Impaired Arterial Pressure Regulation During Exercise Due to Enhanced Muscular Vasodilatation in Calponin Knockout Mice

Authors

  • Shizue Masuki,

    1. Department of Sports Medical Sciences, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto 390–8621, Japan
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  • Michiko Takeoka,

    1. Department of Molecular Oncology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto 390–8621, Japan
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  • Shun'ichiro Taniguchi,

    1. Department of Molecular Oncology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto 390–8621, Japan
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  • Minesuke Yokoyama,

    1. Mouse Genome Technology Center, Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194–8511, Japan
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  • Hiroshi Nose

    Corresponding author
    1. Department of Sports Medical Sciences, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto 390–8621, Japan
    • Corresponding author
      H. Nose: Department of Sports Medical Sciences, Shinshu University Graduate School of Medicine, 3-1-1 Asahi Matsumoto 390-8621, Japan. Email: nosehir@sch.md.shinshu-u.ac.jp

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Abstract

Calponin is known to be an actin binding protein in smooth muscle, inhibiting actomyosin ATPase activity in vitro. We previously reported that α-adrenergic vasoconstriction in calponin knockout (KO) mice was reduced compared with that in wild-type C57BL/6J (WT) mice and, as a compensation, arterial baroreflex sensitivity in KO mice was enhanced at rest. In the present study, we assessed arterial pressure regulation in WT and KO mice during graded treadmill exercise at 5, 10, and 15 m min-1. Mean arterial pressure (MAP) in KO mice fluctuated more than that in WT mice at every speed of exercise with two-fold higher variances (P < 0.001). The baroreflex sensitivity (ΔHR/ΔMAP) in WT mice (n= 6), determined from the heart rate response (δHR) to spontaneous change in MAP (δMAP), was -5.1 ± 0.6 beats min-1 mmHg-1 (mean ±s.e.m.) at rest and remained unchanged at -5.0 ± 0.9 beats min-1 mmHg-1 during exercise (P < 0.01), while that in KO mice (n= 6) was -9.9 ± 1.7 beats min-1 mmHg-1 at rest, significantly higher than that in WT mice (P < 0.001), and was reduced to -4.7 ± 0.4 beats min-1 mmHg-1 during exercise (P < 0.01), not significantly different from that in WT mice. In another experiment, we measured muscle blood flow (MBF) in the thigh by laser-Doppler flowmetry, electromyogram (EMG), and MAP during voluntary locomotion in KO (n= 7) and WT (n= 7) mice. Muscle vascular conductance, MBF/MAP, started to increase immediately after locomotion, judged from EMG, and reached 50 % of the maximum after the time of 2.3 ± 0.2 s in KO mice, shorter than 5.8 ± 0.6 s in WT mice (P < 0.001). Prior administration of α-adrenergic blockade (phentolamine) shortened the time in WT mice to that in KO mice (P < 0.001), but did not shorten the time in KO mice. Thus, impaired MAP regulation in KO mice during exercise was caused by a blunted muscle vascular α-adrenergic contractile response and by the attenuated HR response to spontaneous change in MAP due to reduced baroreflex sensitivity.

Calponin, referred herein to basic calponin or calponin h1 has been reported to reduce unloaded isometric forces and to shorten velocity (Jaworowski et al. 1995; Obara et al. 1996; Matthew et al. 2000; Takahashi et al. 2000) by the inhibition of actomyosin ATPase activity in a reconstituted isolated filament system (Winder & Walsh, 1990). On the other hand, there have been several studies suggesting that calponin increases the contractile response to noradrenaline (norepinephrine (NE)) (Nigam et al. 1998) or phenylephrine (PE) (Parker et al. 1994; Menice et al. 1997; Je et al. 2001) by facilitating agonist-induced signal transduction in isolated vascular smooth muscle.

Masuki et al. (2003) recently reported in genetically calponin-deficient mice (knockout (KO) mice) that the increase in mean arterial pressure (MAP) after a bolus intra-arterial injection of PE was reduced to half that in the wild-type (WT) mice during rest. Despite this, they found that the level of MAP in the KO mice remained the same as that in the WT mice while that of heart rate (HR) was significantly lower. On the other hand, the variability of HR was two-fold higher in the KO mice than that in the WT mice while that of MAP was controlled within the same range as in the WT mice, suggesting that the baroreflex control of HR compensates well for the impaired peripheral α-adrenergic vasoconstriction. Indeed, the arterial baroreflex sensitivity in the KO mice was two-fold higher than that in the WT mice as a compensatory adaptation (Masuki et al. 2003). These results suggest that in the KO mice the fast or dynamic vascular response to sympathetic nerve activity was impaired whereas the slow or static response remained intact. Thus, calponin may play an important role in enhancing the dynamic vasoconstrictive response to sympathetic nerve activity during rest.

