David J. Lefer - US grants

DESCRIPTION (provided by applicant): Hydrogen sulfide (H2S) is a newly recognized signaling molecule with very potent cytoprotective actions. The fields of H2S physiology and pharmacology have been rapidly growing in recent years, but a number of fundamental issues must be addressed to advance our understanding of the biology and clinical potential of H2S in the future. It is important to study the chemistry and pharmacology of exogenous H2S, to be aware of the limitations associated with the choice of chemical agents used to generate H2S in vitro and in vivo. In this regard, synthetic H2S-releasing agents (i.e. H2S donors) are important research tools and potentially very valuable therapeutic candidates for drug development. However, currently available H2S donors are very limited in terms of research use or clinical development since those compounds are very short-lived and the timing and amount of H2S release is largely uncontrollable. To solve these problems, we have recently developed four types of new H2S donors based on N-mercapto, perthiol, gem-dithiol, and phosphorodithioate templates. H2S generations from these donors can be controlled by different mechanisms and the rates of H2S generation can be regulated upon structural modifications. We also found that the administration of H2S donors at the time of reperfusion significantly decreased infarct size and preserved left ventricular function in an in vivo murine model of myocardial ischemia/reperfusion injury. In this project, we plan to develop a toolbox of long-lasting and controllable H2S releasing agents and apply them to explore the pharmacology of H2S under pathological disease states in in vitro and in vivo model systems. Three Specific Aims will be pursued: 1) to design, synthesize, and evaluate controllable H2S donors, 2) to screen the activities of synthetic H2S donors under in vitro conditions; and 3) to examine the cardioprotective actions of donors in acute myocardial ischemia/reperfusion (MI/R) injury and chronic heart failure. We believe that the proposed research will expand our understanding of the chemistry/pharmacology of H2S and provide valuable tools and information to facilitate H2S biomedical research.

Abstract Coronary artery disease is the leading cause of death and morbidity world-wide. Metabolic shifts and oxidative stress that occur in the myocardium during the phases of ischemia and reperfusion cause myocardial injury, and play a pivotal role in the development and progression of myocardial damage and heart failure (HF). Humanin (HN), a novel small peptide generated by mitochondria, has been shown to exhibit strong cytoprotection in many diseases with increased oxidative stress including Alzheimer?s disease, atherosclerosis, myocardial and cerebral ischemia, and type 2 diabetes. Our group has demonstrated that administration of HN results in a decrease in infarct size and preservation of cardiac function in a mouse model of myocardial ischemia-reperfusion (MI-R) injury. Additionally, our preliminary data presented in this grant submission shows that HN administration results in: 1) infarct size reduction following MI-R injury in clinically relevant swine model; 2) increased glucose metabolism and decreased fatty acid oxidation in primary cardiomyocytes, heart lysates and perfused hearts; 3) decreased complex II activity and 4) improved survival of cardiomyocytes following hypoxia and oxidative stress. Based on these data, we hypothesize that HN treatment will improve cardiac function in mouse and swine models of MI-R injury through its unique ability to induce metabolic adaptations to favor glucose utilization in cardiac myocytes, and decrease ROS through a combination of decreased FAO and complex II activity, thereby limiting acute myocardial cell death, and preventing the progression to HF. In this grant application, we will delineate the optimal dose and cardioprotective efficacy of HN in clinically relevant swine and murine models of MI-R injury and HF, and elucidate the mechanisms that underlie HN?s cardioprotective effects on the I-R myocardium. Results from these experiments may potentially have a tremendous impact in treating cardiovascular diseases. HN may provide a much-needed therapeutic option for patients to protect the myocardium from I-R injury, and prevent the progression to HF.

