Deoxycorticosterone Acetate Mice Research Articles

Tumor Necrosis Factor Alpha Deficiency Improves Endothelial Function and Cardiovascular Injury in Deoxycorticosterone Acetate/Salt-Hypertensive Mice.

It has been shown that the inflammatory cytokine tumor necrosis factor α (TNFα) plays a role in the development of hypertension and end-stage renal diseases. We hypothesize that TNFα contributes to endothelial dysfunction and cardiac and vascular injury in deoxycorticosterone acetate (DOCA)/salt-hypertensive mice. The wild-type or TNFα-deficient mice were uninephrectomized and implanted with DOCA pellet treatment for 5 weeks; the mice were given either tap water or 1% NaCl drinking water. DOCA mice developed hypertension (systolic blood pressure (SBP): 167 ± 5 vs. 110 ± 4 mmHg in control group, p < 0.05), cardiac and vascular hypertrophy, and the impairment of endothelium-dependent relaxation to acetylcholine (EDR). TNFα deficiency improved EDR and lowered cardiac and vascular hypertrophy with a mild reduction in SBP (152 ± 4 vs. 167 ± 5 mmHg in DOCA group, p < 0.05) in DOCA mice. The mRNA expressions of the inflammatory cytokines, including TNFα, interleukin 1β (IL1β), monocyte chemotactic protein 1 (MCP1), and monocyte/macrophage marker F4/80 were significantly increased in the aorta of DOCA-hypertensive mice; TNFα deficiency reduced these inflammatory gene expressions. DOCA-hypertensive mice also exhibited an increase in the vascular oxidative fluorescence intensities, the protein expressions of gp91phox and p22phox, and the fibrotic factors transforming growth factor β and fibronectin. TNFα deficiency reduced oxidative stress and fibrotic protein expressions. The DOCA mice also showed a decrease in the protein expression of eNOS associated with increased miR155 expression; TNFα deficiency prevented a decrease in eNOS expression and an increase in miR155 expression in DOCA mice. These results support the idea that TNFα significantly contributes to vascular inflammation, vascular dysfunction, and injury in hypertension.

A0407 Astragaloside IV Reduces Cardiac Oxidation and Improves Left Ventricular Diastolic Dysfunction

Objectives: Astragaloside IV (ASI), which is a saponin, has been reported to enhance the activity of eNOS and scavenge reactive oxygen species. We hypothesized that diastolic dysfunction may be associated with reduced eNOS activity that can be reversed by ASI treatment. Methods: We used an established model of isolated diastolic dysfunction caused by deoxycorticosterone acetate (DOCA)-induced mild hypertension. Mice were divided into four groups: sham and DOCA mice with and without ASI treatment. ASI was administrered by intraperitoneal injection of 0.02 mg/kg/day for 7 days. Diastolic function was assessed in vivo by echocardiography and ex vivo in isolated myocytes. eNOS monomer, dimer ratios and phosphorylation were assayed with Western blot analysis. High performance liquid chromatography analysis was used to measure cardiac superoxide anion and biopterins. Results: DOCA mice exhibited diastolic dysfunction (E/E¢:SHAM vs. DOCA 23.90 ± 4.58 vs. 38.83 ± 4.37 p = 0.001) that was reversed after ASI treatment. ASI had no effect on sham animals. Isolated myocytes from the DOCA-salt mice demonstrated impaired relaxation, diastolic sarcomere length and re-lengthening (relaxation constant, τ), which were restored by ASI treatment. The DOCA mice showed increased cardiac superoxide production (2HO-ET level:DOCA vs. DOCA+ASI 0.36 ± 0.19 μmol/mg tissue vs. 0.20 ± 0.05 μmol/mg tissue p = 0.05 and reduced NO production alleviating by ASI. (nitrite/ nitrate content: DOCA vs. DOCA+ASI 2.58 ± 0.49 mmol/mg protein vs. 3.33 ± 0.30 mmol/mg protein p = 0.04). Active eNOS as measured by eNOS dimers andeNOS-S1177 was significantly decreased in DOCA Conclusion: Hypertension induced diastolic dysfunction is associated with increased cardiac oxidation and measures of active eNOS. ASI treatment reversed these changes and improved diastolic dysfunction.

Abstract 440: The Cardiomyocyte Diastolic Stiffness and Contractility are Inversely Related to the Size of Titin: A Cellular Work Loop Study of Single Intact Cardiomyocytes

