Postnatal Changes in Mitochondrial Protein Mass and Respiration in Skeletal Muscle from the Newborn Pig

Comp. Biochem. Physiol. Vol. 118B, No. 3, pp. 639–647, 1997 Copyright  1997 Elsevier Science Inc. All rights reserved.

ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00268-X

Postnatal Changes in Mitochondrial Protein Mass and Respiration in Skeletal Muscle from the Newborn Pig Isabelle Schmidt and Patrick Herpin INRA, Station de Recherches Porcines, 35590 Saint Gilles, France ABSTRACT. Quantitative and functional changes occurring in mitochondria were studied in pig skeletal muscle between birth and 5 days of life. Postnatal changes were followed separately on intermyofibrillar and subsarcolemmal mitochondria isolated from rhomboı¨deus (RH) and longissimus dorsi (LD) muscles. The integrity and purity of the isolated mitochondria was checked by electron microscopic observations. The mass of mitochondrial protein was not different between muscles at birth. It increased tremendously during the first 5 days of life, by 49% in LD (P , 0.001) and 93% in RH (P , 0.001) muscle and was 30% higher in RH than in LD muscle at 5 days of life (P , 0.05). Mitochondria isolated from RH muscle exhibited 30% higher oxidative and phosphorylative capacities than those from LD muscle at 5 days of life (P , 0.05). Intermyofibrillar (IM) mitochondria had high respiration rate, enzyme activities and coupling parameters (respiratory control ratio, phosphorus-oxygen ratio) from birth. Subsarcolemmal (SS) mitochondria were less active than IM mitochondria; their respiration rate and enzyme activities were 60% lower (P , 0.01) and increased with age, particularly in LD muscle (P , 0.05). Short-term cold exposure had no effect on mitochondrial mass and activity. These results suggest that muscle mitochondria are functional from birth and are changing primarily quantitatively. SS and IM mitochondria exhibit specific changes that are probably involved in the postnatal acquisition of skeletal muscle oxidative metabolism. comp biochem physiol 118B;3:639–647, 1997.  1997 Elsevier Science Inc. KEY WORDS. Age, creatine kinase, cytochrome oxidase, mitochondria, muscle, piglet

INTRODUCTION At birth, the newborn pig experiences a dramatic and sudden 15–20°C decrease in its thermal environment. Because it has no brown adipose tissue (41), it relies essentially on muscular shivering thermogenesis for thermoregulatory purposes (6). Biochemical and physiological mechanisms involved in the postnatal maturation of skeletal muscle energy metabolism, including provision of energy substrates and oxygen, maturation of metabolic pathways and changes in mitochondrial density and activity, should therefore be of utmost importance for successful adaptation to extrauterine life. Previous studies have shown that whole body energy metabolism (7) and muscle oxidative capacities are greatly enhanced within the first 48–54 hr of life (5,22) in pigs, the magnitude of these postnatal changes depending on both muscle type and environmental temperature. However, to our knowledge, there is no available information on the quantitative and qualitative changes occurring in muscle Address reprint requests to: P. Herpin, INRA, Station de Recherches Porcines, 35590 Saint Gilles, France. Tel. 02-99-28-50-87; Fax 02-99-28-5080; E-mail: [email protected]. Abbreviations–IM, intermyofibrillar; SS, subsarcolemmal; RH, rhomboı¨deus; LD, longissimus dorsi; TN, thermoneutral; C, cold; CO, cytochrome oxidase; CK, creatine kinase; RCR, respiratory control ratio; ADP/ O, phosphorus-oxygen ratio. Received 24 March 1997; revised 3 July 1997; accepted 23 July 1997.

mitochondria early after birth. In pig liver, Mersman et al. (29) have shown functional changes in isolated mitochondria within 6–12 hr after birth and an increased mitochondrial number within 2 weeks. Similarly, in rats, the specific activity of several enzymes from the Krebs cycle and the respiratory chain increased after birth (19,36), and the respiratory control and phosphorus - oxygen ratio rose gradually to those observed in adult liver mitochondria within 5–7 days after birth (30). As part of an ongoing program on the effect of age and cold exposure on skeletal muscle energy metabolism in the piglet, we first examined the quantitative and functional changes occurring at the mitochondrial level between birth and 5 days of life in pigs exposed to thermal neutrality or to the cold. Postnatal changes were followed separately on intermyofibrillar (IM) and subsarcolemmal (SS) mitochondria isolated from a slow-oxidative rhomboı¨deus (RH) and a fast-glycolytic longissimus dorsi (LD) muscle.

MATERIALS AND METHODS Animals Forty-eight Pie´train 3 Large White cross-bred halothaneinsensitive piglets from the INRA (Institut National de la Recherche Agronomique) herd were used in the experiment. Parturition was induced with an intramuscular injec-

