INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

SEVERAL NOVEL SIGNALING PATHWAYS of pathological cardiac hypertrophy have been unveiled in the powerful model of mechanical overloading by thoracic aorta constriction in genetically modified mice (8, 22). Thus far, the molecular mechanisms of adaptive cardiac hypertrophy in response to physiological stimuli remain virtually unexplored because of a paucity of effective and well-controlled experimental models in this species. Ideally, the outcome should mimic typical results of exercise training in humans, e.g., increased maximal oxygen uptake (O_2 max) and work economy (21). In healthy individuals, increased exercise capacity is dependent on functional and structural adaptations in skeletal muscle, blood vessels, and heart. Cardiac adaptation includes increased ventricular chamber volumes and weights, cardiomyocyte hypertrophy, increased cardiac output, and improved contractile function measured in isolated cardiomyocytes and in the intact heart (2, 3).

At present, there are few well-controlled endurance training studies in mice. Training is usually carried out as voluntary exercise regimens, with little control of exercise intensity and the individual's fitness level. Voluntary wheel running is most frequently used. O_2 max has been shown to increase by 12-30% after a 7- to 12-wk period of free wheel running (11, 40). The latter study also demonstrated a 6% decreased respiratory exchange ratio (RER) at O_2 max in trained mice. Exercise capacity and adaptations have also been measured indirectly, e.g., as maximal exercise duration and total distance run (26). A 4-wk regimen led to 15% increased cardiac mass as well as increased expression of hypertrophy markers, i.e., atrial and brain natriuretic peptides, by 74 and 52%, respectively (1). Myosin heavy chain redistribution toward greater expression of type IIa and IId/x occurred, whereas skeletal muscle weights remained unchanged. Four weeks of swimming led to cardiac hypertrophy, with a 10% increase in heart weight and a 16% increase in heart-to-body weight ratio (24). Although some studies have reported problems with both acute and chronic mice training, the large number of training studies in mice indicates the opposite. However, procedures for reliable exercise capacity measurements have been difficult to establish in mice (e.g., Refs. 11, 26).

In contrast, rat studies on training effects are numerous, with well-defined protocols for exercise capacity evaluation, intensity-controlled treadmill running, and robust cardiovascular outcomes. Until recently, findings appeared somewhat contradictory. Treadmill running with fixed exercise intensity induced only minor increments in ventricular mass and cardiomyocyte dimensions (2, 3, 28, 29) or did not induce any effects (16, 18). However, 10-20% improvements in O_2 max and work economy had been demonstrated (16, 30). Because swim training had produced up to 33% ventricular enlargement in rats, some studies concluded that the mode of exercise is crucial for eliciting cardiac training adaptations (16, 18). Even in rats, few studies had accounted for the fact that the load intensity required to induce physical conditioning increases as the performance improves during the course of training (4). However, a recent study from our laboratory demonstrated that intensity-controlled treadmill running induced some of the largest and most consistent effects of exercise training (42).

The purpose of the present study was to establish an experimental model of intensity-controlled treadmill running in mice. The specific aims were to 1) establish a valid and reliable treadmill protocol for evaluating endurance capacity in mice; 2) establish a standardized, long-term, intensity-controlled treadmill running protocol that is appropriately adjusted to the individual mouse endurance capacity by weekly O_2 max assessments; and 3) determine the effects of exercise training on endurance capacity and cardiac adaptations, as assessed by echocardiography and postmortem characteristics in male and female mice.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study population and design. A total of 40 female and 36 male adult C57BL/6J mice, 19-23 g, 7-8 wk old (Møllegaards Breeding Center, Lille Skensved, Denmark), were included in the study. Animals were maintained in a 12:12-h light-dark cycle, 22.5 ± 1.4°C temperature, and 55.6 ± 4.0% relative humidity. The mice were fed a pellet rodent diet and water ad libitum. Strewment was changed every third day. All experimental protocols were performed during the mice dark cycle, and the animals were rewarded with chocolate (Crispo, Nidar Bergene, Norway) after each training or test session. Sedentary mice were given the same amount of chocolate. None of the mice was excluded in the study because they avoided treadmill running, and the same person handled the mice during the study. The experimental protocol was approved by the Norwegian Council for Animal Research, and the experimental procedures conformed with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes and the Guiding Principles for Research Involving Animals and Human Beings from the American Physiological Society. The mice were assigned into groups as described in Table 1.

