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A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca(2+) Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.

Lee JH, Ha JM, Leem CH - Korean J. Physiol. Pharmacol. (2015)

Bottom Line: With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM.However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+).Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Ulsan College of Medicine/Asan Medical Center, Seoul 138-736, Korea.

ABSTRACT
Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca(2+)]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca(2+)], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca(2+)] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca(2+)], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca(2+)] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM. This 1 µM cytosolic Ca(2+) could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+). Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

No MeSH data available.


Identification of isosbestic points. (A) The isosbestic point at 450-nm emission (red arrow). Ca2+ in the non-bound state (- -) and Ca2+ in the bound state (-) (B) The isosbestic point at 500-nm emission for Ca2+ in the non-bound state (- -) and Ca2+ in the bound state (-) (C) The graph shows subtracted data. Ca2+ in the non-bound state was subtracted from that in the bound state at 450 nm. (D) The graph shows subtracted data. Ca2+ in the non-bound state was subtracted from that in the bound state at 500 nm. (E) Standard deviation data from graph C. (F) Standard deviation data from graph D.
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Figure 4: Identification of isosbestic points. (A) The isosbestic point at 450-nm emission (red arrow). Ca2+ in the non-bound state (- -) and Ca2+ in the bound state (-) (B) The isosbestic point at 500-nm emission for Ca2+ in the non-bound state (- -) and Ca2+ in the bound state (-) (C) The graph shows subtracted data. Ca2+ in the non-bound state was subtracted from that in the bound state at 450 nm. (D) The graph shows subtracted data. Ca2+ in the non-bound state was subtracted from that in the bound state at 500 nm. (E) Standard deviation data from graph C. (F) Standard deviation data from graph D.

Mentions: The excitation wavelength and the concentration of NADH can affect the emission intensity but not the emission spectral curve. Changes in [Ca2+] did not affect the emission and excitation spectrums of NADH (data not shown). Thus, the 450 and 500 nm ratio of NADH was constant at any excitation wavelength. The same principle can be applied to Fura-2-FF if NADH or [Ca2+] does not affect the emission and excitation spectrums of Fura-2-FF. However, the Fura-2-FF emission spectrum was shifted to the left by Ca2+ (Fig. 3B). Therefore, only isosbestic excitation could generate a Ca2+-independent emission spectrum. The isosbestic point was different for each emission wavelength (i.e., 450 and 500 nm). To minimize NADH contamination during isosbestic point acquisition, 10 µM FCCP and 10 µM ADP were added in the absence of mitochondrial substrates. The residual emission intensity was measured, and the relationship between the intensity and the cell area was obtained for correction of subsequent experiments. After myocytes were loaded with Fura-2-FF, the cell area was measured in each cardiac myocyte to correct the residual signals. The excitation spectrums at 450 and 500 nm emission were obtained by changing [Ca2+] from 0 to 10 mM under FCCP-free and mitochondrial substrate-free conditions (Fig. 4A and B). The excitation spectrum of the Ca2+-bound state was subtracted from the excitation spectrum of Ca2+-free conditions. Those subtracted curves are shown in Fig. 4C and D. To obtain an isosbestic point, we chose the wavelength showing the minimum standard deviation value (Fig. 4E and F). The isosbestic points were 361 at 450-nm emissions and 353 at 500-nm emissions, respectively. Using isosbestic excitation, the following equations were valid: (1)F361,450=F361,450,NADH+F361,450,fura(2)F353,500=F353,500,NADH+F353,500,fura(3)F400,500=F400,500,NADH+F400,500,fura where Fx,y is the measured emission intensity at y nm by x nm excitation, Fx,y,NADH represents the pure NADH-dependent emission intensity, and Fx,y,fura represents the pure Fura-2-FF dependent emission intensity. In addition, the Rf, RN1, and RN2 values must be constant because of the consistent shape of the emission spectrum: (4)Rf=F361,450,fura/F353,500,fura(5)RN1=F400,500,NADH/F361,450,NADH(6)RN2=F353,500,NADH/F361,450,NADH With these constants, equations (1), (2), and (3) can be changed as follows: (7)F361,450=F361,450,NADH+Rf*F353,500,fura(8)F353,500=RN2*F361,450,NADH+F353,500,fura(9)F400,500=RN1*F361,450,NADH+F400,500,fura


A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca(2+) Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.

