Pan-tissue mitochondrial respiration atlas overview, work-flow, and analysis pipeline. A.

Study overview highlighting the 33 different tissues collected from four different groups of mice (n = 10 / group) for respirometry analysis and mitochondrial content quantification. The four groups of mice are young male, young female, old male, and old female. Young = 10-week-old; old = 80-week-old. B. General schematic showing the preparation of samples for respirometry analysis. C. General representation of the electron transport chain to illustrate the key components of the respirometry assay used to assess mitochondrial function, the associated data, and examples of subsequent data analysis. AA, Antimycin A; Rot, Rotenone; TMPD, N, N, N’, N’-tetramethyl-p-phenylenediamine; Asc, Ascorbate; NADH, nicotinamide adenine dinucleotide.

Tissue-by-tissue analysis of mitochondrial function in male and female mice. A.

Young male mitochondrial respiration through complex I (CI, NADH-stimulated), complex II (CII, Succinate stimulated in the presence of Rotenone, a CI inhibitor), and at complex IV (CIV, via TMPD + Ascorbate). B. Young female mitochondrial respiration through CI, CII, and at CIV. C. Old male mitochondrial respiration through CI, CII, and at CIV. D. Old female respiration through CI, CII, and at CIV, respectively. n = 10 mice per tissue. All oxygen consumption rates (OCR) are normalized to mitochondrial content (based on MTDR). All data is represented as the mean with standard error, and organized highest (left) to lowest (right). Young = 10 weeks; Old = 80 weeks. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles.

Tissue-by-tissue analysis of young male and female mitochondrial function. A.

(left) Systems-level view of mitochondrial respiration (NADH-stimulated) through complex I (CI). (right) Mitochondrial respiration through CI across all young tissues. B. (left) Systems-level view of mitochondrial respiration through complex II (CII, Succinate-stimulated in the presence of rotenone to inhibit CI). (right) Mitochondrial respiration through CII across all tissues. C. (left) Systems-level view of mitochondrial respiration at complex IV (CIV) in the presence of rotenone and antimycin A to inhibit CI and CIII, respectively. (right) Mitochondrial respiration at CIV across all tissues. All data is presented as the mean with standard error, and organized highest to lowest for young male values. D. Heat map view of mitochondrial function across all tissues assayed (omitting reproductive organs). Data is represented as young male/female. Tissues with elevated respiration in males appear red while the same for females appear blue. Data is organized highest to lowest by summation of young male CI, CII, and CIV respiration values. n = 10 young male (YM, 10 weeks) and 10 young female (YF, 10 weeks) per tissue assayed. All oxygen consumption rates (OCR) are normalized to mitochondrial content (based on MTDR). Statistical significance is represented as: a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles.

Tissue-by-tissue analysis of young and old male mitochondrial function. A.

(left) Systems- level view of mitochondrial respiration (NADH-stimulated) through complex I (CI). (right) Mitochondrial respiration through CI across all male tissues. B. (left) Systems-level view of mitochondrial respiration (Succinate-stimulated in the presence of rotenone to inhibit CI) through complex II (CII). (right) Mitochondrial respiration through CII across all male tissues. C. (left) Systems-level view of mitochondrial respiration at complex IV (CIV) in the presence of rotenone and antimycin A to inhibit CI and CIII, respectively. (right) Mitochondrial respiration at CIV across all male tissues. All data is presented as the mean with standard error and organized highest to lowest for old male values. D. Heat map view of mitochondrial function across all male tissues assayed. Data is presented as old/young male. Tissues with elevated respiration in old males appear red while the same for young males appear blue. Data is organized highest to lowest by summation of old male CI, CII, and CIV respiration values. n = 10 old male (OM, 80 weeks) and 10 young male (YM, 10 weeks) per tissue assayed. All oxygen consumption rates (OCR) are normalized to mitochondrial content (based on MTDR). Statistical significance is represented as, a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles.

