Mechanisms of Ageing and Development, 52 (1990) 1-- 10
1
Elsevier Scientific Publishers Ireland Ltd.
N E O N A T A L H Y P E R T H Y R O I D I S M E N H A N C E S G R O W T H OF THE H E A R T B U T N O T OF S K E L E T A L M U S C L E
R. MOUSSAVI* Department of Physiology-Anatomy, University of California, Berkeley, California, 94720 (U.S.A.)
(Received May 19th, 1989)
SUMMARY Comparison of growth and biochemical development of the heart and skeletal muscle (gastrocnemius) in normal rats and those treated with triiodothyronine (T3) (0.1/ag/g bw, s.c.) daily, f r o m birth until weaning (day 25), revealed that hyperthyroidism selectively enhanced cardiac growth as shown by marked increases in total content (per heart) of D N A (25o7o), RNA (22%), protein (31%), and myofibrillar protein (30%), as well as the activity of myosin ATPase (11--20°70). On the contrary, the above parameters were significantly decreased in the skeletal muscle of hyperthyroid rats as was body weight. Termination of T3 treatment on day 25 led to resumption of rapid body growth which was not complete even by day 90. Cardiac growth decelerated in rehabilitating rats, however, resulting in final reduction of all growth parameters, except D N A content which was equal in 90-day control and rehabilitated animals. Muscle growth in terms of D N A and RNA content failed to respond to rehabilitation and continued to be lower than control animals while protein synthesis was relatively enhanced. It is concluded that neonatal hyperthyroidism markedly enhanced growth of the heart but not of skeletal muscle. This condition not only inhibited growth of the muscle during the T3 treatment period but also led to permanently reduced D N A content and other growth parameters even two months after discontinuation of treatment.
words: Hyperthyroidism; Cardiac muscle; Skeletal muscle; DNA; RNA; Protein; growth; Myosin ATPase; Na-K-ATPase
Key
INTRODUCTION Thyroid hormone affects adult cardiac and skeletal muscle size. Hyperthyroidism
*Present address: National Institute of Health Building 6, Room 114 Bethesda, Maryland 20892, U.S.A.
0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
induces hypertrophy of the heart mainly by stimulating protein and RNA synthesis [1--5]. It also alters contractile properties of the heart, possibly by increasing myosin ATPase activity [6--11] or myosin isozymes [7,10,12,13]. Conversely, hyperthyroidism produces wasting and weakness in skeletal muscle of both humans and experimental animals [14--17]. Muscle wasting in this condition is attributed to increased protein degradation [18--20]. Hyperthyroidism is also associated with abnormalities in the contractility of skeletal muscle, possibly by altering the fiber type composition or by changing the activity of myosin ATPase in fast and slow fibers or both [21--27]. Indirect effects have also been suggested for thyroid hormones via a neurally mediated mechanism [28]. Although the role of thyroid hormone in adult animals has been studied widely, its mechanism of action in developing animals is less well understood. The goal of this study was to compare the effects of neonatal hyperthyroidism, as induced by treatment with triiodothyronine (T3) from birth, on growth and differentiation of the heart and gastrocnemius muscle, and the ability of these organs to normalize their growth patterns after discontinuance of the hyperthyroid treatment. The experiments were carried out in male rats. Cardiac and skeletal muscle growth was assessed by measuring the changes in total content of DNA, RNA and protein. Effects on biochemical differentiation were determined by assaying for the activity of Na*-K*-ATPase and myosin ATPase as markers for excitability and contractility of the heart and muscle tissue. MATERIALS AND METHODS
Animals Animal care and distribution of pups have been previously described [29,30]. Newborn Long-Evans rats (8 pups per litter) were assigned randomly to normal (control), hyperthyroid, or hyperthyroid-rehabilitated groups. All animals were weaned on postnatal day 25. Special care was taken that soft food and water were accessible to the smaller and weaker postweaning hyperthyroid animals. Mortality among the hyperthyroid animals was 25--35°7o, leaving only 5 - - 6 survivors in each litter. Hyperthyroidism and rehabilitation Hyperthyroidism was induced by daily subcutaneous injections of T3 (0.1 /ag/g body weight dissolved in 0.01 N N a O H ) from birth until sacrifice at 12 days or weaning (25 days). Control animals were injected with 0.01 N N a O H (1 ml/Kg) equal in volume to the T3 dose. T3 is believed to be the active intracellular form of the hormone and is routinely used to induce hyperthyroidism [31 ]. Rehabilitation (recovery) from the hyperthyroid state was induced by discontinuing the T3 treatment at day 25. Animals were allowed to recover up to day 90, when, in normal rats, growth of the heart is almost complete and skeletal muscle development is close to its plateau in terms of cell proliferation [29,32,33].
