Regional effects of voluntary exercise on cell size and contraction-frequency responses in rat cardiac myocytes

Repeated bouts of aerobic exercise elicit positive adaptive responses in the cardiovascular system. The cardiac adaptations to such training include bradycardia associated with increases in left ventricular end-diastolic volume, myocardial mass and maximal stroke volume. These changes result in improved myocardial contractile function (for reviews, see Moore and Korzick, 1995; Schaible and Scheuer, 1985).

At the cellular level, endurance training has been demonstrated to induce hypertrophy in ventricular myocytes. For example, treadmill running in rats increased the length of myocytes from the left ventricle (Moore et al., 1993; Palmer et al., 1998) and cell capacitance (Mokelke et al., 1997). The effect of training on contractility is more varied, with studies showing increased contractility (Moore et al., 1993) or little change (Laughlin et al., 1992; Palmer et al., 1998; Palmer et al., 1999).

It is possible that, in animal models of exercise training where exercise is enforced, the cardiovascular responses to training may be combined with stress-induced responses associated with the coercion (Rupp, 1989). To assess the cardiovascular responses to exercise alone, it might be preferable to employ voluntary training regimes. Studies have shown that rats will run voluntarily during their normal hours of activity (Mondon et al., 1985; Munoz et al., 1994; Shyu et al., 1984). Running occurs in short bouts (1–5 min) at speeds of 48–68 m min⁻¹, covering 2–20 km day⁻¹, with heart rates reaching over 500 beats min⁻¹ (Henriksen et al., 1994; Overton et al., 1986; Rodnick et al., 1989; Rodnick et al., 1992; Russel et al., 1987).

Exercise causes an increase in heart rate, and it is possible that any exercise-induced changes in contractility are more pronounced at the higher rates of stimulation that occur during exercise. The contractility of cardiac muscle is modulated by stimulation frequency (for reviews, see Koch-Weser and Blinks, 1963; Bers, 1993). For example, in teleost (trout) ventricular myocytes, increased stimulation frequency decreases contractility (Harwood et al., 2000), while in many mammalian species contractility is increased with frequency. Rat ventricular myocytes, in contrast, have been shown to respond to an increase in stimulation frequency with either an increase or a decrease in contractility (Capogrossi et al., 1986; Frampton et al., 1991). It seems that these different contraction–frequency relationships (CFRs) reflect different changes in the intracellular Ca²⁺ concentration transient (Frampton et al., 1991).

Studies on the response of myocytes to exercise usually employ cell populations drawn from the whole ventricular wall. However, it is now known that there are regional differences in the electrical (Antzelevitch et al., 1991) and mechanical (Cazorla et al., 2000) properties of myocytes from the sub-epicardium (EPI) and sub-endocardium (ENDO). Furthermore, it has been shown that the responses to certain stimuli are dependent upon the regional origin of the myocyte (Anversa et al., 1986; Gerdes and Capasso, 1995; Shipsey et al., 1997). It is therefore possible that both the CFR of rat myocytes and the response of myocytes to exercise are dependent upon myocyte regional origin. One reason for the variation in the effects of exercise on contractility reported in previous studies (see above) may be the grouping together of cell populations that, on the basis of their CFR and regional origin, possess different responses to exercise.

The purpose of this study was therefore to test the following hypotheses: (i) that voluntary exercise leads to adaptation of ventricular myocyte size and contractility; (ii) that contractile adaptation to voluntary exercise is more pronounced at higher stimulation frequencies; (iii) that the response to voluntary exercise is dependent upon the regional origin of the myocytes; and (iv) that the CFR of rat ventricular myocytes is dependent upon their regional origin.

