Nicotinamide Adenine Dinucleotide Fluorescence Spectroscopy and Imaging of Isolated Cardiac Myocytes
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Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes John Eng, Ronald M. Lynch, and Robert S. Balaban Laboratory of Cardiac Energetics, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892 ABSTRACT Nicotinamide adenine dinu- individual myocytes. The spectra tures were observed that correlated cleotide (NADH) plays a critical role in showed a broad fluorescence centered with the distribution of a mitochondrial oxidative phosphorylation as the pri- at 447 ± 0.2 nm, consistent with mito- selective fluorescent probe (DASPMI), mary source of reducing equivalents to chondrial NADH. Addition of cyanide the mitochondrial distribution seen in the respiratory chain. Using a modified resulted in a 100 ± 10% increase in published electron micrographs, and a fluorescence microscope, we have fluorescence, while the uncoupler metabolic digital subtraction image of obtained spectra and images of the FCCP resulted in a 82 + 4% decrease. the cyanide fluorescence transition. blue autofluorescence from single rat These two transitions were consistent These data are consistent with the cardiac myocytes. The optical setup with mitochondrial NADH and implied notion that the blue autofluorescence permitted rapid acquisition of fluores- that the myocytes were 44 ± 6% of rat cardiac myocytes originates from cence emission spectra (390-595 nm) reduced under the resting control con- mitochondrial NADH. or intensified digital video images of ditions. Intracellular fluorescent struc- INTRODUCTION originates primarily from bound NADH and reduced nicotinamide adenine dinucleotide phosphate (NADPH) Mitochondrial reduced nicotinamide adenine dinucleo- in the mitochondria, with insignificant contributions from tide (NADH) is the primary electron source for the free cytosolic NADH and NADPH (7). In isolated heart electron transport chain in oxidative phosphorylation. It is mitochondria, NADH is the predominant reduced pyri- currently believed that the mitochondrial NADH/NAD+ dine nucleotide (8) and has as much as a fourfold greater redox state may be a primary control site in cellular fluorescence yield than NADPH (9). respiration (1,2,3), so that measurement of the cellular In contrast to biochemical determinations, the moni- NADH redox state and its compartmentation is critical toring of NADH autofluorescence provides a rapid, non- toward developing an understanding of metabolic regula- destructive measure of relative NADH levels and allows tion in the intact cell. each experimental sample to act as its own control. Using Cellular NADH (reduced) and NAD+ (oxidized) lev- fluorescence techniques, it has also been possible to els have been measured using enzymatic assays on tissue characterize the NADH distribution of subcellular mito- extracts. The determination of the NADH and NAD+ chondrial aggregates in intact cells (10,11). compartmentation has relied on the measurement of The purpose here was to characterize the blue auto- specific redox couples participating in localized enzy- fluorescence of single rat cardiac myocytes using micro- matic reactions, such as the lactate dehydrogenase system spectrophotometry and video microscopy techniques. The in the cytosol (4) and the glutamate dehydrogenase blue autofluorescence was characterized by analyzing its system in the mitochondrial matrix (5). Surface fluores- fluorescence emission spectrum, its response to known cence has been used to measure the relative level of inhibitors of oxidative phosporylation, and its topological NADH in both perfused (3) and in vivo tissues (6). distribution within the cell. The single-cell preparation NADH absorbs ultraviolet light with a maximum at 340 eliminates a number of problems accompanying whole- nm and emits a broad-band blue fluorescence centered at heart preparations: organ motion, cell environment inho- - 450 nm. Oxidized nicotinamide adenine dinucleotide mogeneity, and inner filter effects from tissue and blood (NAD+) does not absorb or fluoresce significantly at chromophores-such as hemoglobin, myoglobin, and these wavelengths. Data from biochemical determina- cytochromes that can alter the spectral characteristics of tions show that the blue fluorescence observed in the heart the fluorescence signal. Preliminary results of this work were presented at Dr. Lynch's present address is Department of Physiology, University of the Biophysical Society Meeting, Phoenix, AZ, USA Massachusetts, Worcester, MA 01605. (1988). volumeVolume 55 April 1989 621-63062 1-630 Biophysical Journal 621 shutter, passed through a 360 nm bandpass filter (transmission 330-380 METHODS AND MATERIALS nm), reflected by a dichroic mirror (reflectance 420 nm), and focused on