Acta Physiol Scand 1979, 107: 39-46
Skeletal muscle metabolism, morphology and function in sedentary smokers and nonsmokers JAN ORLANDER, KARL-HEINZ KIESSLING and LARS LARSSON' Institute of Zoophysiology, University of Uppsala, the Department of Animal Nutrition, Swedish University of Agricultural Sciences, Uppsala, and the Laboratory for Human Performance, National Defence Research Institute, Stockholm, Sweden
ORLANDER, J . , KIESSLING, K-H. & LARSSON, L.: Skeletal muscle metabolism, morphology and function in sedentary smokers and nonsmokers. Acta Physiol Scand 1979, 107: 3946. Received 29 Dec. 1978. ISSN 0001-6772. Institute of Zoophysiology, University of Uppsala, the Department of Animal Nutrition, Swedish University of Agricultural Sciences, Uppsala, and the Laboratory for Human Performance, National Defence Research Institute, Stockholm, Sweden. Smokers and nonsmokers of a homogeneous population of sedentary men have been compared with respect to skeletal muscle (vastus lateralis) morphological, metabolic and functional characteristics. The percentage type I fibres was lower and that of type IIB fibres higher in the smokers. Fibre areas were almost equal in the two groups. Muscle oxidative capacity was lowered in the smokers, as judged from decreased mitochondria1 enzyme activities and a lowered fibrillar space mitochondria1 volume fraction. Isometric and dynamic strengths were lower in the smokers, except at the highest velocity of movement studied. Dynamic strengths expressed in relation to isometric strength were similar at all velocities except the highest, where the smokers were relatively stronger. Muscular endurance, measured in short-term isometric and dynamic tests, was not different. It is suggested that the lowered muscle oxidative capacity and strength in the smokers may be partly a consequence of the different fibre type distribution. A possibly lower physical activity level, and tobacco smoke constituents (e.g. carbon monoxide) may also be instrumental. It is not clear whether the different fibre type distribution in the smokers is an effect of smoking per se, or if background factors are responsible. Key words: Muscle metabolism, muscle morphology, smoking, strength, tobacco
Tobacco smoking is known to markedly impair the capacity for prolonged exercise, as revealed by performance tests and/or measurements of maximal oxygen uptake (Cooper et al. 1968, Peterson & Kelley 1%9, McDonough et al. 1970, Hrubes & Battig 1970, Shaver 1973, Raven et al. 1974, IngemannHansen & Halkjaer-Kristensen 1977). The impaired working capacity has been attributed to increased oxygen debt and lactate production in smokers (Chevalier et al. 1963, Krumholz et al. 1964, Krumholz & Hedrick 1972, Reece & Ball 1972), although there are conflicting data (Raven et al. 1974). Carbon monoxide has been implicated as a major etiological agent (Chevalier et al. 1%6). The physical working capacity and the metabolic capacity of the working muscles are closely interrelated (Holloszy & Booth 1976). It would therefore seem conceivable that part of the impairment of the
working capacity in smokers might be related to effects on muscle. There is but scanty information in the literature concerning possible effects of tobacco smoking on skeletal muscle characteristics. Some early studies (reviewed by Fischer et al. 1%0) on the effect of smoking on muscle strength or efficiency showed variable results (increases, decreases or no effect at all). Apparently, no studies on muscle morphology or metabolism in relation to smoking have appeared so far. In the present study, smokers and nonsmokers of a homogenous population of sedentary men were compared with respect to skeletal muscle morphological, metabolic and functional characteristics.
' Present address: Dept. of Physiology 111, Karolinska Institute at Gymnastik- och idrottshogskolan, Stockholm, Sweden. Actti P/z?siol Sctind 107
J . Orlander et a / .
40
Table I . Anthropometricaf characteristics Values are means k S. E . FFS, fat free soft tissue weight. n.s., not significant ~~
Group Smokers Nonsmokers P
No. of subjects
Age (y.)
