97 Molecular and Cellular Biochemistry 174: 97–100, 1997. © 1997 Kluwer Academic Publishers. Printed in the Netherlands. Detection of mitochondrial defects by laser fluorimetry Wolfram S. Kunz,1 Kirstin Winkler,1 Andrey V. Kuznetsov,1 Hartmut

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Molecular and Cellular Biochemistry 174: 97–100, 1997.

Detection of mitochondrial defects by laser fluorimetry

Wolfram S. Kunz,

¹

Kirstin Winkler,

¹

Andrey V. Kuznetsov,

¹

Hartmut Lins,

¹

Elmar Kirches

²

and Claus W. Wallesch

¹

1Neurobiochemisches Labor der Klinik für Neurologie; ²Institut für Neuropathologie, Universitätsklinikum Magdeburg, Magdeburg, Germany

Abstract

The mitochondrial function in skeletal muscle biopsies of three patients with chronic progressive external ophthalmoplegia, having deletions of the mitochondrial DNA, was studied by laser-excited fluorescence measurements of NAD(P)H and flavoproteins in saponin-skinned fibers. We detected substantially elevated steady state redox states of the mitochondrial NAD- system in the muscle fibers of these patients. Moreover, the respiratory chain-linked autofluorescence changes in the muscle fibers of these patients were larger in comparison to controls indicating substantial alterations of the mitochondrial content.

These results are in line with the presence of elevated numbers of partially respiratory chain inhibited mitochondria in the skeletal muscle of chronic progressive external ophthalmoplegia patients. (Mol Cell Biochem 174: 97–100, 1997)

Key words: skinned muscle fibers (human), NAD(P)H fluorescence, flavoprotein fluorescence, mitochondrial myopathies and encephalomyopathies, mtDNA deletions

Introduction

Up to now an increasing number of diseases has been iden- tified which are directly related to mitochondrial dysfunction [1–3]. Among these diseases affecting skeletal muscle, chronic progressive external ophthalmoplegia (CPEO) is rather common and caused by deletions of the mitochondrial genome in most of the reported cases [4], by np 3243 MELAS mutation in tRNA^Leu [5] or the rare np 5692 mutation in tRNA^Asn [6]. The functional implications of these genetic defects, however, are still a matter of speculation. To eluci- date changes of mitochondrial function in chronic progressive ophthalmoplegia we investigated saponin-skinned muscle fibers. This method of plasma membrane removal results in an unobstructed access to the mitochondria for substrates or ADP and allows the determination of the maximal oxidation capacities using low amounts of biological material [7]. Moreover, the mitochondrial function can be elucidated in these fibers using laser-excited fluorescence measurements of NAD(P)H and fluorescent flavoproteins [8].

Using this novel method we report the detection of partially

respiratory chain inhibited mitochondria in biopsy samples of skeletal muscle of patients with chronic progressive external ophthalmoplegia.

Materials and methods

Patients

S.B. is a 55 year old woman with mild myopathy and severe ptosis. M.T. is a 34 year old woman with ptosis and myopathy. K.G. is a 32 year old woman with ptosis and severe myopathy. All three patients fulfill the clinical criteria of chronic progressive external ophthalmoplegia. In the muscle biopsy of all patients fibers with a very strong and spot-like stain for succinate dehydrogenase were found in combination with glycogen and lipid accumulation. In all cases deletions of the mitochondrial DNA in skeletal muscle were detected: S.B.

harbored in 67% of mtDNA in muscle a 4.7 kb deletion (including the positions np 6188 and np 10877); M.T. harbored in 53% of muscle mtDNA a 2.3 kb deletion (located between

Address for offprints: W.S. Kunz, Neurobiochemisches Labor, Klinik für Neurologie, Universitätsklinikum der Otto-von-Guericke Universität Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany

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the Hind III site at np 11680 and the Xho I site at np 14956) and K.G. harbored in 84 % of muscle mtDNA a 4.6 kb deletion including the Hind III site at np 11680 and the Bam HI site at np 14258.

Methods

Bundles of fibers between 10–15 mg wet weight of M. vas- tus lateralis were obtained from the biopsy samples of my- opathy patients and orthopedic patients (controls, age between 50–70 years) and treated with saponin as described in [7]. The respiration measurements were performed at 25°C using a high resolution Oroboros-oxygraph (Anton Paar, Graz; [9]) in a medium consisting of 110 mM mannitol, 60 mM KCl, 10 mM KH₂PO₄, 5 mM MgCl₂, 0.5 mM Na₂EDTA and 60 mM Tris-HCl (pH = 7.4). The measurements of enzyme activities in the muscle homogenate were performed at 30°C using a Cary 1 spectrophotometer with standard methods in the medium for oxygraphic measurements [10].

For the measurements of fluorescence of NAD(P)H and of fluorescent flavoproteins in saponin-skinned muscle fibers, the experimental setup described in [8] was used.

