Indiana University-Purdue University Indianapolis (IUPUI) / Background: Pulmonary Hypertension (PH) is a devastating disease characterized by pulmonary arterial remodeling, right ventricular dysfunction and ultimately right heart failure. Increased emphasis has been given to skeletal muscle dysfunction in PH, and to its implication in the severe exercise intolerance that is a hallmark of the condition. In this dissertation, skeletal muscle blood flow was measured via the microsphere technique at rest and during exercise (Aim 1), with an acute dose of dietary nitrate via beetroot juice (BRJ) gavage used to determine if supplementation could improve muscle blood flow and alter energetics (Aim 2). VO2max, voluntary running and grip strength tests were used to determine the effect of disease on performance, and to test for an ergogenic effect of BRJ vs. placebo (PL) in healthy and PH rats (Aim 3). Methods: A prospective, randomized, counterbalanced, placebo-controlled trial was used to examine the aforementioned aims across four groups; PH rats (induced with monocrotaline, MCT, 60mg/kg, s.q., 4 weeks) supplemented with BRJ (MCT BRJ, n=9); PH rats supplemented with placebo (MCT PL, n=9); healthy control rats (vehicle, s.q.) supplemented with BRJ (CON BRJ, n=8); healthy control rats supplemented with placebo (CON PL, n=9). Results: Monocrotaline induced a severe PH phenotype evidenced by increased RV wall thickness, RV hypertrophy, RVSP and reduced cardiac output and stroke volume compared to controls (p=<0.001). MCT rats demonstrated lower muscle blood flow at rest, and more prominently during exercise compared to controls (p=0.007-0.047), regardless of supplementation. MCT rats displayed a greater reliance on anaerobic metabolism, demonstrated by increased blood lactate accumulation (p=<0.001), and this was significantly related to reduced blood flow during exercise (r=-0.5879, p=0.001). BRJ supplementation resulted in increased plasma nitrate and nitrite compared to PL (p=<0.001), but at the skeletal muscle level, only nitrate was increased after BRJ. BRJ did not have a significant effect on blood flow, with no improvement during exercise shown vs. PL. Similarly, BRJ did not significantly improve exercise function in MCT or CON rats. Conclusion: MCT rats demonstrated a reduction in muscle blood flow, with BRJ supplementation not resulting in improved flow or exercise performance.

Doctor of Philosophy / Department of Kinesiology / David C. Poole / The onset of skeletal muscle contractions induces rapid and robust increases in metabolic rate (V̇O₂) and blood flow (Q̇) in order to supply the energetic demands of the muscle. In young healthy populations, these variables increase proportionally to maintain oxygen flux into the myocyte for both sexes. However, while the resultant changes in V̇O₂ and Q̇ conflate to establish adequate driving pressures of oxygen (PO₂), it appears that the underlying control processes express distinct sexual dimorphism. Estrogen is crucial for cardiovascular control for young women through its relationship with nitric oxide (NO) and results in lower blood pressure and risk of cardiovascular disease for women. However, in post-menopausal women and some disease states, such as heart failure (HF), these protections are lost due to reductions in estrogen and NO bioavailability which causes women to catch and surpass men in rates of hypertension and cardiovascular disease. The purpose of this dissertation is to explore the mechanisms responsible for establishing the oxygen delivery-to-utilization matching (Q̇O₂/V̇O₂) necessary for skeletal muscle contractions in health and disease. In the first investigation (Chapter 1), we explored the effect of altered NO bioavailability on spinotrapezius muscle interstitial space PO₂ (PO₂is; determined by Q̇O₂/V̇O₂) of healthy male and female rats. We show that both sexes regulate PO₂is to similar levels at rest and during skeletal muscle contractions. However, modulating NO bioavailability exposes sex differences in this regulation with females having greater reliance on basal NO bioavailability and males having greater responsiveness to exogenous NO. In the second investigation (Chapter 2), we sought to determine whether measures of central and peripheral function in HF rats predicted exercise tolerance (as critical speed (CS)). We showed for the first time, that CS can be resolved in HF animals and that decrements in central cardiac (echocardiography) and peripheral skeletal muscle function (PO₂is) predicted CS. Building upon these findings, the third investigation (Chapter 3) aimed to determine if the sex differences in the control of PO₂is seen in healthy rats translated to greater deficits in HF for females. Furthermore, this investigation sought to determine if five days of dietary nitrate supplementation (an exogenous NO source) would raise PO₂is in HF rats, with a greater effect seen in females. We revealed that HF reduces PO₂is at rest and during skeletal muscle contractions and this negative effect is exacerbated for females. However, elevating NO bioavailability with dietary nitrate increases resting PO₂is and alters the dynamic response during contractions with females potentially being more responsive than males. The results herein reveal the importance of NO in the control of Q̇O₂/V̇O₂ in health. The onset of HF results in deleterious declines in exercise tolerance, which are mediated through reductions in central and peripheral function, due, in part, to attenuated NO bioavailability. This creates intensified Q̇O₂/V̇O₂ dysfunction in females with HF; however, this can potentially be countered with dietary supplementation of inorganic nitrate. Altogether, the present dissertation suggests that targeting NO bioavailability, particularly in female HF patients, could be a beneficial non-pharmaceutical therapeutic strategy.