Tissue sampling and chemical assays Tissues were removed and chemical analyses were performed according to previously described methods [29,30]. DNA, RNA and proteins were measured at 12, 25, and 90 days of age; myofibrillar proteins, Mg 2÷ and CaE*-activated myofibrillar ATPase activity and Na*-K*-ATPase activity were measured only at 25 days. Statistics The Student's t-test for unpaired sample means was used to determine the significance of differences between the means of control and experimental groups. P values of less than 0.05 were considered statistically significant. RESULTS
Hyperthyroidism The hyperthyroid pups showed the classic signs of accelerated bodily development and differentiation characteristic of postnatal hyperthyroidism including 2--3 day earlier eye-opening, appearance of fur and ear growth, and maturation of skull size and shape. As shown elsewhere [34,35], the body weights were significantly lower (30%) in the 25-day-old hyperthyroid rats compared to controls (Fig. 1).
- 400
Body Weight (g) i-Start Ta
f /
I-Stop T3
/
":560
-320 * -280
60-240 50C.°ntr°l.--...x / : N ° t m ° ' / ~ o ..'"'i(50%)
40-
/i ~I5°1o) i/~...I~
Rehob, i i l., ,e d
-200 -160
30
li
/.'" u6 lb i; 2b 25 25 3L~ 4's 5's 6'5 z'5 a'~ 9's Age (days)
Fig. 1. Effects of T3 administration from birth (hyperthyroidism) and its discontinuance at day 25 (arrows) on body growth and weight in male rats. Asterisks (*) indicate a signficant difference (P < 0.01) between experimental and control groups.
2300-
Heart
400(? ¸
~ 1500'
o
(~)
,~ 1200
o
180
2400
120~L
(z2)
~ 1600,
1
Elsevier Scientific Publishers Ireland Ltd.
N E O N A T A L H Y P E R T H Y R O I D I S M E N H A N C E S G R O W T H OF THE H E A R T B U T N O T OF S K E L E T A L M U S C L E
R. MOUSSAVI* Department of Physiology-Anatomy, University of California, Berkeley, California, 94720 (U.S.A.)
(Received May 19th, 1989)
SUMMARY Comparison of growth and biochemical development of the heart and skeletal muscle (gastrocnemius) in normal rats and those treated with triiodothyronine (T3) (0.1/ag/g bw, s.c.) daily, f r o m birth until weaning (day 25), revealed that hyperthyroidism selectively enhanced cardiac growth as shown by marked increases in total content (per heart) of D N A (25o7o), RNA (22%), protein (31%), and myofibrillar protein (30%), as well as the activity of myosin ATPase (11--20°70). On the contrary, the above parameters were significantly decreased in the skeletal muscle of hyperthyroid rats as was body weight. Termination of T3 treatment on day 25 led to resumption of rapid body growth which was not complete even by day 90. Cardiac growth decelerated in rehabilitating rats, however, resulting in final reduction of all growth parameters, except D N A content which was equal in 90-day control and rehabilitated animals. Muscle growth in terms of D N A and RNA content failed to respond to rehabilitation and continued to be lower than control animals while protein synthesis was relatively enhanced. It is concluded that neonatal hyperthyroidism markedly enhanced growth of the heart but not of skeletal muscle. This condition not only inhibited growth of the muscle during the T3 treatment period but also led to permanently reduced D N A content and other growth parameters even two months after discontinuation of treatment.
words: Hyperthyroidism; Cardiac muscle; Skeletal muscle; DNA; RNA; Protein; growth; Myosin ATPase; Na-K-ATPase
Key
INTRODUCTION Thyroid hormone affects adult cardiac and skeletal muscle size. Hyperthyroidism
*Present address: National Institute of Health Building 6, Room 114 Bethesda, Maryland 20892, U.S.A.
