Dysfunctional Mitochondria in the Cardiac Fibers of a Williams–Beuren Syndrome Mouse Model

Figures

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  Figure 1. CD mice cardiac fibers exhibit reduced oxygen consumption and ATP production (A). Mitochondrial respiration was measured in permeabilized cardiac fibers (n = 5–6). Oxygen consumption was measured under various mitochondrial states and normalized to fiber weight: LEAK conditions (malate, pyruvate, and glutamate as substrates); OXPHOS (routine respiration rate) through complex I, I + II, II, or IV); (B) respiratory control ratio (RCR, measure of mitochondrial efficiency). (C) ATP production in primary CD and WT MEFs. The cells were seeded and analyzed with the Luciferase assay. ATP levels were normalized to their respective cell numbers. n = 3 experiments with three replicates each. The data were analyzed using a multiple (A) or unpaired t-test (B,C) and are represented by the mean ± SD; statistical significance was set at a threshold of * p < 0.05; ** p < 0.01; *** p < 0.001.

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  Figure 2. CD mice show reduced levels of OXPHOS protein complexes. The mitochondrial copy number was measured relative to the genomic copy number in cardiac tissue (A) and MEFs (B). (C) Representative Western blot of mitochondrial OXPHOS respiratory complex protein levels. TOM20 was used as a loading control. An antibody cocktail comprising the following subunits of respiratory complex proteins was used: NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8 (NDUFB8; complex I), succinate dehydrogenase complex, subunit B, iron sulfur (SDHB/Ip; complex II), ubiquinol-cytochrome c reductase core protein II (UQCR2; complex III), ATP synthase 5A (ATP 5A, Complex V), and cytochrome c oxidase subunit 2 (COXII; complex IV). (D) Quantitative analysis of the protein levels of each of the aforementioned subunits, respectively. The intensity of each protein was normalized to TOM20 in the same sample. The data were analyzed using an unpaired (A,B) or multiple comparisons (D) t-test following the Holm–Šídák method and are represented by mean ± SD; n= 4–6 mice per group. The cells were analyzed in n = 3 experiments with three replicates each. Statistical significance was set at a threshold of * p < 0.05;…

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  Figure 3. The mitochondrial structure and dynamics were altered in the CD cardiac tissue. (A) Representative TEM images of mitochondria distribution along cardiac fibers. Scale bars = 2 μm (top) and 0.1 μm (bottom), respectively. (B) (Top), representative confocal images of mitochondria distribution in MEFs (scale bars = 10 μm) showing increased mitochondrial density with a reduced average size and increased circularity (bottom). The value of circularity ranges between 0 and 1, with the less functional mitochondria closer to 1. The cells were analyzed in n = 4 experiments with three replicates each. The data were analyzed using an unpaired t-test and are represented by the mean ± SD. Statistical significance was set at a threshold of * p < 0.05.

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  Figure 4. Reduced expression levels of proteins controlling mitochondrial dynamics. (A) Quantification (right) and representative Western blot (left) of OPA 1; (B) fission protein FIS1 and fusion proteins, (C) MFN1, and (D) MFN2. GAPDH was used as a loading control. The data were analyzed using multiple comparisons (A) or an unpaired (B–D) t-test following the Holm–Šídák method and represented by the mean ± SD; n = 4–5 mice per group. The cells were analyzed in n = 4–5 mice/genotype. Statistical significance was set at a threshold of ** p < 0.01.