Kurt W. Saupe - US grants

Aging impairs the functioning of cardiomyocyte mitochondria in two ways that likely contribute to the increase in heart disease with aging. First, aging impairs myocardial oxidation of free fatty acids which are the main source of ATP for the heart. Second, aging increases mitochondrial production of free radicals. It is our overall hypothesis that these two age- associated changes in mitochondrial function cause much of the age-associated decline in cardiac physiology, and that reversing the impaired mitochondrial function would "rejuvenate" the heart in a clinically significant way. Many age-associated changes in mitochondrial function can be reversed with dietary supplementation of carnitine derivatives such as acetyl-L- carnitine, a critical co-factor in fatty acid metabolism. To date, carnitine's ability to reverse age-associated deterioration of mitochondrial function has only been documented on a sub- cellular level. With aging, blood, skeletal muscle and heart concentrations of carnitine decreases, but can be restored with dietary supplementation. Our Specific Aims are to test the hypotheses that dietary carnitine supplementary will improve three process that deteriorate with aging: 1) the heart's ability to consume oxygen and increase work output 2) the heart's tolerance to ischemia and reperfusion 3) maximal exercise capacity. Specific Aims 1 and 2 will be conducted from 3 months old and 24 months old Fisher 344 rats using a unique isolated heart preparation. Studies will be conducted at Boston University School of Medicine and at Brigham and Women's Hospital. Combining the resources of these two laboratories allows us to study the effect of aging on the heart in an integrative way, from a biochemical level to the whole animal level.

DESCRIPTION (from the application): The enzyme 5'AMPK is increasingly regarded as a master controller of cellular metabolism. In the senescent heart, there appear to be changes in this enzyme system, either in the upstream regulation of 5'AMPK, the amount of the enzyme, or the downstream responses to activation of the enzyme. The overall goal of the proposed research is to define on a molecular level how this enzyme is regulated in the heart, how that regulation changes with age, and what role changes in this enzyme with age play in causing the insulin resistance that occurs with aging. The initial step in accomplishing this goal is to determine how the amount of mRNA and protein of this enzyme change with age, and define more completely what regulates 5'AMPK activity. The second step is to determine whether changes in the 5'AMPK system with age contribute to insulin resistance, and whether age-associated insulin resistance can be reversed with chronic pharmacological stimulation of 5'AMPK. This second step will be accomplished using a unique preparation where the following key steps in the 5'AMPK pathway can be measured in isolated hearts: left ventricular contractile function, the concentrations of known and putative allosteric regulators of 5'AMPK, and cardiac uptake of oxygen, glucose, lactate and free fatty acids. The applicant's long-term career goal is to become an independent investigator studying age-associated changes in myocardial metabolism, and their role in heart disease. In the applicant's recent studies it has become apparent that accomplishing this goal will require learning cellular and molecular techniques that can be applied to studying myocardial metabolism during senescence. The techniques in the proposed research are designed to complement the applicant's experience in cardiac energetics/NMR spectroscopy. Through interactions with Dr. Lakatta and the strong biogerontology research community at Boston University School of Medicine (two training grants and a Gerontology Center), the applicant will be instructed in the methodological issues central to the study of biogerontology research.

