Korean J Physiol Pharmacol.  2015 Jul;19(4):373-382. 10.4196/kjpp.2015.19.4.373.

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat

• Lee JH
• Ha JM
• Leem CH

• KMID: 2285581
• DOI: http://doi.org/10.4196/kjpp.2015.19.4.373

Abstract

Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca2+]. 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 [Ca2+], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca2+] 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, [Ca2+], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca2+] and TMRE for Psi(m) or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca2+] concentration was 1.03 microM. This 1 microM cytosolic Ca2+ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca2+] increase was limited to ~30 microM in the presence of 1 microM cytosolic Ca2+. 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.

Keyword

Calcium; Fura-2-FF; Mitochondrial membrane potential; NADH; pH

MeSH Terms

Animals
Calcium
Coloring Agents
Cytosol
Fura-2
Hydrogen-Ion Concentration
Membrane Potential, Mitochondrial
Membrane Potentials
Mitochondria
Muscle Cells*
NAD*
Rats*
Calcium
Coloring Agents
Fura-2
NAD

Figure

• 

  Fig. 1 A microfluorometry system for multiparameric measurement. The diagram shows the instrumentation used to measure NADH (emission, 450 nm; excitation, 361 nm), Fura-2-FF (emission, 500 nm; excitation, 353 and 400 nm), and TMRE (emission, 590 nm; excitation, 530 nm) or carboxy-SNARF-1 (emission, 590 and 640nm; excitation, 539 nm). The light from a Xenon arc lamp excited the fluoroprobes in a myocyte, and then the emission passed through a dichroic mirror and band-pass filters to measure the intensity using photon-counting devices. Near infrared from a microscope was used as a light source to visualize the myocytes during experiments using a CCD camera and monitor.

• 

  Fig. 2 The excitation spectrum of NADH-Fura-2-FF. The spectrum shows the interference between NADH and Fura-2-FF at each emission (450 and 500 nm). (A) The NADH excitation spectrum at 450-nm emission. (B) The NADH excitation spectrum at 500-nm emission. (C) The Fura-2-FF excitation spectrum at 450-nm emission under Ca2+-free and 1 mM Ca2+ conditions. (D) The Fura-2-FF excitation spectrum at 500-nm emission under Ca2+-free and 1 mM Ca2+ conditions.

• 

  Fig. 3 NADH and Fura-2 excitation and emission spectra. (A) The NADH excitation spectrum was obtained at 465-nm emission and the emission spectrum was obtained at 365-nm excitation. (B) The Fura-2 excitation spectrum was obtained at 500-nm emission and the emission spectrum was obtained at 340-nm excitation. The emission spectrum was shifted to the left by an increase in Ca2+. Ex is excitation. Em is emission.

• 

  Fig. 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.

• 

  Fig. 5 Measurement of RN factors. (A) The NADH signal in Fura-2-FF-free myocytes at 361-nm excitation and 450-nm emission was measured in the presence of various mitochondrial substrates. (B) F400,500 (●) and F353,500 (-) were measured simultaneously to monitor the NADH contribution to the Fura-2-FF signal. (C) The graph shows the relationships between F361,450,NADH and F353,500,NADH (●) and between F361,450,NADH and F400,500,NADH (○). Linear relationships are evident in the graph.

• 

  Fig. 6 Cellular area-dependent autofluorescent background. ● (F361,450) was used for NADH signal correction. ○ (F353,500) and ▾ (F400,500) were used for Fura-2-FF signal correction, respectively. ▵ (F530,590) was used for TMRE signal correction.

• 

  Fig. 7 Simultaneous measurement of NADH, [Ca2+]m, and pH. Using carboxy-SNARF- and Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and pH was measured simultaneously. The mitochondrial pH changes were investigated following Ca2+ changes. The increase in [Ca2+]m did not affect mitochondrial pH. Mitochondrial pH was 7.504±0.047 (mean±S.E., n=13).

• 

  Fig. 8 Results of NADH and Fura-2-FF interference correction. The left panels show the signals prior to correction and the right panels show the signals after correction. NADH and the ratio of Fura-2-FF signals were changed following correction. The calculated [Ca2+]m was substantially decreased. (A) NADH signals at 450-nm emission. (B) Fura-2-FF signals at 500-nm emission. The figure shows F400,500 (- -), F353,500 (----), and the ratio of Fura-2-FF (-). (C) The mitochondrial calcium concentration calculated from the Fura-2-FF ratio.

• 

  Fig. 9 Resting and maximal [Ca2+]m at l µM cytosolic Ca2+. The resting [Ca2+]m was 1.03±0.13 µM (mean±S.E., n=32) and the maximal [Ca2+]m was 29.6±1.61 µM (mean±S.E., n=33).

• 

  Fig. 10 Simultaneous measurement of NADH, [Ca2+]m, and Ψm. Using Fura-2-FF-loaded myocytes, NADH, [Ca2+]m, and Ψm were measured simultaneously. The resting Ψm was assumed to be -150 mV.

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