Aerobic capacity. Oxygen uptake (O₂) in mice was tested during treadmill running in a metabolic chamber. Ambient air was led through the chamber at a rate of 0.5 l/min, and 200 ml/min samples of gas were extracted to the paramagnetic oxygen analyzer (type 1155, Servomex) and the carbon dioxide analyzer (LAIR 12, M & C Instruments). The gas analyzers have an accuracy of measurements of ±2% and were calibrated with standardized gas mixtures before every test session. The single-lane test treadmill was custom made and placed in the metabolic chamber; for training, mills with multiple lanes were used. Stainless steel grids at the end of the lines provided an electrical stimulus of 0.25 mA, 1 Hz, and 200-ms length, to keep the mice running, and brushes avoided the mice from pinching feet between grid and treadmill. Test-retest assessments of O₂ and RER were performed during submaximal running at fixed standardized treadmill velocities at 25° (47%) inclination of the treadmill. After having been exposed to the equipment and treadmill running twice, six mice of both sexes were assigned to test-retest procedures. Mice were placed in the metabolic chamber for 10 min, and then they went through a 10-min warm-up at 0.08 m/s before running for 5 min at four different velocities (0.1, 0.14, 0.18, and 0.22 m/s). Each mouse ran twice, with the velocity order low to high or high to low set randomly, and performed both. With modifications, this method was adopted from previous studies in rats (6, 42).

Training. For the investigations of prolonged training adaptations, 28 female and 24 male mice were randomized into either treadmill running or sedentary control groups. When O_2 max remained unchanged for at least 3 consecutive wk, which occurred after 8 wk of training, the mice were killed. Training mice exercised on treadmills 2 h/day for 5 days/wk. At the start of every week, O_2 max was measured as described and workloads adjusted accordingly. In training mice, exercise intervals alternated between 8 min at 85-90% of O_2 max and 2 min at 50-60%. Before the first interval, each mouse performed a regular warm-up as described earlier. At the day when O_2 max was tested, trained mice performed eight intervals after the test. In sedentary mice, treadmill running skills were maintained by treadmill running for 15 min on a flat treadmill at 0.15 m/s for 3 days/wk. This latter activity did not seem to alter either O_2 max or work economy (Fig. 3).

Echocardiography. Echocardiography was performed in 16 female mice, after sedation with ketamine hydrochloride (100 mg/kg, Pfizer) and xylazine (5 mg/kg, Bayer) intraperitoneally, by using a GE Vingmed System FiVe ultrasound scanner with an epicardial probe at 10 MHz and 250 frames/s (GE Vingmed Ultrasound, Norway). After the mouse chest was shaved, the transducer was gently placed on it for recordings. Heart rate, systolic and diastolic left ventricular wall thickness, lumen diameters, and fractional shortening were calculated as the mean of 10 consecutive cardiac cycles in two-dimensional M-mode long-axis recordings following the recommendations of the American Society of Echocardiography (35). Mitral inflow deceleration time, peak velocity of early and late component of mitral inflow, and isovolumetric relaxation time were calculated as the mean of 10 consecutive cardiac cycles of pulsed wave Doppler spectra recordings.

Cardiac and skeletal muscle mass. After the training, mice were anestethized with diethyl ether and anticoagulated (0.1 ml heparin, 1,000 IU/ml; Novo Nordisk, Cophenhagen, Denmark). Hearts were rapidly removed, and left and right ventricles were carefully dissected and weighed. Extensor digitorum longus and soleus muscles from both hindlimbs were dissected out carefully and weighed after tendon removal.

