Genetically encoded biosensors for evaluating NAD+/NADH ratio in cytosolic and mitochondrial compartments

Abstract

SUMMARY The ratio of oxidized to reduced NAD (NAD + /NADH) sets intracellular redox balance and antioxidant capacity. Intracellular NAD is compartmentalized and the mitochondrial NAD + /NADH ratio is intricately linked to cellular function. Here, we report the monitoring of the NAD + /NADH ratio in mitochondrial and cytosolic compartments in live cells by using a modified genetic biosensor (SoNar). The fluorescence signal of SoNar targeted to mitochondria (mt-SoNar) or cytosol (ct-SoNar) responded linearly to physiological NAD + /NADH ratios in situ . NAD + /NADH ratios in cytosol versus mitochondria responded rapidly, but differently, to acute metabolic perturbations, indicating distinct NAD pools. Subcellular NAD redox balance regained homeostasis via communications through malate-aspartate shuttle. Mitochondrial and cytosolic NAD + /NADH ratios are influenced by NAD + precursor levels and are distinctly regulated under pathophysiological conditions. Compartment-targeted biosensors and real-time imaging allow assessment of subcellular NAD + /NADH redox signaling in live cells, enabling future mechanistic research of NAD redox in cell biology and disease development.

Tissue‐specific effects of exercise as NAD+‐boosting strategy: Current knowledge and future perspectives

“…47 As indicated by cell culture experiments on beating cardiomyocytes supplemented with either fatty acids and glucose, or glucose only, the addition of βoxidation as a metabolic pathway reversed the sarcoplasmic accumulation of NADH and led to an increased sarcoplasmic NAD + /NADH ratio compared to glucose-only conditions. 56 In line with these results from cell culture models, decreased NADH levels are also observed in skeletal muscle cell lysates after low-intensity exercise, as indicated by two investigations on men cycling at 40% VȮ 2max . 52,53 Additionally, increased NAD + levels were found when measurements were not performed immediately after exercise, but in the subsequent recovery period, 63,64 underlining the characteristic switch from glucose to fatty acid oxidation after exercise cessation.…”

“…While such approaches are lacking in in‐vivo experiments so far, they are a well‐established method in cell culture models. In a recent analysis of sarcoplasmic NAD + /NADH ratios of beating cardiomyocytes supplemented with glucose, 56 the high glycolytic rate resulting from glucose supplementation led to a sarcoplasmic accumulation of NADH and a decreased sarcoplasmic NAD + /NADH ratio. In contrast, mitochondrial NAD + /NADH ratios increased, indicating that the oxidation of NADH to NAD + on mitochondrial complex I outweighs the reduction to of NAD + in β‐oxidation, and TCA cycle.…”

Cystathionine γ lyase S-sulfhydrates Drp1 to ameliorate heart dysfunction

“…7 C–D). Recently we developed a genetic fluorescent indicator, mt-SoNar, to evaluate mitochondrial NADH/NAD + ratio that is closely linked to mitochondria function [ 24 ]. Confocal images showed that ISO decreased mt-SoNar fluorescence, which was prevented by H 2 S or VBIT-4 incubation in Drp1 WT, not in C607A, overexpression adult cardiomyocytes.…”

“…Adult cardiomyocytes were isolated from Sprague Dawley rats (200–250 g, male, Shanghai Jiesijie) according to the standard enzymatic technique as described previously [ 24 ]. In brief, the heart was excised quickly from anesthetized rat, cannulated through the ascending aorta, mounted on a Langendorff equipment, and perfused with oxygenated Krebs-Henseleit Buffer (KHB) with collagenase II (Worthington, Cat No.…”

Circadian cardiac NAD+ metabolism, from transcriptional regulation to healthy aging

“…Thus, a measurement of flux between precursors, NAD + , and NADH may be more informative on supplementation efficacy, and NAD + deficiency in heart disease than simple steady-state observations of NAD + alone. Though potentially difficult or expensive to perform injudiciously, labeling studies for NAD + synthesis have been successfully executed previously ( 57 ), and genetic tools have been made to successfully distinguish cytosolic and mitochondrial measurements of NAD + and NADH in isolated cardiomyocytes ( 70 ). In the case of NR, there is a significant clinical appeal in its oral availability ( 71 ), but investigations into muscle and heart NR supplementation find that much of the orally given NR is absorbed by the liver ( 36 , 72 , 73 ).…”