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Treatment of hypertension-mediated cardiac damage with left ventricular (LV) hypertrophy (LVH) and heart failure remains challenging. To identify novel targets, we performed comparative transcriptome analysis between genetic models derived from stroke-prone spontaneously hypertensive rats (SHRSP). Here, we identified carboxypeptidase X 2 (Cpxm2) as a genetic locus affecting LV mass. Analysis of isolated rat cardiomyocytes and cardiofibroblasts indicated Cpxm2 expression and intrinsic upregulation in genetic hypertension. Immunostaining indicated that CPXM2 associates with the t-tubule network of cardiomyocytes. The functional role of Cpxm2 was further investigated in Cpxm2-deficient (KO) and wild-type (WT) mice exposed to deoxycorticosterone acetate (DOCA). WT and KO animals developed severe and similar systolic hypertension in response to DOCA. WT mice developed severe LV damage, including increases in LV masses and diameters, impairment of LV systolic and diastolic function and reduced ejection fraction. These changes were significantly ameliorated or even normalized (i.e., ejection fraction) in KO-DOCA animals. LV transcriptome analysis showed a molecular cardiac hypertrophy/remodeling signature in WT but not KO mice with significant upregulation of 1234 transcripts, including Cpxm2, in response to DOCA. Analysis of endomyocardial biopsies from patients with cardiac hypertrophy indicated significant upregulation of CPXM2 expression. These data support further translational investigation of CPXM2., Differential increased cardiac expression of Cpxm2 was assigned to cardiac hypertrophy in SHRSP rats. Cpxm2 knock-out (KO) in mice reduced cardiac hypertrophy and remodeling in DOCA salt hypertension

Background: High blood pressure is the primary risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly clinically resembles salt-resistant essential hypertension and causes death by stroke before 50 years of age. We recently implicated the gene encoding phosphodiesterase 3A ( PDE3A ); however, in vivo modeling of the genetic defect and thus showing an involvement of mutant PDE3A is lacking. Methods: We used genetic mapping, sequencing, transgenic technology, CRISPR-Cas9 gene editing, immunoblotting, and fluorescence resonance energy transfer. We identified new patients, performed extensive animal phenotyping, and explored new signaling pathways. Results: We describe a novel mutation within a 15 base pair (bp) region of the PDE3A gene and define this segment as a mutational hotspot in hypertension with brachydactyly. The mutations cause an increase in enzyme activity. A CRISPR/Cas9-generated rat model, with a 9-bp deletion within the hotspot analogous to a human deletion, recapitulates hypertension with brachydactyly. In mice, mutant transgenic PDE3A overexpression in smooth muscle cells confirmed that mutant PDE3A causes hypertension. The mutant PDE3A enzymes display consistent changes in their phosphorylation and an increased interaction with the 14-3-3θ adaptor protein. This aberrant signaling is associated with an increase in vascular smooth muscle cell proliferation and changes in vessel morphology and function. Conclusions: The mutated PDE3A gene drives mechanisms that increase peripheral vascular resistance causing hypertension. We present 2 new animal models that will serve to elucidate the underlying mechanisms further. Our findings could facilitate the search for new antihypertensive treatments.

Spinal cord injury (SCI) above mid-thoracic levels leads to autonomic dysfunction affecting both the cardiovascular system and thermoregulation. The renin-angiotensin system (RAS) which is a potent regulator of blood pressure, including its novel beneficial arm with the receptor Mas could be an interesting target in post-SCI hemodynamics. To test the hypothesis that hemodynamics, activity and diurnal patterns of those are more affected in the Mas deficient mice post-SCI we used a mouse model of SCI with complete transection of spinal cord at thoracic level 4 (T4-Tx) and performed telemetric monitoring of blood pressure (BP) and heart rate (HR). Our data revealed that hypothermia deteriorated physiological BP and HR control. Preserving normothermia by keeping mice at 30°C prevented severe hypotension and bradycardia post-SCI. Moreover, it facilitated rapid return of diurnal regulation of BP, HR and activity in wild type (WT) mice. In contrast, although Mas deficient mice had comparable reacquisition of diurnal HR rhythm, they showed delayed recovery of diurnal rhythmicity in BP and significantly lower nocturnal activity. Exposing mice with T4-Tx (kept in temperature-controlled cages) to 23°C room temperature for one hour at different time-points post-SCI, demonstrated their inability to maintain core body temperature, Mas deficient mice being significantly more impaired than WT littermates. We conclude that Mas deficient mice were more resistant to acute hypotension, delayed nocturnal recovery, lower activity and more severely impaired thermoregulation. The ambient temperature had significant effect on hemodynamics and, thus it should be taken into account when assessing cardiovascular parameters post-SCI in mice.

Background and Purpose— Chronic stress is associated with increased stroke risk. However, the underlying pathophysiological mechanisms are poorly understood. We examined the effects of chronic stress on endothelial function and ischemic brain injury in a mouse model. Methods— 129/SV mice were treated with glucocorticoid receptor antagonist mifepristone (25 mg kg −1 /d) or vehicle and exposed to 28 days of chronic stress consisting of exposure to rat, restraint stress, and tail suspension. Heart rate and blood pressure were continuously recorded by telemetry. Endothelial nitric oxide synthase mRNA and protein expression as well as superoxide production and lipid hydroperoxides were quantified. Endothelium-dependent vasorelaxation was measured in aortic rings. Ischemic lesion volume was quantified after 30 minutes filamentous middle cerebral artery occlusion and 72 hours reperfusion. Results— Chronic stress caused a significant increase in heart rate, impaired endothelium-dependent vasorelaxation, increased superoxide production, and reduced aortic and brain endothelial nitric oxide synthase levels. Animals exposed to chronic stress showed major increases in ischemic lesion size. These deleterious effects of stress were completely reversed by treatment with mifepristone. Conclusions— Chronic stress increases stroke vulnerability likely through endothelial dysfunction, which can be reversed by a glucocorticoid receptor antagonist.

Background— Angiotensin (Ang) II–induced target-organ damage involves innate and acquired immunity. Mice deficient for the helix-loop-helix transcription factor inhibitor of differentiation (Id2 −/− ) lack Langerhans and splenic CD8a+ dendritic cells, have reduced natural killer cells, and have altered CD8 T-cell memory. We tested the hypothesis that an alteration in the number and quality of circulating blood cells caused by Id2 deletion would ameliorate Ang II–induced target-organ damage. Methods and Results— We used gene-deleted and transgenic mice. We conducted kidney and bone marrow transplants. In contrast to Ang II–infused Id2 +/− , Id2 −/− mice infused with Ang II remained normotensive and failed to develop albuminuria or renal damage. Bone marrow transplant of Id2 +/− bone marrow to Id2 −/− mice did not restore the blunted blood pressure response to Ang II. Transplantation of Id2 −/− kidneys to Id2 +/− mice also could not prevent Ang II–induced hypertension and renal damage. We verified the Ang II resistance in Id2 −/− mice in a model of local tissue Ang II production by crossing hypertensive mice transgenic for rat angiotensinogen with Id2 −/− or Id2 +/− mice. Angiotensinogen-transgenic Id2 +/− mice developed hypertension, albuminuria, and renal injury, whereas angiotensinogen-transgenic Id2 −/− mice did not. We also found that vascular smooth muscle cells from Id2 −/− mice showed an antisenescence phenotype. Conclusions— Our bone marrow and kidney transplant experiments suggest that alterations in circulating immune cells or Id2 in the kidney are not responsible for Ang II resistance. The present studies identify a previously undefined role for Id2 in the pathogenesis of Ang II–induced hypertension.

