Abstract 205: Recovery from Ischemia-Reperfusion Injury Is Metabolic Substrate-Dependent

Abstract

Introduction: The adult heart utilizes mostly fat for energy production, with adaptation to different fuels (“metabolic plasticity”) being a hallmark of the healthy heart. However, metabolic maladaptation is known to occur in heart failure. As such, the ability of the heart to metabolize specific substrates could impact the outcome of pathological insults, such as ischemia-reperfusion (IR) injury. The aim of this study was to develop a system whereby adult mouse cardiomyocytes (AMC) subjected to IR injury could be supplied with different fuels, and metabolism measured in real-time. Methods: AMC were divided in 3 groups, supplied either with glucose (GLU, 5mM), palmitate/fat free BSA (FAT, 100µM) or GLU+FAT. A previously developed method for in-vitro IR injury using a Seahorse XF24 [1], was adopted for ACM. IR comprised 60 min. ischemia and 60 min. reperfusion, and additional metabolic parameters were measured separately using mitochondrial inhibitors and uncouplers [2]. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were simultaneously measured during the IR protocol, followed by a cell death assay. Results: FAT cells showed higher baseline OCR and lower ECAR rates compare to GLU cells, although uncoupled OCR was lower in FAT group, suggesting a lower metabolic reserve capacity for cells respiring on fat. Upon IR, the drop in pH was significantly greater in GLU compare to FAT, indicating faster lactate production. During reperfusion, both OCR and ECAR recovered to pre-ischemic levels in GLU cells but failed to do so in FAT cells. Post-IR cell death was significantly higher in FAT vs. GLU. Surprisingly, GLU+FAT (modeling a “physiologic” substrate mix) replicated the same metabolic profile and cell death as GLU. Conclusions: (i) AMC had better recovery from IR injury using glucose as fuel. (ii) Lower cell viability in FAT (vs. GLU) correlated with smaller metabolic reserve capacity and with a smaller pH drop during ischemia. This is consistent with a known protective role for acidification during IR injury. (iii) Mixed substrates (GLU+FAT) gave a similar response to glucose alone, suggesting that fat may not be toxic, rather glucose is protective, in IR injury. [1] Circ Res. (2012), 110. 948-57. [2] J Vis Exp. (2010), 46. pii: 2511.