However, there has been no study to show the role of calponin in arterial pressure regulation during exercise. During exercise, the vasoconstrictive response to muscle sympathetic nervous activity (MSNA) was reportedly reduced by the inhibition of NE release from sympathetic nerve terminals, which is caused by local vasodilatory factors released from contracting muscles, known as ‘functional sympatholysis’ (Laughlin et al. 1996). This would make MAP unstable during treadmill exercise in the KO mice by enhancing the impairment of the dynamic vasoconstrictive response to MSNA because perivascular concentrations of the local factors would dynamically change with rhythmical muscle contractions. Moreover, we were interested in the question of whether the baroreflex sensitivity compensates well for the impaired dynamic vasoconstrictive response during exercise as it does during rest in the KO mice (Masuki et al. 2003) because the sensitivity has been reported to be altered during exercise (Burger et al. 1998). Based on these results, we hypothesized that the impaired dynamic vasoconstrictive response, functional sympatholysis, and insufficient compensation by baroreflexes would make MAP unstable during exercise, resulting in a reduced exercise capacity in the KO mice.

To test these hypotheses, we continuously measured MAP in the KO and WT mice during treadmill exercise and determined the baroreflex sensitivity from the change in HR (▵HR) in response to the spontaneous change in MAP (▵MAP). In addition, to assess the sympathetic nerve control of muscle vasculature during exercise in the KO mice, we measured muscle blood flow (MBF) in the thigh at the onset of voluntary locomotion before and after α-adrenergic blockade administration, and compared the results with those in the WT mice. Because rapid and high muscle vasodilatation occurs at the onset of locomotion (Masuki & Nose, 2003), we thought we would be able to assess the effects of impaired dynamic vasoconstrictive response in the KO mice on MBF by the measurements made at this time.

Methods

Animals

The calponin-deficient mice used in the present study were generated from the same cell line used in the previous studies (Yoshikawa et al. 1998; Matthew et al. 2000; Takahashi et al. 2000; Masuki et al. 2003). We compared MAP regulation during exercise between C57BL/6J mice lacking the calponin h1 gene (KO mice) and mice carrying a normal calponin h1 gene (WT mice). Absence of calponin h1 expression in the KO mice was reconfirmed by RT-PCR (Masuki et al. 2003) and by Western blotting with a calponin h1-specific antibody (Yoshikawa et al. 1998; Matthew et al. 2000). Adult male mice aged 7-15 weeks (WT, n= 14, 27.4 ± 1.0 g body weight, and KO, n= 14, 28.4 ± 0.9 g body weight) were housed at 25 °C with food and water ad libitum and illuminated from 7:00 to 19:00. The mice were closely monitored to ensure that none experienced undue stress or discomfort. The procedures used here were in accordance with the Guiding Principles in the Care and Use of Animals in the Fields of Physiological Sciences published by the Physiological Society of Japan (1988) with the prior approval of the Animal Ethics Committee of Shinshu University School of Medicine.

Surgical procedures

Implantation of arterial and other catheters.

Details were reported elsewhere (Mattson, 1998; Masuki et al. 2003). Briefly, after a mouse was anaesthetized with pentobarbital sodium (50 mg (kg body weight)−1i.p.), a polyethylene catheter to measure MAP and HR was inserted into the left femoral artery to place the tip 5 mm below the left renal artery. A polyethylene catheter for i.p. administration of α-adrenergic blockade was also placed in the abdominal cavity of a mouse in which MBF was measured. The catheters were secured to the surrounding muscles, tunnelled subcutaneously and exteriorized between the scapulae. The exteriorized catheter was connected to a cannula swivel (model TCS2-21, Tsumura; Tokyo, Japan), and the mouse was placed in a home cage with a free moving system (model FM-1121, Tsumura). The arterial catheter was flushed everyday with 100 i.u. heparin in 0.2 ml saline. The surgery was performed at least 4 days before MAP and HR measurements.

Implantation of laser-Doppler flow probe and electromyogram (EMG) electrodes.

The details in MBF measurements have been reported elsewhere (Masuki & Nose, 2003). Briefly, following the arterial catheter implantation, a laser-Doppler flow probe (model FLO-C1 BV, Omegawave; Tokyo) was implanted under light ether anaesthesia in the right hindlimb with EMG electrodes in the WT and KO mice. The probe consisted of two glass fibres each of which was covered with a plastic sheet; one for insertion of the laser light, and the other for detection of the reflection. The tips of the fibres were stripped of their cover, glued to be 4 mm in length and 0.5 mm in diameter, and inserted into the intermuscular space of the vastus medialis and lateralis muscles. A pair of EMG electrodes was also implanted in the vastus lateralis muscle. The glass fibres and electric wires from EMG electrodes were secured to surrounding leg muscles, tunnelled subcutaneously, and pulled out from the interscapulae. The surgery was performed at least 4 days before the measurement.