Height (cm)
Weight 0%)
FFS (kg)
Skeletal weight (kg)
Body fat weight (kg)
Thigh circumference (cm)
18
44k3 43k3 n.s.
179k2
78.4k2.6 76.8k1.6 n.s.
5 3 . 9 t 1.7 53.7k1.3 n.s.
12.4k0.5 12.8k0.2 n.s.
10.0k0.8 9.2k0.5 n.s.
54.7k0.9 54.2k0.7 n.s.
25
180k1
n.s.
MATERIAL AND METHODS Subjects. 43 apparently healthy men (18 smokers and 25 nonsmokers) volunteered to participate in the study. They were all white-collar workers (employees of an insurance company), and engaged in little or no physical activity during their spare time. The basis for acceptance of a subject was the desire to get a subject population as homogeneous as possible with regard to physical activity level. Smoking habits were not considered in this selection. An oral consent was obtained from each subject after he had been informed of the procedure and possible risks of the experiments. Informations regarding smoking habits were obtained by using a questionnaire. Each subject was asked to specify his weekly tobacco consumption. 11 were cigarette smokers and smoked 91 (10-200) cigarettes, with or without filters, per week. 4 subjects smoked the pipe and consumed 62 (25-125) g of tobacco per week. 3 had “mixed” habits, smoking both the pipe and cigars or cigarettes. It was decided to consider all smokers together as one group. For each subject, age, height, weight, skeletal width and skinfold thickness were recorded. Body fat weight, skeletal weight and fat free soft tissue weight (FFS) were calculated according to von Dobeln (1964). The circumference of the left thigh was measured in a horizontal plane just under the gluteal furrow. As shown in Table 1, the two groups were very similar with regard to all studied anthropometrical variables. All experiments were performed at 8-11 a.m., and followed a fixed protocol with biopsies preceding strength measurements. No restrictions regarding smoking were given, but no subject smoked during at least 20 min preceding the experiments. Muscle biopsies. Muscle tissue was obtained from the left vastus lateralis by the percutaneous needle technique (Bergstrom 1%2). The specimens were used for histochemistry, enzyme assays and electron microscopy. Hisrochemistry. The muscle samples for fibre typing and area determination were trimmed, mounted, frozen in isopentane cooled by liquid nitrogen, and stored at -80°C until analysis. Serial transverse sections (10 p m ) were cut with a cryotome at -20°C and the myofibrillar ATPase staining method (Gomori 1941, Padykula & Herman 1955) was used for muscle fibre classification. Photographs of the stained sections were taken and classification into type I (slow twitch) and type I1 (fast twitch) (Engel 1962) was A< ta Phv\iol Jccind 107
made in all 43 subjects. In 34 of the subjects, the type I1 fibres were subclassified into A, B and C subgroups (Brooke & Kaiser 1970, Dubowitz & Brooke 1973). The average number of counted fibres for classification into main groups (type I and type 11) and subgroups (type I , type I I A , B, and C) was 585252 and 251235, respectivelYt The fibre areas were calculated from the “lesser fibre diameter” of each fibre type (type I and type 11) using an eyepiece micrometer (Dubowitz & Brooke 1973). Assuming each fibre to have a circular cross-section with a diameter equal to the “lesser fibre diameter”, the average cross-sectional area of each fibre type was calculated. The fibre areas were determined from the NADH tetrazolium reductase staining (Novikoff et al. l%l) and an average of 207k13 diameters were measured per subject. The mean fibre area was calculated according to the formula presented by Haggmark et al. (1978), i.e. fraction type I x type I fibre area + fraction type 11 x type I1 fibre area. Biochemical analyses. Five enzymes were chosen to represent the major pathways in energy metabolism. Glycolysis was represented by phosphofructokinase (PFK; E.C.2.7.1.1 l), lactate fermentation by lactate dehydrogenase (LDH; E.C. 1.1.1.27), fatty acid p-oxidation by 3-hydroxyacyl-CoA dehydrogenase (HAD; E.C. 1.1.1.35), the citric acid cycle by citrate synthase (CS; E.C.4.1.3.7) and the respiratory chain by cytochrome oxidase (cytox; E.C.1.9.3.1). In addition, two enzymes related to the contraction process and the short-term supply of ATP were investigated: Mg*+-stimulated ATPase (E.C.3.6.1.4) and myokinase (MK; E.C.2.7.4.3). For PFK, LDH, HAD, CS and cytox, homogenates were prepared and assays performed as described previously (Orlander e t al. 1977). PFK was assayed according to Shonk & Boxer (1964), LDH and HAD by the methods of Bass et al. (l%9), CS as described by Srere (1%9),and cytox according to Whereat et al. (1969). For the assays of Mg2+-stimulated ATPase and MK, the procedures described by Thorstensson (1976) were employed. LDH isoenzyrnes were separated by disc-electrophoresis, and relative quantities of M and H subunits were determined as described by Sjodin (1976). Protein was estimated by the method of Lowry et al. (1951). Elecfron microscopy. Small pieces of muscle tissue were processed for electron microscopy, and micrographs were taken for stereological analysis by methods described by Weibel(1969). An account of the procedure has been given before (Orlander et al. 1977). It is of note that the values on mitochondria1 mean volume and number per
Human muscle and smoking unit volume are approximations (see Orlander et al. 1977). As the electron microscopical technique is very laborious, this part of the investigation was limited to 26 subjects. Functional measuremenfs. Maximum isometric and dynamic strengths were measured in the left knee-extensor muscles using an isokinetic dynamometer (Cybex 11, Lumex Inc., New York). The subjects were seated in an adjustable chair with support for the back, shoulders and hips. The hip angle was fixed at n-12 rad (w") and the lower leg moved the lever of the dynamometer. The lever was kept at a constant length and the centre of the dynamometer's axis of rotation was aligned with the anatomical axis of rotation, i.e. the knee joint. The angular movement of the knee joint was from 0 . 5 5 ~to 0 rad (100" to O", i.e. full knee extension). The angular velocities studied were ~ 1 3 , P rad x s-', or 60", 120". and 180" X s-'. In 2 ~ / 3 and , addition, comparisons were made with maximum isometric force measured with the same equipment with knee angles of nl6, r t 3 , and ~ 1 rad, 2 or 30", 60".and 90". The peak isometric value irrespective of knee angle was defined as the individual maximum isometric strength. Two attempts were allowed at each knee angle and velocity, and the highest values were noted. The measurements were made in sequence from slow to fast speeds with 30 s recovery between each contraction. The following se3 quence was used: ~ l rad, 6 -13 rad x s-l, rrl3 rad, 2 ~ / rad x s-', ~ / rad, 2 and P rad x ss'. High precision and accuracy for the torque registrations have been reported by others (Moffroid et al. 1969, Thorstensson et al. 1976). The influence of muscular fatigue due to the testing procedure may be considered negligible (Torstensson 1976). Muscular endurance was assessed in both isometric and dynamic terms. Maximum isometric strength (MIS) and isometric endurance, respectively, were determmed for both legs simultaneously (Karlsson & Ollander 1972) by recording the force exerted when the subjects pressed their feet against a stiff bar, equipped with a force transducer. MIS was taken as the highest force value obtained during a series of 5 contractions. After 3 min rest, isometric endurance time was recorded, measured as the maximum time during which a tension level of 50% of MIS could be maintained. Dynamic endurance was measured as the ability to maintain tension output during repeated maximal dynamic contractions performed on the isokinetic dynamometer. The angular velocity was pre-set at P rad x s-I (180" x s-') with the subject seated as described above. Dynamic endurance was calculated from measuring 50 maximal contractions and determining the relative (%) decline in peak torque from the mean of the three initial contrac-
41
118
IIA
Fig. 1 . Fibre type distribution. Values are means **denotes P