Studying cardiac titin under physiological conditions can be at the level of the left ventricular (LV) chamber and the multicellular muscle level. However, those preparations are influenced by neurohormonal reflex, preload-afterload status and extracellular matrix. In this study, cellular work loop force measurement of the single intact cardiomyocyte was conducted under physiological conditions. Intact cell experiments were performed at 37°C under field-stimulation at 2 Hz frequency. The cell was attached to the force transducer and the piezo length controller which modulates the cell length by the feedback control system based on developed force. The force length relation (cellular work loop) of the single cell was generated. The end diastolic force length relation (EDFLR), the end systolic force length relation (ESFLR) and the preload recruitable stroke work (PRSW) were obtained. The Ttn ΔIAjxn -/- ( IA -/- ) mice, in which the I-band/A-band region of titin’s spring has been deleted were studied in comparison to α-MHC-Cre ; cRbm20 ΔRRM +/- ( cRbm20 +/- ) mice which are deficient in titin splicing and express large titin isoforms in the heart. The cellular diastolic stiffness (slope of EDFLR) was increased in IA -/- and was reduced in cRbm20 +/- (151.3, 104.0 and 91.5 μN/mm 2 in IA -/- , WT and cRbm20 +/- respectively). The contractility (slopes of ESFLR and PRSW) were 233.5 and 1215.6 uN/mm 2 in WT, and were increased in IA -/- (371.2 and 2076 μN/mm 2 ) but attenuated in cRbm20 +/- (202.9 and 907.7 μN/mm 2 ). Cellular work loop studies were also conducted in intact cells isolated from the mice that underwent transverse aortic constriction (TAC) surgery with deoxycorticosterone acetate (DOCA) implantation. TAC DOCA mice developed diastolic dysfunction at the LV chamber level. The result showed that the cells from TAC DOCA hearts had increased slopes of EDFLR, ESFLR and PRSW ( 157.9, 378.9 and 1699.0 μN/mm 2 ) compared to the cells from sham mice (98.6, 230.3, and 1203.9 μN/mm 2 ). Conclusions: Cellular work loops recapitulated the cardiac physiology at the LV chamber level. The result showed that the cellular diastolic stiffness and contractility were inversely related to the size of titin and that titin stiffness plays an important roles in both diastolic and systolic function.

Age-Related Differential Structural and Transcriptomic Responses in the Hypertensive Heart.

While aging is a critical risk factor for heart failure, it remains uncertain whether the aging heart responds differentially to a hypertensive stimuli. Here we investigated phenotypic and transcriptomic differences between the young and aging heart using a mineralocorticoid-excess model of hypertension. Ten-week (“young”) and 36-week (“aging”) mice underwent a unilateral uninephrectomy with deoxycorticosterone acetate (DOCA) pellet implantation (n = 6–8/group) and were followed for 6 weeks. Cardiac structure and function, blood pressure (BP) and the cardiac transcriptome were subsequently examined. Young and aging DOCA mice had high BP, increased cardiac mass, cardiac hypertrophy, and fibrosis. Left ventricular end-diastolic pressure increased in aging DOCA-treated mice in contrast to young DOCA mice. Interstitial and perivascular fibrosis occurred in response to DOCA, but perivascular fibrosis was greater in aging mice. Transcriptomic analysis showed that young mice had features of higher oxidative stress, likely due to activation of the respiratory electron transport chain. In contrast, aging mice showed up-regulation of collagen formation in association with activation of innate immunity together with markers of inflammation including cytokine and platelet signaling. In comparison to younger mice, aging mice demonstrated different phenotypic and molecular responses to hypertensive stress. These findings have potential implications for the pathogenesis of age-related forms of cardiovascular disease, particularly heart failure.

RAGE-aptamer attenuates deoxycorticosterone acetate/salt-induced renal injury in mice

The mineralocorticoid receptor (MR) and its downstream signaling play an important role in hypertensive renal injury. The interaction of advanced glycation end products (AGE) with their receptor (RAGE) is involved in the progression of renal disease. However, the pathological crosstalk between AGE–RAGE axis and MR system in kidney derangement remains unclear. We screened DNA-aptamer directed against RAGE (RAGE-apt) in vitro and examined its effects on renal injury in uninephrectomized deoxycorticosterone acetate (DOCA)/salt-induced hypertensive mice. RAGE, GTP-bound Rac-1 (Rac1), and MR were co-localized in the podocytes of DOCA mice. The deletion of RAGE gene significantly inhibited mesangial matrix expansion and tubulointerstitial fibrosis in DOCA mice, which was associated with the reduction of glomerular oxidative stress, MR, Rac1, and urinary albumin excretion (UAE) levels. RAGE-apt attenuated the increase in carboxymethyllysine (CML), RAGE, nitrotyrosine, Rac1, and MR levels in the kidneys and reduced UAE in DOCA mice. Aldosterone (Aldo) increased nitrotyrosine, CML, and RAGE gene expression in murine podocytes, whereas CML stimulated MR and Rac1 levels, which were blocked by RAGE-apt. The present study indicates the crosstalk between the AGE–RAGE axis and Aldo–MR system, suggesting that RAGE-apt may be a novel therapeutic tool for the treatment of MR-associated renal diseases.

Tetrahydrobiopterin improves left ventricular diastolic function possibly through upregulating phosphorylated protein kinase B expression in hypertensive mice induced by deoxycorticosterone acetate