Female Sprague-Dawley rats, mass- and age-matched (100.7±1.3 g, mean ± S.E.M, and 6 weeks old), were assigned to one of two groups: sedentary (N=9) and exercise-trained (N=9). All rats were housed individually in plastic cages (25 cm×38 cm×18 cm, width×length×height; North Kent Plastic, UK) and had standard chow and water ad libitum. Over a period of 14±4 weeks, trained rats had free access to a stainless-steel vertical running wheel (1.12 m in circumference; Lafayette Instruments, West Lafayette, USA) attached to their cages. Running distance was recorded daily, and rats were weighed weekly. All animals were housed in a room maintained at 19–21 °C with a 12 h:12 h light:dark cycle. Given that we were using a voluntary model of exercise, young female rats were chosen for this study because they have been reported to run more than older or male animals (e.g. Eikelboom and Mills, 1987).

After being weighed, rats were killed in accordance with UK Home Office Schedule 1 regulations. The heart was removed rapidly, and extraneous tissue was dissected away. The heart was flushed with a modified Hepes-Tyrode solution of the following composition (in mmol l⁻¹ ): 130 NaCl, 1.43 MgCl₂, 5.4 KCl, 0.75 CaCl₂, 5 Hepes, 10 glucose, 20 taurine and 10 creatine, pH 7.3 at room temperature (22–24 °C). The heart was then blotted dry and weighed before it was mounted on a Langendorff perfusion apparatus.

Single ventricular myocytes were isolated using an enzymatic dispersal technique, as described previously (Frampton et al., 1991). Following perfusion with enzyme solution, tissue was dissected from the sub-epicardium (EPI) and sub-endocardium (ENDO) of the left ventricle. This tissue was placed into small conical flasks with collagenase/protease-containing solution supplemented with 1 % bovine serum albumin. The cells were dispersed by agitating the flasks at 37 °C for periods of 5 min. At the end of a 5 min period, single cells were separated from the non-dispersed tissue by filtration. The resulting cell suspension was centrifuged at 30 g for 45 s and resuspended in Hepes-Tyrode (see above). Non-dispersed tissue was subjected to further enzyme treatment. The isolated cells were stored at 5 °C until use.

Isolated cells were placed in a chamber with a glass coverslip base mounted on the stage of an inverted microscope (Nikon Diaphot). The chamber was perfused with a bicarbonate-buffered Tyrode solution of the following composition (in mmol l⁻¹ ): 135 Na⁺, 5 K⁺, 1 Mg²⁺, 102 Cl⁻, 20 HCO₃⁻, 1 SO₄²⁻, 1 Ca²⁺, 20 acetate, 10 glucose, 5 units l⁻¹ insulin (equilibrated with 5 % CO₂/95 % O₂), pH 7.35 at 37 °C. Bath temperature was maintained at 37 °C by a feedback-controlled heater system. Myocytes were stimulated via platinum bath electrodes with voltage pulses with a duration of 5 ms and an intensity of approximately 1.25 threshold. Stimulation frequency was varied between 1 and 9 Hz to encompass those occurring naturally in rats at rest and during voluntary exercise (Overton et al., 1986).

Rod-shaped myocytes with squared rather than rounded ends displaying regular sarcomeric striation patterns were chosen for study. Cells were also required to be quiescent when not stimulated. Cells were visualised on a television monitor with an NTSC camera (Pulnix TM640) in partial scanning mode. This image was used to measure cell shortening (our index of contractility) in response to electrical stimulation, using a video motion edge detector (Crescent Electronics, Sandy, UT, USA). The cell image was sampled at 200 Hz. Cell shortening was calculated from the output of the edge detector using a CED 1401 A/D converter and Signal Averager 6 software (Cambridge Electronic Design, Cambridge, UK). Once steady state had been achieved at each stimulation frequency, between 8 and 16 consecutive contractions were averaged. Cell shortening (expressed as a percentage of resting cell length), time to peak of the contraction from stimulation and time from peak contraction to half-relaxation were calculated.

where L is cell length (μ m), W is cell width and D is cell depth (μm).

