Effect

of microgravity

individual

muscle

JILL K. MANCHESTER,* IGOR KRASNOV,1 PATTI *Deparnr

of Pharmacology

USA, 1lnstitute Lyons, France

of Biomedical

on metabolic

enzymes

of

fibers

MAGGIE M.-Y. CHI, BEVERLY NORRIS,1 BERNARD FERRIER,S M. NEMETH,t DAVID B. MCDOUGAL, JR., AND OLIVER H. LOWRY and TNeurology, Problems,

Washington

Moscow,

USSR,

University

Sc/zoo!

and

of Medicine,

National

St. Louis,

Missouri

63110,

de Ia Sante et de Ia Recherche M#{233}dicale,

INDIVIDUAL FIBERS OF ANY MUSCLE vary greatly in enzyme composition, a fact that is obscured when enzyme levels of a whole muscle are measured. The purpose of this study was to assess the changes due to weightlessness in the enzyme patterns of the individual fibers within the flight muscles. Segments of individual fibers were dissected from freeze-dried portions of soleus and tibialis anterior (TA)’ muscles, and each segment was analyzed for two to eight different enzymes. This allowed direct comparisons of the covariance of enzymes from the same and different systems of energy metabolism in control and flight fibers. THE

ABSTRACT

Eleven enzymes were measured in individual fibers of soleus and tibialis anterior (TA) muscles from two flight and two control (synchronous) animals. There were five enzymes of glycogenolytic metabolism: phosphorylase, glucose-6-phosphate isomerase, glycerol-3phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase (group GLY); five of oxidative metabolism: citrate synthase, malate dehydrogenase, -hydroxyacyl-CoA dehydrogenase, 3-ketoacid CoA-transferase, and mitochondrial thiolase (group OX); and hexokinase, subserving both groups. Fiber size (dry weight per unit length) was reduced about 35% in both muscles. On a dry weight basis, hexokinase levels were increased 100% or more in flight fibers from both soleus and 1A. Group OX enzymes increased 56-193% in TA without significant change in soleus. Group GLY enzymes increased an average of 28% in soleus fibers but underwent, if anything, a modest decrease (20%) in TA fibers. These changes in composition of TA fibers were those anticipated for a conversion of about half of the originally predominant fast glycolytic fibers into fast oxidative glycolytic fibers. Calculation on the basis of fiber length, rather than dry weight, gave an estimate of absolute enzyme changes: hexokinase was still calculated to have increased in both soleus and TA fibers, but only by 50 and 25%, respectively. Three of the OX enzymes were, on this basis, unchanged in TA fibers, but 3-ketoacid CoA-transferase and thiolase had still nearly doubled, whereas TA GLY enzymes had fallen about 40%. In soleus fibers, absolute levels of OX enzymes had decreased an average of 25% and GLY enzymes were marginally decreased. MANCHESTER, J. K.; CHI, M. M. -Y.; NORRIS, B.; FERRIER, B.; KsNOV, I.; NEMETH, P. M.; MCDOUGAL, D. B., JR.; AND LOWRY, 0. H. Effect of microgravity on metabolic enzymes of individual muscle fibers. FASEBJ. 4: 55-63; 1990. -

Key Words: glycogenolytic enZymes’ oxidative metabolism enzyme hexokinase soleus muscle fiber tibialis anterior muscle

fiber

0892-6638/90/0004-0055/$01.50.

MATERIALS Source

METHODS

of muscles

The treatment received by the flight and synchronous animals is given in the overview paper (1). Preparation

of material

Small portions of soleus (slow-twitch) and TA (fasttwitch) muscles (originally frozen in liquid N2) were freeze-dried at -35#{176}C,and 2-3-mm segments of individual fibers were separated as described by Ess#{233}n et al. (2). These were stored separately under vacuum at - 70#{176}C, under which conditions they are stable indefinitely. Studies were made of more than 100 soleus and 200 tibialis fibers from two synchronous and two flight animals. Each fiber was analyzed in duplicate for two to eight different enzymes, and the size (tg/mm) was determined. This involved more than 2700 quantitative measurements. The work was expedited by a preliminary study which showed that most of the enzymes of interest can be extracted and stored without loss at - 70#{176}C in a spe-

‘Abbreviations: TA, tibialis anterior; LDH, lactate dehydrogenase; DH, dehydrogenase; GPI, glucose-6-phosphate isomerase; MDH, malate dehydrogenase; KACAT, 3-ketoacid CoAtransferase; S, hydroxyacyl-CoA

hydrogenase; PK, pyruvate

© FASEB

AND

synchronous transferase; dehydrogenase; GPDH,

HK, hexokinase; kinase.