Objective: To investigate whether tetrahydrobiopterin (BH4) could improve left ventricular diastolic function through phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) signaling pathway in hypertensive mice. Methods: Ten-week-old male C57BL/6 mice were used to establish the deoxycorticosterone acetate (DOCA)-salt hypertensive model, age matched Sham mice serve as the controls. Mice were divided into four groups: Sham(n=20), Sham+ BH4 (n=20), DOCA (n=22), and DOCA+ BH4 (n=22). On the 14 days after surgery, mice in Sham+ BH4 and DOCA+ BH4 groups received BH4 (0.1 ml/10 g) supplement for 7 days, while mice in Sham and DOCA groups were given equal volume of normal saline.The blood pressure measurements were performed 7 days later.Hemodynamic and echocardiographic parameters were used to assess left ventricular functions.High performance liquid chromatography (HPLC) analysis was used to measure cardiac biopterins BH4 and BH2.The phosphorylated phospholamban (p-PLB) was detected by immunohistochemical staining. PI3K, Akt and phosphorylated Akt were assayed with Western blot analysis. Results: (1) The systolic and diastolic blood pressure of DOCA group were significantly higher than control group (P<0.05). Compared with DOCA group, the systolic blood pressure was lower in DOCA+ BH4 mice (P=0.027). Diastolic blood pressure was similar between the groups. (2) Compared with Sham group, the left ventricular diastolic function indexes such as mitral annulus velocity (E') and E'/A'ratio were significantly lower, while the E/ E'ratio was significantly higher(P<0.05)in DOCA mice. The E/ E'ratio of DOCA+ BH4 group was significantly lower than that of DOCA group (P<0.05). Compared with Sham group, the left ventricular end-diastolic pressure (LVEDP), left ventricular end-diastolic pressure volumetric coefficient (EDPVR) and left ventricular relaxation time constant Tau index were significantly higher in DOCA mice (P=0.002, 0.011 and 0.016, respectively). The EDPVR and Tau index were significantly lower in DOCA+ BH4 group than in DOCA group (P<0.05). (3) Compared with Sham group, the myocardial contents of BH4 and BH2 were significantly lower in DOCA mice (P<0.05). The BH4 levels and BH4/BH2 ratio were significantly higher in Sham+ BH4 and DOCA+ BH4 groups than in the DOCA group (P<0.05), but the BH2 levels were similar between groups. (4) The cGMP content, SOD activity and NO content in the left ventricular myocardial tissue were significantly lower (P<0.05), while the MDA content was significantly higher in DOCA mice than in Sham mice.The NO content and SOD activity in DOCA+ BH4 groups were significantly higher than in the DOCA group (P<0.05). (5) Compared with DOCA group, the expression of p-PLB was significantly higher in Sham mice and lower in DOCA+ BH4 mice (P<0.05). (6) The expression of PI3K, Akt and p-Akt (Ser473 and Thr308) in DOCA mice were significantly lower than in Sham group (P<0.05). The expression of PI3K, Akt and p-Akt (Ser473) was significantly higher in DOCA+ BH4 group than in DOCA group (P<0.05). p-Akt (Thr308) expression was similar between DOCA + BH4 group and DOCA group (all P>0.05). Conclusion: Our results suggest that BH4 could improve left ventricular diastolic function in hypertensive mice, this effect might be mediated by reducing the oxidative stress in ventricular myocardium through modlating the expression of Akt and PLB phosphorylation.

Role of angiotensin II type 1a receptor in renal injury induced by deoxycorticosterone acetate-salt hypertension.

The aim of this study was to investigate the in vivo role of angiotensin II type 1a (AT1a) receptor in renal damage as a result of hypertension by using transgenic mice with AT1a receptor gene disruption. Transgenic mice that express human liver-type fatty acid binding protein (L-FABP) with or without disruption of the AT1a receptor gene (L-FABP+/− AT1a−/−, and L-FABP+/− AT1a+/+, respectively) were used with urinary L-FABP as an indicator of tubulointerstitial damage. Those female mice were administered subcutaneously deoxycorticosterone acetate (DOCA)–salt tablets plus drinking water that contained 1% saline for 28 d after uninephrectomy. In L-FABP+/− AT1a+/+ mice that received DOCA-salt treatment, hypertension was induced and slight expansion of glomerular area, glomerular sclerosis, and tubulointerstitial damage were observed. In L-FABP+/− AT1a−/− mice that received DOCA-salt treatment, hypertension was similarly induced and the degree of glomerular damage was significantly more severe than in L-FABP+/− AT1a+/+-DOCA mice. Urinary L-FABP levels were significantly higher in L-FABP+/− AT1a−/−-DOCA mice compared with those in L-FABP+/− AT1a+/+-DOCA mice. Hydralazine treatment significantly attenuated renal damage that was found in L-FABP+/− AT1a−/−-DOCA mice along with a reduction in blood pressure. In summary, activation of the AT1a receptor may contribute to maintenance of the glomerular structure against hypertensive renal damage.—Hisamichi, M., Kamijo-Ikemori, A., Sugaya, T., Ichikawa, D., Natsuki, T., Hoshino, S., Kimura, K., Shibagaki, Y. Role of angiotensin II type 1a receptor in renal injury induced by deoxycorticosterone acetate–salt hypertension.

OS 02-04 ASTRAGALOSIDE IV IMPROVES LEFT VENTRICULAR DIASTOLIC DYSFUNCTION IN HYPERTENSIVE MICE BY INCREASING THE PHOSPHORYLATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE

Objective: The saponin Astragaloside IV (ASI) reportedly enhances endothelial NOS (eNOS) activity by increasing the scavenging of reactive oxygen species (ROS) and NO production. We hypothesized that oxidative stress may decrease eNOS phosphorylation and result in diastolic dysfunction in DOCA-salt mice, which could be reversed by ASI treatment. Design and Method: We used the deoxycorticosterone acetate (DOCA)-salt mouse model. The mice were divided into four groups; SHAM and DOCA control mice received sterile water, SHAM+ASI and DOCA+ASI received ASI treatment via an intraperitoneal injection of 0.02 mg/kg/day for 7 days. Diastolic function was assessed by echocardiography and IonOptix. The levels of eNOS monomers, dimers and phosphorylation were determined with a Western blot analysis. High performance liquid chromatography was used to measure the cardiac superoxide anions and biopterins. Results: DOCA mice exhibited diastolic dysfunction that was reversed after ASI treatment (E′/A′: SHAM vs. DOCA 1.27 ± 0.13 vs. 0.62 ± 0.07, p < 0.001; DOCA vs. DOCA+ASI 0.62 ± 0.07 vs. 1.27 ± 0.29, p = 0.002. E/E': SHAM vs. DOCA 23.90 ± 4.58 vs. 38.83 ± 4.37, p = 0.001; DOCA vs. DOCA+ASI 23.90 ± 4.58 vs. 25.28 ± 3.40, p = 0.001). Isolated myocytes from the DOCA-salt mice demonstrated impaired relaxation, diastolic sarcomere length and re-lengthening, which were restored by ASI treatment. The DOCA mice showed increased superoxide production and reduced NO production, which were alleviated by ASI. Our results showed that the levels of eNOS dimers, eNOS-S1177 and eNOS-T495 were significantly decreased in the DOCA-salt mice compared with the SHAM mice. The level of eNOS-S1177 was increased in the DOCA+ASI mice compared with the untreated DOCA-salt mice (p = 0.026); however, ASI did not affect the phosphorylation of eNOS-T495. MyBP-C S-glutathionylation increased after ASI treatment in the DOCA+ASI mice compared with the DOCA-salt hypertensive mice (p = 0.046). Conclusions: The decrease in eNOS dimerization and phosphorylation correlated with diastolic dysfunction, which was reversed by ASI in DOCA mice. The anti-oxidative effects of ASI on MyBP-C glutathionylation and eNOS phosphorylation preserved cardiac relaxation.

Abstract 207: Mitochondrial Calcium Flux is Arrhythmogenic in Nonischemic Cardiomyopathy

Introduction: Mitochondria participate in Ca2+ homeostasis and Ca2+ oscillations are thought to be involved in arrhythmogenesis. Nevertheless, the role of mitochondria in arrhythmogenesis is unclear. We have reported spontaneous and induced arrhythmias in a mouse model of nonischemic cardiomyopathy, so we investigated the role of mitochondrial Ca2+ handling in arrhythmias in this model. Methods: Nonischemic cardiomyopathy was induced in C57BL/6 mice by 6 weeks of hypertension evoked after unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and 1% salt water substitution. Sham operated mice were used as controls. ECG telemetry recording was used to monitor arrhythmias from DOCA mice and 7 sham mice at 18 weeks of age. Action potentials (APs) were recorded by perforated current-clamp in isolated mouse ventricular cells. Potassium and L-type Ca2+ currents were measured by voltage-clamp. Changes in cytoplasmic and mitochondrial Ca2+ were determined by fluorescent imaging using Fluo-4 and Rhod-2, respectively, in isolated ventricular cells. Results: Seven DOCA mice and two sham mice had isoproterenol (0.5 mg/kg)-induced premature ventricular contractions (PVCs, p&lt;0.05). Three DOCA mice and no sham mice had isoproterenol (2.5 mg/kg)-induced ventricular fibrillation. QTc was significantly prolonged from 41.9 ±1.4 in sham mice to 52.0 ± 2.1 ms in DOCA mice. At the cellular levels, myocytes had a prominent increase of APD90 from 66.6 ± 21.1 to 311.8 ± 44.9 ms, explained by augmented L-type Ca2+ current and decreased total K+ currents. Two of 12 sham mice ventricular cells and 8 from 12 DOCA mice ventricular cells had EADs (p&lt;0.05). Three of 12 DOCA mice but none of the sham myocytes had PVCs with stimulated APs. Trigger activity could be abolished by 10 µM Ru360, a mitochondrial calcium uniporter specific antagonist. Ru360 had no effect on L-type Ca2+ current, total K+ currents, or APD90. Ru360 inhibited mitochondrial Ca2+ uptake in DOCA mice ventricular cells, implicating mitochondrial Ca2+ release in modulating the arrhythmic risk. Conclusions: Mitochondrial Ca2+ handling appears to play an important role in triggered activity and may be important in the incidence of ventricular arrhythmias in nonischemic cardiomyopathy.

Mitochondrial dysfunction causing cardiac sodium channel downregulation in cardiomyopathy

