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. 2020 Sep 30;37:101740. doi: 10.1016/j.redox.2020.101740

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© 2020 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 1 — Generation, genotyping, and confirmation of SOD2^Δ 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 SOD2^fl and SOD2^Δ mice and reported relative to values in SOD2^fl heart tissue (mean ± SD from n = 5 animals per genotype, *P < 0.001). C, Western blotting of heart lysate demonstrates SOD2 protein expression in SOD2^fl 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 SOD2^fl 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) and Troponin I (green) (upper) from SOD2^fl mice where the arrow indicates SOD2 in cardiomyocytes. SOD2 and Troponin I immunofluorescence (lower) in SOD2Δ heart where the arrow indicates SOD2 in other cells. Nuclei were counterstained with DAPI (blue). Note that SOD2 is not apparent in cardiomyocytes from SOD2Δ hearts. The result is representative of three independent experiments. Scale bar = 50 μm. E, Kaplan-Meier analysis of survival probabilities for the SOD2Δ (n = 85) versus the SOD2^fl (n = 91) mice. F, The heart of a 4-month-old SOD2Δ mouse is significantly larger than that of a SOD2^fl mouse (scale in mm). Quantification of LV mass and HW/BW of SOD2^fl and SOD2Δ mice. G, Quantification of electrophysiology studies, atrial fibrillation (AF), atrial effective refractory period (AERP), ventricular tachycardia (VT), and ventricular effective refractory period (VERP) of SOD2Δ vs. SOD2^fl mice heart (n = 6, *P < 0.01). H, RT-PCR shows fold differences in the mRNA of Nppa and Nppb with the value of SOD2^fl defined as 1 (mean ± SD, n = 5 each). *P < 0.001 compared to control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1 — Generation, genotyping, and confirmation of SOD2^Δ 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 SOD2^fl and SOD2^Δ mice and reported relative to values in SOD2^fl heart tissue (mean ± SD from n = 5 animals per genotype, *P < 0.001). C, Western blotting of heart lysate demonstrates SOD2 protein expression in SOD2^fl 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 SOD2^fl 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) and Troponin I (green) (upper) from SOD2^fl mice where the arrow indicates SOD2 in cardiomyocytes. SOD2 and Troponin I immunofluorescence (lower) in SOD2Δ heart where the arrow indicates SOD2 in other cells. Nuclei were counterstained with DAPI (blue). Note that SOD2 is not apparent in cardiomyocytes from SOD2Δ hearts. The result is representative of three independent experiments. Scale bar = 50 μm. E, Kaplan-Meier analysis of survival probabilities for the SOD2Δ (n = 85) versus the SOD2^fl (n = 91) mice. F, The heart of a 4-month-old SOD2Δ mouse is significantly larger than that of a SOD2^fl mouse (scale in mm). Quantification of LV mass and HW/BW of SOD2^fl and SOD2Δ mice. G, Quantification of electrophysiology studies, atrial fibrillation (AF), atrial effective refractory period (AERP), ventricular tachycardia (VT), and ventricular effective refractory period (VERP) of SOD2Δ vs. SOD2^fl mice heart (n = 6, *P < 0.01). H, RT-PCR shows fold differences in the mRNA of Nppa and Nppb with the value of SOD2^fl defined as 1 (mean ± SD, n = 5 each). *P < 0.001 compared to control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)