Changes in the kinetics of muscle contraction in vitamin D-depleted rats

Kidney International, Vol. 13 (1978), pp. 189—1 93

Changes in the kinetics of muscle contraction in vitamin Ddepleted rats JOHN S. RODMAN and THOMAS BAKER Department of Pharmacology, Cornell University Medical College, and Department of Medicine, Lenox Hill Hospital, New York, New York

Changes in the kinetics of muscle contraction In vitamin Ddepleted rats. Using an in situ rat soleus neuromuscular preparation, changes in the muscle contraction kinetics in response to vitamin D depletion were studied. For a single isometric contraction, the time-to-peak tension (T9) and the time-for-recovery-halfway-to-resting tension (Ti,2r) were recorded. For a 150 Hz, 300 msec tetanus, the T112 was determined. Animals raised on highcalcium, high-phosphate, vitamin D-depleted diets showed prolongation of all parameters. Repletion of vitamin D returned T and

weakness" in 20 of 45 patients with osteomalacia of varying causes. In these patients, strength improved with vitamin D therapy. The myopathy associated with osteomalacia is characterized by a patchy, predominantly proximal involvement, brisk reflexes,

Tiir values to normal. Neither dietary calcium deficiency nor

dence of denervation. Concomitant bone pain makes evaluation of the extent of muscle weakness difficult. Muscle enzymes are usually normal but the alkaline phosphatase is elevated [2]. With the exception of Xlinked hypophosphatemic rickets [101, all forms of osteomalacia are associated with muscle weakness. Hypocalcemia is uncommon in osteomalacia, and the degree of muscle weakness rarely correlates with

thyroparathyroidectomy produced any prolongation of T or Tizr values. Therefore, based upon the experimental data, it appears that vitamin D or one of its metabolites, independent of any effect on the serum calcium or serum phosphate concentration, is necessary for normal muscle relaxation. Modifications des cinetiques de Ia contraction musculaire chez les rats déplétés en vitamine D. Les modifications des cinétiques de Ia contraction musculaire en réponse a une déplétion en vitamine

D ont été étudiées sur une preparation neuromusculaire de

and a waddling gait. The electromyogram shows abnormalities of the myopathic type without evi-

solCaire. Pour une contraction isométrique unique, le temps de tension maximale (T9) et Ic temps de demi-rCcuperation de Ia

the serum calcium [2]. Vitamin D or one of its

tension de repos (T112r) ont Cté enregistrés. Le Tii2r a etC determine pour un tétanos de 300 msec a 150 Hz. Chez les aniniaux élevés avec une alimentation riche en calcium et en phosphate et depourvue de vitamine D, un allongement de tous ces paramètres a Cté observe. L'administration de vitamine D a ramenC T et Ti,2r a Ia normale. Ni Ic deficit alimentaire en calcium ni Ia thyropara-

to study the effect of vitamin D depletion on the

metabolites may therefore be necessary for normal muscle function. These experiments were designed kinetics of muscle contraction.

thyroidectomie n'ont produit d'allongement de T ou de T112.

Methods

Ainsi 11 apparait, de ces rCsultats experimentaux, que Ia vitamine D

Male Sprague-Dawley rats were raised from the weaning stage for 65 to 74 days on the various regimens indicated in Table 1. Special test diets were purchased from Teklad Laboratories. These were: TD 74597 with 1.5% calcium, 1.5% phosphorus, and vitamin D; TD 73446 with 1.5% calcium, 1.5% phosphorus, and no vitamin D; TD 76028 with 0.05%

ou I'un de ses métabolites, indCpendamment du calcium ou du phosphore sCriques, est nCcessaire a une relaxation musculaire normale.

Hemodialysis keeps patients with chronic renal

failure alive, but often these patients develop musculoskeletal problems. Dialysis patients show two patterns of muscle weakness: one affecting more

calcium, vitamin D, and no phosphorus added to casein hydrolysate; and TD 73445 with 0.05% calcium and no phosphorus added to casein hydrolysate and no vitamin D. The animal quarters were free of ultraviolet radiation. Animals in group 2 were thyroparathyroidectom-

distal muscles and associated with neuropathy and the other affecting predominantly proximal muscles [1]. The latter pattern is descriptively identical to that seen with various forms of osteomalacia [2] and may respond to therapy with 1 ,25-dihydroxycholecalciferol [3]. Ample documentation of the association between

ized by cautery 10 days before the experiments. Only those animals of this group with serum calcium concentrations less than 7.0 mgIlOO ml were included in

osteomalacia and muscle weakness exists [4—9]. Smith and Stern [4] found "unequivocal muscle

the final data. Animals in group 5 received 1 mg of

Received for publication May 16, 1977; and in revised form September 16, 1977.

