Determination of skeletal muscle calpain and calpastatin activities during maturation BOR-RUNG Department
OU AND
NEIL
E. FORSBERG
of Animal Science, Oregon State University, Corvallis, Oregon 97331-6702
Ou, BOR-RUNG,AND NEIL E. FORSBERG.Determination of skeletal muscle calpain and calpastatin activities during maturation. Am. J. Physiol. 261 (Endocrinol. Metab. 24): E677-
E683,1991.-Our objectives were to characterize events underlying changesin skeletal musclecalpain and calpastatin activities, using maturation as a model. Muscle sampleswere taken from rabbits of four ages (newborn and 1, 2, and 5 mo old). Concentrations of RNA and protein and activities of calpains I and II and calpastatin were determined. Steady-state concentrations of mRNAs encoding calpain I, calpain II, calpastatin, cy-and P-tubulin, and @-actinwere determined using Northern blot analysis. Calpain and calpastatin activities declined markedly between birth and 1 mo of age and remained unchanged thereafter. Several factors accountedfor the neonatal lossesof calpains and calpastatin. First, muscle protein concentration increasedbetween birth and 1 mo of age and diluted calpain and calpastatin specific activities. Second,there wasa marked reduction of muscle RNA concentration between birth and 1 mo of age, which indicates that protein synthetic capacity declined with age. Finally, calpastatin mRNA concentration declinedbetweenbirth and 1 mo of ageand further contributed to developmental losses of calpastatin activity. Calpain I mRNA concentration was unaffected by age, and although calpain II mRNA concentration declined with age, losseswere not detectedbetweenbirth and 1 mo; henceage-relatedchanges in calpain I and II activities are not mediated at the mRNA level. The age-relatedreductions in calpain II and calpastatin mRNA concentrations resembledage-relatedchangesin CYand P-tubulin and @-actin mRNA concentration. Hence developmental attenuation of calpain II and calpastatin gene expression is associatedwith housekeepinggeneexpressionand does not arise from direct regulation of calpain II and calpastatin geneexpression.
elucidate determinants of muscle calpain and calpastatin activities, using maturation as a model. METHODS
Twenty-four female New Zealand White rabbits were obtained from the Oregon State University Rabbit Research Center. Rabbits were of four different ages as follows: newborn (1 day old, 57.6 g), 1 mo old (weaned, 1,053 g), 2 mo old (market wt, 1,583 g), and 5 mo old (adult, 4,217 g, 6 animals/age group). On arrival, rabbits were killed by a single intraperitoneal injection of T-61 euthanasia solution. After death, muscle samples were taken then frozen between blocks of dry ice and were stored wrapped in aluminum foil and plastic at -9OOC. Biceps femoris (cranial portion) were taken from l-, 2-, and 5-mo-old rabbits. A mixture of hindlimb muscles, which included cranial biceps femoris and caudal biceps femoris, was taken from newborn animals. Calpain and calpastatin assays. Muscle calpain I and II activities were determined after their chromatographic separation, with the use of phenyl-Sepharose column chromatography (13). Muscle samples (3 g) were homogenized using a Polytron (Brinkmann Instrument, Westbury, NY; 0.4 X maximum speed; 30-40 s) in 5 vol of icecold buffer [50 mM tris( hydroxymethyl)aminomethane (Tris) l HCl, pH 7.5, 1 mM EDTA, 10 mM ,&mercaptoethanol (@ME), and 150 nM pepstatin A] and then were centrifuged at 10,000 g for 30 min at 4°C. A sample of the supernatant was assayed for protein content as outlined below. Twenty microliters of leupeptin (1 mM), 0.6 muscleprotein degradation; aging; geneexpression;proteinase; ml of NaCl (5 M), 1 ml of phenyl-Sepharose CL-4B neonate (prewashed with buffer A containing 20 mM Tris HCl, pH 7.5,0.1 mM CaC12, 10 mM @ME, and 20 PM leupeptin) and 0.25 M NaCl were added to the supernatant. DESPITE THE IMPORTANCE OF calpains to myofibrillar This mixture was agitated for 5 min, after which 0.4 ml protein degradation (3, lo), little is known regarding the of 0.1 M CaC12 was added, followed by an additional 10 factors that acutely and chronically modulate calpain min of agitation. This suspension was poured onto a 0.8 activities and the activity of their endogenous inhibitor, x 4-cm plastic column and was washed successively with calpastatin. Maturation is associated with a develop2 ml each of buffer A containing 0.25 M NaCl, buffer A mental decline in rates of myofibrillar protein degradaaione, then buffer A without leupeptin. Calpain II was tion (9, 17, 23, 25, 26), which is independent of muscle eluted with 4 ml of buffer B [(in mM) 20 Tris HCl, pH fiber type (9). Therefore maturation provides a model in 7.5, 1 EGTA, and 10 PME] supplemented with 0.1 M which events that determine changes in calpain and NaCl. The column was then washed with 2 ml buffer B. Calpain I was eluted with 4 ml buffer B. All of the above calpastatin activities may be identified. In earlier studies (24), we determined that sheep muscle calpain and cal- procedures were carried out at 4°C. pastatin activities decline with age; however, molecular Calpain activities were measured using Hammarsten mechanisms underlying these changes were not examcasein (Merck) as a substrate. The reaction mixture ined. Objectives of the present study, therefore, were to contained 2 mM CaClz (final concentration), 3 ml column 0193-1849/91 $1.50 Copyright 0 1991 the American Physiological Society
E677
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E678
MUSCLE
CALPAIN
AND
CALPASTATIN
eluant, and 1 ml casein solution (8 mg/ml casein in 20 mM Tris HCl, pH 7.5, and 10 mM PME). Control samples contained 2 mM EDTA, which replaced CaC12. After incubating at 25°C for 30 min, 1 ml of 36% trichloroacetic acid (TCA; wt/vol) was added. TCA-soluble products were measured as outlined by Bradford (2). One unit (U) of calpain activity was defined as the amount of enzyme that caused a calcium-dependent change of 0.1 unit of absorbance at 595 nm in 30 min at 25OC. Calpastatin assays were determined as outlined by Nakamura et al. (22). Skeletal muscle (0.25 g) was homogenized with a Polytron (0.4 x maximum speed, 3040 s) in 5 vol of ice-cold Tris HCl (20 mM, pH 7.5) containing 5 mM EDTA. Homogenates were centrifuged at 10,000 g for 20 min at 4OC. Supernatants were heated at 100°C for 10 min to inactivate endogenous calpains and other proteinases. After heat treatment, the supernatant was centrifuged at 10,000 g for 10 min at 4°C. Aliquots of the supernatant were added to partially purified stock rabbit muscle calpain II (3 U) containing 2 mM CaC12 and were incubated at 25°C for 5 min. After this, hydrolysis of casein was assessed as outlined for calpain assays. One unit of calpastatin was defined as the amount that inhibited 1 U of rabbit muscle calpain II ‘Validation studies. Buffer volumes required for adequate separation of calpains from calpastatin and of calpain I from calpain II were determined in preliminary studies. The percentage recoveries of muscle calpains I and II were determined by applying known quantities of rabbit muscle calpains I and II to a column and by determining the proportion of their activities that eluted from the column. Protein content was determined (2) using bovine serum albumin as a standard. Calpain and calpastatin activities were expressed on this basis. Muscle RNA content was determined using the method of Munro and Fleck (21). Extraction of total RNA. The protocol for extraction of RNA was described in Chomczynski and Sacchi (4). Muscle (0.5 g) was minced and homogenized at room temperature with 5 ml solution A (4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, and 10 mM @ME). To this 0.5 ml of 2 M sodium acetate (pH 4.0), 5 ml phenol and 1 ml of a chloroformisoamyl alcohol mixture (49: 1) were added sequentially. The mixture was mixed by inversion after each addition and was then centrifuged (10,000 g) for 20 min at 4°C. After centrifugation, the aqueous layer was transferred to a fresh tube, and 5 ml of isopropanol were added. The mixture was stored at -20°C for 1 h then was centrifuged at 10,000 g for 20 min at 4°C. The supernatant was discarded, and the RNA pellet was resuspended in 0.5 ml solution A. One volume of isopropanol was added, and the mixture was stored at -20°C for 1 h. The RNA pellet was recovered by centrifugation at 12,000 g for 10 min at 4°C was washed with 75% ethanol, and was dried under vacuum. The dried RNA pellet was then dissolved in diethyl, pyrocarbonate-treated water and was quantitated by spectrophotometry at a wavelength of 260 nm. Plasmids containing cDNA fragments encoding rabbit calpain I (pLU1001; see Ref. 7), rabbit calpain II l
