Michael Obst

Increased Vascular Smooth Muscle Contractility in TRPC6−/− Mice

ABSTRACT Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6 − / − smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6 −/− smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.

Vascular Endothelial Cell-Specific NF-κB Suppression Attenuates Hypertension-Induced Renal Damage

Nuclear factor kappa B (NF-&kgr;B) participates in hypertension-induced vascular and target-organ damage. We tested whether or not endothelial cell–specific NF-&kgr;B suppression would be ameliorative. We generated Cre/lox transgenic mice with endothelial cell–restricted NF-&kgr;B super-repressor I&kgr;B&agr;&Dgr;N (Tie-1-&Dgr;N mice) overexpression. We confirmed cell-specific I&kgr;B&agr;&Dgr;N expression and reduced NF-&kgr;B activity after TNF-&agr; stimulation in primary endothelial cell culture. To induce hypertension with target-organ damage, we fed mice a high-salt diet and N(omega)-nitro-l-arginine-methyl-ester (L-NAME) and infused angiotensin (Ang) II. This treatment caused a 40-mm Hg blood pressure increase in both Tie-1-&Dgr;N and control mice. In contrast to control mice, Tie-1-&Dgr;N mice developed a milder renal injury, reduced inflammation, and less albuminuria. RT-PCR showed significantly reduced expression of the NF-&kgr;B targets VCAM-1 and ICAM-1, compared with control mice. Thus, the data demonstrate a causal link between endothelial NF-&kgr;B activation and hypertension-induced renal damage. We conclude that in vivo NF-&kgr;B suppression in endothelial cells stops a signaling cascade leading to reduced hypertension-induced renal damage despite high blood pressure.

Clonidine Improves Spontaneous Baroreflex Sensitivity in Conscious Mice Through Parasympathetic Activation

Abstract—&agr;-2 Adrenoceptors are important in baroreflex regulation. We tested the impact of &agr;-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 &agr;-2 adrenoceptor blockade (yohimbine 2 mg/kg) and &agr;-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 <0.01). HRV (total power=4629±465, 7002±440, and 6452±341 ms2 during 1, 10, and 50 mg/kg of clonidine, P <0.01) and BRS were profoundly increased with clonidine (14±1, 13±1, and 10±1 ms/mm Hg, P <0.01). The effects of clonidine were abolished with atropine (2 mg/kg plus 50 mg/kg of clonidine) but not with metoprolol (4 mg/kg plus 50 mg/kg of clonidine). These data suggest that &agr;-2 adrenoceptors exert a regulatory influence on autonomic cardiovascular control and baroreflex function. The effect of clonidine on baroreflex HR regulation is mediated by the parasympathetic nervous system. These murine data fit well with recent human observations regarding parasympathetic activation via &agr;-2 adrenoceptors.

Heart Rate Variability and Baroreflex Function in AT2 Receptor-Disrupted Mice

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 (AT2) receptor–deleted (AT2 −/−) and wild-type (AT2 +/+) mice with either deoxycorticosterone acetate (DOCA)-salt hypertension or N&ohgr;-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 AT2 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 AT2 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 AT2 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 AT2 receptor–deleted mice and AT2 receptor–deleted mice at baseline. Systolic blood pressure variability in the low frequency band was lower in AT2 receptor–deleted mice (0.6±0.1 ms2 versus 3.9±1.3 ms2) than in wild-type mice. Baroreceptor-heart rate reflex sensitivity was significantly increased in AT2 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 AT2 receptor impairs arterial baroreceptor reflex function, probably by a central action. These data support the existence of an inhibitory central effect of the AT2 receptor on baroreflex function.

Novel Role for Inhibitor of Differentiation 2 in the Genesis of Angiotensin II–Induced Hypertension

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.

