Computational and digital analyses in the INSPIRE mouse cohort to define sex-specific functional determinants of biological aging

Abstract

Biological age, which reflects the physiological state of an individual, offers a better predictive value than chronological age for age-related diseases and mortality. Nonetheless, determining accurate functional features of biological age remains challenging due to the multifactorial nature of aging. Here, we established a unique mouse cohort comprising 1576 male and female outbred SWISS mice subjected or not to high-fat, high-sucrose diet to investigate multiorgan/system biological aging throughout adulthood. Comprehensive functional and biological phenotyping at ages of 6, 12, 18, and 24 months revealed notable sex-specific disparities in longitudinal locomotion patterns and multifunctional aging parameters. Topological data analysis enabled the identification of functionally similar mouse clusters irrespective of chronological age. Moreover, our study pinpointed critical functional markers of biological aging such as muscle function, anxiety characteristics, urinary patterns, reticulocyte maturation, cardiac remodeling and function, and metabolic alterations, underscoring muscle function as an early indicator of biological age in male mice.

Fig. 1.. Outline of experimental design.

(A) Schematic overview of mouse features studied in the article. Cross-sectional cohorts of male and female outbred SWISS mice were subjected or not to HFHS diet at 6 months, and comprehensively characterized at 6, 12, 18, or 24 months, before euthanasia. (B) Experimental workflow used to define multifunctional aging trajectories and biological aging parameters. (C) Functional and biological features measured and categorized into eight specific domains, including anxiety, cognitive, cardiac, metabolic, physical performance, urinary, immune, and hematological. Created with BioRender.com. HFHS, high-fat, high-sucrose; mo, months old.

Fig. 2.. Longitudinal home-cage monitoring highlights aging-related progressive decline of locomotion with sex nuances in outbred SWISS mice.

(A) Schematic representation of home-cage monitoring of spontaneous and voluntary activity in SWISS mice. Data were automatically recorded through the DVC system (Tecniplast SpA) from 6 to 24 months. Spontaneous mobility data were collected from an electronic board underneath each cage measuring mouse capacitance. Running wheels were used as an indicator of voluntary activity. Created with Biorender.com. (B) Heatmaps of spontaneous locomotor activity and (C) corresponding quantification of animal locomotion index of male and female SWISS mice between 6 and 24 months (n = 16 to 17 cages, corresponding to 64 to 68 mice). (D to F) Overall animal locomotion index of male and female SWISS mice (n = 16 to 17 cages, corresponding to 64 to 68 mice). (D) Between 6 and 24 months; (E) between 6 and 18 months; and (F) between 18 and 24 months. (G) Heatmaps of voluntary activity and (H) corresponding quantification of running wheel distance of male and female SWISS mice between 6 and 24 months (n = 17 cages, corresponding to 68 mice). (I to K) Overall running wheel distance of male and female SWISS mice (n = 17 cages, corresponding to 68 mice). (I) Between 6 and 24 months; (J) between 6 and 18 months; and (K) between 18 and 24 months. Differences were considered significant when P < 0.05. N, night; D, day; F, females; M, males.

Fig. 3.. Assessment of multiorgan trajectories underlines sex-specific traits during aging.

(A) Schematic representation and (B) functional parameters used for the determination of physical performance in cross-sectional cohorts of naturally aging mice at 6, 12, 18, and 24 months. (C to J) Trajectories of parameters in male and female naturally aging mice at 6, 12, 18, and 24 months including (C) muscular function indicated by Net running time on a treadmill (s); (D) anxiety indicated by periphery-to-center ratio in an open field (%); (E) memory function indicated by the percentage of alternation in a Y-maze (%); (F) metabolic function indicated by body weight (g); (G) urinary function indicated by the total number of urine spots on an absorbent paper; (H) cardiac function indicated by E/A ratio as an index of diastolic function; (I) immune function indicated by lymphocyte proportions (%); and (J) hematological function indicated by the count of red blood cells (M/μl). For all graphs, n = 26 to 42 per group for males and n = 32 to 42 per group for females. Differences were considered significant when P < 0.05. BW, body weight; g, grams; s, seconds.

Fig. 4.. HFHS-induced obesity accentuates aging-related multiple dysfunctions with sex specificities.

(A and B) Heatmaps of spontaneous locomotor activity and corresponding quantification of animal locomotion index of (A) male and (B) female SWISS mice subjected or not to HFHS diet between 6 and 24 months (n = 17 cages corresponding to 68 mice for CT group and n = 27 cages for HFHS group corresponding to 108 mice). (C to F) Trajectories of parameters in male and female mice naturally aging or subjected to HFHS at 6, 12, 18, and 24 months including (C) muscular function indicated by running distance on a treadmill (m); (D) cardiac remodeling indicated by heart weight (mg); (E) hematological function indicated by immature reticulocyte fraction (%); (F) immune function indicated by neutrophils count (K/μl). For all graphs, n = 26 to 42 per group for males and n = 32 to 42 per group for females. Differences were considered significant when P < 0.05. HFHS, High fat high sucrose; F, females; M, males.

Fig. 5.. TDA of multiorgan/system parameters identifies distinct phenoclusters of mice that differentially correlate with physical performance.

(A) Representation of the phenoclusters identified from the overall mouse population (including CT- and HFHS-subjected 6-, 12-, 18-, and 24-month-old mice). (B) Illustration of mouse distribution in phenoclusters based on chronological age. (C) Representation of the phenoclusters identified from mice stratified by each group of age, i.e., 6-, 12-, 18-, and 24-month-old. (C) Illustration of mouse distribution in phenoclusters based on chronological age. (D) Left panel: features, parameters and thresholds used to evaluate physical performance in mice; right panel: physical performance score determination based on the number of failed tests. (E) Illustration of mouse distribution in phenoclusters based on physical performance score Differences were considered significant when P < 0.05.

Fig. 6.. Refined analysis identified markers of biological aging.

(A) Analysis of 18-month-old males showing parameters affected by HFHS regardless of aging (in blue) and features of biological aging (in gray). (B) Analysis of 18-month-old females showing parameters affected by HFHS regardless of aging (in blue) and features of biological aging (in gray). (C) Analysis of 12-month-old males showing parameters affected by HFHS regardless of aging (in blue) and features of biological aging (in gray).