Non-Obese Diabetic (NOD) mice show profound pathomorphological changes in sympathetic ganglia during the development of type 1 diabetes mellitus. We tested the hypothesis that NOD mice represent an experimental model to investigate cardiovascular changes seen in humans with diabetic autonomic neuropathy. Blood glucose (BG) levels were measured once a week. Diabetes mellitus was diagnosed as BG levels exceeded 250 mg/dl twice. NOD mice that did not become diabetic served as control group. Blood pressure (BP) and heart rate (HR) were monitored by telemetry and baroreflex sensitivity (BRS) was calculated with the sequence method or with cross spectral analysis. The measurements were obtained before onset of diabetes and during the 4th week of diabetes. The onset of diabetes was accompanied by a continuous decline in HR (615+/-14 vs. 498+/-23 bpm), whereas BP values remained stable (108+/-2 vs. 111+/-2 mm Hg). The circadian HR rhythm increased in diabetic NOD mice. BRS was higher in diabetic NOD mice than in controls. Atropine reduced BRS more profoundly in diabetic mice compared to non-diabetic mice. Despite pathomorphological similarities of the diabetic autonomic neuropathy between patients with diabetes and diabetic NOD mice, the changes in blood pressure regulation are different. In conclusion the use of diabetic NOD mice as a functional model for human diabetes may be questioned.

We compared the effect n-3 polyunsaturated fatty acids (PUFAs) with direct renin inhibition on electrophysiological remodeling in angiotensin II–induced cardiac injury. We treated double-transgenic rats expressing the human renin and angiotensinogen genes (dTGRs) from week 4 to 7 with n-3 PUFA ethyl-esters (Omacor; 25-g/kg diet) or a direct renin inhibitor (aliskiren; 3 mg/kg per day). Sprague-Dawley rats were controls. We performed electrocardiographic, magnetocardiographic, and programmed electrical stimulation. Dietary n-3 PUFAs increased the cardiac content of eicosapentaenoic and docosahexaenoic acid. At week 7, mortality in dTGRs was 31%, whereas none of the n-3 PUFA- or aliskiren-treated dTGRs died. Systolic blood pressure was modestly reduced in n-3 PUFA-treated (180±3 mm Hg) compared with dTGRs (208±5 mm Hg). Aliskiren-treated dTGRs and Sprague-Dawley rats were normotensive (110±3 and 119±6 mm Hg, respectively). Both n-3 PUFA–treated and untreated dTGRs showed cardiac hypertrophy and increased atrial natriuretic peptide levels. Prolonged QRS and QT c intervals and increased T-wave dispersion in dTGRs were reduced by n-3 PUFAs or aliskiren. Both treatments reduced arrhythmia induction from 75% in dTGRs to 17% versus 0% in Sprague-Dawley rats. Macrophage infiltration and fibrosis were reduced by n-3 PUFAs and aliskiren. Connexin 43, a mediator of intermyocyte conduction, was redistributed to the lateral cell membranes in dTGRs. n-3 PUFAs and aliskiren restored normal localization to the intercalated disks. Thus, n-3 PUFAs and aliskiren improved electrical remodeling, arrhythmia induction, and connexin 43 expression, despite a 70-mm Hg difference in blood pressure and the development of cardiac hypertrophy.

Angiotensin II (Ang II) activates signalling pathways predominantly through the G-protein-coupled Ang II type 1 receptor (AT(1)R). The regulator of G protein signalling 2 (RGS2) is a negative G protein regulator. We hypothesized that RGS2 deletion changes blood pressure regulation by increasing the response to Ang II. To address this issue, we infused Ang II (0.5 mg kg(-1) day(-1)) chronically into conscious RGS2-deleted (RGS2(-/-)) and wild-type (RGS2(+/+)) mice, measured mean arterial blood pressure and heart rate (HR) with telemetry and assessed vasoreactivity and gene expression of AT(1A), AT(1B) and AT(2) receptors. Angiotensin II infusion increased blood pressure more in RGS2(-/-) than in RGS2(+/+) mice, while HR was not different between the groups, indicating a resetting of the baroreceptor reflex. Urinary catecholamine excretion was similar in Ang II-infused RGS2(-/-) and RGS2(+/+) mice, indicating a minor role of sympathetic tone for blood pressure differences. Myogenic tone and vasoreactivity in response to Ang II, endothelin-1 and phenylephrine were increased in isolated renal interlobar arterioles of RGS2(-/-) mice compared with RGS2(+/+) mice. The AT(1A), AT(1B) and AT(2) receptor gene expression was not different between RGS2(-/-) and RGS2(+/+) mice. Our findings suggest that RGS2 deletion promotes Ang II-dependent hypertension primarily through an increase of myogenic tone and vasoreactivity, probably by sensitization of AT(1) receptors.

Animal models of cardiovascular diseases allow to investigate relevant pathogenetic mechanisms in detail. In the present study, the mutations Asp175Asn and Glu180Gly in alpha-tropomyosin (TPM1), known cause familiar hypertrophic cardiomyopathy (FHC) were studied for changes in hemodynamic parameters and spontaneous baroreflex regulation in transgenic rats in comparison to transgenic and non-transgenic controls by telemetry. Heart rate variability (HRV) and blood pressure variability (BPV) were analyzed using time- and frequency domain, as well as non-linear measures. The dual sequence method was used for the estimation of the baroreflex regulation. In transgenic rats harboring mutated TPM1, changes in HRV were detected during exercise, but not at rest. Both mutations, Asp175Asn and Glu180Gly, caused increased low frequency power. In addition, in animals with mutation Asp175Asn a reduced total HRV was observed. BPV did not show any differences between all transgenic animal lines. During exercise, a strong increase in the number of bradycardic and tachycardic fluctuations accompanied with decreased baroreflex sensitivity (BRS) was detected in animals with either TPM1 mutation, Asp175Asn or Glu180Gly. These data suggest, that the analysis of cardiac autonomic control, particularly of baroreflex regulation, represents a powerful non-invasive approach to investigate the effects of subtle changes in sarcomeric architecture on cardiac physiology in vivo. In case of mutations Asp175Asn or Glu180Gly in TPM1, early detection of alterations in autonomic cardiac control could help to prevent sudden cardiac death in affected persons.

