Measurement of nitrate and nitrite in biopsy-sized muscle samples using HPLC

Studies of rats have indicated that skeletal muscle plays a central role in whole-body nitrate ( NO−3 )/nitrite ( NO−2 )/nitric oxide (NO) metabolism. Extending these results to humans, however, is challenging due to the small size of needle biopsy samples. We therefore developed a method to precisely and accurately quantify NO−3 and NO−2 in biopsy-sized muscle samples. NO−3 and NO−2 were extracted from rat soleus samples using methanol combined with mechanical homogenization + ultrasound, bead beating, pulverization at liquid N2 temperature or pulverization + 0.5% Triton X-100. After centrifugation to remove proteins, NO−3 and NO−2 were measured using HPLC. Mechanical homogenization + ultrasound resulted in the lowest NO−3 content (62 ± 20 pmol/mg), with high variability [coefficient of variation (CV) >50%] across samples from the same muscle. The NO−2 / NO−3 ratio (0.019 ± 0.006) was also elevated, suggestive of NO−3 reduction during tissue processing. Bead beating or pulverization yielded lower NO−2 and slightly higher NO−3 levels, but reproducibility was still poor. Pulverization + 0.5% Triton X-100 provided the highest NO−3 content (124 ± 12 pmol/mg) and lowest NO−2 / NO−3 ratio (0.008 ± 0.001), with the least variability between duplicate samples (CV ~15%). These values are consistent with literature data from larger rat muscle samples analyzed using chemiluminescence. Samples were stable for at least 5 wk at -80°C, provided residual xanthine oxidoreductase activity was blocked using 0.1 mmol/l oxypurinol. We have developed a method capable of measuring NO−3 and NO−2 in <1 mg of muscle. This method should prove highly useful in investigating the role of skeletal muscle in NO−3 / NO−2 /NO metabolism in human health and disease. NEW & NOTEWORTHY Measurement of nitrate and especially nitrite in small, i.e., biopsy-sized, muscle samples is analytically challenging. We have developed a precise, accurate, and convenient method for doing so using an affordable commercial HPLC system

Investigating Skeletal Muscle Metabolic Adaptations underlying Aerobic Fitness Gains following High Intensity Interval Training in a Rat Model of Pulmonary Arterial Hypertension

poster abstractRationale: In patients with pulmonary arterial hypertension (PAH) a shift from oxidative to a less efficient non-oxidative (glycolytic) metabolism in skeletal muscle is believed to contribute to the reduced exercise tolerance hallmark of the disease. As seen for other cardiopulmonary diseases, exercise training (ExT) may ameliorate this “glycolytic switch” in PAH and improve exercise capacity. Previous studies in this lab showed an improved metabolic profile of skeletal muscle in PAH rats following an ExT protocol of continuous running at moderate relative intensity, 60 minutes at 75% of maximal aerobic capacity (VO2 Max). This study tests the hypothesis in a PAH rat model that HIIT will also result in preserved aerobic capacity and attenuation of skeletal muscle glycolytic shift. Methods: Male Sprague-Dawley rats received either monocrotaline (MCT, 40 mg/kg) to induce mild PAH (n= 14), or saline, for healthy controls (n=9). After 2 wks, a 6 wkprogram of treadmill HIIT was initiated for a subset of PAH (n= 8) and healthy controls (n=6). The 30 min HIIT sessions alternated between 2 minutes at 85% VO2 max and 3 minutes at ~30% VO2 max. VO2 max was assessed at baseline, and in pre-training and post-training via analysis of expired gases. Preliminary results: MCT-induced decrement in VO2 max was attenuated by HIIT (p0.05). Western blotting of soleus homogenates for cytochromes I-V of the electron transport chain (OXPHOS), and for PGC1α, a potent stimulus for mitochondrial biogenesis, is being performed at present to further investigate potential training-induced adaptations in skeletal muscle metabolis