PHRL,

F, flight; I3OAC, glycerophosphate

glycogen

fide-

phosphorylase;

55

m www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on November 11, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumb

cial glycerol-KCI-detergent medium (20 mM potassium phosphate buffer, pH 7.4, 25% glycerol, 0.6 M KCI, 0.5% Triton X-100, 5 mM /3-mercaptoethanol, 0.5 mM EDTA, and 0.02% bovine serum albumin). Each dry sample, weighing about 0.5 tg (0.5-1 mm long), was added to 5 pJ of this special medium under mineral oil. After incubation for 2 h at room temperature, the samples were transferred to a - 70#{176}C freezer. Since each assay required only 0.1-0.2 tl of extract (equivalent to 10-20 ng of dry fiber), the single 5-tl ex-

histochemistry were mounted on coverslips, air-dried, and stained for ATPase according to Brooke and Kaiser (10), with preincubation at pH 10.0, 4.5, or 4.2. Photomicrographs of the stained sections were used as guides in selecting fibers of designated ATPase type from the freeze-dried sections. Usually duplicate samples of suitable size (5-15 ng dry weight) could be dissected from the selected fiber in one cross section. Moreover, the same fiber could readily be identified in successive

tract

each

was

sufficient

for duplicate

assays

of a large

num-

of different enzymes. One enzyme, glycerophosphate dehydrogenase, was inhibited by the Triton X-100 in this medium and therefore dry samples weighing 15-20 ng were added directly to the specific reagent. Thiolase activity was partially lost after long storage with repeated freezing and thawing. Therefore, definitive assays were made with fresh extracts. For technical details of sampling, weighing, and conducting enzyme assays on this scale, see refs 3 and 4.

sections,

permitting

measurement

of both

enzymes

in

fiber.

ber

Enzyme

assays

The methods were adapted from procedures described previously: malate dehydrogenase (EC 1.1.1.37) in ref 5, hexokinase (EC 2.7.1.1) and 3-ketoacid CoA-transferase (EC 2.8.3.5) in ref 6, thiolase (EC 2.3.1.9) in refs 7 and 8, glucose-6-phosphate isomerase (EC 5.3.1.9) in ref 9, and the rest in ref 3. They all contain a pyridine nucleotide step, which made possible the amplification by enzymatic

cept Fiber

cycling

lactate

that

was

needed

for all the enzymes

ex-

dehydrogenase.

RESULTS The enzymes that were measured fall into two groups: six that are usually most active in slow-twitch and fasttwitch oxidative fibers [hexokinase and five enzymes of oxidative metabolism-citrate synthase (EC 4.1.3.7), malate dehydrogenase (MDH), /3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), 3-ketoacid CoA-transferase, and mitochondrial thiolase] and five that are.rnost active in fast-twitch glycolytic fibers [glycogen phosphorylase (EC 2.4.1.1), glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase (LDH) (EC 1.1.1.27), and glucose-6phosphate isomerase]. Control

activities

In the synchronous animals the fast-twitch glycolytic group of enzymes were 3- to 11-fold higher in TA than soleus muscles (Fig. 1, Table 1). Average fiber size was almost the same for both types of muscle. In contrast,

size

Size IlK CS MOM0AC FT

Fiber sizes were determined by weighing the whole dry fiber segment after measuring its length under the microscope. Sizes are reported here in micrograms per millimeter. These can be converted to cross-sectional area (in m2) by multiplying by 3590 or to the diameter (in tm) of a cylinder of equivalent volume by multiplying (ig/mm)”2 by 67.6; 67.6 (3590 x 4/ir)2 (6). We believe there is no change in fiber length during freeze-drying. However, since the muscles were removed from the body before quick-freezing, there could have been uncontrolled differences in muscle length and hence in fiber size (weight per unit length). =