Tissue-by-tissue analysis of young and old female mitochondrial function. A.

(left) Systems-level view of mitochondrial respiration (NADH-stimulated) through complex I (CI). (right) Mitochondrial respiration through CI across all female tissues. B. (left) Systems-level view of mitochondrial respiration (Succinate-stimulated in the presence of rotenone to inhibit CI) through complex II (CII). (right) Mitochondrial respiration through CII across all female tissues. C. (left) Systems-level view of mitochondrial respiration at complex IV (CIV) in the presence of rotenone and antimycin A to inhibit CI and CIII, respectively. (right) Mitochondrial respiration at CIV across all female tissues. All data is organized highest to lowest for old female values. D. Heat map view of mitochondrial function across all female tissues assayed. Data is presented as old/young female, so tissues with elevated mitochondrial respiration in old females appear red while the same for young females appear blue. Data is organized highest to lowest by summation of old female CI, CII, and CIV respiration values. n = 10 old female (OF, 80 weeks) and 10 young female (YF, 10 weeks) per tissue assayed. All oxygen consumption ratea (OCR) are normalized to mitochondrial content (based on MTDR). Statistical significance is represented as, a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles.

Tissue-by-tissue analysis of old male and female mitochondrial function. A.

(left) Systems- level view of mitochondrial respiration (NADH-stimulated) through complex I (CI). (right) Mitochondrial respiration through CI across all old tissues. B. (left) Systems-level view of mitochondrial respiration (Succinate-stimulated in the presence of rotenone to inhibit CI) through complex II (CII). (right) Mitochondrial respiration through CII across all tissues. C. (left) Systems level view of mitochondrial respiration at complex IV (CIV) in the presence of rotenone and antimycin A to inhibit CI and CIII, respectively. (right) Mitochondrial respiration at CIV across all tissues. All data is presented as the mean with standard error and organized highest to lowest for male values. D. Heat map view of mitochondrial function across all tissues assayed. Data is represented as old male/female, so tissues with elevated mitochondrial respiration in males appear red while the same for females appear blue. Data is organized highest to lowest by summation of male CI, CII, and CIV respiration values. n = 10 old male (OM, 80 weeks) and 10 old female (OF, 80 weeks) per tissue assayed. All oxygen consumption rates (OCR) are normalized to mitochondrial content (based on MTDR). Statistical significance is represented as, a = p < 0.05, b = p < 0.01, c = p < 0.001, and d = p < 0.0001. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; Vent, ventricles.

Mitochondrial respiration is affected by sex but dominated by age. A.

Total number of statistically significant differences across all tissue-by-tissue comparisons, grouped by the component measured (CI = respiration through complex I, CII = respiration through complex II, or CIV = respiration through complex IV). B. Total number of significant differences across tissue-by-tissue comparisons grouped by the specific comparison, colored to represent the mitochondrial component in which respiration began (CI, CII, or CIV), and summed to the right of each histogram to highlight the number of significant findings per comparison. To underscore the number of statistically significant sex- or age- associated differences, the total number of significant findings from YM-by-YF and OM-by-OF (sex effect), and OM-by-YM and OF-by-YF (age effect) were summed. C. Quantification of the total number of tissues affected in each tissue-by-tissue comparison. Data are colored based on the specific comparison and grouped as sex- or age-associated to illustrate the effect-type. D. Cumulative absolute difference of means for CI, CII, and CIV (from left to right) grouped by effect-type, sex (originating from YM-by-YF or OM-by-OF) or age (originating from OM-by-YM or OF-by-YF). These graphs do not indicate directionality of the change, only the absolute cumulative magnitude. Each box within the histogram represents a unique tissue. All histograms are organized lowest (left) to highest (right) in degree of tissue contribution to total change given the comparison type. The top contributors to sex- or age-associated changes are highlighted with non-greyscale colors and their relative percentage of contribution to the total cumulative difference is provided. E. Principal Component Analysis (PCA) of all tissues and groups for CI, CII, and CIV (from left to right). F. Pearson correlation heat maps of all tissues combined from young and old, male and female mice. From top to bottom and left to right the samples are organized by group as follows: YF, YM, OF, and OM (n = 10 mice per group). YM = young male, YF = young female, OM = old male, OF = old female. Young = 10 weeks, Old = 80 weeks. BAT, brown adipose tissue; gWAT, gonadal white adipose tissue; iWAT, inguinal white adipose tissue; Quad, quadriceps muscles; At, atria; Vent, ventricles.

