Published online before print September 27, 2007, doi: 10.1161/CIRCRESAHA.107.154351
© 2007 American Heart Association, Inc.
Molecular Medicine |
Rescue of cGMP Kinase I Knockout Mice by Smooth Muscle–Specific Expression of Either Isozyme
From the Institut für Pharmakologie und Toxikologie der Technischen Universität (S.W., D.B., R.L., P.W., R.W., J.W.W., J.S., F.H.), München; Interfakultäres Institut für Biochemie (N.V., S.F., R.F.), Universität Tübingen, Germany. J.S. is currently at the Institut für Pharmakologie und Toxikologie, Universität Regensburg, Germany.
Correspondence to Dr Franz Hofmann, Institut für Pharmakologie und Toxikologie der Technischen Universität, Biedersteiner Str. 29, D-80802 München, Germany. E-mail hofmann{at}ipt.med.tu-muenchen.de
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Abstract |
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Smooth muscle expresses the I
and the Iß isoforms of cGMP-dependent protein kinase I (cGKI). Inactivation of the murine cGKI gene prkg1 leads to multiple phenotypes and premature death at 
6 weeks. We reconstituted mice with the cGKI or -Iß isozyme to test which isozyme was needed to support basic smooth muscle functions. Mice were generated by gene targeting. The cGKI or the -Iß coding sequences were placed under the control of the SM22 promoter to express either isoform selectively in smooth muscle cells (SM-I or SM-Iß transgene). To generate smooth muscle–specific cGKI or cGKIß rescue mice, the SM-I or SM-Iß transgenes were crossed on a cGKI–/– genetic background. The levels of cGKI or -Iß expression were comparable to endogenous cGKI expression in wild-type aortic and intestinal smooth muscles. In cGKI or -Iß rescue mice, expression of the isozymes was not detectable in non–smooth muscle tissues and cells. Median survival time of the I and Iß rescue mice was 52 weeks. Both isozymes mediated the 8-bromo-cGMP–induced relaxation of precontracted jejunum and aorta muscle strips. Activation of both isozymes reduced hormone- or K+-induced [Ca2+]i levels. The cGKI and cGKIß rescue mice did not show a significant difference in intestinal passage time of BaSO4 in comparison with wild-type animals. Telemetric blood pressure measurements in conscious freely moving animals did not show differences between rescues and control mice in basal blood pressure and its regulation by DETA-NO, sodium nitroprusside, carbachol, or Y-27632. These results show that cGKI in smooth muscle is essential and that either cGKI isozyme alone can rescue basic vascular and intestinal smooth muscle functions.
Key Words: cGMP kinase isozymes • PKG • nitric oxide • smooth muscle • blood pressure
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Introduction |
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The NO/cGMP signaling cascade plays an essential role in vascular smooth muscle (SM) relaxation, and clinical studies indicate that endothelium-derived NO is involved in normal and pathological blood pressure regulation in humans.1–3 The important effector of cGMP, cGMP-dependent protein kinase I (cGKI), is highly expressed in SM.4 Conventional deletion of the gene for cGKI in mice leads to multiple phenotypes, including severe gastrointestinal disturbances and elevated blood pressure, leading to premature death of the animals.5
The cGKI gene generates 2 isoforms, cGKI and cGKIß, that differ only in their individual N termini (the first 90 to 100 residues), which are encoded by 2 alternatively used exons.6,7 Both isoforms are expressed together in various SMs.8,9 Strong evidence has been published that these isozymes interact with different proteins and affect SM relaxation through different mechanisms.10–13 cGKI interacts specifically with MYPT1 (myosin-interacting subunit of myosin phosphatase 1)12 and with RGS-2 (regulator of G protein signaling 2),13 whereas cGKIß shows specificity for inositol 1',4',5'-triphosphate receptor–associated G kinase substrate (IRAG).10,11 However, in vitro data from Feil et al14 and Christensen et al15 have suggested that only the I isozyme is able to restore the ability of NO/cGMP to inhibit hormone-triggered increases of the intracellular Ca2+ concentration ([Ca2+]i). In contrast, the analysis of freshly isolated SM cells and SM tissue from IRAG mouse mutants has led Geiselhöringer et al16 to the conclusion that the cGKIß/IRAG pathway is essential for cGMP-mediated regulation of [Ca2+]i and SM relaxation.