This formula is based on the findings of Satoh et al. (Satoh et al., 1996) that rat ventricular myocytes are essentially elongated ellipses whose volume is equal to 54 % of the volume of a rectangular block defined by cell length × width× depth. We estimated cell depth to be W/(W:D ratio). Measurements of confocal microscopy images of EPI and ENDO cells from untrained and trained rats confirmed that the cell width:cell depth ratios of these groups were not significantly different (P>0.05, two-way ANOVA, mean ratio ± S.E.M. 1.44±0.03, measurements from 153 cells). This finding is in agreement with our previous finding that myocyte length, width and depth from non-exercising rats did not differ significantly between EPI and ENDO cells (Cazorla et al., 2000).

Similar numbers of myocytes from each animal were used in this study (between 13 and 29), and these were pooled for analysis. Significant differences between means at any given stimulation frequency in cells from ENDO and EPI regions from sedentary and trained animals were determined using multifactorial analysis of variance (ANOVA). Differences in cell dimension of ENDO and EPI cells from sedentary (S) and trained (T) animals were analysed by two-way ANOVA followed by Bonferroni-corrected unpaired Student’s t-test. Pairwise comparisons between myocyte groups from the same region (e.g. ENDO T versus ENDO S) or level of activity (e.g. ENDO T versus EPI T) are presented. For body mass, heart mass and their ratio, differences between sedentary and trained animals were assessed using unpaired Student’s t-tests. Statistical significance was set at 0.05.

Fig. 1 illustrates the mean daily running distance for each week of the study for the group of nine animals. The first 2 weeks represented a period of familiarisation for all runners. Daily running reached a peak between weeks 4 and 6 (approximately 10 km day⁻¹ ) and then declined gradually to approximately 4 km day⁻¹ from week 11 onwards. Average weekly running distance during the training period was 47.9±5.4 km (mean ± S.E.M., N=9).

Fig. 2 shows the daily running distance for a typical animal over the course of the training period. Daily running distance was episodic, peaking approximately every 4 days throughout the period of training irrespective of the total weekly distance run (see inset to Fig. 2).

No significant changes in total body mass (sedentary, 247±4 g; trained, 254±7 g) or in whole heart mass (sedentary, 1278±36 mg; trained, 1319±51 mg) were induced by voluntary exercise. There was no significant difference in heart-to-body mass ratio (sedentary, 5.17±0.14 mg g⁻¹ ; trained, 5.20±0.22 mg g⁻¹ ) (means ± S.E.M., N=9) between sedentary and trained rats. Similar observations have been reported using enforced treadmill running (Molkelke et al., 1997; Palmer et al., 1998).

Fig. 3 gives mean myocyte dimensions (see Materials and methods) of ENDO and EPI cells isolated from sedentary and exercised hearts. In sedentary animals, there were no significant differences in length, width or volume between ENDO and EPI cells. In trained animals, however, ENDO cells displayed evidence of hypertrophy: both cell width and volume were greater than those of trained EPI cells and sedentary ENDO cells (by approximately 20 %, P<0.05, two-way ANOVA). The mean volume of trained EPI cells was also approximately 5 % greater than that of sedentary EPI cells, but this difference was not statistically significant. Thus, voluntary exercise training appears to induce selective hypertrophy in ENDO cells.

Fig. 4 illustrates cell shortening at different rates of stimulation (1, 3 and 5 Hz) in representative rat left ventricular myocytes that showed a positive CFR (Fig. 4A) or a negative CFR (Fig. 4B). Table 1 shows that, in the present study, ENDO and EPI myocytes from sedentary and trained animals exhibited both positive and negative CFRs with similar distributions, the majority of cells displaying a positive CFR over the frequency range 1–5 Hz. These observations suggest that the CFR of myocytes does not depend on the regional origin of the myocytes or upon training.