Skeletal muscle metabolism, morphology and function in sedentary smokers and nonsmokers JAN ORLANDER, KARL-HEINZ KIESSLING and LARS LARSSON' Institute of Zoophysiology, University of Uppsala, the Department of Animal Nutrition, Swedish University of Agricultural Sciences, Uppsala, and the Laboratory for Human Performance, National Defence Research Institute, Stockholm, Sweden
ORLANDER, J . , KIESSLING, K-H. & LARSSON, L.: Skeletal muscle metabolism, morphology and function in sedentary smokers and nonsmokers. Acta Physiol Scand 1979, 107: 3946. Received 29 Dec. 1978. ISSN 0001-6772. Institute of Zoophysiology, University of Uppsala, the Department of Animal Nutrition, Swedish University of Agricultural Sciences, Uppsala, and the Laboratory for Human Performance, National Defence Research Institute, Stockholm, Sweden. Smokers and nonsmokers of a homogeneous population of sedentary men have been compared with respect to skeletal muscle (vastus lateralis) morphological, metabolic and functional characteristics. The percentage type I fibres was lower and that of type IIB fibres higher in the smokers. Fibre areas were almost equal in the two groups. Muscle oxidative capacity was lowered in the smokers, as judged from decreased mitochondria1 enzyme activities and a lowered fibrillar space mitochondria1 volume fraction. Isometric and dynamic strengths were lower in the smokers, except at the highest velocity of movement studied. Dynamic strengths expressed in relation to isometric strength were similar at all velocities except the highest, where the smokers were relatively stronger. Muscular endurance, measured in short-term isometric and dynamic tests, was not different. It is suggested that the lowered muscle oxidative capacity and strength in the smokers may be partly a consequence of the different fibre type distribution. A possibly lower physical activity level, and tobacco smoke constituents (e.g. carbon monoxide) may also be instrumental. It is not clear whether the different fibre type distribution in the smokers is an effect of smoking per se, or if background factors are responsible. Key words: Muscle metabolism, muscle morphology, smoking, strength, tobacco
Tobacco smoking is known to markedly impair the capacity for prolonged exercise, as revealed by performance tests and/or measurements of maximal oxygen uptake (Cooper et al. 1968, Peterson & Kelley 1%9, McDonough et al. 1970, Hrubes & Battig 1970, Shaver 1973, Raven et al. 1974, IngemannHansen & Halkjaer-Kristensen 1977). The impaired working capacity has been attributed to increased oxygen debt and lactate production in smokers (Chevalier et al. 1963, Krumholz et al. 1964, Krumholz & Hedrick 1972, Reece & Ball 1972), although there are conflicting data (Raven et al. 1974). Carbon monoxide has been implicated as a major etiological agent (Chevalier et al. 1%6). The physical working capacity and the metabolic capacity of the working muscles are closely interrelated (Holloszy & Booth 1976). It would therefore seem conceivable that part of the impairment of the
working capacity in smokers might be related to effects on muscle. There is but scanty information in the literature concerning possible effects of tobacco smoking on skeletal muscle characteristics. Some early studies (reviewed by Fischer et al. 1%0) on the effect of smoking on muscle strength or efficiency showed variable results (increases, decreases or no effect at all). Apparently, no studies on muscle morphology or metabolism in relation to smoking have appeared so far. In the present study, smokers and nonsmokers of a homogenous population of sedentary men were compared with respect to skeletal muscle morphological, metabolic and functional characteristics.
' Present address: Dept. of Physiology 111, Karolinska Institute at Gymnastik- och idrottshogskolan, Stockholm, Sweden. Actti P/z?siol Sctind 107
J . Orlander et a / .
40
Table I . Anthropometricaf characteristics Values are means k S. E . FFS, fat free soft tissue weight. n.s., not significant ~~
Group Smokers Nonsmokers P
No. of subjects
Age (y.)