Protocol

Exercise capacity test.

Mice exercised on a treadmill (model TMK-001, Melquest; Toyama, Japan) equipped with an electric stimulator (model MSG-001, Melquest) and a couple of infrared beam lamp and sensor (model CX-21, SUNX; Nagoya, Japan) at the back of the treadmill (Fewell et al. 1997). After 4 days for learning, seven WT and seven KO mice were subjected to graded treadmill exercise, initiated at 5 m min−1, followed by 5 m min−1 increase every 5 min. The exercise capacity test was performed in each mouse twice per day for 4 days. The numbers of beam breaks at each exercise speed were averaged for eight trials (2 trials × 4 days) as an indicator of the exercise capacity in each mouse. The maximal exercise speed was determined as the fastest speed at which mice completed 5-min exercise without breaking the infrared beam for longer than 1 min.

Measurement of arterial blood pressure during treadmill exercise.

Six of seven mice for each group of the WT and KO mice after the exercise capacity test were used to measure MAP and HR during graded treadmill exercise. Before the measurements, we confirmed that mice had recovered their exercise capacity as before the implantation of the arterial catheter. After waiting about an hour for a mouse to stay still on the treadmill out of a home cage, the baselines of MAP and HR at rest for 10 min were measured, then the mouse started to exercise on the treadmill at 5 m min−1. The exercise speed was increased by 5 m min−1 every 10 min until they completed 20 m min−1 exercise or did not maintain a given speed due to exhaustion. Exhaustion was judged from consecutive beam breaks lasting longer than 1 min. The measurements were performed during the period randomly chosen from 10:00 to 17:00.

Effects of α-adrenergic blockade on muscle vascular conductance (MVC) during voluntary locomotion.

MAP, HR, MBF and EMG in seven mice for each of the WT and KO groups were continuously measured in a free-moving state for 90 min; mice were randomly chosen from 10:00 to 17:00 in their home cages. The measurements were made 4-6 days after the surgery and the results were compared before and after i.p. administration of α-adrenergic blockade (phentolamine). The administration was performed through the catheter placed in the abdominal cavity as an initial dose of 1 mg (kg body weight)−1 followed by 6 mg (kg body weight)−1 h−1 for continuous administration during the measurements. The effects of the α-adrenergic blockade were confirmed by no increase in MAP by the intra-arterial injection of α-adrenergic agonist (phenylephrine). The increase in MAP from the baseline by the injection of 30 µg ml−1 phenylephrine (3.3 ml (kg body weight)−1) was 34.5 ± 4.3 and 21.8 ± 1.7 mmHg in WT and KO mice, respectively, before the administration of blockade (P < 0.001), but that was abolished to -0.1 ± 1.7 and -0.3 ± 1.5 mmHg after the administration (P > 0.6).

Measurements

MAP and HR.

MAP was measured through the arterial catheter connected to a pressure transducer (model TP-400T, Nihon Kohden; Tokyo, Japan). HR was counted from the arterial pressure pulse with a tachometer (model AT-601G, Nihon Kohden). The response time of the tachometer at a given change in the pressure-pulse rate was short enough to allow discussion of the ▵HR in the present study. MAP and HR were continuously recorded with a digital data recorder (Thermal Arraycorder WR 8500, Graphtec; Yokohama, Japan) at 100 ms intervals through a low-pass filter of edge frequency of 1.5 Hz. The measurements in each mouse were conducted three times on separate days, averaged as a function of time after the start of exercise for each mouse, and the means and s.e.m. values for six mice were determined in the WT and KO groups.

MBF and EMG.

MBF was recorded by laser-Doppler flowmetry with a digital data recorder (Thermal Arraycorder) at 100 ms intervals through a digital low-pass filter of the edge frequency of 0.1 Hz, to remove the artifacts caused by altered relative angles between the tip of the probe and the vascular wall during locomotion (Masuki & Nose, 2003). Briefly, the MBF signal during the periods where the EMG burst was above 200 % of baseline was passed through a low-pass digital filter of edge frequency 0.1 Hz (KC-DF-FIR01, Kissei Comtec; Matsumoto, Japan), while the MBF signal during the remaining periods where the EMG burst was below 200 % of the baseline was not passed through the filter, and the signals during the two periods were ultimately combined and recorded every 100 ms by a computer (OptiPlex GX260, Dell; Kawasaki, Japan). MVC from MBF/MAP was presented as the percentage change from the difference between the baseline and the maximal value by 10 s after the onset of locomotion after the same filtering treatments as in MBF.

