Abstract Body: A cell’s ability to adapt to nutrient stress is fundamental to survival. There is evidence that hepatic cystathionine-γ-lyase (CSE)-derived hydrogen sulfide (H₂S) production mediates the beneficial effect of chronic dietary restriction. The mechanism for the regulation of CSE-derived H₂S production and cellular targets that convey the stress resistance are not fully understood. Additionally, it is not known if changes in nutrient levels alter cardiac CSE-derived H₂S production or if H₂S contributes to the heart’s adaptation to nutrient stress. We, therefore, focused on using changes in nutrient levels to understand the physiological role of CSE-derived H₂S in the heart. To assess the effects of nutrient deprivation on H₂S levels and protein sulfhydration (H₂S-mediated posttranslational modification) at the level of the cardiomyocyte, we isolated adult cardiomyocytes from αMHC-MerCreMer⁺ mice (MCM) and cardiomyocyte-specific CSE knockout mice (cCSE) mice (10 weeks of age). The isolated cells were subjected to nutrient deprivation for up to 8 hours. Analysis of MCM cardiomyocytes revealed an increase in H₂S levels and protein sulfhydration starting as early as 2 hours after the onset of nutrient deprivation. Nutrient deprivation did not alter the expression of CSE. However, we did observe changes in CSE activity and found that CSE deficiency diminished the nutrient deprivation-induced increase in H₂S levels and protein sulfhydration. Further analysis revealed that AMP-activated protein kinase (AMPK) regulated the H₂S-producing activity of CSE during nutrient deprivation via phosphorylation at serine residue 125. We also found that the interaction of lipid droplets with mitochondria following nutrient deprivation is dependent on CSE, suggesting that CSE regulates an important adaptive mechanism aimed at maintaining energy metabolism under conditions of diminished nutrients. Mechanistically, we found that the sulfhydration of Perilipin 5 (Plin5) – protein involved in the recruitment of lipid droplets to the mitochondria - could play a role in this regulation. Together, this data indicates that AMPK induces the H₂S-producing activity of CSE in response to nutrient stress to impart adaptive cellular mechanisms. These findings break new ground in defining a mechanism by which the H₂S-producing activity of CSE is regulated.