Although the exact reason for this discrepancy is not clear, each of these studies has its limitations. For example, some of the experiments supporting a role of cGKI but not cGKIß were performed with constructs encoding cGKI– or cGKIß–green fluorescent protein fusion proteins. Although both fusion proteins displayed cGMP-dependent kinase activity in vitro,14 it cannot be excluded that the fusion partner disturbed the interaction of cGKIß with its anchoring and/or substrate proteins. On the other hand, the study supporting an exclusive role for cGKIß was performed with transgenic mice expressing a mutated IRAG protein that might produce unknown side effects, and the function of cGKIß was not directly tested.16 Another potentially confounding factor could be the use of cultured SM cells. It is well known that SM cells in culture switch from the contractile to the synthetic phenotype,17 which might affect functional studies, because some of cGKI targets could be downregulated. Because some of the abovementioned studies used cultured SM cells, whereas others used acutely isolated SM cells, it is possible, although unlikely, that their different outcomes were related to different phenotypic states of the cultured cells.
To directly analyze the ability of cGKI and cGKIß to affect SM function in vivo, each isozyme was expressed separately and selectively in its native SM environment using conventional cGKI knockout animals as genetic background. Interestingly, individual SM-specific expression of the cGKI as well as the cGKIß isozyme was sufficient to support basic regulatory functions of the NO/cGMP signaling cascade.
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Materials and Methods |
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Generation of SM-I
Rescue and SM-Iß Rescue MiceThe strategy to express cGKI
or cGKIß selectively in SM cells was analogous to a previous approach for SM-specific expression of a tamoxifen-activated Cre recombinase.18 Briefly, the cDNA of bovine cGKI or cGKIß,6 a simian virus (SV40) polyadenylation signal, and a phosphoglycerate kinase promoter-driven neomycin resistance gene cassette were integrated into the murine SM22 gene locus (chromosome 9) by homologous recombination in embryonic stem cells (Figure I in the online data supplement at http://circres.ahajournals.org). The targeting vectors, pSM-I or pSM-Iß, contained the cGKI- or cGKIß-encoding sequence fused in frame to the translation start codon of the SM22 gene, resulting in a mutation of serine 2 of the cGKI to an arginine and mutation of glycine 2 of the cGKIß to an arginine. The AscI-linearized targeting vectors were electroporated into R1 embryonic stem cells (derived from a 129/Sv genetic background), and G418/ganciclovir-resistant clones were screened for correct integrants by PCR (using primers QY83 and RF85) and Southern blot analysis according to published procedures.18 Positive clones were injected into C57BL/6 blastocysts to generate chimeras that transmitted the modified SM22 alleles to the germ line. The corresponding mouse lines, designated SM-I and SM-Iß (genotype: SM22+/I or SM22+/Iß), were bred with heterozygous cGKI knockout mice (genotype: cGKI+/L–)19 to generate "rescue" mice that carried the SM-I or SM-Iß allele on a cGKI-null background. The rescue mice were designated SM-I rescue or SM-Iß rescue (genotype: SM22+/I; cGKIL–/L– or SM22+/Iß; cGKIL–/L–). Genotypes of mice were determined by PCR analysis of tail DNA. The SM22 wild-type, SM-I, and SM-Iß alleles were detected using the following primers: RF67 (5'-CTCAGAGTGGAAGGCCTGCTT-3'); SW16 (5'-CGCAAGGGTTACTCACCACA-3'); SW12 (5'-CCTCCTTGAGCATGAGAATCTTG-3'); SW8 (5'-AACTCCAGCTCCAGCTCG-3'). RF67 and SW16 amplify a 292-bp fragment of the SM22 wild-type allele; RF67 and SW12 amplify a 150-bp fragment of the SM-I allele; RF67 and SW8 amplify a 195-bp fragment of the SM-Iß allele. The cGKI wild-type (+) and knockout (L–) alleles were detected as described.19
Experimental Animals
