Indian Journal of Experimental Biology Vol. 38, June 2000, pp. 575-579

Effect of thyroidectomy on fast and slow muscle fibres of rat gastrocnemius muscle

Nisha Vashi shta & C L Talesara*

Muscle Physiology & Hi stochemistry unit, Department of Zoology, University of Delhi, Delhi II 0007, Indi a

Received 23 July 1998; revised I March 2000

A combined histochemical, biochemi cal and electrophoreti c study with respect to the enzymes succnic dehydroge­nase(S DH ),myofibrillar adenosi ne triphosphatase (rn-A TPase), lactate dehydrogenase (LDH) isozymes and myosin li ght chains was carri ed out to invest igate the response of rat gastrocnemiu s muscle (medial head). Twelve weeks after thyroi­dectomy, the results indicated a shift from fas t to slow type pattern of LDH isozymes, fibre type transformati on from Type II to Type I and a decrease in SOH and m-ATPase activity. The results suggest, possible thyroidal in volvement in determining the phenotypic properties of skeletal muscle.

A good deal of the functional diversity of skeletal muscle derives from differences in their fibre type distribution and this also helps to determine metabolic diversity of skeletal muscle1.2. Mammalian skeletal muscles are broadly composed of red, white and intermediate fibres. Physiologically based on the contraction pattern they are designated as slow twitch (Type I) and fast twitch (Type II) fibres. Slow twitch fibres are also referred as slow oxidative (SO) and are fatigue resistant. Fast twitch fibres are called as Type II - red and white fibres and these are further categorized as Type ITa - red, fast oxidative glycolytic (FOG), fatigue resistant and Type lib - white, fast glycolytic (FG), easily fatiguable fibres3

-7

. Differential response of these fibres to various stress conditions has been studied exhaustivell'9.

Succinic dehydrogenase (SOH) and myofibrillar adenosine triphoshatase (m-ATPase) are two very important enzymes concerned with the musc le study. The relative level of m-ATPase activ ity reveals the contracti le pattern (fast or slow) of a muscle 10

.

Whereas, SOH re flects the oxidative capacity of a muscle 11

•12

• To know the metabolic profile of the muscle type, lactate dehydrogenase (LDH) isozymes distribution pattern was studied 13

'14

• To confirm the physiological contractile patterns, electrophoretic studies of myosin light chains (LC 1,LC2 and LC3) were undertaken1

5.16

. The present work has been undertaken to elucidate the specific correlation of myos in light chains and LDH isozyme pattern, SOH and m-ATPase

*Correspondent author.

activity and fibre type compositions of gastrocnemius muscle following thyroidectomy.

Materials and Methods Adult (I 0-12 weeks) male albino rats, Rattus nor­

vegicus weighing 130-150 g were used as a mam­malian representative for the experiments. Rats were kept under standard laboratory condition s, fed on commercial food (Gold M ohur rat feed, Hindustan Lever Ltd.) , and water ad libitum.

Thyroidectomy was performed asepticall y under general anaesthesia with the he lp of ether17

• The ani­mal s were sacrificed, after 12 weeks of thyroidec­tomy . Six to e ight animals were used in each batch of experimental animals . Gastrocnemius musc le (medial head) from animals of simil ar weight range and age group was used as control. Gastrocnemius mu scle has been selected in particular as a mammalian represen­tative because of its variability in fibre types and its relation between complex function s and associated complex interna l organization . It is an important lo­comotor muscle of fast running rats ass isting in ex­tension of ankle joint and fl ex ion of knee . The mus­cles so exci sed from control and experimental rats were processed for the corre lati ve hi stochemical, bio­chemical, and e lectrophoretic analysis.

For hi stochemical studies the mu sc les were frozen in liquid nitrogen and immedi ately transferred to a cryostat (IEC-Mode l CTD-International Harris Cry­ostat) at -25°C. Cross sections ( I 0 11m thick) were made and stained for SDH 18

• Other sections were stained for alkali and ac id stable forms of myofibril Jar

576 INDIA N J EXP BIOL, JUNE 2000

adenos ine triphosphatase (m-ATPase) bas ically by the method of Padykula and Herman19 as modified by Guth and Samaha20

. For Biochemi cal, myofibril lar -ATPase assay, myofibril s were prepared according to Perry and Gre/1

. Assay was perfo rmed within 6 hr of myofibril preparati ons at pH 7.5 and at 37°C. Values

were expressed as Jl moles of Pi liberated/rug pro­tein/min . Amount of Pi liberated and prote in content present was measured by the method of Rockste in and Herron22 and Gorna ll et al.23 respecti ve ly . SOH activity was measured in musc le homogenate using TNT [2-(p-iodophenyl, 3 (p-nitrophenyl )-5-phenyl tetrazo lium chl oride] as e lectron acceptors and 0.2 M sodium succ inate as a substrate24

. Values were ex­pressed as Jlg formazan I mg prote in I min .

