Double deletion of calponin 1 and calponin 2 in mice decreases systemic blood pressure with blunted length-tension response of aortic smooth muscle

Abstract

Calponin is a family of actin filament-associated regulatory proteins. Among its three isoforms, calponin 1 is smooth muscle specific and calponin 2 is expressed in smooth muscle and certain non-muscle cells. Previous studies showed that calponin 1 knockout mice had detectable changes in the contractility of urogenital smooth muscle whereas other smooth muscles were less affected. To investigate the possibility that calponins 1 and 2 have overlapping functions in smooth muscle, we examined the effect of double knockout of calponin 1 and calponin 2 genes (Cnn1 and Cnn2) on smooth muscle functions. The results showed for the first time that calponin 1 and calponin 2 double knockout in mice does not cause lethality. The double knockout mice showed decreased systemic blood pressure, decreased force development and blunted length tension response in endothelial-removed aortic rings. A compensatory increase of calponin 1 was found in smooth muscle of Cnn2^-/- mice but not vice versa. Cnn1^-/- and Cnn2^-/- double knockout aortic smooth muscle exhibits faster relaxation than that of wild type control. Double deletion or co-suppression of calponin 1 and calponin 2 in vascular smooth muscle to blunt myogenic response may present a novel approach to develop new treatment for hypertension.

Keywords: Blood pressure; Calponin; Cnn1 and Cnn2 double knockout mice; Myogenic response; Smooth muscle contractility.

Figure 1.. Confirmation of Cnn1^−/− single, Cnn2^−/− single and Cnn1^−/−,Cnn2^−/− double knockout mice.

SDS-PAGE and Western blot using calponin 1-specific mAb CP1, calponin 2-specific mAb 1D11, and anti-calponin 2 polyclonal antibody RAH2 that cross-reacts with calponin 1 showed the expression of both isoforms in wild type (WT) mouse aortic smooth muscle. The deletion of calponin 1 or calponin 2 in the single knockout mouse smooth muscles was confirmed in aorta and bladder samples. Both isoforms became undetectable in Cnn1^−/−,Cnn2^−/− double knockout mouse aortic smooth muscle. It is worth noting that the affinity of RAH2 antibody to its immunogen calponin 2 [12] is higher than the cross reaction to calponin 1 when the same loading of purified mouse calponin 1 and calponin 2 was tested in Western blot. Therefore, the actual level of calponin 1 relative to that of calponin 2 is higher than that seen in the RAH2 Western blots, as shown in the Western blot using anti-calponin 1 mAb CP1 although the level of calponin 1 in aorta is much lower than that in bladder.

Figure 2.. Cnn1^−/−,Cnn2^−/− double knockout mice had decreased blood pressure.

Measurements using tail cuff in conscious mice detected significantly lower systolic, diastolic and mean arterial blood pressures in Cnn1^−/−,Cnn2^−/− double knockout mice as compared with wild type (WT) control while such trends were also seen in Cnn1^−/− and Cnn2^−/− single knockout mice. N = 9 in WT, n = 3 in Cnn1^−/− single, n = 5 in Cnn2^−/− single, and n = 5 in Cnn1^−/−,Cnn2^−/− double knockout groups. Values are presented as Mean ± SEM. *P<0.05 and **P<0.01 vs. WT; #P<0.05 vs. Cnn1^−/− single knockout in Student’s t test.

Figure 3.. Decreased contractile force of Cnn1^−/−,Cnn2^−/− double knockout mouse aortic smooth muscle with no change in the sensitivity to norepinephrine.

(A) Ex vivo contractility showed that the norepinephrine-activated, vessel size-normalized maximum tension was lower in Cnn1^−/−,Cnn2^−/− double knockout aortic rings than that of wild type (WT) control. (B) Activation curves normalized to the maximum tension showed nearly identical sensitivities of Cnn1^−/−,Cnn2−/− double knockout aortic rings and WT control. N = 4 mice in WT and 5 mice in Cnn1^−/−,Cnn2^−/− groups. Values are presented as Mean ± SEM. ***P<0.001 in two-way ANOVA.

Figure 4.. Faster relaxation of Cnn1^−/−,Cnn2^−/− double knockout mouse aortic rings.

(A) Representative relaxation curves of aortic rings of Cnn1^−/−,Cnn2^−/− double knockout mice showed a faster relaxation than that of wild type (WT) control after replacing the 50 mM KCl buffer with normal Krebs solution. (B) Quantitative analysis of relaxation time showed significantly shorter TR75 and TR90 in Cnn1^−/−,Cnn2^−/− double knockout aortic ring than that of WT control, indicating increased later phase relaxation. N = 4 mice in WT and 5 mice in Cnn1^−/−,Cnn2^−/− double knockout groups. Values are presented as Mean ± SEM. ***P<0.001 in Student’s t test.