Klotho: An Emerging Factor With Ergogenic Potential

Sarcopenia and impaired cardiorespiratory fitness are commonly observed in older individuals and patients with chronic kidney disease (CKD). Declines in skeletal muscle function and aerobic capacity can progress into impaired physical function and inability to perform activities of daily living. Physical function is highly associated with important clinical outcomes such as hospitalization, functional independence, quality of life, and mortality. While lifestyle modifications such as exercise and dietary interventions have been shown to prevent and reverse declines in physical function, the utility of these treatment strategies is limited by poor widespread adoption and adherence due to a wide variety of both perceived and actual barriers to exercise. Therefore, identifying novel treatment targets to manage physical function decline is critically important. Klotho, a remarkable protein with powerful anti-aging properties has recently been investigated for its role in musculoskeletal health and physical function. Klotho is involved in several key processes that regulate skeletal muscle function, such as muscle regeneration, mitochondrial biogenesis, endothelial function, oxidative stress, and inflammation. This is particularly important for older adults and patients with CKD, which are known states of Klotho deficiency. Emerging data support the existence of Klotho-related benefits to exercise and for potential Klotho-based therapeutic interventions for the treatment of sarcopenia and its progression to physical disability. However, significant gaps in our understanding of Klotho must first be overcome before we can consider its potential ergogenic benefits. These advances will be critical to establish the optimal approach to future Klotho-based interventional trials and to determine if Klotho can regulate physical dysfunction

High Intensity Interval Training Benefits Right Heart Function in a Rat Model of Pulmonary Arterial Hypertension

poster abstractPulmonary Arterial Hypertension (PAH) is a disease of progressive remodeling in pulmonary arteries that elevates pulmonary pressures and eventually leads to right ventricular (RV) failure and death. The purpose of this study was to examine the benefit and detriment of high intensity interval training (HIIT) to the RV in a monocrotaline (MCT) PAH rat model. It is hypothesized that HIIT will improve indicators of RV function without increasing myocardial inflammation or apoptosis. Male Sprague Dawley rats were injected with either MCT (40 mg/kg, n=14)) to induce mild PAH or saline for healthy controls (CON, n=9). A subgroup of MCT (n= 8) and CON rats (n=6) performed a 6 week treadmill HIIT program 5x/week using short bouts of alternating high intensity (2 min, 85-90%VO2max) and low intensity (3 min, ~30%VO2max) running for 30 min/session. Histochemistry/immunohistochemistry was performed on cryofixed or formalin-fixed/paraffin-embedded RV sections to assess indicators of inflammation (CD45+ cells), apoptosis (TUNEL), fibrosis (trichrome) and was imaged using epifluorescence or brightfield microscopy. Image quantification was performed using ImageJ. For the HIIT rats, a reduction in MCTinduced RV hypertrophy was observed, as measured echocardiographically, and by the calculated ratio of RV mass relative to LV+Septum mass. RV function was better preserved for HIIT vs. sedentary MCT, as indicated by stroke volume and cardiac index (cardiac output normalized by body weight) in echocardiography. MCT-induced RV fibrosis as measured by trichrome staining was lower for HIIT, also indicating a healthier myocardium. HIIT did not prompt greater counts per field of CD45+ cells and TUNEL+ cells in HIIT vs. sedentary MCT RV myocardium. In conclusion, in the monocrotaline rat model of PAH, HIIT appears to be a beneficial exercise approach that improves RV function without exacerbating RV inflammation or apoptosis. Future work will examine effects in other PAH models and ultimately in patients with disease

2004 Portland Metro Area Arts Atlas

This atlas was conceptualized and exucuted by RACC and a Senior Capstone class of Portland State University

Voluntary Wheel Running Has Beneficial Effects in a Rat Model of CKD-Mineral Bone Disorder (CKD-MBD)

Background Reduced bone and muscle health in individuals with CKD contributes to their higher rates of morbidity and mortality. Methods We tested the hypothesis that voluntary wheel running would improve musculoskeletal health in a CKD rat model. Rats with spontaneous progressive cystic kidney disease (Cy/+ IU) and normal littermates (NL) were given access to a voluntary running wheel or standard cage conditions for 10 weeks starting at 25 weeks of age when the rats with kidney disease had reached stage 2–3 of CKD. We then measured the effects of wheel running on serum biochemistry, tissue weight, voluntary grip strength, maximal aerobic capacity (VO2max), body composition and bone micro-CT and mechanics. Results Wheel running improved serum biochemistry with decreased creatinine, phosphorous, and parathyroid hormone in the rats with CKD. It improved muscle strength, increased time-to-fatigue (for VO2max), reduced cortical porosity and improved bone microarchitecture. The CKD rats with voluntary wheel access also had reduced kidney cystic weight and reduced left ventricular mass index. Conclusions Voluntary wheel running resulted in multiple beneficial systemic effects in rats with CKD and improved their physical function. Studies examining exercise interventions in patients with CKD are warranted