Left ventricular myocytes. After the training, mice were anestethized and heparinized, and hearts were removed as described above. Hearts were then submerged in oxygenated perfusion buffer, cannulated via the aorta under a microscope, and connected to a standard Langendorff retrograde perfusion system adapted to mice. To balance variation of the left ventricle myocyte isolation method, one heart from both trained and sedentary group was taken each day. Left ventricular and septal myocytes were isolated by using a modified protocol from Christensen et al. (9). The heart was perfused at 2.2 ml/min for 10 min with a modified Joklik's minimum essential medium (Life Technologies) mixed in 2,000 ml deionized water containing (in mM) 1.2 MgSO₄, 1.0 DL-carnitine (Sigma Chemical), and 23.8 NaHCO₃. The buffer was equilibrated with 5% CO₂-95% O₂ for 30 min (37°C, pH 7.2). Subsequently, 150 U/ml collagenase type II (Worthington) and 0.1% (wt/vol) bovine serum albumin (Sigma Chemical) were added, and the heart was perfused until it became palpably flaccid, which usually occurred after 6-8 min. The heart was then cut down into the Joklik's medium containing 0.8% (wt/vol) bovine serum albumin (Sigma Chemical) and 0.75 mM CaCl₂. The left ventricle was separated and minced and was shaken for 15-20 min in the collagenase type II and bovine serum albumin buffer (37°C, 5% CO₂-95% O₂, 100 rpm). The solution with the myocytes and unsolved tissue was then gently filtered through a nylon mesh (250 µm); added to HEPES buffer containing (in mM) 135 NaCl, 5 KCl, 1 MgCl₂ · 6H₂O, 1.2 CaCl₂ · 2H₂O, 10 HEPES, 8 C₆H₁₂ · H₂O (37°C, pH 7.2, 5% CO₂-95% O₂); and centrifuged (600 rpm, 45 s, 21°C). The supernatant was then removed, and HEPES buffer was added. Cardiomyocytes were deposited on laminin-coated coverslips (10 mg/ml, Life Technologies) and placed in a cell chamber on an inverted microscope (Diaphot-TMD, Nikon) and measured for length and midpoint width.

Allometric scaling. As demonstrated in Table 3, the body mass increased more in males than in females. Changes in ventricular and skeletal muscle weights and O₂ may not be entirely due to training regimens but also may be due to growth-related changes in body mass. It was therefore necessary to normalize O₂ and cardiac and skeletal muscle weights to the body dimensions (4). According to dimensional analysis and empirical studies, O₂ should be expressed in relation to body mass raised to the power of 0.75, over a wide range of body weights (e.g., Ref. 41). Dividing for instance organ weight by body weight is the usual approach, but this has previously been shown to be valid only if body weight is expressed as lean body mass (5), because fat has very low metabolic activity (4). When lean body mass is undefined, ventricular mass should be expressed in relation to body mass raised to the power of 0.78, which is demonstrated empirically as the best approximation (5). Because no empirical studies relate the hindlimb muscles of the mice to body weight, the reduced exponent was calculated in the present training and sedentary control population by allometric equations to determine the relationship between body and extensor digitorum longus and soleus muscle mass. Skeletal muscle mass = a · m^b, where a is the mass coefficient, m is the body mass, and b is the reduced exponent. The numerical value of b can be obtained from the log-log plot of the experimental data because the logarithmic expression is a straight line (e.g., log soleus mass = log a + b · log m). It was found that skeletal muscle mass should be expressed to body mass raised to the power of 0.75. A detailed introduction to allometric scaling is presented elsewhere (4).

