Nutraceutical and pharmaceutical cocktails did not preserve diaphragm muscle function or reduce muscle damage in D2-mdx mice.

NEW FINDINGS: What is the central question of this study? We previously demonstrated that quercetin transiently preserved respiratory function in dystrophin-deficient mice. To gain lasting therapeutic benefits, we tested quercetin in combination with nicotinamide riboside, lisinopril and prednisolone in the D2-mdx model. What is the main finding and its importance? We demonstrated that these quercetin-based cocktails did not preserve respiratory or diaphragmatic function or reduce histological damage after 7 months of treatment starting at 4 months of age. ABSTRACT: Duchenne muscular dystrophy is characterized by the absence of dystrophin protein and causes muscle weakness and muscle injury, culminating in respiratory failure and cardiomyopathy. Quercetin transiently improved respiratory function but failed to maintain long-term therapeutic benefits in mdx mice. In this study, we combined quercetin with nicotinamide riboside (NR), lisinopril and prednisolone to assess the efficacy of quercetin-based cocktails. We hypothesized that quercetin, NR and lisinopril independently would improve respiratory function and decrease diaphragmatic injury and when combined would have additive effects. To address this hypothesis, in vivo respiratory function, in vitro diaphragmatic function and histological injury were assessed in DBA (healthy), D2-mdx (dystrophic) and D2-mdx mice treated with combinations of quercetin, NR and lisinopril from 4 to 11 months of age. Respiratory function, assessed using whole-body plethysmography, was largely similar between healthy and dystrophin-deficient mice. Diaphragm specific tension was decreased by ∼50% in dystrophic mice compared with healthy mice (P < 0.05), but fatigue resistance was similar between groups. Contractile area was decreased by ∼10% (P < 0.05) and fibrotic area increased from 3.5% in healthy diaphragms to 27% (P < 0.05) in dystrophic diaphragms. Contrary to expectations, these functional and histological parameters of disease were not offset by any intervention. These data suggest that quercetin, NR and lisinopril, independently and in combination, did not prevent diaphragmatic injury or preserve respiratory function.

Autophagy in the heart is enhanced and independent of disease progression in mus musculus dystrophinopathy models.

BACKGROUND: Duchenne muscular dystrophy is a muscle wasting disease caused by dystrophin gene mutations resulting in dysfunctional dystrophin protein. Autophagy, a proteolytic process, is impaired in dystrophic skeletal muscle though little is known about the effect of dystrophin deficiency on autophagy in cardiac muscle. We hypothesized that with disease progression autophagy would become increasingly dysfunctional based upon indirect autophagic markers. METHODS: Markers of autophagy were measured by western blot in 7-week-old and 17-month-old control (C57) and dystrophic (mdx) hearts. RESULTS: Counter to our hypothesis, markers of autophagy were similar between groups. Given these surprising results, two independent experiments were conducted using 14-month-old mdx mice or 10-month-old mdx/Utrn± mice, a more severe model of Duchenne muscular dystrophy. Data from these animals suggest increased autophagosome degradation. CONCLUSION: Together these data suggest that autophagy is not impaired in the dystrophic myocardium as it is in dystrophic skeletal muscle and that disease progression and related injury is independent of autophagic dysfunction.

Autophagic dysfunction and autophagosome escape in the mdx mus musculus model of Duchenne muscular dystrophy.