DESCRIPTION (provided by applicant): Poor ischemic tolerance and loss of ischemic preconditioning are hallmarks of the aged heart, and contribute to the fact that age is the strongest risk factor for cardiovascular disease. However, the metabolic adjustments to a diet that is calorie restricted (CR) prevents these deleterious effects of aging. Our overall hypothesis is that CR changes the abundance and proliferative state of: 1) resident progenitor cells in the heart, and 2) adipose- derived stem cells that are used to treat peripheral vascular disease, ischemic heart disease and intractable angina. This hypothesis is supported by data demonstrating that CR not only retards aging, but also increases the abundance and proliferative capacity of resident stem cells in some tissues. The long term goal of this research is to exploit the established anti-aging effects of CR to improve cardiac health in patients. These studies will also provide insight into the broader questions of whether diet might influence health via effects on resident stem cells. The first aim is to test whether CR increases the proliferative state of a specific type of cardiac progenitor cell, cardiac side population cells that are Sca1? . We focus on this specific cell type because it is a distinct population that consists of progenitors for a cell type (cardiomyocytes) that likely play a central role in the poor ischemic tolerance and other deleterious aspects of cardiac aging. The second aim is to define the impact of CR on the abundance, proliferative state, and colony forming of adipose-derived stem cells. We have chosen to focus on adipose tissue (epididymal fat pad) as a source of donor cells because it undergoes extensive remodeling during CR, and because of the literature demonstrating the efficacy of these cells to revascularize ischemic tissue and treat myocardial infarctions. Male C57Bl/6 mice will receive a control diet or a CR diet (60% of ad libitum calories) from 6 months of age until they are sacrificed at middle age (12 months) or senescence (24 months of age). This will allow us to determine the impact singly, and in combination, of aging and CR. Fluorescence activated cell sorting will be used to identify cardiac side population cells that are lineage negative/Sca1? (cardiomyocyte progenitor cells), and adipose-derived cells that are lineage negative/CD34? (stem cells with cardiomyogenic potential). The effects of age and CR on the proliferative state of these cells will be assessed by measuring their rates of BrdU incorporation, telomerase activity, and the abundance of molecular markers of cellular division (Ki67) and cell cycle arrest (p16INK4a). To assess the overall proliferative state of cardiomyocyte precursors the rate of new cardiomyocyte formation (BrdU incorporation into cardiomyocytes) and cardiomyocyte age (telomerase activity) will be measured. If CR increases the number and proliferative state of these two types of resident stem/progenitor cells, the proposed studies will form the basis for future studies determining how best to exploit these effects of CR for clinical use. PUBLIC HEALTH RELEVANCE Aging can be slowed and lifespan extended by placing animals on a diet that is very low in calories and enriched in vitamins and minerals. How this type of diet slows aging and prevents age-associated diseases is not known. Our goal is to determine if this diet "rejuvenates" stem cells located in the adult heart and fat.

[unreadable] DESCRIPTION (provided by applicant): While a role for adenosine monophosphate activated protein kinase (AMPK) in regulating body energy balance via effects in non-adipose tissues has gained acceptance, little is known about the biological function of AMPK in white and brown adipose tissues. The proposed studies will test two hypotheses, the first being that adrenergic signaling regulates AMPK activity in white adipose tissue, which in turn plays a role in controlling white adipocyte insulin sensitivity and adipokine production. The second hypothesis is that AMPK contributes to the regulation of body weight and temperature by controlling the thermogenic potential and amount of non-shivering thermogenesis in brown adipose. To test these hypotheses, three specific aims will be accomplished. First, the adrenergic receptor sub-type(s) necessary and sufficient to upregulate 11 AMPK activity in white adipose tissue, as well as the cell type(s) in which this occurs, will be determined. This will be done in vivo by exposing wildtype and 23-adrenergic receptor knockout mice to 14 days of cold exposure or adrenergic receptor agonists administered via micro-osmotic pumps, and fractionating the white adipose tissue using differential centrifugation. The second aim is to determine what role sympathetic modulation of 11 AMPK activity has in "healthful" and "harmful" remodeling of white adipose tissue. In mice, the extent to which 23-adrenergic induced healthful remodeling of WAT occurs in AMPK knockout mice will be determined. In metabolic syndrome patients and age/sex matched controls, the hypothesis that desensitization of adrenergic signaling in white adipocytes is associated with a downregulation of 11 AMPK activity will be tested. The third aim is to test the hypothesis that chronic weight gain increases caloric expenditure of BAT via an AMPK-mediated increase in brown adipocyte mitochondrial biogenesis. This will be tested in wildtype and 11 AMPK knockout mice in vivo, as well as in vitro using pharmacological activation and inhibition of 11 AMPK in primary cultures of brown adipocytes. The long term goal of this research is to manipulate the amount, and "health" of white adipose tissue by targeting the AMPK signaling system in white and brown adipocytes. PUBLIC HEALTH RELEVANCE: Efforts to combat the "obesity epidemic" by encouraging exercise and dieting have been largely unsuccessful. Our studies examine a way of metabolizing calories into heat instead of storing them as fat, and the possibility that the "health" of fat is regulated by a branch of the autonomic nervous system. [unreadable] [unreadable] [unreadable]