EMG was also recorded through a band-pass filter of 53-1000 Hz (Bioelectric Ampl 4124, NEC, Tokyo, Japan) after integrating with a time constant of 100 ms (i:EMG:), and expressing as the percentage change from the resting value. The data during locomotion were adopted according to the criteria of an i:EMG: burst greater than 200 % above the baseline lasting longer than 10 s. MVC and i:EMG: during three 10 s periods after the start of locomotion were averaged in each mouse, and the means and s.e.m.s for seven mice were determined in the WT and KO groups.

Analyses

Variance and frequency distribution analyses of ▵MAP and ▵HR.

To exclude any effects of transient change in exercise intensity and electrical stimulations on the analyses, we removed the data during the following two periods of exercise: (1) 90 s before reaching every graded speed of exercise, and (2) 30 s after the last consecutive beam break to exclude any influence of electrical stimulation on the measurements. To standardize the number of data for analyses, we removed the data during the first 90 s of the 10 min resting period. The total number of data adopted for the analyses are shown in Table 1.

Table 1. MAP and HR at rest and during treadmill running
 Rest5 m min−110 m min−115 m min −1
  1. Values are means ±s.e.m. Number of data was represented as % of total measurements during resting and exercise at 5, 10, and 15 m min−1 periods. ** and *** Significant differences from wild-type (WT) mice, P < 0.01 and P < 0.001, respectively, †, ††, and ††† Significant differences from the values at rest in each group, P < 0.05, P < 0.01, and P < 0.001, respectively. § Variance was calculated from the change in MAP (ΔMAP) or HR (ΔHR) from mean values every 4 s.

  WT (n= 6) 
MAP
  Mean (mmHg)90.2 ± 2.7117.1 ± 2.9†††115.4 ± 2.7†††115.5 ± 2.8†††
  Variance§ (mmHg2)3.1 ± 0.84.9 ± 0.64.9 ± 0.82.9 ± 0.2
HR
  Mean (beats min−1)492 ± 9671 ± 16†††673 ± 17†††683 ± 15††
  Variance§ (beats2 min−2)98 ± 14305 ± 30†††281 ± 48†††238 ± 27††
  Number of data (%)10098 ± 198 ± 193 ± 3††
  KO (n= 6) 
MAP
  Mean (mmHg)93.0 ± 3.0114.3 ± 3.1†††113.6 ± 3.0†††114.3 ± 3.2†††
  Variance§ (mmHg2)3.4 ± 0.610.2 ± 1.5***†††9.0 ± 0.9***††7.1 ± 1.6***†
HR
  Mean (beats min−1)463 ± 20647 ± 14†††661 ± 14†††679 ± 11†††
  Variance§ (beats2 min−1)296 ± 48**389 ± 69331 ± 51299 ± 67
  Number of data (%)10092 ± 3***83 ± 4***†73 ± 10***††

The variances of changes in HR (▵HR) and MAP (▵MAP) from the values averaged every 4 s were determined on the data during the periods of 10 min rest and 10 min exercise at 5, 10, and 15 m min−1. ▵MAP and ▵HR were calculated from the same equations as in the determination of spontaneous baroreflex sensitivity described below. Since the measurements were conducted three times in each mouse on separate days, three variances of ▵MAP and ▵HR were averaged in each mouse, and the means and s.e.m.s for six mice were determined in the WT and KO groups. Similarly, the frequency distributions of ▵MAP and ▵HR during resting and exercise at 10 m min−1 periods, 8.5 min × 3 times for each period, were also determined in each mouse and expressed as percentage of the total number of measurements adopted for the analyses.

Spontaneous baroreflex sensitivity analyses.

Spontaneous baroreflex sensitivity was determined in each mouse at rest (25.5 min, 8.5 min × 3 times, n= 15 300) and during exercise (≈23.1 min, ≈7.7 min × 3 times, n=≈13 800) at 10 m min−1 using the same data used for the frequency distribution analyses. Since spontaneous change in MAP and consecutive change in HR were observed at 5-15 cycles min−1 (4-12 sec cycle−1) as shown in Fig. 3, the relationship between ▵MAP and the consecutive ▵HR from values averaged every 4 s was analysed at 4 s intervals using a cross-correlation function given in the following formulae (Basar & Weiss, 1981).

display math

Where R(t) is a cross-correlation coefficient between x (= MAP) and y (= HR) at the given time of t after correction for the delay time (▵t= 0.6 s) in response to HR change. x-(t) and y-(t) were averaged values of MAP and HR, respectively, from time t - τ/2 to t+τ/2 (τ= 4 s). The detailed numerical analyses have been reported previously (Masuki et al. 2003). The slope (▵HR/▵MAP), an index of the spontaneous baroreflex sensitivity, was calculated by standard y-minimized regression analyses on the data pooled every 4 s at rest and during exercise where ▵MAPwas negatively correlated with ▵HR (P < 0.05).

