The heme oxygenase-1 (Hmox1; HO-1) pathway was tested for defense of mitochondrial quality control in cardiomyocyte-specific Hmox1 KO mice (HO-1[CM]–/–) exposed to oxidative stress (100% O2). After 48 hours of exposure, these mice showed persistent cardiac inflammation and oxidative tissue damage that caused sarcomeric disruption, cardiomyocyte death, left ventricular dysfunction, and cardiomyopathy, while control hearts showed minimal damage. After hyperoxia, HO-1(CM)–/– hearts showed suppression of the Pgc-1α/nuclear respiratory factor-1 (NRF-1) axis, swelling, low electron density mitochondria by electron microscopy (EM), increased cell death, and extensive collagen deposition. The damage mechanism involves structurally deficient autophagy/mitophagy, impaired LC3II processing, and failure to upregulate Pink1- and Park2-mediated mitophagy. The mitophagy pathway was suppressed through loss of NRF-1 binding to proximal promoter sites on both genes. These results indicate that cardiac Hmox1 induction not only prevents heme toxicity, but also regulates the timing and registration of genetic programs for mitochondrial quality control that limit cell death, pathological remodeling, and cardiac fibrosis.
Hagir B. Suliman, Jeffrey E. Keenan, Claude A. Piantadosi
(A) Photomicrographs of immunofluorescence staining of citrate synthase (CS, green) in the hearts. Control WT/Cre heart shows normal mitochondrial labeling with CS in the cardiomyocytes; WT/Cre heart 8d after hyperoxia shows increased CS labeling and mitochondrial fluorescence intensity; HO-1(CM)–/– mouse heart kept on air only shows modest decreases in mitochondrial density; HO-1(CM)–/– mouse hearts 8d after hyperoxia show decreased CS labeling and loss of intensity of mitochondrial fluorescence, suggesting that hyperoxia had caused persistent mitochondrial damage and cell degeneration in HO-1 deficiency. Original magnification, 200×; scale bar = 100 μm. (B) By Western blotting, mitochondrial CS and Tfam expression were significantly increased 8d after hyperoxia in WT/Cre mice but not in HO-1(CM)–/– mice (mean ± SEM; horizontal bars represent mean values.*P < 0.05 for pre- vs. posthyperoxia; n = 6 per group). (C) Electron microscopic findings of myocardial ultrastructure. Electron photomicrographs of cardiomyocytes of control WT/Cre mice showing linear myofibril arrangement and normal mitochondria; WT/Cre mouse exposed to hyperoxia showing enhanced autophagy, and arrows indicating mitophagosomes; electron micrograph from HO-1(CM)–/– mouse showing dilated intramembranous spaces (long arrows); HO-1(CM)–/– after hyperoxia have severe injury, including disoriented myofilaments and aggregates of enlarged mitochondria (M). The images reveal swollen and disorganized mitochondria with abnormal cristae structure (short arrow) and fragmented mitochondria (long arrow), suggesting poor respiratory capacity (n = 6/group). Note lack of mitophagosomes. Original magnification, 12,000×; scale bar = 1 μm. (D) Drp1 mRNA expression in hearts determined by qPCR. (E) Opa1 mRNA expression in hearts determined by qPCR. Values normalized to 18S levels (mean ± SEM; horizontal bars represent mean values.*P < 0.05 for pre- vs. posthyperoxia; n = 6/group; 2-way ANOVA).