Fig. 5 shows mean cell shortening for positive (Fig. 5A,B) and negative (Fig. 5C,D) CFRs for the four cell types. ENDO cells isolated from sedentary animals that showed a positive CFR exhibited a significantly greater percentage shortening than sedentary EPI cells (Fig. 5A) when stimulated at 1 Hz (4.9±0.2 % compared with 4.2±0.3 %, respectively), at 3 Hz (6.4±0.3 % compared with 5.3±0.2 %, respectively) and at 5 Hz (7.6±0.3 % compared with 6.6±0.3 %, respectively) (means ± S.E.M., N=46–87 cells). Significant differences were not present at the higher stimulation rates of 7 and 9 Hz. This regional difference was not present in myocytes isolated from trained rats (Fig. 5B). There were no differences in the amplitude of contraction of EPI and ENDO cells from sedentary and trained animals that displayed negative CFRs (Fig. 5C,D).

ENDO and EPI myocytes from sedentary and trained animals that showed a negative CFR had significantly greater percentage cell shortening than those that exhibited a positive CFR when stimulated at 1 Hz (ENDO, 9.4±0.8 % compared with 4.9±0.2 %; EPI, 9.5±0.8 % compared with 4.2±0.2 %, N=16–87) and at 3 Hz (ENDO, 8.6±0.8 % compared with 6.4±0.3 %; EPI, 8.1±0.7 % compared with 5.3±0.2 %; means ± S.E.M., N=16–87; Fig. 6). For brevity, data are presented for sedentary animals only. However, all types of cell showed a similar percentage shortening when stimulated at the higher frequencies (5–9 Hz) that reflect the heart rate of rats in vivo. Furthermore, when stimulation rate was increased from 7 to 9 Hz, all myocytes showed a decrease in percentage shortening (N=4–77 cells, P<0.05). In summary, the two main observations from Fig. 6 are that, at lower stimulation frequencies (1–3 Hz), rat myocytes do not present a homogeneous population in terms of the magnitude of cell shortening, and that, at physiological stimulation rates (5–9 Hz), all rat cells demonstrate a negative CFR irrespective of tissue location.

Fig. 7 shows the effects of stimulation frequency on the time to peak contraction and Fig. 8 on the time to half-relaxation for the four cell populations. As stimulation frequency increased, both time to peak and half-time of relaxation fell in all cell types. Although there were no consistent regional effects on the time courses of contraction, Figs 7 and 8 do show a consistent effect of training: the data for EPI cells in trained animals (Figs 7B,D, 8B,D) are all shifted upwards relative to the ENDO data when compared with the corresponding data from sedentary animals (Figs 7A,C, 8A,C). This observation may indicate that the time course of contraction of EPI cells, relative to ENDO cells, is lengthened in response to exercise (see Discussion).

Our observations add weight to the argument that enforced training regimes are not essential for the study of exercise-induced changes in myocyte physiology because these responses can be provoked by voluntary exercise models. The pattern of running activity and the distances run by rats in the present study are in agreement with those from previous studies (Henriksen et al., 1994; Munoz et al., 1994; Overton et al., 1986; Rodnick et al., 1992). Daily running showed peaks of activity approximately every 4 days (Fig. 2). The rat oestrus cycle has a period of approximately 5 days, and it is possible, therefore, that running activity may be related to or influenced by the oestrus cycle (Chatterton et al., 1995).

Our observation that body mass was not influenced by voluntary running exercise is consistent with previously documented studies using female rats (Henriksen et al., 1994; Munoz et al., 1994; Rodnick et al., 1989). In the present study, the whole heart mass was slightly, but not significantly, increased (by 3 %). Heart-to-body mass ratio was increased, but not significantly. Similar findings have been reported in running models that have also reported cellular hypertrophy (e.g. Palmer et al., 1998). An increase in ventricular mass of 9.8 % was induced by exercise training in humans (Wolfe et al., 1986).

