In recent years, the mouse has gained tremendous interest for biomedical research because transgenic and gene targeting experiments can be performed with relative ease in mice and because the mouse genome is sequenced. Because of the increasing experimental use of genetically manipulated mice in cardiovascular research (for review see Refs.1,2) it is imperative to expand our knowledge on mouse physiology including the effects of strain and sex, 2 hitherto somewhat neglected aspects.
There are some reports on functional differences between inbred mouse strains in the literature. Platelet function (as measured by the bleeding time after injury) is different between strains of inbred mice.3 Insulin signaling, analyzed by gene expression of insulin receptor and its substrates, is strain dependent.4 Even ventricular cardiomyocyte nuclear number differed greatly between independent, inbred strains of mice.5 The hemodynamic parameters in isolated mouse hearts exhibited strain differences.6 Differences between strains in heart rate and blood pressure were reported.7,8 The transcriptional overexpression of a transgene can be influenced by the genetic background. For instance, DBA/2 mice and C57BL/6 mice exhibited low- and high-level expression, respectively, of a transgene (manganese superoxide dismutase) which was accompanied by a different phenotype.9 Moreover, the genetic background may be important for knock out studies. The severity of the phenotype (early death vs. more normal life span) of a superoxide dismutase was dependent on which strain was used for the gene ablation (129Sv vs. C57BL/6). Strain differences in mice can be regarded as models for polymorphisms or mutation that exist in human populations. These genetic differences between human groups and/or individuals are gaining interest (because of technical progress in automated sequencing reveals more differences that alter gene function) and are expected to change the practice of drug therapy in the years to come.
Sex differences in mice could also be observed. For instance, sex differences in the behavior of mouse lines commonly used in transgenic studies exist.10 Insulin signaling and blood pressure are not only strain dependent as mentioned above, but also sex dependent.8,4 Even in cardiovascular parameters sex differences have been noted. For example, the genetically induced increased expression of the Na+/Ca2+-exchanger resulted in a more marked rise in [Ca2+]i during metabolic inhibition of myocytes from male than from female mice.11
Sex differences are also of clinical relevance. In the last decade, many reports have noted the influence of sex on cardiovascular disease in humans. These include hypertension, coronary heart disease, atrial fibrillation, and cardiomyocyte apoptosis.12-15
However, data are currently lacking whether strain differences or sex differences are relevant for the study of arterial smooth muscle function in mice.
Initially, we hypothesized that no difference due to strain and sex would exist in isolated vascular preparations. Therefore, we investigated the contractile response in isolated arterial vessels (aorta and pulmonary artery) of mice from different strains (CD1, BL6, and DBA) often used in the generation of transgenic mice. We studied both aorta (as typical conductance vessel) and pulmonary artery (as a model of resistance vessel). We tested several pathways of vasoconstriction16 because they might be differently affected by sex and strain. Therefore, the contractile response in isolated vessels due to depolarization by KCl, α1-adrenoceptor stimulation by phenylephrine (PE) and protein phosphatase inhibition by cantharidin (Cant),17 was investigated. The main conclusions of our work are that strain and sex do have an influence on the contractile behavior in isolated vessels from mice.
CD1, C57BL/6 (BL6), and DBA/2 (DBA) mice were purchased from Harlan Winkelmann (Borchen, Germany). Six to 7-month old mice were killed by CO2 gas inhalation. Thoracic aortae and pulmonary arteries were dissected and rinsed in cold phosphate-buffered salt solution (mM: NaCl 137, KCl 13.4, Na2HPO4 8.1, KH2PO4, pH 7.45, 4°C), and loose fat and connective tissue were removed under a light microscope. Size and morphology of the vessel preparations were not different between mouse strains and sex.