Cardiomyopathy is associated with cardiac Na+ channel downregulation that may contribute to arrhythmias. Previously, we have shown that elevated intracellular NADH causes a decrease in cardiac Na+ current (INa) signaled by an increase in mitochondrial reactive oxygen species (ROS). In this study, we tested whether the NADH–mitochondria ROS pathway was involved in the reduction of INa in a nonischemic cardiomyopathic model and correlated the findings with myopathic human hearts. Nonischemic cardiomyopathy was induced in C57BL/6 mice by hypertension after unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and salt water substitution. Sham operated mice were used as controls. After six weeks, heart tissue and ventricular myocytes isolated from mice were utilized for whole cell patch clamp recording, NADH/NAD+ level measurements, and mitochondrial ROS monitoring with confocal microscopy. Human explanted hearts were studied using optical mapping. Compared to the sham mice, the arterial blood pressure was higher, the left ventricular volume was significantly enlarged (104.7±3.9 vs. 87.9±6.1μL, P<0.05), and the ejection fraction was reduced (37.1±1.8% vs. 49.4±3.7%, P<0.05) in DOCA mice. Both the whole cell and cytosolic NADH level were increased (279±70% and 123±2% of sham, respectively, P<0.01), INa was decreased (60±10% of sham, P<0.01), and mitochondrial ROS overproduction was observed (2.9±0.3-fold of sham, P<0.01) in heart tissue and myocytes of myopathic mice vs. sham. Treatment of myocytes with NAD+ (500μM), mitoTEMPO (10μM), chelerythrine (50μM), or forskolin (5μM) restored INa back to the level of sham. Injection of NAD+ (100mg/kg) or mitoTEMPO (0.7mg/kg) twice (at 24h and 1h before myocyte isolation) to animals also restored INa. All treatments simultaneously reduced mitochondrial ROS levels to that of controls. CD38 was found to transduce the extracellular NAD+ signal. Correlating with the mouse model, failing human hearts showed a reduction in conduction velocity that improved with NAD+. Nonischemic cardiomyopathy was associated with elevated NADH level, PKC activation, mitochondrial ROS overproduction, and a concomitant decrease in INa. Reducing mitochondrial ROS by application of NAD+, mitoTEMPO, PKC inhibitors, or PKA activators, restored INa. NAD+ improved conduction velocity in human myopathic hearts.

Abstract 139: Doca-salt Induced Vascular Dysfunction and Elevated Arginase Activity is Prevented in Haploinsufficient Rho Kinase 1 Mice

Endothelial dysfunction is an established phenomenon in deoxycorticosterone acetate (DOCA)-salt hypertension. It has been reported that increased vascular arginase activity is involved with hypertension. We previously reported that the small GTPase, RhoA and Rho kinase (ROCK) are involved with activation of arginase in diabetic and angiotensin II hypertensive models. In this study, we hypothesized that partial knockdown of ROCK1 would prevent vascular endothelial dysfunction in DOCA-salt hypertension by blocking the rise in vascular arginase activity. We tested this concept using wild type and haploinsufficient ROCK1 knockout (R1 +/- ) mice, which were treated by DOCA pellet (50 mg) for 6 weeks and fed by 1% saline. Systolic blood pressure (SBP) was measured before and during 6 week treatment from wild type sham (WT), wild type DOCA (WT-D), ROCK1 sham (R1 +/- ) and ROCK1 DOCA (R1 +/- -D) mice (N = 6). SBP in WT-D mice rose over the 6 weeks treatment (from 104.6.6±2.6 mmHg to 124.4±5.6 mmHg) but not in WT mice (from 108.9±1.2 mmHg to 104.60±4.27 mmHg, p&lt;0.05). At 6 weeks, SBP in R1 +/- -D mice (108.4±2.8 mmHg) was slightly higher than R1 +/- mice (98.9±1.6 mmHg, p&lt;0.05). However, SBP in R1 +/- -D mice is significantly lower than WT-D mice during the whole treatment period. Additionally, vascular function was assessed in aortic segments (N = 4-6). We found WT-D mice exhibited a marked reduction of maximum endothelium-dependent relaxation (MER) to acetylcholine (57.6±4.3%, p&lt;0.05) compared to WT mice (70.6±1.6%). Impairment of relaxation was absent in R1 +/- -D mice (MER: 82.9±2.8%, p&lt;0.05). Arginase activity in aorta of WT-D mice (152.7±18.7 units) was ∼45% higher than in WT mice (105.3±2.2 units) and ∼225% higher than in R1 +/- -D mice (67.1±5.8 units). These data demonstrate involvement of ROCK1 in enhanced arginase activity and impaired endothelium dependent vasorelaxation in DOCA-salt hypertensive mice.

Impact of salt on cardiac differential gene expression and coronary lesion in normotensive mineralocorticoid-treated mice

We previously reported that excess of deoxycorticosterone-acetate (DOCA)/salt-induced cardiac hypertrophy in the absence of hypertension in one-renin gene mice. This model allows us to study molecular mechanisms of high-salt intake in the development of cardiovascular remodeling, independently of blood pressure in a high mineralocorticoid state. In this study, we compared the effect of 5-wk low- and high-salt intake on cardiovascular remodeling and cardiac differential gene expression in mice receiving the same amount of DOCA. Differential gene and protein expression was measured by high-density cDNA microarray assays, real-time PCR and Western blot analysis in DOCA-high salt (HS) vs. DOCA-low salt (LS) mice. DOCA-HS mice developed cardiac hypertrophy, coronary perivascular fibrosis, and left ventricular dysfunction. Differential gene and protein expression demonstrated that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation and extracellular matrix remodeling (e.g., Col3a1, Col1a2, Hmox1, and Lcn2). A major subset of downregulated genes encoded for transcription factors, including myeloid differentiation primary response (MyD) genes. Our data provide some evidence that vascular remodeling, fibrosis, and inflammation are important consequences of a high-salt intake in DOCA mice. Our study suggests that among the different pathogenic factors of cardiac and vascular remodeling, such as hypertension and mineralocorticoid excess and sodium intake, the latter is critical for the development of the profibrotic and proinflammatory phenotype observed in the heart of normotensive DOCA-treated mice.