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tion of a prostaglandin analogue on day 112 of gestation to ensure farrowing on day 113. Sows and piglets were kept in the usual farrowing house conditions until the beginning of the experiment. At birth, 4 hr, 1 day and 5 days of life, piglets of average body weight were anesthetized by halothane inhalation and then killed by exsanguination. LD and RH muscles were immediately removed and stored in ice-cold isolation buffer A (see below) for further determination of the changes in IM and SS mitochondria respiration, yield of mitochondria extraction, mitochondrial mass and cytochrome oxidase and creatine kinase activities. At birth, piglets (n 5 10) were killed immediately, but in the other groups, they were first maintained for 4 hr in a respiratory chamber in thermoneutral (TN) or cold (C) conditions as follows. To follow the decrease of the lower critical temperature with age, TN piglets were exposed to 34°C at 4 hr (n 5 6), 33°C at 1 day (n 5 6) and 30°C at 5 days of life (n 5 10). C piglets were exposed to 24°C at 4 hr (n 5 6) and 20°C at 5 days of life (n 5 10). Newborns were provided with 40 g fresh sow colostrum/kg body weight at 1 hr of life, whereas 1- and 5-day-old piglets were removed from the sow immediately after suckling. Whole Body Heat Production Heat production was measured continuously by indirect calorimetry in an open-circuit temperature-controlled chamber as previously described (7). Briefly, the piglet O 2 consumption and CO 2 production were determined from the difference in O 2 and CO 2 concentrations between the air entering and leaving the chamber and the flow rate of the extracted air during successive 180-sec periods, a correction being applied for any change in the O 2 and CO 2 content of the chamber during this period. Oxygen and carbon dioxide concentrations were measured using a paramagnetic oxygen analyzer (Oxygor 6 N, Maihak, Colombes, France) and an infrared carbon dioxide analyzer (Finor, Maihak). The flow rate of the extracted air was measured continuously using a mass flowmeter (HFM, Hastings, Nozay, France). Heat production was expressed in kJ/min and /kg body weight. Isolation of Mitochondria Muscle IM and SS mitochondria are known to exhibit marked differences in respiration intensity and coupling state and were isolated separately, as previously described (22). Immediately post-mortem, muscle samples were homogenized in buffer A (see below) in an ice-cold Teflonpestle-glass Potter-Elvehjem homogenizer (B. Braun, Melsungen, Germany), and mitochondria were obtained by differential centrifugation using a Sorvall Superspeed RC-5b refrigerated centrifuge (Du Pont Instruments, LeBlanc Menil, France) and suspended in storage buffer B (see below). Gentle use of Nagarse (Serva, Heidelberg, Germany)

was necessary (5 min at 0°C with immediate dilution and rinsing of the homogenate with buffer A) to digest the myofibrils and to liberate IM mitochondria. Isolation Buffer and Storage Medium Muscles were homogenized in an isolation buffer A, pH 7.4, consisting of 100 mM sucrose, 180 mM KCl, 50 mM Trisbase, 10 mM EDTA, 5 mM MgCl 2 and 1 mM ATP. The isolated mitochondria were kept in a storage medium B, pH 7.4, containing 250 mM sucrose, 2 mM EDTA and 20 mM Tris-base. Yield of Mitochondrial Extraction Mitochondrial protein concentration of the final pellets was determined by the Biuret method with BSA as standard. Isolated mitochondria were then diluted to 20 mg mitochondrial protein/ml with buffer B. The yield of mitochondrial extraction (Yd ) was calculated from mitochondrial protein concentrations (M prot , mg/ml) and from the difference in cytochrome oxidase (CO) activity between mitochondria homogenates (Hx; nAtoms O/ min/g tissue) and the corresponding final mitochondria pellets (Mx; nAtoms O/min/mg mitochondrial protein), according to the following formula: Yd 5

COMx ⋅ [M prot ] ⋅ Vf , COHx ⋅ M t

where x 5 SS or IM, V f being the final volume of the mitochondrial pellet (ml), and M t the mass of tissue used for the isolation procedure (g). The mass of IM and SS mitochondria per g of muscle was then calculated as follows: Mass 5

[M prot ] ⋅ Vf . M t ⋅ Yd

H SS was sampled from the supernatant after the first centrifugation, whereas H IM was collected after treatment with Nagarse. In addition, an aliquot of the whole muscle homogenate H M was taken at the beginning of the isolation procedure for the measurement of muscle CO activity. Collection of samples was performed under continuous stirring of the homogenate. Preliminary experiments have shown that using citrate synthase activity instead of CO activity as a mitochondrial marker had no effect on those calculations. Electron Microscopy To confirm the purity and the integrity of the isolated mitochondria, some pellets were checked by electron microscopy. The pellets were carefully removed from the tubes and quickly placed in ice-cold fixative (0.5% paraformaldehyde in 100 mM Na-K phosphate buffer, pH 7.4) for 2 hr. They were then washed in ice-cold 175 mM Na-K phosphate

Muscle Mitochondria in the Newborn Pig

buffer (pH 7.4) for a night at 4°C and postfixed (2% osmium tetroxide in 100 mM Na-K phosphate buffer, pH 7.4) for 30 min at room temperature. The fixed pellets were quickly dehydrated in ice-cold ethanol and embedded in Epon as described by Barre´ et al. (4). Thin sections for electron microscopy were stained with uranyl acetate and lead citrate and studied under an Hitachi HU-12-A electron microscope.