GENE
EXPRESSION
(pLM28; see Ref. 7), and rabbit calpastatin (pC1413; see Ref. 6) were obtained from Drs. Y. Emori and K. Suzuki of the Tokyo Metropolitan Institute for Medical Science. Plasmids containing cDNA fragments encoding chick brain cu-tubulin (pT1; see Refs. 5 and 29) and ,&tubulin (pT2; see Refs. 5 and 29) were obtained from Dr. Donald Cleveland (The Johns Hopkins University). A plasmid encoding rat ,&actin (pR@-Al) was obtained from Dr. Larry Kedes (University of Southern California). Plasmids were amplified and recovered using standard techniques (19). Plasmid DNA was purified after treatment with ribonuclease (US Biochemical, Cleveland, OH) using Sepharose-4B chromatography. cDNA probes encoding calpains I and II were prepared from unique 3’ends of pLUlOO1 (865-bp Rsa I/Dde I fragment; see Ref. 7) and pLM28 (530-bp Rsa I fragment; see Ref. 7), respectively. cDNA probes encoding calpastatin (1,260bp EcoR I fragment of pC1413; see Ref. 6), chick atubulin (1,500-bp Hind III fragment of pTl), chick /3tubulin (1,700-bp Hind III fragment of pT2), and rat pactin (l,OOO-bp Hind IIIIpuu II fragment of pRP-Al) were also prepared. After restriction digestion, cDNA fragments were separated by agarose gel electrophoresis and were recovered by electrophoresis onto a dialysis membrane (l), followed by recovery and purification by ethanol precipitation. cDNA fragments (50 ng) were labeled to high specific activity using dCTP (3,000 Ci/ mmol; New England Nuclear, Boston, MA) and a random primer kit (US Biochemical). Northern blot hybridization. RNA samples were denatured at 55°C for 15 min, were applied to a 1.1% agaroseformaldehyde gel (30 pg/lane), then were electrophoresed at 30 V for 12 h. RNA was transferred onto a nitrocellulose membrane (BAS-85; Schleicher and Schuell, Keene, NH) and was immobilized by baking under vacuum for 2 h at 8OOC. Membranes were prehybridized in Stark’s buffer (30 ml; see Ref. 19) containing 0.02% sheared salmon sperm DNA (Sigma Chemical, St. Louis, MO) at 42°C overnight then were hybridized at 42°C in Stark’s buffer (8 ml) containing 20% dextran sulfate (wt/ vol), 0.02% salmon sperm DNA, and [32P]cDNA (500,000 counts min-l *ml-‘) for 48 h. After hybridization, membranes were washed twice with x0.1 standard sodium citrate and 0.1% sodium dodecyl sulfate at room temperature for 15 min and were washed three times at 51°C in the same buffer. Membranes were exposed to Kodak XOmat film with intensifying screen for l-2 days. Quantitation of exposures on autoradiographic films was performed using a Bio-Rad model 1650 scanning densitometer and a Hoefer GS-350H scanning program. VaZidation studies. A dose-response analysis was performed in preliminary studies to determine the quantity of RNA that could be routinely processed for Northern blot analysis (M. A. Ilian, unpublished observations). Variable quantities of total RNA (12.5, 25, and 50 pg) were processed using Northern blot analysis, and concentrations of calpain I and II and calpastatin mRNAs were examined. Intensity of exposure on autoradiographic film increased linearly between 12.5 and 50 pg RNA. Hence 30 pg RNA were routinely electrophoresed for assays of calpain and calpastatin mRNAs in this study. l
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MUSCLE
CALPAIN
AND CALPASTATIN
StatisticaL analysis. Data were examined for equal variance and for a normal distribution before statistical analysis. Differences among different experimental groups were assessed using one-way analysis of variance (28). Individual treatment differences were examined with Tukey’s multiple-range test (28). RESULTS
The effects of maturation on muscle weights and on muscle protein and RNA concentrations are shown in Table 1. Protein concentration increased as the rabbits aged. Muscles taken from l-, 2-, and 5mo-old rabbits all had greater (P < 0.05) protein concentrations than newborn rabbits. However, no differences (P > 0.05) in protein concentrations were detected among l-, 2-, and 5mo-old rabbits. Muscle RNA concentrations declined progressively (P c 0.05) as rabbits aged. Five-month-old rabbits contained only 17% of the RNA concentration detected in newborn muscle samples. Recoveries of rabbit muscle calpains I and II from phenyl-Sepharose were 55.4 t 6.9 and 50.6 & 3.9%, respectively. Data presented (Fig. 1) are not corrected for recovery. Specific activities of calpains I and II and calpastatin declined rapidly after birth (Figs. 1 and 2). For calpains and calpastatin, significant changes in activities were not detected beyond 1 mo of age. Irrespective of age, calpain II activity was slightly in excess of calpain I activity, and calpastatin activity exceeded combined activities of calpains I and II. Relative activities of calpains to their inhibitor, expressed as the ratio of calpain I plus calpain II activities divided by calpastatin activity, 1. Effects of chronological age on rabbit muscle weights, protein concentration, and total RNA concentration TABLE
Newborn
1 MO
2Mo
5Mo
57.6kl.2 1,053&96 1,583&65 4,217+108 Body d, g Biceps femoris, g ND 4.64t0.33 9.90t0.51 26.8rtl.8 Protein, mg/g 58.lt5.1” 127k4t 108&7t 143+lOt 5.83t0.29* 2.70+0.2lt 1.81+0.15$ 0.98+0.09$ RNA, n-%/g Values are means t SE. Protein and RNA are expressed as a proportion of wet muscle weight. ND, not determined. Values in same row, which do not share a common superscript, differ (P < 0.05). 50 -
0 0
I
I
1
2
1
I
I
I
4
5
6
Age (rknthd +-
Calpain
I
-f-
Calpain
II
FIG. 1. Effects of maturation on specific activities of muscle calpain I and calpain II. Values are means rf: SE of 6 animals and are expressed as a proportion of muscle protein.
E679
GENE EXPRESSION 1600 4
>r .Z .->
3 .-c s G 2 z
800 6004002000’ 0
I
I
1
2
I
I
I
J
4
5
6
Age (m30nths)
2. Effects of maturation on specific activities of muscle calpastatin. Values are means t SE of 6 observations and are expressed as a proportion of muscle protein. FIG.
changed during maturation. Calpain-to-calpastatin ratios in newborn and in l-, 2-, and 5mo-old rabbits were 0.049 t 0.005 and 0.053 t O.OO2,0.075 t 0.007, and 0.048 Z!Z0.003, respectively. The elevation in calpain activities relative to calpastatin in 2mo-old rabbits was significant (P c 0.05) compared with all other age groups. This effect was due to a greater proportional loss of calpastatin activity. Other differences (P c 0.05) in this ratio among treatment groups were not detected. The effects of age on calpain I and II mRNA concentrations are shown in Fig. 3. Because calpain I and II mRNA levels were assayed independently, their relative concentrations cannot be determined from this analysis. Calpain I mRNA concentration tended to decline during maturation; however, these changes were not significant (Fig. 3, A and C; P > 0.05). Calpain II mRNA concentration declined (P < 0.05) during maturation (Fig. 3, B and C). Calpain II mRNA concentration in muscle taken from Lmo-old (adult) animals was significantly lower (Fig. 4B; P < 0.05) than calpain II mRNA levels in newborn animals; however, no differences (P > 0.05) in calpain II mRNA were detected in l-mo-old rabbits compared with newborn rabbits. Other differences (P < 0.05) between treatment groups were also not detected. The effects of maturation on calpastatin mRNA concentrations are shown in Fig. 4. Calpastatin mRNA was expressed either as a long form (band I) or as a short form (band 11). Calpastatin band II mRNA was the only mRNA form present in newborn and l-mo-old rabbit muscle. Its concentration declined (P < 0.05) between birth and weaning and thereafter remained unchanged (P > 0.05). Although calpastatin band I mRNA was not detected in newborn or l-mo-old rabbits, it was detected in two 2mo-old rabbits and in all 5-mo-old rabbits. Total calpastatin mRNA (bands I and II) increased (P < 0.05) between 2 and 5 mo of age. The effects of maturation on constituitively expressed genes were examined. cu-Tubulin mRNA concentration declined (P < 0.05) between birth and l-2 mo of age and thereafter remained unchanged (P > 0.05; Fig. 5). Concentrations of ,&tubulin and @-actin mRNAs also changed with age in a manner that resembled cu-tubulin (data not shown).