Several formulae have been used in previous studies to calculate cell volume. We have based our calculation on work by Satoh et al. (Satoh et al., 1996), who used confocal microscopy to perform detailed volume rendering of optically sectioned rat ventricular myocytes. Satoh et al. (Satoh et al., 1996) suggested that cell volume could be predicted from the measurement of cell length times width times a calibration factor (7.59×10⁻³ pl μm⁻² ). However, applying this formula to a population of cells (ENDO trained) that increased in width and maintained the width:depth ratio of the other myocyte groups might underestimate the exercise-induced change in volume. The calculated cell volume we present therefore included our confocal microscopy measurement of the width:depth ratio for the cell population as a whole (1.44) and a correction factor (0.54, from Satoh et al., 1996) to account for the elliptical rather than rectangular cross section of myocytes. For comparison, cell volumes were also calculated using the formula from Satoh et al. (Satoh et al., 1996); these volumes were smaller by between 2.8 % (EPI sedentary) and 8.8 % (ENDO trained), but identical statistical differences between myocyte types were observed whichever volume calculation was used.

We observed no difference between the size of EPI and ENDO myocytes of sedentary rats. This observation is consistent with a previous study using these animals (Cazorla et al., 2000) and other studies on rats (e.g. Smith et al., 1990). However, some studies (e.g. Gerdes et al., 1986) have reported that rat ENDO myocytes are larger than EPI myocytes. It may be that the differences in regional myocyte sizes between studies result from the techniques used to calculate cell volume and the strain of rat used.

Our voluntary running model resulted in an increase in ENDO volume of approximately 20 % compared with ENDO cells from sedentary animals and EPI cells from trained animals (Fig. 3). Previous studies measuring the effect of exercise on isolated myocyte size have not distinguished between EPI and ENDO cells; however, an exercise-induced increase in cell length has been reported (Mokelke et al., 1997; Moore et al., 1993; Palmer et al., 1998). From measurements in fixed preparations, White et al. (White et al., 1988) reported that the cross-sectional area of EPI myocytes, but not of ENDO myocytes, increased with swimming exercise in male and female rats. However, it should be noted that direct comparisons between fixed preparations and living, isolated single cells are difficult to make (Gerdes and Capasso, 1995). Our observation of significant cellular hypertrophy in the absence of significant tissue hypertrophy has been reported before (e.g. Palmer et al., 1998) and would appear contradictory in nature. However, because we observed cellular hypertrophy (20 % increase in volume) in a region of the heart that may constitute approximately 15–20 % of the total mass of the heart, it is possible that the overall increase in whole heart mass (we observed a 3 % increase) is too small to be detected, given the variability of heart and body mass.

A major cardiac adaptation to exercise is thought to be an increase in end-diastolic volume. This may come about via several mechanisms, e.g. increased filling time due to bradycardia, increased chamber dimensions (Moore and Korzick, 1995) or increased myocardial compliance (Woodiwiss and Norton et al., 1995). An increase in myocyte size would facilitate an increase in chamber dimensions without a large increase in wall stress (Moore and Korzick, 1995). The increased (ENDO) cell width we observed is usually associated with pressure overload and increased wall thickness (Moore and Korzick, 1995; Gerdes and Capasso, 1995). Consistent with our observation in single cells, an exercise-induced increase in wall thickness has been reported in rats (Woodiwiss and Norton, 1995). Wall stress is known to be greater at the ENDO than the EPI surface of the heart (Yin, 1981), and preservation of this gradient may be the reason for selective ENDO hypertrophy. Regional hypertrophy in response to other stimuli has been reported before (for reviews, see Anversa et al., 1986; Gerdes and Capasso, 1995).

Cells may respond to exercise by changes in in vivo strain (sarcomere length). Such an effect would not be detectable in isolated myocytes because previous studies have shown that, although EPI and ENDO myocytes have different sarcomere lengths in vivo, they have similar sarcomere lengths following isolation (see Cazorla et al., 2000).