SM-I rescue and SM-Iß rescue mice on a 129/Sv genetic background (genotype: SM22+/I; cGKIL–/L– or SM22+/Iß; cGKIL–/L–) were compared with litter matched control mice (genotype: SM22+/+; cGKI+/L– or SM22+/+; cGKI+/+). For blood pressure measurements, male rescue and litter-matched control animals were analyzed at an age of 10 to 20 weeks. For measurements of Ca2+ transients and contraction experiments litter-matched male and female mice were used with an age >10 weeks. The cGKI knockout mice (genotype: cGKIL–/L–) had a 129/Sv background and were analyzed at an age of 4 to 6 weeks. Experiments were approved by the local governmental committee on animal care and welfare in Munich. Following breeding scheme was used. Crossing heterozygous cGKI (genotype: SM22+/I; cGKI+/L–) with heterozygous cGKI+/– (genotype: SM22+/+; cGKI+/L–) always yields SM22-I (SM22+/Ia; cGKIL–/L–), cGKI–/– (SM22+/+, cGKIL–/L–), heterozygous SM22-I (SM22+/Ia; cGKI+/L–), and wild type ([SM22+/+; cGKI+/L–] or [SM22+/+; cGKI+/+]). The same breeding scheme was used for the SM-22Iß mouse line. For additional information, see the online data supplement.
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Results |
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To express the cGKI
or cGKIß isoform selectively in SM, homologous recombination in embryonic stem cells was used to "knock in" the cDNA sequences of the bovine cGKI or cGKIß directly into the start ATG of the endogenous SM22 gene (supplemental Figure I), which is expressed exclusively in SM cells of adult mice.20,21 The resulting mouse lines, designated SM-I (genotype: SM22+/I) and SM-Iß (genotype: SM22+/Iß), were crossed to mice carrying a cGKI-null allele (cGKI+/L–)19 to generate rescue mice that express either the SM-I or the SM-Iß"knock in" allele on a cGKI-null background. The rescue mice were designated SM-I rescue (genotype: SM22+/I; cGKIL–/L–) or SM-Iß rescue (genotype: SM22+/Iß; cGKIL–/L–) and were compared with littermate controls (genotype: [SM22+/+; cGKI+/L–] or [SM22+/+; cGKI+/+]). SM-I and SM-Iß rescue mice were viable and had a greatly improved general health status and body size compared with cGKI-null mutants. Males and females of both rescue lines were fertile, but females had a slightly reduced litter size (3 to 6 pups) and a reduced success rate of pregnancy as compared with control mice. As anticipated, the rescue mice expressed the correctly spliced mRNA as well as the protein of the respective cGKI isozyme exclusively in SM (Figure 1). Western blot analysis with an antibody detecting both cGKI isoforms (cGKI common antibody) as well as with isoform-specific antibodies demonstrated that expression of cGKI or cGKIß was restored in SM tissues but not in the brain of the rescue mice (Figure 1A). Immunohistochemistry indicated that isozyme expression was restored in vascular and visceral SM cells but not in chondrocytes or megakaryocytes (Figure 1B and 1C). Further analyses confirmed the absence of cGKI in non-SM cells of the rescue mice, such as blood cells and cardiomyocytes, which normally express the enzyme (data not shown). Western blots and histological sections of aorta and duodenum indicated that the rescue mouse lines expressed the respective isozyme at approximately physiological concentrations in these tissues (Figure 1A and 1C). Semiquantitative analysis of Western blots from adult mice showed that the expression level of the isozymes in aorta, jejunum, and colon was very similar to each other and <2-fold greater than that of wild-type tissues (supplemental Figure II). The determination of protein kinase activity in aortic extracts in the presence of 10 µmol/L cGMP supported this conclusion (activities in pmol/minxmg protein: 52±6.4 [control], 46±9.6 [SM-Iß rescues], and 114±16 [SM-I rescues]; n=3 to 4 for each genotype). The expression of several known substrates of the cGKI isozymes, such as MYPT1, IRAG, RhoA, and RGS-2, was not altered in the aorta of rescue mice as compared with control or cGKI-null mice (supplemental Figure IIIA and IIIB). Taken together, these results indicated that adult SM-I and SM-Iß rescue mice express the respective isoform exclusively in vascular and visceral SM but not in non-SM cells. The cGKI isoforms as well as all tested cGKI substrates are expressed at approximately physiological concentrations.
