Electron Microscopy and Multi‐Omics Reveal Mitochondrial Dysfunction and Structural Remodeling in the Hearts of Elderly Mice

Figures

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  FIGURE 1. (A–E) (A) The Clinical Frailty Index (CFI) shows a progressive and statistically significant increase with age, with a highly significant trend (p for trend < 0.0001). Multiple comparisons indicate significant differences between all groups (Young, Adult, Middle Age, Elderly), ****p < 0.0001. The Young group is shown for reference, to illustrate that values between Young and Adult mice are comparable and that no significant differences are present at this early stage. This highlights that the major changes emerge only at later ages, whereas the transition from Young to Adult remains stable. (B) Ejection Fraction (EF) was also unchanged among groups, confirming the absence of systolic dysfunction associated with aging. (C) Fractional Shortening (FS) did not differ across age groups, indicating preserved left ventricular systolic function. (D) E/A ratio Across multiple tests (Welch t‐test, Student t‐test, Mann–Whitney U, permutation test, log‐transformed Welch, and winsorized Welch), the obtained results showed lower E/A in 30 m mice. p‐values ranged from 0.009 to 0.020, reaching statistical significance in all but the Kolmogorov–Smirnov distributional test. Effect sizes were large…

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  FIGURE 2. (A–D) Transmission electron microscopy (TEM) images illustrate mitochondrial ultrastructure in cardiomyocytes from adult (12‐month‐adult; A and C panels) and elderly mice (30‐month‐old; B and D panels). In adult hearts, mitochondria exhibit dense, well‐organized cristae and preserved matrix architecture. In contrast, aged cardiomyocytes display marked mitochondrial remodeling, with evident swelling, disrupted cristae morphology and reduced cristae density (highlighted by yellow arrows panel D) Scale bars = 2 μm.

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  FIGURE 3. (A–D) Transmission electron microscopy (TEM) images comparing subcellular morphology in the hearts of adult (12 months; panels A and C) and elderly mice (30 months; panels B and D). (A, C) In the adult heart, lipid droplets (LDs) with canonical morphology (yellow arrows) are regularly distributed among the myofibrils. Mitochondria display normal morphology with well‐organized cristae. (B, D) In the elderly heart, normal lipid droplets become less frequent and are replaced by numerous concentric lamellar whorls (LWs; yellow arrows), indicative of depleted lipid content. Scale bars: 2 μm (A, B); 200 nm (C, D).

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  FIGURE 4. (A–E) (A) Schematic flowchart illustrating the workflow from left ventricle isolation followed by single‐cell RNA sequencing (scRNA‐seq) to assess transcriptomic changes. (B) UMAP (Uniform Manifold Approximation and Projection) visualization of the main cell types identified across in left ventricle slices of adult, middle age and elderly mice. Cells were clustered into 8 distinct subtypes, shown here with different colors. (C) Bar graph shows relative percentage of major cell types. (D) DEG heatmap showing relative trend of statistically significant genes. (E) Ingenuity Pathway Analysis using as background the terms “aging” and “heart with main terms highlighted and gene relative trend.

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  FIGURE 5. (A–E) Hierarchical clustering showing statistically significant proteins from ANOVA, (A) STRING network of downregulated proteins, (B), with corresponding enrichment (C), and STRING network of upregulated proteins, (D), with enrichment of main terms, (E).

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  FIGURE 6. (A–D) (A) Hierarchical clustering showing the top 90 statistically significant metabolites from ANOVA expression across the aged heart; (B) SMPDB enrichment; (C) main lipid subclasses remodeling across the aged heart Ceramides (Cer), Diacylglycerols (DG), Cholesterol Esters (CE), (D) characteristics of double bonds and chain length for Cer and DG. Low: < 20 carbons, Medium: 20 < 40 carbons, High: > 40 carbons.