Measurement of Maximum Velocity of the Succinate Dehydrogenase Reaction in Isolated Cardiomyocytes
Authors: Jensen Cole , Ruiz-Soto Juan Pablo , Mahadev Bhat Sanjana , Han Young Soo , Sieck Gary 
Published: May 2023

Succinate dehydrogenase (SDH) is a key enzyme in the tricarboxylic acid (TCA) cycle, as well as Complex II of the electron transport chain (ETC). In the SDH reaction, succinate is oxidized to fumarate in the TCA cycle, producing FADH2 contributing to the proton gradient that drives the reduction of ubiquinone to ubiquinol in the ETC. Thus, measurement of the maximum velocity of the SDH reaction (SDHmax) provides insight into both TCA cycle and ETC contributions to the maximum respiratory capacity of mitochondria. The goal of the present study was to use a quantitative histochemical technique, which we previously developed in skeletal muscle fibers, to quantify the SDHmax in permeabilized (45 μg/mL saponin) cardiomyocytes isolated from male Sprague-Dawley rats. In the SDH reaction, the oxidation of succinate (80 mM succinate; determined to be the maximum substrate concentration) to fumarate is coupled to the reduction of nitroblue tetrazolium (NBT; 1.5 mM) to its diformazan (NBTdfz) using 1-methoxyphenazine methosulphate (mPMS; 1 mM) as an exogenous electron carrier and azide (0.75 mM) to inhibit Complex IV. The progressive accumulation of NBTdfz as the reaction proceeds is measured by repeated densitometric (at 570 nm, the peak absorbance wavelength for NBTdfz) measurements. The optical density (OD) measurements are converted to mM fumarate using the Beer-Lambert-Bouger equation based on pathlength for light absorbance and the molar extinction coefficient for NBTdfz. Importantly, the pathlength for light absorbance must be controlled. In cross-sections of skeletal muscle fibers, pathlength was controlled by cryosectioning at 6 μm. In the present study, pathlength was controlled by optical sectioning using a confocal microscope. The isolated cardiomyocytes were imaged in 3D using a Nikon Eclipse A1 laser scanning confocal system with a ×40x/1.3 NA oil-immersion lens with an optical slice set at 0.5 μm. Changes in OD within a series of 10 optical slices were measured every 15 s over a 5 min period and expressed as mM fumarate/min using the Beer-Lambert-Bouger equation. We found that the SDH reaction was linear across 5 min, and that the change in OD was consistent throughout all 10 optical slices within a cardiomyocyte, suggesting that NBTdfz accumulates in the cytoplasm. Our results confirm that this assay of SDHmax is reproducible in cardiomyocytes and offers a single cell alternative to high-resolution respirometry. The ability to measure SDHmax in single cells is in contrast to respirometry, which only measures the average O2 consumption of aggregates of cells (isolated or in tissue). Thus, a direct measurement of SDHmax can provide valuable physiological insight into variation in mitochondrial function across a population of cardiomyocytes, e.g., as might occur with ischemic injury. Supported by the Mayo Clinic This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.