Figure 3.

Original traces of arterial pressure (AP) and HR during treadmill exercise at 10 m min−1 for 30 s

The panels on the right are enlarged from the parts indicated by the solid bars in the left panels.

Statistics

Values are expressed as means ±s.e.m. The difference in beam breaks during graded treadmill exercise between the WT and KO groups was tested by a 2 (WT, KO) × 3 (5, 10, 15 m min−1) ANOVA (Fig. 1), which was also used to test the differences in the means and variances of MAP and HR between the groups (Table 1). The differences in MAP and HR during graded exercise between the WT and KO groups were tested by a two-way (WT, KO) ANOVA for repeated measures (Fig. 2), which was also used to test the differences in the change in MVC and i:EMG: after the start of locomotion (Fig. 6). The effects of α-adrenergic blockade on the time to reach 50 % of the maximum MVC (T50 %) were tested by a 2 (WT, KO) × 2 (control, α-adrenergic blockade) ANOVA (Fig. 7). The difference in spontaneous baroreflex sensitivity was tested by a 2 (WT, KO) × 2 (rest, exercise) ANOVA (Table 2). Specific trend analysis for each group was performed with a one-way ANOVA for repeated measures (Fig. 2 and Table 1). Subsequent post hoc tests to determine significant differences in the various pairwise comparisons were performed using Fisher's LSD. The null hypothesis was rejected at P < 0.05.

Figure 1.

The number of beam breaks detected with an infrared beam sensor equipped at the back of the treadmill during exercise for 5 min

Means and s.e.m. bars are presented for the 7 WT and 7 KO mice. Significant differences between the two groups, P < 0.05 and ⋆⋆P < 0.01.

Figure 2.

Mean arterial pressure (MAP) and heart rate (HR) responses to graded treadmill exercise

Means and s.e.m. bars for the 6 WT and 6 KO are presented every 15 s and every 1 min, respectively. ⋆⋆⋆ Significant differences between the two groups, P < 0.001.

Figure 6.

Percentage changes in muscle vascular conductance (MVC) and integral electromyogram (i:EMG:) at the onset of voluntary locomotion

Means and s.e.m. bars are presented for 7 mice in each group. s.e.m. bars are presented every 0.5 s. A, control. B, after administration of α-adrenergic blockade (phentolamine).

Figure 7.

The time to reach 50 % of the maximum muscle vascular conductance at the onset of voluntary locomotion before and after administration of α-adrenergic blockade (phentolamine)

Means and s.e.m. bars are presented for 7 mice in each group. ⋆⋆⋆ Significant differences from the control group of the WT mice, P < 0.001.

Table 2. Spontaneous baroreflex sensitivity (ΔHR/ΔMAP) in WT and KO mice
 RestExercise
  1. ΔHR/ΔMAP, heart rate response to the spontaneous change in mean arterial pressure, beats min−1 mmHg−1. **Significant differences between two groups at rest, P < 0.01. ††Significant differences between at rest and during exercise in KO mice, P < 0.01. §ΔHR was highly correlated with ΔMAP in all mice at the level of P < 0.00001 (n= 4964–11906).

Mouse numberΔHR/ΔMAP, R 2§ΔHR/ΔMAP, R 2§
WT1−4.80.44−4.90.29
WT2−6.80.58−8.50.28
WT3−5.30.42−5.80.40
WT4−3.50.43−2.90.33
WT5−6.80.41−5.70.41
WT6−3.10.39−2.20.37
Mean ±s.e.m.−5.1 ± 0.6 −5.0 ± 0.9 
KO 1−7.30.36−3.70.35
KO 2−9.00.48−5.20.40
KO 3−11.50.45−5.20.26
KO 4−6.40.35−4.40.42
KO 5−7.70.57−6.20.42
KO 6−17.40.61−3.30.35
Mean ±s.e.m.−9.9 ± 1.7** −4.7 ± 0.4†† 

Results

Exercise capacity test

Figure 1 shows the beam breaks during 5-min exercise at 5, 10 and 15 m min−1. As the exercise speed increased, the number of beam breaks increased; 1.4 ± 0.3, 10.6 ± 1.7 and 22.4 ± 3.1 breaks in the KO mice, significantly higher than 0.5 ± 0.2 (P < 0.05), 3.6 ± 1.4 (P < 0.01), and 8.3 ± 2.6 breaks (P < 0.01) in the WT mice at 5, 10, and 15 m min−1, respectively. The maximal exercise speed in the KO mice was 18.9 ± 0.9 m min−1, significantly lower than 27.8 ± 1.7 m min−1 in the WT mice (P < 0.001).

