2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Cardiovascular disease is the leading cause of death in this country, with the direct annual cost projected to approach $250 billion in 2005. Basic research and epidemiological studies have indicated that inadequate dietary intakes of mineral elements such as calcium, copper, magnesium, zinc, as well as overload of iron are associated with altered functions of the heart and circulation. Furthermore, dietary surveys indicate that appreciable numbers of people have sub-optimal intakes of at least some of these minerals. However, we presently have little definitive proof that changing dietary practices with regard to these minerals will benefit cardiovascular health. A clear understanding of how these minerals, particularly at marginal intakes, contribute to cardiovascular function will provide the basis for dietary recommendations that improve the health of the general public. The current project will focus on the contribution of dietary copper to cardiovascular health. The overall objective is to determine, using animal models, whether copper (Cu)intakes consistent with those observed in humans can adequately support cardiovascular functions. This objective will be pursued through the mechanistic tether of oxidative stress/altered nitric oxide metabolism by which Cu functions, and includes the following specific objectives:. 1)to develop a strategy for assessment of marginal copper deficiency in animals; to use this strategy to determine biomarkers of copper status that are suitable for assessment of marginal status in humans,. 2)to determine the contribution of oxygen- and nitrogen-derived reactive species to the cardiomyopathy (metabolic, contractile) induced by Cu deficiency, and the dietary intakes at which this pathology occurs,. 3)to determine whether low Cu intakes consistent with those observed in humans can impair nitric oxide-dependent control of blood vessels and blood pressure regulation,. 4)to determine whether the oxidative stress induced by Cu deficiency affects homocysteine metabolism and, thereby, cardiovascular function, and whether such effects influence nitric oxide-dependent signal transduction and/or other mechanisms that affect atherosclerosis,. 5)to determine whether marginal Zn deficiency can exacerbate or unmask cardiovascular effects of sub-optimal Cu status by virtue of its role in oxidative/nitrosative metabolism. Research will address components of National Program 107, Human Nutrition (100%). From component 1, Nutrition Requirements, objectives A (Biomarkers), B (Mechanism of Action), C (Nutrient Interactions), E (Genetic Variability) and G (Function and Performance) will be addressed. From component 2, Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease, objective A (Identify nutritional, environmental and genetic factors that modify the effects of nutrient intake and metabolism on health outcomes) will be addressed. 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2005) Assess relationships between organ copper and marginal intakes of dietary copper; select organ with best discrimination of copper intake. Determine if copper deficiency causes reduced mitochondrial respiratory complex activity. Identify mitochondrial respiratory complexes causing increased hydrogen peroxide production during copper deficiency. Determine nitric oxide effect on mitochondrial respiration in copper deficiency. Determine effect of copper deficiency on homocysteine metabolism. Year 2 (FY 2006) Determine signaling pathway for induction of inducible nitric oxide synthase during copper deficiency. Determine nature of effect of altered nitric oxide on blood pressure during copper deficiency. Determine effect of copper deficiency on bilirubin and biliverdin reductase. Determine if low zinc acts to exaggerate cardiovascular effects of marginal copper. Year 3 (FY 2007) Correlate organ copper content with semi-direct indicators of copper status; select best potential single biomarker(s). Identify respiratory complexes affected by developmental copper deficiency. Determine extent of oxidative modification of mitochondrial DNA by copper deficiency. Determine role of nitric oxide in impaired contractile function of copper deficiency. Determine relationship between nitric oxide, oxidative stress, homocysteine in atherosclerotic symptoms of copper deficiency. Clarify role of oxidative/nitrosative stress in zinc/copper interaction. Year 4 (FY 2008) Determine nature of effect of altered nitric oxide on coronary vessels during copper deficiency. Determine role of heme oxygenase in atherosclerotic effects of copper deficiency. Determine the ability of a diet high in antioxidants to ameliorate oxidant stress induced by low copper/low zinc. [This is a new milestone (positive contingency) that anticipates the finding of a role for oxidative/nitrosative stress in the detrimental interaction between zinc and copper.] Year 5 (FY 2009) Correlate organ copper content with combinations of indicators of copper status; select best combination of indicators as biomarker. Identify oxidized, nitrated mitochondrial proteins in copper deficiency. Identify mitochondrial DNA mutations of copper deficiency. Relate mitochondrial DNA mutations to expression of respiratory complexes caused by copper deficiency. Determine whether elevation of inducible nitric oxide synthase preconditions copper-deficient hearts [contingency]. 