The purpose of this study was to investigate whether 3 days of dietary nitrate supplementation has positive effects on exercise performance and energy metabolism at rest and during strenuous knee exercise, and exercise recovery. The experimental protocol was a double blind, placebo-controlled, two-period, and within-subjects design. Fourteen healthy nonsmoking males (22.71 ± 0.72yr, 72.93 ± 2.35kg, and 47.67 ± 1.56 ml/kg/min VO2max) participated in the study. Two supplements (nitrate lozenge; NO-L or placebo; PLA) were orally administrated randomly at 48 and 24 hours before each trial day and again 40 minutes before each exercise trial. Total work, peak torque, and respiratory gases were automatically collected during repeated knee extensions/flexions (isokinetic concentric contractions with dominant leg; 4 sets of 28 repetitions at 180°/sec with 30 sec rest intervals) and recovery (6 contractions of 1 repetition maximum with 120 vii sec between contractions). For these results, peak torque, rate of fatigue, work efficiency, and rate of recovery were calculated. Blood specimens were collected at rest before and after the treatment, post exercise, and end of recovery to track the changes in blood glucose and lactate concentrations. There was a significant inverse correlation for total work during knee extension exercise and oxygen consumption (PLA: r = -.560 and NO-L: r = -.546; p < .01, respectively). During the exercise, RER was significantly higher for PLA compared with NO-L (PLA: 1.42 ± 0.02 vs. NO-L: 1.38 ± 0.02: p = .03). Work within each set and total work performed were higher for NO-L, but these differences were not significant. However, NO-L enhanced exercise efficiency by 3.3% when compared with PLA (PLA: 2497.5 ± 134.56 ft-lb/L/min vs. NO-L: 2578.7 ± 132.24 ftlb/L/min; p = .05). Extensor peak torque recovery rate slope was not significant, but meaningfully faster for NO-L (PLA: 2.39 ± 0.52% vs. NO-L: 3.06 ± 0.54%; p = .09) and significant interaction effect (p = .02) was found during recovery contractions, especially from contractions 2 to 3 (p = .03). It is also worth noting that differences in time to peak torque during knee extension exercise for NO-L and PLA approached significances (PLA: 0.213 ± 0.01 sec vs. NO-L: 0.200 ± 0.01 sec; p = .08). The results suggest that 3 days of dietary nitrate supplementation improves rate of exercise recovery and enhances work efficiency during vigorous resistance exercise. / text

<p>The human stomach normally contains high levels of bioactive nitric oxide (NO). This NO derives from salivary nitrate (NO₃^-) that is converted to nitrite (NO₂^-) by oral bacteria and thereafter non-enzymatically reduced in the acidic gastric lumen to NO. Nitrate is a common component in vegetables, and after ingestion it is absorbed in the small intestine. Interestingly, circulating nitrate is then concentrated by the salivary glands. Hence, intake of nitrate-rich vegetables results in high levels of NO in the stomach. The physiological effects of the high concentration of NO gas normally present in the gastric lumen have been hitherto unknown, and the present investigations were therefore conducted to address this issue.</p><p>NO produced in the gastric lumen after nitrate ingestion increased gastric mucosal blood flow and the thickness of the firmly adherent mucus layer in the stomach. The blood flow and mucus layer are essential defense mechanisms that protect the mucosa from luminal acid and noxious agents. Nonsteroidal antiinflammatory drugs (NSAID) are commonly prescribed and effective drugs for treating pain and inflammation, but are associated with severe gastrointestinal side effects. We demonstrated that a nitrate-rich diet protects against NSAID-induced gastric damage, as a result of the increased formation of NO in the stomach. We also showed that the gastroprotective effect attributed to nitrate depended completely on conversion of nitrate to nitrite by the bacterial flora colonizing the tongue, and that the oral microflora is therefore important in regulating physiological conditions in the stomach.</p><p>In summary, this thesis challenges the current dogma that nitrate intake is hazardous, and on the contrary suggests that dietary nitrate plays a direct role in regulating gastric homeostasis. It is likely that a sufficient supply of nitrate in the diet together with the oral microflora is essential for preventing pathological conditions in the gastrointestinal tract.</p>

