Pressure-independent enhancement of cardiac hypertrophy in natriuretic peptide receptor A-deficient mice.

Mice lacking natriuretic peptide receptor A (NPRA) have marked cardiac hypertrophy and chamber dilatation disproportionate to their increased blood pressure (BP), suggesting, in support of previous in vitro data, that the NPRA system moderates the cardiac response to hypertrophic stimuli. Here, we have followed the changes in cardiac function in response to altered mechanical load on the heart of NPRA-null mice (Npr1-/-). Chronic treatment with either enalapril, furosemide, hydralazine, or losartan were all effective in reducing and maintaining BP at normal levels without affecting heart weight/body weight. In the reverse direction, we used transverse aortic constriction (TAC) to induce pressure overload. In the Npr1-/- mice, TAC resulted in a 15-fold increase in atrial natriuretic peptide (ANP) expression, a 55% increase in left ventricular weight/body weight (LV/BW), dilatation of the LV, and significant decline in cardiac function. In contrast, banded Npr1+/+ mice showed only a threefold increase in ANP expression, an 11% increase in LV/BW, a 0.2 mm decrease in LV end diastolic dimension, and no change in fractional shortening. The activation of mitogen-activated protein kinases that occurs in response to TAC did not differ in the Npr1+/+ and Npr1-/- mice. Taken together, these results suggest that the NPRA system has direct antihypertrophic actions in the heart, independent of its role in BP control.

Angiotensin-converting enzyme and male fertility.

The angiotensin-converting enzyme (ACE; EC 3.4.15.1) gene (Ace) encodes both a somatic isozyme found in blood and several other tissues, including the epididymis, and a testis-specific isozyme (testis ACE) found only in developing spermatids and mature sperm. We recently used gene targeting to disrupt the gene coding for both ACE isozymes in mice and reported that male homozygous mutants mate normally but have reduced fertility; the mutant females are fertile. Here we explore the male fertility defect. We demonstrate that ACE is important for achieving in vivo fertilization and that sperm from mice lacking both ACE isozymes show defects in transport within the oviducts and in binding to zonae pellucidae. Males generated by gene targeting that lack somatic ACE but retain testis ACE are normally fertile, establishing that somatic ACE in males is not essential for their fertility. Furthermore, male and female mice lacking angiotensinogen have normal fertility, indicating that angiotensin I is not a necessary substrate for testis ACE. Males heterozygous for the mutation inactivating both ACE isozymes sire wild-type and heterozygous offspring at an indistinguishable frequency, indicating no selection against sperm carrying the mutation.

Intraocular pressure in inbred mouse strains.

PURPOSE: To develop a protocol to measure the intraocular pressure (IOP) of living mice and to determine the IOP of genetically different mouse strains. METHODS: Eyes of anesthetized animals were cannulated with a very fine fluid-filled glass microneedle. The microneedle was connected to a pressure transducer, and the pressure signal was analyzed with a computer system. Intraocular pressures of male C3H/He iota, C57BL/ 6 iota, A/iota, and BALB/c iota mice were determined. RESULTS: Differences in IOP were detected between genetically distinct mouse strains maintained in virtually identical environments. C3H/He iota was the strain with the highest average IOP (13.7 +/- 0.8 mm Hg). This strain average was 1.4 mm Hg higher than that for C57BL/6 iota (12.3 +/- 0.5 mm Hg; P = 0.14), 4.3 mm Hg higher than that for A/iota (9.4 +/- 0.5 mm Hg; P < 0.001), and 6 mm Hg higher than that for BALB/c iota (7.7 +/- 0.5 mm Hg; P < 0.001). CONCLUSIONS: The authors have developed an accurate and reliable procedure for measuring intraocular pressure in living mice. This procedure can detect IOP differences between groups of mice that differ by genotype.

Single-copy transgenic mice with chosen-site integration.

We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters. In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells. In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter.

Male-female differences in fertility and blood pressure in ACE-deficient mice.

Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxy-peptidase that generates the vasoconstricting peptide angiotensin II and inactivates the vasodilating peptide bradykinin. The gene encoding ACE is composed of two homologous regions and codes for both a somatic and testis isoenzyme. Experiments with hypertensive rats and some, but not other, studies of humans suggest that sequences at or linked to the gene influence blood pressure. The testis-specific form of ACE has its own promoter within intron 12 (ref. 14), is encoded by the 3' region of the gene, and is found only in postmeiotic spermatogenic cells and sperm. Its function is unknown. Here we investigate the role of the Ace gene in blood pressure control and reproduction using mice generated to carry an insertional mutation that is designed to inactivate both forms of ACE. All homozygous female mutants were found to be fertile, but the fertility of homozygous male mutants was greatly reduced. Heterozygous males but not females had blood pressures that were 15-20 mm Hg less than normal, although both male and female heterozygotes had reduced serum ACE activity.

A noninvasive computerized tail-cuff system for measuring blood pressure in mice.