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min and per mg mitochondrial protein or per g muscle. CK activity, a useful index of the phosphorylative potential of the isolated mitochondria, was measured spectrophotometrically at 30°C and 340 nm by the procedure of Foster et al. (16). Disappearance of β-NADH was followed for 30 min and enzyme activity was expressed in mU/mg mitochondrial protein. Statistical Analysis

Mitochondrial Oxygen Consumption The respiration of isolated mitochondria (0.5 mg mitochondrial protein/ml for IM and 1 mg mitochondrial protein/ml for SS) was measured polarographically at 25°C using a Clark O 2 electrode (Oxygraph Hansatech Ltd, London, U.K.) in 1 ml of respiratory medium, pH 7.4, containing 75 mM sucrose, 30 mM KCl, 20 mM KH 2 PO 4 , 1 mM EDTA, 6 mM MgCl 2 . With each type of mitochondria, state IV and state III respiration rates (11) were initiated by the addition of 10 µl of malate 1 pyruvate (final concentration 5 mM) in the presence of 1% BSA and, 2 or 3 min later, by the addition of 10 µl ADP (final concentration 100 µM), respectively. Addition of ADP was repeated two or three times in each assay and the respiratory control ratio (RCR) and the phosphorus-oxygen ratio (ADP/O) were calculated according to Estabrook (15). CO and Creatine Kinase (CK) Activity CO activity was determined polarographically at 25°C using a Clark oxygen electrode in both muscle homogenates and isolated mitochondria (3) to estimate muscle CO activity, mitochondria CO specific activity and the recovery of mitochondria extraction. Preparations were diluted in a modified Chapell-Perry medium, and lubrol (100 mg/g mitochondrial protein) was used to unmask enzyme activity in mitochondria or homogenates standing in ice for 30 min. Enzyme activity was expressed in nAtoms O 2 consumed/

Results are presented as means 6 SEM. The effect of age and cold stress were tested for each parameter using ANOVA (38). RESULTS In Vivo Measurements Body weight of the piglets averaged 1.59 6 0.11, 1.53 6 0.07, 1.81 6 0.11 and 2.21 6 0.10 kg at birth, 4 hr, 1 day and 5 days of life, respectively. Between 4 hr and 5 days of age, heat production and O 2 consumption increased (P , 0.001) by 70% and 35% in TN and C animals, respectively (Table 1). As expected, heat production was higher (P , 0.001) in C than in TN piglets, increasing by 100 and 59% in the cold at 4 hr and 5 days of age, respectively. The respiratory quotient was higher (P , 0.05) in C than in TN animals at 4 hr of life, decreased (P , 0.001) in both groups with age and was not different between groups at 5 days of life. Muscle CO Activity At birth, muscle CO activity was similar in LD and RH muscles, averaging 13.4 6 0.7 nAtoms O/min/mg tissue and about 2.3-fold higher in IM than in SS homogenates (Fig. 1). Between birth and 5 days of age, CO activity increased by 31 (P , 0.06) and 69% (P , 0.001) in LD and RH muscles, respectively. This enhancement is observed in

TABLE 1. Effect of age and 4-hr cold exposure on whole body heat production, respiratory quotient and O2 consumption of

the newborn pig

Heat production TN* C Respiratory quotient TN C O2 consumption TN C

4 hr

1 day

5 days

Effect of age

0.19 6 0.01 0.39 6 0.02***

0.29 6 0.01 ND

0.32 6 0.02 0.51 6 0.02***

P , 0.001 P , 0.001

0.86 6 0.01 0.90 6 0.01*

0.82 6 0.01 ND

0.77 6 0.01 0.78 6 0.01

P , 0.001 P , 0.001

9.3 6 0.5 18.6 6 0.8***

14.4 6 0.4 ND

15.9 6 0.6 26.2 6 1***

P , 0.001 P , 0.001

Values are means 6 SEM for six determinations. Heat production (kJ/min/kg BW), respiratory quotient and O2 consumption (ml/min/kg BW) were measured as described in Materials and Methods. ND, not determined. *Thermoneutral (TN) piglets were exposed to 34°C at birth, 33°C at 1 day and 30°C at 5 days of life for a 4-hr period. In the cold group (C), the ambient temperature was 10°C lower. Effect of cold for a given age: *P , 0.05; ***P , 0.001.

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Short-term cold exposure had no effect on muscle CO activity (data not shown). Mitochondrial Protein Mass

FIG. 1. Effect of age on cytochrome oxidase activity in LD

and RH muscles of the newborn pig. Values are means 6 SEM (n 5 6). ■, H M , whole muscle homogenate; h, H IM , intermyofibrillar mitochondria homogenate; , H SS , subsarcolemmal mitochondria homogenate. Effect of age: **P , 0.01; ***P , 0.001.

both types of mitochondria (P , 0.01), with CO activity in IM and SS homogenates increasing by 104 and 73% in LD muscle and by 94 and 97% in RH muscle, respectively. This enhancement occurred mainly during the first day of life in SS homogenates (140% and 71% in LD and RH muscles, respectively; P , 0.05) but persisted over the 5day period in IM homogenates (P , 0.001 between 1 and 5 days of life in both muscles). Differences in CO activity between muscles increased progressively with age, not significant at birth and 4 hr of life, significant at 1 day in IM homogenates (145% in RH muscle, P , 0.05) and at 5 days in IM and SS homogenates (135% in RH muscle, P , 0.05). Therefore, muscle CO activity was 50% higher (P , 0.001) in RH than in LD muscle at 5 days of age.