Systemic hemodynamics in non-anesthetized L-NAME- and DOCA-salt-treated mice

Objective Long-term cardiac output measurements in non-anesthetized mice are now possible. We used this technology to study two different hypertensive models in mice. Design We combined telemetric blood pressure and heart rate recordings with Doppler flow probe cardiac output measurements in mice during treatment with Nω-nitro-l-arginine methyl ester (l-NAME) and deoxycorticosterone acetate (DOCA)–salt. Method The mice received a flowprobe around the ascending aorta and, 10–18 days later, blood pressure telemetry. After recovery, baseline values were recorded and the mice were given l-NAME (5 mg/10 ml tap water), l-NAME followed by valsartan (50 mg/kg per day per gavage), or DOCA–salt (50 mg DOCA-pellet, 0.9% saline to drink, uninephrectomy). Mean arterial pressure, heart rate, stroke volume and cardiac output were recorded daily and total peripheral resistance was calculated. Results l-NAME resulted in an abrupt increase in mean arterial pressure caused solely by an increase in total peripheral resistance. Cardiac output was decreased. Valsartan treatment decreased blood pressure and total peripheral resistance, while cardiac output was restored to normotensive values. DOCA–salt required 3 days before hypertension developed. Contrary to the volume expansion, increased cardiac output, autoregulation hypothesis, the blood pressure increase was only associated with increased total peripheral resistance, while cardiac output was not changed. Conclusion Both l-NAME and DOCA–salt increased blood pressure by increasing total peripheral resistance. Comprehensive hemodynamics can be done in non-anesthetized, free-moving mice. The methods provide new perspectives for studying mouse models in the long-term.

Pressure Natriuresis in AT2 Receptor–Deficient Mice with L-NAME Hypertension

AT(2) receptor-disrupted (AT(2) -/-) mice provide a unique opportunity to investigate the cardiovascular and BP-related effects of NO depletion. This study compared the pressure-diuresis-natriuresis relationship in (AT(2) -/-) and wild-type (AT(2) +/+) mice after treating the animals with L-NAME (130 mg/kg body wt per day) for 1 wk. L-NAME increased mean arterial pressure (MAP) more in AT(2) -/- than in AT(2) +/+ mice (118 +/- 2 versus 108 +/- 4 mmHg). This difference occurred even though L-NAME-treated AT(2) +/+ mice had a greater sodium excretion than AT(2) -/- mice (10.9 +/- 0.5 versus 8.0 +/- 1.0 micro mol/h). The pressure-natriuresis relationship in conscious AT(2) -/- mice was shifted rightward compared with controls. RBF was decreased in AT(2) -/- compared with AT(2) +/+ mice. L-NAME decreased RBF in these mice further from 4.08 +/- 0.43 to 2.79 +/- 0.15 ml/min per g of kidney wt. GFR was not significantly different between AT(2) +/+ and AT(2) -/- mice (1.09 +/- 0.08 versus 1.21 +/- 0.09 ml/min per g of kidney wt). L-NAME reduced GFR in AT(2) -/- to 0.87 +/- 0.07 ml/min per g of kidney wt. Fractional sodium (FE(Na)) and water (FE(H2O)) curves were shifted more strongly to the right by L-NAME in AT(2) -/- mice than in AT(2) +/+ mice. AT(1) receptor blocker treatment lowered BP in both L-NAME-treated strains to basal values. It is concluded that the AT(1) receptor plays a key role in the impaired renal sodium and water excretion induced by NO synthesis blockade. Changes in RBF, GFR, and tubular sodium and water reabsorption are involved and may be also responsible for the greater BP increase in L-NAME-treated AT(2) -/- mice.

Cardiac hypertrophy and fibrosis in chronic L-NAME-treated AT2 receptor-deficient mice.

Background The role of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in cardiac hypertrophy and fibrosis is incompletely understood. The availability of AT2 receptor-deficient mice (AT2 −/y) makes it possible to study the effects of AT1 receptors without the confounding influence of AT2 receptor activity. Objective To test the hypothesis that the AT2 receptor affords protection from left ventricular hypertrophy and fibrosis in chronic hypertension induced by Nω-nitro-l-arginine methyl ester (l-NAME). Design Four groups of mice were studied over a period of 3 weeks: AT2 −/y mice with and without l-NAME, and AT2 +/y mice with and without l-NAME. Methods Blood pressure and heart rate were monitored by telemetry in groups of AT2 +/y and AT2 −/y mice for 4 weeks. l-NAME groups received the compound in drinking water for the last 3 weeks. We determined left ventricular AT1 receptor expression, cardiac hypertrophy and fibrosis, with and without l-NAME treatment. We used a miniaturized conductance-manometer system to measure pressure–volume loops at the time when the animals were killed. Results AT2 −/y mice treated with l-NAME showed worse left ventricular hypertrophy, more perivascular fibrosis and greater concentrations of brain natriuretic peptide than did AT2 +/y mice treated with l-NAME. The end-systolic pressure–volume relationship, an index of left ventricular contractility, was decreased in AT2 −/y mice treated with l-NAME. Conclusions The AT2 receptor is not essential for development of l-NAME-induced cardiac hypertrophy, fibrosis and concomitant changes in left ventricular performance. In contrast, the AT2 receptor offers a protective effect.

