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Muscle Characteristics in Career Breath-Hold Divers: Effect of Water Temperature

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Muscle Characteristics in Career Breath-Hold Divers: Effect of Water Temperature

Authors: Park, Jeong Bae; Kim, Hyo Jeong; Kim, Jae Cheol; Lutan, Rusli; Kim, Chang Keun
Source: Aviation, Space, and Environmental Medicine, Volume 76, Number 12, December 2005 , pp. 1123-1127(5)
Publisher: Aerospace Medical Association

Abstract:

Park JB, Kim HJ, Kim JC, Lutan R, Kim CK. Muscle characteristics in career breath-hold divers: effect of water temperature. Aviat Space Environ Med 2005; 76:1123–1127.

Introduction: In a previous study we reported that Korean female breath-hold divers (BHD) with life-long experience of diving in cold water (10–12°C in winter and 25–27°C in summer) had reduced muscle fiber size and increased capillary density. The hypothesis tested in the present study was whether prolonged habitual diving at a moderate water temperature (MWT, 29–30°C all year round) similarly caused a reduction in muscle fiber size. Methods: The subjects were 14 Indonesian BHDs with long experience of diving at MWT, and a control group of 10 age-matched non-diving Indonesian men (CON), selected from the same tribe among which the BHDs lived. Muscle samples obtained from the middle portion of the vastus lateralis muscle were analyzed for muscle morphology by histochemical analysis and the levels of vascular endothelial growth factor (VEGF) protein by Western blotting. Results: Muscle fiber type composition was identical in both groups, and no difference in cross-sectional area (CSA), VEGF protein, or capillarity between the BHD and the CON was observed. Conclusion: The present study demonstrated that prolonged habitual breath-hold diving at MWT does not cause any alteration in muscle fiber composition, fiber size, or capillarity.

Keywords: muscle fiber size; capillarity; water immersion; water temperature
Document Type: Research article

ENVIRONMENTAL CONDITIONS affecting muscle temperature modify both functional and metabolic properties of muscle ber. These include changes in enzyme activities affecting the muscle contractile speed, including the maximum velocity of shortening and tension development (19). Cold exposure causes an increase of thyroid hormone and catecholamine levels. Sayen et al. (26) reported that hyperthyroidism increased the level of fast sarcoplasmic endoplasmic reticulum Ca2+-ATPase type I mRNA in the rat soleus, suggesting that hyperthyroidism causes a reduction of 1/2 relaxation time in the soleus. Previous investigations in rodent and chick muscles have demonstrated that intermittent cold-water immersion and hyperthyroidism cause a shift in fiber type expression from fast-twitch ber to an increasing percentage of slow-twitch bers, or vice versa (14,19).

Although studies in humans have investigated the effects of cold immersion on diverse adaptations in physiological systems using whole body or head-out immersion with acute or intermittent cold-water exposure (16), only a few studies have reported the physiological changes in human skeletal muscle after prolonged habitual cold-water immersion (5). For several generations Korean female breath-hold divers (BHDs) have harvested seafood from the coastal waters of Korea. They begin their profession at age 12–13 and continue to dive throughout most of their lives. They diverepeatedly to depths of 5–7 m in waters that range from 10°C in winter to 27°C in summer (25).

Recently we investigated the effect of prolonged intermittent cold-water immersion on skeletal muscle in Korean female divers, and observed that all divers, without exception, exhibit a smaller fiber size and increased capillarity (5). We observed similar changes in rodents in a recent study of 20 wk of head-out cold-water (18°C) immersion (17). Such changes in fiber cross-sectional area (CSA) and capillarity with prolonged cold-water immersion are possibly related to a reduction in the number of myonuclei (17) and increased expression of vascular endothelial growth factor (VEGF) mRNA and VEGF protein (15).

However, these apparent adaptations in the skeletal muscle of the rodent may be species- and fiber type-speciec, and further, the results obtained from rodents may not compare directly to humans (5,15,17). Even though the hypothesis that a diving response may also occur in humans stems essentially from observations made, such as strong peripheral vasoconstriction (6), with a reduction of peripheral blood flow (3), and an increase in carotid artery blood flow (21) during diving in cold water or face immersion, diving in a moderate water temperature may reduce blood ow less than a cold water temperature (28).

