SOD2 deficiency in cardiomyocytes defines defective mitochondrial bioenergetics as a cause of lethal dilated cardiomyopathy

Figures

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  Fig. 1. Generation, genotyping, and confirmation of SOD2Δ mice. A, strategy for generating cardiac-specific alpha myosin-heavy chain SOD2 knockout mice. i) The SOD2 genomic structure containing exon 3 (upper) was replaced by a targeting Neo cassette in the expected homologous recombination (lox allele) (lower). ii) After complete Cre-mediated recombination (SOD2Δ), the Neo cassette and exon 3 were excised from the genome. B, PCR of DNA (left) obtained from mouse tail snips was screened for the totally recombined allele; orphan loxP, 350 bp (fl), and the Cre allele, 200 bp. SOD2 mRNA expression (right) was measured in heart tissue from SOD2fl and SOD2Δ mice and reported relative to values in SOD2fl heart tissue (mean ± SD from n = 5 animals per genotype, *P < 0.001). C, Western blotting of heart lysate demonstrates SOD2 protein expression in SOD2fl and SOD2Δ with β-actin used as a loading control. SOD2 expression was normalized to β-actin staining (n = 3 animals) and presented as mean ± SD in arbitrary units in which the density of SOD2 in the SOD2fl samples was set to 1. *P < 0.05 by t-test vs. control. D, Immunofluorescence staining of heart section stained with antibody to SOD2 (red)…

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  Fig. 2. Cardiac dysfunction was exhibited in SOD2Δ mice. A, Representative Hematoxylin and Eosin-stained heart tissue (LV) from SOD2Δ as compared to their SOD2fl littermates. Sarcomere disruption is indicated by *. Scale bar = 100 μm. B, Masson's trichrome-staining image (upper) of SOD2fl and SOD2Δ heart section (LV). Arrow indicates fibrosis in heart tissue. Scale bars = 100 μm. Quantification of cardiac fibrosis area from Masson's trichrome-stained heart sections (lower). All analyses were carried out in 4-month-old mice. Values are the means ± SD (n = 5). *P < 0.05 vs. control group. C, Transmission electron micrograph of 4-month-old myocardium (LV) from SOD2Δ mice as compared to their SOD2fl littermates. SOD2Δ myocardium showed damaged mitochondria (Mi) with disorganization of cristae (C) and vacuole formation (V) in SOD2Δ as compared to SOD2fl. Scale bar = 500 nm. D, Quantification of superoxide dismutase (SOD2), CuZnSOD, catalase, and glutathione peroxidase activity (n = 5, *P < 0.001). Assessment of GSH, GSSG, and GSH/GSSG from heart tissue of SOD2Δ vs. SOD2fl mice. E, Superoxide generation was analyzed using HPLC after staining myocardial tissue with a dihydroethidium…

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  Fig. 3. Echocardiographic indices of contracting SOD2Δ mouse hearts (n = 6) (A–I). A, Representative M-mode images of SOD2fl and SOD2Δ heart. B, systolic left ventricular internal diameter (LVID; s). C, diastolic left ventricular internal diameter (LVID; d). D, percentage fractional shortening (%FS). E, left ventricular systolic volume (LV Vol; s). F, left ventricular diastolic volume (LV Vol; d). G, % ejection fraction. H, systolic intraventricular septum diameter (IVS; s). I, diastolic intraventricular septum diameter (IVS; d).*P < 0.05 compared to control.

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  Fig. 4. Mitochondrial bioenergetics in SOD2Δ mouse hearts. A and B, the graphs represent oxygen consumption rate (OCR) measurements in isolated mitochondria of SOD2Δ vs. SOD2fl hearts at baseline and after sequential addition of oligomycin, FCCP, and Antimycin A + Rotenone. The OCR value is expressed as pmol/min/μg of protein. C, spare respiratory capacity, ATP turnover, and maximum respiration were significantly reduced in SOD2Δ mice. All values are mean ± SD (n = 6). *P < 0.001 as compared to control (SOD2fl).

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  Fig. 5. OCR and ECAR assay in neonatal cardiomyocytes from of SOD2Δ and SOD2fl mice. A and B, OCR measurement after sequential addition of oligomycin, FCCP, and Antimycin A + Rotenone. C, Mitochondrial respiration, including spare respiratory capacity, ATP turnover, and maximum respiration, were significantly decreased in SOD2Δ mice. D, ECAR was measured in neonatal cardiomyocytes over time; the graph represents glycolysis and glycolytic reserve. All values are mean ± SD (n = 5). *P < 0.001 as compared to control (SOD2fl).

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  Fig. 6. Representative immunoblots showing the interaction of 4HNE with the proteins of the respiratory complex and TCA cycle. A, murine heart homogenates from SOD2fl and SOD2Δ mice were immunoprecipitated with mouse 4-HNE antibody (lanes 1 & 2) or mouse IgG antibody (lanes 5 & 6) as a control. Total heart homogenates (20 μg) from IP samples were loaded in lanes 3 and 4. The immunoprecipitates were immunoblotted (IB) with antibodies specific for the indicated proteins (upper). Quantification of immunoprecipitate with mouse 4-HNE antibody (lower). All values are mean ± SD (n = 4). *P < 0.001 as compared to control (SOD2fl).