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Background Structural and functional mitral valve abnormalities are commonly encountered in the general population. In order to determine which functional protein classes may be important to normal valve function we performed the first reported comprehensive proteomic profiling of structurally normal human mitral valves. Methods We harvested mitral valve leaflets from 5 patients who underwent orthotopic cardiac transplantation for cardiomyopathy. The morphologic appearance of mitral valves was normal with only mild functional mitral regurgitation on echocardiogram. High resolution 2D gel electrophoresis was performed to separate proteins. Gel spots were excised from 2 representative samples and protein identification was carried out using liquid chromatography tandem mass spectrometry. Results We identified 190 unique proteins with >99% probability and at least 2 unique peptides. The proteins were from various cellular locations, including cell surface, cytoskeleton, endoplasmic reticulum, extracellular matrix, mitochondrion, nucleus, cytoplasm, and plasma membrane. Functional clustering revealed that the identified proteins were involved in major molecular pathways including, energy along with fatty acid metabolism, calcium signaling acute phase response & oxidative stress, and anti‐apoptosis pathways. Conclusion Proteomic profiling of the normal human mitral valve demonstrates a pattern of expression suggesting multiple biological functions. Further quantification of protein levels and comparison with diseased mitral valves will provide new insights into the etiology and therapy of mitral valve disease in humans.

Leucine (Leu) is the most potent branched‐chain amino acid that selectively enhances protein synthesis via intracellular signaling pathways. Since protein synthesis is an energy consuming process, we hypothesized that Leu may enhance mitochondrial biogenesis and functions. We determined the effect of Leu at mitochondrial mRNA levels, enzyme activities, and ATP production rate (MAPR) in cultured human liver cell line THLE‐3. Cells were pre‐incubated in a low‐Leu (0.1mM), serum‐free media for 24 hours and gene transcript profiles were measured using Affymetrix HG‐U133A GeneChips at 0, 30, 60, 120 and 480 minutes after increasing the Leu concentrations from to 2.0 mM. Thirty mitochondrial gene transcripts, both mitochondrial DNA and nuclear‐encoded, were significantly increased at various time points with treatment of higher concentrations of Leu. The mRNA levels of 6 mitochondrial genes including two ATP synthase, cytochrome P450, cytochrome C oxidase III, and NADH dehydrogenase 4 were confirmed by real‐time PCR measurements. Western blot analysis showed that 8 hours of high concentrations of Leu increased protein levels of key mitochondrial proteins, citrate synthase and cytochrome c oxidase I, by 42% and 20% respectively. In addition, the enzyme activity of citrate synthase was significantly increased by 25%, and MAPR by 30–40% using various substrates after 8 hours of high Leu. In conclusion, Leu stimulated mitochondrial biogenesis and oxidative phosphorylation in liver cells. This work is supported by NIH grants T32 DK7352, and RO1 DK41972.

Whole body and mixed muscle protein synthesis rate declines with age in healthy people. In response to a 4‐month aerobic exercise program we showed that mixed muscle protein synthesis rate increased 22%. Several gene transcripts that encode mitochondrial (mito) proteins increased in response to exercise but it remains to be determined whether these are translated to protein synthesis. In this study we tested the hypothesis that mito protein synthesis also increased in response to aerobic exercise training. Healthy men and women (N = 42) from 30–74 y were studied before and after a 4‐month program of stationary bicycle training (3d/wk, up to 40min/d at 80% HRmax) or control activity. Muscle protein synthesis rate was measured using [ 15 N] phenylalanine as a tracer. Mito and sarcoplasmic (sarco) proteins were separated from muscle biopsies for analysis. At baseline there was a decline with age (p<0.05) in both mito (6%/decade, r= −0.32) and sarco (5.5%/decade, r= −0.42) protein synthesis rate. Muscle protein synthesis rates did not change in control subjects, but in the exercise group mito synthesis increased 27% (0.046 ± 0.003 %/h pre vs. 0.058 ± 0.005 post, p=0.017, N=25) and there was a trend for 10% higher sarco protein synthesis (0.036 ± 0.002 %/h pre vs. 0.040 ± 0.003 post, p=0.066, N=33). These effects of exercise did not vary with age. We conclude that despite a decline in mito protein synthesis and function with advancing age, the potential for mito biogenesis in response to aerobic exercise remains robust in older muscle, as demonstrated by enhanced mito protein synthesis, gene expression, and enzyme action. Support: NIH RO1DK41973, MO1RR00585.