Southern blots

One µg of total DNA from muscle biopsy samples was di- gested with the appropriate restriction enzymes and separated on 0.7% agarose gels. Southern blots [11] were hybridized with a digoxigenin-labeled human mtDNA probe and devel- oped by chemiluminescence (Tropix, Heidelberg). Sequence specific probes were generated by PCR-amplification of short mtDNA sequences (300–400 bp).

Results

Mitochondrial defects in skeletal muscle are usually determined by measurements of enzyme activities of the mitochondrial electron transport chain in homogenates of the biopsy sample. We additionally measured the maximal oxy- gen consumption rates of saponin-skinned muscle fibers, reported to be indicative for mitochondrial defects [7, 12].

In Table 1 the results of these determinations are summarized for three patients suffering from chronic progressive external ophthalmoplegia with deletions of the mtDNA in skeletal muscle. It is remarkable that only for the patient with a very high percentage of deleted mtDNA (K.G.) the mitochondrial defect is easily detectable using these methods. This is most probably caused by large adaptive changes in the mitochondrial content of the muscle (cf. the elevated citrate

synthase activities) which complicates the classical detection of the mitochondrial defects.

Next, we analyzed the mitochondrial function in saponin- skinned fibers of these patients using measurements of fluorescence of NAD(P)H and fluorescent flavoproteins [8]. This method allows the determination of redox state changes of mitochondrial pyridine nucleotides on substrate, ADP and inhibitor additions. In Fig. 1 responses of NAD(P)H fluorescence to the addition of octanoylcarnitine + malate and ADP, glutamate and cyanide are shown for saponin-skinned fibers from the human vastus lateralis muscle of an orthopedic patient and of the patients S.B and K.G. The addition of the mitochondrial substrates octanoylcarnitine + malate led in all cases to an increase in NAD(P)H fluorescence. ADP, which stimulates the flux through the mitochondrial respiratory chain, caused in control fibers a remarkable reoxidation of NAD(P)H. A much lowered reoxidation of NAD(P)H was observed for the patient S.B. For K.G. almost no reoxidation of NAD(P)H on ADP addition was detected. The following addition of glutamate, which improves the supply of reducing equivalents and stimulates the mitochondrial respiration, caused in control fibers a reduction of NAD(P)H to a new steady state. For S.B. this steady state is in comparison to the controls a more reduced one, and for K.G. the NAD-system remained nearly fully reduced. The maximal reduction of NAD(P)H was reached by the addition of the cytochrome c oxidase inhibitor cyanide. The observed differences in the oxidation-reduction properties of the mitochondrial NAD- system in the fibers of the CPEO patients can be interpreted to be caused by a partial inhibition of the respiratory chain.

This should lead to a lowered efflux of reducing equivalents from the mitochondrial NADH and therefore to elevated

Table 1. Enzyme activities in the muscle homogenates and maximal rates of respiration of saponin-skinned muscle fibers of patients with deletions of mtDNA

Controls M.T. K.G. S.B.

Citrate synthase 5.7 ± 2 12.5 ± 0.2 34.8 ± 1.7 11.4 ± 0.1 (n = 25)

NADH:cytochrome c 3.0 ± 0.8 3.6 ± 0.2 3.0 ± 0.1 1.5 ± 0.2

reductase (n = 25)

Cytochrome c oxidase 3.8 ± 1.2 4.8 ± 0.2 3.2 ± 0.1 0.34 ± 0.01 (n = 25)

Glutamate supported 8.6 ± 1.9 6.2 ± 0.8 3.4 ± 0.6 6.6 ± 0.1 respiration (n = 18)

Succinate supported 9.2 ± 1.9 8.3 ± 2.1 5.6 ± 1 5.3 respiration (n = 18)

mtDNA deletion size – 2.3 kb 4.6 kb 4.7 kb

Degree of mtDNA – 53% 84% 67%

heteroplasmy

The enzyme activity values are expressed in U/g wet weight determined at 30°C (means ± SD of a triplicate determination). n = number of orthopedic controls. The maximal rates of respiration (in nmol O₂/min/mg dry weight) were determined at 25°C using the substrates 10 mM succinate + 10 µM rotenone or 10 mM glutamate + 5 mM malate in the presence of 1 mM ADP.

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steady state redox states of the NAD-system.

To verify this redox behavior of the mitochondrial NAD- system we additionally performed measurements of the flavoprotein fluorescence signals, shown in Fig. 2. This fluorescence, measured at 454 nm excitation and 520 nm emis- sion, originates mostly from the oxidized form of the FAD- containing α-lipoamide dehydrogenase, being in redox contact with the mitochondrial NAD pool [8]. Again, in the muscle fibers of S.B. and K.G. a much lower reoxidation of flavoproteins (visible in this case as fluorescence increase) on ADP addition was detected. This finding strongly supports the interpretation of a partial inhibited respiratory chain for both cases. In Table 2, the results of NAD(P)H and flavoprotein fluorescence measurements with different mitochondrial substrates are summarized for saponin-skinned fibers of all three patients and the orthopedic controls. For all CPEO patients elevated steady state redox states of the mitochondrial NAD-system were detected.