Electrophoretic methods and gel scanning­Isozymes were separated and stained for LOH by 7% polyacrylamide gel e lectrophores is (PAGE) according to Oavis25

. Samples were prepared in chi lled saline EOTA buffer (pH 8). Gels were stored in 7% acetic

ac id and scanned in cecil (CE570) ge l scanner. Myo­fibril s were used for SOS PAGE according to the method of Laemmli26

. Ge ls were stained in Coomasie brilliant blue and preserved in 7% acetic ac id , scanned in LKB (Bromma) 2202 ultra scan laser den­sitometer.

Statistical analysis All the data were analyzed with two way ANOV A.

P<0.05 was considered significant.

Results and Discussion P . k 27-29 h d . f rev10us wor ers ave reporte convers tons o

various muscle properties fo ll owing ex perimental hyper and hypothyroidism in rats. The results of the present study confirm the earlier findings and further show remarkable consistency in the directi on of change of several corre lated musc le properties. In the present study the large scale transformati ons in histo­chemical fibre types, SOH and m-ATPase acti vities,

Table !-Effect of thyroidectomy ( 12 weeks postoperati vely) on the adult rat gastrocnemius muscle [Values are mean±SE]

A Parameters

Normal Gas­trocnemius Muscle (cont ro l)

Thyroidectomized Gastrocnemius muscle (experimental)

Body wt (g)

134.25 ± 1.84

102.25 ±2.8'

Serum T4 (}lg/dl)

10. 1 ±1.8

0.65 ±0. 11 '

Mean fi bre diameter (}lm)

Type I Type II

57.92 64.52 ±0.06 ±0.1 9

32.6 34.46 ±0.06' ±0.03'

Percentage composition SOH acti vity m-ATPase of fi bre types (}lg formazan/ acti vity

mg protein/min. ) (}lmoles pi/mg protein/min.)

Type I Type II

33 67 4.51 0.44 ±0.12 ±0.02

59' 4 1' 2.66 0.27 ±0.23' ±0.01'

' P<0.05; SOH = succinate dehydrogenase; m-ATPase = myolibrill ar adenosine triphosphatase; LOH = lactate dehydrogenase

B

Parameters

Normal Gastrocnemius Muscle (cont ro l)

Thyroidectomized Gastrocnemius muscle (ex peri ­mental)

+

+++

Amount of LOH subunits based on densitometry

MH, M2H2 M1H M4

++ +++ ++++ +++++

+++ ++++ +++ ++

LOH, Lactate dehydrogenase- +++++ very high, ++++ high, +++ moderate, ++ low, + least Myosin light chai n pattern - +++ high, ++ moderate, +low

Myosin light chai n pattern based on densi tometry

+ ++ +++

+ ++ ++

VASHISHTA & TALESARA: THYROIDECTOMY & MUSCLE FIBRES OF RAT GASTROCNEMIUS 577

LDH and myosin light chain patterns were observed in the fast to slow direction with hypothyroidism.

Thyroidectomy resulted in an expected reduction in the body weightin g and in T4 serum level in (!-Lg/d l; Table I ) . Figs I and 2 sh0w hi stochemical changes in the thyroidectomized rat gastrocnemjus muscle as compared with the control gastrocnemius muscle. Type I and Type II [Fig.2 (Band C)] fibres underwent decrease in their mean diameters (Table I; P< 0.05) . It has previously been shown that fibre size increases as a function of muscle weighe0

. Therefore, keeping the decrease in body weight as described above in

.. -, ·········-~···. ~\-T: ••• ' • \ •" ., ' , f

' ,.. .,, ' ~ ' • • . , ,' ~

,..' \ ... -~·

. ~

~ I .. .. ~· ~ ~ · \

view it seems likely that such reduced fibre diameters are at least partly due to an Inhibition of muscle growth in the hypothyroidectomized rat.

