Comparative phenotyping of C57BL/6J substrains reveals distinctive patterns of cardiac aging

Figures

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  Fig. 1. Cardiac aging is associated with structural cardiac changes in two B6J substrains. a, b Body weight (BW) and heart weight per BW were measured in young (5 months) and old (24 months) mice (n = 9–27). c Collagen was stained by picrosirius red and representative pictures of the total heart cross section and cardiac vessels are shown. d, e Collagen area was divided by total area to quantify total collagen and perivascular collagen area was quantified (n = 6–10). f, g CD45 was stained in heart tissue samples. The number of CD45-positive cells was calculated by dividing the CD45-positive nuclei by the total amount of nuclei indicated by DAPI (n = 5–6). Data represent mean ± SD. Statistical significance was tested with Student’s t-test (normality passed) or Mann–Whitney U test (normality not passed) and p-values are shown. B6, C57BL/6

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  Fig. 2. Age-associated decline in heart function is more pronounced in B6JRj than in B6JCrl mice. a, b, c, d, e Left ventricular (LV) mass per body weight (BW), stroke volume, cardiac output, and ejection fraction were analyzed by echocardiography in young (5 months) and old (24 months) anesthetized mice (n = 4–10). f, g Plasma norepinephrine and n-terminal pro b-type natriuretic peptide (NT-proBNP) were determined using commercial-available ELISAs (n = 4–8). Statistical significance was tested with unpaired t-test (normality passed) or Mann–Whitney U test (normality not passed) and p-values are shown. B6, C57BL/6

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  Fig. 3. Proteomics revealed age-related differences in the cardiac oxidative phosphorylation pathway between B6JRj and B6JCrl mice. a, b Gene-set enrichment analysis (GSEA) was performed with heart proteomics data. Only cardiac relevant pathways with a q-value lower than 0.25 are presented (n = 3–4). a Young B6JRj mice are compared to old B6JRj mice. b Young B6JCrl mice are compared to old B6JCrl mice. c, d Western blot was done using OXPHOS antibody cocktail to stain for mitochondrial complex I-V, signal was normalized to total lane protein using Ponceau S staining. Differences in protein levels are demonstrated as changes compared to the young group (n = 7–9). Statistical significance was tested with unpaired t-test (normality passed) or Mann–Whitney U test (normality not passed) and p-values are shown. B6, C57BL/6; C, complex. Max, maximal; O, old; Y, young OXPHOS, oxidative phosphorylation

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  Fig. 4. Random plasma metabolites show slight changes during aging in both substrains. a, b, c, d Glucose, insulin, non-esterified fatty acids (NEFAs), and triglycerides were measured in plasma samples of young and old mice from both substrains (n = 8–12). Statistical significance was tested with unpaired t-test (normality passed) or Mann–Whitney U test (normality not passed) and p-values are shown. B6, C57BL/6

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  Fig. 5. Cardiomyocyte contraction was impaired during aging in B6JRj but not in B6JCrl mice. a, b, c, d, e Cardiomyocyte function was analyzed directly after cardiomyocyte isolation using the IonOptix system. Contraction was measured during pacing from at least 10 cells per sample with at least 10 transients per cell (n = 5–9). f Results are presented in a scheme showing the changes in sarcomere length per time. Statistical significance was tested with unpaired t-test (normality passed) or Mann–Whitney U test (normality not passed) and p-values are shown. B6, C57BL/6; V, velocity; T, time