Lee JH, Ha JM, Leem CH - Korean J. Physiol. Pharmacol. (2015)

Identification of isosbestic points. (A) The isosbestic point at 450-nm emission (red arrow). Ca2+ in the non-bound state (- -) and Ca2+ in the bound state (-) (B) The isosbestic point at 500-nm emission for Ca2+ in the non-bound state (- -) and Ca2+ in the bound state (-) (C) The graph shows subtracted data. Ca2+ in the non-bound state was subtracted from that in the bound state at 450 nm. (D) The graph shows subtracted data. Ca2+ in the non-bound state was subtracted from that in the bound state at 500 nm. (E) Standard deviation data from graph C. (F) Standard deviation data from graph D.
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Figure 4: Identification of isosbestic points. (A) The isosbestic point at 450-nm emission (red arrow). Ca2+ in the non-bound state (- -) and Ca2+ in the bound state (-) (B) The isosbestic point at 500-nm emission for Ca2+ in the non-bound state (- -) and Ca2+ in the bound state (-) (C) The graph shows subtracted data. Ca2+ in the non-bound state was subtracted from that in the bound state at 450 nm. (D) The graph shows subtracted data. Ca2+ in the non-bound state was subtracted from that in the bound state at 500 nm. (E) Standard deviation data from graph C. (F) Standard deviation data from graph D.
Mentions: The excitation wavelength and the concentration of NADH can affect the emission intensity but not the emission spectral curve. Changes in [Ca2+] did not affect the emission and excitation spectrums of NADH (data not shown). Thus, the 450 and 500 nm ratio of NADH was constant at any excitation wavelength. The same principle can be applied to Fura-2-FF if NADH or [Ca2+] does not affect the emission and excitation spectrums of Fura-2-FF. However, the Fura-2-FF emission spectrum was shifted to the left by Ca2+ (Fig. 3B). Therefore, only isosbestic excitation could generate a Ca2+-independent emission spectrum. The isosbestic point was different for each emission wavelength (i.e., 450 and 500 nm). To minimize NADH contamination during isosbestic point acquisition, 10 µM FCCP and 10 µM ADP were added in the absence of mitochondrial substrates. The residual emission intensity was measured, and the relationship between the intensity and the cell area was obtained for correction of subsequent experiments. After myocytes were loaded with Fura-2-FF, the cell area was measured in each cardiac myocyte to correct the residual signals. The excitation spectrums at 450 and 500 nm emission were obtained by changing [Ca2+] from 0 to 10 mM under FCCP-free and mitochondrial substrate-free conditions (Fig. 4A and B). The excitation spectrum of the Ca2+-bound state was subtracted from the excitation spectrum of Ca2+-free conditions. Those subtracted curves are shown in Fig. 4C and D. To obtain an isosbestic point, we chose the wavelength showing the minimum standard deviation value (Fig. 4E and F). The isosbestic points were 361 at 450-nm emissions and 353 at 500-nm emissions, respectively. Using isosbestic excitation, the following equations were valid: (1)F361,450=F361,450,NADH+F361,450,fura(2)F353,500=F353,500,NADH+F353,500,fura(3)F400,500=F400,500,NADH+F400,500,fura where Fx,y is the measured emission intensity at y nm by x nm excitation, Fx,y,NADH represents the pure NADH-dependent emission intensity, and Fx,y,fura represents the pure Fura-2-FF dependent emission intensity. In addition, the Rf, RN1, and RN2 values must be constant because of the consistent shape of the emission spectrum: (4)Rf=F361,450,fura/F353,500,fura(5)RN1=F400,500,NADH/F361,450,NADH(6)RN2=F353,500,NADH/F361,450,NADH With these constants, equations (1), (2), and (3) can be changed as follows: (7)F361,450=F361,450,NADH+Rf*F353,500,fura(8)F353,500=RN2*F361,450,NADH+F353,500,fura(9)F400,500=RN1*F361,450,NADH+F400,500,fura

Bottom Line: With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM.However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+).Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Ulsan College of Medicine/Asan Medical Center, Seoul 138-736, Korea.

ABSTRACT
Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca(2+)]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca(2+)], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca(2+)] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca(2+)], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca(2+)] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM. This 1 µM cytosolic Ca(2+) could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+). Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected.

No MeSH data available.