The effects of age on mitochondrial respiration occur in sex-specific ways. A-B.

Systems- level view of the total relative change (sum of all relative changes) present at each mitochondrial parameter (respiration via CI, CII, or CIV) for males (M) and females (F), respectively. All change is old (O) compared to young (Y). C. Heat maps of O / Y data for each mitochondrial parameter per tissue across lifespan. Male and female values are grouped and organized by greatest (top) to least (bottom) sum of relative change per tissue. Tissues with the largest positive relative change as a result of aging are located on the top-most region of the heat maps, while those with the largest negative relative change are at the bottom. The middle of each heat map represents tissues with little relative response to aging or with opposite effect directions across sex. D. Graphs showing magnitude of sex-effect with age across all tissues and mitochondrial parameters (CI, CII, or CIV). The blue line and circles represent log2(old male/young male) data and the red line and boxes represent log2(old female/young female) data; both linked to the left y-axis. The black line and triangles represent the absolute difference of male and female relative change data (linked to the right y-axis). All data is organized from left to right by highest to lowest absolute difference of relative change across sex. Tissues with the greatest relative difference across sex are located to the left-most region of the graph, while those with the most similar response to aging are located to the right. A hollow blue circle, red circle, and black triangle represent a tissue with a divergent sex-specific age response. E. Summary diagrams classifying the relative trends of each mitochondrial parameter assayed per tissue as age- or age-and-sex-specific. Age-specific effects have a shared relative change direction across sex. Tissues in the red “M + F Increased” box have a positive relative change for both males and females. Tissues in the blue “M + F Decreased” box have a negative relative change for both males and females. Tissues in the Purple box labeled “Divergent” have opposing log2(O/Y) directions (signs, + or -) for male and female values; these tissues display relative mitochondrial changes that are age-and-sex-specific. F. Summary diagram showing tissues with a shared increase (red box) or decrease (blue box) consistent across all complexes (CI, CII, and CIV). Tissues within the shared increase or decrease classifications are grouped by their tissue-type, for example. PL, DI, SL, and HV are all muscle, CO, HI, HY, and CE are all brain and so on. CI = respiration measured through complex I stimulated by NADH; CII = respiration through complex II stimulated by succinate in the presence of rotenone; CIV = respiration at complex IV stimulated by TMPD and ascorbate in the presence of rotenone and antimycin A. Tissues: BT = brown adipose tissue, CC = cecum, CE = cerebellum, CO =brain cortex, DI = diaphragm, DC = distal colon, DU = duodenum, EY = eyes, GS = gastrocnemius, GW = gonadal white adipose tissue, HS = hamstring, HA = heart atria, HV = heart ventricles, HI = hippocampus, HY = hypothalamus, IL = ileum, IW = inguinal white adipose tissue, JE = jejunum, KC = kidney cortex, KM = kidney medulla, LV = liver, LN = lung, MW = mesenteric white adipose tissue, PN = pancreas, PL = plantaris, PC = proximal colon, QD = quadriceps, SK = skin, SL = soleus, SP = spleen, ST = stomach, TN = tongue. Reproductive organs are omitted from cross-sex analysis.

Assay parameters for Seahorse-based respirometry analysis across all tissues.

Table shows tissues used, approximate size per tissue homogenized for analysis, amount of 1X MAS buffer used for homogenization, and the amount of protein used for respiration analysis across all tissues.