INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX REFERENCES

CALPONIN, also called basic calponin or calponin H1, was first isolated from the chicken gizzard as an actin-binding protein, which was reported to be involved in the regulation of smooth muscle contraction (22). Calponin was reported to reduce unloaded isometric forces and shortening velocity (8, 14, 19, 23) by the inhibition of actomyosin ATPase activity in a reconstituted isolated filament system (26). On the other hand, there have been several studies suggesting that calponin increases the contractile response to norepinephrine (NE) (18) or phenylephrine (PE) (9, 16, 20) by facilitating agonist-induced signal transduction.

With the exception of these in vitro studies, however, there have been few studies investigating the role of calponin in arterial pressure regulation in the whole body. Because arterial pressure is determined from the product of cardiac output and total peripheral resistance, the dysfunction of vascular smooth muscle contraction itself and/or the impaired -adrenergic response to sympathetic nervous activity in genetically calponin-deficient mice [knockout (KO) mice] would make arterial pressure unstable. Alternatively, baroreflex control would compensate for the impaired arterial pressure regulation by changing the heart rate (HR) and/or cardiac output in the group.

To examine these hypotheses, we measured mean arterial pressure (MAP) and HR in the free-moving KO mice and determined the baroreflex sensitivity from the change in heart rate (HR) in response to the spontaneous change in MAP (MAP) and compared the results with those in wild-type (WT) mice. We also determined the baroreflex sensitivity by intravascular administration of -adrenergic agonist of PE or sodium nitroprusside (SNP) and compared the results between the groups. Moreover, we assessed the effects of carotid denervation on MAP to examine the compensatory effects of baroreflexes, if any, in the KO mice.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX REFERENCES

Animals

View larger version (17K): [in this window] [in a new window]   Fig. 1.   RT-PCR analysis of calponin H1 (1,295 bp) and GAPDH (731 bp) using mRNA extracted from an aorta. Compared with the wild-type (WT) mouse, calponin H1 is lacking in the knockout (KO) mouse.

The procedures used here were in accordance with the American Physiological Society's "Guiding Principles for Research Involving Animals and Human Beings" and conducted with permission from the Animal Ethics Committee of Shinshu University School of Medicine.

Surgical Procedures

Intact condition. Details were reported elsewhere (15). Briefly, the arterial catheter was fashioned from a 450-mm segment of polyethylene tube (SP31, ID: 0.5 mm, OD: 0.8 mm, Natsume; Tokyo, Japan) welded to a 22-mm polyethylene tube segment (SP8, ID 0.2 mm, OD 0.5 mm, Natsume) with a tip that was tapered to ~0.3 mm in diameter over hot air. At least 4-6 days before the experiment, the mice were anesthetized with pentobarbital sodium (50 mg/kg body wt ip), and the hair over the anterior left lower limb and between the scapulas was shaved. The skin was sterilized with povidon iodine (Meiji; Tokyo, Japan). The surgery was performed on a warming pad kept at 37°C (model 39, DP Scientific; Braintree, MA). The left femoral artery was exposed, and a small incision was made in the arterial wall with microspring scissors. The tip of the catheter was introduced through the incision and advanced by ~20 mm so that the tip was placed 5 mm below the bifurcation of the left renal artery. The catheter was secured to the leg muscle and tunneled subcutaneously and exteriorized between the scapulas. The exteriorized catheter was connected to a cannula swivel (model TCS2-21, Tsumura; Tokyo, Japan), and the mice were placed in a home cage with a free-moving system (Tsumura). A few hours after the preparation, the mice recovered to move the catheterized limb freely. The catheter was flushed everyday with 500 IU heparin in 0.2 ml of saline. The mice were allowed to recover for 4-6 days after catheter implantation before the measurements were taken.