Figure 5.. Cnn1 single knockout produced lower contractile force and faster relaxation in mouse aortic smooth muscle.

(A) Cnn1^−/− single but not Cnn2^−/− single knockout mouse aortic rings produced lower maximum force than wild type (WT) control, similar to that of Cnn1^−/−,Cnn2^−/− double knockout aortae. (B) Analysis of relaxation time parameters showed that Cnn1^−/− single knockout mouse aortic rings produced shorter TR50, TR75 and TR90 than WT control, similar to the effect of Cnn1^−/−,Cnn2^−/− double knockout. No change in relaxation time was found for Cnn2^−/− single knockout aortae except a longer TR25 indicating slower early relaxation. Values are presented as Mean ± SEM. N = 10 mice in WT, 5 mice in Cnn1^−/−,Cnn2^−/− double knockout, 5 mice in Cnn1^−/− single knockout and 5 mice in Cnn2^−/− single knockout groups. * P<0.05 vs. WT; #P<0.05 vs. Cnn1^−/−,Cnn2^−/− double knockout; ^&P<0.05 vs. Cnn1^−/− single knockout in Student’s t test.

Figure 6.. Decreases of myofilament contents in aorta of Cnn1^−/− single but not Cnn2^−/− single or Cnn1^−/−,Cnn2^−/− double knockout mouse aortae.

(A) Representative SDS-PAGE and Western blots using anti-SMA mAb of aortae of wild type (WT), Cnn1^−/− single, Cnn2^−/− single and Cnn1^−/−,Cnn2^−/− double knockout mice. (B) Densitometry quantification of SDS-gel bands showed no change in the level of MHC vs. total muscle protein in the calponin knockout groups as compared to WT control. (C) Normalized to total protein, SMA was decreased in the aortae of Cnn1^−/− single knockout, but not Cnn2^−/− single or Cnn1−/−,Cnn2−/− double knockout mouse aortae. Values are presented as Mean ± SEM. N = 7 in WT vs. n=3 in Cnn2^−/− groups and n = 5 mice in Cnn1^−/− group. N=4 in WT vs. n=4 in Cnn1^−/−,Cnn2^−/− group. *P<0.05 vs. WT in Student’s t test.

Figure 7.. Resting tension and length-dependence of active tension development of mouse aortic smooth muscle.

(A) The resting tension-muscle length relationship were similar in the calponin knockout and wild type (WT) mouse aortic rings. (B) The active tension-muscle length relationship derived from total tension subtracting resting tension showed lower maximum force development in Cnn1^−/− single and Cnn1^−/−,Cnn2^−/− double knockout aortic smooth muscle in which the double knockout group had blunted responses to the increase in muscle length. N = 11 mice in WT, 5 mice in Cnn1^−/−, 5 mice in Cnn2^−/− and 4 mice in Cnn1^−/−,Cnn2^−/− double knockout groups. Values are presented as Mean ± SEM. ***P<0.001 in Cnn1^−/− vs. WT. #P<0.05 in Cnn1^−/−,Cnn2^−/− double knockout vs. WT. Statistics was performed by two-way ANOVA with Bonferroni test for mean comparisons.

Figure 8.. Compensatory increase of calponin 1 in Cnn2^−/− mouse aortic smooth muscle.

(A) SDS-gel/Western blot and densitometry quantification normalized to actin or MHC showed significantly increased level of calponin 1 in Cnn2^−/− mouse aortic smooth muscle. (B) No significant increase of calponin 2 was found in Cnn1^−/− mouse aortic smooth muscle. Values are presented as Mean ± SEM. N = 4 mice each in WT and Cnn2^−/− groups, n = 3 mice in Cnn1^−/− group. *P<0.05 and **P<0.01 vs. WT in Student’s t test.

Figure 9.. Compensations between calponin 1 and calponin 2 in urinary bladder and large intestine smooth muscles of Cnn1^−/− and Cnn1^−/− single knockout mice.

(A) The ratios between calponin 1 and calponin 2 determined by densitometry quantification of SDS-gel and Western blot in aortic, bladder and large intestinal smooth muscles of wild type (WT) showed significant differences. (B) SDS-gel, Western blot and densitometry quantification of bladder smooth muscle showed a significant increase of calponin 1 in Cnn2^−/− mice as compared to WT control. (C) Large intestinal smooth muscle of Cnn2^−/− mice also showed a significant upregulation of calponin 1 as compared to that of WT control. A trend of calponin 2 increases was seen in Cnn1^−/− mouse bladder and large intestine but statistical significance was not established. Values are presented as Mean ± SEM. N = 3 mice each in WT, Cnn1^−/− and Cnn2^−/− groups. **P<0.01; ***P<0.001 vs. WT in Student’s t test.