Arterial blood pressure and HR at rest and during treadmill exercise

Figure 2 shows MAP and HR during graded treadmill exercise at 5, 10, 15 and 20 m min−1. Each speed lasted for 10 min. As shown in the upper panel of Fig. 2, MAP in the WT and KO mice increased sharply after the start of exercise and remained at this level throughout the graded exercise (P < 0.001), with no significant differences between the WT and KO mice (P > 0.05). All the WT mice accomplished 20 m min−1 exercise while no KO mice did.

As shown in the lower panel of Fig. 2, HR at rest was significantly lower than that in the WT mice (P < 0.001). HR increased sharply after the start of exercise in the WT and KO mice, gradually increased during graded exercise and reached 709 ± 22 beats min−1 at the maximal exercise speed of 15 m min−1 in the KO mice and 735 ± 18 beats min−1 at that of 20 m min−1 in the WT mice, with no significant differences between the WT and KO mice during exercise (P > 0.05).

Figure 3 shows original traces of MAP and HR during exercise at 10 m min−1 for a WT mouse in the upper panel and for a KO mouse in the lower panel. As shown in the figure, MAP in a WT mouse was controlled within a narrow range, whereas that in a KO mouse fluctuated more. The attached panels on the right were enlarged from the parts indicated by the solid bars in the left panels, showing that a fall in arterial pressure caused a rise in HR after a 0.6 s delay in both mice.

Variance and frequency distribution of ΔMAPand ΔHR

Table 1 summarizes the means and variances in MAP and HR at rest and during exercise at 5, 10, and 15 m min−1. The variance in ▵HR at rest was significantly higher in the KO mice than that in the WT mice (P < 0.01), but with no significant difference in the variance in ▵MAP at rest between the groups (P > 0.1). On the other hand, the variances in ▵MAP during exercise at 5, 10 and 15 m min−1 were significantly higher in the KO mice than those in the WT mice (P < 0.001), suggesting that MAP fluctuated more in the KO mice as shown in Fig. 3 and Fig. 4B, but with no significant differences in the variance in ▵HR during graded exercise between the groups (P > 0.1). The number of data during exercise used for these analyses were slightly but significantly lower in the KO mice than that in the WT mice (P < 0.001), due to higher beam breaks during exercise as shown in Fig. 1.

Figure 4.

ΔMAP and ΔHR frequency distributions at rest (A) and during treadmill exercise at 10 m min−1 (B)

Means and s.e.m. bars are presented for 6 WT and 6 KO mice.

▵MAP and ▵HR frequency distributions at rest are shown in Fig. 4A, and those during exercise at 10 m min−1 in Fig. 4B. At rest, as shown in Fig. 4A, ▵HR was distributed with a wider range in the KO mice than that in the WT mice with significantly higher standard deviation of 16.9 ± 1.3 beats min−1 in the KO mice compared with 9.8 ± 0.7 beats min−1 in the WT mice (P < 0.001), but with no significant differences in the distribution of ▵MAP between the groups (P > 0.7). On the other hand, during exercise at 10 m min−1, as in Fig. 4B, ▵MAP was distributed with a wider range in the KO mice than that in the WT mice with significantly higher standard deviation of 3.0 ± 0.2 mmHg in KO mice compared with 2.2 ± 0.2 mmHg in the WT mice (P < 0.01) but with no significant differences in the distribution of ▵HR between the groups (P > 0.5).

Spontaneous baroreflex sensitivity

The spontaneous baroreflex sensitivity was determined by using the results showing that ▵MAP and ▵HR were significantly and negatively correlated with each other (P < 0.05). The number of data with a significant negative correlation was 41 ± 3 % and 52 ± 7 % of the total measurements adopted for the analyses (n= 10, 800-15 300) at rest and 39 ± 3 % and 42 ± 2 % during exercise in the WT and KO mice, respectively, with no significant differences between the groups (P > 0.1). Figure 5 shows typical examples of the regression analysis on pooled data with a significant negative correlation in a WT and KO mouse at rest and during the treadmill exercise at 10 m min−1. As summarized in Table 2, the negative slope of ▵HR/▵MAP at rest was twofold steeper in the KO mice than that in the WT mice (P < 0.01), but during exercise the slope in the KO mice decreased to 50 % of that at rest (P < 0.01), while that in the WT mice remained unchanged, with no significant differences between the groups during exercise (P > 0.5).

Figure 5.