Carotid denervated condition. At least 3 days after arterial catheter implantation, mice were anesthetized again as described above. A midline incision was made in the anterior neck and the area of the carotid bifurcation was exposed. The internal, external, and common carotid arteries were stripped of connective tissues, and the region was painted with 10% phenol in ethanol, as reported previously in rats (12, 24). The recovery period from the carotid denervation was at least 4 days before the measurements of HR and MAP. The success of the denervation was confirmed by the increased fluctuation in MAP and no HR response to the fluctuation as in Table 1 (4).

View this table: [in this window] [in a new window]   Table 1.   MAP, HR, and spontaneous baroreflex sensitivity (HR/MAP) in WT and KO mice

Measurements

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Mean arterial pressure (MAP) and heart rate (HR) during measuring period of 60 min in a WT mouse. The three criteria to judge as resting state were the following: 1) virtually not moving, 2) <10 mmHg increase in MAP, and 3) <90 beats/min increase in HR.

Spontaneous Baroreflex Sensitivity Analyses

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Typical examples of MAP and HR at rest for 16 min in a WT mouse (A) and in a KO mouse (B). The sections of the traces with short bars are shown in detail in Fig. 4.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   HR and MAP in a WT mouse (A) and a KO mouse (B) from the parts indicated by the solid bars in Fig. 3, A and B, respectively. A rise in MAP caused a fall in HR and inversely a fall in MAP caused a rise in HR after a 0.6-s delay in both groups. The amplitude of the HR response at a given change in MAP was greater in the KO mouse than that in the WT mouse.

Drug-Induced Baroreflex Sensitivity Analyses

The baroreflex curves expressed as the relationship between MAP and HR were analyzed with a logistic sigmoid function according to the following equation (11, 17)
where  is the range between the upper and lower plateau, e is exponential function,  is a coefficient to calculate the gain as a function of pressure,  is the MAP at midrange of the curve (midpoint), and  is the lower plateau. Each parameter and the threshold pressure (lowest pressure that products a significant decline in HR) and the saturation pressure (pressure necessary to achieve maximal inhibition of HR) were determined by fitting the equation to minimize the sum of y distance between the experimental data (HR) and the y value (HR) obtained by substituting x value (MAP) for the equation.

Statistical Analysis

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX REFERENCES

Table 1 shows MAP and HR at rest in the WT and KO mice in carotid innervated and denervated conditions. The resting period in both groups was 70-80% of the total measuring period with no significant differences between the groups or between innervated and denervated conditions. In the innervated condition, HR was significantly lower in the KO mice than in the WT mice (P < 0.05), whereas MAP was not significantly different between the groups (P > 0.9). After carotid sinus denervation, MAP increased significantly (P < 0.05) in the WT mice but not in the KO mice (P > 0.4), whereas HR in the KO mice increased significantly (P < 0.001) but not in the WT mice (P > 0.2). In the innervated condition, there were no significant differences in the CV of MAP between the groups, whereas that of HR was twofold higher in the KO mice than that in the WT mice (P < 0.001). After carotid denervation, the CV of MAP increased by twofold in both groups (P < 0.001), whereas the CV of HR in the KO mice decreased to one-half that in the innervated mice (P < 0.001), which was not significantly different from that in the innervated WT mice. There was no significant change in the CV of HR for the WT mice after denervation.

Figure 3 shows typical examples of MAP and HR during the resting period of 16 min in one typical WT mouse (Fig. 3A) and one typical KO mouse (Fig. 3B). Although the variability in MAP was similar between the groups, that in HR was significantly higher in the KO mice than that in the WT mice.

Figure 4, A and B, is enlarged from the parts indicated by closed bars in Fig. 3, A and B, respectively. As shown in Fig. 4, a rise in MAP caused a fall in HR and inversely a fall in MAP caused a rise in HR after a 0.6-s delay.