We, therefore, investigated the effects of prolonged habitual diving at a moderate water temperature (MWT, 29–30°C all year round) on human skeletal muscle in Indonesian BHDs. Subsequently, in this study we have compared these results to findings in Korean BHDs who have been diving under similar conditions and depths, and for the same period of time, except that this was in cold water (10–12°C in winter and 25–27°C in summer).

METHODS

Subjects

We recruited 14 Indonesian BHDs, who had been diving more than 24 yr, to serve as subjects. We re cruited 10 age-matched non-diving Indonesian men from the same tribe among which the BHDs lived to serve as controls (CONs). Indonesian BHDs are practiced in the art of gathering pebbles and small stones from the ocean floor for construction. They wear only a swimming suit during diving since the year-round water temperature is 29–30°C. The BHDs dive repeatedly ( 150 dives • d-¹ ) to depths of 5–10 m and dive for up to 5h • d-¹ , which includes a short rest on the boat after several repeated dives.

All subjects were fully informed of the nature of the experiment and of the risks and discomfort associated with the experimental procedures before they volunteered to participate in the study. The Ethics Committee of the Korea National Sport University approved the study.

Anthropometric Measurement

The anthropometric measurements performed on the subjects included bodyweight, height, body fat composition, and the girth of the middle portion of the thigh. Body density was determined from skinfold measurements of seven sites on the triceps, subscapular, midaxilla, iliac crest, abdominal, front thigh, and medial calf as described by Thorland et al. (30) using Lange calipers (10 g • mm-¹ constant pressure, Cambridge Science Industries, Cambridge, MD). The percentage of body fat was determined as described by Siri (27) and all measurements were performed on the day of the muscle biopsy.

For BHDs, the mean age, height, and weight were 40 (range 33–49) yr, 158 (range 153–162) cm, and 50 (range 44–56) kg, respectively. Thigh girth, body fat, and fat free mass were 5.4 ± 2.9 cm, 9.8 ± 2.8%, and 46.3 ± 3.3 kg, respectively.

For the CONs mean age, height, and bodyweight were 39 (ranges 31–43) yr, 162 (range 157–166) cm, and 57 (range 50–64) kg, respectively. Thigh girth, body fat, and fat free mass were 42.6 ± 0.9 cm, 8.2 ± 1.5%, and 45.8 ± 4.6 kg, respectively.

The Wingate Anaerobic Test

All of the subjects participated in the 30-s Wingate test protocol on a bicycle ergometer (10). The height of the saddle and handle bar, and the distance from the saddle to handle bar were adjusted for each subject before the test. The subjects were given a 2-min warm-up exercise with 1 kilo-pound (KP) of resistance and then rested for 5 min before the test. Each subject exercised against a resistance of 0.075 KP • kg-¹ body-weight (10). During the test, subjects were asked to maintain maximal pedaling speed throughout the 30s and encouragement was given verbally during performance. Pedaling revolutions were recorded by video camera. The mean and maximal powers were recorded and power relative to bodyweight was calculated every 5s. The formula for calculating power output was as follows:

Relative mean power (kgm • min-¹ • kg-¹ ) = [Revolution (during 30 s) x Circumference (6 m) x Resistance (kg) x 2]/bodyweight

Relative peak power (kgm • min • kg ) = [max Revolution (during 5 s) x Circumference (6 m) x Resistance (kg) x 12]/bodyweight

Fatigue index (%) = [(Relative peak power - Relative lowest power)/Relative peak power] x 100.

Muscle Biopsy and Blood Sampling

Muscle samples were obtained under local anesthesia from the middle portion of the vastus lateralis of the quadriceps femoris muscle using the percutaneous needle biopsy technique (7). Muscle samples were immediately mounted in an embedding medium [optimal cutting temperature (O.C.T.) compound Tissue-Tek, Lab-tek Products, Naperville, IL], frozen in isopentane pre-cooled with liquid nitrogen, and stored at70°C until analyzed.

Histochemistry: Serial transverse sections (10µm) were cut at -20°C using a Cryocut. The sections were mounted on a cover slide and stained for myofibrillar ATPase at pH 9.4 after both alkaline and acidic (pH 10.3 and pH 4.6) preincubations (8). In each muscle sample, muscle fiber (range 253–439) were analyzed and characterized as type I, type IIa, and type IIx. The capillary supply surrounding the different types of fiber was visualized using the amylase-periodic acid-Schiff method (2). Muscle fiber type composition, CSA, capillary density, the capillary to fiber ratio, and diffusional area, where fiber CSA was divided by the number of surrounding capillaries, were determined using an image analyzer (COMFAS, Hadsund, Denmark).