Typical examples of the relation between ΔMAP and ΔHR at rest and during treadmill exercise at 10 m min−1 in a WT mouse and a KO mouse

The number of data was ∼6400 in each mouse at rest or during exercise.

MVC at the onset of voluntary locomotion

Figure 6 shows MVC and i:EMG: after the start of voluntary locomotion before (Fig. 6A) and after (Fig. 6B) administration of phentolamine. MVC started to increase within 1 s after voluntary locomotion in the WT and KO mice. As presented in Fig. 7, the time to reach 50 % of the maximum MVC (T50 %) was determined in each mouse and the results for seven mice in the WT and KO mice. T50 % of 2.3 ± 0.2 s in the KO mice was significantly shorter than the value of 5.8 ± 0.6 s in the WT mice (P < 0.001). Prior administration of phentolamine shortened T50 % in the WT mice to that in the KO (P < 0.001), but did not change T50 % in the KO mice (P > 0.1). There were no significant differences in i:EMG: throughout measurements either between the groups or between before and after administration of phentolamine (P > 0.1).

Discussion

The major findings in the present study were, (1) MAP during exercise fluctuated more in KO mice than in WT mice; (2) the spontaneous baroreflex sensitivity during exercise in the KO mice was reduced to half that during rest, but with no significant change in the WT mice; (3) muscular vasodilatation at the onset of voluntary locomotion was enhanced in the KO mice compared with that in the WT mice; (4) the vasodilatation in the WT mice was accelerated by α-adrenergic blockade compared to that in the KO mice, but with no effect of the blockade in the KO mice; and (5) the maximal exercise speed in the KO mice was reduced compared with that in the WT mice.

The baroreflex sensitivity during exercise was determined from the HR response to the spontaneous change in MAP (Table 2, Fig. 5). The reason for adopting treadmill exercise to determine spontaneous baroreflex sensitivity was to exclude any effects induced by change in exercise intensity during voluntary locomotion. In the previous study (Masuki et al. 2003), the baroreflex sensitivity in the KO mice during the resting period of 135 min was -13.1 beats min−1 mmHg−1, twofold higher than the value of -6.2 beats min−1 mmHg−1 in the WT mice, which was identical to the results determined during rest for 25.5 min of 8.5 min × 3 times in the present study (Table 2). Also in the previous study (Masuki et al. 2003), the period during which ▵MAP was negatively correlated with ▵HR was 51 % of the total measuring period in the WT mice, and 69 % of that in the KO mice, which was identical to the results not only at rest but also during exercise in the present study. The reproducibility in the percentage period of the negative correlation suggests that the baroreflex sensitivity determined by this method is reliable enough to enable discussion of the change in the sensitivity during exercise. The reasons for determining baroreflex sensitivity during exercise only at 10 m min−1 were that the majority of the mice kept running continuously at this speed without intermittent back and forth on the treadmill lane as observed at 5 m min−1 and that there were smaller differences in the number of data for analyses between the KO and WT mice at 10 m min−1 than at 15 m min−1 (Table 1).

The greater variability in MAP during exercise in the KO mice (Fig. 3, Table 1) than that in the WT mice may be partially explained by the reduced arterial baroreflex sensitivity compared with that at rest (Fig. 5, Table 2) where the enhanced baroreflex sensitivity was reported to compensate well for the blunted α-adrenergic vasoconstrictive response (Masuki et al. 2003). It appears to be broadly known that the baroreflex sensitivity is not altered during exercise compared with that at rest although the operation point of the baroreflex function curve moves to higher arterial pressure (O'Leary & Seamans, 1993; Krieger et al.1998). In addition, we reconfirmed that the baroreflex sensitivity remained unchanged during exercise in the WT mice as in Fig. 5 and Table 2. Thus, precise reasons for the reduction in arterial baroreflex sensitivity in the KO mice are unclear. However, Burger et al. (1998) recently reported that the baroreflex sensitivity from the change in HR response to spontaneous beat-by-beat change in systolic pressure was reduced during exercise compared with that at rest in spontaneous hypertensive female rats in which sympathetic nervous outflow was enhanced. In the present study, since the sympathetic nervous outflow at rest in the KO mice was expected to be enhanced due to increased baroreflex sensitivity, similar mechanisms to those in spontaneous hypertensive rats may be involved in the reduction in baroreflex sensitivity during exercise.