Figure 5 shows an example of R(t) between HR and MAP at the given time of t in a WT mouse, demonstrating a significantly positive or negative correlation above or below the dotted lines of P < 0.05, respectively. As summarized in Table 1, the negative correlation period between HR and MAP was 69% of the total measuring period in the KO mice, which was significantly >51% in the WT mice (P < 0.01). On the other hand, the significant positive correlation period was 4% in the KO mice, which was significantly <11% in the WT mice (P < 0.05). After carotid sinus denervation, the negative correlation period decreased to 40% of the predenervated level in the WT mice (P < 0.001) and to 20% in the KO mice (P < 0.001), whereas that of the positive correlation increased threefold in the WT mice (P < 0.01) and sixfold in the KO mice (P < 0.01).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   R(t), a cross-correlation function between a change in MAP (MAP) and HR, in a WT mouse, is presented as a function of time. MAP and HR during the period where R(t) was higher than the level of P < 0.05 were adopted for regression analyses to determine spontaneous baroreflex sensitivity (shaded area). A more detailed explanation is given in the text.

The significant negative correlations were pooled for regression analyses and the results are presented in Fig. 6A for the WT mice and in Fig. 6B for the KO mice. As summarized in Table 1, the negative slope of HR/MAP was twofold steeper in the KO mice than that in the WT mice in the innervated condition (P < 0.001). After carotid sinus denervation, the negative slope decreased to one-third that in the innervated condition for the WT mice (P < 0.001) and to one-sixth in the KO mice (P < 0.001), and the significant difference between the groups in the innnervated condition disappeared (P > 0.7). The positive slope was not significantly different between the groups in the innervated condition, but after denervation it decreased to 53% of the predenervated value in the WT mice (P < 0.05) and to 30% in the KO mice (P < 0.001) with no significant difference between the groups (P > 0.9).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   The relation between MAP and HR adopted during the significant negative correlation period in the WT mice (A) and KO mice (B). The data number in each mouse was ~42,000 in the WT mice and ~56,000 in the KO mice.

Figure 7 shows the changes in MAP after the injections of PE and SNP. The increases in MAP by 5 and 10 µg/kg of PE in the KO mice were 9.3 ± 0.4 and 18.2 ± 1.4 mmHg, respectively, which were significantly <23.0 ± 1.5 and 28.8 ± 2.7 mmHg in the WT mice, respectively (P < 0.001). On the other hand, there were no significant differences in vascular response to SNP between the two groups (P > 0.07).

View larger version (10K): [in this window] [in a new window]   Fig. 7.   Change in MAP after injection of phenylephrine (PE) at 5, 10, and 50 µg/kg and sodium nitroprusside (SNP) at 15 and 30 µg/kg. Means ± SE bars on 5 WT and 5 KO mice. *** P < 0.001, significant differences between the WT and KO mice.

Figure 8A shows a typical example of the relation between MAP and HR after injection of PE and SNP into a WT mouse with a best-fit sigmoid baroreflex curve generated, as described above. Figure 8B shows the best-fit curves to the mean values of the midpoint, threshold, saturation, maximum, and minimum determined in WT and KO mice. There were no significant differences in the parameters between the two groups as summarized in Table 2.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   The relation between MAP and HR after injection of PE and SNP. A: typical example of a WT mouse. A total of 85 measurements are presented. The best-fit sigmoid baroreflex curve generated as described in the text is also presented. B: composite baroreflex curves and baroreflex parameters for WT and KO mice are presented. a, Maximum; b, threshold; c, midpoint; d, saturation; e, minimum. Means ± SE bars refer to 5 WT and 5 KO mice.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION APPENDIX REFERENCES

In this study, we first chronically implanted catheters in the calponin KO mice and measured MAP and HR under free-moving conditions. The major findings were the following: 1) the variability of HR in the KO mice during rest was twofold higher than that in the WT mice, whereas that in MAP was not significantly different between the two groups; 2) baroreflex sensitivity, determined by the HR response to spontaneous change in MAP, was twofold higher in the KO mice than that in the WT mice; 3) there was no significant difference in the sensitivity determined by intra-arterial administration of the PE or SNP; 4) MAP response to the PE in the KO mice was reduced to one-half that in the WT mice but with no significant difference in the response to SNP; and 5) the higher variability of HR and the higher baroreflex sensitivity in the KO mice disappeared after carotid sinus denervation.