Myofibrillar on the same

ATPase fibers

typing

and

enzyme

Vol. 4

Jan. 1990

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soleus

4O-

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20

assays

In addition to enzyme measurements on samples from the isolated fiber segments, assays for pyruvate kinase and glycerol-3-phosphate dehydrogenase were made on single fiber samples dissected from freeze-dried transverse cryostat sections. This allowed direct correlation of enzyme levels with fiber type, determined by myofibrillar ATPase staining of adjacent sections. The sections were cut sequentially at - 20#{176}C; those for enzyme assay were dried under vacuum at -35#{176}C;those for

56

100

KCT

I

0

I

I

I

I

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Figure 1. Comparison of average size and enzyme levels of the fibersof the synchronous soleus and TA muscles of Tables 1 and 2. Abbreviations: HK, hexokinase; CS, citrate synthase; MDH, malate dehydrogenase; I3OAC, /3-hydroxyacyl-CoA dehydrogenase; KACT, 3-ketoacid CoA-transferase; TH, mitochondrial thiolase;

PHRL,

glycogen phosphorylase;

GPI, glucose-6-phosphate

isomer-

ase;GPDH, glycerophosphate dehydrogenase; PK, pyruvate kinase; LDH, lactate dehydrogenase.

The FASEB Journal

MANCHESTER

ET AL.

m www.fasebj.org by Kaohsiung Medical University Library (163.15.154.53) on November 11, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumb

the averages for five of the six enzymes of the oxidativehexokinase group were 45-50% higher in soleus than in TA muscle (Fig. 1; Table 1). Citrate synthase was the exception, having similar activities in the two muscles. The enzyme variability among the fibers of each muscle type are of some interest (Fig. 2). The coefficient of variation (CV) differs markedly among the different enzymes and between the two muscle types. In the synchronous muscles, all of the CVs were much higher (64-900%) for slow-twitch enzymes of TA than for those of soleus muscles, whereas the reverse was true for fast-twitch glycolytic enzymes. (Analytical errors were in the order of 5% and were, therefore, almost negligible relative to these large CVs.) Note the greater variability in TA for f3-hydroxyacyl-CoA dehydrogenase (a key enzyme of fatty acid oxidation) than for the two members of the citrate cycle. Variations in fiber size were almost the same for both muscles. Effects

of weightlessness

on average

values

Since data are available, in most cases, for only two synchronous and two flight muscles, caution must be observed in interpreting small average differences. However, examination of individual fiber patterns is helpful in this regard. Tables 1 and 2 and Figs. 3 and 4 compare average enzyme activity and fiber size for each synchronous and flight muscle studied. The average size (dry weight per unit length) was about 35% lower in flight than in synchronous muscles of both types (Table 1). All the enzyme activities in the tables are based on dry weight. TABLE

1.

Levels

of five enzymes

characteristic

size HK CS MOMKCT TH $OACPHRLGPI GRDH I I’

Phosphorylase

0.84

0.60

(16)

S9

± 0.06

-

I

60

x

S

S

40

S

x /

i’soleus

S S

20

S..

F9

0.03 0.50 (16) ±

±

Tibialis S8

0.02

- -

I

0

I

I

x

--

I

I

Figure

2. Coefficients of variation of enzyme levels of individual fibersof the synchronous soleus and TA muscles of Tables 1 and 2. Abbreviations as in Fig. 1.

Therefore the absolute enzyme contents of the fibers from flight muscles are on the average 35% lower than would appear from these data (if it is assumed that synchronous and flight muscles were frozen at equal length). This will be discussed later. In soleus muscle, the only conclusive enzyme change with flight was in hexokinase, which increased an average of 137% on the dry weight basis (Table 1; Fig. 3). Four of the enzymes of oxidative metabolism were clearly unchanged. 3-Ketoacid CoA-transferase activities averaged 29% lower in the flight fibers but the difference between muscles was too great for this to be meaningful. The five enzymes associated with glyco-

GPI

(10)

0.70

Glycerol-P DH

(16)

± 0.05

Pyruvate kinase

7.72

(16)

± 0.43 10.1 (10) ± 1.5

LDH

24.0

(16)

± 1.1 23.0 (16) ± 1.6

(8) 1.7

(10)

± 0.12 1.46 (10) ± 0.38

(dry) at 20#{176}C

kg

0.47

±

(16)

x S

13.6

0.61

S.

“1