We have recently discovered fundamental biological regulatory mechanisms of the gaseous biological mediator hydrogen sulfide (H2S). First and foremost, we have discovered that an intramitochondrial H2S generating process, driven by the enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) produces H2S in physiological amounts, which, in term, drives and balances oxidative phosphorylation and maintains cellular bioenergetics. Next, we have also discovered that H2S-mediated bioenergetic functions follow a bell-shaped dose-response, where low (endogenous) levels of H2S support cellular energetics, while high levels of H2S become inhibitory and deleterious. In addition, we have recently demonstrated that H2S plays a critical role in the regulation of vascular tone, at least in part by acting as a physiological (endogenous) inhibitor of vascular phosphodiesterase 5 (PDE5). Regulation of blood flow and oxygen delivery to tissues is a critical determinant of cellular metabolism; hence, the in vivo bioenergetic role of H2S is the consequence of a combination of its effects on regional blood flow and its direct regulatory actions on cellular energetic processe (oxidative phosphorylation, glycolysis). The first overall goal of the current project is to characterize the bioenergetic roles of H2S in vitro and in vivo. These processes are likely to play important regulatory roles in normal physiology. In addition, the second goal of the project is to characterize the bioenergetic roles of H2S in selected models of critical illness (circulatory shoc of various etiologies). This second overall goal is guided by the clinical need for novel approaches for the experimental therapy of circulatory shock, as well as by preliminary data demonstrating that there are marked alterations in H2S homeostasis in critical illness. Our investigations will delineate the role of (a) changes in the expression of H2S-producing enzymes; (b) changes in redox status and reactive oxygen/nitrogen species and (c) changes in the degree of tissue hypoxia/acidosis in the biological functions of H2S in circulatory shock. Our project will culminate in the formulation of a unifying concept that will resolve current controversies in the field, and will define the key molecular determinants, which render the metabolic and vascular effects of H2S beneficial vs. detrimental in circulatory shock. In Aim 1, we will determine the regulation of mitochondrial function and cellular bioenergetics by H2S under resting in vitro conditions, as well as during conditions, when various relevant metabolic aspects of circulatory shock are modeled in vitro. In Aim 2, we will determine the role of H2S imbalance in the regulation of vascular tone during circulatory shock. In Aim 3, we will define the molecular mechanisms by which H2S imbalance contributes to the development of multiple organ failure in murine models of circulatory shock. Finally, in Aim 4, we will investigate the rol of H2S in changes in cellular bioenergetics in leukocytes from patients with circulatory shock ex vivo. These studies are fundamentally relevant for both the field of bioenergetics, as well as for the pathogenesis of critical illness.