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Both the SM-I and SM-Iß rescue mice had a greatly extended life expectancy in comparison with cGKI-null mutants. Median survival time of animals of both rescue lines was 52 weeks, whereas, as expected,5 50% of the conventional cGKI knockout mice died within the first 6 weeks after birth (Figure 2). Factors contributing to the relative longevity of the rescue mice might be the improved intestinal properties, as indicated by a normal intestinal passage time (Figure 3), and normal relaxation of carbachol contracted jejunum strips (Figure 4A), as well as normal relaxation of precontracted aortic strips (Figure 4B). Interestingly, the IC50 for 8-bromo-cGMP (8-Br-cGMP) was not significantly different for SM strips of wild-type or SM-I or SM-Iß rescue mice (jejunum: wild type, 8.3 µmol/L; cGKI, 5.0 µmol/L; cGKIß, 9.0 µmol/L; aorta: wild type, 0.37 mmol/L; cGKI, 0.1 mmol/L; cGKIß, 0.4 mmol/L). Activation of cGKI or cGKIß reduced norepinephrine-induced increases in [Ca2+]i to the same extent as activation of cGKI in wild-type cells, whereas no reduction was observed in cGKI–/– cells (Figure 5C). Identical results were obtained when increases in [Ca2+]i were elicited by potassium-induced depolarization (Figure 5D).
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The above results suggested that expression of either cGKI isozyme in SM cells of cGKI knockout mice was sufficient to restore the basic regulation of SM function to the wild-type level. To validate this hypothesis in a more physiological context, an extensive analysis of blood pressure regulation in awake, freely moving animals was conducted using a telemetric recording system. Mean arterial blood pressure (MAP) and pulse pressure values, which depend on cardiac output and aortic elasticity, did not differ significantly between adult (10- to 20-week-old) control, SM-I rescue, and SM-Iß rescue mice, regardless of whether measurements were taken continuously for 24 hours (Figure 6) or only during the resting (day) or activity (night) period of the animals (supplemental Figure IV). Similar results were obtained when juvenile (3- to 6-week-old) mice were studied by the same technique as used for the adult. As expected,5 MAP was elevated in cGKI–/– mice as compared with wild-type and the SM-I and SM-Iß mice (supplemental Figure V). Identical results were obtained in conscious and unconscious mice (supplemental Figure VA and VB). Blood pressure was not determined in older cGKI–/– mice, because it was already reported that these animals have "normal" MAP, most likely resulting from their severe phenotype.5
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The notion that blood pressure regulation is normal in SM-I and SM-Iß rescue mice is further supported by the finding that the acute blood pressure–lowering effect of NO-releasing drugs that is lost in cGKI knockout mice (see elsewhere5,22 and supplemental Figure VC and VD) was efficiently restored in juvenile animals of both rescue lines but not in the cGKI–/– mice (Figure VC and VD). Similarly, intraperitoneal injection of adult animals with diethylenetriamine NONOate (DETA/NO) or sodium nitroprusside induced comparable pressure drops in control, SM-I rescue, and SM-Iß rescue mice (Figure 7). Additional experiments showed that injection of carbachol or the Rho kinase inhibitor Y-27632 reduced MAP in control, SM-I rescue, and SM-Iß rescue mice to the same extent (supplemental Figure VI).