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E680
MUSCLE
A,
W
M
CALPAIN BNWMA 12
A
----
T C m P 5.
-18s
AND CALPASTATIN
34
56
7
8
GENE EXPRESSION
9101112
FIG. 3. A: Northern blot analyses showing effects of maturation on steady-state mRNA concentrations encoding calpain I in rabbit muscle. Thirty micrograms of total RNA were applied to each lane and were analyzed for calpain I mRNA as outlined in text. Ages included newborn (N), weaned (W, 1 mo old), market weight (M, 2 mo old), and adult (A, 5 mo old). Because of large numbers of RNA samples that needed to be processed simultaneously, equal numbers of RNA samples from each treatment (n = 3) were processed on each of the 2 blots shown. The two blots were processed together. Six animals from each age group are therefore represented on the 2 blots. 2% and 18s ribosomal RNA positions are indicated. B: Northern blot analyses showing effects of maturation on steady-state mRNA concentrations encoding calpain II in rabbit muscle. Thirty micrograms of total RNA were applied to each lane and were analyzed for calpain II mRNA as outlined in text. Ages and allocation of RNA samples for the two blots are described in A. C: scanning densitometry of Northern blots shown in A and B. Values are expressed as arbitrary densitometer units/mg total RNA + SE.
u” -“.
2
t 0’ 0
I
I
I
I
I
I
1
2
3
4
5
6
Age (months) +
Calpain
I
-t
Calpain
II
DISCUSSION
The objectives of the study were to evaluate factors underlying changes in calpain and calpastatin activities, using the maturation-dependent attenuation of myofibrillar protein degradation as a model. The activities of
calpain and calpastatin declined as rabbits matured. Losses of activity occurred between birth and 1 mo of age. Hence the neonate provides a model in which regulation of calpain and calpastatin activities and therefore regulation of myofibrillar protein degradation may be elucidated. Several general observations regarding the
A N 123456
W
A
M --7
8
9 1011
12
-18s
0
4
2
Age (months) f
Band
I
-k-
Band
II
FIG. 4. A: Northern blot analyses showing effects of maturation on steady-state mRNA concentrations encoding calpastatin in rabbit muscle. Two forms of calpastatin mRNA were detected, and these are identified as bands Z and ZZ,which corresponded to bands Z and ZZ reported by Emori et al. (6). Thirty micrograms of total RNA were applied to each lane and were analyzed for calpastatin mRNA as outlined in text. Ages and allocation of RNA samples for the 2 blots are described in legend to Fig. 3A. B: scanning densitometry of Northern blots shown in A. Band Z was not detectable in newborn or l-mo-old rabbits. Values are expressed as arbitrary densitometer units/mg total RNA + SE.
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MUSCLE
AN--P
w
M
CALPAIN
AND CALPASTATIN
E681
GENE EXPRESSION
A
123456769101112
0
1
2
4
5
6
Age (nkths) FIG. 5. A: Northern blot analyses showing effects of maturation on steady-state mRNA concentrations encoding a-tubulin in rabbit muscle. Thirty micrograms of total RNA were applied to each lane and were analyzed for ol-tubulin mRNA as outlined in text. Ages and allocation of RNA samples for the 2 blots are described in legend to Fig. 3A. B: scanning densitometry of Northern blots shown in A. Values are expressed as arbitrary densitometer units/mg total RNA f SE. cz-Tubulin mRNA level in lane 1 of A, bottom, was very low and was not included in scanning densitometry.
expression of muscle calpains and calpastatin were made. First, calpain and calpastatin activities both declined during maturation. In other studies we determined that calpains and calpastatin respond similarly to fasting (15 and M. A. Ilian and N. E. Forsberg, unpublished observations) and to P-adrenergic agonist treatments (8). This indicates that common elements may underlie the expression of calpains and calpastatin. Despite this possibility, the change in the ratio of calpain to calpastatin activities during maturation indicates that the regulation of calpain expression is, to a certain extent, independent of the regulation of calpastatin expression. Although this ratio may be interpreted as “calpain potential,” the biological significance of this ratio is not known. Second, the observation that calpastatin activity relative to calpains did not increase during maturation indicates that age-related attenuation of myofibrillar protein degradation is not mediated indirectly by increased calpastatin expression. Third, irrespective of age, calpastatin activity exceeded the combined activities of calpains I and II. This has been reported in our other studies (8, 24) and implies that mechanisms must exist to allow for the expression of calpain activity in the presence of calpastatin. Kapprell and Go11 (18) have proposed that uncharacterized posttranslational factors circumvent the inhibition of calpains by calpastatin. Z-disk lipids may permit calpain activity in the presence of calpastatin (3). Fourth, although this study does not resolve the issue regarding which proteinase is rate limiting to myofibrillar protein degradation, we can surmise that, if calpains are rate limiting to this process, the age-related changes in their activities may underlie age-related changes in fractional rates of muscle protein degradation. Several factors contributed to the changes in calpain and calpastatin activities. First, muscle protein concentrations increased in the first month of age. This may be due to synthesis of a more stable pool of proteins relative to synthesis of the calpains and calpastatin or to increased synthesis of muscle proteins relative to calpains and calpastatin, both of which would dilute calpain and
calpastatin specific activities. If the ratio of calpain to muscle protein (calpain specific activity) is a determinant of the fractional rate of muscle protein degradation, factors that influence muscle protein concentration may indirectly influence the fractional rate of protein degradation. Age-related dilution of calpain activities by muscle proteins may contribute to the age-related attenuation of myofibrillar protein degradation. Second, fractional rates of muscle protein synthesis decline with age (9, 17,23, 25,26). These changes result from age-related reductions in muscle ribosome concentration (9, 20), which has been defined as protein synthetic capacity (20, 27). Additionally, protein synthesis per ribosome (protein synthetic efficiency) declines with age (9). In this study RNA concentration declined markedly (54%) between birth and 1 mo of age, and it is likely therefore that synthesis of calpains and calpastatin were indirectly reduced. Third, calpastatin band II mRNA concentration declined between birth and 1 mo of age. This would probably further contribute to neonatal losses of muscle calpastatin activity. Although calpain II mRNA concentration declined with age, significant differences in calpain II mRNA concentration were not detected between birth and 1 mo of age, the period during which losses of calpain II activity occurred. Hence changes in calpain I and II mRNA concentrations do not contribute to neonatal attenuation of calpain activities. Loss of calpastatin mRNA concentration in the neonate may account for the greater proportional loss in activity by calpastatin compared with calpains, which occurred between birth and 2 mo of age. Although total calpastatin mRNA (bands I and II) increased twofold between 2 and 5 mo of age, an increase in calpastatin activity was not detected at this time. The increase in calpastatin mRNA that occurred at this time may have been countered by the 46% reduction in muscle RNA content, which occurred between 2 and 5 mo of age. Changes in calpain II and calpastatin mRNA concentrations did not result from direct regulation of calpain and calpastatin gene expression but instead resembled the changes in house-
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E682
MUSCLE
CALPAIN
AND
CALPASTATIN
keeping gene expression relative to total RNA. In support of the possibility that calpain gene expression is determined, at least in part, by factors that affect housekeeping gene expression, Hata et al. (14) reported that the human calpain II V-flanking region, in addition to containing four negative enhancer-like elements, possesses similarities to housekeeping promoters. However, in other studies (15 and M. A. Ilian and N. E. Forsberg, unpublished observations) we determined that calpain gene expression is governed by factors other than those that affect housekeeping genes. Fasting increased rabbit biceps femoris calpain I and II mRNA concentrations severalfold but reduced ,&actin mRNA concentration. Therefore we propose that, under normal physiological circumstances, calpains are expressed as housekeeping proteins, but in stressful conditions nonhousekeeping regulatory elements also influence in calpain gene expression. Perhaps the negative regulatory &-elements detected by Hata et al. (14) account for this feature of calpain II gene expression. Three sizes of calpastatin mRNA (bands I, II, and 111), which arise from three 3’-processing sites of the calpastatin gene, have been reported in rabbit heart (6). Of interest in this study was the age-related appearance of band I. In other studies we determined that fasting causes the expression of band III in rabbit skeletal muscle (M. A. Ilian and N. E. Forsberg, unpublished observations). Although we are uncertain of the physiological consequences to expression of multiple calpastatin mRNA species and to age-related changes in proportions of calpastatin mRNA bands, we propose that maturation either modifies recognition of the 3’-processing sites of the calpastatin gene or selectively alters susceptibilities of the three bands to degradation. This study demonstrates that changes in muscle calpain and calpastatin activities, at least in the neonate, arise indirectly from regulation of common cellular processes. These processes include age-related accumulation of muscle proteins, age-related attenuation of muscle protein synthesis, and, for calpastatin, changes in housekeeping gene expression relative to total RNA. None of these changes could account for the acute changes in proteolysis that occur in skeletal muscle; hence we propose that the regulation detected in this study is directed toward maintenance of calpain concentrations in muscle at concentrations that are appropriate to the physiological needs of normal animals rather than toward acute regulation of calpain activities, which are required during stress or disease. Acute regulation of existing calpains may be brought about at the posttranslational level. We are very grateful to Drs. Y. Emori and K. Suzuki (Tokyo Metropolitan Institute of Medical Science), Dr. D. Cleveland (Massachusetts Institute of Technology), and Dr. L. Kedes (University of Southern California) for gifts of cDNAs. This work was supported in part by United States Department of Agriculture Grant 88-37265-3896. Address reprint requests to B.-R. Ou. Received
10 December
1990; accepted
in final
form
9 July
1991.
