Hyperoxia but not AOX expression mitigates pathological cardiac remodeling in a mouse model of inflammatory cardiomyopathy

Figures

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  Figure 1. Heart failure due to Mcp1 overexpression in heart is suppressed by hyperoxia. (A) Summary of the experimental set-up. (B) Transmission electron micrographs of heart tissue from 26-week old animals of the indicated genotypes and treatments. Yellow scale-bars – 2 μm, N – nucleus, red arrows indicate structures resembling autophagosomes. The gross tissue disruption produced by Mcp1 overexpression in the heart is strongly mitigated by hyperoxia. (C) Cardiac ejection fraction (EF, %) of mice of the indicated genotypes (wt – wild-type, Mcp1 – overexpressing Mcp1 in cardiomyocytes), data shown as mean ± SD, n ≥ 5 for all groups. Horizontal lines denote significantly different groups (two-way ANOVA, post hoc Tukey HSD test, p < 0.05; see Table S1). Computed from the data shown in Fig. S1C,D, using MRI conducted at 26 weeks.

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  Figure 2. SNAPf-AOX mice express AOX when activated by tissue-specific recombination. (A) Schematic map of the SNAPf-AOX insert at the Rosa26 locus (flanking regions, grey). The SNAP-tag coding sequence (black), followed by a threefold reiteration of the SV40 poly(A) signal (SV40 pA, orange) is flanked by loxP sites (green, directionality as shown). Cre-mediated recombination removes the entire SNAP-tag cassette, juxtaposing the synthetic CAG promoter (blue) with the AOX coding sequence (yellow), followed by the β-globin intron and poly(A) signal (β-G intron/pA, white). (B,C) Western blots of protein extracts from tissues of progeny mice of the indicated genotypes, from crosses between SNAPf-AOX and hemizygous mice expressing cre recombinase under the control of (B) the ubiquitously active β-actin (ACTB-cre) or (C) cardiomyocyte-specific myosin heavy-chain 6 (Myh6-cre) promoters. Tissues denoted as follows: H – heart, Lu – lung, Li – liver, B – brain, K – kidney, S – spleen, P – pancreas, Mt – thigh muscle, Mm – masseter muscle, E – eye, T – testis. Molecular weight of the detected polypeptides (kDa) inferred from the electrophoretic mobility of markers.

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  Figure 3. AOX expression modifies the cardiac phenotype of Mcp1 overexpressing mice. (A) Survival curves of mice of the indicated genotypes (wt – wild-type, Mcp1 – overexpressing Mcp1 in cardiomyocytes under the control of the Myh6 promoter; global AOX – expressing AOX constitutively; cardio AOX – expressing AOX specifically in cardiomyocytes, after activation of SNAPf-AOX by Myh6-cre, Myh6-cre – expressing only cre in adult cardiomyocytes, and combinations as shown, n ≥ 10 for all groups except those including Myh6-cre, where n ≥ 5). The survival curve for Mcp1 plus cardiomyocyte-specific AOX was significantly different from that of Mcp1 alone, or together with global AOX (p < 0.0001, Log-rank (Mantel-Cox) or Gehan-Breslow-Wilcoxon test, see Table S2). (B) Systolic and diastolic volume of the cardiac left ventricle (LV) of mice of the indicated genotypes, measured by ultrasound at 12 and 16 weeks, as indicated (means ± SD, n ≥ 5 for all groups). (C) Cardiac ejection fraction (EF, %) computed for individual mice of the indicated genotypes and ages, means ± SD. Horizontal lines marked with asterisks indicate significant differences (one-way ANOVA within an age point, applying Tukey’s post…

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  Figure 4. Modification of gene expression due to Mcp1/AOX expression. qRTPCR analysis of levels of the indicated RNAs in cardiac left ventricle of 12-week old mice of the genotypes shown, in each case normalized to the value for wild-type (means ± SD, n = 5 for each genotype). Horizontal lines marked with asterisks indicate significant differences between wild-type and other groups (one-way ANOVA with Tukey post hoc HSD test; **, ***p < 0.01, 0.001, respectively). For clarity, significant differences between other groups is not shown, but is summarized in full in Table S3.

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  Figure 5. Compromised mitochondrial functions in Mcp1 mice are minimally modified by AOX. Transmission electron micrographs of cardiac left ventricle from 12-week old animals of the indicated genotypes and treatments. Scale bars as indicated: bottom images represent higher magnification of portions of those shown in the top line. The dark inclusions in close proximity to mitochondria, seen in cardiomyocytes from the Mcp1-overexpressing mice, are interpreted as secondary lysosomes. >12 such sections were analysed from mice of each genotype (2–3 sections from each of 2 blocks from 3 individuals). Multiple such inclusions were seen in the sections from Mcp1 mice, but not from controls or from mice expressing global AOX. Note also the less densely packed cristae in mitochondria from the Mcp1-overexressing mice, which was evident in all sections analysed. (B) Respirometry of mitochondrial suspensions from heart tissue of mice of the indicated genotypes, driven by cI- or cII-linked substrates as shown. (C) Representative Western blots of heart protein extracts from 12-week old mice of the indicated genotypes, probed as shown. Note that each lane represents a sample from an individual mouse,…

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  Figure 6. Cardiomyocyte-specific AOX expression alters cardiac metabolism of Mcp1 mice. Principal component analysis of metabolome data (13C-glucose tracer) from cardiac left ventricles of 12-week old mice of the indicated genotypes. The plot is based on 17 glucose metabolites that passed statistical filtering, corrected for low fractional contribution from the labelled glucose.

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  Figure 7. Minimal phenotypic modification by AOX, of Cox10 heart knockout. (A,B) Survival curves of mice of the indicated genotypes: Cox10−/− – cardiomyocyte-specific knockout of Cox10, i.e. mice were Cox10fl/fl plus Myh6-cre+/−; Cox10−/− + cardio AOX – cardiomyocyte-specific knockout of Cox10 plus activation of SNAPf-AOX by Myh6-cre (both heterozygous); Myh6-cre – Myh6-cre+/− only, no cre – control mice lacking Myh6-cre, cardio AOX – cardiomyocyte-specific activation of SNAPf-AOX by Myh6-cre, both hemizygous; Cox10+/− – cardiomyocyte-specific heterozygous knockout of Cox10, i.e. mice were Cox10+/fl and Myh6-cre+/−; Cox10+/− + cardio AOX – cardiomyocyte-specific heterozygous knockout of Cox10, i.e. mice were Cox10+/fl and heterozygous for both Myh6-cre and SNAPf-AOX. n ≥ 6 for all groups. (C) Cardiac ejection fraction (EF, %) computed for individual mice of the indicated genotypes and ages, means ± SD, by echocardiography. Horizontal lines marked with asterisks indicate significant differences (one-way ANOVA within an age point, applying Tukey’s post hoc HSD test; *, **, ***p < 0.05, 0.01, 0.001, respectively).