In this study, we aimed to investigate the adaptation of blood pressure (BP), heart rate (HR), and locomotor activity (LA) circadian rhythms to light cycle shift in transgenic rats with a deficit in brain angiotensin [TGR(ASrAOGEN)]. BP, HR, and LA were measured by telemetry. After baseline recordings (bLD), the light cycle was inverted by prolonging the light by 12 h and thereafter the dark period by 12 h, resulting in inverted dark-light (DL) or light-dark (LD) cycles. Toward that end, a 24-h dark was maintained for 14 days (free-running conditions). When light cycle was changed from bLD to DL, the acrophases (peak time of curve fitting) of BP, HR, and LA shifted to the new dark period in both SD and TGR(ASrAOGEN) rats. However, the readjustment of the BP and HR acrophases in TGR(ASrAOGEN) rats occurred significantly slower than SD rats. The LA acrophases changed similarly in both strains. When light cycle was changed from DL to LD by prolonging the dark period by 12 h, the reentrainment of BP and LA occurred faster than the previous shift in both strains. The readjustment of the BP and HR acrophases in TGR(ASrAOGEN) rats occurred significantly slower than SD rats. In free-running conditions, the circadian rhythms of the investigated parameters adapted in TGR(ASrAOGEN) and SD rats in a similar manner. These results demonstrate that the brain RAS plays an important role in mediating the effects of light cycle shifts on the circadian variation of BP and HR. The adaptive behavior of cardiovascular circadian rhythms depends on the initial direction of light-dark changes.

The regulator of G protein signaling (RGS) 2, a GTPase-activating protein, is activated via the nitric oxide (NO)-cGMP pathway and thereby may influence blood pressure regulation. To test that notion, we measured mean arterial blood pressure (MAP) and heart rate (HR) with telemetry in Nω-nitro-l-arginine methyl ester (l-NAME, 5 mg l-NAME/10 ml tap water)-treated RGS2-deficient (RGS2−/−) and RGS2-sufficient (RGS2+/+) mice and assessed autonomic function. Without l-NAME, RGS2−/− mice showed during day and night a similar increase of MAP compared with controls. l-NAME treatment increased MAP in both strains. nNOS is involved in this l-NAME-dependent blood pressure increase, since 7-nitroindazole increased MAP by 8 and 9 mmHg ( P < 0.05) in both strains. The l-NAME-induced MAP increase of 14–15 mmHg during night was similar in both strains. However, the l-NAME-induced MAP increase during the day was smaller in RGS2−/− than in RGS2+/+ (11 ± 1 vs. 17 ± 2 mmHg; P < 0.05). Urinary norepinephrine and epinephrine excretion was higher in RGS2−/− than in RGS2+/+ mice. The MAP decrease after prazosin was more pronounced in l-NAME-RGS2−/−. HR variability parameters [root mean square of successive differences (RMSSD), low-frequency (LF) power, and high-frequency (HF) power] and baroreflex sensitivity were increased in RGS2−/−. Atropine and atropine plus metoprolol markedly reduced RMSSD, LF, and HF. Our data suggest an interaction between RGS2 and the NO-cGMP pathway. The blunted l-NAME response in RGS2−/− during the day suggests impaired NO signaling. The MAP increases during the active phase in RGS2−/− mice may be related to central sympathetic activation and increased vascular adrenergic responsiveness.

Natriuretic peptides (NP) mediate their effects by activating membrane-bound guanylyl cyclase-coupled receptors A (NPR-A) or B (NPR-B). Whereas the pathophysiological role of NPR-A has been widely studied, only limited knowledge on the cardiovascular function of NPR-B is available. In vitro studies suggest antiproliferative and antihypertrophic actions of the NPR-B ligand C-type NP (CNP). Because of the lack of a specific pharmacological inhibitor, these effects could not clearly be attributed to impaired NPR-B signaling. Recently, gene deletion revealed a predominant role of NPR-B in endochondral ossification and development of female reproductive organs. However, morphological abnormalities and premature death of NPR-B-deficient mice preclude detailed cardiovascular phenotyping. In the present study, a dominant-negative mutant (NPR-BΔKC) was used to characterize CNP-dependent NPR-B signaling in vitro and in transgenic rats. Here we demonstrate that reduced CNP- but not atrial NP-dependent cGMP response attenuates antihypertrophic potency of CNP in vitro . In transgenic rats, NPR-BΔKC expression selectively reduced NPR-B but not NPR-A signaling. NPR-BΔKC transgenic rats display progressive, blood pressure-independent cardiac hypertrophy and elevated heart rate. The hypertrophic phenotype is further enhanced in chronic volume overload-induced congestive heart failure. Thus, this study provides evidence linking NPR-B signaling to the control of cardiac growth.

Recently, a receptor for renin was described that may be important for vascular uptake and activation of (pro)renin, thus leading to local generation of angiotensin II. To assess the in vivo relevance of this protein, we generated transgenic rats overexpressing the human renin receptor gene in smooth muscle tissue, under the control of a 16-kb fragment of the mouse smooth muscle myosin heavy chain gene [TGR(SMMHC-HRR)]. Four lines of transgenic animals were obtained. The correct pattern of expression of the transgene was confirmed by RNase protection assay and in situ hybridization. TGR(SMMHC-HRR) rats are fertile and develop normally. After 6 months of age, transgenic rats develop a cardiovascular phenotype with an elevated systolic blood pressure (137.8±5 versus 118.9±3.7 mm Hg; P =0.008), and an augmentation in heart rate (349.1±7.7 versus 303.1±16.16 bpm; P =0.023) in TGR(SMMHC-HRR) and controls, respectively. These alterations are progressively increasing with aging. Although kidney function and plasma renin were normal in TGR(SMMHC-HRR), an increase in plasma aldosterone [TGR(SMMHC-HRR) 428±64.9 versus 207.3±73.24 pg/mL in control; P =0.02] and in aldosterone/renin ratio [TGR(SMMHC-HRR) 8.04±2.2 versus 2.8±0.55 in control; P =0.03] was observed. This suggests that renin receptor overexpression has resulted in increased intraadrenal angiotensin II, thereby provoking enhanced aldosterone generation in the absence of changes in plasma renin. The rise in aldosterone may underlie, at least in part, the observed cardiovascular phenotype of TGR(SMMHC-HRR).

Antibodies against HLA antigens cause refractory allograft rejection with vasculopathy in some, but not all, patients.We studied 33 kidney-transplant recipients who had refractory vascular rejection. Thirteen had donor-specific anti-HLA antibodies, whereas 20 did not. Malignant hypertension was present in 16 of the patients without anti-HLA antibodies, 4 of whom had seizures. The remaining 17 patients had no malignant hypertension. We hypothesized that activating antibodies targeting the angiotensin II type 1 (AT1) receptor might be involved.Activating IgG antibodies targeting the AT1 receptor were detected in serum from all 16 patients with malignant hypertension and without anti-HLA antibodies, but in no other patients. These receptor-activating antibodies are subclass IgG1 and IgG3 antibodies that bind to two different epitopes on the second extracellular loop of the AT1 receptor. Tissue factor expression was increased in renal-biopsy specimens from patients with these antibodies. In vitro stimulation of vascular cells with an AT1-receptor-activating antibody induced phosphorylation of ERK 1/2 kinase and increased the DNA binding activity of the transcription factors activator protein 1 (AP-1) and nuclear factor-kappaB. The AT1 antagonist losartan blocked agonistic AT1-receptor antibody-mediated effects, and passive antibody transfer induced vasculopathy and hypertension in a rat kidney-transplantation model.A non-HLA, AT1-receptor-mediated pathway may contribute to refractory vascular rejection, and affected patients might benefit from removal of AT1-receptor antibodies or from pharmacologic blockade of AT1 receptors.