Another possible explanation for the greater fluctuation of MAP during exercise was that the α-adrenergic vasoconstrictive response was attenuated in the KO mice. Masuki et al. (2003) reported that the vasoconstrictive response to a bolus intra-arterial injection of phenyleprine in the KO mice was reduced to half that in the WT mice. Je et al. (2001) reported in an in vitro study that the contractile response to phenyleprine was significantly decreased in the ferret aorta after calponin antisense treatment, and suggested that calponin facilitated the α-adrenergic vasoconstrictive response. As shown in Fig. 6, the increasing rate of MVC after the onset of spontaneous locomotion was significantly greater in the KO mice than that in the WT mice. Moreover, α-adrenergic blockade enhanced the increasing rate of MVC in the WT mice but not in the KO mice (Fig. 7). These results suggest that the α-adrenergic vasoconstrictive response was impaired in the KO mice, which may be involved in the greater fluctuation of MAP during treadmill exercise.

However, baroreflex control of MVC during treadmill exercise would be different from that at the onset of voluntary locomotion. During treadmill exercise, MAP was reportedly reduced by administration of sympathetic ganglionic blockade in dogs (Sheriff et al. 1993; Buckwalter & Clifford, 1999) and/or by baroreceptor-denervation in rabbits (DiCarlo & Bishop, 1992). Also, Masuki & Nose (2003) reported that the enhanced increasing rate of MVC and the consecutive fall in MAP occurred at the onset of locomotion in carotid sinus-denervated or α- adrenergic blockade-administered mice. These results suggest that muscular vasodilatation was suppressed by baroreflexes not only during treadmill exercise but also at the onset of voluntary locomotion, which is probably one of the feedback mechanisms to help maintain MAP by counteracting the vasodilatory effects of local factors released from contracting muscles.

In the present study, although the level of MAP during treadmill exercise in the KO mice was maintained the same as that in the WT mice (Table 1 and Fig. 2), the fluctuation was enhanced in the KO mice despite the same sensitivity of ▵HR/▵MAPas in the WT mice (Fig. 3), suggesting that the fast or dynamic vasoconstrictive response of the vascular smooth muscle to MSNA was impaired in the KO mice while the slow or static response was preserved. As shown in Fig. 7, the time to reach 50 % of the maximum MVC (T50 %) after the onset of voluntary locomotion was approximately 3 s in the KO mice, which was not shortened by administration of α-adrenergic blockade, different from the case in the WT mice, suggesting that the vasoconstrictive response to MSNA did not work to suppress such a sharp increase in MVC for the KO mice. During treadmill exercise, the spontaneous change in MAP occurred at 4-12 s cycle−1 in the KO mice (Fig. 3), suggesting the speed of muscular vasodilatation by local factors during treadmill exercise was as rapid as that observed at the onset of voluntary locomotion (Fig. 6). Thus, the greater fluctuation of MAP during treadmill exercise in the KO mice may be caused not only by reduced arterial baroreflex sensitivity but also by an attenuated dynamic α-adrenergic vasoconstrictive response to MSNA.

The variances in MAP at every speed during graded exercise were significantly higher in the KO mice than those in the WT mice (Table 1). Since the MAP for 30 s after the last consecutive beam break was removed from the analyses, the higher MAP variances in the KO mice were not likely to be caused by the higher frequency of electric stimulation to mice. This idea was supported by the findings that there were no significant differences in the variances and mean values of HR between the WT and KO mice, because they suggested no different effects of electric stimulation on physical and mental stress to mice between the groups. Moreover, the higher fluctuation of MAP in the KO mice was almost always accompanied by a consecutive change in HR through baroreflexes (Fig. 3 and Fig. 5, Table 2), suggesting that higher fluctuation was not caused by mechanical artifacts on an arterial catheter due to more frequent movement as indicated by the higher beam breaks in the KO mice (Fig. 1). Thus, the higher variances in MAP during graded exercise for the KO mice were not caused by the higher frequency of electric stimulation, or by more frequent movement, but by impaired MAP regulation.

The numbers of beam breaks during graded exercise were significantly higher in the KO mice than in the WT mice (Fig. 1) and the maximal exercise speed was 32 % lower in the KO mice than in the WT mice. Since the variances in MAP during graded exercise were significantly higher in the KO mice than those in the WT mice (Table 1), the unstable MAP during exercise may be involved in the reduced maximal exercise speed in the KO mice.

Summarizing these results, the fluctuation of MAP was enhanced during exercise in the KO mice. This may be caused by the reduced baroreflex sensitivity during exercise compared with that at rest and the blunted muscle vascular α-adrenergic contractile response during exercise in the KO mice. Thus, calponin may play an important role in regulating MAP by increasing the α-adrenergic vasoconstrictive response to sympathetic nervous activity during exercise.

Acknowledgements

This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and was also supported by Ground-based Research Announcement for Space Utilization from the Japan Space Forum. For this study, S. Masuki was a recipient of the Recognition Award for Meritorious Research at Experimental Biology 2003 from the American Physiological Society, Environmental and Exercise Physiology Section.

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