SUMMARY The altered metabolism of tumor cells confers a selective advantage for survival and proliferation, and studies have shown that targeting such metabolic shifts may be a useful therapeutic strategy. We developed an intensely fluorescent, rapidly responsive, pH-resistant, genetically encoded sensor of wide dynamic range, denoted SoNar, for tracking cytosolic NAD+ and NADH redox states in living cells and in vivo. SoNar responds to subtle perturbations of various pathways of energy metabolism in real-time, and allowed high-throughput screening for new agents targeting tumor metabolism. Among > 5,500 unique compounds, we identified KP372-1 as a potent NQO1-mediated redox cycling agent that produced extreme oxidative stress, selectively induced cancer cell apoptosis and effectively decreased tumor growth in vivo. This study demonstrates that genetically encoded sensor-based metabolic screening could serve as a valuable approach for drug discovery.

SUMMARY We have developed genetically encoded fluorescent sensors for reduced nicotinamide adenine dinucleotide (NADH), which manifest a large change in fluorescence upon NADH binding. We demonstrate the utility of these sensors in mammalian cells by monitoring the dynamic changes in NADH levels in subcellular organelles as affected by NADH transport, glucose metabolism, electron transport chain function, and redox environment, and we demonstrate the temporal separation of changes in mitochondrial and cytosolic NADH levels with perturbation. These results support the view that cytosolic NADH is sensitive to environmental changes, while mitochondria have a strong tendency to maintain physiological NADH homeostasis. These sensors provide a very good alternative to existing techniques that measure endogenous fluorescence of intracellular NAD(P)H and, owing to their superior sensitivity and specificity, allow for the selective monitoring of total cellular and compartmental responses of this essential cofactor.

Rationale: Lipid overload-induced heart dysfunction is characterized by cardiomyocyte death, myocardial remodeling, and compromised contractility, but the impact of excessive lipid supply on cardiac function remains poorly understood. Objective: To investigate the regulation and function of the mitochondrial fission protein Drp1 (dynamin-related protein 1) in lipid overload-induced cardiomyocyte death and heart dysfunction. Methods and Results: Mice fed a high-fat diet (HFD) developed signs of obesity and type II diabetes mellitus, including hyperlipidemia, hyperglycemia, hyperinsulinemia, and hypertension. HFD for 18 weeks also induced heart hypertrophy, fibrosis, myocardial insulin resistance, and cardiomyocyte death. HFD stimulated mitochondrial fission in mouse hearts. Furthermore, HFD increased the protein level, phosphorylation (at the activating serine 616 sites), oligomerization, mitochondrial translocation, and GTPase activity of Drp1 in mouse hearts, indicating that Drp1 was activated. Monkeys fed a diet high in fat and cholesterol for 2.5 years also exhibited myocardial damage and Drp1 activation in the heart. Interestingly, HFD decreased nicotinamide adenine dinucleotide (oxidized) levels and increased Drp1 acetylation in the heart. In adult cardiomyocytes, palmitate increased Drp1 acetylation, phosphorylation, and protein levels, and these increases were abolished by restoration of the decreased nicotinamide adenine dinucleotide (oxidized) level. Proteomics analysis and in vitro screening revealed that Drp1 acetylation at lysine 642 (K642) was increased by HFD in mouse hearts and by palmitate incubation in cardiomyocytes. The nonacetylated Drp1 mutation (K642R) attenuated palmitate-induced Drp1 activation, its interaction with voltage-dependent anion channel 1, mitochondrial fission, contractile dysfunction, and cardiomyocyte death. Conclusions: These findings uncover a novel mechanism that contributes to lipid overload-induced heart hypertrophy and dysfunction. Excessive lipid supply created an intracellular environment that facilitated Drp1 acetylation, which, in turn, increased its activity and mitochondrial translocation, resulting in cardiomyocyte dysfunction and death. Thus, Drp1 may be a critical mediator of lipid overload-induced heart dysfunction as well as a potential target for therapy.

Highlights d FiNad responds to subtle changes of NAD + metabolism in live cells and animals d The role of NAD + precursors in boosting NAD + levels is mapped in various organisms d Increased NAD + synthesis controls morphofunctional changes of activated macrophages d FiNad enables live-cell and in vivo imaging of NAD + decline during aging

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