Aortic rings, about 2 mm in length, were individually mounted in a dual wire myograph (Model A 410, Danish Myo Technogy A/S, Aarhus, Danmark). Two wires were pulled through the lumen of the aortic segments and mounted according to Mulvany and Halpern.18 Aortic rings were equilibrated in oxygenated physiologic salt solution (PSS) containing (mM) NaCl 118, NaHCO3 25, CaCl2 2.5, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, glucose 5.5, ethylene diamine tetraacetate 0.026 for about 30 minutes at 37°C and subsequently stretched to their optimal lumen diameter for active tension development. This basal tension was determined on the basis of internal circumference-wall tension ratio of the rings by setting their internal circumference to 90% to 100% of what the circumference of the vessels would have been if they were exposed to a passive tension equivalent to a transmural pressure of 100 mm Hg,18 that is, 30 to 35 mN.19,20
Force Measurement-Pulmonary Artery
The experiments were performed with vessels approximately 1.3 mm long when measured between the heart and its first branching point. The passive tension was set to 4 to 7 mN, which is equivalent to a transmural pressure of 25 mm Hg (right ventricular systolic pressure was reported to be ∼15 mm Hg21 or rather ∼31 mm Hg22; transmural pressure 30 mm Hg).23
Vessels were washed with PSS and contracted 3 times by exposure to a high potassium solution (K-PSS) composed of (mM): NaHCO3 25, CaCl2 2.5, KCl 122.7, KH2PO4 1.2, MgSO4 1.2, glucose 5.5 mM, ethylene diamine tetraacetate 0.026 mM. Thus, K-PSS was the same as PSS except that KCl was substituted by NaCl. After washout (PSS) rings were first exposed to increasing concentrations of PE (1 nM to 100 μM; 10 min for each concentration). Subsequently, for relaxation carbachol (1 μM) was added. After washout (PSS) rings were exposed to Cant (dissolved in dimethyl sulfoxide and diluted in PSS; 10 μM for 60min and 100 μM for further 120 min) and changes in force of contraction were recorded for at least 180 minutes. Force was calculated as active wall tension (=increase in vessel wall force about basal tension divided by twice the vessel segment length), that is, mN/mm.18
Cant, PE, and carbachol were obtained from Sigma (Taufkirchen, Germany). All other chemicals used were of analytical or best grade commercially available.
Results are expressed as mean ± standard error of the mean (SEM). Students t test (comparison between sexes of a given strain) or analysis of variance (for multiple comparison) with Bonferroni post-hoc test was used for the statistical analysis of the data. A P value smaller than 0.05 was deemed to be statistically significant. In addition logistic regression analysis and χ2 tests used where indicated.
Effects of KCl, PE, and Cant on Force of Contraction in Intact Vessel Rings of Aorta
We investigated the effects of KCl, PE, and Cant on force of contraction in aorta from male and female DBA, CD1, and BL6 mice. Isolated rings were contracted 3 times by high potassium solution (123 mM KCl: K-PSS). After reaching resting tension again, isolated rings were exposed to increasing concentrations of PE (1 nM to 100 μM). Rings were subsequently relaxed until the basal tension was reached again. Then, rings were challenged with 10 μM Cant for 60 minute and 100 μM Cant for additional 120 minutes. A typical recording is given in Figure 1.
The effect of increasing concentrations of PE in aortic rings from male and female mice are depicted in Figure 2 (A, DBA mice; B, CD1 mice; C, BL6 mice). Data of maximum effects of all experiments with aortae from male and female mice of each strain are summarized in Table 1.
Effects of KCl
In aorta segments from CD1 mice, force induced by KCl was significantly higher in CD1 male compared with BL6 male, DBA male, and DBA female mice. In addition, force was greater in CD1 female and BL6 female compared with DBA male. Thus, great differences in the contractile response due to KCl between different strains exist. For instance, maximum force due to treatment with KCl was about 3 times higher in aortic preparations from CD1 male mice compared with DBA male mice (Table 1). In direct statistical comparison, sex-specific difference in the contractile response due to depolarization by KCl could be observed in CD1 and DBA mice (Table 1).
Effects of PE
The maximum effect of PE was greater in CD1 male compared with CD1 female, BL6 male, BL6 female and DBA female mice. In direct statistical comparison there was a sex difference in CD1 and DBA but not in BL6 mice. Thus, we observed sex and strain-specific differences after α1-adrenoceptor stimulation (Table 1 and Fig. 2).
Effects of Cant
After 60 minutes of exposure to 10 μM Cant, force of contraction was significantly higher in CD1 male compared with DBA male. In addition, force was higher in CD1 female compared with DBA female. Thus, strain-specific differences exists (Table 1). The maximum effect of Cant (100 μM) was significantly greater in CD1 male mice compared with DBA male mice. In addition, force was greater in BL6 female mice compared with DBA female mice. Moreover, in direct comparison more force was generated in CD1 males compared with CD1 females. No difference in sex was observed in BL6 and DBA mice. Thus, we observed strain and sex-specific differences (Table 1).