The mass of mitochondrial protein was calculated from the amount of isolated mitochondria and the yield of mitochondrial extraction. Whereas the yield of extraction of SS mitochondria remained essentially constant, amounting to 14% between birth and 5 days of age, it decreased from 18 to 13% in IM mitochondria from both muscles. Simultaneously, the amount of isolated mitochondria slightly decreased in SS mitochondria from LD muscle, remained constant in RH muscle and was enhanced (P , 0.01) in IM mitochondria from both muscles (Table 2). As a consequence, the mass of mitochondrial protein increased (P , 0.001) tremendously in both muscles with age. In LD muscle, this increase (149%, P , 0.001) was only supported by the proliferation of IM mitochondria (1130%, P , 0.001). In RH muscle, this increase was much higher and was observed for both type of mitochondria (193%, P , 0.001). Therefore, mitochondrial protein mass was not different between muscles at the time of birth but was 30% higher in RH than LD muscle at 5 days of age (P , 0.05). This increase in mitochondrial protein mass was only seen between 1 and 5 days of age. There was no effect of short-term cold exposure on mitochondrial protein mass. Electron microscopic examination of the final pellets confirm the purity and the integrity of SS and IM mitochondria, showing only intact mitochondria and a few unidentified membranous profiles (Fig. 2). Moreover, SS mitochondria seem to be smaller than IM mitochondria. Respiration of Isolated Mitochondria Direct polarographic tracing showed good reproducibility after each addition of ADP, which confirm that neither type of mitochondria was significantly damaged. IM MITOCHONDRIA. Respiration intensity of isolated IM mitochondria was maximum and similar in LD and RH

TABLE 2. Effect of age on the yield of IM, SS and total mitochondrial proteins in LD and RH muscles of the newborn pig

LD muscle IM SS Total RH muscle* IM SS Total

0 hr

4 hr

1 day

5 days

Effect of age

2.25 6 0.12 2.29 6 0.09 4.54 6 0.18

1.74 6 0.18 2.32 6 0.09 4.06 6 0.25

2.63 6 0.21 1.56 6 0.17 4.18 6 0.31

4.88 6 0.61 1.71 6 0.16 5.96 6 0.85

P , 0.001 P , 0.001 P , 0.05

2.93 6 0.23* 1.98 6 0.13 4.91 6 0.32

2.63 6 0.32* 2.32 6 0.20 4.94 6 0.44

3.54 6 0.38 1.87 6 0.11 5.42 6 0.42*

4.31 6 0.46 2.57 6 0.35* 6.32 6 0.69

P , 0.05 NS NS

Values are means 6 SEM for six determinations. Yield of IM, SS and total mitochondrial proteins is expressed in mg isolated mitochondrial protein/g muscle. *Difference between muscles for a given age: *P , 0.05.

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A

B

FIG. 2. Electron micrographs of mitochondria isolated from RH muscle at birth. (A) IM mitochondria; (B) SS mitochondria. Original magnification 315,000.

muscle at birth (Table 3). It remained constant in RH muscle but decreased by about 30% (P , 0.05) in LD muscle between 1 and 5 days of age. Thus, at 5 days of age, intensity of respiration was higher (state III, 126%, P , 0.05; state IV, 121%, P , 0.01) in RH than in LD muscle. RCR and ADP/O ratios were closed to 4 and 2.98, respectively, and

did not changed with age, indicating that mitochondria were well coupled from birth. SS MITOCHONDRIA. Respiration intensity of SS mitochondria (Table 4) was 70% lower (P , 0.001) than that of IM mitochondria. The RCR was also slightly lower (about

TABLE 3. Effect of age on respiration of muscle IM mitochondria from the newborn pig

0 hr LD muscle State III* State IV RCR ADP/O RH muscle† State III State IV RCR ADP/O

4 hr

1 day

5 days

79.4 19.0 4.26 3.03

6 6 6 6

7.1 1.2 0.39 0.09

87.3 23.0 3.96 2.90

6 6 6 6

5.7 2.2 0.46 0.05

91.8 24.0 3.80 2.78

6 6 6 6

14.3 1.4 0.54 0.11

60.2 15.8 4.13 3.02

6 6 6 6

91.4 19.6 4.87 3.17

6 6 6 6

10.3 1.6 0.65 0.08

93.7 24.3 4.25 3.01

6 6 6 6

4.1 3.3 0.72 0.08

92.7 22.7 4.25 2.93

6 6 6 6

12.3 1.7 0.76 0.10

81.1 6 6.5** 20.7 6 2.5* 4.18 6 0.43 3.06 6 0.07

5.6 1.4 0.60 0.12

Effect of age P , 0.05 P , 0.01 NS NS NS NS NS NS

Values are means 6 SEM. Number of animals: n 5 10 at birth, n 5 6 at 4 hr and 1 day and n 5 10 at 5 days of age. *State III and state IV respiration rates were determined as described in Materials and Methods and expressed in nAtoms O/min/mg mitochondrial protein. †Difference between muscles for a given age: *P , 0.05; **P , 0.01.