The baroreflex sensitivity was determined from the HR response to spontaneous change in MAP (Table 1) and from induced change in MAP by drug injections (Table 2). Recently, the baroreflex sensitivity was evaluated from the pulse interval response to spontaneous change in arterial pressure in conscious cats (2), humans (6, 7), and anesthetized mice (25). It was determined that the sensitivity from the slope of the pulse intervals and the systolic pressure, based on the findings that changes in three or more consecutive pulse intervals in response to the preceding systolic arterial pressure were positively correlated. In the present study, although we failed to measure precisely the systolic and diastolic pressures because of the damper effect of the measuring system, MAP fluctuated at the frequency of 5-15 cycles/min, followed by an almost similar frequency of fluctuation of HR after a short delay time. When analyzed by a cross-correlation function at 4-s intervals, HR was negatively correlated with MAP during 51% of the total resting period in the WT mice and 69% in the KO mice in the innervated condition. Moreover, after sinus denervation, the percentage period of the negative correlation was markedly diminished in both groups. In addition, the baroreflex sensitivity determined by the spontaneous method in the WT mice agreed well with previously reported findings in mice by the drug-induced method (13) and spontaneous method (25). Thus the baroreflex sensitivity by the spontaneous method in the present study was reliable enough to clarify the difference in the sensitivity between the WT and KO mice.

In the KO mice, MAP was controlled in such a narrow range as in the WT mice, whereas the HR variability was significantly higher than that in the WT mice (Fig. 3 and Table 1), which was adequately explained by the enhanced spontaneous baroreflex sensitivity in the KO mice (Fig. 6B and Table 1). Moreover, in the KO mice, the negative correlation period between HR and MAP was 35% longer than that in the WT mice and the positive correlation period was 50% shorter (Table 1). In addition, all of these differences between the groups disappeared after carotid sinus denervation (Table 1). These results suggest that MAP in the KO mice was more strongly controlled by baroreflexes than that in the WT mice.

The enhanced baroreflex sensitivity in the KO mice may be associated with vascular characteristics due to calponin deficiency in the KO mice. Regarding the molecular aspects of calponin function in the smooth muscle, Winder and Walsh (26) suggested in a reconstituted isolated filament system that calponin inhibits actomyosin ATPase by binding to the actin filament. Takahashi et al. (23) suggested that calponin reduced the shortening velocity in the tonic phase of contraction by regulating of cross-bridge cycling. On the other hand, calponin was reported to undergo an agonist-induced signal transduction in ferret vascular smooth muscle (16, 20). Recently, Je et al. (9) reported that the contractile response to PE was significantly decreased in the ferret aorta after calponin antisense treatment, suggesting that calponin facilitates -adrenergic contractions in tonic smooth muscle. Nigam et al. (18) reported that the calponin-positive rat aorta was slightly but significantly more sensitive in the contractile response to NE compared with that in the calponin-negative rat aorta. In the present study, we confirmed in the KO mice that the vascular response to PE was reduced to 40-63% of that in the WT mice (Fig. 7).

Together, these results suggest that the impaired contraction/relaxation functions in the vascular smooth muscle itself and/or the reduced -adrenergic contractile response to the sympathetic nervous activity makes MAP unstable in the KO mice and that the enhanced baroreflex sensitivity compensates well for the impairments by increasing HR variability.

We hypothesized that baroreceptor denervation in the KO mice would induce higher fluctuations in MAP than that in the WT mice if the baroreflexes in the KO mice compensate for the MAP fluctuation due to impaired contraction/relaxation functions of the vascular smooth muscle itself. Against our hypotheses, however, there was no higher increase in the fluctuation of MAP for the KO mice than that for the WT mice (Table 1). Moreover, we found the vascular response to PE was reduced (Fig. 7) and that MAP in the KO mice did not increase significantly after denervation despite the possible increase in sympathetic nervous outflow, whereas MAP in the WT mice significantly increased (Table 1). These results suggest that the enhanced baroreflex sensitivity in the KO mice was a compensatory adaptation to the blunted -adrenergic response of the vascular smooth muscles to sympathetic nervous activity elicited by baroreflexes rather than by impaired contraction/relaxation functions of the muscle itself (14, 23).