AIM: Duchenne muscular dystrophy is caused by the absence of functional dystrophin protein and results in a host of secondary effects. Emerging evidence suggests that dystrophic pathology includes decreased pro-autophagic signalling and suppressed autophagic flux in skeletal muscle, but the relationship between autophagy and disease progression is unknown. The purpose of this investigation was to determine the extent to which basal autophagy changes with disease progression. We hypothesized that autophagy impairment would increase with advanced disease. METHODS: To test this hypothesis, 7-week-old and 17-month-old dystrophic diaphragms were compared to each other and age-matched controls. RESULTS: Changes in protein markers of autophagy indicate impaired autophagic stimulation through AMPK, however, robust pathway activation in dystrophic muscle, independent of disease severity. Relative protein abundance of p62, an inverse correlate of autophagic degradation, was dramatically elevated with disease regardless of age. Likewise, relative protein abundance of Lamp2, a lysosome marker, was decreased twofold at 17 months of age in dystrophic muscle and was confirmed, along with mislocalization, in histological samples, implicating lysosomal dysregulation in this process. In dystrophic muscle, autophagosome-sized p62-positive foci were observed in the extracellular space. Moreover, we found that autophagosomes were released from both healthy and dystrophic diaphragms into the extracellular environment, and the occurrence of autophagosome escape was more frequent in dystrophic muscle. CONCLUSION: These findings suggest autophagic dysfunction proceeds independent of disease progression and blunted degradation of autophagosomes is due in part to decreased lysosome abundance, and contributes to autophagosomal escape to the extracellular space.

Lymphocyte enzymatic antioxidant responses to oxidative stress following high-intensity interval exercise.

The purposes of this study were to 1) examine the immune and oxidative stress responses following high-intensity interval training (HIIT); 2) determine changes in antioxidant enzyme gene expression and enzyme activity in lymphocytes following HIIT; and 3) assess pre-HIIT, 3-h post-HIIT, and 24-h post-HIIT lymphocyte cell viability following hydrogen peroxide exposure in vitro. Eight recreationally active males completed three identical HIIT protocols. Blood samples were obtained at preexercise, immediately postexercise, 3 h postexercise, and 24 h postexercise. Total number of circulating leukocytes, lymphocytes, and neutrophils, as well as lymphocyte antioxidant enzyme activities, gene expression, cell viability (CV), and plasma thiobarbituric acid-reactive substance (TBARS) levels, were measured. Analytes were compared using a three (day) × four (time) ANOVA with repeated measures on both day and time. The a priori significance level for all analyses was P < 0.05. Significant increases in superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities were observed in lymphocytes following HIIT. No significant increases in lymphocyte SOD, CAT, or GPX gene expression were found. A significant increase in TBARS was found immediately post-HIIT on days 1 and 2. Lymphocyte CV in vitro significantly increased on days 2 and 3 compared with day 1. Additionally, there was a significant decrease in CV at 3 h compared with pre- and 24 h postexercise. These findings indicate lymphocytes respond to oxidative stress by increasing antioxidant enzyme activity. Additionally, HIIT causes oxidative stress but did not induce a significant postexercise lymphocytopenia. Analyses in vitro suggest that lymphocytes may become more resistant to subsequent episodes of oxidative stress. Furthermore, the analysis in vitro confirms that lymphocytes are more vulnerable to cytotoxic molecules during recovery from exercise.

Testosterone administration induces protection against global myocardial ischemia.

We tested the hypothesis that chronic testosterone treatment would promote a cardioprotective phenotype against ischemia/reperfusion (I/R) injury. For this study, 3-month-old F344 male rats underwent sham-surgery, orchiectomy (ORX), or ORX plus 21 days testosterone treatment (1.0 mg testosterone/day). At sacrifice, cardiac performance was assessed in a working heart model of I/R (25 min of global ischemia and 45 min of reperfusion). ORX reduced serum testosterone by approximately 98% and testosterone administration elevated serum testosterone to a concentration of 4.6-fold over that of Sham-operated controls (p<0.05). ORX did not significantly impair recovery of cardiac performance following I/R, but did increase cardiac release of lactate dehydrogenase (LDH) during pre- and post-ischemia (p<0.05). Testosterone administration prevented the ORX-induced increase in LDH during both pre- and post-ischemia and increased post-ischemic recovery of aortic flow, cardiac output, cardiac work, left ventricular developed pressure, and contractility (p<0.05) during reperfusion. Testosterone administration also increased left ventricular expression of catalase, but did not affect the expression of manganese superoxide dismutase, glutathione peroxidase, or sarcolemmal K (ATP) channel protein Kir6.2. Neither circulating nor cardiac concentrations of estradiol were altered by either treatment. We conclude that administration of high-dose testosterone confers cardioprotection through yet to be identified androgen-dependent mechanism(s).