We have validated a noninvasive computerized tail-cuff system for measuring blood pressure in mice. The system was designed to perform all functions automatically, including a programmable routine of cuff inflation and deflation, analysis and assignment of pulse rate and blood pressure, and recording of data electronically. To evaluate this system over a range of blood pressures, we gave groups of mice enalapril or NG-nitro-L-arginine methyl ester in their drinking water. For each of these groups, an equal number of control mice were given nothing in their drinking water. Tail-cuff blood pressures were recorded as the means of blood pressures determined on at least 3 days after at least 7 days of training. Tail-cuff enalapril and control group means were measured both 3 and 4 months after enalapril (or no drug) was begun; the group means at 3 months were not significantly different from the group means at 4 months. These results demonstrate that the system gives reproducible results. After the tail-cuff measurements were completed, intra-arterial blood pressures were attempted in all mice under unrestrained, unanesthetized conditions, and individual mouse (n = 22) blood pressures with the use of the two methods were compared. The blood pressures from individual mice by tail-cuff and intra-arterial methods were highly correlated (r = .86, P < .01). The means for the four mouse groups were also highly correlated (r = .98, P < .02).(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic control of blood pressure and the angiotensinogen locus.

Variants of the human angiotensinogen gene have been linked in some studies to increased circulating angiotensinogen levels and essential hypertension. To test for direct causality between genotypes at the angiotensinogen locus and blood pressures, we have studied mice carrying zero, one, two, three, or four functional copies of the murine wild-type angiotensinogen gene (Agt) at its normal chromosomal location. Plasma angiotensinogen levels increase progressively, although not linearly, from zero in the zero-copy animals to 145% of normal in the four-copy animals. Mice of all genotypes are normal at birth, but most zero-copy animals die before weaning. The kidneys of the zero-copy animals show pathological changes as adults, but the kidneys are normal in the other genotypes. One adult zero-copy male tested was fertile. The blood pressures of the one-copy through four-copy animals show significant and almost linear increases of approximately 8 mmHg per gene copy despite their normal compensatory mechanisms being intact. These results establish a direct causal relationship between Agt genotypes and blood pressures.

Genetic decreases in atrial natriuretic peptide and salt-sensitive hypertension.

To determine if defects in the atrial natriuretic peptide (ANP) system can cause hypertension, mice were generated with a disruption of the proANP gene. Homozygous mutants had no circulating or atrial ANP, and their blood pressures were elevated by 8 to 23 millimeters of mercury when they were fed standard (0.5 percent sodium chloride) and intermediate (2 percent sodium chloride) salt diets. On standard salt diets, heterozygotes had normal amounts of circulating ANP and normal blood pressures. However, on high (8 percent sodium chloride) salt diets they were hypertensive, with blood pressures elevated by 27 millimeters of mercury. These results demonstrate that genetically reduced production of ANP can lead to salt-sensitive hypertension.

Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.

We have inactivated the endogenous apolipoprotein E (apoE) gene by using gene targeting in mouse embryonic stem (ES) cells. Two targeting plasmids were used, pJPB63 and pNMC109, both containing a neomycin-resistance gene that replaces a part of the apoE gene and disrupts its structure. ES cell colonies targeted after electroporation with plasmid pJPB63 were identified by the polymerase chain reaction (PCR) followed by genomic Southern analysis. Of 648 G418-resistant colonies analyzed, 9 gave a positive signal after PCR amplification, and 5 of them were confirmed as targeted by Southern blot analysis. The second plasmid, pNMC109, contains the negatively selectable thymidine kinase gene in addition to the neomycin-resistance gene. After electroporation with this plasmid, 177 colonies resistant both to G418 and ganciclovir were analyzed; 39 contained a disrupted apoE gene as determined by Southern blotting. Chimeric mice were generated by blastocyst injection with 6 of the targeted lines. One of the lines gave strong chimeras, three of which transmitted the disrupted apoE gene to their progeny. Mice homozygous for the disrupted gene were produced from the heterozygotes; they appear healthy, even though they have no apolipoprotein E in their plasma.

A scanning electron microscopic and photon absorptiometric study of the development, prolongation, and pattern of recovery from lactation-induced osteopenia in rats.

Measurements by scanning electron microscopy (SEM) of femoral hemisections confirmed and amplified results by single-photon absorptiometry that had shown a marked increase in lactation osteopenia in rats fed a low-calcium diet (LCD, 0.04% Ca) as compared with a medium-(adequate) calcium diet (ACD, 0.4% Ca). SEM of bones from rats at the end of lactation on either diet showed a large loss of trabecular bone, increased porosity of endosteal surfaces, and cortical thinning. These changes were much more striking in LCD rats than in ACD rats. Backscattered electron imaging of cross sections of the femora revealed marked cortical thinning at midshaft after lactation, especially in rats on the LCD; this method also showed a marked increase in newly formed, less dense diaphyseal bone on the endosteal surface when dietary calcium had been made available to the LCD rats after lactation ceased. Unlike the rats fed the ACD after lactation, the rats continued on the LCD for the first 3 weeks postlactation failed to recover bone mineral, even though there was a marked decrease in resorbing surfaces of the femora as revealed by morphologic examination. When the diet was changed from the LCD to the ACD for the second 3 weeks postlactation (week 4-6), the bone mineral increased substantially. Overall, these results demonstrate the marked loss of bone during lactation, especially severe in rats fed a low-calcium diet, and the rapid postlactational recovery of bone when adequate dietary calcium was made available, even if the recovery had been delayed for the first 3 weeks by feeding a diet very low in calcium.