REFERENCES 1. AUSUBEL, SEIDMAN,
F. M., R. BRENT, R. E. KINGSTON, J. A. SMITH, AND K. STRUHL.
D. D. MOORE, J. G. Current Protocols in
GENE
EXPRESSION
2MoLecuLar Biology. New York: Wiley, 1987, vol. 1. M. M. A rapid and sensitive method for the quantita2. BRADFORD, tion of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72: 248-254, 1976. 3. BULLARD, B., G. SAINSBURY, AND N. MILLER. Digestion of proteins with the Z-disc by calpain. J. Muscle Res. Cell Motil. 11: 271279,199o. 4. CHOMCZYNSKI, P., AND N. SACCHI. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. 5. CLEVELAND, D. W., M. A. LOPATA, R. J. MACDONALD, N. 9. COWAN, W. J. RUTTER, AND M. W. KIRSCHNER. Number and evolutionary conservation of CY- and P-tubulin and cytoplasmic @and cu-actin genes using specific cloned cDNA probes. CeZZ 20: 95105,198O. 6. EMORI, Y., H. KAWASAKI, S. IMAJOH, K. IMAHORI, AND K. SUZUKI. Endogenous inhibitor for calcium-dependent cysteine protease contains four internal repeats that could be responsible for its multiple reactive sites. Proc. Natl. Acad. Sci. USA 84: 3590-3595, 1987. 7. EMORI, Y., H. KAWASAKI, H. SUGIHARA, S. IMAJOH, S. KAWASHIMA, AND K. SUZUKI. Isolation and sequence analysis of cDNA clones for the large subunits of two isozymes of rabbit calciumdependent protease. J. BioZ. Chem. 261: 9465-9471, 1986. 8. FORSBERG, N. E., M. A. ILIAN, A. ALI-BAR, P. R. CHEEKE, AND N. B. WEHR. Effects of cimaterol on rabbit growth and myofibrillar protein degradation and on calcium-dependent proteinase and calpastatin activities in skeletal muscle. J. Anim. Sci. 67: 33133321,1989. 9. GARLICK, P. J., C. A. MALTIN, A. G. S. BAILLIE, M. I. DELDAY, AND D. A. GRUBB. Fiber type composition of nine rat muscles. II. Relationships to protein turnover. Am. J. Physiol. 257 (Endocrinol. Metab. 20): E828-E832, 1989. 10. GOLL, D. E., W. C. KLEESE, AND A. SZPACENKO. Skeletal muscle proteases and protein turnover. In: AnimaZ Growth Regulation, edited by D. R. Campion, G. J. Hausman, and R. J. Martin. New York: Plenum, 1989, chapt. 8, p. 141-182. 11. GOODMAN, M. N. Myofibrillar protein degradation in skeletal muscle is diminished in rats with streptozotocin-induced diabetes. Diabetes 36: 100-105, 1987. 12. GOODMAN, M. N. Differential effects of acute changes in cell Ca2’ concentration on myofibrillar and non-myofibrillar protein degradation in the rat EDL muscle in vitro. Biochem. J. 241: 121-127, 1987. 13. GOPALAKRISHNA, R., AND S. H. BARSKY. Quantitation of tissue calpain activity after isolation by hydrophobic chromatography. Anal. Biochem. 148: 413-423, 1985. 14. HATA, A., S. OHNO, Y. AKITA, AND K. SUZUKI. Tandemly-reiterated negative enhancer-like elements regulate transcription of a human gene for the large subunit of calcium-dependent protease. J. BioZ. Chem. 264: 6404-6411, 1989. 15. ILIAN, M. A., AND N. E. FORSBERG. Proteinase mRNAs in skeletal muscle are selectively upregulated by fasting (Abstract). J. Cell Biol. 111: 371a, 1990. 17. KANG, C. W., M. L. SUNDE, AND R. W. SWICK. Characteristic of growth and protein turnover in skeletal muscle of turkey poults. Pod. Sci. 64: 380-387, 1985. 18. KAPPRELL, H. P., AND D. E. GOLL. Effect of calcium on binding of the calpains to calpastatin. J. BioZ. Chem. 264: 17888-17896, 1989. 19. MANIATIS, T., E. F. FRISCH, AND J. SAMBROOK. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor, 1982. 20. MILLWARD, D. J., P. J. GARLICK, W. P. T. JAMES, D. Y. NNANYELUGO, AND J. S. RYATT. Relationship between protein synthesis and RNA content in skeletal muscle. Nature Lond. 241: 204-205, 1973. H. N., AND A. FLECK. Analysis of nitrogenous constitu21. MUNRO, ents. Measurements of nucleic acids in tissues. In: Mammalian Protein Metabolism, edited by H. N. Munro. New York: Academic, 1969, vol. 3, p. 477-481. M., K. IMAHORI, AND S. KAWASHIMA. Tissue distri22. NAKAMURA, bution of an endogenous inhibitor of calcium-activated neutral protease and age-related changes in its activity in rats. Comp. Biochem. Physiol. B Comp. Biochem. 89: 381-384, 1988. 23. ODDY, V. H., D. B. LINDSAY, P. J. PARKER, AND A. J. NORTHROP.
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MUSCLE
CALPAIN
AND CALPASTATIN
Effect of insulin on hind-limb and whole-body leucine and protein metabolism in fed and fasted lambs. Br. J. Nutr. 58: 437-452,1987. 24. Ou, B. R., H. H. MEYER, AND N. E. FORSBERG. Effects of age and castration on activities of calpains and calpastatin in sheep skeletal muscle. J. Anim. Sci. 69: 1919-1924, 1991. 25. REEDS, P. J., A. CADENHEAD, M. F. FULLER, G. E. LOBLEY, AND J. D. MCDONALD. Protein turnover in growing pigs. Effects of age and food intake. Br. J. Nutr. 43: 445-455, 1980. 26. SIEBRITS, F. K., AND P. M. BARNES. The change in the rate of muscle protein metabolism of rats from weaning to 90 days of age. Comp.
Biochem.
Physiol.
A Comp.
Physiol.
92: 485-488,
1989.
E683
GENE EXPRESSION
D., B. H. L. CHUA, N. L. BELSER, AND H. E. MORGAN. Faster protein and ribosome synthesis in thyroxine-induced hypertrophy of rat heart. Am. J. Physiol. 248 (Cell Physiol. 17): C309C319,1985. 28. STEEL, R. G. D., AND J. H. TORRIE. Principles and Procedures of Statistics. New York: McGraw-Hill, 1980. 29. VALENZUELA, P., M. QUIROGA, J. ZALDIVAR, W. J. RUTTER, M. R. KIRSCHNER, AND D. W. CLEVELAND. Nucleotide and corresponding amino acid sequences encoded by CY-and @-tubulin mRNAs. 27. SIEHL,
Nature
Land.