α-2 Adrenoceptors are important in baroreflex regulation. We tested the impact of α-2 adrenoceptors on heart rate variability (HRV) and spontaneous baroreflex sensitivity (BRS) in conscious mice with telemetry (TA11PA-C20). Baseline beat-to-beat measurements (2 hours between 8:00 am to 12:00 pm ) were compared with measurements after intraperitoneal α-2 adrenoceptor blockade (yohimbine 2 mg/kg) and α-2 adrenoceptor stimulation (clonidine 1, 10, and 50 mg/kg). Blood pressure (BP) was 128±6/87±6 mm Hg and heart rate (HR) was 548±18 bpm at baseline. BRS, calculated with the cross-spectral method, was 1.2±0.1 ms/mm Hg at baseline. BP increased 20±2/13±2 mm Hg with yohimbine. HR increased by 158±23 bpm. BRS did not change. BP decreased 16±7/5±4 mm Hg with 1 mg/kg of clonidine and did not change with a higher dose. HR decreased with clonidine (176±28, 351±21, 310±29 bpm during 1, 10, and 50 mg/kg of clonidine, P 2 during 1, 10, and 50 mg/kg of clonidine, P P

We have used inbred and congenic rat strains in F(2) segregation studies to discover epistasis in a polygenic model of hypertension. Previously, we have found evidence that the presence of a blood pressure quantitative trait locus (QTL) on chromosome 1 is conditional upon the allele status of chromosome 10. To prove the existence of an epistatic interaction we have analyzed congenic strains for chromosome 1 and 10 carrying high blood pressure QTL alleles from the spontaneously hypertensive rat on a normotensive background of the Wistar-Kyoto (WKY) rat. Additionally, a double congenic strain was developed with both chromosome 1 and 10 high blood pressure QTL alleles on the WKY background. Analysis of variance for blood pressure phenotypes as determined by radiotelemetry showed a significant effect for chromosome 10 but not chromosome 1 QTL alleles and demonstrated a significant interaction between the two loci (P

Physical training induces numerous physiological adaptations in the cardiovascular and skeletal muscle system related to changes in gene expression including modulation of the renin angiotensin sys...

Abstract —Telemetric blood pressure determinations, heart rate measurements, and pressure-natriuresis-diuresis experiments were used to characterize cardiovascular and renal function in bradykinin B 2 receptor knockout mice fed mouse chow containing 0.25% NaCl or mouse chow containing 4% NaCl. In B 2 receptor knockout mice fed usual mouse chow, the mean arterial blood pressure leveled between 108±1 and 110±3 mm Hg, and the heart rate leveled between 520±26 and 525±29 bpm, values that were not different from those measured in B 1 receptor knockout mice or 129Sv/J control mice. Increasing dietary salt intake did not affect mean arterial blood pressure and heart rate. Accordingly, pressure-natriuresis curves, pressure-diuresis curves, renal blood flow, and glomerular filtration rate were not different between B 2 receptor knockout and 129Sv/J mice. Increasing dietary salt intake to 4% increased renal blood flow to levels between 8.41 and 9.50 mL/min per gram kidney wet weight in 129Sv/J mice, whereas in B 2 receptor–deficient mice, renal blood flow was not affected and ranged between 6.85 and 7.88 mL/min per gram kidney wet weight. Other renal function parameters were not affected. Absence of B 2 receptor function was verified in B 2 receptor knockout mice with bradykinin infusion. These data suggest that the absence of B 2 receptor function does not necessarily make B 2 receptor knockout mice hypertensive or induce salt sensitivity. Presumably, differences in the genetic background or an adaptation to the loss of B 2 receptor function may account for these results, in contrast with earlier reports involving B 2 receptor knockout mice. We hold the latter possibility to be more likely and to be a fruitful possibility for future research.

Leptin is a hormone related to metabolism. It also influences blood pressure, but the mechanisms triggered in this process are not yet elucidated. Angiotensin-I converting enzyme (ACE) regulates cardiovascular functions and recently has been associated with metabolism control and obesity. Here, we used ob/ob mice, a model lacking leptin, to answer the question whether ACE and leptin could interact to influence blood pressure, thereby linking the renin-angiotensin system and obesity. These mice are obese and diabetic but have normal 24 h mean arterial pressure. Our results show that plasma and lung ACE activities as well as ACE mRNA expression were significantly decreased in ob/ob mice. In agreement with these findings, the hypotensive effect produced by enalapril administration was attenuated in the obese mice. Plasma renin, angiotensinogen, angiotensin I, bradykinin, and angiotensin 1-7 were increased, whereas plasma angiotensin II concentration was unchanged in obese mice. Chronic infusion of leptin increased renin activity and angiotensin II concentration in both groups and increased ACE activity in ob/ob mice. Acute leptin infusion restored ACE activity in leptin-deficient mice. Moreover, the effect of an ACE inhibitor on blood pressure was not changed in ob/+ mice during leptin treatment but increased four times in obese mice. In summary, our findings show that the renin-angiotensin system is altered in ob/ob mice, with markedly reduced ACE activity, which suggests a possible connection between the renin-angiotensin system and leptin. These results point to an important interplay between the angiotensinergic and the leptinergic systems, which may play a role in the pathogenesis of obesity, hypertension, and metabolic syndrome.

Receptor-activator of NF-kappa B ligand (TNFSF11, also known as RANKL, OPGL, TRANCE and ODF) and its tumour necrosis factor (TNF)-family receptor RANK are essential regulators of bone remodelling, lymph node organogenesis and formation of a lactating mammary gland(1-4). RANKL and RANK are also expressed in the central nervous system(5,6). However, the functional relevance of RANKL/RANK in the brain was entirely unknown. Here we report that RANKL and RANK have an essential role in the brain. In both mice and rats, central RANKL injections trigger severe fever. Using tissue-specific Nestin-Cre and GFAP-Cre rank(floxed) deleter mice, the function of RANK in the fever response was genetically mapped to astrocytes. Importantly, Nestin-Cre and GFAP-Cre rank(floxed) deleter mice are resistant to lipopolysaccharide-induced fever as well as fever in response to the key inflammatory cytokines IL-1 beta and TNF alpha. Mechanistically, RANKL activates brain regions involved in thermoregulation and induces fever via the COX2-PGE(2)/EP3R pathway. Moreover, female Nestin-Cre and GFAP-Cre rank(floxed) mice exhibit increased basal body temperatures, suggesting that RANKL and RANK control thermoregulation during normal female physiology. We also show that two children with RANK mutations exhibit impaired fever during pneumonia. These data identify an entirely novel and unexpected function for the key osteoclast differentiation factors RANKL/RANK in female thermoregulation and the central fever response in inflammation. Austrian Ministry of Sciences Austrian Academy of Sciences GEN-AU ( AustroMouse) European Commission European Research Council (ERC) European Commission Uehara Memorial Foundation Japan Foundation for Applied Enzymology Austrian Ministry for Science and Research Wiener Wissenschafts-, Forschungs- und Technologiefonds (WWTF) IMBA