Height (cm)
Weight 0%)
FFS (kg)
Skeletal weight (kg)
Body fat weight (kg)
Thigh circumference (cm)
18
44k3 43k3 n.s.
179k2
78.4k2.6 76.8k1.6 n.s.
5 3 . 9 t 1.7 53.7k1.3 n.s.
12.4k0.5 12.8k0.2 n.s.
10.0k0.8 9.2k0.5 n.s.
54.7k0.9 54.2k0.7 n.s.
25
180k1
n.s.
MATERIAL AND METHODS Subjects. 43 apparently healthy men (18 smokers and 25 nonsmokers) volunteered to participate in the study. They were all white-collar workers (employees of an insurance company), and engaged in little or no physical activity during their spare time. The basis for acceptance of a subject was the desire to get a subject population as homogeneous as possible with regard to physical activity level. Smoking habits were not considered in this selection. An oral consent was obtained from each subject after he had been informed of the procedure and possible risks of the experiments. Informations regarding smoking habits were obtained by using a questionnaire. Each subject was asked to specify his weekly tobacco consumption. 11 were cigarette smokers and smoked 91 (10-200) cigarettes, with or without filters, per week. 4 subjects smoked the pipe and consumed 62 (25-125) g of tobacco per week. 3 had “mixed” habits, smoking both the pipe and cigars or cigarettes. It was decided to consider all smokers together as one group. For each subject, age, height, weight, skeletal width and skinfold thickness were recorded. Body fat weight, skeletal weight and fat free soft tissue weight (FFS) were calculated according to von Dobeln (1964). The circumference of the left thigh was measured in a horizontal plane just under the gluteal furrow. As shown in Table 1, the two groups were very similar with regard to all studied anthropometrical variables. All experiments were performed at 8-11 a.m., and followed a fixed protocol with biopsies preceding strength measurements. No restrictions regarding smoking were given, but no subject smoked during at least 20 min preceding the experiments. Muscle biopsies. Muscle tissue was obtained from the left vastus lateralis by the percutaneous needle technique (Bergstrom 1%2). The specimens were used for histochemistry, enzyme assays and electron microscopy. Hisrochemistry. The muscle samples for fibre typing and area determination were trimmed, mounted, frozen in isopentane cooled by liquid nitrogen, and stored at -80°C until analysis. Serial transverse sections (10 p m ) were cut with a cryotome at -20°C and the myofibrillar ATPase staining method (Gomori 1941, Padykula & Herman 1955) was used for muscle fibre classification. Photographs of the stained sections were taken and classification into type I (slow twitch) and type I1 (fast twitch) (Engel 1962) was A< ta Phv\iol Jccind 107
made in all 43 subjects. In 34 of the subjects, the type I1 fibres were subclassified into A, B and C subgroups (Brooke & Kaiser 1970, Dubowitz & Brooke 1973). The average number of counted fibres for classification into main groups (type I and type 11) and subgroups (type I , type I I A , B, and C) was 585252 and 251235, respectivelYt The fibre areas were calculated from the “lesser fibre diameter” of each fibre type (type I and type 11) using an eyepiece micrometer (Dubowitz & Brooke 1973). Assuming each fibre to have a circular cross-section with a diameter equal to the “lesser fibre diameter”, the average cross-sectional area of each fibre type was calculated. The fibre areas were determined from the NADH tetrazolium reductase staining (Novikoff et al. l%l) and an average of 207k13 diameters were measured per subject. The mean fibre area was calculated according to the formula presented by Haggmark et al. (1978), i.e. fraction type I x type I fibre area + fraction type 11 x type I1 fibre area. Biochemical analyses. Five enzymes were chosen to represent the major pathways in energy metabolism. Glycolysis was represented by phosphofructokinase (PFK; E.C.2.7.1.1 l), lactate fermentation by lactate dehydrogenase (LDH; E.C. 1.1.1.27), fatty acid p-oxidation by 3-hydroxyacyl-CoA dehydrogenase (HAD; E.C. 1.1.1.35), the citric acid cycle by citrate synthase (CS; E.C.4.1.3.7) and the respiratory chain by cytochrome oxidase (cytox; E.C.1.9.3.1). In addition, two enzymes related to the contraction process and the short-term supply of ATP were investigated: Mg*+-stimulated ATPase (E.C.3.6.1.4) and myokinase (MK; E.C.2.7.4.3). For PFK, LDH, HAD, CS and cytox, homogenates were prepared and assays performed as described previously (Orlander e t al. 1977). PFK was assayed according to Shonk & Boxer (1964), LDH and HAD by the methods of Bass et al. (l%9), CS as described by Srere (1%9),and cytox according to Whereat et al. (1969). For the assays of Mg2+-stimulated ATPase and MK, the procedures described by Thorstensson (1976) were employed. LDH isoenzyrnes were separated by disc-electrophoresis, and relative quantities of M and H subunits were determined as described by Sjodin (1976). Protein was estimated by the method of Lowry et al. (1951). Elecfron microscopy. Small pieces of muscle tissue were processed for electron microscopy, and micrographs were taken for stereological analysis by methods described by Weibel(1969). An account of the procedure has been given before (Orlander et al. 1977). It is of note that the values on mitochondria1 mean volume and number per
Human muscle and smoking unit volume are approximations (see Orlander et al. 1977). As the electron microscopical technique is very laborious, this part of the investigation was limited to 26 subjects. Functional measuremenfs. Maximum isometric and dynamic strengths were measured in the left knee-extensor muscles using an isokinetic dynamometer (Cybex 11, Lumex Inc., New York). The subjects were seated in an adjustable chair with support for the back, shoulders and hips. The hip angle was fixed at n-12 rad (w") and the lower leg moved the lever of the dynamometer. The lever was kept at a constant length and the centre of the dynamometer's axis of rotation was aligned with the anatomical axis of rotation, i.e. the knee joint. The angular movement of the knee joint was from 0 . 5 5 ~to 0 rad (100" to O", i.e. full knee extension). The angular velocities studied were ~ 1 3 , P rad x s-', or 60", 120". and 180" X s-'. In 2 ~ / 3 and , addition, comparisons were made with maximum isometric force measured with the same equipment with knee angles of nl6, r t 3 , and ~ 1 rad, 2 or 30", 60".and 90". The peak isometric value irrespective of knee angle was defined as the individual maximum isometric strength. Two attempts were allowed at each knee angle and velocity, and the highest values were noted. The measurements were made in sequence from slow to fast speeds with 30 s recovery between each contraction. The following se3 quence was used: ~ l rad, 6 -13 rad x s-l, rrl3 rad, 2 ~ / rad x s-', ~ / rad, 2 and P rad x ss'. High precision and accuracy for the torque registrations have been reported by others (Moffroid et al. 1969, Thorstensson et al. 1976). The influence of muscular fatigue due to the testing procedure may be considered negligible (Torstensson 1976). Muscular endurance was assessed in both isometric and dynamic terms. Maximum isometric strength (MIS) and isometric endurance, respectively, were determmed for both legs simultaneously (Karlsson & Ollander 1972) by recording the force exerted when the subjects pressed their feet against a stiff bar, equipped with a force transducer. MIS was taken as the highest force value obtained during a series of 5 contractions. After 3 min rest, isometric endurance time was recorded, measured as the maximum time during which a tension level of 50% of MIS could be maintained. Dynamic endurance was measured as the ability to maintain tension output during repeated maximal dynamic contractions performed on the isokinetic dynamometer. The angular velocity was pre-set at P rad x s-I (180" x s-') with the subject seated as described above. Dynamic endurance was calculated from measuring 50 maximal contractions and determining the relative (%) decline in peak torque from the mean of the three initial contrac-
41
118
IIA
Fig. 1 . Fibre type distribution. Values are means **denotes P