289: 650-655,
1981.
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OU AND
NEIL
E. FORSBERG
of Animal Science, Oregon State University, Corvallis, Oregon 97331-6702
Ou, BOR-RUNG,AND NEIL E. FORSBERG.Determination of skeletal muscle calpain and calpastatin activities during maturation. Am. J. Physiol. 261 (Endocrinol. Metab. 24): E677-
E683,1991.-Our objectives were to characterize events underlying changesin skeletal musclecalpain and calpastatin activities, using maturation as a model. Muscle sampleswere taken from rabbits of four ages (newborn and 1, 2, and 5 mo old). Concentrations of RNA and protein and activities of calpains I and II and calpastatin were determined. Steady-state concentrations of mRNAs encoding calpain I, calpain II, calpastatin, cy-and P-tubulin, and @-actinwere determined using Northern blot analysis. Calpain and calpastatin activities declined markedly between birth and 1 mo of age and remained unchanged thereafter. Several factors accountedfor the neonatal lossesof calpains and calpastatin. First, muscle protein concentration increasedbetween birth and 1 mo of age and diluted calpain and calpastatin specific activities. Second,there wasa marked reduction of muscle RNA concentration between birth and 1 mo of age, which indicates that protein synthetic capacity declined with age. Finally, calpastatin mRNA concentration declinedbetweenbirth and 1 mo of ageand further contributed to developmental losses of calpastatin activity. Calpain I mRNA concentration was unaffected by age, and although calpain II mRNA concentration declined with age, losseswere not detectedbetweenbirth and 1 mo; henceage-relatedchanges in calpain I and II activities are not mediated at the mRNA level. The age-relatedreductions in calpain II and calpastatin mRNA concentrations resembledage-relatedchangesin CYand P-tubulin and @-actin mRNA concentration. Hence developmental attenuation of calpain II and calpastatin gene expression is associatedwith housekeepinggeneexpressionand does not arise from direct regulation of calpain II and calpastatin geneexpression.
elucidate determinants of muscle calpain and calpastatin activities, using maturation as a model. METHODS
Twenty-four female New Zealand White rabbits were obtained from the Oregon State University Rabbit Research Center. Rabbits were of four different ages as follows: newborn (1 day old, 57.6 g), 1 mo old (weaned, 1,053 g), 2 mo old (market wt, 1,583 g), and 5 mo old (adult, 4,217 g, 6 animals/age group). On arrival, rabbits were killed by a single intraperitoneal injection of T-61 euthanasia solution. After death, muscle samples were taken then frozen between blocks of dry ice and were stored wrapped in aluminum foil and plastic at -9OOC. Biceps femoris (cranial portion) were taken from l-, 2-, and 5-mo-old rabbits. A mixture of hindlimb muscles, which included cranial biceps femoris and caudal biceps femoris, was taken from newborn animals. Calpain and calpastatin assays. Muscle calpain I and II activities were determined after their chromatographic separation, with the use of phenyl-Sepharose column chromatography (13). Muscle samples (3 g) were homogenized using a Polytron (Brinkmann Instrument, Westbury, NY; 0.4 X maximum speed; 30-40 s) in 5 vol of icecold buffer [50 mM tris( hydroxymethyl)aminomethane (Tris) l HCl, pH 7.5, 1 mM EDTA, 10 mM ,&mercaptoethanol (@ME), and 150 nM pepstatin A] and then were centrifuged at 10,000 g for 30 min at 4°C. A sample of the supernatant was assayed for protein content as outlined below. Twenty microliters of leupeptin (1 mM), 0.6 muscleprotein degradation; aging; geneexpression;proteinase; ml of NaCl (5 M), 1 ml of phenyl-Sepharose CL-4B neonate (prewashed with buffer A containing 20 mM Tris HCl, pH 7.5,0.1 mM CaC12, 10 mM @ME, and 20 PM leupeptin) and 0.25 M NaCl were added to the supernatant. DESPITE THE IMPORTANCE OF calpains to myofibrillar This mixture was agitated for 5 min, after which 0.4 ml protein degradation (3, lo), little is known regarding the of 0.1 M CaC12 was added, followed by an additional 10 factors that acutely and chronically modulate calpain min of agitation. This suspension was poured onto a 0.8 activities and the activity of their endogenous inhibitor, x 4-cm plastic column and was washed successively with calpastatin. Maturation is associated with a develop2 ml each of buffer A containing 0.25 M NaCl, buffer A mental decline in rates of myofibrillar protein degradaaione, then buffer A without leupeptin. Calpain II was tion (9, 17, 23, 25, 26), which is independent of muscle eluted with 4 ml of buffer B [(in mM) 20 Tris HCl, pH fiber type (9). Therefore maturation provides a model in 7.5, 1 EGTA, and 10 PME] supplemented with 0.1 M which events that determine changes in calpain and NaCl. The column was then washed with 2 ml buffer B. Calpain I was eluted with 4 ml buffer B. All of the above calpastatin activities may be identified. In earlier studies (24), we determined that sheep muscle calpain and cal- procedures were carried out at 4°C. pastatin activities decline with age; however, molecular Calpain activities were measured using Hammarsten mechanisms underlying these changes were not examcasein (Merck) as a substrate. The reaction mixture ined. Objectives of the present study, therefore, were to contained 2 mM CaClz (final concentration), 3 ml column 0193-1849/91 $1.50 Copyright 0 1991 the American Physiological Society
E677
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E678
MUSCLE
CALPAIN
AND
CALPASTATIN
eluant, and 1 ml casein solution (8 mg/ml casein in 20 mM Tris HCl, pH 7.5, and 10 mM PME). Control samples contained 2 mM EDTA, which replaced CaC12. After incubating at 25°C for 30 min, 1 ml of 36% trichloroacetic acid (TCA; wt/vol) was added. TCA-soluble products were measured as outlined by Bradford (2). One unit (U) of calpain activity was defined as the amount of enzyme that caused a calcium-dependent change of 0.1 unit of absorbance at 595 nm in 30 min at 25OC. Calpastatin assays were determined as outlined by Nakamura et al. (22). Skeletal muscle (0.25 g) was homogenized with a Polytron (0.4 x maximum speed, 3040 s) in 5 vol of ice-cold Tris HCl (20 mM, pH 7.5) containing 5 mM EDTA. Homogenates were centrifuged at 10,000 g for 20 min at 4OC. Supernatants were heated at 100°C for 10 min to inactivate endogenous calpains and other proteinases. After heat treatment, the supernatant was centrifuged at 10,000 g for 10 min at 4°C. Aliquots of the supernatant were added to partially purified stock rabbit muscle calpain II (3 U) containing 2 mM CaC12 and were incubated at 25°C for 5 min. After this, hydrolysis of casein was assessed as outlined for calpain assays. One unit of calpastatin was defined as the amount that inhibited 1 U of rabbit muscle calpain II ‘Validation studies. Buffer volumes required for adequate separation of calpains from calpastatin and of calpain I from calpain II were determined in preliminary studies. The percentage recoveries of muscle calpains I and II were determined by applying known quantities of rabbit muscle calpains I and II to a column and by determining the proportion of their activities that eluted from the column. Protein content was determined (2) using bovine serum albumin as a standard. Calpain and calpastatin activities were expressed on this basis. Muscle RNA content was determined using the method of Munro and Fleck (21). Extraction of total RNA. The protocol for extraction of RNA was described in Chomczynski and Sacchi (4). Muscle (0.5 g) was minced and homogenized at room temperature with 5 ml solution A (4 M guanidinium isothiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, and 10 mM @ME). To this 0.5 ml of 2 M sodium acetate (pH 4.0), 5 ml phenol and 1 ml of a chloroformisoamyl alcohol mixture (49: 1) were added sequentially. The mixture was mixed by inversion after each addition and was then centrifuged (10,000 g) for 20 min at 4°C. After centrifugation, the aqueous layer was transferred to a fresh tube, and 5 ml of isopropanol were added. The mixture was stored at -20°C for 1 h then was centrifuged at 10,000 g for 20 min at 4°C. The supernatant was discarded, and the RNA pellet was resuspended in 0.5 ml solution A. One volume of isopropanol was added, and the mixture was stored at -20°C for 1 h. The RNA pellet was recovered by centrifugation at 12,000 g for 10 min at 4°C was washed with 75% ethanol, and was dried under vacuum. The dried RNA pellet was then dissolved in diethyl, pyrocarbonate-treated water and was quantitated by spectrophotometry at a wavelength of 260 nm. Plasmids containing cDNA fragments encoding rabbit calpain I (pLU1001; see Ref. 7), rabbit calpain II l