The leptin-deficient ob/ob mice are insulin resistant and obese. However, the control of blood pressure in this model is not well defined. The goal of this study was to evaluate the role of leptin and of the renin-angiotensin system in the cardiovascular abnormalities observed in obesity using a model lacking leptin. To this purpose, we measured blood pressure in ob/ob and control animals by radiotelemetry combined with fast Fourier transformation before and after both leptin and enalapril treatment. Autonomic function was assessed pharmacologically. Blood pressure during daytime was slightly higher in the ob/ob compared to control mice, while no difference in heart rate was observed. Blood pressure response to trimetaphane and heart rate response to metoprolol were greater in ob/ob mice than in control littermates indicating an activated sympathetic nervous system. Heart rate response to atropine was attenuated. Baroreflex sensitivity and heart rate variability were blunted in ob/ob mice, while low frequency of systolic blood pressure variability was found increased. Chronic leptin replacement reduced blood pressure and reversed the impaired autonomic function observed in ob/ob mice. Inhibition of angiotensin-converting enzyme by enalapril treatment had similar effects, prior to the loss of weight. These findings suggest that the renin-angiotensin system is involved in the autonomic dysfunction caused by the lack of leptin in ob/ob mice and support a role of this interplay in the pathogenesis of obesity, hypertension, and metabolic syndrome.

Growth arrest–specific protein 6 (Gas 6) is involved in inflammatory kidney diseases, vascular remodeling, cell adhesion, and thrombus formation. We explored a role for Gas 6 in aldosterone-induced target organ damage. We observed that Gas 6 was upregulated in rats with high aldosterone levels. Mineralocorticoid receptor blockade prevented target organ damage and decreased the elevated Gas 6 expression. Vascular smooth muscle cells given aldosterone increased their Gas 6 expression in vitro. To test the pathophysiological relevance, we investigated the effects of deoxycorticosterone acetate (DOCA) on Gas 6 gene-deleted ( −/− ) mice. After 6 weeks DOCA, Gas 6 −/− mice developed similar telemetric blood pressure elevations compared to wild-type mice but were protected from cardiac hypertrophy. Cardiac expression of interleukin 6 and collagen IV was blunted in Gas 6 −/− mice, indicating reduced inflammation and fibrosis. Gas 6 −/− mice also had an improved renal function with reduced albuminuria, compared to wild-type mice. Renal fibrosis and fibronectin deposition in the kidney were also reduced. Gas 6 deficiency reduces the detrimental effects of aldosterone on cardiac and renal remodeling independent of blood pressure reduction. Gas 6 appears to play a role in mineralocorticoid receptor-mediated target organ damage. Furthermore, because warfarin interferes with Gas 6 protein expression, the findings could be of clinical relevance for anticoagulant choices.

Serotonin synthesis in mammals is initiated by 2 distinct tryptophan hydroxylases (TPH), TPH1 and TPH2. By genetically ablating TPH2, we created mice ( Tph2 −/− ) that lack serotonin in the central nervous system. Surprisingly, these mice can be born and survive until adulthood. However, depletion of serotonin signaling in the brain leads to growth retardation and 50% lethality in the first 4 weeks of postnatal life. Telemetric monitoring revealed more extended daytime sleep, suppressed respiration, altered body temperature control, and decreased blood pressure (BP) and heart rate (HR) during nighttime in Tph2 −/− mice. Moreover, Tph2 −/− females, despite being fertile and producing milk, exhibit impaired maternal care leading to poor survival of their pups. These data confirm that the majority of central serotonin is generated by TPH2. TPH2-derived serotonin is involved in the regulation of behavior and autonomic pathways but is not essential for adult life.

Background— Hypertensive target organ damage, especially cardiac hypertrophy with heart failure and arrhythmia, is a major source of morbidity and mortality. Angiotensin II, a major mediator of hypertension and cardiac damage, has proinflammatory properties. Inflammation and activation of the immune system play a pivotal role in pathogenesis of hypertensive target organ damage. However, the role of immunosuppressive CD4 + CD25 + regulatory T (Treg) cells in the pathogenesis of hypertensive target organ damage is unexplored. Methods and Results— We conducted adoptive transfer of Treg cells into angiotensin II–infused hypertensive mice. Treg cell recipients exhibited improved cardiac hypertrophy and less cardiac fibrosis despite sustained hypertension. Amelioration of cardiac morphology was accompanied by an improvement in arrhythmogenic electric remodeling, indicating the functional significance of the enhanced cardiac morphology. Delocalization of the connexin 43 gap junction protein is one of the major pathomechanisms in electric remodeling. Pronounced connexin 43 immunoreactivity was found at the lateral borders of cardiomyocytes in angiotensin II–treated mice. In contrast, connexin 43 was restricted to the intercalated disk regions in sham controls. Surprisingly, angiotensin II+Treg–treated mice showed normal connexin 43 gap junction protein localization. Adoptive Treg cell transfer resulted in a marked reduction in cardiac CD4 + , CD8 + , and CD69 + cell and macrophage infiltration. Conclusions— Immunosuppressive effects of transferred Treg cells ameliorated cardiac damage and accounted for the improved electric remodeling independently of blood pressure–lowering effects. Our results provide new insights into the pathogenesis of hypertensive cardiac damage and could therefore lead to new therapeutic approaches that involve manipulation of the immune system.

Leptin receptor-deficient db/db mice develop human type 2 diabetes mellitus, hypertension, and obesity with disrupted circadian blood pressure (BP) rhythm. Whether leptin is the sole mechanism mediating autonomic imbalance and hypertension is unclear. To explore this notion further, we measured BP by radiotelemetry combined with fast Fourier transformation and assessed autonomic function pharmacologically before and after renin-angiotensin system blockade with enalapril. the resting period BP (117 +/- 3 versus 108 +/- 1.0 mm Hg) and heart rate (HR; 488 +/- 12 versus 436 +/- 8 bpm) were higher in db/db mice compared with db/+ mice. BP and HR amplitudes were lower in db/db mice compared with db/+ mice. BP response to trimetaphan (-43 +/- 5 versus -27 +/- 3 mm Hg) and HR response to metoprolol (-59 +/- 12 versus -5 +/- 4 bpm) were greater in db/db mice than in db/+ mice. the HR response to atropine was blunted in db/db mice (59 +/- 17 versus 144 +/- 24 bpm), as were baroreflex sensitivity and HR variability. Enalapril improved autonomic regulation in db/db mice. Stimulation of central alpha-2 adrenoreceptors enhanced both parasympathetic HR control and baroreflex sensitivity in db/db mice. We suggest that functional, rather than structural, alpha-2 adrenoceptor changes and the renin-angiotensin system are involved in the increased sympathetic and decreased parasympathetic tones in db/db mice. Our data suggest that db/db mice exhibit features found in humans with type 2 diabetic autonomic neuropathy and could serve as a model for this complication. (Hypertension. 2009; 53[part 2]: 387-392.) Max Delbruck Ctr Mol Med, D-13125 Berlin, Germany Hannover Med Sch, Inst Clin Pharmacol, D-3000 Hannover, Germany Universidade Federal de São Paulo, Dept Biophys, Escola Paulista Med, São Paulo, Brazil Vanderbilt Univ, Sch Med, Dept Med, Auton Dysfunct Serv,Div Clin Pharmacol, Nashville, TN 37212 USA HELIOS Klin, Franz Volhard Clin, Fac Med Charite, Berlin, Germany Universidade Federal de São Paulo, Dept Biophys, Escola Paulista Med, São Paulo, Brazil Web of Science