The result showed an increase in the percentage composition of Type I (slow twitch) fibres with the concomitant decrease in the percent composition of Type l1 (fast twitch) fibres [Figs I and 2 (B and C), Table I] . As in the histochemical reaction the basis for resolving fast twitch and slow twitch myos in ATPase is its property of being alkali resistnnt and susceptible20

. Increase in the proportion of Type I fibres following thyroidectomy in the present work is

Figs I and 2-Cross sections of normal (Fig. I) and thyroidectomized (Fig. 2) gastrocnemius muscle in adult rats, stained for SOH (A), m-ATPase after acid preincubation (B) and after alkali ne preincubation (C) X 100. Normal muscle showing three type of fibres on the basis of SOH and two types of fibres on the basis of m-A TPase staining. Thyroidectomized rat muscle ( 12 weeks postoperatively) showed decrease in the percentage composi tion of Type II fibres , fibre diameter and staining intensity as compared to their respective control s.

578 INDIAN J EXP BIOL, JUNE 2000

consistent with the increase observed in alkali lability and thi s change in the alkaline stability clearly shows that the property of the myosin has been transformed 12 weeks after thyroidectomy in fast to slow direc­tion. The extent of the changes in myosin ATPase activity during hypothyroidi sm was simjlar to that of the changes observed in fast muscle following long term cross innervation31

• The reduction in the m­ATPase activity and fast light chain contents [Fig 3(D) and Table I] paralleled closely the conversion of fast twitch into a slow twitch fibres of the gastrocne­mius muscle in hypothyroidectomized rats. The ex is­tence of multiple isoforms of the myofibril tar proteins is a major factor in fibre type di versity and it is the myofibrillar isozymes composition of the fibre which

~ - + 1

+

:. _j _ ~ s;

. I h .I h . . 12 11 matn y governs t e contractt e c aractenstlcs· ·· ·. On the basis of polyacrylamide gel electrophoresis

five forms of LDH isozymes have been resolved in the normal adult gastrocnemius muscle (Fig. 3A). The differences in the isozyme composition are presuma­bly correlated with the metabolic differences in vari-

. t4 14 I h I . I ous tt ssues ·· . n t e norma gastrocnemiUs muse e, the slowest moving band (towards the cathode) is designated as LDH-5 or the M-type, M-LDH and the successive band towards the anode are numbered as LDH-4, LDH-3, LDH-2 and LDH-1. The normal ga -trocnemius muscle shows maximum enzyme activity of LDH-5 (M-LDH). LDH has been previously shown to be a sensitive marker for muscle transformation as it is known to undergo virtually complete transforma-

I I I LC3 LC2 Lc1

+

Fi g. 3-Lactate dehydrogenase isozymes and myosin li ght chains of normal and thyro idectomi zed ( 12 weeks postoperat ively) gastrocne­mius muscle of adult rats as separated on polyacrylamide gel electrophoresis (PAGE) and SDS-polyacrylamide gel electrophoresis (S DS­PAGE), respectively wi th thei r densitometric scans. A, LDH isozymes of normal gastrocnemi us mu scle; 8 , LDH isozymes of 12 weeks thyroidectomi zed gastrocnemius mu scle; C, myosi n li ght chains of normal gastrocnemius muscle; D, Myosin light chains of 12 weeks thyroidectomi zed gastrocnemius muscle. [ signs + and - indicate the direction of current tl ow. Number I , 2, 3, 4 and 5 represent LD H isozymes H4, H1M. H2M2, HM3 and M4, respectively. LC 1, LC2 and LC3 represent myosin light chai ns].

YASHISHTA & TALESARA: THYROIDECTOMY & MUSCLE FIBRES OF RAT GASTROCNEMIUS 579

tion in short term cross innervation studies29. The pre­

sent study also showed the redistribution in selective proportion of LDH isozymes after 12 weeks of thy­roidectomy . There was a decrease in M subunit indi­cating the change in isozy '11e pattern from fast to s low type (Fig. 3B and Table I).

The results clearly indicate that the thyroidecto­mized rat gastrocnemius muscle results in fibre type transformation from Type II (fast twitch) to Type I (s low twitch) in gastrocnemius muscle of rats . Al­though the present study gives very little ins ight into the mechani sm by which the thyroid state alters the character of skeletal mu scle, it clearly suggests the possible thyroidal involvement in determining the phenotypic properties of the skeletal muscle .

Acknowledgement Thanks are due to UGC, New Delhi for the award

of SRF to NV and to Mr. E .A. Daniel s for ass istance in photomicrography .

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