Statistics. Data are expressed as means ± SD. The Friedman test and appropriate procedures for multiple comparisons were used to determine changes in O₂, RER, and body weights throughout the experimental period, as well as differences in O₂ by using different inclinations of the treadmill. The Mann-Whitney U-test was used to evaluate differences between groups and sexes. The relationship between O₂ and speed was calculated by linear regression analysis. P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Test protocols. The procedures for measuring O₂ and RER were found to be reproducible for measuring O₂ and RER during exercise. Measurements of O₂ in both female and male mice running at four submaximal velocities were similar on 2 different days (Fig. 1). Test-retest correlation O₂ was 0.98, and the coefficient of variation was 9.1%. Inclination of the treadmill affected the highest O₂ measured, as demonstrated in Table 2. This was evident in both trained and untrained mice of both sexes. Peak O₂ was found at a range of inclinations between 15 and 35°. RER reached the highest values in the same range of inclinations, although in untrained females the maximum value occurred at 25° (47%) inclination (Table 2). Testing procedures for O_2 max were reliable and consistent with previous results. As shown in Fig. 2, O₂ increased linearly with increasing running velocity, and reached a plateau despite increased velocity, in both trained and untrained mice of both sexes. Linear regression analysis was performed until O₂ leveled off for both sexes [i.e., for trained female mice, O₂ = 110.3 · speed + 13.6 (r = 0.98, P < 0.001), and for sedentary controls, O₂ = 49.2 · speed + 33.1 (r = 0.97, P < 0.001)]. The respective linear regression for trained male mice was O₂ = 51.9 · speed + 36.7 (r = 0.99, P < 0.001), and for their respective sedentary controls, O₂ = 29.3 · speed + 45.2 (r = 0.96, P < 0.001).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Test-retest of oxygen uptake (O2) during treadmill running at submaximal intensities (0.10, 0.14, 0.18, and 0.22 m/s) in female (n = 6) and male (n = 6) mice. No differences were found between tests or sexes. CV, coefficient of variation. Individual data are shown.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   O2 at increasing intensity during treadmill running at 25° inclination in female (A) and male (B) trained (Tr; n = 8 female and 8 male) and sedentary (Sed; n = 8 female and 8 male) mice. Running was performed first in 5-min bouts at 0.15, 0.20, and 0.25 m/s and then with the speed increased from 0.25 m/s with 0.03 m/s every 3 min until exhaustion. Note that O2 levels off before exhaustion, despite increasing intensity. Values are means ± SD.

Training effects. After 6 wk of training, both female and male mice reached a plateau 49 ± 7 and 29 ± 5% above sedentary controls in female and male mice, respectively. However, O_2 max appeared to increase progressively in both sexes when it was not corrected for body mass. As shown in Fig. 3, female mice had a higher O_2 max than males, except in baseline recordings and in sedentary controls. This was evident both when expressed as milliliters per kilogram per minute and expressed as milliliters per kilogram raised to the power of 0.75 per minute but not when expressed as milliliters per minute. Body mass increased in all mice during the training regimen by 14 ± 3 and 17 ± 3% in trained and sedentary female mice, respectively, and by 29 ± 5 and 34 ± 6% in male counterparts (all P < 0.01; Table 3). Body mass did not develop differently between sedentary and trained mice within any sex, but increased significantly more in male mice than in females. When properly scaled for body mass, O_2 max (expressed ml · kg^0.75 · min¹) in female mice was 15% higher than in males mice after the training regimen. The maximal running speed when O_2 max was tested increased from 0.36 ± 0.03 to 0.63 ± 0.03 m/s in female mice during the training regimen, whereas the respective increase in male mice was from 0.37 ± 0.02 to 0.65 ± 0.04 m/s.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Time course of maximal O2 during training period in Tr (n = 14 female and 12 male) and Sed (n = 14 female and 12 male) mice. A: maximal O2 without normalization to body mass. B: maximal O2 normalized to body mass. C: maximal O2 normalized to body mass raised to the power of 0.75 according to allometric scaling. Tr mice ran 2 h/day for 5 days/wk in intervals of 8 min at 85-90% and 2 min at 50-60% of maximal O2, whereas Sed mice ran 15 min at 0° inclination at 0.15 m/s for 3 days/wk. Tr mice were tested at the beginning of every week, whereas Sed mice only performed pre- and posttraining tests. Note that maximal O2 levels off after 6 wk. Values are means ± SD. a Difference between trained and sedentary mice within the same sex, P < 0.01. Difference between sexes: b P < 0.01, c P < 0.05.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Work economy in Tr (n = 14 female and 12 male) and Sed (n = 14 female and 12 male) mice after 8 wk, i.e., O2 (A), and respiratory exchange ratio (B) during treadmill running at 25° inclination and 0.15, 0.20, and 0.25 m/s. Tr mice ran 2 h/day for 5 days/wk in intervals of 8 min at 85-90% and 2 min at 50-60% of maximal O2, whereas Sed mice ran 15 min at 0° inclination at 0.15 m/s for 3 days/wk. CO2, carbon dioxide production. Values are means ± SD. Note that Tr mice differed significantly from Sed in either sex. a Difference between trained and sedentary mice within the same sex, P < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present study documents robust and well-controlled protocols for exercise training of mice of both sexes. Reliable O_2 max assessments were obtained in treadmill running at 25° (47%) inclination, whereas peak O₂ measurements at grades below 15° or above 35° underestimated aerobic exercise capacity. Within 6 wk, intensity-controlled interval running at this inclination induced the largest increments in O_2 max, myocardial weights, and cardiomyocyte dimensions reported in this species. As discussed in Aerobic capacity after training, allometric scaling significantly facilitated comparison across sexes and varying body weights. We found that noninvasive evaluation of cardiac dimensions by echocardiography was less sensitive in detecting myocardial hypertrophy than postmortem chamber weights and assessments of isolated cardiomyocyte length and width by video microscopy.