Protein preparation and Western blot analysis: Muscle samples were homogenized in a buffer containing 150 mmol • L-¹ NaCl, 5 mmol • L-¹ EDTA, 50 mmol • L-¹ Tris-HCl, 1%-Ethylphenyl-polyethylene glycol (NP40), 1 mmol L aprotinin, 0.1 mmol L leupeptin, and mmol • L-¹ pepstatin (pH 8.0). After being centrifuged at 14,000 g for 30 min, the Triton X-100 soluble fractions (20µg protein) were resolved by electrophoresis in 8% SDS-polyacrylamide gels under non-reducing conditions. Two identical sets of gels were run simultaneously along with pre-stained molecular mass markers in each set of gels. A total of eight sets of gels were performed. After an overnight electrotransfer to polyvinyl difluoride membranes, the membranes were blocked using 5% skim milk in phosphate-buffered sa line (PBS, pH 7.4) and incubated with polyclonal anti VEGF antibody (Santa Cruz, CA) at 2µg • ml-¹ dilution in PBS for 1 h at room temperature. This was followed by 1 x 15 min and 2 x 5 min washes with PBS plus 0.1% Tween 20. The membranes were then incubated with goat anti-rabbit IgG conjugated with horseradish per-oxidase at 1:3000 dilutions in PBS for 1 h at room temperature. After the final wash, the immunoreactive bands were detected by enhanced chemiluminescence with Kodak film.

Triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) analysis: Venous blood samples were taken from a forearm vein at rest in the sitting position using a vacuum tube with clotting activator. Whole blood samples were kept at room temperature for 2 h and centrifuged (3000 rpm) for 15 min to harvest serum. T3, T4, and TSH were measured by radioimmunoassay (Cobra 5010 II, Quantum).

Conventional statistical methods were used to calculate mean and SD. Comparisons between groups were performed using Student’s t-test. A level of p < 0.05 was considered to be significant for differences between mean values.

RESULTS

Although both mean and peak power were greater in the BHDs than in the CONs (p < 0.05, Fig. 1), relative peak power was only greater in the BHDs (p < 0.05) when it was normalized with respect to body mass. The fatigue index was not different between the groups 44.4 ± 11.1 in the CONs vs. 44.7 ± 8.7% in the BHDs).

 

Fig. 1. Anaerobic A) relative mean and peak power and B) mean and peak power outputs between the breath-holding divers (BHDs) and the controls (CONs). * Significantly different at p<0.05 (black bar = BHDs, white bar = CONs).

 

Fig. 2. Diffusional area of different fiber types between the groups. * Significantly different from the CON at p<0.05 (black bar = BHDs, white bar = CONs).

The percentage of different fiber types was identical between the two groups (51.5 ± 16.6 in the CONs vs. 51.3 ± 10.8% in the BHDs). There was no difference in CSA (3591 ± 732 vs. 4173 ± 1518m in total) between the CONs and BHDs and no significant difference in capillary density in the CONs compared with the BHDs (377.3 ± 51.7 vs. 402.5 ± 86.2 capillaries • mm-²).

The BHDs had smaller diffusional areas compared with those of the CONs in type IIx fiber only (1041.9 ± 165.6 vs. 1257.3 ± 41.9µm², respectively, p<0.05), whereas no difference was found in type I (1085.6 ± 263.1 vs. 1012.0 ± 35.9µm², respectively) and type IIa fibers (1089.6 ± 209.0 vs. 1170.4 ± 53.0µm², respectively) (Fig. 2).

There were no differences in serum level of T3 and T4 between the CONs and the BHDs (1.16 ± 0.24 ng • dl-¹ and 7.18 ± 1.88 ng • dl-¹ vs. 1.20 ± 0.10 ng • dl-¹ and 7.30 ± ng • dl-¹, respectively), even though the BHDs had a slightly higher TSH (17%) than the CONs.

Although the protein levels of VEGF increased by 67% in the BHDs compared with the CONs, it failed to reach statistical significance (p>0.05, Fig. 3).

DISCUSSION

Recently, we reported that muscle fiber from Korean diving women had a smaller cross-sectional area and higher capillarity (5) than fiber from non-diving controls. We also demonstrated that cold-water immersion reduced the number of myonuclei and that this was closely related to the reduction in muscle fiber size in the rodent (17), as was a higher gene expression for blood vessel remodeling (15). In the present study there were no differences in body composition and muscle function between groups, even though the BHDs were greater in relative peak power. In contrast to the study of Korean BHDs, there was no difference in the present study in fiber size between BHDs and CONs. The diving conditions of the Indonesian BHDs were identical to those of the Korean BHDs with the one difference being that diving occurred at MWT (29–30°C all year round).