GENE
EXPRESSION
(pLM28; see Ref. 7), and rabbit calpastatin (pC1413; see Ref. 6) were obtained from Drs. Y. Emori and K. Suzuki of the Tokyo Metropolitan Institute for Medical Science. Plasmids containing cDNA fragments encoding chick brain cu-tubulin (pT1; see Refs. 5 and 29) and ,&tubulin (pT2; see Refs. 5 and 29) were obtained from Dr. Donald Cleveland (The Johns Hopkins University). A plasmid encoding rat ,&actin (pR@-Al) was obtained from Dr. Larry Kedes (University of Southern California). Plasmids were amplified and recovered using standard techniques (19). Plasmid DNA was purified after treatment with ribonuclease (US Biochemical, Cleveland, OH) using Sepharose-4B chromatography. cDNA probes encoding calpains I and II were prepared from unique 3’ends of pLUlOO1 (865-bp Rsa I/Dde I fragment; see Ref. 7) and pLM28 (530-bp Rsa I fragment; see Ref. 7), respectively. cDNA probes encoding calpastatin (1,260bp EcoR I fragment of pC1413; see Ref. 6), chick atubulin (1,500-bp Hind III fragment of pTl), chick /3tubulin (1,700-bp Hind III fragment of pT2), and rat pactin (l,OOO-bp Hind IIIIpuu II fragment of pRP-Al) were also prepared. After restriction digestion, cDNA fragments were separated by agarose gel electrophoresis and were recovered by electrophoresis onto a dialysis membrane (l), followed by recovery and purification by ethanol precipitation. cDNA fragments (50 ng) were labeled to high specific activity using dCTP (3,000 Ci/ mmol; New England Nuclear, Boston, MA) and a random primer kit (US Biochemical). Northern blot hybridization. RNA samples were denatured at 55°C for 15 min, were applied to a 1.1% agaroseformaldehyde gel (30 pg/lane), then were electrophoresed at 30 V for 12 h. RNA was transferred onto a nitrocellulose membrane (BAS-85; Schleicher and Schuell, Keene, NH) and was immobilized by baking under vacuum for 2 h at 8OOC. Membranes were prehybridized in Stark’s buffer (30 ml; see Ref. 19) containing 0.02% sheared salmon sperm DNA (Sigma Chemical, St. Louis, MO) at 42°C overnight then were hybridized at 42°C in Stark’s buffer (8 ml) containing 20% dextran sulfate (wt/ vol), 0.02% salmon sperm DNA, and [32P]cDNA (500,000 counts min-l *ml-‘) for 48 h. After hybridization, membranes were washed twice with x0.1 standard sodium citrate and 0.1% sodium dodecyl sulfate at room temperature for 15 min and were washed three times at 51°C in the same buffer. Membranes were exposed to Kodak XOmat film with intensifying screen for l-2 days. Quantitation of exposures on autoradiographic films was performed using a Bio-Rad model 1650 scanning densitometer and a Hoefer GS-350H scanning program. VaZidation studies. A dose-response analysis was performed in preliminary studies to determine the quantity of RNA that could be routinely processed for Northern blot analysis (M. A. Ilian, unpublished observations). Variable quantities of total RNA (12.5, 25, and 50 pg) were processed using Northern blot analysis, and concentrations of calpain I and II and calpastatin mRNAs were examined. Intensity of exposure on autoradiographic film increased linearly between 12.5 and 50 pg RNA. Hence 30 pg RNA were routinely electrophoresed for assays of calpain and calpastatin mRNAs in this study. l
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MUSCLE
CALPAIN
AND CALPASTATIN
StatisticaL analysis. Data were examined for equal variance and for a normal distribution before statistical analysis. Differences among different experimental groups were assessed using one-way analysis of variance (28). Individual treatment differences were examined with Tukey’s multiple-range test (28). RESULTS
The effects of maturation on muscle weights and on muscle protein and RNA concentrations are shown in Table 1. Protein concentration increased as the rabbits aged. Muscles taken from l-, 2-, and 5mo-old rabbits all had greater (P < 0.05) protein concentrations than newborn rabbits. However, no differences (P > 0.05) in protein concentrations were detected among l-, 2-, and 5mo-old rabbits. Muscle RNA concentrations declined progressively (P c 0.05) as rabbits aged. Five-month-old rabbits contained only 17% of the RNA concentration detected in newborn muscle samples. Recoveries of rabbit muscle calpains I and II from phenyl-Sepharose were 55.4 t 6.9 and 50.6 & 3.9%, respectively. Data presented (Fig. 1) are not corrected for recovery. Specific activities of calpains I and II and calpastatin declined rapidly after birth (Figs. 1 and 2). For calpains and calpastatin, significant changes in activities were not detected beyond 1 mo of age. Irrespective of age, calpain II activity was slightly in excess of calpain I activity, and calpastatin activity exceeded combined activities of calpains I and II. Relative activities of calpains to their inhibitor, expressed as the ratio of calpain I plus calpain II activities divided by calpastatin activity, 1. Effects of chronological age on rabbit muscle weights, protein concentration, and total RNA concentration TABLE
Newborn
1 MO
2Mo
5Mo
57.6kl.2 1,053&96 1,583&65 4,217+108 Body d, g Biceps femoris, g ND 4.64t0.33 9.90t0.51 26.8rtl.8 Protein, mg/g 58.lt5.1” 127k4t 108&7t 143+lOt 5.83t0.29* 2.70+0.2lt 1.81+0.15$ 0.98+0.09$ RNA, n-%/g Values are means t SE. Protein and RNA are expressed as a proportion of wet muscle weight. ND, not determined. Values in same row, which do not share a common superscript, differ (P < 0.05). 50 -
0 0
I
I
1
2
1
I
I
I
4
5
6
Age (rknthd +-
Calpain
I
-f-
Calpain
II
FIG. 1. Effects of maturation on specific activities of muscle calpain I and calpain II. Values are means rf: SE of 6 animals and are expressed as a proportion of muscle protein.
E679
GENE EXPRESSION 1600 4
>r .Z .->
3 .-c s G 2 z
800 6004002000’ 0
I
I
1
2
I
I
I
J
4
5
6
Age (m30nths)
2. Effects of maturation on specific activities of muscle calpastatin. Values are means t SE of 6 observations and are expressed as a proportion of muscle protein. FIG.
changed during maturation. Calpain-to-calpastatin ratios in newborn and in l-, 2-, and 5mo-old rabbits were 0.049 t 0.005 and 0.053 t O.OO2,0.075 t 0.007, and 0.048 Z!Z0.003, respectively. The elevation in calpain activities relative to calpastatin in 2mo-old rabbits was significant (P c 0.05) compared with all other age groups. This effect was due to a greater proportional loss of calpastatin activity. Other differences (P c 0.05) in this ratio among treatment groups were not detected. The effects of age on calpain I and II mRNA concentrations are shown in Fig. 3. Because calpain I and II mRNA levels were assayed independently, their relative concentrations cannot be determined from this analysis. Calpain I mRNA concentration tended to decline during maturation; however, these changes were not significant (Fig. 3, A and C; P > 0.05). Calpain II mRNA concentration declined (P < 0.05) during maturation (Fig. 3, B and C). Calpain II mRNA concentration in muscle taken from Lmo-old (adult) animals was significantly lower (Fig. 4B; P < 0.05) than calpain II mRNA levels in newborn animals; however, no differences (P > 0.05) in calpain II mRNA were detected in l-mo-old rabbits compared with newborn rabbits. Other differences (P < 0.05) between treatment groups were also not detected. The effects of maturation on calpastatin mRNA concentrations are shown in Fig. 4. Calpastatin mRNA was expressed either as a long form (band I) or as a short form (band 11). Calpastatin band II mRNA was the only mRNA form present in newborn and l-mo-old rabbit muscle. Its concentration declined (P < 0.05) between birth and weaning and thereafter remained unchanged (P > 0.05). Although calpastatin band I mRNA was not detected in newborn or l-mo-old rabbits, it was detected in two 2mo-old rabbits and in all 5-mo-old rabbits. Total calpastatin mRNA (bands I and II) increased (P < 0.05) between 2 and 5 mo of age. The effects of maturation on constituitively expressed genes were examined. cu-Tubulin mRNA concentration declined (P < 0.05) between birth and l-2 mo of age and thereafter remained unchanged (P > 0.05; Fig. 5). Concentrations of ,&tubulin and @-actin mRNAs also changed with age in a manner that resembled cu-tubulin (data not shown).