Test protocols. The most common method for testing aerobic capacity in mice is recording volume and velocity of voluntary wheel running (15, 17, 19, 34, 44). Others have estimated aerobic capacity by total time of standardized incremental treadmill running (e.g., Ref. 26), O₂ during swimming (25), or by the cold-exposure method (e.g., Refs. 20, 25, 38). However, these approaches have several problems. Seeherman et al. (38) found that the cold-exposure method yielded 23% lower O_2 max than measurements during treadmill running, whereas swimming differs from running because of relatively inactive forelegs (24). Aerobic capacity may further be underestimated if motivation is low.

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Aerobic capacity after training. The training regimen in the present study is probably the largest and most efficient reported. Within 6 wk, O₂ reached a plateau 50% above sedentary controls in female mice and 30% above controls in males. Niebauer et al. (31) found that O_2 max increased 10% in C57BL/6J females after treadmill running for 4 wk, 1 h twice each day, 6 days/wk, at an intensity of ~85% of O_2 max. Increments of 5-6% in C57BL/6J males (37) and 12% occurred in male Hsd:ICR house mice (40) after 6 wk of treadmill running and 7-8 wk of voluntary wheel running, respectively. Differences in training adaptations reported in other studies are probably due to different training regimens and protocols and to insufficient control of exercise intensity. However, comparison of the results in many previous studies may be confounded by different methods for normalizing O₂ to body weight. As shown in Fig. 3, allometric scaling greatly enhances comparison of O₂ in animals with different weights. Reports of long-term running in mice have reported decreased (34, 40, 44) or unchanged (1, 19, 31, 33, 37, 39) body mass, whereas swimming yielded an increase (24). In a survey of 100 recent transgenic mice studies at PubMed, body mass ranged between 17 and 74 g and age between 26 days and 12 mo.

Cardiac structure and function. In the present study, ventricular weights increased more than previously reported in mice and about the same as in rats exercising for 7 wk with the same regimen (42). Other studies in mice report no change (31) or an increase between 10 and 15% in cardiac mass (1, 24), after 4 wk of training in C57BL/6J mice, treadmill, voluntary wheel, and swimming exercise, respectively. These studies lack a rigorous control of exercise intensity, and cardiovascular load seemed lower.

Skeletal muscle. The weights of the hindlimb extensor digitorum longus and soleus muscles increased substantially. Males had larger extensor digitorum longus muscles, but soleus muscle weight was similar. Other studies report varying results: 20-24% increased (39) and unchanged (1, 19) skeletal muscle mass, and increased cross-sectional area (19, 33) and GATA-2 expression (hypertrophy marker), after 4-12 wk of training (19, 33). Interestingly, skeletal muscle growth occurs in mice in response to endurance training, contrary to observations in humans (4).

Conclusions. In summary, the present study documents a procedure for reliable testing of O_2 max in mice. Graded treadmill running seems to be a sensitive method to assess changes in work capacity and work economy in response to exercise training and probably also in disease states such as heart failure. The protocol for intensity-controlled interval treadmill running provides a well-defined model for exercise training that yields large and robust effects that mimic central results in humans. We anticipate that future experiments in this model will unveil novel molecular, cellular, and integrative mechanisms of adaptation to exercise, e.g., differences in signaling pathways between training-induced adaptive myocyte enlargement and pathological hypertrophy observed in heart failure. Understanding the mechanisms of training-induced amelioration of myocardial function may help identify molecular targets for the treatment and prevention of cardiovascular disease.