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Discussion |
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Transcription of the cGKI gene generates 2 isozymes that differ in their first 100 amino acids.6,7 Both isozymes are expressed in various SMs.8,9 Extensive work has shown that the amino terminus of the I
isozyme interacts specifically with MYPT112 and RGS-2,13 whereas the amino terminus of the Iß isozyme interacts with IRAG,10,11 a protein expressed in SM cells and platelets, closely associated with 1',4',5'-triphosphate receptor 1.11,23 The significance of cGKIß for the regulation of SM tone was questioned because only the reintroduction of the cGKI isozyme into cGKI–/– SM cells was able to reconstitute the 8-Br-cGMP–mediated reduction of norepinephrine-stimulated [Ca2+]i transients.14 Furthermore, thrombin-increased [Ca2+]i levels were reduced more efficiently by the I than the Iß isozyme in CHO cells.15 Similar results were reported for the coronary SM cell line Co403.15 However, both groups did not report whether or not the used cells expressed the respective target proteins.
The present report shows quite convincingly that both cGKI isozymes can compensate for each other in the regulation of vascular tone in the awake mouse. This notion is based on several findings. cGKI and cGKIß were expressed at roughly "physiological" levels in all SMs analyzed. Both rescue lines showed identical regulation of intestinal and vascular SM functions in vitro and in vivo. Extensive studies on blood pressure regulation did not reveal any difference among wild-type, cGKI rescue, and cGKIß rescue mice.
These results indicate that the functional outcome of either isoform-dependent signaling in SM is similar regardless of the specific cGKI targets involved. The basically identical phenotype could be caused by an unlikely but possible alternative mechanism. The target specificity of the cGKI isozymes could be much lower in vivo than determined predominantly by in vitro methods. Each isoform might localize to the different subcellular compartments to perform the respective functions of the other isoform. An example supporting this notion is shown in supplemental Figure VII. Vasodilator-stimulated phosphoprotein (VASP), a known substrate for cGKI,24 is phosphorylated by each isozyme if high enough concentrations of 8-Br-cGMP are provided. The difference in the 8-Br-cGMP concentration required in SM-I and SM-Iß rescue cells for half-maximally shifting VASP to phospho-VASP may be caused by the distinct activation constants for cGMP of cGKI and cGKIß,25,26 by differences in the substrate affinity, by differences in the kinetics of the phosphorylation sites of VASP, and by differences in the association of each isozyme with its substrate. Thus, we cannot exclude completely the possibility that each isozyme can replace the other isozyme, if expressed alone in vivo. Certainly, a substantial reconsideration of the in vivo target specificity needs further evidence.
However, the functional equivalence of both isozymes can be reconciled easily with the known regulatory pathways, if we assume that each pathway is sufficient to mediate basic NO/cGMP regulation of SM tone. It is likely that cGKI lowers vascular tone by increasing myosin phosphatase activity27–29 and by increasing the inactivation of phospholipases C through increased hydrolysis of GqGTP to GqGDP.13,30,31 Phosphorylation of IRAG by cGKIß decreases the release of Ca2+ from the 1',4',5'-triphosphate stores and thereby may reduce very efficiently cytosolic increases in [Ca2+]i and SM tone.16
The results presented in this report were obtained in the intact animal or with tissues and cells isolated from the rescue mice. The respective cGKI isozyme was expressed during embryogenesis and postnatal development. This distinguishes the SM cells that were studied in the present report from the cultured and transfected cGKI–/– cells used previously to test the effects of each isozyme.14 It is well known that cultured SM cells lose their contractile response because they switch to the synthetic phenotype. Recently, it was shown that murine aortic SM cells lose some cGMP-induced effects already after the first passage.32 The previous experiments of Feil et al14 were performed after the first passage of the SM cells. The potentially rapid change of the phenotype of the cultured cells has not been appreciated previously. It is therefore likely that previous experiments with cultured cells14,33,34 did not reproduce accurately the situation prevailing in an intact animal.
In conclusion, the present work shows that both isozymes of cGKI contribute significantly to the regulation of SM tone by NO and cGMP. It is not surprising that each cGKI isozyme can function as a backup system for the other isozyme because they mediate an important signaling pathway, the NO/cGMP-dependent regulation of vascular tone.
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Acknowledgments |
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We thank Sabine Brummer for excellent support.
Sources of Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. R.L. and D.B. were fellows of the GRK333 and GRK438, respectively.
Disclosures
None.
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Footnotes |
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Original received April 16, 2007; revision received September 4, 2007; accepted September 13, 2007.
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[Abstract/Free Full Text]
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