrial quality control.

Figure 1. Heme oxygenase-1 overexpression prevents doxorubicin-induced cardiac toxicity.

(A) Schematic of the treatment protocol utilized in this study. Mice were administered three 6.0 mg/kg doses of intravenous doxorubicin (DOX) every third day for a week and euthanized for tissue collection. To assess cardiac function, mice underwent serial echocardiography the day before DOX administration and up to 14 days after treatment. (B) Heme oxygenase-1 (HO-1) overexpression in humanized HO-1 overexpressing (HBAC) mice was confirmed by Western blot on tissue lysates generated from the left ventricle (LV). GAPDH was used as a loading control. (C) Ejection fraction (EF), (D) end diastolic diameter (EDD), and (E) end systolic diameter (ESD) were measured by serial echocardiography. Black bars depict data from WT mice, white bars depict data from HBAC mice. Data are presented as mean ± SEM. *P < 0.05, 2-tailed paired t test was used to determine statistically significant changes after DOX treatment, relative to baseline values; n = 5–8 per group.

Figure 2. Heme oxygenase-1 overexpression protects cardiomyocytes from doxorubicin-mediated damage.

(A and B) Representative micrographs of midsagittal sections of cardiac left ventricle (LV) from WT and humanized heme oxygenase-1 (HO-1) overexpressing (HBAC) mice 14 days after doxorubicin (DOX) treatment. Cross sections stained with H&E from (A) WT or (B) HBAC mice. Scale bar: 1 mm. (C–F) H&E-stained sections demonstrating that HO-1 overexpression prevents cardiomyocyte vacuolization (black arrows), hypereosinophilia (red arrows), and interstitial edema (asterisks) observed in WT mice. Scale bar: 100 μm. (G and H) Trichrome staining for fibrosis in the LV of WT and HBAC mice. Scale bar: 100 μm. Slides were assessed by a pathologist blinded to the different groups. n = 3–6 per group. (I) Mononuclear phagocyte (CD45⁺CD11b⁺Gr-1^–MHCII⁺) infiltration into the LV of WT and HBAC mice treated with DOX or vehicle was quantified by flow cytometry and expressed as a proportion of CD45⁺ cells. Horizontal bars represent mean values. *P < 0.05, **P < 0.01, ANOVA and the Newman-Keuls post-test were used to determine statistically significant changes; n = 3–5 per group.

Figure 3. Heme oxygenase-1 overexpression promotes mitochondrial biogenesis.

(A) Representative Western blot of heme oxygenase-1 (HO-1) expression in the left ventricle (LV) of mice with cardiac-specific overexpression of HO-1 (cs-HO-1). GAPDH was used as a loading control. (B) Mitochondrial DNA (mtDNA) was quantified in WT (black circles) and cs-HO-1 (white circles) LV obtained from mice treated with vehicle or doxorubicin (DOX) using real-time PCR. (C–E) Densitometric quantification from Western blots (n = 5 per group) to assess expression of the (C) mtDNA polymerase (Polγ) or (D and E) components of the mitochondrial electron transport chain (NADH dehydrogenase 1 [ND1] in D and cytochrome c oxidase III [COX3] in E). (F–H) Densitometric quantification from Western blots (n = 5 per group) to assess expression of (F and G) nuclear (nuclear respiratory factor 1 [NRF1] and peroxisome proliferator-activated receptor γ coactivator 1 [PGC1α]) and (H) mitochondrial (mitochondrial transcription factor A [TFAM]) transcription factors necessary for mitochondrial biogenesis. Band density was quantified relative to β-actin. Original blots can be seen in Supplemental Figure 1. Data are expressed in arbitrary units as average ± SEM after normalization to β-actin. P < 0.05, ANOVA and the Newman-Keuls post-test were used to determine statistically significant changes; n = 5 per group.

Figure 4. Doxorubicin-induced mitochondrial abnormalities at day 14.

Transmission electron micrographs of the left ventricle (LV) 14 days after treatment with (A, C, and E) vehicle or (B, D, and F) doxorubicin (DOX) in (A and B) WT, (C and D) humanized heme oxygenase-1 (HO-1) overexpressing (HBAC) mice, or (E and F) mice with cardiac-specific overexpression of HO-1 (cs-HO-1). White arrows depict dilated sarcoplasmic reticulum. (G–I) HO-1 overexpression in cs-HO-1 mice prevents DOX-induced disorganization of intercalated disks (IDs). Original magnification, ×4,400. Scale bar: 2 μm. n = 3–5 mice per group, with at least 5 images evaluated per mouse. Images were assessed by a reviewer blinded to the different groups.

Figure 5. Doxorubicin-induced mitochondrial abnormalities at day 60.

Transmission electron micrographs of the left ventricle (LV) 60 days after treatment with (A–C) vehicle or (D–F) doxorubicin (DOX) in (A and D) WT mice, (B and E) mice with cardiac-specific overexpression of heme oxygenase-1 (cs-HO-1), and (C and F) HO-1 knockout (HO-1^–/–) mice. White Vs mark nonmembrane-bound vacuoles, and white arrows mark mitochondria engaged with a membrane-bound vacuole. Original magnification, ×4,400. Scale bar: 2 μm. (G) Mitochondrial number and (H) area were quantified on images collected at an original magnification of ×1,650. Image evaluation and mitochondrial quantification were performed by a reviewer blinded to the different groups (n = 3–5 mice per group) and averaged from at least 5 images per mouse. Data presented as mean ± SEM. P < 0.05, ANOVA and the Newman-Keuls post-test were used to determine statistically significant changes.

Figure 6. Heme oxygenase-1 overexpression increases fusion and inhibits fission.

Expression of protein markers for mitochondrial fusion and fission was assessed in the hearts of WT mice and mice with cardiac-specific overexpression of heme oxygenase-1 (cs-HO-1) 14 days after treatment with vehicle or doxorubicin (DOX) by Western blot. Data are presented as protein density relative to β-actin. Black circles depict data from WT mice, and white circles depict data from cs-HO-1 mice. Expression of the fusion markers (A) mitofusin 1 (Mfn1) and (B) mitofusin 2 (Mfn2). Expression of the fission markers (C) dynamin-related protein 1 (DRP1) and (D) mitochondrial fission 1 (Fis1). Data are expressed in arbitrary units as an average ± SEM after normalization to β-actin. Original blots are shown in Supplemental Figure 2. P < 0.05, ANOVA and the Newman-Keuls post-test used to determine statistically significant changes; n = 5 per group.

Figure 7. Heme oxygenase-1 overexpression prevents dynamic changes in mitophagy over time after mitochondrial insult.

(A) WT mice were treated with doxorubicin (DOX) and euthanized 2, 5, 8, or 14 days after treatment. Levels of key protein mediators of mitophagy (PINK1 and parkin) and apoptosis (caspase-3 [C3]) were assessed by Western blot in tissue lysates prepared from the LV. GAPDH and Ponceau S (Pon S) were used as loading controls. Control mice were untreated. n = 3–4 per group. Densitometry plots of this blot are depicted in Supplemental Figure 3. (B) Mitophagy was indexed by PINK1 expression in untreated WT mice and mice with cardiac-specific overexpression of heme oxygenase-1 (cs-HO-1) before as well as 5 and 14 days after DOX treatment. Black vertical lines indicate where the blot was cropped. All lanes in this representative image are from the same blot. GAPDH and Ponceau S were used as loading controls. n = 3 mice per group.