Mitochondrial DNA damage in iron overload

Abstract

Chronic iron overload has slow and insidious effects on heart, liver, and other organs. Because iron-driven oxidation of most biologic materials (such as lipids and proteins) is readily repaired, this slow progression of organ damage implies some kind of biological "memory." We hypothesized that cumulative iron-catalyzed oxidant damage to mtDNA might occur in iron overload, perhaps explaining the often lethal cardiac dysfunction. Real time PCR was used to examine the "intactness" of mttDNA in cultured H9c2 rat cardiac myocytes. After 3-5 days exposure to high iron, these cells exhibited damage to mtDNA reflected by diminished amounts of near full-length 15.9-kb PCR product with no change in the amounts of a 16.1-kb product from a nuclear gene. With the loss of intact mtDNA, cellular respiration declined and mRNAs for three electron transport chain subunits and 16 S rRNA encoded by mtDNA decreased, whereas no decrements were found in four subunits encoded by nuclear DNA. To examine the importance of the interactions of iron with metabolically generated reactive oxygen species, we compared the toxic effects of iron in wild-type and rho(o) cells. In wild-type cells, elevated iron caused increased production of reactive oxygen species, cytostasis, and cell death, whereas the rho(o) cells were unaffected. We conclude that long-term damage to cells and organs in iron-overload disorders involves interactions between iron and mitochondrial reactive oxygen species resulting in cumulative damage to mtDNA, impaired synthesis of respiratory chain subunits, and respiratory dysfunction.

FIGURE 1.

Morphology of cardiac myocyte H9c2 cells and H9c2-rho^o cells were grown in the absence or presence of 300 μm FAC. A, control H9c2 cells. B, H9c2 cells grown in the presence of 300 μm FAC for 4 days. C, control H9c2 rho^o cells grown in the absence of 300 μm FAC. D, H9c2 rho^o cells grown in the presence of 300 μm FAC for 4 days. Note that FAC appears to be preferentially toxic to the wild-type H9c2 cells versus the rho^o cells.

FIGURE 2.

Calcein staining of H9c2 cells grown in the absence (A) or presence (B) of 200 μm FAC for 24 h. Substantial intracellular iron was present following iron exposure as shown by the quenching of calcein fluorescence (B).

FIGURE 3.

8-Oxo-dG accumulation in purified mtDNA measured by enzyme-linked immunosorbent assay in H9c2 cells before and 3–7 days after exposure to 300 μm FAC. Results are expressed as nanograms of 8-oxo-dG/mg of total mtDNA ± 1 S.D. differ from control cells not exposed to iron. *, p < 0.05; **, p ≤ 0.001 (Student's t test, two tailed). n = 3 separate preparations in each case.

FIGURE 4.

Iron loading causes progressive loss of near full-length (PCR-amplifiable) mtDNA. H9c2 cells were exposed 300 μm iron for 7 and 14 days. The total DNA was isolated that was amplified with qPCR. Full-length mtDNA product was significantly decreased after 7 and 14 days (A), whereas no changes were observed in the PCR product of an equally long segment of the transferrin receptor nuclear gene (B). p < 0.01 for mitochondrial versus nuclear long length products (Student's t test, two-tailed). n = 3 separate preparations in each case.

FIGURE 5.

Progressive loss of full-length (PCR-amplifiable) mtDNA during exposure of cultured H9c2 cells to 300μm FAC. Results shown are the mean ± 1 S.D. of the ratio of near full-length versus 200-bp amplification products. All values differ significantly from untreated controls at p < 0.05 (Student's t test, two-tailed). n = 4 separate preparations for each point.

FIGURE 6.

Growth of wild-type and rho^o H9c2 cells in the absence and presence of iron. Control and rho^o H9c2 cells were cultured for up to 4 days in normal medium or medium containing 300 μm FAC. At each time point, trypan blue excluding cells were counted using a hemocymeter. Each point represents the mean of four independent assays ± 1 S.D. Iron-exposed rho^o H9c2 cells grew at the same rate as the control rho^o cells, whereas wild-type H9c2 cells exposed to iron grew very little over the first 3 days and then began dying. For iron-exposed wild-type versus rho^o cells exposed to iron, cell numbers differ significantly on days 3 and 4 at p < 0.001 (n = 4 in all cases).

FIGURE 7.

Iron-induced ROS generation in wild-type and rho^o H9c2 cells. ROS production was examined after 24 h of culture in the absence or presence of 200 and 300 μm iron using oxidation of the fluorescent probe DCF-DA. Whereas ROS production in wild-type H9c2 cells (filled bars) was significantly increased by iron exposure, no changes were observed in rho^o H9c2 cells (open bars). p < 0.05 for rho^o versus wild type cells (Student's t test, two-tailed). n = 4 in all cases.

FIGURE 8.

Decreased oxygen consumption by H9c2 cells exposed to 300 μm iron for 7 or 11 days. Respiration rates were measured using 3–5 × 10⁶ viable cells (determined by trypan blue exclusion) and results were adjusted mathematically to rate of oxygen consumption per 5 × 10⁶ viable cells. A starting O₂ concentration of 240 μm was assumed based on O₂ solubility at sea level at 37 °C. Compared with untreated H9c2 cells, maximal oxygen consumption rates in the presence of the uncoupler CCCP were significantly decreased following iron exposure at both 7 and 11 days (A). Similar iron-mediated decrements in respiration were also observed in the absence of an uncoupler (B). p < 0.01 untreated versus iron-treated cells (Student's t test, two-tailed). n = 3 separate cell preparations in each case.

FIGURE 9.

Decreased mRNA for respiratory chain components (and 16 S rRNA) following iron exposure of H9c2 cells. H9c2 cells were exposed to 300 μm iron for 7 days. Real-time PCR was used to detect levels of mRNA expression of mitochondrial genes (16 S rRNA, Nd4, Cox1, and NdI) and nuclear genes (GADPH, Cyc1, Sdh subunit b, Cox vb, and Nrf1). Results suggest 50–90% loss of mRNAs encoded by mtDNA but no decreases in those encoded by nuclear genes. Gapdh mRNA was used as an input control. **, p < 0.001 untreated versus iron-treated H9c2 cells (Student's t test, two-tailed). n = 3 in each case.