Ranolazine prevents heart failure with preserved ejection fraction, diastolic dysfunction, and left ventricular hypertrophy in a hypertensive murine model

Background: Recent in vitro studies on failing human heart muscle has suggested that ranolazine (RAN), a novel antianginal drug known to inhibit late sodium current, may improve diastolic dysfunction (DD) caused by hypertension (HTN)-induced oxidative stress. By improving DD, RAN can improve heart failure with preserved ejection fraction (HFPEF). Methods: Amouse model of DDwas developed by inducing mild HTN by unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and substituting drinking water with 1% NaCl. Control animals underwent sham operation and drank regular tap water. RAN 50 (mg/kg) was given orally twice a day. Weekly blood pressures (BPs) and heart rates (HRs) were measured using tail-cuff plethysmography. Cardiac function and left ventricular posterior wall thickness (LVPWd) were measured by echocardiogram under light anesthesia using isoflurane. Treatment durationwas for 3weeks and 7weeks. Pulmonary congestion was assessed by lung to body weight (L/BW) ratio. Results: Compared to controls, DOCA mice were hypertensive (BP 112 ±4 vs. 89±2 mmHg) and had DD evidenced by a low LV inflow propagation velocity Vp (28.1±0.5 vs. 47.2±1 cm/s⁎), a low mitral annulus early longitudinal velocity E' (2.70 vs. 4.40 cm/s⁎), and a high E/E' ratio (29.3±1.5 vs. 18.0±0⁎). DOCA mice had LVH as evidenced by high heart/BW ratio (6.22±0.15 vs. 4.90±0.22 mg/g⁎) and increased LVPWd (0.82±0.22 vs. 0.74±0.01 mm⁎). There was no systolic dysfunction in either group. RAN pretreatment effectively prevented DD and LVH in DOCA mice as evidenced by high E' (3.9 Vs 2.70 cm/s⁎), low E/E' (18.8±1.0⁎), high Vp (41±2Vs 28.1±0.5 cm/s⁎), lowheart/BW ratio (4.79±0.11 mg/g⁎), and low LVPWd thickness (0.76±0.01 vs. 0.82±0.02 mm⁎). RAN had no effect on HR [625±18 vs. 630±20 bpm, P=not significant (NS)]. RAN pretreatment effectively prevented development of heart failure in DOCA mice at 7 weeks as evidenced by significantly lower L/BW ratios when compared with untreated DOCA mice (0.41±0.002 vs. 0.52±0.02 mg/g⁎). Conclusions: RANhas prevented DD, LVH, and HFPEF in a hypertensive murine model of DD. Prevention of heart failure in our model warrants additional animal andhuman studies to further evaluate the therapeutic potential of RAN in HFPEF (Mean±S.E.M., ⁎Pb.05).

PLATINUM Japan SV: platinum chromium everolimus-eluting stent in small vessels

Background: Recent in vitro studies on failing human heart muscle has suggested that ranolazine (RAN), a novel antianginal drug known to inhibit late sodium current, may improve diastolic dysfunction (DD) caused by hypertension (HTN)-induced oxidative stress. By improving DD, RAN can improve heart failure with preserved ejection fraction (HFPEF). Methods: Amouse model of DDwas developed by inducing mild HTN by unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and substituting drinking water with 1% NaCl. Control animals underwent sham operation and drank regular tap water. RAN 50 (mg/kg) was given orally twice a day. Weekly blood pressures (BPs) and heart rates (HRs) were measured using tail-cuff plethysmography. Cardiac function and left ventricular posterior wall thickness (LVPWd) were measured by echocardiogram under light anesthesia using isoflurane. Treatment durationwas for 3weeks and 7weeks. Pulmonary congestion was assessed by lung to body weight (L/BW) ratio. Results: Compared to controls, DOCA mice were hypertensive (BP 112 ±4 vs. 89±2 mmHg) and had DD evidenced by a low LV inflow propagation velocity Vp (28.1±0.5 vs. 47.2±1 cm/s⁎), a low mitral annulus early longitudinal velocity E' (2.70 vs. 4.40 cm/s⁎), and a high E/E' ratio (29.3±1.5 vs. 18.0±0⁎). DOCA mice had LVH as evidenced by high heart/BW ratio (6.22±0.15 vs. 4.90±0.22 mg/g⁎) and increased LVPWd (0.82±0.22 vs. 0.74±0.01 mm⁎). There was no systolic dysfunction in either group. RAN pretreatment effectively prevented DD and LVH in DOCA mice as evidenced by high E' (3.9 Vs 2.70 cm/s⁎), low E/E' (18.8±1.0⁎), high Vp (41±2Vs 28.1±0.5 cm/s⁎), lowheart/BW ratio (4.79±0.11 mg/g⁎), and low LVPWd thickness (0.76±0.01 vs. 0.82±0.02 mm⁎). RAN had no effect on HR [625±18 vs. 630±20 bpm, P=not significant (NS)]. RAN pretreatment effectively prevented development of heart failure in DOCA mice at 7 weeks as evidenced by significantly lower L/BW ratios when compared with untreated DOCA mice (0.41±0.002 vs. 0.52±0.02 mg/g⁎). Conclusions: RANhas prevented DD, LVH, and HFPEF in a hypertensive murine model of DD. Prevention of heart failure in our model warrants additional animal andhuman studies to further evaluate the therapeutic potential of RAN in HFPEF (Mean±S.E.M., ⁎Pb.05).