Aging is associated with decreasing muscle mass and protein synthesis. Amino acid administration is reported to increase muscle protein synthesis and balance. We determined whether increasing dietary protein content from adequate (1.5g/kgFFM/d, AP) to high (3g/kgFFM/d, HP) had beneficial or adverse effects on whole body and muscle protein metabolism, renal function and insulin sensitivity in healthy young (24±1y) and older (70±2y) men and women. Subjects were randomized to a crossover design of 10 d of AP or HP protein diets, without changing caloric intake. Measurements of postabsoptive whole body leucine appearance rate (protein breakdown), oxidation, and non‐oxidative leucine disposal (protein synthesis) rates were performed using L‐[1‐ 13 C]‐leucine and fractional synthesis rate (FSR) of mixed muscle proteins was done using [ 15 N]‐lysine as tracers. Glomerular filtration rate (GFR) and insulin sensitivity were determined by iodothalamate and intravenous glucose tolerance test, respectively. HP diet increased protein breakdown in both young and old (p<0.05) and increased leucine oxidation in both age groups with greater increase in young (p<0.005). WBPS, muscle protein FSR and insulin sensitivity were unaffected by diet. GFR was lower (p<0.01) in older than in young during both diets. HP diet increased GFR in young (p<0.01) but decreased GFR in older people (p<0.01). In conclusion, increasing dietary protein content had no detectable beneficial effects on muscle or whole body protein metabolism in healthy older people and may cause deleterious effects on renal function. Support: NIH R01AG09531.

The effects of different modes of exercise on mixed muscle protein synthesis (MMPS) in young (Y) and older (O) adults are not fully understood. We determined the effects of ET, RT, and CT on MMPS in 33Y (25±1y) and 31O (70±1y) adults who were randomized to one of three 8wk interventions: (i) Control: no exercise, (ii) ET: cycling 1h @ 65% VO 2 peak, 5x/wk, (iii) RT: RT 4x/wk. After completing the control period, the controls completed an 8wk (iv) CT program (ET & RT). Subjects were studied at baseline and 48h following the last training bout. [ 13 C 6 ]Phe was infused for 8h, with serial v. lateralis biopsies. Isotopic enrichments (IE) were measured by LC/MS for free Phe from tissue fluid (TF) and from that incorporated into MMP. MMPS (%/h) = (ΔIE MMP )/(IE TF )/t*100. A rank sum test was used to test baseline differences between Y and O. 2‐way ANCOVA was used to test for differences between groups by treatment, with baseline values serving as the covariate. MMPS (%/h) tended to be higher in Y (0.060±0.003) compared to O (0.053±0.003) (P=0.06). ET [57%, P<0.0001], RT [21%, P=0.042] and CT [42%, P<0.0001] increased MMPS independently of age (P>0.4). The ET‐induced increase in MMPS was greater than the RT‐induced increase (P<0.05), although ET unlike RT does not increase muscle mass suggesting that muscle protein turnover was increased. In conclusion, ET, RT and CT were effective at increasing MMPS independently of age. R01AG09531, KL2RR024151

