Acta Physiol Scand 1981, 112: 89-95

Effects of age and prolonged running on proteolytic capacity in mouse cardiac and skeletal muscles

A. SALMlNEN and V. VIHKO

Division of Muscle Research, Department of Cell Biology, University of Jyvaskyla, Jyvaskyla. Finland

SALMINEN, A. & VIHKO, V.: Effects of age and prolonged running on proteolytic capacity in mouse cardiac and skeletal muscles. Acta Physiol. Scand. 1981, 112:89-95. Received 25 Sept. 1980. ISSN 0001-6772. Division of Muscle Research, Department of Cell Biology, University of Jyvaskyla, SF-40100 Jyvaskyla 10, Finland. Male NMRI-mice, aged 3, 6 , 9 , and 12 months, were made to run for a period of 4 h at a speed of 13.5 mlmin on a motordriven treadmill. 5 days after exertion, selected enzymatic estimates of acid and alkaline proteolytic as well as energy metabolic capacities were analyzed from the cardiac muscle and from the red and white parts of m. quadriceps femoris (MQF). The activities of alkaline and myofibrillar proteases increased most consid- erably in skeletal muscles with age. Cathepsin D and P-glucuronidase activities were less affected in both muscles. Prolonged running increased the activities of cathepsin D, dipep- tidy1 aminopeptidase I and P-glucuronidase in the white and, especially, in the red part of MQF. This stimulation of acid hydrolytic capacity was more prominent at the ages of 3 and 6 months than in the older animals. The estimates of alkaline proteolytic or energy metabolic capacities were not affected by prolonged running. In cardiac muscle, no sig- nificant changes were recorded in acid hydrolytic or energy metabolic capacity. Histologi- cal observation showed no necrosis or other pathological phenomena in the proximal part of m. rectus femoris after exertion. We suggest that the increased acid proteolytic capacity is involved in subcellular regenerative processes of skeletal muscle fibres. The smaller lysosomal response of older mice may indicate a reduced potential capacity for cellular repair. Key words: Aging, exertion, muscles (enzymology, pathology), myocardium (enzymology), proteins (metabolism), lysosomes (enzymology)

Strenuous exercise causes reversible and irreversi- ble skeletal muscle fibre injuries (e.g. Gollnick & King 1%9, Hecht et al. 1975, Vihko et al. 1978~). Both maximal exhaustive and submaximal pro- longed exertions demand efficient energy produc- tion in exercising skeletal muscles. Lack of energy is one possible reason for cell damage (see Trumpet al. 1976). lrreversible injuries manifest themselves by focal fibre necrosis and by invasion of inflam- matory phagocytes (e.g. Vihko et al. 1978~) . In surviving, reversibly injured, skeletal muscle fibres the acid hydrolytic capacity is highly increased dur- ing the 2-7 days after strenuous exercise, suggest- ing an enhanced autolytic breakdown (Vihko et al. 1978~. 6 ) .

During ageing the capacity for cellular respiration is reduced in skeletal and cardiac muscles (see e.g. Basset al. 1975, Hams 1975). This retardation has a

negative effect on the energy balance during exer- cise and it delays recovery processes after exertion (Ermini et al. 1971). These phenomena might in- crease the susceptibility of muscle fibres to cellular damage. The present investigation was aimed at clarifying whether age affects the proteolytic capacity of mouse skeletal and cardiac muscles at rest and after prolonged exercise.

METHODS

Animals and prolonged running Male NMRI-mice, aged 3 , 6 , 9 and 12 months, were used in this study. Animal care was undertaken as described earlier (Vihko et al. 1978~) . The mice of diffenmt ages were randomly divided into control and exercising ani- mals. All exercising mice were made to run for 4 h on a motor-driven treadmill with 6" uphill tracks. The running speed was 13.5 mlmin. In the middle of the exertion there

90 A . Salminen and V . Vihko

Table I . Enzyme octivities and protein contents in red and white parts of m. quadriceps femoris (MQF) of control (C) and exercised (Ex) mice of different ages

3 months 6 months 9 months

C Ex C Ex C Ex Variables (12) (12) (12) (12) (12) (12)

Red part of MQF Cathepsin D Dipeptidyl amino- peptidase I

P-Glucuronidase Alkaline protease Leucine arylamidase Malate dehydrogenase Citrate synthase Protein content