Abstract P240: Mitochondrial Reactive Oxygen Species Regulate the Cardiac Na + Channel in Heart Failure

Background: Previously, we have shown that elevated intracellular NADH causes a decrease in cardiac Na + current ( I Na ) signaled by an increase in mitochondrial reactive oxygen species (ROS). The decrease in I Na can be ameliorated by NAD + or a mitochondrial specific anti-oxidant, mitoTEMPO. It is known that cardiomyopathy is associated with reduced I Na . Therefore, we tested whether the NADH-mitochondrial ROS pathway was involved in the reduction in I Na in cardiomyopathic mice. Methods: Nonischemic cardiomyopathy was induced in C57BL/6 mice 6 weeks after unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and salt water substitution. Sham operated mice were used as controls. Ventricular myocytes isolated from mice were utilized for whole-cell patch clamp recording, intracellular NADH/NAD + level measurements, and mitochondrial ROS monitoring with confocal microscopy. Results: Compared to the sham mice, the left ventricular volume was significantly enlarged (105.5 ± 4.4 µL vs. 91.6 ± 4.0 µL, P&lt;0.05), and the ejection fraction was significantly reduced (39.2 ± 1.6% vs. 48.7 ± 1.6%, P&lt;0.05) in DOCA mice. Intracellular NADH level was increased (2.78-fold; P&lt;0.01), and I Na was decreased (62±8%; P&lt;0.01) in myocytes of DOCA mice vs. sham. NAD + (500 μ M) and mitoTEMPO (10 μ M) recovered I Na (90±9% and 103±9% of sham, respectively, P&gt;0.05). Mitochondrial ROS overproduction was observed with DOCA mice myocytes by MitoSOX Red (∼4.8-fold of sham; P&lt;0.01). NAD + and mitoTEMPO decreased ROS in DOCA mice myocytes (∼20±6% of DOCA for both, P&lt;0.01). Conclusions: NADH and mitochondrial ROS were elevated, and I Na was decreased in nonischemic cardiomyopathy. Maneuvers that reduced mitochondrial ROS restored I Na . Since reduced I Na and the subsequent slow conduction velocity are thought to contribute to arrhythmic risk in this condition, NAD + and mitochondrial anti-oxidants may have anti-arrhythmic activity in cardiomyopathy.

Mineralocorticoid Accelerates Transition to Heart Failure With Preserved Ejection Fraction Via “Nongenomic Effects”

Mechanisms promoting the transition from hypertensive heart disease to heart failure with preserved ejection fraction are poorly understood. When inappropriate for salt status, mineralocorticoid (deoxycorticosterone acetate) excess causes hypertrophy, fibrosis, and diastolic dysfunction. Because cardiac mineralocorticoid receptors are protected from mineralocorticoid binding by the absence of 11-beta hydroxysteroid dehydrogenase, salt-mineralocorticoid-induced inflammation is postulated to cause oxidative stress and to mediate cardiac effects. Although previous studies have focused on salt/nephrectomy in accelerating mineralocorticoid-induced cardiac effects, we hypothesized that hypertensive heart disease is associated with oxidative stress and sensitizes the heart to mineralocorticoid, accelerating hypertrophy, fibrosis, and diastolic dysfunction. Cardiac structure and function, oxidative stress, and mineralocorticoid receptor-dependent gene transcription were measured in sham-operated and transverse aortic constriction (studied 2 weeks later) mice without and with deoxycorticosterone acetate administration, all in the setting of normal-salt diet. Compared with sham mice, sham plus deoxycorticosterone acetate mice had mild hypertrophy without fibrosis or diastolic dysfunction. Transverse aortic constriction mice displayed compensated hypertensive heart disease with hypertrophy, increased oxidative stress (osteopontin and NOX4 gene expression), and normal systolic function, filling pressures, and diastolic stiffness. Compared with transverse aortic constriction mice, transverse aortic constriction plus deoxycorticosterone acetate mice had similar left ventricular systolic pressure and fractional shortening but more hypertrophy, fibrosis, and diastolic dysfunction with increased lung weights, consistent with heart failure with preserved ejection fraction. There was progressive activation of markers of oxidative stress across the groups but no evidence of classic mineralocorticoid receptor-dependent gene transcription. Pressure-overload hypertrophy sensitizes the heart to mineralocorticoid excess, which promotes the transition to heart failure with preserved ejection fraction independently of classic mineralocorticoid receptor-dependent gene transcription.

Pyk2 mediates increased adrenergic contractile responses in arteries from DOCA-salt mice — Vasoactive Peptide Symposium

The calcium-dependent proline-rich tyrosine kinase (Pyk2), a nonreceptor protein activated by tyrosine phosphorylation, links G protein-coupled receptors to vascular responses. We tested the hypothesis that enhanced vascular reactivity in deoxycorticosterone acetate (DOCA)-salt hypertensive mice is due to increased activation of Pyk2. Aorta and small mesenteric arteries from DOCA-salt and uninephrectomized (UNI) male C57Bl/6 mice were used. Systolic blood pressure (mm Hg) was higher in DOCA (126 ± 3) vs. UNI (100 ± 4) mice. Vascular responses to phenylephrine (1 nM to 100 μM) were greater both in aorta and small mesenteric arteries from DOCA-salt than UNI, but treatment with Tyrphostin A-9 (0.1 μM, Pyk2 inhibitor) abolished the difference among the groups. Pyk2 levels, as well as phospho-Pyk2 Tyr402, paxillin, and phospho-paxillin Tyr118 were increased in DOCA-salt aorta. Incubation of vessels with Tyrphostin A-9 restored phosphorylation of Pyk2 and paxillin. Increased activation of Pyk2 contributes to increased vascular contractile responses in DOCA-salt mice.