Renin initiates angiotensin II formation and has no other known functions. We observed that transgenic rats (TGR) overexpressing the human renin gene (hREN) developed moderate obesity with increased body fat mass and glucose intolerance compared with nontransgenic Sprague-Dawley (SD) rats. The metabolic changes were not reversed by an angiotensin-converting enzyme inhibitor, a direct renin inhibitor, or by (pro)renin receptor blocker treatment. The obese phenotype in TGR(hREN) originated from higher food intake, which was partly compensated by increases in resting energy expenditure, total thermogenesis (postprandial and exercise activity), and lipid oxidation during the first 8 weeks of life. Once established, the difference in body weight between TGR(hREN) and SD rats remained constant over time. When restricted to the caloric intake of SD, TGR(hREN) developed an even lower body weight than nontransgenic controls. We did not observe significant changes in the cocaine and amphetamine-regulated transcript, pro-opiomelanocortin, both anorexigenic, or neuropeptide Y, orexigenic, mRNA levels in TGR(hREN) versus SD controls. However, the mRNA level of the agouti-related peptide, orexigenic, was significantly reduced in TGR(hREN) versus SD controls at the end of the study, which indicates a compensatory mechanism. We suggest that the human renin transgene initiates a process leading to increased and early appetite, obesity, and metabolic changes not related to angiotensin II. The mechanisms are independent of any currently known renin-related effects.

BACKGROUND To study the regulation of a naturally occurring genetic variant of high angiotensin-converting enzyme (ACE) gene (Ace in rat) expression, i.e., the Ace allele of the normotensive Wistar-Kyoto (WKY) rat, in the hypertensive background of stroke-prone spontaneously hypertensive (SHRSP) rats. METHODS We analyzed a congenic strain termed SHRSP.WKY-Ace derived from SHRSP in which a chromosomal fragment of rat chromosome 10 including Ace was replaced by the WKY locus. We compared blood pressures by radiotelemetry, measured plasma ACE activity, tissue ACE messenger RNA (mRNA) and enzyme activities in lung, kidney, and left ventricle (LV) of the heart in adult animals. RESULTS Congenic animals demonstrated a twofold increase in plasma ACE activity in comparison to SHRSP (P < 0.05) and thus similar levels to WKY. The increased tissue expression of ACE mRNA and enzyme activities in lung, kidney, and LV observed in WKY were similarly found in congenic animals when compared to SHRSP (P < 0.05, respectively). Systolic and diastolic blood pressures were not different between congenic and SHRSP animals. Analysis of renin in plasma and angiotensin peptides in LV tissues indicated the induction of compensatory mechanisms by downregulation of renin and angiotensin I (Ang I) concentrations in congenic animals. CONCLUSIONS We demonstrated that genetically determined high ACE expression linked to WKY Ace remains unchanged in the hypertensive background of SHRSP.WKY-Ace. Our data indicate that buffering mechanisms in the renin-angiotensin system contribute to the finding that the development of spontaneous hypertension is not affected, despite an average twofold higher expression of ACE in congenic animals.

Angiotensin II (Ang II) activates signalling pathways predominantly through the G-protein-coupled Ang II type 1 receptor (AT(1)R). The regulator of G protein signalling 2 (RGS2) is a negative G protein regulator. We hypothesized that RGS2 deletion changes blood pressure regulation by increasing the response to Ang II. To address this issue, we infused Ang II (0.5 mg kg(-1) day(-1)) chronically into conscious RGS2-deleted (RGS2(-/-)) and wild-type (RGS2(+/+)) mice, measured mean arterial blood pressure and heart rate (HR) with telemetry and assessed vasoreactivity and gene expression of AT(1A), AT(1B) and AT(2) receptors. Angiotensin II infusion increased blood pressure more in RGS2(-/-) than in RGS2(+/+) mice, while HR was not different between the groups, indicating a resetting of the baroreceptor reflex. Urinary catecholamine excretion was similar in Ang II-infused RGS2(-/-) and RGS2(+/+) mice, indicating a minor role of sympathetic tone for blood pressure differences. Myogenic tone and vasoreactivity in response to Ang II, endothelin-1 and phenylephrine were increased in isolated renal interlobar arterioles of RGS2(-/-) mice compared with RGS2(+/+) mice. The AT(1A), AT(1B) and AT(2) receptor gene expression was not different between RGS2(-/-) and RGS2(+/+) mice. Our findings suggest that RGS2 deletion promotes Ang II-dependent hypertension primarily through an increase of myogenic tone and vasoreactivity, probably by sensitization of AT(1) receptors.

Hypertension is an important disease with polygenic inheritance. In order to identify the genes involved in blood pressure regulation, hypertensive rat and mouse models have been developed either by selective breeding or by transgenic technology. The most essential technological prerequisite in these studies is a reliable assessment of the blood pressure in rodents. Three methods are used most frequently for this purpose: tail cuff plethysmography, intra-arterial catheters, and radiotelemetry. Plethysmography is noninvasive, relatively simple, and suitable for a large number of animals, but also imprecise. Intra-arterial catheters are more precise, but require surgery. And both methods restrain and thereby stress the animals, which leads to alterations in blood pressure. Therefore, the telemetric blood pressure measurement, which allows the study of conscious, freely moving animals, has become the gold standard for measuring blood pressure in rodents. However, this method is extremely expensive. Thus, for each experiment the costs have to be put in relation to the quality of data required. This chapter will describe blood pressure measurement methods in technical detail.

Hypertension is an important disease with polygenic inheritance. In order to identify the genes involved in blood pressure regulation, hypertensive rat and mouse models have been developed either by selective breeding or by transgenic technology. The most essential technological prerequisite in these studies is a reliable assessment of the blood pressure in rodents. Three methods are used most frequently for this purpose: tail cuff plethysmography, intra-arterial catheters, and radiotelemetry. Plethysmography is noninvasive, relatively simple, and suitable for a large number of animals, but also imprecise. Intra-arterial catheters are more precise, but require surgery. And both methods restrain and thereby stress the animals, which leads to alterations in blood pressure. Therefore, the telemetric blood pressure measurement, which allows the study of conscious, freely moving animals, has become the gold standard for measuring blood pressure in rodents. However, this method is extremely expensive. Thus, for each experiment the costs have to be put in relation to the quality of data required. This chapter will describe blood pressure measurement methods in technical detail.