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E680
MUSCLE
A,
W
M
CALPAIN BNWMA 12
A
----
T C m P 5.
-18s
AND CALPASTATIN
34
56
7
8
GENE EXPRESSION
9101112
FIG. 3. A: Northern blot analyses showing effects of maturation on steady-state mRNA concentrations encoding calpain I in rabbit muscle. Thirty micrograms of total RNA were applied to each lane and were analyzed for calpain I mRNA as outlined in text. Ages included newborn (N), weaned (W, 1 mo old), market weight (M, 2 mo old), and adult (A, 5 mo old). Because of large numbers of RNA samples that needed to be processed simultaneously, equal numbers of RNA samples from each treatment (n = 3) were processed on each of the 2 blots shown. The two blots were processed together. Six animals from each age group are therefore represented on the 2 blots. 2% and 18s ribosomal RNA positions are indicated. B: Northern blot analyses showing effects of maturation on steady-state mRNA concentrations encoding calpain II in rabbit muscle. Thirty micrograms of total RNA were applied to each lane and were analyzed for calpain II mRNA as outlined in text. Ages and allocation of RNA samples for the two blots are described in A. C: scanning densitometry of Northern blots shown in A and B. Values are expressed as arbitrary densitometer units/mg total RNA + SE.
u” -“.
2
t 0’ 0
I
I
I
I
I
I
1
2
3
4
5
6
Age (months) +
Calpain
I
-t
Calpain
II
DISCUSSION
The objectives of the study were to evaluate factors underlying changes in calpain and calpastatin activities, using the maturation-dependent attenuation of myofibrillar protein degradation as a model. The activities of
calpain and calpastatin declined as rabbits matured. Losses of activity occurred between birth and 1 mo of age. Hence the neonate provides a model in which regulation of calpain and calpastatin activities and therefore regulation of myofibrillar protein degradation may be elucidated. Several general observations regarding the
A N 123456
W
A
M --7
8
9 1011
12
-18s
0
4
2
Age (months) f
Band
I
-k-
Band
II
FIG. 4. A: Northern blot analyses showing effects of maturation on steady-state mRNA concentrations encoding calpastatin in rabbit muscle. Two forms of calpastatin mRNA were detected, and these are identified as bands Z and ZZ,which corresponded to bands Z and ZZ reported by Emori et al. (6). Thirty micrograms of total RNA were applied to each lane and were analyzed for calpastatin mRNA as outlined in text. Ages and allocation of RNA samples for the 2 blots are described in legend to Fig. 3A. B: scanning densitometry of Northern blots shown in A. Band Z was not detectable in newborn or l-mo-old rabbits. Values are expressed as arbitrary densitometer units/mg total RNA + SE.
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MUSCLE
AN--P
w
M
CALPAIN
AND CALPASTATIN
E681
GENE EXPRESSION
A
123456769101112
0
1
2
4
5
6
Age (nkths) FIG. 5. A: Northern blot analyses showing effects of maturation on steady-state mRNA concentrations encoding a-tubulin in rabbit muscle. Thirty micrograms of total RNA were applied to each lane and were analyzed for ol-tubulin mRNA as outlined in text. Ages and allocation of RNA samples for the 2 blots are described in legend to Fig. 3A. B: scanning densitometry of Northern blots shown in A. Values are expressed as arbitrary densitometer units/mg total RNA f SE. cz-Tubulin mRNA level in lane 1 of A, bottom, was very low and was not included in scanning densitometry.
expression of muscle calpains and calpastatin were made. First, calpain and calpastatin activities both declined during maturation. In other studies we determined that calpains and calpastatin respond similarly to fasting (15 and M. A. Ilian and N. E. Forsberg, unpublished observations) and to P-adrenergic agonist treatments (8). This indicates that common elements may underlie the expression of calpains and calpastatin. Despite this possibility, the change in the ratio of calpain to calpastatin activities during maturation indicates that the regulation of calpain expression is, to a certain extent, independent of the regulation of calpastatin expression. Although this ratio may be interpreted as “calpain potential,” the biological significance of this ratio is not known. Second, the observation that calpastatin activity relative to calpains did not increase during maturation indicates that age-related attenuation of myofibrillar protein degradation is not mediated indirectly by increased calpastatin expression. Third, irrespective of age, calpastatin activity exceeded the combined activities of calpains I and II. This has been reported in our other studies (8, 24) and implies that mechanisms must exist to allow for the expression of calpain activity in the presence of calpastatin. Kapprell and Go11 (18) have proposed that uncharacterized posttranslational factors circumvent the inhibition of calpains by calpastatin. Z-disk lipids may permit calpain activity in the presence of calpastatin (3). Fourth, although this study does not resolve the issue regarding which proteinase is rate limiting to myofibrillar protein degradation, we can surmise that, if calpains are rate limiting to this process, the age-related changes in their activities may underlie age-related changes in fractional rates of muscle protein degradation. Several factors contributed to the changes in calpain and calpastatin activities. First, muscle protein concentrations increased in the first month of age. This may be due to synthesis of a more stable pool of proteins relative to synthesis of the calpains and calpastatin or to increased synthesis of muscle proteins relative to calpains and calpastatin, both of which would dilute calpain and
calpastatin specific activities. If the ratio of calpain to muscle protein (calpain specific activity) is a determinant of the fractional rate of muscle protein degradation, factors that influence muscle protein concentration may indirectly influence the fractional rate of protein degradation. Age-related dilution of calpain activities by muscle proteins may contribute to the age-related attenuation of myofibrillar protein degradation. Second, fractional rates of muscle protein synthesis decline with age (9, 17,23, 25,26). These changes result from age-related reductions in muscle ribosome concentration (9, 20), which has been defined as protein synthetic capacity (20, 27). Additionally, protein synthesis per ribosome (protein synthetic efficiency) declines with age (9). In this study RNA concentration declined markedly (54%) between birth and 1 mo of age, and it is likely therefore that synthesis of calpains and calpastatin were indirectly reduced. Third, calpastatin band II mRNA concentration declined between birth and 1 mo of age. This would probably further contribute to neonatal losses of muscle calpastatin activity. Although calpain II mRNA concentration declined with age, significant differences in calpain II mRNA concentration were not detected between birth and 1 mo of age, the period during which losses of calpain II activity occurred. Hence changes in calpain I and II mRNA concentrations do not contribute to neonatal attenuation of calpain activities. Loss of calpastatin mRNA concentration in the neonate may account for the greater proportional loss in activity by calpastatin compared with calpains, which occurred between birth and 2 mo of age. Although total calpastatin mRNA (bands I and II) increased twofold between 2 and 5 mo of age, an increase in calpastatin activity was not detected at this time. The increase in calpastatin mRNA that occurred at this time may have been countered by the 46% reduction in muscle RNA content, which occurred between 2 and 5 mo of age. Changes in calpain II and calpastatin mRNA concentrations did not result from direct regulation of calpain and calpastatin gene expression but instead resembled the changes in house-
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keeping gene expression relative to total RNA. In support of the possibility that calpain gene expression is determined, at least in part, by factors that affect housekeeping gene expression, Hata et al. (14) reported that the human calpain II V-flanking region, in addition to containing four negative enhancer-like elements, possesses similarities to housekeeping promoters. However, in other studies (15 and M. A. Ilian and N. E. Forsberg, unpublished observations) we determined that calpain gene expression is governed by factors other than those that affect housekeeping genes. Fasting increased rabbit biceps femoris calpain I and II mRNA concentrations severalfold but reduced ,&actin mRNA concentration. Therefore we propose that, under normal physiological circumstances, calpains are expressed as housekeeping proteins, but in stressful conditions nonhousekeeping regulatory elements also influence in calpain gene expression. Perhaps the negative regulatory &-elements detected by Hata et al. (14) account for this feature of calpain II gene expression. Three sizes of calpastatin mRNA (bands I, II, and 111), which arise from three 3’-processing sites of the calpastatin gene, have been reported in rabbit heart (6). Of interest in this study was the age-related appearance of band I. In other studies we determined that fasting causes the expression of band III in rabbit skeletal muscle (M. A. Ilian and N. E. Forsberg, unpublished observations). Although we are uncertain of the physiological consequences to expression of multiple calpastatin mRNA species and to age-related changes in proportions of calpastatin mRNA bands, we propose that maturation either modifies recognition of the 3’-processing sites of the calpastatin gene or selectively alters susceptibilities of the three bands to degradation. This study demonstrates that changes in muscle calpain and calpastatin activities, at least in the neonate, arise indirectly from regulation of common cellular processes. These processes include age-related accumulation of muscle proteins, age-related attenuation of muscle protein synthesis, and, for calpastatin, changes in housekeeping gene expression relative to total RNA. None of these changes could account for the acute changes in proteolysis that occur in skeletal muscle; hence we propose that the regulation detected in this study is directed toward maintenance of calpain concentrations in muscle at concentrations that are appropriate to the physiological needs of normal animals rather than toward acute regulation of calpain activities, which are required during stress or disease. Acute regulation of existing calpains may be brought about at the posttranslational level. We are very grateful to Drs. Y. Emori and K. Suzuki (Tokyo Metropolitan Institute of Medical Science), Dr. D. Cleveland (Massachusetts Institute of Technology), and Dr. L. Kedes (University of Southern California) for gifts of cDNAs. This work was supported in part by United States Department of Agriculture Grant 88-37265-3896. Address reprint requests to B.-R. Ou. Received
10 December
1990; accepted
in final
form
9 July
1991.