White purr of MQF Cathepsin D Dipeptidyl aminopep

P-Glucuronidase Alkaline protease Leucine arylamidase Malate dehydrogenase Citrate synthase Protein content

tidase 1

12.lf0.5

1.82f0.05 0.17k0.01 2.77f0.19 12.5f0.2 I1.3f0.5 0.98f0.04 224f2

8.7k0.4

1.52f0.04 0.1 1 fO.01 2.00f0.14 7.5fO. I 4.4f0.1

0.27f0.01 227f3

15.6+0.9**

3.15f0.27*** 0.44f0.06*** 2.93 f 0.20 14.3f0.3*** 10.6fO.5 0.92f0.05 214+3*

10.6f0.4**

1.97+0.09*** 0.20~0.02*** 2.03f0.11 8.1 +0.2** 4.7f0.2

0.32f0.02* 225 f 4

12.8f0.5

1.62f0.07 0.I7fO.Ol 3.47 f0.20 12.3f0.2 1 l.7f0.5 0.97f0.04 214f3

9.5f0.3

1.32f0.04 0.11 f O . O 1 2.30f0.27 7.3f0.1 4.9f0.3

0.28f0.01 219f3

16.7+0.8***

3.23f0.19*** 0.53f0.05*** 3.30f0.24 14.lf0.3*** 1 I .2+0.5 0.87f0.05 211k4

11.1 f0.3**

1.92+0.07*** 0.22f0.01*** 2.20f0.17 8.0+0.2** 4.950.2

0.30f0.02 219f4

13.7k0.5

1.82f0.07 0.17f0.01 3.73f0.24 12.1f0.1 11.1 f0 .3 1.02f0.06 216f5

10.9f0.3

1 S2f0.07 0.12f0.01 2.73 f 0.20 7.3f0.1 4.9f0.2

0.3 I f0.02 225f4

15.7k0.8

2.32+0.18* 0.35f0.05** 3.53f0.21 12.6f0.2 11.2f0.4 1.01 f0.05 217f4

12.0f0.4*

1.82+0.07** 0.21 f0.02*** 2.50f0.23 7.6f0. I 4.7f0.2

0.29f0.02 213f5

*p<0.05, **p<O.OI, ***p<O.OOl. Enzyme activities are expressed as pmol reaction products X s - l X kg-' fresh muscle (dipeptidyl aminopeptidase 1, P- glucuronidase and leucine arylamidase) or mmol XS-' xkg-' fresh muscle (malate dehydrogenase and citrate synthase). Cathepsin D and alkaline protease activities are expressed as proteinase units, one unit corresponding to enzyme activity which during 1 s at 37°C liberates per kg fresh muscle TCA-soluble hydrolysis products with a colour value equivalent to 1 pmol of tyrosine. Protein contents are given as g proteinlkg fresh muscle. Values are means f S.E.

was a 15 min pause, during which the mice had free access to drinking water. All the animals were able to run for 4 h. After the exertion the mice lived for 5 days under normal cage conditions before sacrifice. The weight of 3 months old mice was 39.9f0.6 g (fS.E.) and it increased with age (Table 4). No significant differences were observed be- tween control and exercised groups.

samples Mice were killed by cervical dislocation. The cardiac muscle was first removed and washed free of blood. The predominantly white and red skeletal muscle samples were detached from both the m. quadriceps femori (MQF). The white muscle sample was composed of the distal head of m. vastus lateralis. The red muscle sample

Table 2. Enzyme activities and rate of acid autolysis in rn. rectus fernoris of control ( C ) and exercised ( E x ) mice of different ages

Rate of acid autolysis and myofibrillar protease activity are expressed as proteinase units (see Table I ) . Myofibrillar protease activity is expressed per kg protein in myofibrillar fraction. Other explanations are as in Table 1

3 months 6 months 9 months

C Ex C Ex C Ex Variables (12) (12) (12) (12) (12) (12)

Acid autolysis 1.33f0.05 1.42f0.09 1.29f0.03 1.38f0.03* 1.47f0.04 1.52f0.06 P-Glucuronidase 0.12f0.01 0.20f0.03* 0.12f0.01 0.19f0.01*** 0.12f0.01 0.16+0.01* Myofibrillar protease 8.7f0.4 9.2k0.4 11.0f0.5 11.5f0.7 12.6f0.5 12.6f0.5 Protein content 184f3 181f4 175f4 176f4 182f4 177f3

Muscle proteolytic capacity 91

12 months

C Ex (7) (7)