We previously identified a quantitative trait locus (QTL) for stroke proneness between the kallikrein ( Klk) and Mt1pa markers on rat chromosome 1. To gain functional insights, we constructed congenic strains by introgressing either the whole or selected chromosomal segments from the stroke-prone (SHRsp) onto the stroke-resistant (SHRsr) spontaneously hypertensive rat genome and vice versa. The phenotype was the latency to develop stroke under a Japanese high-salt, low-potassium diet for 3 mo [known as Japanese diet (JD)]. Blood pressure (BP) was measured by tail cuff throughout the experiment. Urinary protein excretion was monitored in all lines under JD. The SHRsp-derived lines carrying the SHRsr allele, and particularly the D1Rat134- Mt1pa chromosomal segment, had a significant delay of stroke occurrence and improved survival compared with SHRsp ( P < 0.001). On the other hand, a significant occurrence of stroke events (20%) was detected in the reciprocal lines by the end of the 3-mo treatment with JD ( P = 0.003). The stroke phenotype was also associated with increased proteinuria. Our results underscore the functional importance of the Chr 1 stroke QTL. Furthermore, they underscore the utility of stroke/congenic lines in dissecting the genetics of stroke.

Regulator of G protein signaling (RGS2) deletion in mice prolongs signaling by G protein-coupled vasoconstrictor receptors and increases blood pressure. However, the exact mechanism of the increase in blood pressure is unknown. To address this question we tested autonomic nervous system function and blood pressure regulation in RGS2-deficient mice (RGS2 −/−). We measured arterial blood pressure and heart rate (HR) with telemetry, computed time and frequency-domain measures for blood pressure and HR variability (HRV) as well as baroreflex sensitivity [BRS-low frequency (LF)], and assessed environmental stress sensitivity. Mean arterial blood pressure (MAP) was ∼10 mmHg higher in RGS2 −/− compared with RGS2 +/+ mice, while HR was not different between the groups, indicating a resetting of the baroreceptor reflex. Atropine increased MAP more in RGS2 −/− than in RGS2 +/+ mice while HR responses were not different. Urinary norepinephrine excretion was higher in RGS2 −/− than in RGS2 +/+ mice. The blood pressure decrease following prazosin was more pronounced in RGS2 −/− mice than in RGS2 +/+ mice. The LF and high-frequency (HF) power of HRV were reduced in RGS2 −/− compared with controls while BRS-LF and SBP-LF were not different. Atropine and atropine + metoprolol markedly reduced the HRV parameters in the time (RMSSD) and frequency domain (LF, HF, LF/HF) in both strains. Environmental stress sensitivity was increased in RGS2 −/− mice compared with controls. We conclude that the increase in blood pressure in RGS2 −/− mice is not solely explained by peripheral vascular mechanisms. A central nervous system mechanism might be implicated by an increased sympathetic tone. This state of affairs could lead to a baroreceptor-HR reflex resetting, while BRS remains unimpaired.

To study the mechanisms by which missense mutations in α-tropomyosin cause familial hypertrophic cardiomyopathy, we generated transgenic rats overexpressing α-tropomyosin with one of two disease-causing mutations, Asp175Asn or Glu180Gly, and analyzed phenotypic changes at molecular, morphological, and physiological levels. The transgenic proteins were stably integrated into the sarcomere, as shown by immunohistochemistry using a human-specific anti-α-tropomyosin antibody, ARG1. In transgenic rats with either α-tropomyosin mutation, molecular markers of cardiac hypertrophy were induced. Ca2+sensitivity of cardiac skinned-fiber preparations from animals with mutation Asp175Asn, but not Glu180Gly, was decreased. Furthermore, elevated frequency and amplitude of spontaneous Ca2+waves were detected only in cardiomyocytes from animals with mutation Asp175Asn, suggesting an increase in intracellular Ca2+concentration compensating for the reduced Ca2+sensitivity of isometric force generation. Accordingly, in Langendorff-perfused heart preparations, myocardial contraction and relaxation were accelerated in animals with mutation Asp175Asn. The results allow us to propose a hypothesis of the pathogenetic changes caused by α-tropomyosin mutation Asp175Asn in familial hypertrophic cardiomyopathy on the basis of changes in Ca2+handling as a sensitive mechanism to compensate for alterations in sarcomeric structure.

Linkage analyses in experimental crosses of stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar-Kyoto (WKY) rats have strongly suggested the presence of quantitative trait loci (QTL) influencing blood pressure and ACE levels on rat chromosome 10, which have been confirmed in multiple independent studies. Analysis of the orthologous region on human chromosome 17 also revealed significant linkage to blood pressure in several populations. Wnk4 , a gene previously identified to cause pseudohypoaldosteronism type II, a rare mendelian form of arterial hypertension, is located on human chromosome 17. The hypothesis has been advanced that molecular variants of this gene might contribute to common polygenic forms of hypertension, since Wnk4 is located in a region of conserved synteny that demonstrates an overlap between quantitative trait loci for primary hypertension in humans and rats. In this report, we describe the confirmation of the blood pressure QTL on rat chromosome 10 by congenic approaches, spanning the Wnk4 locus. Comparative analysis of the complete coding sequence of Wnk4 in SHRSP and WKY strains revealed no mutation and demonstrated high conservation between rat and human proteins. Furthermore, comparison of mRNA levels in the kidney showed no differences between SHRSP and WKY. Additionally, we excluded a secondary effect of blood pressure on the transcriptional regulation of Wnk4 . Our results fail to support a material contribution of Wnk4 to blood pressure regulation in this model of polygenic hypertension. Thus, Wnk4 is likely not to represent the underlying disease gene for the QTL captured in chromosome 10 congenic animals.

We adapted telemetry and sequence analysis employed in humans to mice and measured heart rate variability and the spontaneous baroreflex sensitivity in angiotensin II type 2 (AT 2 ) receptor–deleted (AT 2 −/−) and wild-type (AT 2 +/+) mice with either deoxycorticosterone acetate (DOCA)-salt hypertension or Nω-nitro- l -arginine methylester hydrochloride (L-NAME) hypertension. Mean arterial pressure leveled during the day at 101±1 mm Hg and during the night at 109±1 mm Hg in AT 2 receptor–deleted mice, compared with 98±2 mm Hg (day) and 104±2 mm Hg (night) in wild-type mice. Mean arterial pressure increased in AT 2 receptor–deleted mice with L-NAME to 114±1 mm Hg (day) and 121±1 mm Hg (night), compared with 105±2 mm Hg (day) and 111±2 mm Hg (night), respectively. DOCA-salt also increased day and night blood pressures in AT 2 receptor–deleted mice to a greater degree than in wild-type mice. Heart rate variability in the time and frequency domain was not different between AT 2 receptor–deleted mice and AT 2 receptor–deleted mice at baseline. Systolic blood pressure variability in the low frequency band was lower in AT 2 receptor–deleted mice (0.6±0.1 ms 2 versus 3.9±1.3 ms 2 ) than in wild-type mice. Baroreceptor-heart rate reflex sensitivity was significantly increased in AT 2 receptor–deleted mice compared with wild-type mice (3.4±0.6 versus 2.1±0.5 ms/mm Hg). These differences remained after DOCA-salt and L-NAME treatments. We conclude that activation of the AT 2 receptor impairs arterial baroreceptor reflex function, probably by a central action. These data support the existence of an inhibitory central effect of the AT 2 receptor on baroreflex function.