Axl Mediates Vascular Remodeling Induced by Deoxycorticosterone Acetate–Salt Hypertension

Axl, a receptor tyrosine kinase, was recently identified as a novel candidate gene in a genetic model of salt-sensitive hypertension (Sabra rat). Our group first reported that Axl plays a significant role in vascular remodeling in response to injury. Here we investigated the role of Axl in the pathogenesis of hypertension in a deoxycorticosterone acetate (DOCA)-salt model. Hypertension was induced in Axl wild-type (Axl(+/+)) mice and Axl-deficient (Axl(-/-)) mice by uninephrectomy and DOCA-salt for 6 weeks. Controls were uninephrectomized and received tap water and regular chow ad libitum. DOCA-salt treatment increased systolic blood pressure by 25 mm Hg in both genotypes after 1 week. Systolic blood pressure remained significantly elevated in Axl(+/+) DOCA, whereas systolic blood pressure levels in Axl(-/-) DOCA mice were the same as controls at 6 weeks. DOCA-salt increased relative kidney weight and glomerular hypertrophy by 40% compared with controls in both genotypes. Consistent with levels of systolic blood pressure, endothelium-dependent vasorelaxation was impaired in Axl(+/+) DOCA mice compared with Axl(+/+) controls, whereas in Axl(-/-) DOCA mice relaxation responses were similar to Axl(-/-) controls. In addition, endothelium-independent vasorelaxation was improved in Axl(-/-) DOCA mice compared with Axl(+/+) DOCA mice. Nitrotyrosine and phospho-Akt immunoreactivity was significantly reduced in arteries from Axl(-/-) DOCA mice compared with Axl(+/+) DOCA mice. The remodeling index of the mesenteric artery (media:lumen ratio) was significantly increased in Axl(+/+) DOCA mice compared with Axl(-/-) DOCA mice. Finally, increased vascular apoptosis in the Axl(-/-) DOCA mice suggests a likely mechanism for Axl-dependent effects on hypertension. These data strengthen the pathogenic role for Axl in salt-sensitive hypertension.

Preserved expression of GLUT4 prevents enhanced agonist-induced vascular reactivity and MYPT1 phosphorylation in hypertensive mouse aorta

We previously showed that GLUT4 expression is decreased in arterial smooth muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats and that GLUT4-knockout mice have enhanced arterial reactivity. Therefore, we hypothesized that increased GLUT4 expression in vascular smooth muscle in vivo would prevent enhanced arterial reactivity and possibly reduce blood pressure in DOCA-salt hypertensive mice. Adult wild-type (WT) and GLUT4 transgenic (TG) mice were subjected to DOCA-salt hypertension with uninephrectomy or underwent uninephrectomy and remained normotensive. GLUT4 expression was increased more than twofold in the aortas of GLUT4 TG mice compared with WT aortas. Eight weeks after implantation of the DOCA pellets, GLUT4 expression decreased by 75% in aortas of WT hypertensive mice, but not in GLUT4 TG hypertensive aortas. Systolic blood pressure was significantly and similarly increased in WT and GLUT4 TG DOCA-salt mice compared with their respective sham-treated controls (159 vs. 111 mmHg). Responsiveness to the contractile agonist 5-HT was significantly increased in aortic rings from WT DOCA-salt mice but remained normal in GLUT4 TG DOCA mice. Phosphorylation of the myosin phosphatase targeting subunit MYPT1 was significantly enhanced in aortas of WT DOCA-salt mice, and this increase was prevented in GLUT4 TG mice. MYPT1 phosphorylation was also increased in nonhypertensive GLUT4-knockout mice. Myosin phosphatase, a major negative regulator of calcium sensitivity, is itself negatively regulated by phosphorylation of MYPT1. Therefore, our results show that preservation of GLUT4 expression prevents enhanced arterial reactivity in hypertension, possibly via effects on myosin phosphatase activity.

Effect of the angiotensinogen genotype on experimental hypertension in mice

Polymorphisms of the angiotensinogen (Agt) gene may affect blood pressure. We used a mouse model to test for the role of the Agt genotype in low-renin or high-renin forms of hypertension. Mice bearing one, two, three, or four copies of the Agt gene underwent renal artery clipping to induce high-renin two-kidney, one-clip renovascular hypertension (2K1C), or uninephrectomy, salt loading, and application of deoxycorticosterone-acetate (DOCA) pellets to induce low-renin mineralocorticoid hypertension. Appropriate control animals were also studied. Blood pressure was measured by tail cuff as well as by direct intra-arterial recordings. There was a small effect of the Agt genotype on baseline blood pressure before induction of hypertension. The extent of 2K1C hypertension was not affected by the genotype. In contrast, there was a marked gene-dose effect on DOCA-hypertension (21.2 mmHg over all genotypes). Treatment of DOCA mice with the angiotensin II type 1 receptor antagonist abolished the genotype effect on blood pressure and left ventricular hypertrophy. There was a trend towards less suppression of endogenous aldosterone by DOCA treatment with increasing number of Agt gene copies. We conclude that the Agt genotype exerts a marked effect on blood pressure in a low-renin form of hypertension but no effect in the face of stimulated renin, at least in mice.