0085—2538/78/0013—0189 $01.00

© 189

1978,

by the International Society of Nephrology.

Rodman and Baker

190

Table 1. Regimens for all groups of rats studied Group no. I

2 3 4 5 6

Single contraction

Description

Normal diet (Purina Rat Chow) Normal diet, parathyroidectomy performed two weeks prior to experiment High-calcium (1.5%), high-phosphate (1.5%), with vitamin D diet High-calcium (1.5%), high-phosphate (1.5%), without vitamin D diet High-calcium (1.5%), high-phosphate (1.5%), without vitamin D diet but animals were supplemented with vitamin D Low-calcium (0.05%), low-phosphate, with vitamin D

p

diet 7

8

Tetanus (150 Hz)

Low-calcium (0.05%), low-phosphate, with vitamin D diet, and with calcium gluconate in drinking water Low-calcium (0.05%), low-phosphate, without vitamin D diet, and with calcium gluconate in drinking water

T1 r time to recovery me

PT

crystalline cholecalciferol dissolved in peanut oil by

\1avasel

tube feeding 6 to 8 days before the experiments. Groups 7 and 8 were given drinking water containing 1.5% calcium gluconate. For all animal groups the following dissection was made: The left sciatic nerve was sectioned at the hip,

the popliteal fossa was dissected and all nerves except those innervating the soleus, and gastrocnemius muscles were cut. The leg was rigidly mounted in a myograph with appropriately placed steel pins. The gastrocnemius tendon was cut just above the calcaneus, and the soleus tendon with a remnant of the calcaneal spur was attached to a strain guage. Thus, only the isometric tension developed by the soleus muscle was measured. The exposed tissues were covered with liquid paraffin. The stimuli were rectangular pulses of 0.1 msec duration and were delivered via an isolation unit by

bipolar platinum electrodes placed on the sciatic nerve. The threshold voltage was determined, and then the voltage was set approximately four times that voltage. The frequency of stimulation was 0.1 Hz.

peak tension = time to peak

300

msec —..

2

t

Display on

FIg. 1. Scheme of the various contractile parameters measured. Upper tracing shows the time-to-peak contractile tension (Tn) and

the time-to-half-relaxation-from-peak tension (Tjj2r) of a single maximal muscle contraction. Lower tracing shows the time-tohalf-relaxation-from-peak tension (T112r) of a maximal, tetanic muscle response.

collected from the right femoral artery. Total serum calcium was determined by fluorometry and serum phosphate by a microcolorometric method. Results

Experimental results are summarized in Tables 2 and 3. The striking finding is the prolongation of Tijtr

for both the single and the tetanic contractions in vitamin D-depleted animals on a high-calcium, highphosphorus diet (group 4). The time-to-peak tension

The profiles of isometric contractions, developed in response to single stimuli applied to the nerve,

(T0) also appears to be lengthened. As is evident

were displayed on an oscilloscope and photo-

for the single contraction is altered in group 4' animals. Repletion with vitamin D (group 5) restored all parameters to normal. In animals which were vitamin D-depleted but raised on a high-calcium, lowphosphorus diet (group 8), T112 for the tetanus is not

graphed. Next, a 150-Hz train for 300 msec was delivered to the nerve. The oscilloscope display was triggered by the end of the train, enabling the recording of the relaxation of the tetanic contraction of the soleus muscle. For the single contraction, the time-to-peak tension (Tn) and the time-for-recovery-halfway-to-thebaseline (Tiitr) were recorded (Fig. 1). For the tetanus, the T112r was determined (Fig. 1). At the conclusion of each experiment, blood was

from Figure 2, the shape of the tension vs. time curve

significantly increased. In animals made hypocalcemic by dietary deficien-

cy of calcium (group 6), there was no significant change in any of the parameters tested. Curiously, the animals made hypocalcemic by thyroparathyroidectomy (group 2) showed a tendency toward short-