Matthew et al. (14) reported that the amount of actin was reduced by 25-50% in smooth muscle in the same line of KO mice used in the present study. The possibility may not be excluded that the decreased actin filament in the smooth muscle for the KO mice rather than the absence of calponin was responsible for the reduced vascular sensitivity to PE. However, no difference in the response to SNP between the two groups was observed, suggesting that the reduced sensitivity to PE was more associated with -adrenergic signal transduction in the smooth muscle than with decreased actin filament.

As shown in Table 2 and Fig. 8, drug-induced baroreflex sensitivity was not significantly different between the WT and KO mice and not identical to the higher spontaneous baroreflex sensitivity in the KO mice (Table 1). There are no known studies explaining the mechanisms. However, the baroreflex sensitivity may vary in various conditions in free-moving animals by efferent control to the cardiovascular center in the brain stem from the higher central nervous system (21). For example, the baroreflex sensitivity was reported to decrease during exercise (3, 4) and after exercise (5) compared with that before exercise. Recently, Kajeker et al. (10) studied the role of rostral ventrolateral medulla neurons in controlling arterial baroreflex sensitivity and reported that GABA signaling in the brain stem increased after a bout of exercise to decrease arterial baroreflex sensitivity, leading to hypotension. Moreover, microinjection of the NO synthetase inhibitor into this area increased and the NO donor SNP reduced sympathetic outflow, perhaps via an inhibitory effect on sympathoexcitatory neurons in the brain stem (28). These results suggest that the enhanced spontaneous baroreflex sensitivity in the KO mice was caused by central modulation to the cardiovascular center in the brain stem and the enhancement was diminished by the administration of the drugs, as in the present study.

As shown in Table 1, the spontaneous baroreflex sensitivity was one-third and one-half of the drug-induced baroreflex gain in the WT and KO mice, respectively, despite the same amplitude of change in MAP (± ~5 mmHg) from the baseline (Fig. 8 and Table 2). To determine spontaneous baroreflex sensitivity, MAP and HR were analyzed every 4 s, much shorter than the 30 s to determine drug-induced baroreflex sensitivity, a period for HR and/or MAP to return to the baseline after the injections. However, the spontaneous changing rate of MAP was 1-7 mmHg/s, identical to that of drug-induced MAP of 1-8 mmHg/s, suggesting that baroreflex sensitivity in both methods was determined from the HR response similar to the changing rate of MAP. The discrepancy in the baroreflex sensitivity between the two methods may be due to the difference in the regression analyses: a linear regression (spotaneous) and a curvilinear regression (drug induced). In the present study, we found the spontaneous baroreflex sensitivity in the KO mice was twofold higher than that in the WT mice.

The reason for intra-arterial injection of SNP in the present study was because in preliminary experiments in KO mice, HR increased immediately after the intravenous injection of SNP before MAP started to decrease. The precise mechanism for this phenomenon in the KO mice is unclear. However, the high concentration of SNP in circulating blood after a bolus injection into the central vein may modulate baroreflex sensitivity in the brain stem (28) or affect the pacemaker of the heart through direct or indirect effects. Although this immediate response of HR was not observed after intravenous injection of PE, the intra-arterial injection of PE was performed to standardize the procedure.

In summary, the vascular -adrenergic response was impaired in the KO mice. Although there was no significant difference in the drug-induced baroreflex sensitivity between the WT and KO mice, the enhance spontaneous baroreflex sensitivity and the higher frequency of negative correlation between MAP and HR in the KO mice suggested that the baroreflex may work as a compensatory adaptation to the impairment of the vascular -adrenergic response. Thus calponin may play an important role in controlling MAP by increasing the vascular -adrenergic response to sympathetic nervous activity.