Rescuing the heart in type 1 diabetes with methylene blue

Grant Details

Description

ObjectiveWe hypothesize that in early diabetes a specific mitochondrial defect develops and causes both an increased reactive oxygen species (ROS generation), and a decrease in ATP production in the heart. We further hypothesize that methylene blue (MB) is able to correct the mitochondrial function by bypassing this defect. This effect of MB has two direct beneficial consequences: 1) decreases the mitochondrial ROS production, oxidative damage of mitochondrial proteins and prevents further mitochondrial defects; 2) improves ATP production. Thus, the goal of this proposal is to establish the direct effect of MB to prevent mitochondrial dysfunction and the decrease in myocardial diastolic and systolic functions, characteristic of the diabetic heart. We will test this hypothesis following two specific aims. The first aim is to determine the effect of MB on mitochondrial function and cardiac phenotype (contractile function and structural changes) during the progression of type 1 diabetes in rats. The experiments will be performed with heart mitochondria isolated from control and diabetic rats treated with low doses (500 micrograms/gram/day) of MB injected intraperitonealy. We will assess: a) mitochondrial integrated function (oxidation of substrates coupled with formation of ATP) as oxidative phosphorylation; b) the individual activities and amount of mitochondrial complexes involved in this process; c) the rate of ROS generation; d) myocardial ATP content and the ATP generating capacity of heart mitochondria; e) myocardial lipid accumulation and fibrosis; f) cardiac diastolic and systolic functions. Therefore, this aim is to gain insight into the effect of mitochondrial defect on ATP and ROS production, and to determine if MB rescues the mitochondrial dysfunction and prevents cardiac damage during the progression of diabetes. The second aim is to investigate the mechanisms by which MB rescues the defective heart mitochondria. This specific aim will: a) Identify the mitochondrial components that are directly related with the effect of MB; b) Evaluate the mitochondrial ability to form ROS when incubated with MB (low concentrations,1 nanogram-1000 nanograms); c) How does MB increase ATP production in heart mitochondria?Background/RationaleBecause heart functions are directly dependent on energy in the form of ATP, derangements in cardiac energy metabolism play an important role in the pathogenesis of diabetic cardiomyopathy (DC). Mitochondria produce the energy that is continuously required and used by the heart during both contraction (systolic period) and relaxation (diastolic period). This energy is in the form of adenosine triphosphate (ATP) which is produced by oxidative phosphorylation (OXPHOS). In this process oxidation of reduced compounds (i.e., NADH) derived from fuel substrates (glucose, fatty acids) is coupled with the phosphorylation of adenosine diphosphate (ADP) to ATP. Reactive species of oxygen (ROS) are less desired end-products of mitochondrial OXPHOS. ROS are thought to contribute to the initiation and progression of DC, since strategies to scavenge ROS have been shown to alleviate cardiac dysfunction. Mitochondrial dysfunction manifested by increased generation of ROS and decreased energy production are involved in the development of main characteristics of DC: death of cardiac cells, accumulation of fibrotic tissue and fat in the heart, abnormalities of calcium handling (less for contraction and more in storage areas in the cells), low ATP content necessary for the heart contractile apparatus; all these alterations cause first diastolic dysfunction followed by systolic dysfunction and heart failure in patients with type 1 diabetes. In a rat model of type 1 diabetes we found a decrease in OXPHOS due to a specific defect in heart mitochondria. Methylene blue (MB) is a pharmacological agent used for more than a century to treat various clinical conditions such methemoglobinemia, septic shock, and to prevent the side effects of chemotherapy. There are no side effects reported for the therapeutic dose of 2 mg/kg/day. The therapeutic potential of MB also has been shown in animal models of diseases associated with oxidative stress: MB extends the life span in mice, protects organs from ischemia-reperfusion injury, increases brain cognitive function, and has anxiolytic properties. MB improves mitochondrial function and reverses premature senescence in cultured cells. It is believed that MB delays mitochondrial dysfunction associated with aging and Alzheimer disease. Intriguingly, MB provides protection of brain damage in animal models with a experimentally-induced mitochondrial defect similar to that found by us and others in the diabetic heart. MB was able to rescue the defective mitochondria by normalizing ATP production and decreasing ROS generation. The recent discovery that MB improves mitochondrial function correlated with our observation that type 1 diabetes induces a specific mitochondrial dysfunction in the heart prompts us to design this proposal aimed to investigate the benefit of chronic treatment with low doses of MB in correcting mitochondrial dysfunction and, in this way, preventing the decrease in cardiac function.Description of ProjectDiabetic cardiac disease (diabetic cardiomyopathy, DC) is a main cause of heart failure. Approximately 70% of asymptomatic diabetic patients have cardiac diastolic (relaxation) dysfunction; 30% of these patients have type 1 diabetes. The heart has a continuous requirement for energy in the form of ATP. Therefore, derangements in cardiac energy metabolism play an important role in the pathogenesis of DC. Mitochondrial oxidative phosphorylation (OXPHOS) is the main ATP producer in the heart. In this process oxidation of fuel substrates (glucose, fatty acids) is coupled with the phosphorylation of adenosine diphosphate (ADP) to produce ATP. Reactive species of oxygen (ROS) are less desired end-products of mitochondrial OXPHOS. ROS are thought to contribute to the initiation and progression of DC, since strategies to scavenge ROS have been shown to alleviate cardiac modifications in diabetes: death of cardiac cells, accumulation of fibrotic tissue and fat, abnormalities of calcium handling, low ATP