Double Deletion of Angiotensin II Type 2 and Mas Receptors Accelerates Aging‐Related Muscle Weakness in Male Mice

Figures

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  Figure 1. Schematic overview and aging‐induced changes in body weight and grip strength of mice. A, Schematic overview of the experimental protocol. Body weight (B) and grip strength (C) in mice during the experimental period. The numbers of WT, AT2 KO, and AT2/Mas KO mice were 5, 8, and 6, respectively. (Several mice died before the final excision of tissues.) Difference was analyzed with 1‐way ANOVA, using a Bonferroni post hoc test. *P<0.05, WT vs AT2/Mas KO. † P<0.05, AT2 KO vs AT2/Mas KO. ACE2 indicates angiotensin‐converting enzyme 2; AT1, angiotensin II type 1 receptor; AT2, angiotensin II type 2 receptor; AT2 KO, AT2 knockout; AT2/Mas KO, AT2/Mas double knockout; RT‐qPCR, quantitative real‐time polymerase chain reaction; n.s., not significant and WT, wild‐type.

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  Figure 2. Histological analysis of the vastus muscle in mice. A, Hematoxylin and eosin (H&E)‐stained sections. Representative H&E‐stained sections of the vastus muscle in each group of mice. B, Average value and distribution of cross‐sectional area (CSA). The frequency of skeletal muscles was adjusted as a percentage of the total number of fibers and displayed as a histogram. C, Approximate number of muscle fibers in the vastus muscle, as estimated by dividing the muscle area by the CSA of a single muscle fiber in each mouse. D, Central nucleated fibers expressed as a percentage of total myofibers. E, Leucocyte infiltration determined as infiltrated nucleus expressed as a percentage of myofibers. *P<0.05. AT2 indicates angiotensin II type 2 receptor; AT2 KO, AT2 knockout; AT2/Mas KO, AT2/Mas double knockout; n.s., not significant; and WT, wild‐type.

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  Figure 3. Histological analysis of the vastus muscle in mice. A, Masson's trichrome–stained sections. Representative Masson's trichrome–stained sections of the vastus muscle in each group of mice. Percentage of fibrosis area (B) and number of vessels (C) in muscle fibers. Differences were analyzed with 1‐way ANOVA using a Bonferroni post hoc test. Circles indicate the centrally located nuclei. Arrows indicate the infiltrated leucocytes. Scale bars: 200 μm. AT2 indicates angiotensin II type 2 receptor; AT2 KO, AT2 knockout; AT2/Mas KO, AT2/Mas double knockout; n.s., not significant; and WT, wild‐type.

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  Figure 4. Quantitative real‐time polymerase chain reaction analysis of age‐induced changes in genes associated with the renin‐angiotensin system (RAS), aging, inflammation, mitochondrial function, and fibrosis in the vastus muscle in mice. Quantitative estimation of the expression levels of genes associated with RAS (A), aging and proinflammatory senescence‐associated secretory phenotype (B), and muscle‐specific gene markers of senescence, mitochondrial function, and fibrosis (C), relative to GAPDH expression in the vastus muscle. Differences were analyzed with 1‐way ANOVA using the Bonferroni post hoc test. No significant difference was observed among the groups unless otherwise indicated. *P<0.05. AT2 indicates angiotensin II type 2 receptor; AT2 KO, AT2 knockout; AT2/Mas KO, AT2/Mas double knockout; and WT, wild‐type.

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  Figure 5. Angiotensin II content in the gastrocnemius muscle. Angiotensin II content was adjusted by protein concentration of tissue lysate. Differences were analyzed with 1‐way ANOVA using the Bonferroni post hoc test. *P<0.05. AT2 indicates angiotensin II type 2 receptor; AT2 KO, AT2 knockout; AT2/Mas KO, AT2/Mas double knockout; and WT, wild‐type.