The human stomach normally contains high levels of bioactive nitric oxide (NO). This NO derives from salivary nitrate (NO3-) that is converted to nitrite (NO2-) by oral bacteria and thereafter non-enzymatically reduced in the acidic gastric lumen to NO. Nitrate is a common component in vegetables, and after ingestion it is absorbed in the small intestine. Interestingly, circulating nitrate is then concentrated by the salivary glands. Hence, intake of nitrate-rich vegetables results in high levels of NO in the stomach. The physiological effects of the high concentration of NO gas normally present in the gastric lumen have been hitherto unknown, and the present investigations were therefore conducted to address this issue. NO produced in the gastric lumen after nitrate ingestion increased gastric mucosal blood flow and the thickness of the firmly adherent mucus layer in the stomach. The blood flow and mucus layer are essential defense mechanisms that protect the mucosa from luminal acid and noxious agents. Nonsteroidal antiinflammatory drugs (NSAID) are commonly prescribed and effective drugs for treating pain and inflammation, but are associated with severe gastrointestinal side effects. We demonstrated that a nitrate-rich diet protects against NSAID-induced gastric damage, as a result of the increased formation of NO in the stomach. We also showed that the gastroprotective effect attributed to nitrate depended completely on conversion of nitrate to nitrite by the bacterial flora colonizing the tongue, and that the oral microflora is therefore important in regulating physiological conditions in the stomach. In summary, this thesis challenges the current dogma that nitrate intake is hazardous, and on the contrary suggests that dietary nitrate plays a direct role in regulating gastric homeostasis. It is likely that a sufficient supply of nitrate in the diet together with the oral microflora is essential for preventing pathological conditions in the gastrointestinal tract.

Forskning om människans fysiologi och hur den fungerar uppdateras dagligen. Inom idrottens värld testas nya som gamla, naturliga som onaturliga preparat och träningsmetoder kontinuerligt, allt för att optimera en idrottares prestation. Ett ämne som det forskas mycket om idag är kvävemonoxid och dess påverkan i kroppen. Från att ha tolkats som ett skadligt ämne i kroppen har det gått till att möjligen kunna hjälpa hjärtsjuka patienter och även optimera idrottsutövande. Kvävemonoxid bildas i kroppen naturligt med hjälp av syre, men det kan även bildas utan syre genom intag av nitrat som hittas i många grönsaker. Studier om nitrat inom idrottsområdet har koncentrerats till dess påverkan vid uthållighetsidrott och effekten har visat på ökad verkningsgrad och blodflöde till muskulaturen. Senare fann forskarna även hur dietärt nitrat sänkte ens syreupptagningsförmåga (Vo2max) tillsammans med en ökad uthållighet. Detta är en intressant upptäckt då sänkt Vo2max ofta leder till en försämrad arbetsprestation. Dessa studier ger en bild av hur nitrat fungerar vid aerobt arbete, det vill säga med syre. Det som forskningen inte tagit upp ännu är hur nitrat påverkar anaerobt arbete och maximala prestationer som förekommer vid styrketräning. Syftet med denna studie var därför att undersöka hur nitrat påverkar muskelfunktion och uthållighet vid styrketräning.  I en randomiserad, dubbelblind, korsande studie, konsumerade åtta män (ålder 19-26, 23 (±2, 3)) nitrat eller placebo (0,1 mmol/kg kroppsvikt/dag) under tre dagar. Under fjärde dagen testades männens prestation i fyra olika styrketest. Laktat och glukosvärden mättes för att se hur den laktacida systemet påverkades. Studien gav inget stöd till att dietärt nitrat påverkar styrketräning. Resultaten från tillfället med nitrat respektive placebo förblev i stort sett oförändrade. Slutsatsen blev att ett intag av nitrat inte har någon större betydelse för denna modell av styrketräning. Den främsta förklaringen till detta kan tänkas vara att nitratet ger störst inverkan vid långvariga arbetsdurationer och främst under aerobt arbete. I detta fall används mestadels lagrad energi i kroppen, och energisystemen där syre krävdes är troligen inte av större betydelse. / Research on human physiology and how it is working is updated daily. In the world of sports they are testing new as old, natural as unnatural preparations and different training methods continuously in order to optimize athletic performance. A substance that´s been research on, up till today is nitric oxide and its influence in the body. From being interpreted as a harmful substance in the body, it went to possibly help heart disease patients, and also optimize the physic in sport performance. Nitric oxide is formed in the body naturally by oxygen, but it can also be formed without oxygen through the ingestion of nitrates found in many vegetables. Studies on nitrate in the sport field have concentrated on the effect on endurance sports and the effect has been shown to increase the efficiency and the blood flow to the muscles. Later on they also found that nitrate supplementation seems to give a lower Vo2max together with an increased time to exhaustion. These findings are really interesting because normally a reduction in Vo2max leads us to a decrease in workability. All these studies give an idea on how nitrate works aerobic, that is with oxygen. The research has not yet an explanation on how nitrate affect anaerobic work and maximum performance that occurs in weight training. The purpose of this study was to investigate how nitrate affects muscle function and endurance in strength training. In a randomized, double-blind, crossover trial, eight men (age 19-26, 23 (±2, 3)) consumed nitrate or a placebo (0.1 mmol/kg bodyweight/day) for three days. During the fourth day the test persons were tested in four different strength tests to see how they performed. Lactate and glucose concentrations were measured to see how the laktacid system was influenced. The study gave no support that dietary nitrate affects weight training. The results from the occasion with nitrate respectively placebo remained essentially unchanged. It was concluded that an intake of nitrate not will give any significant effects on the model of strength training. The main reason for this may be that nitrate provides the greatest impact on long-term work-duration and mainly during aerobic work. In this case the main use is mostly stored energy in the body, and the energy systems in which oxygen is required will probably not be of major importance. / Pilotstudie i samarbete med Åstrands laboratoriet och Karolinska Institutet