Germ-line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells.

Embryonic stem cells (derived from 129/Ola mice) containing a mutant hypoxanthine phosphoribosyltransferase gene that had been corrected in vitro in a planned manner by homologous recombination were injected into blastocysts obtained from C57BL/6J mice. The injected blastocysts were introduced into pseudopregnant female mice to complete their development. Eleven surviving pups were obtained. Nine were chimeras: six males and three females. Two of the males transmitted the embryonic stem cell genome containing the alteration in the hypoxanthine phosphoribosyltransferase gene to their offspring at high frequencies. These experiments demonstrate that a preplanned alteration in a chosen gene can be made in the germ line of an experimental animal by homologous recombination in an embryonic stem cell.

Ethanol-induced changes in morphology and strength of femurs of rats.

Chronic ingestion of ethanol resulted in ultrastructural and mechanical changes in rat femurs. Scanning electron microscopy of the distal end of the femur revealed that the trabeculae of bones from ethanol-fed rats were thinner, more columnar, and more extensive than those from control rats. Three-point bending tests of the rat femurs showed that the maximum force or so-called "strength" required to break the bone was less in ethanol- than in control-fed animals. A significant inverse correlation was observed between the strength required to break the femur and the dose of ethanol calculated on a body weight basis. For the first time our study presents quantitative proof that a relationship exists between bone strength and the consumption of ethanol in rats. The study revealed that ethanol consumption resulted in a weaker femur compared to controls. We suggest that a common mechanism may be responsible for the decreased bone strength of ethanol-fed rats and the increased incidence of fractures in human alcoholics.

Reduced bone mass in calcitonin-deficient rats whether lactating or not.

Calcitonin deficiency was produced in lactating and age-matched nonmated rats by thyroidectomy (TX) after transplantation of the parathyroid glands to a thigh muscle. At the end of lactation and a comparable period in the nonlactating rats, this condition resulted in femurs, vertebrae, and tibiae that weighed less than those in the thyroid-intact controls. Furthermore, the femurs of the CT-deficient rats were narrower at midshaft and shorter, indicating reduced bone growth. The reduction in bone mass in CT-deficient rats, although highly significant, was much smaller than that caused by lactation. Adequate thyroid hormone replacement therapy was provided by giving all the TX rats L-thyroxine (T4) sc or in the drinking water. The body weights of the lactating rats were heavier than those of their nonmated controls but TX had no significant effect on the mean body weight of either group. The previously observed lower concentration of serum calcium in lactating rats than in nonlactating thyroid-intact rats was also seen in TX rats, indicating that CT is not responsible for the relatively low serum calcium during lactation. Our results showing that the bones of TX rats (with T4 replacement) were smaller and lighter than those from thyroid-intact controls whether lactating or not do not support the concept that CT has a special physiological function to protect the skeleton during lactation.

The effect of lactation on the mineral distribution profile of the rat femur by single photon absorptiometry.

The distribution of bone mineral in excised femurs from lactating and nonlactating control rats was localized and quantitated using single photon absorptiometry. Bone mineral content was measured proximodistally in nine sequential 0.32 cm increments along the femur length and then plotted as a function of the distance from the proximal end of the femur. The plots described a mineral distribution profile and revealed that bone loss following lactation was site specific, being greater in the metaphyseal regions (23-36%) than in the diaphyseal area (14-20%). The change in total bone mineral of femur caused by lactation was estimated by integrating the mineral distribution profile plots. The 28% lower bone mineral determined by this method was in close agreement with the 27% difference determined from femur dry weight measurements. Reproducibility of measurements with 10 repositionings of a single femur was within 4% at all but the 2 most distal sites. Variation in the estimated total bone mineral was 1.3%.

In vivo bone mineral analysis throughout skeletal growth in rats: differences due to sex or vitamin D deficiency.

This study reports a new technique for the measurement of bone mineral content (BMC) in live rats. A single photon absorptiometric instrument has been adapted for rapid, reproducible measurement of BMC. Four bone sites were selected for use, based on ease of positioning and reproducibility of measurement; these were as follows: proximal femur, midfemur, proximal seventh caudal vertebra, and midseventh caudal vertebra. Significant increases were detected when BMC was measured at biweekly intervals from weaning to 18 weeks of age in normal male rats. Comparison of body growth and changes in BMC of male and female rats with age showed that body weight gain of female rats slowed earlier than that of male rats whereas BMC increased at similar rates in both sexes. Vitamin D deprivation from day 24 of life resulted in decreased BMC at all four measurement sites compared with such measurements in normal control rats. Differences were detectable after 8 weeks of age and occurred despite the maintenance of serum calcium levels within normal range and only slight reduction in body weights of vitamin D deprived rats. These studies demonstrate that single photon absorptiometry can be used to monitor changes in BMC in live rats on a routine basis without harm to the animals. Changes in BMC such as those due to growth or vitamin D deprivation can easily be quantitated using this technique.