Altered protein homeostasis may explain the increased risk of degenerative and cardiometabolic disorders with aging. Here, we investigated the impact of chronological age and obesity on the accumulation (ratio of old to de novo synthesized proteins) of haptoglobin, fibrinogen, and apolipoprotein A‐1 (ApoA‐1) in 7 each of elderly lean (69.8 ± 5.0 years, 23.2 ± 1.8 kg/m 2 ), elderly obese (72.6 ± 5.6 years, 30.6 ± 2.3 kg/m 2 ), young lean (23.1 ± 3.2 years, 23.4 ± 1.9 kg/m 2 ), and young obese (25.2 ± 2.4 years, 30.6 ± 2.4 kg/m 2 ). Proteins were labeled with [ 13 C 6 ]‐phenylalanine, separated by 2‐dimentional gel electrophoresis, and identified by mass spectrometry. Insulin sensitivity was lower in the obese groups, while percent body fat was higher, and lean mass was lower, in the elderly. Haptoglobin accumulation was greater in the obese compared to lean (0.512 ± 0.229 vs. 0.272 ± 0.114; p = 0.008) across all ages, with no age effect. Fibrinogen trended toward higher accumulation in the obese compared to lean, with no age effect, but did not reach statistical significance (1.263 ± 2.108 vs. 0.598 ± 0.197; p = 0.094). ApoA‐1 accumulation showed no obesity or age variation. Associations remained unchanged when controlled for gender, age, and BMI. Thus, adiposity, and not chronological age, promotes protein accumulation, potentially contributing to degenerative and cardiometabolic disease risk. Supported by NIH grant RO1‐AG09531.

Omega‐3 polyunsaturated fatty acids (n‐3 PUFAs) protect insulin sensitivity and glucose homeostasis in rodent models of insulin resistance. These effects of n‐3 PUFAs are linked with anti‐inflammatory properties, but emerging data suggests that mechanisms may also converge on mitochondria. We evaluated the influence of dietary n‐3 PUFAs on mitochondrial physiology in the context of high‐fat diet (HFD) in mice. We hypothesized that the insulin‐sensitizing effects of n‐3 PUFAs would be accompanied by enhanced mitochondrial function in skeletal muscle, leading to attenuated levels of lipid metabolites known to interfere with insulin signaling. We fed mice HFD with or without fish oil for 10 weeks. HFD decreased glucose tolerance, but not in the presence of fish oil. Skeletal muscle long chain acyl coenzyme‐As and ceramides were significantly elevated with HFD, but attenuated in the presence of fish oil. Mitochondrial oxidative capacity was similarly increased in mice fed HFD with and without fish oil. These results indicate that n‐3 PUFAs protect insulin sensitivity, in part, by preventing the accumulation of lipid intermediated that interfere with insulin action. Furthermore, the respiratory function of skeletal muscle mitochondria does not appear to be a major factor in ectopic lipid accumulation, diet‐induced insulin resistance, or the protective effects of n‐3 PUFAs on insulin sensitivity.

Mitochondrial function declines with age but increases with caloric restriction (CR). Mitochondria and muscle fibers were prepared from young (8–11mo) and old (22–24mo) AL‐fed and old CR mice. State 3 respiration was lower in OAL vs. YAL, but preserved in OCR (). Normalizing to protein content eliminated the age‐effect in AL mice but amplified the effect of CR, indicating that CR enhances the intrinsic mito function. ROS production in isolated fibers increased with age in AL but not CR mice (). Coupling was reduced in OAL but not OCR. These results indicate that mito capacity declines with age in situ and in vitro, apparently driven largely by decreased mitochondrial protein content. CR increases mito capacity and efficiency independently of mito protein content. YAL OAL OCR Fibers (pmol/s/m g tiss) CI 54.0±4.1 38.9±1.6 50.8±3.0 CII 44.4±3.4 36.9±1.4 45.0±2.3 CI+II 63.4±5.5 51.8±2.5 67.0±3.4 Mito (pmol/s/m g tiss) CI 38.9±1.8 21.5±2.6 30.7±2.0 CII 30.6±0.9 19.6±1.2 24.7±1.4 CI+II 59.0±1.6 34.0±1.9 46.7±2.7 Mito (pmol/s/m g prot) CI 8350±635 7071±1082 10422±863 CII 6536±414 6309±506 8408±676 CI+II 12601±762 11063±1156 15949±1381 H 2 O 2 (pM/s/mg tiss) CI 10.0±1.8 30.8±3.7 15.0±2.0 CI+II 141.4±20.9 187.2±53.8 101.1±13.6 P:O State IV to III 2.94±0.03 2.71±0.02 2.92±0.05 Means±SEM. P<0.05 OAL vs YAL, P<0.05 OCR vs OAL, P<0.05 OCR vs. OAL & YAL.