14.4f0.7

I .78+0.03 0.19f0.01 4.00f0.22 12.1 f0.4 I I.0f0.8 1.06f0.08 219f5

1 1 . 1 f0.6

1.52f0.04 0.13f0.01 2.57f0.20 7.7f0.2 4.7t0.3

0.30f0.03 221k5

15. I f0.4

2.45tO.14*** 0.33f0.03** 3.90f0.26 12.5f0.4 9.6f0.5

0.97f0.06 205f4

1 I .6f0.7

1.92 f 0. I 1 * 0.19+0.02* 2.73f0.12 7.8f0.2 4.4f0.3

0.32t0.02 211t7

consisted of the red parts of the proximal heads of m. vastus lateralis and m. vastus medialis and the red fibres of m. vastus intermedius. M. rectus femoris was excised from the left MQF for enzymatic study.

The cardiac muscle of the 3 months old controls weighed 140f3 mg. The weights of hearts increased with age (Table 4). No statistically significant differences be- tween control and exercised groups were observed. The red and white muscle samples of 3 months old controls weighed 55.6f0.1 mg and 62.3t0.2 mg. respectively. M. rectus femoris weighed 1 1 2 f 2 mg. No significant differ- ences were recorded between any of the groups.

The muscle samples were quickly prepared, weighed, frozen, and kept at -80°C until analyzed. The cardiac muscle samples were homogenized in distilled water using an all-glass Potter-Elvehjem homogenizer (670 rpm) and

12 months

C Ex (7) (7)

1.47f0.05 1.45f0.04 0.13_+0.01 0.19+0.02** 12.95 I . 1 13.3f0.9 174f6 175f2

diluted to 2% (w/v). The white and red skeletal muscle samples were homogenized in distilled water with an all- glass microhomogenizer and diluted to 3% (wlv). M. rec- tus femoris was homogenized in 0.01 M potassiumphos- phate buffer, pH 7.7, using a Teflon pestle (670 rpm).

Assay of enzyme activities and autolytic rates The activities of cathepsin D (EC 3.4.23.5), dipeptidyl aminopeptidase I (EC 3.4.14.1) and /3-glucuronidase (EC 3.2.1.31) were assayed as described by Barrett (1972). The rate of acid autolysis was estimated at pH 3.8 essentially as described by Stauber et al. (1976). using I mM MgCI2 as the activator. Leucine arylamidase (leucine 4-nitroanilide as substrate) activity was assayed according to the method of Pluskal & Pennington (1976) using 50 mM KHzP04- NaZHFQ4 buffer, pH 7.3. Alkaline protease activity was determined at pH 10.0 using denatured casein as substrate (Pennington 1977). The activity of myofibrillar protease was assayed as described by Mayer et al. (1974). The estimates of energy metabolic capacities, malate dehyd- rogenase (EC 1. I . I .37), citrate synthase (EC 4.1.3.7) and lactate dehydrogenase (EC 1 . I . 1.27). together with pro- tein contents were measured as described earlier (Vihko et al. 19786, 1979).

Histological methods Serial cryostate sections for performing a routine hema- toxylin-eosin staining were cut from the proximal part of m. rectus femoris of the right MQF (Vihko et at. 1978a). Histological sections were used to evaluate pathological changes in skeletal muscle.

RESULTS

Effect of age on the proteolytic capacity

In skeletal muscle the activity of cathepsin D in- creased prominently with age (Table 4). p- Glucuronidase activity and the rate of acid autolysis increased slightly between the ages of 3 and 12 months. In cardiac muscle P-glucuronidase activity was highest at the age of 12 months (Table 4).

The activities of alkaline and myofibrillar pro- teases increased highly significantly with age in skeletal muscles (Table 4). Leucine arylamidase ac- tivity was unchanged.

No significant changes were observed in the estimates of energy metabolism in skeletal and car- diac muscles. Protein content decreased slightly with age.