Heart failure (HF) is a leading cause of cardiovascular mortality and morbidity in the United States. Despite current treatments, patients with HF suffer from a poor quality of life and reduced lifespan. An improved understanding of the critical pathological mechanisms of HF is required for the development of novel therapies. Hydrogen sulfide (H2S) is a potent endogenous, gaseous signaling molecule that critically regulates cardiovascular homeostasis. H2S regulates blood pressure, inhibits apoptosis and inflammation, protects mitochondria, and exerts powerful antioxidant actions. Previous work from our group has shown that exogenously administered H2S produces robust cardioprotective effects in animal models of heart failure. We have shown that gene-targeted mice that overexpress endogenous H2S producing enzymes are protected in the setting of HF. H2S is generated endogenously by three enzymes cystathionine ?-lyase (CSE), cystathionine ?-synthase (CBS) and 3-mercaptopyruvate sulfurtranseferase (3-MST). CSE, CBS and 3-MST are all expressed in the heart and circulation, but exhibit significant differences in their regulation and cellular localization. Our Central Hypothesis for the proposed studies is that H2S derived from different enzymes, in different cell populations (endothelial cells, cardiac myocytes, fibroblasts) exerts distinct cardioprotective effects in the pathogenesis of HF. Although, we have demonstrated that H2S levels are reduced in the heart and circulation of both laboratory animals and patients with heart failure, the causes and consequences of reduced H2S availability are poorly characterized. We have developed novel gain and loss of function mouse models that will provide mechanistic insights regarding the contribution of CSE, CBS and 3-MST to HF development and progression. We will employ a multifaceted approach that includes physiological, molecular, biochemical, genetic, and pharmacological approaches to elucidate the role of endogenous H2S in heart failure. The proposed studies will evaluate left ventricular structure and function, cardiac fibrosis, exercise capacity, vascular function, mitochondrial bioenergetics, and molecular signaling to evaluate the role of endogenous H2S on the pathobiology of HF. Specifically, we will: (1) determine the time course of expression of all three endogenous H2S generating enzymes as well as the levels of H2S bioavailability in pressure overload and myocardial infarction induced HF; (2) directly investigate the contribution of H2S-producing enzymes in the development and progression of HF pathology through the use of cell type-specific gene-targeted mouse models with gain and loss of function for CSE, CBS, and 3-MST; (3) identify novel endogenous cytoprotective signal cascades mediated via endogenous H2S producing enzymes in the early and late stages of pressure overload and MI induced HF. Successful completion of these studies will further our understanding of the pathogenesis of HF and will provide critical information required for the development of improved pharmacological strategies to harness H2S therapy for the benefit of patients with HF.

SUMMARY Sarcolemmal ATP-sensitive K+ (KATP) channels are abundantly expressed in the heart. Several groups have now identified a key role for these channels in mediating cardioprotection against ischemic injury and their participation in the protective mechanism of ischemic preconditioning. In the heart there several different subtypes of KATP channels and little is known about the roles during ischemia and reperfusion. Of particular interest are the KATP channel subtypes present in the coronary smooth muscle (SM) and coronary endothelial cells (EC). There is increasing focus on these coronary channels as a target for blood flow regulation and cardioprotection, yet they are relatively poorly understood. The SM and EC KATP channels are distinct from ventricular KATP channels and they are also distinct from each other. A major barrier to our understanding of their respective roles during a complex event such as myocardial ischemia is the lack of currently available resources specifically to study these two channel subtypes. We have generated novel genetic mouse models that can distinguish these subtypes of KATP channels and show with one of these that EC KATP channels strongly participate in myocardial protection during ischemia/reperfusion. The goal of the proposed studies is systematically to examine the role(s) of the EC and SM KATP channel subtypes in the regulation of coronary blood flow, protection during ischemia and the protective response to ischemic preconditioning. We hypothesize that both EC and SM KATP channel subtypes contribute to the regulation of coronary blood flow and cardioprotection, but through distinctly different mechanisms. Using novel and validated conditional knockout mice, we will specifically target EC or SM KATP channel subtypes. The proposed studies have three Aims. In Aim 1, we will investigate the roles of these two coronary KATP channel subtypes in blood flow during ischemia. We will use isolated, pressurized microvessels and isolated, perfused hearts under normal, hypoxic and ischemic conditions. We will additionally investigate the role of EC and SM KATP channels in the myocardial ?no-reflow? phenomenon. Aim 2 will investigate the roles of EC and SM KATP channels in myocardial protection using an in vivo murine I/R model and investigate pathways that regulate infarct development. Aim 3 will investigate the contribution of EC and SM KATP channel subtypes during ischemic preconditioning using an in vivo murine I/R model and cellular assays. We will also examine trafficking of these KATP channel subtypes as a potential protective mechanism and investigate molecular signaling pathways involved. This multi-investigator proposal combines the expertise of three highly established investigators; Dr. Lefer?s extensive expertise with in vivo cardiac ischemia/reperfusion models, and Dr. Coetzee?s track record of studying KATP channels with electrophysiological, biochemical and molecular approaches and Dr. Tinker?s expertise in studying molecular signaling pathways in vascular KATP channels. The proposed studies will provide important molecular insights into the unique functions of coronary KATP channel subtypes under pathophysiological conditions.