Vitamin D and muscle

191

Table 2. Results of contractile responses of all groups studied

Tetanic contraction Time to /2

Single contraction Time to '/z Time to peak (Tn) msec

N

Group

I (normals)

11

17.1

0.5

9 8

0.7 0.8 0.6 0.7

2 (parathyroidectomized)

P — <0.1

decay (T112r)

0.2 0.5

NS

1.0

NS

<0.1

20.7

<0.001

NS

15.6

0.8 0.8

NS NS <0.001

4 (high-Ca, high-P. no vit. D)

17

5 (high-Ca, high-P, no vit. D

8

diet but vit, D supplements) 6 (low-Ca, low-P) 7 (high-Ca, low-P) 8 (high-Ca, low-P, no vit. D) a N = number of animals.

5

17.5

0.8

11

16.9

1.0

NS NS

14.4

0.8 0.8

8

17.2

0.6

NS

19.2

1.2

15.3

decay (T112r)

P

msec

— <0.05

14,8 13.6 14.6

15.4 17.0 18.7 16.6

3 (high-Ca, high-P)

P

msec

NS

34.9 31.6 31.2 40.7 32.0

1.0 1.5

— <0.1

2.7

NS <0.02 NS

34.0 33.0

3.3 2.0

32.8

1.9

1.5

2.0

Single contraction

NS NS NS

Tetanus

Table 3. Serum calcium and serum phosphate concentrations and body weights for all groups studied

Group

I (normals) 2 (parathyroidectomized)

N

Serum calcium

9.4 9 6.0

8 3 (high-Ca, high-P) 4 (high-Ca, high-P, no vit. 17 D) 5 (high-Ca, high-P, no vit. D diet but vit. D 8 supplements) 5 6 (low-Ca, low-P) Il 7 (high-Ca, low-P) 8 (high-Ca, low-P, no vit. 8 D) a N = number of animals.

g

mg/lOO ml mg/lOO ml 0.1

7.0

0.6 373

21

0.2 0.2

12.3

0.7 331

26

9.5

6.5

0.7 344

10

4.6

0.2

9.1

0.4 243

11

11

Control

Serum Animal phosphate weight

T, l5msec; T-r'

Group 4 (Hi'Ca.

9.4 5.1

0.2 0.5

9.7 7.4

8.8

0.3

7.9 6.9

1.3 323 0.5 210 0.4 255

0.2

3.6

0.1

217

13 15

24

7

l4msec

Hi-P. no vit. Dl

T, 21

msec; Tir 22 msec

Tr 53 msec

Fig. 2. Upper tracings: Control muscle tensions for single and tetanic responses in rats fed normal diet. Lower tracings: Muscle

tensions for single and tetanic responses in rats fed a highcalcium, high-phosphate, and no vitamin D diet.

ening of all of the time parameters measured. It therefore appears that the changes in Tl/2r are independent Of the serum calcium concentration. Neither dietary depletion of phosphate (group 7) nor supplementation (group 3) produced any significant change in any of the time parameters measured. As expected, parathyroidectomy increased the serum phosphate but, as noted above, certainly did not increase the Tiizr for either the single contraction or the tetanus. The increase in Tl/2r'5 in group 4 are

therefore not the result of changes in the serum

tures of the vertebral column. Phosphorus depletion and vitamin D depletion, both separately and together, produced growth retardation. Because the ani-

mals in the various groups were of such different sizes, comparison of single tensions and tetanic tensions was not possible. Discussion

These studies show that, in the rat, depletion of vitamin D will prolong the relaxation phase of a muscle contraction. The effect is independent of the serum calcium concentration, since both thyropara-

phosphate per se. The tracings obtained with groups of animals on diets different from group 4 are shown in Figure 3. The rats used in these experiments were growing animals. It is therefore not surprising that there were considerable differences in body weights in animals

thyroidectomy and dietary depletion of calcium

of the same age. The smallest animals were those

produce myopathy [11], our results might be

raised on diets low in both calcium and phosphorus. Several of these animals died of spontaneous frac-

explained by a decrease in intracellular phosphate in the vitamin D-depleted animals. If that were the total

cause hypocalcemia but do not prolong the Tj/2r.