an individual cardiac myocyte by a 63 x Neofluar; Carl Zeiss Inc., Materials Thornwood, NY (oil immersion, NA 1.25) objective lens. The 360 nm Collagenase (type 2) was purchased from Worthington Biochemical Co. fluorescence excitation filter was used for all experiments. Fluorescence Freehold, NJ; lot 67040M; bovine serum albumin (BSA, fraction V; lot emission light from the entire field of view was transmitted by the 349) was from Miles Laboratories, Inc., Naperville, IL. Calcium-free dichroic mirror to reach ocular objectives, a diffraction grating mono- Minimum Essential Medium (MEM) was prepared locally (National chromator, an image intensifier screen, or a 35 mm film camera. Institutes of Health Media Unit lots EVS80025 and EVS80064). The respiratory uncoupler carbonylcyanide-p-trifluoromethoxy phenylhy- drazone (FCCP) was obtained from Boehringer-Mannheim Diagnostics Oxygen consumption Inc., Mannheim, F.R.G. The fluorescent dyes 5(and 6)-carboxy fluores- cein diacetate, succinimidyl ester, and 2-(4-dimethyl aminostyryl)- Oxygen consumption (Q°2) was determined using a Clarke-type 02 N-methylpyridinium iodide (DASPMI) were obtained from Molecular electrode (Yellow Springs Instrument, Yellow Springs, OH) mounted in Probes, Inc. Eugene, OR. Glassware in contact with the isolated a 1 ml stirred chamber maintained at 370C. The electrode was myocytes was siliconized with Sigmacote (Sigma Chemical Co., St. calibrated with air-equilibrated solution C. Louis, MO). Spectroscopy Preparation of cardiac myocytes The monochromator (Instruments SA, Inc., Metuchen, NJ) output spectrum was imaged by a silicon intensified target (SIT) camera Calcium-tolerant cardiac myocytes were prepared according to a (model 1254; Princeton Applied Research, Princeton, NJ). With this method modified from that of Montini et al. (12). Adult Sprague- arrangement, a 390-595 nm spectrum was dispersed over 500 camera Dawley rats (200-300 g) were anesthetized by intraperitoneal injection pixels in the horizontal direction, providing a spectral resolution of 0.4 of 1.0 g sodium pentobarbital (Richmond Vetinary Co., Rich- Supply nm/pixel horizontally. The SIT camera scanning was controlled by a mond, VA). Concomitantly, 1,500 U heparin sodium (Elkins-Sinn, Inc., multichannel controller (model 1216; Princeton Applied Research) was to inhibit blood Cherry Hill, NJ) injected intraperitoneally coagula- interfaced to a DEC 11/23 MINC minicomputer (Digital Equipment tion. Hearts were excised and mounted on an for quickly apparatus Corp., Marlboro, MA). The 1216 controller was programmed to sum retrograde The hearts were with Langendorff perfusion (13). perfused the SIT camera pixels in the vertical direction and send the resulting 30-40 ml calcium-free MEM (solution A) at 8 ml/min to remove blood. string of 500 values to the minicomputer, where these values were Solution A was then with 100 ml solution B replaced (1 mg/ml digitized by analog to digital converters. A single 390-595 nm fluores- collagenase and 1 mg/ml BSA in solution A). Perfusion was maintained cence emission spectrum was obtained in one 33 ms SIT scan. To at 8 for 22 min while the solution B. All solutions ml/min recirculating improve signal-to-noise, 10 SIT scans were summed to obtain each data were maintained at pH 7.4 and 370C while bubbling with 95% 02-5% acquisition. Each data acquisition was corrected for the camera dark CO2. The BSA in solution B is believed to protect cell membranes by binding fatty acids which may cause membrane disruption through a detergent action (14). The ventricular tissue was diced to 2 mm pieces, added to 10 ml solution B in a small siliconized flask, and placed in an orbital shaker (90 rpm, 25 mm orbit). After 10 min of shaking, 10 ml solution C (1 mg/ml BSA in solution A) were added to the flask, and the supernatant cell suspension was poured into a siliconized centrifuge tube. This suspen- sion was centrifuged at low speed (-30 g, 45 s), resuspended and centrifuged in 10 ml solution C, resuspended and sedimented (5-6 min) in 5 ml solution C, and finally resuspended in 5 ml solution C. This process was repeated for the remaining tissue fragments in the flask, after additional 10 min shaking cycles, until a majority of the myocytes were dispersed. The collagen-coated superfusion chamber was loaded with 20-40 ml of the final cell suspension. The cells were allowed to settle and adhere to the chamber bottom. For all experiments, the myocytes in the chamber were constantly superfused at 3 ml/min with a pH 7.4 buffer solution containing Hepes (10 mM), glucose (10 mM), NaCl (120 mM), KCI (4.7 mM), KH2PO4 (1 mM), MgSO4 (1 mM), NaHCO3 (24 mM), creatine (1 mM), and taurine (1 mM) (solution D). The superfusate was equilibrated under room air and maintained at 370C. For the single-cell spectroscopy experiments,