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TABLE 4. Effect of age on respiration of muscle SS mitochondria from the newborn pig

0 hr LD muscle State III* State IV RCR ADP/O RH muscle† State III State IV RCR ADP/O

4 hr

1 day

5 days

Effect of age

15.7 5.18 3.06 2.74

6 6 6 6

1.5 0.37 0.25 0.09

18.7 5.65 3.45 2.69

6 6 6 6

1.9 0.65 0.37 0.06

25.8 8.59 3.10 2.55

6 6 6 6

2.5 0.88 0.25 0.06

21.4 5.93 3.67 2.56

6 6 6 6

2.1 0.52 0.30 0.08

P , 0.05 P , 0.01 NS NS

25.1 6.78 3.79 2.74

6 6 6 6

2.7** 0.68* 0.29 0.08

29.7 8.6 3.66 2.73

6 6 6 6

2.1** 1.03* 0.46 0.06

31.1 10.9 2.98 2.66

6 6 6 6

4.6 2.0 0.31 0.10

19.5 5.86 3.40 2.43

6 6 6 6

1.6 0.36 0.30 0.05

P , 0.05 P , 0.01 NS P , 0.05

Values are means 6 SEM. Number of animals: n 5 10 at birth, n 5 6 at 4 hr and 1 day and n 5 10 at 5 days of age. *State III and state IV respiration rates were determined as described in Materials and Methods and expressed in nAtoms O/min/mg mitochondrial protein. †Difference between muscles for a given age: *P , 0.06; **P , 0.01.

3.35, P , 0.001). State III and IV respiration rates increased by about 65% (P , 0.01) during the first day of life in LD muscle, whereas they remained constant in RH muscle. Thereafter, respiration intensity decreased by about 43% (P , 0.01) in RH muscle and 27% (P , 0.05) in LD muscle. There was no difference between RH and LD muscles at 5 days of age. The ADP/O ratio averaged 2.74 at birth and tended to decreased with age, this decrease being significant in RH muscle (P , 0.05). There was no effect of short-term cold exposure on respiration intensity (data not shown). Oxidative and Phosphorylative Capacities In IM mitochondria, CO and CK activities changed differently with age. CO activity increased during the first day (Fig. 3) and decreased by about 35% (P , 0.01) between 1 and 5 days of life in both muscles. CK activity increased progressively with age (P , 0.001), reaching its maximum at 1 day in RH muscle (771 mU/mg mitochondrial protein) and at 5 days of life in LD muscle (521 mU/mg mitochondrial protein). Thus, the CK/CO ratio increased with age, this increase being significant (130%, P , 0.05) in RH muscle. CO and CK activities and the CK/CO ratio were in average 25% higher (P , 0.001) in RH than in LD muscle at all ages. In SS mitochondria, CO and CK activities increased (P , 0.001) simultaneously with age, and the CK/CO ratio did not changed. In LD muscle, this increase occurred progressively up to 5 days of age (1158%, P , 0.001), but in RH muscle, maximal activities were already achieved at 1 day of life. Similarly to what was observed for IM mitochondria, CO and CK activities and the CK/CO ratio were always higher (P , 0.01) in RH than in LD muscles at all ages. Finally, CO and CK activities were much lower (P , 0.05) in SS than in IM mitochondria. DISCUSSION Our results clearly demonstrate that muscle mitochondria are functional from birth and are changing primarily quanti-

FIG. 3. Effect of age on mitochondrial CO and CK activities

in IM and SS mitochondria from the newborn pig. Values are means 6 SEM (n 5 5–6). ■, LD muscle; h, RH muscle. Effect of age: *P , 0.06; **P , 0.01; ***P , 0.001.

tatively during the first 5 days of life. SS and IM mitochondria exhibit specific changes that are probably involved in the postnatal acquisition of skeletal muscle biochemical properties. These changes in muscle oxidative potential parallel the age-related changes in whole body metabolism. Muscle Mitochondrial Mass Increases During the Early Neonatal Period One of the main features of this work is probably the finding of the postnatal increase in mitochondrial protein mass in LD and RH muscles of the piglet between birth and 5 days of age. No changes are shown before 1 day of age, which is consistent with the slowness of the mitochondrial biogene-

Muscle Mitochondria in the Newborn Pig

sis process, mitochondrial DNA and total mitochondrial proteins turning over with a half-life of about 10 days in rat liver (18). The present postnatal enhancement of mitochondrial mass is in good agreement with studies showing a considerable rise in mitochondrial volume density within 3 to 5 days after birth in oxidative and glycolytic muscles of the pig (20) and a fast increase in mitochondrial number per unit tissue volume between birth and 2 days postpartum in pig liver (29) and rat liver (33) and brain (37). In our study, postnatal changes in muscle mitochondrial mass were determined indirectly, using CO activity as a mitochondrial marker to calculate the yield of extraction. Preliminary experiments have shown no difference in the calculations when citrate synthase activity was used. In fact, Davies et al. (12) compared several enzymes and demonstrated that the use of a single marker, such as CO activity, is suitable to perform such calculations. In addition, only intact mitochondria and a few unidentified membranous profiles are seen under electron microscopic examination, attesting the purity of the isolated mitochondria. However, the nature of the postnatal rise in mitochondrial mass (i.e., an increased mitochondrial size or of the surface of the inner mitochondrial membrane and (or) an enhanced mitochondrial division and proliferation) cannot be assessed directly from the present study because electron microscopic examination was not performed on muscle fibers. In addition, in adult mammals, the possibility that all mitochondrial material was interlinked into a complex reticulum has been raised by Kirkwood et al. (26) in rats. Despite the fact that this hypothesis was not confirmed by the threedimensional reconstruction study of Kayar et al. (25) and the study of Bakeeva et al. (2) on newborn rats, ultrastructural examination of skeletal muscle fibers and mitochondria from the newborn pig will be necessary to clarify this challenging aspect of skeletal muscle mitochondrial biogenesis. The plasticity of mitochondrial density and activity according to age, physical activity, thyroid status or cold acclimation is well documented (12,14,32,43). Thyroid hormones are probably involved in this early postnatal enhancement because, first, they are known to be implicated in the long-term control of mitochondrial biogenesis (32) and, second, circulating levels of T3 present a dramatic surge at 6 hr and 1 day of life in pigs (22). In addition, in the four types of mitochondria, respiration rates were maximal 1 day after birth (i.e., at a time period where T3 levels were the highest), which is in good accordance with the shortterm control of mitochondrial respiration by thyroid hormones proposed by various authors (8,21). A specific shortterm stimulation of CO biosynthesis and activity by T3 has also been proposed by Horrum et al. (24). Whatever it might be, our results suggested that mitochondrial maturation and growth (i.e., biosynthesis of respiratory and oxidative units) is a very active process during the first 5 days of life in pig muscle. Simultaneously, there is essentially no change in the coupling parameters (RCR and ADP/O), indicating