To study whether the brain renin-angiotensin system plays a role in the long-term and short-term control of blood pressure and heart rate variability, we examined in transgenic rats [TGR(ASrAOGEN)] with low brain angiotensinogen levels the 24-hour variation of blood pressure and heart rate. Telemetry recordings were made during basal and hypertensive conditions induced by a low-dose subcutaneous infusion of angiotensin II for 7 days. Short-term blood pressure and heart rate variability were evaluated by spectral analysis, and as a measure of baroreflex sensitivity, the average transfer gain between the pressure and heart rate variations was calculated. During the angiotensin II infusion in control but not TGR(ASrAOGEN) rats, the 24-hour rhythm of blood pressure was inverted (5.8±2 versus −0.4±1.8 mm Hg/group of day-night differences of blood pressure, P P P >0.05). These results demonstrate that the brain renin-angiotensin system plays an important role in mediating the effects of angiotensin II on the circadian variation of blood pressure. Furthermore, these data indicate that a permanent deficiency in the brain renin-angiotensin system alters the reflex control of heart rate in rats.

Objectives The hypertension in AT 2 receptor knockout mice is imperfectly defined. Therefore, we investigated the influence of dietary salt loading and deoxycorticosterone (DOCA)-salt treatment on blood pressure and diurnal patterns of blood pressure in these mice by radiotelemetry. Methods We used telemetry in AT 2 receptor knockout and wild-type mice to measure blood pressure, heart rate, aortic pressure dp/dt, locomotor activity, and circadian rhythms. Salt-related effects were studied by increasing the salt in chow to 4%, adding 1% saline in drinking water, and by DOCA-salt treatment. Results Baseline blood pressures were higher in AT 2 receptor knockout than in wild-type mice and were not affected by increasing the salt intake. The blood pressure increase was steeper and greater in AT 2 receptor knockout than in wild-type mice after DOCA-salt treatment A circadian rhythm of blood pressure and heart rate, with higher values during the night, was seen in wild-type, but not in AT 2 receptor knockout mice. In AT 2 receptor knockout mice, this rhythm was only significant when daily salt intake was increased or when DOCA-salt hypertension was induced. The acrophase of blood pressure and heart rate was found between 2000 and 2400 h and was in accordance with the maximum physical activity. Conclusion These data suggest that AT 2 knockout mice display slight hypertension which is not salt-sensitive. On the other hand, the susceptibility to develop DOCA-salt hypertension is increased. The study also illustrates the power of telemetry in monitoring long-term cardiovascular changes and circadian blood pressure and heart rate rhythms in genetically engineered mice.

P191 The brain renin-angiotensin system (RAS) system may play a functional role in the long-term and short-term control of blood pressure (BPV) and heart rate variability (HRV). To study this we recorded in transgenic rats TGR(ASrAOGEN) with low brain angiotensinogen levels the 24-h variation of BP and HR during basal and hypertensive conditions, induced by a low-dose s.c. infusion of angiotensin II (Ang II, 100 ng/kg/min) for 7 days. Cardiovascular parameters were monitored by telemetry. Short-term BPV and HRV were evaluated by spectral analysis and as a measure of baroreflex sensitivity the transfer gain between the pressure and heart rate variations was calculated. During the Ang II infusion, in SD but not TGR(ASrAOGEN) rats, the 24-h rhythm of BP was inverted (5.8 ± 2 vs. -0.4 ± 1.8 mm Hg/group of day-night differences of BP, p< 0.05, respectively). In contrast, in both the SD and TGR(ASrAOGEN) rats, the 24-h HR rhythms remained unaltered and paralleled those of locomotor activity. The increase of systolic BP was significantly reduced in TGR(ASrAOGEN) in comparison to SD rats as previously described, while the HR was not altered in TGR(ASrAOGEN) nor in SD rats. The spectral index of baroreflex sensitivity (FFT gain between 0.3-0.6 Hz) was significantly higher in TGR(ASrAOGEN) than SD rats during control (0.71 ± 0.1 vs. 0.35 ± 0.06, p0.05). These results demonstrate that the brain RAS plays an important role in mediating the effects of Ang II on the circadian variation of BP. Furthermore these data are consistent with the view that the brain RAS modulates baroreflex control of HR in rats, with AII having an inhibitory role.

The aim of the study was to characterize the genetic basis for the early onset of increased urinary albumin excretion (UAE) observed in the salt-sensitive Dahl rat (SS). We first characterized blood pressures and UAE values in adult SS compared with the spontaneously hypertensive rat (SHR) strain. Blood pressure measurements by radiotelemetry at 14 wk demonstrated similar spontaneous hypertension in both strains on a low-sodium diet containing 0.2% NaCl by weight, whereas UAE was markedly increased in SS compared with SHR (253.07 ± 68.39 vs. 1.65 ± 1.09 mg/24 h, P < 0.0001). Analysis of UAE in young animals of both strains fed a low-sodium diet demonstrated that UAE is elevated in SS as early as 4 wk of age ( P < 0.0001), when ultrastructural evaluation of glomeruli by electron microscopy appears still normal. At 8 wk SS demonstrated a 280-fold elevated UAE compared with SHR ( P < 0.0001). Consequently, to identify quantitative trait loci (QTLs) contributing to salt-independent early manifestation of increased UAE in the SS rat, we performed genome-wide linkage and QTL mapping analysis in a young F2population derived from the two contrasting strains. UAE was determined in 539 F2animals at 8 wk. We identified seven suggestive or significant UAE QTLs on rat chromosomes (RNO) RNO2, RNO6, RNO8, RNO9, RNO10, RNO11, and RNO19, accounting together for 34% of the overall variance of UAE in this F2population. Thus early onset albuminuria in the SS rat is under polygenetic influence and independent from salt loading.

To evaluate the cardiovascular actions of kinins, we established a transgenic rat line harboring the human tissue kallikrein gene, TGR(hKLK1). Under the control of the zinc-inducible metallothionein promoter, the transgene was expressed in most tissues including the heart, kidney, lung, and brain, and human kallikrein was detected in the urine of transgenic animals. Transgenic rats had a lower 24-h mean arterial pressure in comparison with control rats, which was further decreased when their diet was supplemented with zinc. The day/night rhythm of blood pressure was significantly diminished in TGR(hKLK1) animals, whereas the circadian rhythms of heart rate and locomotor activity were unaffected. Induction of cardiac hypertrophy by isoproterenol treatment revealed a marked protective effect of the kallikrein transgene because the cardiac weight of TGR(hKLK1) increased significantly less, and the expression of atrial natriuretic peptide and collagen III as markers for hypertrophy and fibrosis, respectively, were less enhanced. The specific kinin-B2 receptor antagonist, icatibant, abolished this cardioprotective effect. In conclusion, the kallikrein-kinin system is an important determinant in the regulation of blood pressure and its circadian rhythmicity. It also exerts antihypertrophic and antifibrotic actions in the heart.