Fig. 3. VEGF protein concentration (black bar = BHDs, white bar = CONs).

On this basis it would seem that prolonged habitual breath-hold diving at MWT does not cause any alteration in muscle fiber composition, fiber size, and capillarity. Korean BHDs, on the other hand, who have been diving in cold water have a higher proportion of type II fiber and smaller fiber size (5) compared with their controls. The present results support our previous hypothesis that it is cold stress, rather than diving per se, which is responsible for the reduction in fiber size. Whole body muscular adaptation to different temperature and environmental conditions may necessitate different changes in different muscles. Walters and Constable (32) observed a transformation of slow twitch to fast twitch ?ber in the soleus muscle following cold water immersion, whereas a shift in the contractile properties of fast-twitch extensor digitorium longus (EDL) muscle toward the slow twitch type was observed by Nomura et al. (19). Further, Vornanen (31) reported that in crucian carp heart muscle only fast myosin heavy chain was present in winter but about one-half of the fast myosin heavy chain was replaced by slow myosin heavy chain in summer.

Cold-induced hyperthyroidism results in the increased expression of the MHC II at the expense of a reduction in the slow myosin isoform. Caiozzo et al. (9) demonstrated that thyroid hormones are a potent stimulus for the alteration of ?ber type. They reported the distributions of type I and type II ?ber in rat soleus were significantly decreased, or increased following T3 treatment for 4 wk. This agreed with the Korean BHDs who exhibited hyperthydroidism (1) as well as a higher proportion of type II ?bers than controls. However, clearly this was not the case for Indonesian BHDs, where fiber compositions were the same (51% of type I fiber in both groups) as the plasma levels of thyroid hormones.

The effects of increased capillarity will be to increase blood flow as well as both circumference wall and shear stress (11,23). Park et al. (22) reported that blood flow in limbs during cold-water immersion was clearly greater in Korean diving women than ordinary Korean women (non-divers) in a given water temperature and Korean BHDs showed increased capillarity after long-term intermittent cold-water diving (5). Qvist et al. (24) demonstrated that arterial blood gas tensions during breath-hold diving remained within normal ranges during usual diving durations ( 30 s) in Korean sea divers and no adaptive changes that could increase the tolerance of Korean divers to the hypoxic or hypothermic conditions associated with repetitive diving occurred. Further, diving in MWT may reduce blood flow less than in cold-water temperatures (28).

In the process of angiogenesis, VEGF is dominant among the regulatory factors, and may play an important role in the primary mechanism responsible for increasing the capacity of aerobic metabolism within cardiac and skeletal muscles under cold environments (15). Further blood flow response to muscle contraction is more closely related to metabolic rate than contractile work performed (13). In the present study the VEGF protein tended to increase, but not significantly (67%, p>0.05), as was true in capillarity. These observations may be explained by both hypoxic and hyperemic actions during breath-hold diving and the sudden redistribution of blood after reaching the water’s surface, which may stimulate the growth of small vessels, especially on the arteriolar side.

Hyperbaric pressure may be an important factor affecting the response of skeletal muscle in BHDs. It is possible that the hyperbaric condition during diving itself contributes to a change of muscle morphology. The BHDs are exposed to 1.5–2 atmospheric pressure during diving activity at the depth of 5–10 m. Oh (20) demonstrated that exposure to a hyperbaric ambient of 2 ATA pressure can cause an increase in cross-sectional area and capillarity in rodents. However, this was not the case in either Korean female BHDs or in Indonesian BHDs. Possible questions may be raised regarding gender differences under environmental stress between the Korean and Indonesian BHDs. Arngrimsson et al. (4) measured VO2max and physical performance at various temperatures (25°C–45°C) and found that the effect of hyperthermia on VO2max and physical performance in men and women were almost identical.

In summary, prolonged habitual breath-hold diving at MWT does not cause any alteration in muscle fiber composition, ?ber size, and capillarity. The present study supports our previous ?nding that the reduced fiber size observed in Korean BHDs may not be caused by diving per se, but most likely by cold stress, which was the probable stimulus for adaptation in skeletal muscle.

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