Post-160-km race illness rates and decreases in granulocyte respiratory burst and salivary IgA output are not countered by quercetin ingestion.

This study measured the influence of the flavonoid quercetin on immune changes and incidence rates of upper respiratory tract infections in ultramarathoners competing in the 160-km Western States Endurance Run. Sixty-three runners were randomized to quercetin and placebo groups, and under double-blinded methods ingested 1000 mg/day quercetin for 3 wks before, during, and 2 wks after the race. Thirty-nine of the 63 subjects (n = 18 for quercetin, n = 21 for placebo) finished the race and provided blood and saliva samples the morning before the race and 15 - 30 min postrace. Upper respiratory tract infections were assessed during the week before and the 2-wk period after the race using an illness symptom checklist. Race times did not differ significantly between quercetin and placebo groups. Significant pre- to postrace decreases were measured for natural killer cells (43 %), granulocyte respiratory burst activity (55 %), and salivary IgA output (48 %), and increases for neutrophil (288 %) and monocyte (211 %) cell counts, with no significant group differences. Postrace illness rates did not differ between groups. In conclusion, quercetin supplementation for 3 wks before and 2 wks after the Western States Endurance Run had no effect on illness rates, perturbations in leukocyte subset counts, or decreases in granulocyte respiratory burst activity and salivary IgA.

Effect of pharmacological lowering of plasma urate on exercise-induced oxidative stress.

Urate is a metabolic end product of purine metabolism that contributes about 66% of the antioxidant capacity of plasma. The objective of this study was to evaluate the importance of plasma urate as an antioxidant using pharmacological lowering and examining the impact on plasma antioxidant capacity and oxidative stress after intense exercise. Fifteen subjects ran for 45 min at approximately 80% VO2 max under the influence of probenecid (1 g/d) (PRO) or placebo (PLA) in a double-blind, crossover design. Blood samples obtained at baseline, pre-exercise, and immediately post-exercise were analyzed for F2-isoprostanes, lipid hydroperoxides (LHs), ferric-reducing ability of plasma (FRAP), urate, ascorbate (AA), and nitrite. A 2 (group)x2 (time) repeated-measures analysis of variance (ANOVA), one-way ANOVA, Tukey-Kramer multiple comparison tests, and Student's t tests were used for statistical analysis. PRO exhibited lowered urate and FRAP compared with baseline (p

Intermittent hyperthermia enhances skeletal muscle regrowth and attenuates oxidative damage following reloading.

Skeletal muscle reloading following disuse is characterized by profound oxidative damage. This study tested the hypothesis that intermittent hyperthermia during reloading attenuates oxidative damage and augments skeletal muscle regrowth following immobilization. Forty animals were randomly divided into four groups: control (Con), immobilized (Im), reloaded (RC), and reloaded and heated (RH). All groups but Con were immobilized for 7 days. Animals in the RC and RH groups were then reloaded for 7 days with (RH) or without (RC) hyperthermia (41-41.5 degrees C for 30 min on alternating days) during reloading. Heating resulted in approximately 25% elevation in heat shock protein expression (P < 0.05) and an approximately 30% greater soleus regrowth (P < 0.05) in RH compared with RC. Furthermore, oxidant damage was lower in the RH group compared with RC because nitrotyrosine and 4-hydroxy-2-nonenol were returned to near baseline when heating was combined with reloading. Reduced oxidant damage was independent of antioxidant enzymes (manganese superoxide dismutase, copper-zinc superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase). In summary, these data suggest that intermittent hyperthermia during reloading attenuates oxidative stress and improves the rate of skeletal muscle regrowth during reloading after immobilization.

Exercise and myocardial tolerance to ischaemia-reperfusion.