Effects of KCl, PE, and Cant on Force of Contraction in Intact Vessel Rings of Pulmonary Artery
For comparison, we investigated the effects of KCl, PE, and Cant on force of contraction in pulmonary arteries from male and female DBA, CD1, and BL6 mice. Isolated rings were contracted by high potassium solution (123 mM KCl: K-PSS). After washout and reaching resting tension again isolated rings were exposed to increasing concentrations of PE (PE; 1 nM to 100 μM). Rings were subsequently relaxed until basal tension was reached again. Then, rings were challenged with 10 μM Cant for 60 minutes and 100 μM Cant for additional 120 minutes. The effects of KCl, PE, and Cant in pulmonary arteries from male and female mice of each strain are summarized in Table 2.
Effects of KCl
In pulmonary arteries from CD1 mice, force induced by KCl was significantly higher in CD1 male compared with CD1 female, BL6 male, BL6 female, DBA male, and DBA female mice. In direct statistical comparison, no differences could be found between sexes in BL6 and DBA mice. As in aorta, great differences in the contractile response due to KCl between different strains exist. In addition, a sex-specific difference in the contractile response due to depolarization by KCl could be observed between CD1 male and CD1 female (Table 2).
Effects of PE and Cant
No differences between strains (of same sex) and sex (same strain) could be observed even in direct statistical comparison in preparations of isolated pulmonary arteries (Table 2 and Fig. 3).
Half-maximal Stimulation by PE
We compared the concentration of PE necessary to induce half-maximal contraction (EC50) of aorta and pulmonary artery. In direct comparison, aortic EC50-values were lower for BL6 and DBA female mice than for their male counterparts. In vessel segments of aorta and pulmonary artery from DBA male mice significant differences could be observed as compared with CD1 male mice (Fig. 4). Hence differences between sexes and between strains exist (Fig. 4).
Stimulation of tonic smooth muscle with, for example, α1-adrenoceptor-agonists is often accompanied by phasic contractions making analysis of measurements difficult. Thus, we compared the occurrence of phasic contractions between all groups tested (Table 3). For BL6 male mice, 10 of 11 aorta segments tested showed phasic contractions after stimulation by PE. In contrast, none of the 6 aortae from DBA male mice tested exhibited phasic contractions. Representative original recordings for BL6 male and DBA male mice are depicted in Figure 5. In pulmonary arteries, neither in DBA male nor in BL6 male, phasic contractions occurred. Data on phasic contractions observed during stimulation by 10 μM PE are summarized in Table 3 and were statistically evaluated. Aortae from BL6 and CD1 mice were more likely to exhibit phasic contractions than from DBA mice (according to logistic regression analysis). The same holds true for pulmonary arteries. χ2 values between P = 0.05 and 0.1 were found when sex differences between the incidences in phasic contraction of DBA (aorta), CD1 (pulmonary artery), and BL6 (pulmonary artery) were compared. P values larger than 0.1 were found in comparison between all the other groups studied.
Molecular genetics has an increasing impact on cardiovascular research. The relatively easy manipulation of the mouse genome make the mouse a very attractive model to analyze genetic modifications and their phenotype, for instance, in cardiovascular research. However, the role of sex and strain differences has not yet been thoroughly considered when generating mice which overexpress a gene of interest in arterial smooth muscle or when gene ablation studies are reported.
Human Sex Differences
Sex differences are also clinically relevant, because these differences exist also in the cardiovascular system of humans. To better understand their causes, there will be an increased search for appropriate transgenic mouse model. Examples for sex differences in humans include: the peripheral blood pressure is higher in men than in premenopausal women. This may be due, in part, to the high estrogen levels in premenopausal women (for review see Ref.12). Indeed, estrogen can relax isolated arteries from rabbits and can attenuate KCl dependent vasoconstriction. The mechanism of the relaxation may involve an estrogen-mediated inhibition of currents through L-type Ca2+-channels in smooth muscle cells by estrogen.24 Moreover, not only the mean blood pressure but also plasma norepinephrine and epinephrine levels are higher in men than in women.25 This might indicate that α-adrenoceptor-meditated vasoconstriction in the arteries is higher in men than women. An additional involvement of the vagus in sex differences of the circulatory function is likely. For instance, vagal stimulation in the heart rate is larger in women and indices of ventricular ectopy are less in women compared with men.12 Hence, a number of hypotheses are conceivable which need to be rigorously tested, not only in human subjects but also in more closely controlled transgenic mouse models.
