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) Representative Western blots of Parkin (Park2) and PINK1 in mitochondrial fractions of the heart from WT/Cre and HO-1(CM)–/– mice prepared before and after hyperoxia. Porin was used as a loading control. (B) Deletion of HO-1 in HO-1(CM)–/– mice decreases cardiac expression of Park2 mRNA. (C) Lack of HO-1 in the heart of HO-1(CM)–/– mice decreased expression of PINK1 mRNA. (D) Schematic diagrams (–500 to +200 bp) of the Pink1 (ENSMUST00000030536) and Park2 (ENSMUST00000191124) promoter regions. Sequences were aligned between human and mouse using rVISTA 2.0. A search for NRF-1 binding sites upstream of the transcription start site (TSS) identified multiple consensus motifs for NRF-1 in human and mouse Pink1 and Park2 gene promoters by Genomatix and DNAsis software. (E) NRF-1 occupancy of promoters in vivo was investigated by ChIP analysis. Input lanes show the PCR product derived from chromatin prior to immunoprecipitation. Antibodies against NRF-1 used for immunoprecipitation, while IgG was used as negative control. Precipitated DNA was analyzed by PCR with primer sets specific for the 3 promoters (mean ± SEM; horizontal bars represent mean values. *P < 0.05 for pre- vs. posthyperoxia; n = 6 per group; 2-way ANOVA).