4a.List the single most significant research accomplishment during FY 2006. Reduced cardiac respiratory enzyme activity in the offspring of moderately Cu-deficient rats results from low dietary Cu during pregnancy, not during lactation: Our previous studies showed that low Cu intake by rats during pregnancy and lactation reduced cytochrome c oxidase activity in cardiac mitochondria of the offspring. The reduction in enzyme activity was first observed late in the lactation phase of heart development and persisted in the offspring even after nine months of repletion with adequate dietary Cu. To determine whether this defect originated before or after birth, pups from moderately Cu-deficient dams were nursed by Cu-adequate dams and pups from Cu-adequate dams were nursed by moderately Cu-deficient dams from one day after birth. This cross-fostering did not reverse the reduction of heart cytochrome c oxidase activity in pups from Cu-deficient dams nor did it cause a defect in those from Cu-adequate dams. This indicates that the long-term loss of cytochrome c oxidase activity in the offspring of moderately Cu-deficient dams is caused by a defect in heart development before birth. IMPACT: Dietary surveys indicate that pregnant women often do not meet the current recommended daily intake for dietary Cu. Our finding suggests that moderately low Cu intake by pregnant women could cause a developmental defect in the fetal heart that may increase the risk of heart disease in their children, particularly as the children become older, because of long-term impairment of mitochondrial function. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] 4b.List other significant research accomplishment(s), if any. Copper deficiency lowers plasma homocysteine and affects mRNA expression of several enzymes involved in homocysteine/methionine metabolism: Homocysteine was found to be decreased in copper deficient rats. Homocysteine, a risk factor in cardiovascular disease, can be metabolized (removed) in one of two ways. It can enter the irreversible transsulfuration pathway or it can be remethylated to form methionine. Our data suggest the remethylation of homocysteine by methionine synthase is increased in copper deficient rats. Further, mRNA for methylenetetrahydrofolate reductase, an enzyme that makes one of the substrates for methionine synthase, is also increased in copper deficiency. IMPACT: These results are contrary to published results and suggest that the risk of heart disease by copper deficiency is not mediated by elevation of homocysteine. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] Dietary copper deficiency selectively reduces genetic expression of only one of five respiratory enzyme complexes in the heart: Dietary copper (Cu) deficiency is known to impair mitochondrial respiratory function, which is catalyzed by five membrane-bound multiple protein complexes. However, few studies have simultaneously analyzed the effect of Cu deficiency on the subunit protein expression of all five protein complexes. Examination of expression of subunits in each of the five respiratory complexes in Cu-deficient rat hearts revealed that only in the cytochrome c oxidase complex was the subunit expression reduced. IMPACT: This study further characterizes the defect in the ability of the heart to use oxygen under Cu-deficient conditions and thus contributes to our knowledge of heart failure in dietary Cu deficiency. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] Dietary Cu deficiency increases nitric oxide production in rat hearts: Nitric oxide is a signaling molecule that, depending on how it is produced, can be either harmful or beneficial to heart function. We found that two enzymes that produce nitric oxide, one that initiates a pathway that leads to cell death and the other that leads to cell protection, are both elevated in dietary copper deficiency in rats. IMPACT: These findings contribute to the view that dietary copper deficiency promotes both heart failure and actions that compensate for this failure and suggest signaling mechanisms by which this may take place. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] 4c.List significant activities that support special target populations. None. 4d.Progress report. None. 5.Describe the major accomplishments to date and their predicted or actual impact. Long-term marginal copper deficiency in adult animals causes heart and blood vessel pathology: Dietary surveys indicate that many humans consume less than the recommended amount of copper. Although studies in animals indicate that dietary copper deficiency causes defects in cardiovascular (heart and blood vessel) structure and function, copper intake in these studies is generally too low to be relevant to human consumption. In a collaborative study with the University of Louisville, scientists at the GFHNRC found that if adult rats are fed marginally-deficient levels of copper, but for sufficiently long periods of time, they too exhibit defects in cardiovascular function and structure that are similar to those observed in severely-deficient young rats. IMPACT: These findings, reported in two papers (Li et al, 2005; Falcone et al, 2005), illustrate that low copper intakes in animals that are comparable to those observed in humans can impair heart and blood vessel function. This provides a rationale for testing for such a possibility in humans. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] Pinto beans provide a bio-available source of dietary copper: Dry edible beans have been shown to be beneficial to cardiovascular health, but the active ingredient(s) for this benefit is(are) unknown. Because copper is known to be essential for cardiovascular health and beans are known to be a good source of copper, we hypothesize that copper from beans could support cardiovascular health. In order to determine this, we first tested whether the copper from dry edible beans was bio-available. We found that when copper was fed to copper-deficient rats in the form of pinto beans, it was equally as effective as inorganic copper in restoring copper status indices (e.g., organ copper, activity of copper-dependent enzymes) to those rats. IMPACT: These findings indicate that the bean’s structure does not impede the delivery of its copper to the animal, which paves the way for testing of effectiveness of bean copper on cardiovascular function in both animals and humans. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] Copper deficiency causes elevation of a structural elastic protein in the heart: We have demonstrated for the first time an 85% increase in fibulin-5 (also known as DANCE/EVEC) and a 71% decrease in cytochrome C oxidase (CCO) VIb subunit, but no change in succinate dehydrogenase complex (also known as complex II) IP subunit in Cu-deficient rat heart when compared with that of Cu-adequate rats. IMPACT: The elevation of fibulin-5, in particular, is important, because it implies that survival mechanisms have been initiated, which indirectly confirms that dietary copper deficiency leads to heart failure. [NP 107 Action Plan Component 4: Nutrient Requirements; ARS Strategic Plan Performance Measures 4.1.2: Define functions, bioavailability, interactions, and human requirements (including effects such as genetic, health status, and environmental factors) for known, emerging, and new classes of nutrients in the food supply and provide that information in databases.] 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The usual routine transfer of nutritional knowledge about the nutritional, beneficial, and non-beneficial effects of trace elements was made through direct contact with industry representatives and the public and with other scientists through presentations at national and international meetings and professional publications. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Jack T. Saari wrote an article which appeared in 2006 in the nutrition section of the Grand Forks Herald titled “Add Color to Your Diet with Fruits, Veggies.” W. Thomas Johnson wrote an article which appeared in September 2006 in the nutrition section of the Grand Forks Herald titled “DASH Away High Blood Pressure.” W. Thomas Johnson presented a talk in February 2006 at the Department of Anatomy and Cell Biology, University of North Dakota School of Medicine and Health Sciences titled “Maternal Copper Deficiency Has Negative Cardiac Effects in the First Generation.” W. Thomas Johnson presented a talk in August 2006 at the East Grand Forks Senior Citizen Center titled “DASH Away High Blood Pressure.” Review Publications Johnson, W.T., Brown-Borg, H.M. 2006. Cardiac cytochrome-c oxidase deficiency occurs during late postnatal development in progeny of copper-deficient rats. Experimental Biology and Medicine. 231:172-180. Wang, J., Song, Y., Elsherif, L., Song, Z., Zhou, G., Prabhu, S.D., Saari, J.T., Cai, L. 2006. Cardiac metallothionein induction plays the major role in the prevention of diabetic cardiomyopathy by zinc supplementation. Circulation. 113:544-554. Relling, D.P., Esberg, L.B., Fang, C.X., Johnson, W.T., Murphy, E.J., Carlson, E.C., Saari, J.T., Ren, J. 2006. High-fat diet-induced juvenile obesity leads to cardiomyocyte dysfunction and upregulation of Foxo3a transcription factor independent of lipotoxicity and apoptosis. Journal of Hypertension. 24:549-561. Johnson, W.T., Newman, Jr., S.M. 2006. Cardiac mitochondrial function is altered in the adult offspring of copper-deficient dams [abstract]. Journal of Federation of American Societies for Experimental Biology. 20(5):A1065. Schuschke, D.A., Williams, C., Kang, Y.J., Saari, J.T. 2006. Cu-repletion promotes angiogenesis in the Cu-deficient rat heart [abstract]. Journal of Federation of American Societies for Experimental Biology. 20(4):A553. Saari, J.T., Reeves, P.G. 2006. Pinto beans are a good source of dietary copper [abstract]. Journal of Federation of American Societies for Experimental Biology. 20(4):A554. Zeng, H., Saari, J.T. 2006. New findings on protein expression on copper deficient rat heart with proteomic approach [abstract]. FASEB J. 20(4):A553. Zeng, H., Saari, J.T., Dahlen, G.M. 2005. Copper deficiency increases fibulin-5 (dance/evec) but decreases cytochrome c oxidase vib expression in rat heart. Inorganic Biochemistry. 100:186-91. Reeves, P.G., Saari, J.T. 2005. Bioavailability of copper from cooked dry beans [abstract]. Annals of Nutrition and Metabolism. 49(Suppl 1). p.2.3. Combs, G.F. 2006. Indications of magnesium and calcium deficiency in populations [abstract]. International Symposium on Health Aspects of Calcium and Magnesium in Drinking Water, Program and Abstracts, p. 41. Nielsen, F.H. 2006. Arsenic. In: Klasing, K.C., editor. Mineral Tolerance of Animals. 2nd Revised Edition. Washington DC; National Academies Press. p. 31-41. Johnson, W.T., Newman Jr, S.M. 2007. Hearts in adult offspring of copper-deficient dams exhibit decreased cytochrome c oxidase activity, increased mitochondrial hydrogen peroxide generation and enhanced formation of intracellular residual bodies. Journal of Nutritional Biochemistry. 18:97-104.