Discussion

Chronic progressive external ophthalmoplegia is reported to be caused either by deletions of mitochondrial DNA [4], by np 3243 MELAS mutation in tRNA^Leu [5] or by np 5692 mutation in tRNA^Asn [6]. As a consequence of point mutations, an impaired synthesis of mitochondrially encoded pro- teins, most probably due to the competition of mutated and nonmutated tRNAs is expected [6]. This concept rather easily explains the presence of partially respiratory chain inhibited mitochondria. Much more problematic seems to be the explanation of the presence of a partially inhibited respiratory chain in cases with rather big deletions of the mtDNA [13, 14]. As a matter of fact, mitochondria which contain exclusively genomes harboring a large scale deletion would be totally inactive from the functional point of view. Apply- ing laser-excited fluorescence measurements we should de- tect under these circumstances a higher background NAD(P)H fluorescence and lower background flavoprotein fluorescence

Fig. 1. Fluorescence changes of NAD(P)H in saponin-skinned human muscle fibers. Between 4 mg and 2 mg wet weight skinned fibers from M.

vastus lateralis were attached to glass wool and perfused as described in [8]. The sample was excited at 325 nm, the fluorescence was registrated at 450 nm. Left trace – control; right traces – patients S.B. and K.G. Additions to the perfusion medium: Octanoylcarnitine (OC) – 1 mM; malate (MAL) – 5 mM; ADP – 1 mM; glutamate (GLU) – 10 mM; KCN – 4 mM.

Fig. 2. Fluorescence changes of fluorescent flavoproteins in saponin- skinned human muscle fibers. About 3 mg wet weight skinned fibers from M. vastus lateralis were attached to glass wool and perfused as described in [8]. The sample was excited at 454 nm, the fluorescence was registrated at 520 nm. Left trace – control; right traces – patients S.B. and K.G.

Additions to the perfusion medium: Octanoylcarnitine (OC) – 1 mM; malate (MAL) – 5 mM; ADP – 1 mM; glutamate (GLU) – 10 mM; KCN – 4 mM.

Table 2. Redox state and content of NAD(P)H and fluorescent flavoproteins in saponin-skinned muscle fibers

Controls M.T. K.G. S.B.

NADH FP NADH FP NADH FP NADH FP

glu + mal (%) 76 ± 5 (n = 32) 79 ± 8 (n = 12) 83 ± 2 88 ± 5 96 ± 2 95 ± 2 80 ± 1 82

pyr + mal (%) 35 ± 9 (n = 12) 62 ± 11 (n = 8) 44 n.d. 70 92 54 n.d.

oc + mal (%) 38 ± 9 (n = 28) 42 ± 11 (n = 12) 60 ± 3 66 ± 3 96 ± 2 90 ± 1 55 61

Content (arb.u.) 12 ± 3 (n = 31) 137 ± 24 (n = 10) 18 ± 2 175 ± 15 18 ± 3 235 ± 65 8 ± 1 110

The redox states are given in % of the fluorescence change between the fully oxidized state (in the absence of substrates) and the completely reduced state (in the presence of substrates and 4 mM cyanide). They were determined in the presence of 1 mM ADP and different combinations of substrates: 10 mM glutamate + 5 mM malate (glu + mar), 5 mM pyruvate + 5 mM malate (pyr + mal) or 1 mM octanoylcarnitine + 5 mM malate (oc + mar). The content of NAD(P)H and fluorescent flavoproteins (in arbitrary units) was determined from the absolute respiratory chain dependent autofluorescence changes calibrated with NADH or riboflavin as fluorescence standards, respectively. n = number of orthopedic controls.

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(the mitochondrial NAD-system should remain reduced be- cause of almost complete block of respiratory chain). We observed, however, fluorescence changes which can be ex- plained only by a larger population of at least partially ‘in- tact’ mitochondria. To provide this partial intactness of respiratory chain the detectable by fluorometry mitochondria should contain as minimum one copy of the wild-type genome. Therefore a competition between wt- and ∆-mRNAs for protein biosynthesis seems to be a correct explanation for the observed functional defect of mitochondria, similar to the effects suggested for point mutations of tRNA genes [6].

Moreover, it is remarkable that the highest steady state reduction of the mitochondrial NAD-system, being indicative for a substantial respiratory chain inhibition, was observed for the patient with the largest degree of heteroplasmy of the mtDNA mutation (K.G.). This finding is in line with the low mitochondrial oxidation rates of the saponin-skinned muscle fibers from this patient.

Summarizing, the determination of steady state redox states of the mitochondrial NAD-system by detection of laser-excited autofluorescence changes of saponin-skinned muscle fibers proved to be a reliable method for the sensitive detection of respiratory chain defective mitochondria in small human biopsy samples. Considering the fact that these reduction levels do not depend on the amount of mitochondria in the muscle fibers this method can be especially useful in cases with large adaptive changes in the mitochondrial content.

Acknowledgments

The excellent technical assistance of Mrs. K. Kaiser and I.

Schellhase is gratefully acknowledged. This work was supported by grants of the Land Sachsen-Anhalt (1795A/0084 and 1919A/0025).

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