Microscopy-Based Assessment of Fatty Acid Uptake and Lipid Accumulation in Cultured Cells.

The heart relies predominantly on the use of fatty acids to derive energy. Metabolic disorders such as obesity, insulin resistance, and diabetes pose a major risk factor for the development of heart failure. Dysregulation of lipid metabolism observed in these diseases manifests as cardiac lipotoxicity, and is associated with cardiac dysfunction. The alarming rise in the incidence of these metabolic disorders warrants the need for tools to investigate the underlying molecular mechanisms. In this article, we describe a confocal microscopy-based approach to monitor fatty acid uptake and lipid accumulation in vitro, in neonatal murine cardiomyocytes and H9c2 cells. The protocol for assessment of fatty acid uptake relies on the use of BODIPY FL C 12™ to study the kinetics of fatty acid uptake via real-time imaging of fatty acid uptake in live cells. Importantly, it circumvents the need for radioactive labeling of fatty acids to evaluate their uptake. Similarly, the protocol for assessment of lipid accumulation relies on the use of BODIPY™ 493/503 to stain the cytosolic neutral lipid population in fixed cells. We couple these confocal microscopy-based approaches with fluorescence intensity analysis using FIJI to quantify fatty acid uptake and lipid accumulation in vitro. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Assessment of fatty acid uptake Basic Protocol 2: Assessment of lipid accumulation.

Emerging Roles of SIRT3 in Cardiac Metabolism.

The heart is a highly metabolically active organ that predominantly utilizes fatty acids as an energy substrate. The heart also derives some part of its energy by oxidation of other substrates, including glucose, lactose, amino acids and ketones. The critical feature of cardiac pathology is metabolic remodeling and loss of metabolic flexibility. Sirtuin 3 (SIRT3) is one of the seven mammalian sirtuins (SIRT1 to SIRT7), with NAD+ dependent deacetylase activity. SIRT3 is expressed in high levels in healthy hearts but downregulated in the aged or diseased hearts. Experimental evidence shows that increasing SIRT3 levels or activity can ameliorate several cardiac pathologies. The primary deacetylation targets of SIRT3 are mitochondrial proteins, most of which are involved in energy metabolism. Thus, SIRT3 improves cardiac health by modulating cardiac energetics. In this review, we discuss the essential role of SIRT3 in regulating cardiac metabolism in the context of physiology and pathology. Specifically, we summarize the recent advancements that emphasize the critical role of SIRT3 as a master regulator of cardiac metabolism. We also present a comprehensive view of all known activators of SIRT3, and elaborate on their therapeutic potential to ameliorate energetic abnormalities in various cardiac pathologies.