0047-6374/90/$03.50 Printed and Published in Ireland
© 1990 Elsevier Scientific Publishers Ireland Ltd.
induces hypertrophy of the heart mainly by stimulating protein and RNA synthesis [1--5]. It also alters contractile properties of the heart, possibly by increasing myosin ATPase activity [6--11] or myosin isozymes [7,10,12,13]. Conversely, hyperthyroidism produces wasting and weakness in skeletal muscle of both humans and experimental animals [14--17]. Muscle wasting in this condition is attributed to increased protein degradation [18--20]. Hyperthyroidism is also associated with abnormalities in the contractility of skeletal muscle, possibly by altering the fiber type composition or by changing the activity of myosin ATPase in fast and slow fibers or both [21--27]. Indirect effects have also been suggested for thyroid hormones via a neurally mediated mechanism [28]. Although the role of thyroid hormone in adult animals has been studied widely, its mechanism of action in developing animals is less well understood. The goal of this study was to compare the effects of neonatal hyperthyroidism, as induced by treatment with triiodothyronine (T3) from birth, on growth and differentiation of the heart and gastrocnemius muscle, and the ability of these organs to normalize their growth patterns after discontinuance of the hyperthyroid treatment. The experiments were carried out in male rats. Cardiac and skeletal muscle growth was assessed by measuring the changes in total content of DNA, RNA and protein. Effects on biochemical differentiation were determined by assaying for the activity of Na*-K*-ATPase and myosin ATPase as markers for excitability and contractility of the heart and muscle tissue. MATERIALS AND METHODS
Animals Animal care and distribution of pups have been previously described [29,30]. Newborn Long-Evans rats (8 pups per litter) were assigned randomly to normal (control), hyperthyroid, or hyperthyroid-rehabilitated groups. All animals were weaned on postnatal day 25. Special care was taken that soft food and water were accessible to the smaller and weaker postweaning hyperthyroid animals. Mortality among the hyperthyroid animals was 25--35°7o, leaving only 5 - - 6 survivors in each litter. Hyperthyroidism and rehabilitation Hyperthyroidism was induced by daily subcutaneous injections of T3 (0.1 /ag/g body weight dissolved in 0.01 N N a O H ) from birth until sacrifice at 12 days or weaning (25 days). Control animals were injected with 0.01 N N a O H (1 ml/Kg) equal in volume to the T3 dose. T3 is believed to be the active intracellular form of the hormone and is routinely used to induce hyperthyroidism [31 ]. Rehabilitation (recovery) from the hyperthyroid state was induced by discontinuing the T3 treatment at day 25. Animals were allowed to recover up to day 90, when, in normal rats, growth of the heart is almost complete and skeletal muscle development is close to its plateau in terms of cell proliferation [29,32,33].
Tissue sampling and chemical assays Tissues were removed and chemical analyses were performed according to previously described methods [29,30]. DNA, RNA and proteins were measured at 12, 25, and 90 days of age; myofibrillar proteins, Mg 2÷ and CaE*-activated myofibrillar ATPase activity and Na*-K*-ATPase activity were measured only at 25 days. Statistics The Student's t-test for unpaired sample means was used to determine the significance of differences between the means of control and experimental groups. P values of less than 0.05 were considered statistically significant. RESULTS
Hyperthyroidism The hyperthyroid pups showed the classic signs of accelerated bodily development and differentiation characteristic of postnatal hyperthyroidism including 2--3 day earlier eye-opening, appearance of fur and ear growth, and maturation of skull size and shape. As shown elsewhere [34,35], the body weights were significantly lower (30%) in the 25-day-old hyperthyroid rats compared to controls (Fig. 1).
- 400
Body Weight (g) i-Start Ta
f /
I-Stop T3
/
":560
-320 * -280
60-240 50C.°ntr°l.--...x / : N ° t m ° ' / ~ o ..'"'i(50%)
40-
/i ~I5°1o) i/~...I~
Rehob, i i l., ,e d
-200 -160
30
li
/.'" u6 lb i; 2b 25 25 3L~ 4's 5's 6'5 z'5 a'~ 9's Age (days)
Fig. 1. Effects of T3 administration from birth (hyperthyroidism) and its discontinuance at day 25 (arrows) on body growth and weight in male rats. Asterisks (*) indicate a signficant difference (P < 0.01) between experimental and control groups.
2300-
Heart
400(? ¸
~ 1500'
o
(~)
,~ 1200
o
180
2400
120~L
(z2)
~ 1600,