Dysferlin-deficient Muscular Dystrophy: Gadofluorine M Suitability at MR Imaging in a Mouse Model

PURPOSE To compare the usefulness of gadofluorine M with that of Gadomer in assessment of dysferlin-deficient muscular dystrophy at 7.0-T magnetic resonance (MR) imaging. MATERIALS AND METHODS All experiments were approved by local review boards. SJL/J mice (n = 24) with dysferlin-deficient muscular dystrophy and C57BL/6 control mice (n = 24) were imaged at 12-15 weeks (young) or older than 30 weeks (old) by using dynamic contrast material-enhanced imaging with inversion-prepared steady-state free-precession sequence before, during, and after administration of gadofluorine M at 2 micromol or Gadomer at 4 micromol intravenously. After imaging, regions of interest were determined from the upper extremity and left ventricular chamber; fractional extravascular extracellular volume, v(e), and permeability surface tissue density product, PS rho, were measured by using a two-compartment pharmacokinetic model. The natural history of muscular dystrophy was assessed histologically in 70 mice (seven five-mouse groups each of SJL/J mice and of control mice) at 4-week intervals from 8 to 32 weeks. In addition, three SJL/J mice and three control mice at age 33 weeks were sacrificed, and fluorescence microscopy was performed for visualization of intravenously administered carbocyanine-labeled gadofluorine M in muscle cells. Statistical analysis was performed by using the t test. RESULTS Gadofluorine M enhancement was significantly greater in skeletal muscle of 30-week-old mice with dysferlin-deficient muscular dystrophy, compared with control mice. Gadofluorine M demonstrated both increased rate of enhancement (PS rho sec(-1) +/- standard error of the mean: 0.004 e(-)(4) +/- 3 vs 0.002 e(-)(4) +/- 3; P < .05) and increased level of enhancement (v(e) +/- standard error of the mean: 0.035 +/- 0.004 vs 0.019 +/- 0.004; P < .05). Gadomer showed no differential enhancement in the two mouse groups. Histologic examination confirmed the presence of labeled gadofluorine M in muscle cells. CONCLUSION Gadofluorine M-enhanced MR imaging may be of value in monitoring dysferlin-deficient muscular dystrophy disease progression in this animal model and could prove to be a useful tool in following the course of chronic muscle diseases in humans.

Sympathetic nerve traffic and circulating norepinephrine levels in RGS2-deficient mice.

Regulator of G protein signaling 2 (RGS2-/-) deficient mice feature an increased resting blood pressure and an excessive pressor response to stress. We measured renal sympathetic nerve activity (RSNA) directly to test the hypothesis that RSNA is increased in RGS2-/- mice, compared to RGS2+/+ mice. Seventeen mice (RGS2-/-, n=9; RGS2+/+, n=8) were anesthetized with isoflurane. We cannulated the left jugular vein for drug administration. Renal sympathetic nerve activity (RSNA) was recorded using bipolar electrodes. Arterial blood pressure (BP) from the femoral artery, ECG (needle electrodes), and RSNA were recorded (sample rate 10 kHz) simultaneously. RSNA was analysed off-line using a modified wavelet de-noising technique and the classical discriminator method. RSNA detected during phenylephrine bolus injections or after the animals death was subtracted from baseline values. Mean arterial blood pressure, norepinephrine plasma levels, the responsiveness to vasoactive drugs, and the sympathetic baroreflex gain were similar in anesthetized RGS2+/+ and RGS2-/- animals. RSNA was lower in RGS2-/- mice compared to wild-type controls (wavelet: spike rate in Hz: RGS2+/+ 25.5+/-5.1; RGS2-/- 17.4+/-4.0; discriminator method: RGS2+/+ 41.4+/-5.7, RGS2-/- 22.0+/-4.3, p<0.05). Thus, the expected result proved not to be the case. Our data suggest a mismatch between sympathetic nerve traffic and plasma norepinephrine concentrations. This observation may depend on altered coupling between electrical nerve activity and norepinephrine release and/or a changed norepinephrine uptake in RGS2-/- mice.