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that muscle mitochondria are functional from birth and are changing primarily quantitatively. These results confirm that the oxidative potential of pig muscle increased gradually during the first 5 days of life and paralleled the age-related changes in whole body metabolism. On one hand, from in vivo O 2 consumption data and making the assumption that skeletal muscles represent 30% of body mass and about 50% of whole body O 2 consumption at thermal neutrality and 67–79% in the cold at 4 hr and 5 days of age, respectively (G. Lossec and P. Herpin, unpublished observations), one can calculate that skeletal muscles consume 4.7 and 8.1 ml O 2 /min/kg BW at 4 hr and 5 days of age at thermal neutrality and 12.5 and 20.7 ml O 2 /min/ kg BW at 4 hr and 5 days of age in the cold, respectively. On the other hand, if we estimate mitochondrial O 2 consumption capacities of skeletal muscles from mitochondrial mass and state III respiration and a correcting factor of 2.4 for the Q10 effect (9), we come out with 13.7 and 24.5 ml O 2 /min/kg BW at 4 hr and 5 days of age, respectively, these values being not affected by short-term cold exposure. It follows that mitochondrial O 2 consumption capacity of skeletal muscles increased by 79% during the first 5 days of life, which is fairly consistent with the 70% increase of skeletal muscle O2 consumption calculated from in vivo data, and is probably high enough to sustain the enhanced metabolic rate imposed by cold exposure without short-term adaptations of mitochondrial respiration. Alternatively, this confirms that mitochondria in situ are able to operate close to their maximal O 2 consumption capacity determined in vitro (39). Postnatal Acquisition of Oxidative Metabolism in LD and RH Muscles Adult vertebrate skeletal muscle fibers are classified physiologically as fast or slow, depending on the activity of myosin ATPase, and oxidative or glycolytic, depending on the prevailing mechanisms regenerating ATP during muscular contraction. Oxidative fibers are necessarily very rich in mitochondria, and ultrastructural discrimination between fibers has also been performed using the mitochondrial profile (20,34). We recently described RH and LD muscles of 8week-old piglets as typically slow-oxidative (63% of type I fibers) and fast-glycolytic (89% of type IIb fibers) muscles, respectively, with mitochondrial protein mass being correspondingly much higher in the former (23). In the present study, there is no clear difference between LD and RH muscles at birth in terms of mitochondrial mass or activity, but differences between muscles increased progressively with age, probably in relation with the progressive postnatal acquisition of adult metabolic type. By 5 days postpartum, whole muscle CO activity was 50% higher, mitochondrial mass was 30% higher, mitochondrial CO and CK activities were 25% higher in both types of mitochondria and respiration rate was also 25% higher in IM mitochondria from RH than from LD muscle. The CK/CO ratio, an index of mito-

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chondrial phosphorylative capacities, was also higher in RH than in LD muscle. As a whole, this suggests that acquisition of optimal oxidative and ATP synthesis capacities occurs very rapidly after birth in the most oxidative muscles. This result is consistent with previous data from our laboratory attesting that oxidative capacities were higher in RH than in LD muscles at 24 (22) and 54 hr (5) of life. Similarly, in pig semitendinosus muscle, the highest increase in mitochondrial volume percentage after birth has been observed in oxidative fibers (20) and the proportion of type I myofibers increased between 12 hr and 3 days of age (1). In rat diaphragm, fiber types can be distinguished using their mitochondrial profile by 8 days postpartum (35). Differences Between IM and SS Mitochondria The two populations of muscle mitochondria differ not only in their respective locations into the cell but also in various biochemical properties (34). Our results show that IM mitochondria exhibit higher respiratory rates, enzyme activities, CK/CO ratio, RCR and ADP/O ratio than SS mitochondria. Opposite results have been obtained on mitochondria isolated from human muscle (17), but our results are in good accordance with data obtained on other species [rats (28) and ducklings (3)]. However, it cannot be excluded that in our study, as well as in those realized on rats, ducklings or humans, part of the functional and quantitative differences observed between those two mitochondrial subpopulations were related, at least in part, to differences in the isolation procedure. These higher oxidative and phosphorylative capacities of IM mitochondria were more or less maintained during the whole experimental period. However, the SS mitochondrial population is involved in the postnatal acquisition of LD and RH muscle metabolic properties because its mass was specifically enhanced in RH and not in LD muscle. Similarly, during cold acclimation, alterations of mitochondrial metabolism, such as the loose coupling of oxidative phosphorylation, have been shown to occur specifically in SS mitochondria from the most oxidative muscles (13,23). Despite their lower mass and activity, SS mitochondria should therefore play a key role in skeletal muscle metabolism. This possibility is also supported by the biological functions attributed to both populations. Indeed, IM mitochondria are mainly involved in the supply of ATP and the regulation of sarcoplasmic Ca 11 during the contractionrelaxation cycle (10), whereas SS mitochondria supply energy for active ion (40) and metabolite transport (31) across the sarcolemma and the phosphorylation of substrates and sarcolemmal proteins (27,42). In fact, both types of mitochondria probably play complementary roles in skeletal muscle metabolism, and the postnatal maturation and proliferation of SS mitochondria is probably necessary to optimize the activity of IM mitochondria. Further studies are needed to clarify the regulatory mechanisms involved in the biogenesis of skeletal muscle IM and SS mitochondria during the neonatal period.