Effect of prolonged running on rhe proteolytic capacity The activities of cathepsin D, dipeptidyl aminopep- tidase I and p-glucuronidase were highly increased

A d o Pliysiol S w t i d I12

92 A. Sulminen und V . Vihko

Table 3. Etizyme uctivities in cardiac muscle of control (C) nnd exercised (Ex) mice of different uges

Variables

Cathepsin D P-Glucuronidase Leucine arylamidase Malate dehydrogenase Lactate dehydrogenase Protein content

3 months 6 months 9 months

21.3f0.7 20.6k0.7 0.22k0.01 0.23f0.02 17.2f0.4 18.2f0.5 1 1 . 1 f 0 . 2 11.2f0.3 0.92f0.03 0.94f0.05 18853 191 +4

21.6f0.5 21.5f0.5 0.25f0.01 0.23f0.01 18.1 f 0 . 6 17.7f0.3 10.5f0.3 10.9f0.2 0.92f0.04 0.90f0.03 188f4 190f4

22.7f0.7 22.9k0.8 0.23f0.01 0.25f0.01 18. l t0 .6 17.5f0.5 10.7f0.3 10.7k0.5 0.88f0.04 0.93f0.05 186f4 182f4

Lactate dehydrogenase activity is expressed as mmol XS-' x kg-I fresh muscle. Other explanations are as in Table 1

in the red and white skeletal muscles on the fifth day after prolonged running (Table I ) . This increase was more pronounced in the red part of MQF than in the white part or in MRF (Tables 1 and 2).

Prolonged running did not affect the activities of alkaline and myofibrillar proteases in skeletal mus- cle (Tables 1 and 2). Leucine arylamidase activity was slightly increased.

Prolonged running did not change the activities of cathepsin D, P-glucuronidase and leucine arylamidase in cardiac muscle (Table 3). The esti- mates of energy metabolic capacity were also un- changed in cardiac and skeletal muscles.

EfliJc.1 ($'age on the proteolytic response

The acid hydrolytic response to prolonged running was more prominent in the younger than in the older mice (Table 1). No corresponding response was observed in cardiac muscle (Table 3).

Histological observations

No atrophy was observed in the MRF of even the oldest mice. Necrotic lesions or regenerative skeletal muscle fibres were not noticed in the MRF of exercised mice more frequently than in the con- trols.

DISCUSSION

The work capacity of skeletal and cardiac muscles is diminished at old age (see e.g. Harris 1975, Er- mini 1976). The efficiency of energy production, especially the aerobic oxidation of carbohydrates, is reduced (Ermini et al. 1971, Bass et al. 1975, Harris 1975). Atrophy and loss of muscle fibres decrease the strength of skeletal muscles (see Hanzlikova & Gutmann 1975, Ermini 1976). De-

generative processes thus prevail in senescent mus- cles (Travis & Travis 1972, Hanzlikova & Gutmann 1975). Our oldest mice, 12 months old, were not very senescent, because some of our mice may reach the age of 2 years. Senile mice are, however, inconvenient for exercise studies because of their remarkably reduced running ability. In the present study the estimates of oxidative energy production were not affected by age between 3 and 12 months in skeletal muscle, and no atrophy was observed in skeletal muscle, although the proteolytic capacity was increased with age.

The activities of acid hydrolases in skeletal mus- cle originate either in muscle fibres or in interstitial cells, such as fibroblasts, endothelial cells or in- vaded macrophages (Vihko et al. 1 9 7 8 ~ ) . Histologi- cal examination revealed no signs of inflammation in a thigh muscle. Thus it is probable that the in- crease in acid hydrolase activity of red and white muscle after exertion originates in skeletal muscle fibres, especially in red oxidative fibres. This con- clusion is in agreement with our earlier direct his- tochemical findings, which showed that most of the activity increase was in skeletal muscle fibres (Vih- ko et al. 1978~) . A similar lysosomal response has been observed in skeletal muscle after experimental ischaemia (Shannon et al. 1974).

Transient mitochondria1 swelling together with intracellular edema found after heavy exercise indi- cate that prolonged running produces reversible skeletal muscle fibre injuries (Gollnick & King 1969). In addition to these changes, formation of autophagic vacuoles, e.g. in hypoxic hepatic paren- chymal cells, is a general indication of sublethal cell injuries (Glinsmann & Ericsson 1966). Focal cyto- plasmic iqjuries may stimulate the autophagic path- way to autodigest damaged cell structures. Our own

Muscle proteolytic. ropcicity 93

12 months

C Ex (7) (7)

23.0f0.8 22.9t I .O 0.26f0.01 0.26 f 0.02 18.6k0.5 18.2f0.2 10.7f0.3 10.3f0.6 0.92f0.05 0.87t0.06 178k4 178k3

unpublished observations have revealed an in- creased number of autophagic vacuoles in skeletal muscle fibres after prolonged running. The ob- served autophagic response coincides with the in- creased activities of acid hydrolases during the 2-7 days after exertion. The autophagic response is ex- ceptionally slow in skeletal muscle in comparison with e.g. hypoxic hepatic parenchymal cells, in

which the process is completed within 24 h (Glinsmann & Ericsson 1966).