We studied the effects of voluntary exercise on regional myocyte contractility. Cells were stimulated at 1–9 Hz at 37 °C to encompass the physiological heart rates of rats (Overton et al., 1986). We found no difference in cell shortening between (ENDO and EPI) left ventricular myocytes isolated from trained and sedentary rat hearts at any frequency of stimulation. This means that the exercise model employed in the present study did not affect the amplitude of myocyte contraction. These results are in agreement with those of Laughlin et al. (Laughlin et al., 1992), who demonstrated no effect of training on myocyte contractility, but are in contrast with those reported by Moore et al. (Moore et al., 1993), who demonstrated an increased cell shortening in myocytes isolated from trained rat hearts (at an external Ca²⁺ concentration of 2 mmol l⁻¹ and a stimulation frequency of 0.067 Hz).

It is interesting to note that we used mechanically unloaded cell shortening as our index of contractility. This index is routinely used in single myocyte studies although, in vivo, myocytes are coupled to each other (i.e. mechanically loaded) and develop force (White et al., 1995). Force development is dependent upon cell cross-sectional area, and our observation that cell width and, therefore, cross-sectional area are increased in ENDO cells from trained animals suggests that in vivo these cells may develop more force than ENDO cells from sedentary animals.

ENDO cells from sedentary rat hearts showing a positive CFR did have a greater percentage shortening than equivalent EPI cells at the lower frequencies of stimulation (1, 3 and 5 Hz). This difference was not observed between EPI and ENDO cells from trained rat hearts (Fig. 5), possibly suggesting an increase in the contraction of EPI cells relative to ENDO cells. One explanation for such a trend would be an increased duration of the trained EPI action potential (see below).

The majority of myocytes showed a positive CFR between 1 and 5 Hz, and this is in agreement with previous studies on single rat myocytes (e.g. Frampton et al., 1991; see also Schouten and ter Keurs, 1986). Our observations on CFRs (Figs 5, 6: Table 1) present several important new findings. We can now reject the hypothesis that positive and negative CFRs are based upon the regional origin of the cells: both types of CFR appeared in EPI and ENDO cells with similar relative distributions. Second, it is interesting to note that, at stimulation frequencies similar to those seen in vivo (5–9 Hz), all rats display a negative CFR.

Finally, lower stimulation frequencies (1–3 Hz) are typically used in studies of myocyte contractility. At these frequencies, cells displaying negative CFRs show significantly greater cell shortening than cells with positive CFRs (Fig. 6). Therefore, contractile studies based on unpaired data are likely to increase data variance greatly if cells with positive and negative CFRs are combined.

Although there were no consistent effects of region on the time course of contraction, training appeared to lengthen the duration of contraction of EPI cells relative to ENDO cells (Figs 7, 8). An increase in the duration of contraction would be consistent with a prolongation of the action potential. Shipsey et al. (Shipsey et al., 1997) reported hypertrophy and selective lengthening of EPI action potential duration in response to chronic isoprenaline treatment. Indeed, our preliminary data from animals exercised for a period of 7 weeks suggest similar selective lengthening of EPI action potential duration in response to voluntary exercise (Natali et al., 2000).

In conclusion, we initially proposed to test four hypotheses, and we have shown under the conditions of our study (i) that voluntary exercise does lead to changes in myocyte size, but has little effect on contractility (when measured as percentage cell shortening); (ii) that the contractile response following voluntary exercise is not more pronounced at higher stimulation frequencies; (iii) that the hypertrophic response to voluntary exercise is dependent upon regional origin, being restricted to ENDO cells; and finally (iv) that the CFR of rat ventricular myocytes is not dependent upon regional origin.

In addition, it appears that all left ventricular myocytes show a negative CFR over the ‘natural’ range of stimulation frequencies (i.e. between 5 and 9 Hz). We conclude that increased cell size may be a more important adaptive response to exercise than a modification of excitation–contraction coupling.

A.J.N. is supported by CAPES (Brazilian Government, Brasilia, Brazil).