MitoRACE: evaluating mitochondrial function in vivo and in single cells with subcellular resolution using multiphoton NADH autofluorescence

Abstract

Key points We developed a novel metabolic imaging approach that provides direct measures of the rate of mitochondrial energy conversion with single‐cell and subcellular resolution by evaluating NADH autofluorescence kinetics during the mitochondrial redox after cyanide experiment (mitoRACE). Measures of mitochondrial NADH flux by mitoRACE are sensitive to physiological and pharmacological perturbations in vivo. Metabolic imaging with mitoRACE provides a highly adaptable platform for evaluating mitochondrial function in vivo and in single cells with potential for broad applications in the study of energy metabolism. Abstract Mitochondria play a critical role in numerous cell types and diseases, and structure and function of mitochondria can vary greatly among cells or within different regions of the same cell. However, there are currently limited methodologies that provide direct assessments of mitochondrial function in vivo, and contemporary measures of mitochondrial energy conversion lack the spatial resolution necessary to address cellular and subcellular heterogeneity. Here, we describe a novel metabolic imaging approach that provides direct measures of mitochondrial energy conversion with single‐cell and subcellular resolution by evaluating NADH autofluorescence kinetics during the mitochondrial redox after cyanide experiment (mitoRACE). MitoRACE measures the rate of NADH flux through the steady‐state mitochondrial NADH pool by rapidly inhibiting mitochondrial energetic flux, resulting in an immediate, linear increase in NADH fluorescence proportional to the steady‐state NADH flux rate, thereby providing a direct measure of mitochondrial NADH flux. The experiments presented here demonstrate the sensitivity of this technique to detect physiological and pharmacological changes in mitochondrial flux within tissues of living animals and reveal the unique capability of this technique to evaluate mitochondrial function with single‐cell and subcellular resolution in different cell types in vivo and in cell culture. Furthermore, we highlight the potential applications of mitoRACE by showing that within single neurons, mitochondria in neurites have higher energetic flux rates than mitochondria in the cell body. Metabolic imaging with mitoRACE provides a highly adaptable platform for evaluating mitochondrial function in vivo and in single cells, with potential for broad applications in the study of energy metabolism.

The Functional Impact of Mitochondrial Structure Across Subcellular Scales

“…Mitochondrial network morphology is not only specific to cell types but also demonstrates intracellular heterogeneity. Within a cell, mitochondria positioned at different site-specific regions display different morphology and biochemical properties (Romashko et al, 1998;Kuznetsov et al, 1998Kuznetsov et al, , 2004aKuznetsov et al, , 2006Collins et al, 2002;Collins and Bootman, 2003;Glancy et al, 2015;Benador et al, 2018;Porat-Shliom et al, 2019;Willingham et al, 2019). For example, mitochondrial populations close to the plasma membrane play an important role in functional coupling of ATP guided ion channels (e.g., Ca 2+ entry) (Lawrie et al, 1996).…”

“…In pancreatic acinar cells, mitochondria are organized into three functionally distinct groups located at the peripheral basolateral region near the plasma membrane, surrounding the nucleus, and between the granular area and basolateral region (Park et al, 2001). Specific subsets of mitochondrial networks and mitochondrial populations within cell types display dissimilar responses to substrates and inhibitors, as well as varied resistance or sensitive to oxidative stress, apoptosis or pathology (Romashko et al, 1998;Kuznetsov et al, 2004b;Willingham et al, 2019). A wide spectrum of cells (hepatocytes, HUVEC, astrocytes, HL-1 cells, fibroblasts and cultured human carcinoma cells) and tissues (cardiac, skeletal muscles and liver) display heterogeneous mitochondrial networks (Collins et al, 2002;Kuznetsov et al, 2004aKuznetsov et al, , 2006Bleck et al, 2018;Glancy, 2020).…”

“…Long-distance mitochondrial transport is particularly critical in large, asymmetric cells with highly specialized subregions. In neurons, mitochondria are transported down axons along MTs to provide cellular free energy and calcium buffering support at sites of synaptic transmission and neurite growth Hollenbeck, 1993, 1995;Hollenbeck, 2003, 2004), resulting in greater energy demands in neurite mitochondria compared to the cell body (Willingham et al, 2019). Indeed, the importance of mitochondrial-MT interactions in neural biology and pathology has been widely recognized (Saxton and Hollenbeck, 2012), and studies have demonstrated that altering mitochondria-MT interactions through deletion of the Miro1 adaptor protein in mice arrests mitochondrial motility and results in severe neurological disease (Nguyen et al, 2014).…”

Mitochondrial light switches: optogenetic approaches to control metabolism

“…For example, substrate and oxygen availability [10,14] or energetic demand from exercise [15–17] can alter the PMF. Metabolic conditions can vary between tissues, and accordingly, there are tissue differences in mitochondrial PMF [18,19]. The PMF can also be differentially regulated within individual mitochondria, with separate matrix cristae compartments able to experience different driving forces [20].…”

Assessing mitochondrial energetics in vivo with molecular detail: the best of both worlds using mitoRACE

“…Fortunately for the field of bioenergetics, in this issue of the Journal of Physiology , Willingham and colleagues present a new innovative in vivo technique, mitoRACE, which assesses mitochondrial energetics through measurements of NADH flux with high spatial and temporal resolution using multiphoton microscopy (Willingham et al . ). By harnessing the autofluorescence of NADH and subcellular resolution microscopy, the authors demonstrate the ability to measure mitochondrial NADH redox status (NADH/NAD + ) in murine skeletal muscle in vivo .…”