We gratefully acknowledge F. Cohen-Adad and C. Duchamp for electron microscopic examination of the mitochondrial pellets and M. Fillaut and J. C. Hulin for their technical assistance during the experiment.

References 1. Arberle, E.D. Myofiber differentiation in skeletal muscles of newborn runt and normal weight pigs. J. Anim. Sci. 59:1651– 1656;1984. 2. Bakeeva, L.E.; Chentsov, Y.S.; Skulachev, V.P. Mitochondrial framework (reticulum mitochondriale) in rat diaphragm muscle. Biochim. Biophys. Acta 501:349–369;1978. 3. Barre´, H.; Berne, G.; Brebion, P.; Cohen-Adad, F.; Rouanet, J.L. Loose coupled mitochondria in chronic glucagon treated hyperthermic ducklings. Am. J. Physiol. 256:R1192–R1199; 1989. 4. Barre´, H.; Cohen-Adad, F.; Duchamp, C.; Rouanet, J. L. Multilocular adipocytes from muscovy ducklings differentiated in response to cold acclimation. J. Physiol. 375:27–38;1986. 5. Berthon, D.; Herpin, P.; Bertin, R.; De Marco, F.; Le Dividich, J. Metabolic changes associated with sustained 48-h shivering thermogenesis in the newborn pig. Comp. Biochem. Physiol. 114B:327–335;1996. 6. Berthon, D.; Herpin, P.; Le Dividich, J. Shivering thermogenesis in the neonatal pig. J. Therm. Biol. 19:413–418;1994. 7. Berthon, D.; Herpin, P.; Duchamp, C.; Dauncey, M.J.; Le Dividich, J. Modification of thermogenic capacity in neonatal pigs by changes in thyroid status during late gestation. J. Dev. Physiol. 19:253–261;1993. 8. Brand, M.D.; Steverding, D.; Kadenbach, B.; Stevenson, P.M.; Hafner, R. The mechanism of the increase in mitochondrial proton permeability induced by thyroid hormones. Eur. J. Biochem. 206:775–781;1992. 9. Byrne, E.; Trounce I. Oxygen electrode studies with human skeletal muscle mitochondria in vitro. J. Neurol. Sci. 69:319– 333;1985. 10. Carafoli, E. Mitochondria Ca 21 transport and the regulation of heart contraction and metabolism. J. Mol. Cell. Cardiol. 7: 83–89;1975. 11. Chance, B.; Williams, G.R. The respiratory chain and oxidative phosphorylation. Adv. Enzymol. 17:65–134;1956. 12. Davies, K.J.A.; Packer, L.; Brooks, G.A. Biochemical adaptation of mitochondria, muscle, and whole-animal respiration to endurance training. Arch. Biochem. Biophys. 209:539–554; 1981. 13. Duchamp, C.; Cohen-Adad, F.; Rouanet, J.L.; Barre´, H. Histochemical arguments for muscular non-shivering thermogenesis in muscovy ducklings. J. Physiol. 457:27–45;1992. 14. Eppley, Z.A.; Russell, B. Perinatal changes in avian muscle: Implication from ultrastructure for the development of endothermy. J. Morphol. 225:357–367;1995. 15. Estabrook, R.W. Mitochondrial respiratory control and the polarographic measurement of ADP/O ratios. In: Colowick, S. P.; Kaplan, N. O. (eds). Methods in Enzymology. New York: Academic Press; 1967:41–47. 16. Foster, G.; Bernt, E.; Bergmeyer, U. Creatine kinase. In: Bergmeyer, H.U. (ed). Methods of enzymatic analysis. New York: Academic Press, 1974:147–151. 17. Frederico, A.; Manneschi, L.; Paolini, E. Biochemical difference between intermyofibrillar and subsarcolemmal mitochondria from human muscle. J. Inher. Metab. Dis. 10(Suppl. 2):242–246;1987. 18. Gross, N.J. Control of mitochondrial turnover under the influence of thyroid hormone. J. Cell Biol. 48:29–40;1971. 19. Hallman, M. Changes in mitochondrial respiratory chain proteins during perinatal development. Evidence of the impor-

Muscle Mitochondria in the Newborn Pig

20. 21. 22.

23. 24. 25.

26. 27.

28. 29.

30.

