Systemic AV.RSV.MCAT delivery significantly enhances treadmill performance in both male and female mice.

<p>A, results from male mice; B, results from female mice. Right panel, absolute running distance; Middle panel, body weight; Left panel, body weight-normalized running distance. Asterisk, significantly different from that of the uninfected group (p<0.04); Double asterisks, significantly higher than that of the uninfected group (p<0.001).</p

Characterization of the EDL muscle.

<p>Characterization of the EDL muscle.</p

The contractility of the isolated EDL muscle is not altered following systemic AV.RSV.MCAT infection.

<p>A to C, results from male mice; D to F, results from female mice. A and D, absolute twitch (left panel) and tetanic forces (right panels). B and E, cross-sectional area normalized specific twitch (left panel) and tetanic forces (right panels). C and F, fatigue response (left panel) and eccentric contraction profile (right panel). Filled bar/circle, AV.RSV.MCAT infected mice; Open bar/circle, uninfected mice.</p

Characterization of AV.RSV.MCAT vector.

<p>A, Schematic outline of the AAV vector. The flanking hairpin structures denote AAV inverted terminal repeats. RSV, Rose sarcoma virus promoter; OTC, mitochondrial targeting sequence from the ornithine transcarbamylase gene; pA, polyadenylation signal from SV40 virus. Not drawn to scale. B, Determination of catalase expression from AAV infected mice by whole muscle lysate western blot and the in-gel zymography assay. Photomicrographs are the representative results from three independent experiments. Uninf., mice not infected by AV.RSV.MCAT. MCAT, mice received systemic AV. RSV.MCAT infection. Dotted lines, images were spliced together from the same gel but were run on noncontiguous lanes. Arrow, endogenous murine catalase; Arrowhead, mitochondrial expressed human catalase from AAV vector. TA, tibialis anterior muscle; Gastro, gastrocnemius muscle, EDL, extensor digitorium longus muscle. C, Catalase activity in whole muscle lysate. Asterisk, significantly higher than that of the uninfected group (p<0.05); Double asterisks, significantly higher than that of the uninfected group (p<0.005). D, Western blot analysis of mitochondrial and whole muscle lysate preparations from the gastrocnemius muscle. Prohibitin is a mitochondria marker. Uninf., mice not infected by AV.RSV.MCAT. MCAT, mice received systemic AV. RSV.MCAT infection.</p

Systemic AV.RSV.MCAT infection leads to mosaic catalase expression in the mitochondria in striated muscles.

<p>A, Representative catalase immunofluorescence staining photomicrographs of AV.RSV.MCAT infected and uninfected skeletal muscle (top panels) and heart (bottom panels). Arrow, AAV transduced skeletal muscle myofiber. B, Representative double immunofluorescence staining photomicrographs of AV.RSV.MCAT infected heart. Bottom panels are high magnification images of the boxed region in respective top panels. Cytochrome C marks mitochondria (red color). Catalase is in green color. Yellow color in merged images reveals mitochondrial catalase expression.</p

A. Disruption of the RIIβ subunit of Protein Kinase A increases lifespan in male mice on the C57BL/6 background.

<p>A. Both the median and maximum lifespans were significantly increased in mutant males compared to WT littermates. The median lifespan was increased from 884 days to 1005 days (p = 0.006 as determined by the log rank test) and the maximum lifespan (defined here as 80% deaths) was increased from 941 days to 1073 days (p = 0.004 as determined by the Wang-Allison test). B. There was no difference in either median or maximum lifespan in female genotypes.</p

Old RIIβ^−/− male mice have attenuated age-related fatty livers.

<p>A. Old (18 month old) WT mice developed livers that were twice the size of, and paler in color than livers from young mice. B,C. Livers from 18 month-old RIIβ^−/− littermates were smaller, darker in color, and 25% lower in weight than those from their WT littermates. N = 5 (WT) and 6 (RIIβ^−/−). D. Liver weight correlated directly with % fat content (R² = 0.7064) N = 4 (WT) and 6 (RIIβ^−/−). For both C and D: Numbers represent means. Error bars represent standard deviations. P's determined by Student's t-test.</p

Both young and old RIIβ^−/− male mice are insulin sensitive compared to WT, and RIIβ^−/− males are resistant to age-induced hyperinsulinemia.

<p>A. RIIβ^−/− blood glucose levels were slightly lower than WT for both old males and females, but both genotypes of both genders fell into an acceptable range. N = 15. B. Male WT mice experienced over 4-fold increases in serum insulin levels with age, while <i>RIIβ^−/−</i> littermates maintained low serum insulin levels between 2 and 16 months of age. 2 month-old mice: N = 5 (WT) and 4 (RIIβ^−/−). 16 month-old mice: N = 8 (WT) and 9 (RIIβ^−/−). C,D. Blood glucose levels of both 2 to 5 month (C) and 18 month (D) RIIβ^−/− males dropped more rapidly and further in response to an inter-peritoneal insulin injection. N = 6; points represent means. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005963#s2" target="_blank">Results</a> were standardized by setting initial blood glucose levels at 100%. Data for all figures: Numbers represent means, and error bars represent standard deviations. **P<0.001; *P<0.05 and b represents borderline significance (as determined by Student's t-test).</p

Old RIIβ^−/− male mice have attenuated cardiac dysfunction.

<p>A. RIIβ^−/− mice were significantly protected from age-related LV hypertrophy, shown by lower LV mass normalized to tibia length in 24 month old WT and RIIβ^−/− mice. B. These RIIβ^−/− mice also had superior Ea/Aa ratios and myocardial performance indices (MPI) compared to WT littermates. N = 6. Numbers represent means. Error bars represent standard deviations. P's determined by Student's t-test.</p

Adiposity correlates with lifespan for males only.

<p>A, C. When lifespan of WT mice was plotted against their maximal body weight, a correlation was found for males, but not females (N = 13 and 15, and R² = 0.4795 and 0.0369, respectively). Males with the longest lifespans (over 1000 days) had body weights between 30 and 35 g. B. Most (82%) of the RIIβ^−/− male mice examined had body weights between 30 and 35 g. 50% of these mice lived over 1000 days, and the 3 mice that fell outside of this weight range had shortened lifespans of under 700 days (Total N = 17). D. Of the 20 female RIIβ^−/− mice measured, only 4 lived to 1000 days or higher. All 5 mice with maximal body weights below 24 g had shortened lifespans equal to or under 800 days, as did the one mouse above 35 g, but lifespans for mice falling between these two body weights were variable.</p