The degree of the exercise-induced acid hyd- rolase response in skeletal muscle was affected by age, the response being much stronger in younger than in older animals. This difference is more prob- ably caused by a partial loss in adaptive capacity of muscle cells during ageing than by increased toler- ance for exertion. Similar reduction of functional capacity during ageing has also been found e.g. in liver, in which susceptibility to the damaging effect of galactosamine is increased (Platt et al. 1978) or the strength of many enzyme inductions is di- minished (see Adelman 1979). Reduced lysosomal response in skeletal muscle after exertion, together with decreased protein biosynthesis (Britton & Sherman 1975), may delay the subcellular regenera- tion and thus also the reattainment of the homeos- tasis of muscle fibres.

In cardiac muscle there was no acid hydrolase response corresponding to that in the skeletal mus-

Table 4. Efikcts ($age on some vcirinbles in skeletal and cardiuc muscles Per cent comparison to 3 months old mice

Variables 6 months 9 months 12 months

Weight of mice

Redlwhite MQF Cathepsin D Dipeptidyl aminopeptidase I P-Glucuronidase Alkaline protease Leucine arylamidase Malate dehydrogenase Citrate synthase Protein content

M. recfus femoris Weight of MRF Acid autolysis P-Glucuronidase Myofibrillar protease Protein content

Cardiac muscle Weight of heart Cathepsin D P-Glucuronidase Leucine arylamidase Malate dehydrogenase Lactate dehydrogenase Protein content

2.5

5.819.2

O.OlO.0

25.3*/15.0 I- 1.61-2.7

3.511 1.4 - 1.013.7 -4.5*1-3.5

- 11.01- 13.2**

0.0 -3.0

0.0 26.4**

-4.9

5.7 1.4

13.6 5.2

-5.4 0.0 0.0

5.0

13.2*125.3** O.OlO.0 0.019.1

34.7**/36.5** - 3.21- 2.7 -1.8111.4*

-3.61-0.9 4.1114.8

6.3 10.5* 0.0

44.8*** - 1 . 1

8.6** 6.6 4.5 5.2

-3.6 -4.3 - 1 . 1

10.5**

19.0*/27.6** -2.210.0 11.8118.2* 44.4***/28.5* -3.212.7 -2.716.8

8.211 I . I - 2.21 - 2.6

2.7 10.5 8.3

48.3*** -5.4

15.0**

8.0 18.2** 8.1 -3.6

0.0 -5.3*

Statistical significances are as in Table I .

Acro Plr?.tiril Scrrrid I I2

94 A . Salminen and V . Vihko

cle. Even more strenuous exhaustive running does not affect the acid hydrolytic capacity Id days after exercise (Salminen & Vihko 1980). Sublethal injuries may also stimulate the autophagic pathway in cardiac muscle, as was found after deprivation of oxygen and glucose from organ culture media of fetal mouse hearts (Sybers et al. 1979). Thus it seems probable that running exertion does not cause sublethal injuries in cardiac muscle cells of mice. However, it must be remembered that au- tophagocytosis could occur in cardiac muscle cells without acid hydrolase response, as has been found in liver (Hirsimaki et al. 1976) or in fibroblasts (Amenta et al. 1978).

The breakdown of muscle protein is catalyzed by acid, neutral and alkaline proteases (see Pennington 1977). The activities of alkaline and myofibrillar proteases are particularly increased under condi- tions of enhanced protein degradation, a s during e.g. starvation (Mayer et al. 1974) or diabetic (Rothig et al. 1978) and glucocorticoid (Mayer et al. 1976) atrophies. Only the proteases of the alkaline pH range are affected in experimentally induced streptozotocin-diabetic atrophy (Rothig et al. 1978). The starvation-induced increase in muscle protein degradation is also non-lysosomal in origin, al- though a small increase in acid protease activity is observed (Jenkins e t al. 1979). In contrast t o these atrophic myopathies the acute exercise myopathy does not affect the activities of alkaline proteases. This different stimulation effect demonstrates the selectivity of intracellular protein degradation.

This study was supported by the Finnish Research Coun- cil for Physical Education and Sport (Ministry of Educa- tion) and the Academy of Finland. We thank Mrs. Arja Mansikkaviita for skillful technical assistance.

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