31.

tance of environmental oxygen tension. Biochem. Biophys. Acta 253:360–372;1971. Handle, S.E.; Stickland, N.C. The effects of low birthweight on the ultrastructural development of two myofibre types in the pig. J. Anat. 150:129–143;1987. Harper, M.E.; Ballantyne, J.S.; Leach, M.; Brand, M.D. Effect of thyroid hormones on oxidative phosphorylation. Biochem. Soc. Trans. 21:785–792;1993. Herpin, P.; Berthon, D.; Duchamp, C.; Dauncey, M.J.; Le Dividich, J. Effect of thyroid status in the perinatal period on oxidative capacities and mitochondrial respiration in porcine liver and skeletal muscle. Reprod. Fertil. Dev. 8:147–155; 1996. Herpin, P.; Lefaucheur, L. Adaptative changes in oxidative metabolism in skeletal muscle of cold-acclimated piglets. J. Therm. Biol. 17:277–285;1992. Horrum, M.A.; Tobin, R.B.; Ecklund, R.E. Thyroid-hormones induced changes in rat liver mitochondrial cytochromes. Mol. Cell. Endocrinol. 68:137–141;1985. Kayar, S.R.; Hoppeler, H.; Mermod, L.; Weibel, E.R. Mitochondrial size and shape in equine skeletal muscle: A threedimensional reconstruction study. Anat. Rec. 22:333–339; 1988. Kirkwood, S.P.; Munn, E.A.; Brooks, G.A. Mitochondrial reticulum in limb skeletal muscle. Am. J. Physiol. 251:C395– C402;1986. Korbl, G.P.; Sloan, I.G.; Gould, M.K. Effect of anoxia, 2,4dinitrophenol and salicylate on xylose transport by isolated rat soleus muscle. Biochim. Biophys. Acta 465:93–109; 1977. Krieger, D.A.; Tate, C.A.; McMillin-Wood, J.; Booth, F.W. Populations of rat skeletal muscle mitochondria after exercise and immobilization. J. App. Physiol. 48:23–28;1980. Mersman, H.J.; Goodman, J.; Houk, J.M.; Anderson, S. Studies on the biochemistry of mitochondria and cell morphology in the neonatal swine hepatocyte. J. Cell Biol. 53:335–347; 1972. Miyahara, M.; Kitazoe, Y.; Hiraoka, N.; Takeda, K.; Watanabe, S.; Sasaki, J.; Osaki, Y.; Yamanoto, H.; Utsumi, K. Developmental changes in mitochondrial components in liver of newborn rats. Biol. Neonate 45:129–141;1984. Muller, W. Sarcolemmal mitochondria and capillarizations of soleus muscle fibres in young rats subjected to endurance

647

32.

33. 34.

35.

36. 37. 38. 39.

40. 41.

42.

43.

training. A morphometric study of semi-thin section. Cell. Tissue Res. 174:367–389;1976. Mutvei, A.; Husman, B.; Andersson, G.; Nelson, B.D. Thyroid hormone and not growth hormone is the principle regulator of mammalian mitochondrial biogenesis. Acta Endocrinol. 121:223–228;1989. Ontko, J.A. Genesis of liver mitochondria in the neonatal rat. Life Sci. 5:817–825;1966. Palmer, J.W.; Tandler, B.; Hoppel, C.L. Biochemical properties of subsarcolemmal and intermyofibrillar mitochondria isolated from rat cardiac muscle. J. Biol. Chem. 252:8731–8739; 1977. Poggi, P.; Marchetti, C.; Scelsi, R.; Calligaro, A.; Casasco, A. An enzyme-histochemical, ultrastructural and morphometric study on fetal and neonatal rat diaphragms. J Submicrosc. Cytol. Pathol. 22:515–521;1990. Pollac, J.K. The maturation of inner membrane of foetal rat liver mitochondria: An example of a positive feedback mechanism. Biochem. J. 150:477–488;1975. Samson, F.E.; Balfour, W.M.; Jacob, R.J. Mitochondrial changes in developing rat brain. Am. J. Physiol. 199:693–696; 1960. SAS. SAS User’s guide. Statistics. Cary, NC: SAS Inst. Inc.; 1988. Schwermann, K.; Hoppeler, H.; Kayar, S.R.; Weibel, E.R. Oxidative capacity of muscle and mitochondria: Correlation of physiological, biochemical, and morphometric characteristics. Proc. Natl. Acad. Sci. USA 86:1583–1587;1989. Sjodin, R.A.; Beauge, L.A. Analysis of the leakages of sodium ion into and potassium ion out of striated muscle cells. J. Gen. Physiol. 61:22–250;1973. Trayhurn, P.; Temple, N.J.; Van Aerde, J.V. Evidence from immunoblotting studies on uncoupling protein that brown adipose tissue is not present in the domestic pig. Can. J. Physiol. Pharmacol. 67:1480–1485;1989. Walaas, O.; Walass, E.; Liptad, E.; Altersen, A.R.; Horn, R.S.; Fossum, S. A stimulatory effect of insulin on phosphorylation of a peptide in sarcolemma-enriched membrane preparation from rat skeletal muscle. FEBS Lett. 80:417–422;1972. Yen, T.C.; Chen, Y.S.; King, K.L.; Yeh, S.H.; Wei, Y.H. Liver mitochondrial respiratory functions decline with age. Biochem. Biophys. Res. Commun. 165:994–1003;1989.