REFERENCES 1. AUSUBEL, SEIDMAN,
F. M., R. BRENT, R. E. KINGSTON, J. A. SMITH, AND K. STRUHL.
D. D. MOORE, J. G. Current Protocols in
GENE
EXPRESSION
2MoLecuLar Biology. New York: Wiley, 1987, vol. 1. M. M. A rapid and sensitive method for the quantita2. BRADFORD, tion of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72: 248-254, 1976. 3. BULLARD, B., G. SAINSBURY, AND N. MILLER. Digestion of proteins with the Z-disc by calpain. J. Muscle Res. Cell Motil. 11: 271279,199o. 4. CHOMCZYNSKI, P., AND N. SACCHI. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. 5. CLEVELAND, D. W., M. A. LOPATA, R. J. MACDONALD, N. 9. COWAN, W. J. RUTTER, AND M. W. KIRSCHNER. Number and evolutionary conservation of CY- and P-tubulin and cytoplasmic @and cu-actin genes using specific cloned cDNA probes. CeZZ 20: 95105,198O. 6. EMORI, Y., H. KAWASAKI, S. IMAJOH, K. IMAHORI, AND K. SUZUKI. Endogenous inhibitor for calcium-dependent cysteine protease contains four internal repeats that could be responsible for its multiple reactive sites. Proc. Natl. Acad. Sci. USA 84: 3590-3595, 1987. 7. EMORI, Y., H. KAWASAKI, H. SUGIHARA, S. IMAJOH, S. KAWASHIMA, AND K. SUZUKI. Isolation and sequence analysis of cDNA clones for the large subunits of two isozymes of rabbit calciumdependent protease. J. BioZ. Chem. 261: 9465-9471, 1986. 8. FORSBERG, N. E., M. A. ILIAN, A. ALI-BAR, P. R. CHEEKE, AND N. B. WEHR. Effects of cimaterol on rabbit growth and myofibrillar protein degradation and on calcium-dependent proteinase and calpastatin activities in skeletal muscle. J. Anim. Sci. 67: 33133321,1989. 9. GARLICK, P. J., C. A. MALTIN, A. G. S. BAILLIE, M. I. DELDAY, AND D. A. GRUBB. Fiber type composition of nine rat muscles. II. Relationships to protein turnover. Am. J. Physiol. 257 (Endocrinol. Metab. 20): E828-E832, 1989. 10. GOLL, D. E., W. C. KLEESE, AND A. SZPACENKO. Skeletal muscle proteases and protein turnover. In: AnimaZ Growth Regulation, edited by D. R. Campion, G. J. Hausman, and R. J. Martin. New York: Plenum, 1989, chapt. 8, p. 141-182. 11. GOODMAN, M. N. Myofibrillar protein degradation in skeletal muscle is diminished in rats with streptozotocin-induced diabetes. Diabetes 36: 100-105, 1987. 12. GOODMAN, M. N. Differential effects of acute changes in cell Ca2’ concentration on myofibrillar and non-myofibrillar protein degradation in the rat EDL muscle in vitro. Biochem. J. 241: 121-127, 1987. 13. GOPALAKRISHNA, R., AND S. H. BARSKY. Quantitation of tissue calpain activity after isolation by hydrophobic chromatography. Anal. Biochem. 148: 413-423, 1985. 14. HATA, A., S. OHNO, Y. AKITA, AND K. SUZUKI. Tandemly-reiterated negative enhancer-like elements regulate transcription of a human gene for the large subunit of calcium-dependent protease. J. BioZ. Chem. 264: 6404-6411, 1989. 15. ILIAN, M. A., AND N. E. FORSBERG. Proteinase mRNAs in skeletal muscle are selectively upregulated by fasting (Abstract). J. Cell Biol. 111: 371a, 1990. 17. KANG, C. W., M. L. SUNDE, AND R. W. SWICK. Characteristic of growth and protein turnover in skeletal muscle of turkey poults. Pod. Sci. 64: 380-387, 1985. 18. KAPPRELL, H. P., AND D. E. GOLL. Effect of calcium on binding of the calpains to calpastatin. J. BioZ. Chem. 264: 17888-17896, 1989. 19. MANIATIS, T., E. F. FRISCH, AND J. SAMBROOK. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor, 1982. 20. MILLWARD, D. J., P. J. GARLICK, W. P. T. JAMES, D. Y. NNANYELUGO, AND J. S. RYATT. Relationship between protein synthesis and RNA content in skeletal muscle. Nature Lond. 241: 204-205, 1973. H. N., AND A. FLECK. Analysis of nitrogenous constitu21. MUNRO, ents. Measurements of nucleic acids in tissues. In: Mammalian Protein Metabolism, edited by H. N. Munro. New York: Academic, 1969, vol. 3, p. 477-481. M., K. IMAHORI, AND S. KAWASHIMA. Tissue distri22. NAKAMURA, bution of an endogenous inhibitor of calcium-activated neutral protease and age-related changes in its activity in rats. Comp. Biochem. Physiol. B Comp. Biochem. 89: 381-384, 1988. 23. ODDY, V. H., D. B. LINDSAY, P. J. PARKER, AND A. J. NORTHROP.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
MUSCLE
CALPAIN
AND CALPASTATIN
Effect of insulin on hind-limb and whole-body leucine and protein metabolism in fed and fasted lambs. Br. J. Nutr. 58: 437-452,1987. 24. Ou, B. R., H. H. MEYER, AND N. E. FORSBERG. Effects of age and castration on activities of calpains and calpastatin in sheep skeletal muscle. J. Anim. Sci. 69: 1919-1924, 1991. 25. REEDS, P. J., A. CADENHEAD, M. F. FULLER, G. E. LOBLEY, AND J. D. MCDONALD. Protein turnover in growing pigs. Effects of age and food intake. Br. J. Nutr. 43: 445-455, 1980. 26. SIEBRITS, F. K., AND P. M. BARNES. The change in the rate of muscle protein metabolism of rats from weaning to 90 days of age. Comp.
Biochem.
Physiol.
A Comp.
Physiol.
92: 485-488,
1989.
E683
GENE EXPRESSION
D., B. H. L. CHUA, N. L. BELSER, AND H. E. MORGAN. Faster protein and ribosome synthesis in thyroxine-induced hypertrophy of rat heart. Am. J. Physiol. 248 (Cell Physiol. 17): C309C319,1985. 28. STEEL, R. G. D., AND J. H. TORRIE. Principles and Procedures of Statistics. New York: McGraw-Hill, 1980. 29. VALENZUELA, P., M. QUIROGA, J. ZALDIVAR, W. J. RUTTER, M. R. KIRSCHNER, AND D. W. CLEVELAND. Nucleotide and corresponding amino acid sequences encoded by CY-and @-tubulin mRNAs. 27. SIEHL,
Nature
Land.
289: 650-655,
1981.
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 13, 2019.