content necessary for the heart contractile apparatus. In a rat model of type 1 diabetes we also found a decrease in OXPHOS due to a specific defect in heart mitochondria. Methylene blue (MB) is a pharmacological agent used for more than a century to treat various clinical conditions, including diseases associated with oxidative stress: aging, ischemia-reperfusion injury, Alzheimer disease. MB provides protection against brain damage in animal models with a experimentally-induced mitochondrial defect similar to that found by us and others in the diabetic heart. MB was able to rescue the defective mitochondria by normalizing ATP production and decreasing ROS generation. These observations prompt us to design this proposal aimed to investigate the benefit of chronic treatment with low doses of MB in correcting mitochondrial dysfunction and, in this way, preventing the decrease in cardiac contractile function in diabetes. We hypothesize that in early diabetes the specific mitochondrial defect causes both an increased ROS generation, and a decrease in ATP production in the heart. We further hypothesize that MB is able to correct the mitochondrial function by bypassing this defect. This effect of MB has two direct beneficial consequences: 1) decreases the mitochondrial ROS production, oxidative damage of mitochondrial proteins and prevents further mitochondrial defects; 2) enhances ATP production. We will test this hypothesis following two specific aims. The first aim is to determine the effect of MB on mitochondrial function and cardiac phenotype (contractile function and structural changes) during the progression of type 1 diabetes in rats. The second aim is to investigate the mechanisms by which MB rescues the defective heart mitochondria. Our study will establish the direct effect of MB to prevent mitochondrial dysfunction and the decrease in myocardial contractile function characteristic of the diabetic heart.Anticipated OutcomeWe propose a time-course study of type 1 diabetes in rat (with three time points: 3 weeks, 8 weeks, and 16 weeks), and compare mitochondrial parameters (integrated function, reactive oxygen species formation, ATP production) and cardiac changes (contractile function: diastolic and systolic function, structural changes) between the following experimental groups: 1) control group; 2) diabetic group; 3) diabetic group with a good-glycemic control (diabetic rats receiving exogenous insulin in appropriate doses to maintain normal plasma glucose level and be glucosuria free); 4) control group with methylene blue; 5) diabetic group treated with methylene blue (interventional therapy). We expect that the diabetic group treated with methylene blue will have mitochondrial and cardiac parameters similar to the good-glycemic control diabetic group and the control group: normal ATP production by mitochondria associated with normal content of ATP in the cardiac muscle; no excessive production of ROS; no changes in the cardiac structure and contractile function. We also propose to investigate the mechanisms of how MB is protective to mitochondria. The mechanism of mitochondrial defect is under investigation in our laboratory. We propose that specific mitochondrial protein subunits are modified by oxidation, glycation or acetylation (currently potential mechanisms involved in chronic diabetic complications). Finding the specific defective protein is crucial to understand the precise mechanism of how MB protects mitochondria by bypassing that defect. A separate group of diabetic rats will receive MB at the onset of diabetes in order to determine if MB inhibits the appearance of mitochondrial defect (preventive therapy). If the treatment with MB initiated at the onset of diabetic hyperglycemia prevents the appearance of the specific mitochondrial defect, we will conclude that the protective effect of MB on mitochondria is due to its extramitochondrial effects. The time-course study we propose also will solve the conundrum of how the cardiac damage is initiated in diabetes. If mitochondrial dysfunction precedes cardiac dysfunction, and the correction of mitochondrial dysfunction with MB prevents the decrease in cardiac function, we will conclude that it is the cardiac mitochondrial abnormalities rather than other factors that are the initial and cardinal manifestation of the diabetic heart.Relevance to Type I DiabetesCardiovascular disease is a common complication of diabetes, responsible for 80% of the mortality in the diabetic population. The cardiac disease that occurs in diabetes and is independent on other diabetic-induced conditions that may indirectly affect the heart (i.e., hypertension, diseases of the cardiac valves, and atherosclerosis) is called diabetic cardiomyopathy (DC). Approximately 70% of diabetic patients have cardiac diastolic (cardiac relaxation) dysfunction even they never experienced any cardiac symptoms, and 30% of these patients have type 1 diabetes. DC is a main cause of heart failure for diabetic patients. According to different studies, the percentage of patients with diabetes and heart failure is between 20-26%. Although hyperglycemia is the driving force leading to cardiac disease in diabetes, the mechanisms of cardiac damage are multifactorial. The release of reactive species of oxygen from mitochondria has been considered the center mechanism of chronic diabetic complications. Because the heart has a continuous need for energy and mitochondria are the powerhouse of the cell consuming oxygen and substrate fuels to produce energy in the form of ATP, abnormalities in mitochondrial function may initiate the cardiac dysfunction in diabetes. In type 2 diabetes, an increase in oxygen consumption without a similar increase in cardiac contraction (reduced cardiac efficiency) was reported. In contrast, in both humans and animal models of type 1 diabetes a decrease in mitochondrial oxygen consumption associated with increased oxidative stress was found. This study is aimed to correct the mitochondrial dysfunction in a model of type 1 diabetes, and in this way, to prevent the decrease in cardiac contractile function.

StatusFinished
Effective start/end date05/1/1210/31/13

Funding

  • Juvenile Diabetes Research Foundation United States of America: $110,000.00

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