The effect is also independent of the phosphate concentration in the serum. It is possible, however, that muscle phosphate transport is altered by vitamin D depletion. Since phosphate depletion in man will

192

Rodman and Baker

explanation for our results, one would anticipate that the hypophosphatemic animals in group 8 would be more affected than those in group 4. In fact, in one of the parameters we measured, the Tiir for the tetanus was normal in group 8. Hemodialysis patients develop a form of muscle dysfunction which is descriptively identical to that seen in various forms of osteomalacia [21. It would be difficult to ascribe their myopathy to a reduction in intracellular phosphate since, in fact, it is normal or increased [12]. The difference between group 4 and group 8 animals warrants further comment. The T112r for the tetanus was significantly prolonged in group 4 but not

group 8. There was also a tendency for the twitch parameters to be shorter in group 8 than group 4, although both were significantly increased over the control and were not significantly different from each other. If 1 ,25-dihydroxycholecalciferol (1 ,25-DHCC)

is the form of vitamin D which is necessary for normal muscle function, these results could be explained. Low phosphate stimulates and high phosphate inhibits the 1-hydroxylation step in the kidney

[13, 14]. Whatever small amount of vitamin D remained in the group 8 animals, it may have been pushed toward 1,25-DHCC by the low phosphate, whereas in group 4 animals, the reaction would have been inhibited. Muscle, in addition to bone, intestine, and kidney, appears to be a target organ for vitamin D metabolites. Several reasons exist for postulating a direct effect of vitamin D on muscle. Tritiated vitamin D localizes to skeletal muscle membranes [15]. If corrections are made for non-cellular elements, physiological doses of vitamin D are found in high concen-

trations in skeletal muscle [161. The uptake of phosphate and leucine by muscle from vitamin Ddepleted rats is augmented by 25-hydroxycholecalciferol [17]. Sarcoplasmic reticulum isolated from the psoas muscle of rabbits fed a vitamin D deficient diet shows a decreased ability to bind calcium, as compared to that isolated from animals receiving a normal diet[18].

The release of calcium from the sarcoplasmic reticulum (SR) in response to an action potential initiates muscle contraction, and active sequestration of calcium by the SR terminates contraction. Anything which disturbs the release or sequestration of calcium by the SR should alter the kinetics of con-

traction. In both skeletal [19] and cardiac muscle [20], caffeine lengthens the time course of an isomet-

ric contraction. Treatment of isolated SR with caffeine causes the release of calcium and the decreased

binding of added calcium [211. Thus, depletion of

vitamin D or one of its metabolites appears to induce a caffeine-like effect on muscle. The action of vitamin D on the SR is different from that of caffeine in that the latter is effective immediately. In studies on SR isolated from the muscle of

uremic rabbits, Matthews et al [22] showed that administration of 1 ,25-DHCC to the animals for five days prior to sacrifice improved the ability of the SR

to concentrate and store calcium. In muscle from untreated animals, 1,25-DHCC was without effect when added to the SR preparation. Thus, a period of

time is needed in vivo for 1,25-DHCC to have an effect. Which metabolite of vitamin D is most important in muscle function remains unanswered. The data of

Matthews et al [22] indicate that 1,25-DHCC will reverse the defect in sequestration of calcium by the sarcoplasmic reticulum induced by experimental ure-

mia. On the other hand, Birge and Haddad [17] showed that addition of a physiologic concentration of 25-hydroxycholecalciferol (25-HCC) to an epitrochlear muscle culture stimulates the incorporation of leucine into protein and raises the ATP content of the muscle. Supraphysiologic doses of 1 ,25-DHCC are without effect. A receptor protein in muscle with a

greater affinity for 25-HCC than l,25-DHCC has been described [23]. Further studies are therefore necessary to determine which of the metabolites is most effective in

reversing the myopathy of vitamin D depletion. When that metabolite is identified, it may be useful in treating dialysis patients with muscle weakness. Acknowledgments

This work was supported by NIH Biomedical Research Grant #5-507-RR-05396. Reprint requests to Dr. T. Baker, Assistant Professor, Department of Pharmacology, Cornell University Medical College, 1300 York Avenue, New York, New York 10021, U.S.A.

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