Mouse Sex Difference
Some examples of sex differences in the cardiovascular system of mice have hitherto been reported. For instance, electrical differences between female and male transgenic mice like inducibility of VT was observed.26 Also the remodeling of the ventricles shows sex differences. Male mice but not female mice developed cardiac dilatation in a mouse model of familial hypertrophic cardiomyopathy, that overexpressed a missense mutation of the α myosin heavy chain gene in the heart.27 Here we found sex-specific differences in aorta of CD1 and DBA mice and pulmonary artery of CD1 mice. CD1 male mice always generated more force compared with all other mice preparations studied. The possible histologic and/or biochemical reasons for these findings need to be addressed. Acute effects like different local levels of vasodilatation by estrogens cannot be an explanation, as the vessels were well washed by exchanges of the buffer, before constriction studies were begun. Chronic effects of sexual hormones or estrous cycle dependent protein expression are more likely to be involved. Otherwise, no sex differences were detected in BL6 mice. Therefore, estrogen effects may either be strain dependent or other additional mechanisms are involved.
Mouse Strain Differences
Differences in smooth muscle function have been noted before, such as interstrain differences in the bronchial responsiveness among inbred mouse strains.28 Some inbred mouse strains were more susceptible to airways constriction to methacholine but the myosin content in the trachealis was not different.28 Considerable differences in physiologic cardiovascular parameters, between mouse strains exist. Differences in ECG between mouse strains were noted early on.29 Finally, in intact mice mean heart rate and heart rate variability exhibited strain differences.30 Mouse strain differences in blood pressure (invasively measured) during anesthesia were described. This was explained by interstrain differences in sensitivity toward anesthetic agents and to opioid agents, not intrinsic properties of the vessels.31
The present study was undertaken to compare the contractile response in various mouse strains often used for the generation of transgenic mice or often cross bred into knock mouse strains. For vasoconstriction we used KCl, PE, and Cant which are representative of different, but important, contractile mechanisms, that is, depolarization, α1-adrenoceptor stimulation and protein phosphatase inhibition.17,32
In aorta, force induced by KCl, PE, and Cant was not different within the groups except for DBA male. Strain differences, in the histology of the adventitia (more smooth muscle cell layers in mouse strains that contract more severely) probably do not account for these differences based on work by others.23 Differences in proteins mediating contraction (actin, myosin, kinases, and phosphatases) are a more plausible explanation for the observed differences but were beyond the scope of the present study, which was intentionally functional.
In addition, phasic contractions of aorta due to stimulation with PE were present in all strains and both sexes studied with the notable exception of aorta from DBA male. This phenomenon was accompanied by the finding that only in aortic segments from DBA male mice the EC50 for PE was higher than in the other vessels studied. Thus, DBA male mice seem to be the most useful to study α1-adrenoceptor stimulation of aortic vessels because phasic contraction will not interfere with the interpretation of the data. The mechanism of tonic versus phasic contraction in smooth muscle preparations and isolated cells (intestine preparations, isolated veins and arteries, isolated smooth muscle cells) are still not completely understood. However, intracellular free Ca2+ levels are involved.16 The phasic component of contraction has been attributed to an inositol trisphosphate-mediated release of Ca2+ from the sarcoplasmic reticulum; the tonic component is explained by Ca2+ entry via the sarcolemma or by inhibition of phosphatase activity.17,33 The elevation of cytosolic Ca2+ leads to activation of myosin light chain kinase and phosphorylation of the myosin light chains which finally triggers the interaction of myosin and actin leading to smooth muscle contraction.16 Myosin is dephosphorylated by a phosphatase, which inhibition (for instance, by phosphorylation of a regulatory subunit) can also lead to contraction.34 In addition, differences in the coupling of smooth muscle cells by connexins may contribute to spontaneous and agonist-induced oscillatory activity. This was demonstrated in a rat model with increased gap junction expression in vascular smooth muscle cells.35 It has also been reported that the probability of triggered cytosolic Ca2+ oscillations increased with the coupling of smooth muscle cells.36 Which pathway actually leads to increased phasic contractions in isolated aortae in strain and sex-specific way still needs to be elucidated.
In summary, our data demonstrate strain and sex-dependent differences in the contractile response of aorta and pulmonary artery of various mouse strains. Thus, when generating new transgenic mouse models in smooth muscle research, the genetic background of mice should be considered.
The skillful technical assistance of Barbara Prystaj and Maria Schulik is gratefully acknowledged.
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