UNLABELLED: It is well established that both short-term (1-5 days) and long-term (weeks to months) high intensity exercise (i.e. 70-75%VO2max) provides cardioprotection against ischaemia-reperfusion injury. However, it is unclear if moderate intensity exercise will also provide cardioprotection. AIM: Therefore, these experiments compared the protective effects of moderate vs. high intensity exercise in providing defense against ischaemia-reperfusion injury. METHODS: Male Sprague-Dawley rats were randomly assigned to one of three-experimental groups: (1) sedentary (control); (2) moderate intensity treadmill exercise (60 min day(-1) at approximately 55%VO2max); or (3) high intensity treadmill exercise (60 min day(-1) at approximately 75%VO2max). Hearts were exposed to 20 min of global ischaemia followed by 30 min reperfusion in an isolated working heart preparation. RESULTS: Compared with sedentary rats, both moderate and high intensity exercised rats maintained a higher (P < 0.05) percentage of pre-ischaemia cardiac output and cardiac work (cardiac output x systolic blood pressure) during reperfusion. No differences in the percent recovery of cardiac output and heart work existed (P > 0.05) between the two exercise groups. CONCLUSIONS: These data reveal that both moderate and high intensity exercise training provide equivalent protection against ischaemia-reperfusion injury.

The Andro Project: physiological and hormonal influences of androstenedione supplementation in men 35 to 65 years old participating in a high-intensity resistance training program.

BACKGROUND: Since the passage of The Dietary Supplement Health and Education Act in 1994, there has been a flood of new "dietary" supplements promoting anti-aging benefits such as the enhancement of growth hormone or testosterone levels. Androstenediol and androstenedione are such products. This study's purpose was to elucidate the physiological and hormonal effects of 200 mg/d of oral androstenediol and androstenedione supplementation in men aged 35 to 65 years while participating in a 12-week high-intensity resistance training program. METHODS: Fifty men not consuming any androgenic-enhancing substances and with normal total testosterone levels, prostate-specific antigen, hemoglobin, and hematocrit, and with no sign of cardiovascular or metabolic diseases participated. Subjects were randomly assigned to a placebo, androstenediol (diol), or androstenedione (dione) group using a double-blind study design. Main outcomes included serum sex hormone profile, body composition assessment, muscular strength, and blood lipid profiles. RESULTS: During the 12 weeks of androstenedione or androstenediol use, a significant increase in the aromatization by-products estrone and estradiol was observed in both groups (P =.03). In the dione group, total testosterone levels significantly increased 16% after 1 month of use, but by the end of 12 weeks, they returned to pretreatment levels. This return to baseline levels resulted from increases in aromatization and down-regulation in endogenous testosterone synthesis based on the fact that luteinizing hormone was attenuated 18% to 33% during the treatment period. Neither androstenediol nor androstenedione enhanced the adaptations to resistance training compared with placebo for body composition or muscular strength. However, both androstenediol and androstenedione supplementation adversely affected high-density lipoprotein cholesterol (HDL-C) levels, coronary heart disease risk (representing a 6.5% increase), and each group's respective (low-density lipoprotein cholesterol [LDL-C]/HDL-C)/(apolipoprotein A/apolipoprotein B) lipid ratio (diol: +5.2%; dione: +10.5%; P =.05). In contrast, the placebo group's HDL-C levels increased 5.1%, with a 12.3% decline in the (LDL-C/HDL-C)/(apolipoprotein A/apolipoprotein B) lipid ratio. These negative and positive lipid effects occurred despite no significant alterations in body composition or dietary intakes in the supplemental groups or placebo group, respectively. CONCLUSIONS: Testosterone precursors do not enhance adaptations to resistance training when consumed in dosages recommended by manufacturers. Testosterone precursor supplementation does result in significant increases in estrogen-related compounds, dehydroepiandrosterone sulfate concentrations, down-regulation in testosterone synthesis, and unfavorable alterations in blood lipid and coronary heart disease risk profiles of men aged 35 to 65 years.